Haematological Findings in Kala Azar, HIV, and Kalaazar

HaematologicalfindingsinKalaazar,HIV,andKala
azar/HIVcoinfectedpatientsincludingT.lymphocytes
differentialcountbybothflowcytometryanddouble
immunocytochemistry
A Thesis Submitted for Fulfillment of Master Degree in Medica Laboratory

Sciences (Hematology & Immunohematology)
By:
Awad Elkareem Abbas Mahmoud
B.Sc. Honor,
Hematology&Immunohematology
Medical Laboratory Sciences, U.of.K.
Supervisor:
Prof. Sayda H. El-Safi
MBBS, MSc., DTM &H,Ph.D.London
Professor of Immunology,
Faculty of Medicine, U. of K.
Co-Supervisor:
Dr. Maria Satti
Associate professor, Department of Pathology
Faculty of Medicine, U. of K.
April 2007
Dedication
To
My Parents
My Brothers,
My Sisters
My Friends
I dedicate my efforts, my work, my love and my live.
Awad Elkareem
Acknowledgment
I would like to express my deep sincere gratitude, appreciation, and
thanks to my supervisor Prof. Sayda Hassan Elsafi for her supervision,
patience, and unlimited help through the whole process of the research.
Special thanks to Dr. Maria satti for her help and guidance.
Lot of thanks to DAAD organization for giving me a scholarship fund
to process this research.
My thanks go to Dr. Ahmed Abdel halim for his unlimtted help.
I would like to thank Us. Ahmed Abdel hafiez Nimer and all the staff
of Hematology; Faculty of Medical Laboratory Sciences for their help and
encouragement.
Special thanks to Dr. Omer Nimeery and all staff of VCT at Om
durman Teaching Hospital for their help and cooperation.
My thanks are also extending to all medical staff of Tabark Alla and
Al-azaza Kalazar centres for their continuous help.
Last but not least, I would like to thank my family for their continuous
support and encouragement.

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Abstract
A descriptive study was conducted in two kalazar centers in Tabarak-Allah
(Gedarif state) and El- Azaza (Singa state), Eastern Sudan.
160 VL patients out of 520 suspected VL cases were included. Diagnoses
was made by direct microscopy using lymph node and/or B.M aspirates and
confirmed serologicaly using DAT and/or Katex. There were 96(60%)
males, 64(40%) females; mean of patient's ages was 14 years.
Full blood count were done for all VL patients. Anemia was found in all,
(mean of Hb: 8.5 g/dl), 80(50%) o them had a normocytic normochromic
anemia, 16(10%) with microcytic hypochromic, 57(35.4%) with dimorphic
anemia, and sickle cells anemia was found in 7 (4.6%) patients. 152 (95%)
patients had a platelet count of < 140X103/ l, 128(80%) patients with
TWBCs < 3X 103/l. The differential WBCs count was a follows:
lymphocytes were > 40% in 112(70%) patients, neutrophils were < 45% in
89(55%) patients, and ESR was > 45 mm/h in 50/50 patients (100%). WBCs
morphology was normal in 110(69%) patients, shift to left was found in
40(25%) patients, and shift to right was found in 6(10%) patients. Prominent
Target cells were found in 54(34%) patients and nucleated RBCs in 24(15%)
patients.
The differential T cells count was done for 5 patients. CD4 T cells were <
250 / l in all 5 patients. The mean of CD4 was 210 / l, the mean of CD8
was 490 / l, and CD4/CD8 ratio was 1/2.3.
II
All parasitologicaly and serologicaly confirmed VL patients were screened

for HIV, 6 (3.5%) patients were co-infected, all of them were males, mean of
patients ages was 25 years.
Full blood counts were done for these patients. All patients were anemic
(mean of Hb: 8.0 g/dl), 3(60%) patients with normocytic normochromic,
2(40%) patients with microcytic hypochromic anemia. Platelets were <
140X103, and TWBCs were < 3X 103/l in all patients. The differential
WBCs count was a follows: lymphocytes were > 40%, neutrophils were <
45%, and ESR > 45 mm/h were found in all patients.
Also differential T cells count were done, CD4 T. cells were < 200/ l in all
patients, mean of CD4 was 180 / l, mean of CD8 was 340 / l, and
CD4/CD8 ration was 1/1.9.
A cross sectional study was done to determine the hematological parameters
and T cell subsets count in 30 known seropositive HIV individual, during
ART at VCT Centre (Om durman Teaching Hospital), Khartoum state.
Anemia was found in 15(50%)patients (mean of Hb: 11g/dl), 12(80%)
patients with normocytic normochromic, 3(20%) patients with microcytic
hypochromic anemia. 4(13.3%) patients had platelets < 140X103, 5(16.7%)
patients with TWBCs < 3X 103/l, 10(33.3%) patients with TWBCs > 6X
103/l. The differential WBCs count; lymphocytes were > 40% in 9(30%)
patients, neutrophils were < 45% in 7(23.3%) patients, neutrophils were >
70% in 3(10%) patients.
In differential T cells count, CD4 T. cells were < 200/ l in 20(66.6%)
patients, mean of CD4 was 255 / l, mean of CD8 was 530 / l, and
CD4/CD8 ration was 1/2.1.
We concluded that normocytic normochromic anemia, leucopenia,
thrombocytopenia were the common hematological changes in VL and
VL/HIV co-infected patients. Relative lymphocytosis and neutropenia were

less common. In HIV seropositive individuals anemia were noted in have of
patients. Luecocytosis, lymphocytosis, Eosinophilia and monocytosis were
noted in third of patients.
The differential T cells count indicated that, the cellular immunity was
depressed among the VL, HIV, and VL / HIV co-infected patients; with
decreased in CD4 number and reversed CD4/CD8 ratio.
Comparison between Flowcytometry & double immunoenzymatic for CD4
counts were done for five HIV seropositive individual. Results indicated that
there is a significant variation between the two methods (P <0.05).
Sensitivity and specificity of double immunoenzymatic staining method
were 100%.
II
List if Tables:
Table. No
Title
Page
Clinical Features of VL patients
86
Hematological parameters of VL patients.
86
WBCs Morphology in VL patient.
87
Poikilocytes in VL patients.
87
Hematological parameters of HIV/VL co-infected
94
patient.
6
T.cell subsets of HIV/VL co-infected patients.
94
Hematological parameters of HIV seropositive
97
individuals.
8
CD4 count in HIV seropositive individuals.
97
Percentages of Males and Females among the three
103
groups of patients.
10
Range and mean of ages among the three groups of
103
patients.
11
Mean of different hematological parameters among the
104
three groups of patients.

12
Percentages of different types of anemia among the

104
13
Mean of CD4 & CD8 count among the three groups of

patients.
105
14
Comparison between flowcytometry and double
107
immunocytochemical for enumeration of CD4.
III
List of Figures:
Fig. No
Title
Page
Frequency of age in VL patients.
88
Number of VL patients.
88
Frequency of WBCs in VL patients.
89
Frequency of Hb among VL patients.
89
Frequency of Platelets in VL patients.
90
Frequency of ESR among VL patients.
90
Percentage of Neutropils in WBCs differential among
91
VL patients.
8
Percentage of Lymphocytes in WBCs differential
91
among VL patients.
9
Percentage of Monocytes in WBCs differential among
92
VL patients.
10
Percentage of Eosinohils in WBCs differential among
92
VL patients.
11
Prevalence of HIV among VL patients.
95
12
Prevalence of HIV among adults VL patients.
95
13
Frequency of age among AIDS patients.
98
14
Number of AIDS patients.
98
15
Frequency of Hb among AIDS patients.
99
16
Frequency of PCV among AIDS paints.
99
IV
Fig. No
Title
Page
17
Frequency of TWBCs among AIDS patients.
100
18
Frequency of Platelets among AIDS patients.
100
19
Percentage of Lymphocytes in WBCs differential
101
among AIDS patients.

20
Percentage of Neutrophils in WBCs differential among
101
AIDS patients.
21
Percentage of MXD in WBCs differential among AIDS
102
patients.
22
Frequency of CD4 among AIDS patients.
102
23
Comparison between mean of CD4 number by two
108
methods.
Abbreviations
ABC: Avidin Biotin Complex
AEC: 3-Amino-9-Ethyl Carbazole
AIDS: Acquired Immune Defficiency Syndrom
AP: Alkaline Phosphatase
APAAP: Alkaline Phosphatase Anti Alkaline Phosphatase
APCs: Antigen Presenting Cells
ART: Anti Retroviral Treatment
BMA: Bone Marrow Aspiration
CD: Cluster Differentiation
CDC: Center for Disease Control
CFT: Complement Fixation Test
CMV: Cyto Megalo Virus
CSF: Cerebro Spinal Fluid
CTL: Cyto Toxic Lymphocyte
DAB: Di- Amino Benzidine
DAT: Direct Agglutination Test
DF: Dilution Factor
DNA: Deoxy Neucleic Acid
EDTA: Ethylene Diamino Tetra- acetic Acid
ELISA: Enzyme Linked Immuno Sorbent Assay
EMRO: Eastern Mediterranean Region
ESR: Erythrocytes Sedimentation Rate
FACS: Flourescence Activated Cells Sorter
GAG: Glucose oxidase Anti-Glucose oxidase

Hb: Hemoglobin
HIV: Human Immunedefficiency Virus
IDPs: Internally Displaced Peoples
IFAT: Immuno Flourescence Antibody Test
IHC: Immuno Histo Chemistry
INF: Interferone
KATEX: Kaazar Agglutination Latex
LAB: Labeled Avidin Biotin
LAT: Latex Agglutination Test
LD: Leishman Donofani
MAC: Mycobacterium Avium Complex
MCL: Muco Cutaneous Leishmaniasis
ME: Mercapto Ethanol
MHC: Major Histo Compatibility
mRNA: Messenger Ribo Nucleic Acid
MW: Molecular Weight
NASBA: Nucleic Acid Sequence Based Amplification
NBT-BCIP: Nitro BlueTetrazolium 5-Bromo-4 Chloro-3 Indolyl Phosphate
PAP: Peroxidase Anti Peroxidase
PBP: Peripheral Blood Picture
PCR: Polymerase Chain Reaction
PCV: Packed Cell Volume
PKDL: Post Kalazar Dermal Leishmaniasis
PMNC: Peripheral Mono Nuclear Cells
RBCS: Red Blood Cells
RIA: Radio Immuno Assay
RNA: Ribo Nucleic Acid

RT-PCR: Reverse Transcriptase Polymerase Chain Reaction
RTU: Ready To Use
SNAP: Sudan National AIDS Program
STD: Standard
TB: Tuberculosis
TBS: Tris Buffer Saline
TCR: T. Cell Receptors
TGF: Tumor Growth Factor
TNF: Tumor Necrosis Factor
tRNA: Transfere Ribo Nucleic Acid
TWBCS: Total White Blood Cells
VL: Visceral Leishmaniasis
WB: Western Blott
WHO: World Health Organization
List if Contents:
Dedication
Acknowledgment
Arabic abstract
English Abstract
II
List of tables
III
List of figures
IV
Abbreviations
CHABTER I: Introduction & literature review.

1.1. Leishmanasis
1.1.1. Leishmanasis Overview
1.1.2. Historical background of VL
1.1.3. Epidemiology of VL
1.1.4. Parasites of VL
1.1.5. Life Cycle
1.1.6. Reservoir hosts
1.1.7. Vectors and transmission
1.1.8. Immunology
1.1.9. Pathology and pathogenicity of Kala-azar
1.1.10. Clinical Presentation
1.1.11. Hematological Features of VL
1.1.12. Diagnosis of VL
10
1.1.12.1. Parasitological diagnosis
10
1.1.12.1.1. Direct microscopy
10
1.1.12.1.2. In vitro culture of visceral Leishmania
11
1.1.12.1.3. Animal inoculation
13
1.1.12.2. Immunological diagnosis
14
1.1.12.2.1. Leishmanin skin test
14
1.1.12.2.2. Non Specific Tests
15
1.1.12.2.2a Formal gel test
15
1.1.12.2.2b Antimony test
15
1.1.12.3. Specific tests
16
1.1.12.3.1. Complement fixation test
16
1.1.12.3.2. Indirect haemagglutination test
16
1.1.12.3.3. Indirect fluorescent antibody test
16
1.1.12.3.4. Enzyme linked immunosorbent assay
17
1.1.12.3.5. Immunoblotting (Western Blot ,WB)
18
1.1.12.3.6. Direct agglutination test
18
1.1.12.3.7. Latex agglutination test
20
1.1.12.3.8. Latex agglutination test (Katex)
20
1.1.12.4. DNA based detection method
21
1.1.13. Treatment
22
1.2. HIV/AIDS
23
1.2.1. Virus morphology & structure
24
1.2.2. Target cells and mechanism of infection
25
1.2.3. Replication Cycle
26
1.2.4. Pathogenesis
28
1.2.5. Transmission
32
1.2.6. Peripheral Blood in AIDS
32
1.2.7. Acquired immunodeficiency syndrome (AIDS)
34
1.2.8. Clinical manifestation
36
1.2.8.1. Primary infection
36
1.2.8.2. Early and Middle stage
37
1.2.8.3. Advanced HIV disease
38
1.2.8.4. Late-stage HIV disease
38
1.2.9. Laboratory diagnosis of HIV
39
1.2.10. T. Lymphocytes
41
1.2.10.1. CD4 cells
41
1.2.10.2. Function of CD4
43
2.10.3. Function of CD8

1.2.11. Methods for CD4&CD8 lymphocyte enumeration
43
44
1.2.11.1. Automated Method
45
1.2.11.1.1 Flow cytometry
45
1.2.11.1.2. Dedicated cytometer
46
1.2.11.2. Manual Method
49
1.2.11. 2.1. TRAx CD4
49
1.2.11.2.2. Dynabeads T4-T8 Quantitative method
50
1.2.11. 2.3.Immunochemistry
50
1.2.11.2. 3.1.Indirect immunohistochemistry
52
1.2.11.2.3.1a. Antibody Bridge Techniques
53
1.2.11.2.3.1b. Affinity Labeling Methods
54
1.2.11.2. 3.2.Detection Systems
56
1.2.11.2. 3.3.Histochemical Enzymes and Substrates
56
1.2.11.2. 3.3a.Horseradish Peroxidase
56
1.2.11.2. 3.3b.Alkaline Phosphatase
58
1.2.11.2. 3.4.Double-or Multiple-Staining Techniques
58
1.2.12. Treatment and control
59
1.3. Visceral Leishmaniasis in Sudan
60
1.4. HIV /AIDS in Sudan
63
1.5. Overiew Leishmania /HIV Co infection

1.5.1. Global of Leishmania /HIV Co infection
66
68
1.5.2. Leishmania /HIV Co infection in Africa

1.5.3. Leishmania /HIV Co infection in Sudan
70
CHABTER II: Justification & Objectives.

2.0. Justification & Objectives
72
2.1. Justification
72
2.2. Objectives
73
CHABTER III: Material & Methods.
3.0. Material & Methods

3.1. Study area
3.1a. VL cases
74
74
74
3.1b. HIV individual
74
3.2. Study Design
75
3.2.1. VL cases
75
3.2.2. HIV individual
75
3.3. Study Subject
75
3.3.1. VL cases
75
75
3.4. Inclusion Criteria
75
3.4.1. VL cases
75
76
3.5. Procedures
76
3.5.1. VL cases
76
3.5.1.1. Data Collection
76
3.5.1.2. Collection of Samples
76
3.5.1.3. Parasitological Test
76
3.5.1.4. Serological Test
77
3.5.1.4.1. Direct agglutination Test
77
3.5.1.4.2. Latex Agglutination Test
78
79
3.5.2.1. Data Collection
79
3.5.2.2. Collection of Samples
79
3.5.1.5. HIV Testing
79
3.5.1.5.1. VL cases
79
3.5.1.5.2. HIV individual
80
3.5.1.6. Hematological parameters
80
1.5.1.6.1. VL cases
80
3.5.1.7. CD4 & CD8 differential count
82
3.5.7.1.1. VL cases
82
3.5.7.1.2. HIV individual
83
3.6. Data Analysis
84
CHABTER IV:
Results.
3.0. Results
85
3.1. VL patients
85
3.1.1. Hematological findings
85
3.1.2. Prevalence of HIV among VL patients
85
4.1.3. CD4, CD8 count
85
4.2. HIV/VL co-infected patients
92
92
4.2.2. CD4 & CD8 count
92
4.3. HIV patients
94
94
4.3.2. CD4
94
4.4. Double immunoenzymatic staining method
104
4.5. Comparison between Flowcytometry and double immunoenzymatic104
CHABTER V: Discussion.
5.0. Discussion
113
CHABTER VI: Conclusions & Recommendations.

6.0. Conclusions & Recommendation
118
6.1. Conclusions
118
6.2. Recommendations
119
References
120
Appendix
154
I. Introduction & Literature review

1.1. Leishmaniasis Overview:
Leishmaniasis is an infection caused by protozoan of the genus
Leishmania of Trypanosomatidae family. The infection is transmitted by
infected female sand flies of the genus Phlebotomous in the Old World and
Lutzomyia in the New World. About 30 species of Leishmania are known to
infect man leading to a wide clinical spectrum. These include three forms of
leishmaniasis; namely the cutaneous (CL), the muco cutaneous (MCL) and
the visceral leishmaniasis (VL) (WHO, 1990).
About 35 million were believed to be at risk of having leishmaniasis
in about 88 countries where as 12 million having been reported to be
currently infected (Salotra et al., 2001). However the actual number of cases
estimated to be three of five times higher than the number reported as
leishmaniasis usually occurs in remote areas where other health problems
are predominant (WHO, 1993).
The World Health Organization (WHO, 1990) recognized the disease
as one of the most important parasitic diseases in the UNDP/World
Bank/WHO Special Program for Research and Training in Tropical Diseases
(TDR). VL affects at least 12 million people world wide with an annual
incidence of 500.000 new cases (WHO, 1996). Previous epidemics of VL
involving hundreds of thousands of people had occurred in India,
Bangladesh and Sudan (WHO, 1996). The disease is fatal, with a mortality
rate reaching approximately 100% among untreated cases (WHO, 1996).
Of the 500,000 new cases of VL which are estimated to occur
annually; 90% come from five countries Bangladesh, Brazil, India, Nepal
and Sudan. VL is caused by L. donovani in the Indian subcontinent and in
1
east Africa, by L. infantum in the Mediterranean region and by L Chagasi,

which is closely related to L. infantum, in the new world mainly in Brazil,
Peru and Paraguay (WHO, 1993).
The disease results from multiplication of the parasites (Leishmania
amastigotes) within the mononuclear phagocytes of the reticulo-endothial
system .The classical clinical features of VL are fever,
weight loss,
pancytopenia and on physical examination, hepatosplenomegaly with the

spleen generally more enlarged than the liver , typically 5-15 cm below the
left costal margin (WHO, 1996). Splenomegaly may be absent in 5% of
cases (Nandy, 1998).Malnutrition is an established risk for development of
VL in childhood (Yousif, 1967). Histologically, the parasites may be found
in the macrophage of the spleen, liver, lymph node and even the skin.
Immunologically VL patients show a selective energy to Leishmania
antigens, first characterized by a negative Leishmanin test, which may fail to
convert even after cure (Elhassan et al., 2001).
1.2. Historical background of VL:
Visceral and cutaneous leishmanaisis were known as far back as the
first century A.D in central Asia. VL has been known in the old world in
India since the 19th century by the name black fever or Kala-azar due to skin
darkening. A mastigotes were first seen in the skin of a patient with "Delhi
boil" in Calacuta by Cunningham in 1885 (WHO, 1984). William Leishman
in 1903 was able to identify the organism in the splenic smear of an Indian
patient who died "with Dum Dum" fever, another name for VL. At the same
year Donovan recognized the same intracellular parasite, seen by Leishman,
in the spleens of three cases in Madras, who presented with fatal disease that
was considered as chronic malaria. Hence the organism was named
Leishman Donovan body in honour of these two pioneers in the field of
2
leishmaniasis. Rogers in 1904 succeeded in culturing the parasite

responsible for VL from splenic aspirates (Dedet et al., 1999).
1.3. Epidemiology of VL:
Leishmaniasis is a worldwide disease which is found in all
continents, except Australia (Manson & Bahr, 1987). However it is most
common in the tropics and sub-tropics. Certain factors contribute to
determine its distribution such as high humidity and its mean diurnal
variation (Napeir, 1946). There is a definite seasonal variation, most
commonly in Autumn following rainy seasons, as in Bengal and Sudan, or in
early summer as in China or in Spring as in Europe .VL is found in Asia ,
Southern Europe, particularly in Greece, Southern France, Spain. In South
America it is found in Brazil and Venezuela. In Africa, the disease is found
in Sudan, Ethiopia, Somalia, northern Kenya, Equatorial West Africa,
Morocco, Algeria and Tunisia, (Manson & Bahr, 1987).
1.4. Parasites of VL:
Leishmaniasis is caused by protozoan parasites of the genus
Leishmania which belongs to the family Trypansomatidae. It has different
species which are responsible for the three clinico-pathological conditions;
(a)Visceral leishmaniasis which is caused by L. donovani
complex (b)
cutaneous-leishmaniasis caused by L. tropic , L. major, and L. mexicana and

(c) muco cutaneous Leishmaniasis which is caused by L. braziliensis and
L.mexicana complexes (WHO, 1990). The L. donovani complex has three
subspecies L. donovani donovani (Sudan, India), L. donovani infantum
(Mediteranian) and L.donovani chagasi (South America) (WHO, 1990).
Infection with L. donovani occurs in three clinico epidemiological patterns
which are Mediterranean type or infantile VL, Indian type VL and African
type.
3
The Leishmania parasites assume two forms, the flagellated form

(infective stage) named promastigote and the non-flagellated form (a
mastigotes). The promastigote form is 15-30 um long found in the gut of the
sand fly (the vector) and can be cultivated in culture media. It has a single
flagellum, arising from a blepharoplast at the anterior end and a nucleus. A
mastigote is the intracellular form which is oval or round measuring about 23, um in length (Manosn & Bahr, 1987). Found in the mammalian host and
can be recovered from all endothelial cells, including reticulo endothelial
system, bone marrow, lymph nods, spleen and liver, as well as from
monocytes, polymorphs and the sub-mucosa of the respiratory and
alimentary tracts (Napier, 1946). It can also be recovered from stool or urine
(WHO, 1997).
1.5. Life Cycle:
L.donovani is an obligatory intracellular parasite which cycles
between the mid gut of sand flies (extracellular promastigotes) and the
phagolysosome of mammalian macrophages (intracellular amastigotes)
(Dedet et al., 1999). Following inoculation of promastigotes into the skin of
mammalian host by a sand fly (Phelebotomns orientalis) while seeking a
blood meal at night, they soon lose their flagella, transform into the
amastigote form and become engulfed by macrophages. Inside the
macrophage they multiply by binary fission. The macrophage eventually
ruptures when heavily parasitized to release parasites which are engulfed
again by other new macrophages. The new amastigotes penetrate the cell
membrane and are ingested by the sandfly, when it seeks its next blood meal
at night (Cheesbrough, 1995). Then each amastigote regains its flagellum,
after going through many development stages. The sandfly becomes
infectious within 10-15 days of the blood meal (Manson & Bahar, 1987).
4
1.6. Reservoir hosts:

There are two types of reservoir hosts: wild reservoirs such as
canines which include foxes, wolves and jackal and domestic animals like
dogs, rodents and rats (WHO, 1990).
1.7. Vectors and transmission:
VL is transmitted from one mammalian host to another including
man by the female sand fly which has different species, geographical and
seasonal distribution. Sandflies are mainly in the tropics and sub-tropics. In
the following countries, the most common species include: P. orientalis in
Sudan, P. martins in Kenya, P. major and P. pernicious in the
Mediterranean, P. chinensis in China and P. argentipes in India.
Other modes of transmission do exist although their occurrence is
rare. These include blood transfusion, direct contact with blood, mucous,
stools or nasal discharge (Manson & Bahr, 1987). Occasionally infection
may occur accidentally in the laboratory (Terry, 1949; Manson & Bahr,
1987), or experimentally through injection of L. donovani intradermally that
can produce nodules containing amastigotes (Manson & Bahr, 1962, 1987).
Congenital transmission has been reported (Manson & Bahr, 1987) as well
as sexual transmission (Symmers, 1960; Manson & Bahr, 1987).
1.8. Immunology:
Leishmania a mastigotes produce variable effects on the elements of
the immune system (WHO, 1990) Leading to variable host responses and
producing a wide spectrum of disease (Manson & Bahr, 1995).
Immunity against leishmania parasite is mediated by cellular
mechanisms (Ghalib, 1993). Antibodies have little if any protective role
against this organism (Probst et al., 2001); thus making the strategies of the
host defense depend on the sequence of the reactions inside the macrophage.
5
The organism protects itself by an electron dense material, thus it

resists macrophage killing by oxygen dependent or non oxygen dependent
mechanism due to oxidative metabolites that interfere with the activity of the
lysosomal enzymes. Therefore macrophage microbicidal activity is reduced
(Hoover et al., 1985).
The parasite produces modulation of the T-cell response. There is
marked depression of the cell-mediated immunity, depressed delayed type
hypersensitivity and low numbers of T-lymphocytes in active VL cases,
which are reversible with successful treatment (Aikat et al., 1979, Ghose et
al., 1979). When CD4 T-cell is activated, the outcome depends on the
balance between T.hl/T. h2 responses (Sundar et al., 1997; Probst et al.,
2001). This balance is thought to be genetically determined which is
important in the pathogenicity of the disease. Resistance and elimination of
the parasite is in favour of the response which mediates the production of
INF. and IL-2. T. h2 response which involves the secretion of IL-4, 1L-5,
IL-6 and IL-10 is associated with disease susceptibility. IL-10 down
regulates the T. h1 response (Ghalib et al., 1993).
Assessment of cell mediated immunity can be done by either in vitro
or in vivo tests. The in vitro tests are lymphocyte proliferative, response and
phenotypic distribution of the peripheral mononuclear cells (PMNC).
Usually there is failure of lymphocytes to respond to leishmamal Ag as well
as mitogens, phytohaemagyhitin and concavalin A, which are reversible with
successful treatment (Ghose et al., 1979; Wyler, 1982). Peripheral
mononuclear cells fail to respond to leishmanial antigen, in active KA, as
well as PKDL (El-Hassan et al., 1992), as compared to the cutaneous form.
The response is specific to leishmanial antigen as it reverts to normal with
commencement of therapy in vivo.
6
Regarding the humoral immune responses there is marked increase

imnmunoglobulin G (IgG) compared to immuno globulin M (IgM) (Manson
& Bahr, 1987; Mure's, 1997). IgGI and IgG3 (sub-classes of IgG) are very
specific and correlated with the disease activity as they decrease after
treatment (EL-Assad et al., 1994).
1.9. Pathology and pathogenicity of Kala-azar:
After the host is bitten by an infected sandfly, the parasites
disseminate in the blood stream and enter macrophages of the spleen, liver,
bone marrow, lymph-nodes, skin and small intestine. The promastigotes
immediately lose their flagella and change their shape into the amastigote
forms which attach themselves to the membrane of the macrophage and
invade it. They spread from local lymph nodes through the blood to different
organs e.g. liver, spleen, bone marrow and lymph glands, rarely the intestine,
adrenals, kidneys and lungs. A leishmanoma is formed in the skin after
injection of the mammalian host with the promastigotes which may pass
unrecognized.
Usually there is anaemia which is commonly normocytic
normochromic, sometimes thrompocytopenia and leucopenia occur which
predispose
the
patient
to
secondary
infection
and
haemorrhagic
manifestations. Nandy et al., (1998) reported a case presenting with renal

haemorrage. Pancytopenia might result from hypersplenism or bone marrow
involvement.
The skin is commonly involved in kala -azar. In fatal cases all the
skin layers are loaded with the parasite. In post kala-azar dermal
leishmaniasis (PKDL), some of the parasites survive for a long time
multiplying slowly in the skin.
Other affected sites include the mucosa of the nasopharynx and oral
cavity (El Hassan et al., 1995), which make the diagnosis of Kalazar
difficult. PKDL patients may be an important source of infection in VL
transmission (Zijistra & El Hassn, 2001).
Histocytic proliferation in these organs produces enlargement with
atrophy or replacement of the normal tissue, therefore kala-azar is a systemic
granulomatous disease of the reticulo-endothelial system (Manson & Bahr,
1987).
1.10. Clinical Presentation:
The natural history of the disease is progressive and it is fatal unless
treated. The incubation period is variable, normally between 3-6 months
(Hommel & Ashford, 1983). It can be as short as 10 days and as long as 10
years. The disease can affect younger age groups especially in the
Mediterranean area (Less than 5 years) and East Africa (between 5-9 years).
Males are more affected than females with a ratio of 4:1 (Manson & Bahr.,
1987). The disease can be endemic, sporadic or epidemic (WHO, 1990). The
disease can be sub-clinical or asymptomatic (Napier., 1946; Manson &
Bahr., 1987). The disease can presents with 3 clinical forms:
l. A primary leishmanoma which is a small papule appearing 4-6 months
before the onset of the disease or may go unnoticed (Manson & Bahr.,
1987).
2. An acute onset occurs either in sporadic or epidemic forms in nonimmune people visiting an endemic area.
3. Chronic form which is the most common presentation in 3 endemic
areas.
The patient presents with general ill health and cachexia (Cole, 1944
and Manson & Bahr, 1987). Fever is almost invariable, it can be high
8
remittent with double peaks and associated with drenching sweating, but no
rigors (Manson & Bahr, 1987); however, it can pass unnoticed by the
patient. It may be intermittent or continuous in the established disease and
may be absent in some cases (Napier, 1946). Fever is accompanied by
progressive splenomegaly which may be very huge in size, reaching the
right iliac fossa within a few days (Manson & Bhar, 1987). In a few cases
the spleen is not palapable. Hepatomegaly is not to the same degree as
splenomegaly. There may be cirrhosis, ascites or jaundice as a late
complication of the disease. Diarrhoea may be one of the presenting
symptoms due to gastro intestinal involvement, as well as epistaxis.
Hyperpigmentation involves the hands, feet and abdomen (Napier, 1946).
The lymph nodes are enlarged, especially in African kalazar.
There may be mild cough and proteinuria, (Manson & Bahr, 1987)
and oedema (Napier, 1946). Amyloidosis is a recognized complication and
may regress with cure. Manson & Bahr, (1987) and Change et al., (1985)
reported a patient who presented with tremors. Other neurological
involvements like delerium and confusional states have also been reported
(Manson & Bahr, 1987). Retinal haemorrage is one of the presenting
features of the disease.
Mucosal and mucocutaneous involvement occurs sporadically in the
Sudan and Eastern Africa. Complications are important causes of death,
these include: tuberculosis, lobar pneumonia, dysentery, liver cirrhosis and
cancrum oris (Manson & Bahr, 1987).
1.11. Hematological Features of VL:
Anaemia is invariable, especially in children and it is usually
normocytic normochromic (Zijistra & El Hassan, 2001), it may be due to
hyper-splenism, auto-immunity with haemolysis or due to ineffective
9
erythropoiesis. Leukopenia is marked and late in the disease. The total white
blood cell count (TWBC) may reach levels below 2000/l, and there may
even be a granulocytosis in fatal cases.
Thrombocytopenia and pancytopenia were reported above. The
erythrocyte sedimentation rate (ESR) may reach a very high level.
The haematological manifestations were reviewed in 94 patients (55
males and 39 females) with visceral leislimaniasis by Nasir, A.M. et al
(1995) in Saudi Arabia. They found that all patients had splenomegaly and
were anaemic, while (73) were neutropenic and (56%) thrombocytopenic.
Coagulation abnormalities were encountered in 10(11 per cent) patients; in
four patients this was associated with disseminated intravascular
coagulopathy. Bone marrow was hypercellular in (90%), normocellular in
(5%),
and
hypocellular
in
(4%).
Also
variable
degrees
of
erythrophagocytosis and leukophagocytos were noted with preponderance of

histiocytes (46%) and granulomatous formation (25%). Low haemosiderin
content in the bone marrow was noted, which together with the finding of
high serum ferritin is consistent with anaemia of chronic inflammation.
1.12. Diagnosis of VL:
There are three main diagnostic approaches for the confirmation of
Kalazar. These include the parasitological, immunological and DNA based
detection methods.
1.12.1. Parasitological diagnosis:
1.12.1.1. Direct microscopy:
The definitive and routine diagnosis of VL leishmaniasis relies on
microscopic detection of leishman donovan bodies in Giemsa stained smears
of lymph node, bone marrow or splenic aspirates (Evans, 1989). These
methods are relatively simple and cheap but there is no possibility to
10
distinguish between Leishmania amastigotes spp (Evans, 1989). Although

spleenic aspiration is generally accepted as the most sensitive method and
can detect more than 90% of cases (Manson & Bahr, 1987), it is contra indicated because bleeding complications are feared. At least three separate
studies showed a sensitivity of at least 94% for splenic puncture in VL
patients (WHO et al., 1948; Siddig et al., 1988; Zijistra et al., 1992). Bone
marrow aspiration unlike splenic aspiration or lymph node aspiration is often
resented by the patient because it is painful especially in children.
Microscopy of lymph node aspirates is the most commonly used procedure
in endemic areas of Kala-azar in the Sudan as it is a safe and simple
procedure (Siddig et al., 1988). However, a variable sensitivity was reported
ranging from 58.3% (Zijistra et al., 1992) to 100% Kirk and Satti, (1940).
Liver aspiration is used infrequently in the diagnosis of VL (Zijistra et al.,
1992). In HIV/ Lieshmania co-infected patients the amastigotes are found in
the peripheral blood in 50% of cases and may be found in unusual location
such as the lungs, larynx, gastro intestinal tract and C.S.F. (Manson & Bahr.,
1987).
1.12.1.2. In vitro culture of visceral leishmania:
Culture of Leishmania parasites for diagnosis of leishmanasis has
been used since the beginning of 20th century. Several culture media have
been used including NNN and various semi-synthetic media, supplemented
with blood or serum (WHO, 1996; Evans, 1989). For primary isolation of
Leishmania parasites, small inocula of clinical material (lymph node, bone
marrow, spleen aspirates ) is inoculated under aseptic condition into the
liquid phase of blood agar biphasic media, . The inoculated media are
incubated at 242c for 7-20 days using cooled incubator. Growth in culture
tubes was detected by turbidity in the liquid phase of the media and
11
confirmed microscopically by presence of motile promstigotes in smear

preparations (WHO, 1996).
Culture media for mass cultivation and primary isolation of old and
new world forms of Leishmania spp include blood agar biphasic medium e.g
NNN medium, sloppy Evans medium, semi solid Lockes blood agar, Evans
modified Tobies medium Usmaru ( Difco BA) MEM: FCS: EBLB medium
and Schneider's Drosophila medium (Evans., 1989). The organisms from
patients with VL can be very difficult to cultivate (Evans, 1989). Therefore
it is wise whenever possible to inoculate experimental animal as well. In
attempt to cultivate Leishmania, it is advisable to use blood agar biphasic
media preferably e.g NNN or USAMRU or modified Tobies medium but
even when the initial isolation is successful the organism may die when subcultured. This seems to be especially common when the initial isolation has
been into rich media such as USMARU or modified Tobies. Often this can
be overcome if sub-cultures are made into less nutritionally rich media such
as NNN or one of the semi solid media like sloppy Evans and semi solid
Locke's blood agar (Evans., 1989& WHO., 1990). The variations of these
media lead to efficacy in the transformation of amastigotes to promastigotes
and in the growth rate of promastigotes. While being easy to carry out,
Leishmania culture needs some expertise and sterile conditions during
collection and processing of material from patients lesion or organs, and for
sub culturing (Dedet et al., 1999).
The purposes of culture of the genus leishmania include: accurate
identification of the organism, to obtain a rich yield of the organism to be
used as antigen in immunological diagnosis for teaching purposes, as a
source for inoculating susceptible experimental animals, for in vitro
screening of drugs and for investigation of the physiology of the pathogens
12
(Cheesbrough, 1995). Initial isolation is the process by which leishmania

organisms are removed from their host and transferred either to an
experimental animal (invivo isolation) eg hamester or into culture media (in
vitro isolation) so that multiplication and bulk cultivation of leishmania is
necessary for isoenzyme identification methods which require relatively a
large number of promastigotes. The choice of isolation methods depends on
the immediate circumstances and to some extent on the technical capabilities
and experience of the staff. In-vitro isolation offers certain advantages over
the in vivo methods as cultures become more rapidly positive after as a short
time as 5-7 days in contrast to a week or a month required for a lesion to
appear in an animal after inoculation the culture materials are less expensive
and cultured organisms can be cryopreserved thus reducing the time and
personnel required for its maintenance. The disadvantages of in-vitro culture
are that some strains of leishmania donovani are extremely difficult to attain
in the field (Evans, 1989). In vitro cultivation is an efficient method for the
parasitological diagnosis of leishmaniasis and for distinguishing between
various strains causing leishmaniasis. The most difficult part is usually the
initial establishment of the organism in culture when the a mastigotes have
to transform to promastigotes to begin multiplication and this step needs
skills and technical capabilities as well as abilities in preparation
specification and sterility during inoculation of the clinical samples into the
different types of media .
1.12.1.3. Animal inoculation (in vivo propagation):
Hamster is considered to be a suitable model for propagation of
Leishmania (Evans, 1989). The animal is inoculated intradermally or
subcutaneously as well as in the nose and hind feet. A positive reaction is
identified by finding an indurations or an ulcer at the injection site. The
13
presence of amastigotes can be verified by the examination of stained

smears. Other animals like guinea pigs and inbred mice can be used (WHO,
1996). Usually laboratory animal inoculation is slower than culture methods.
It takes 3-4 weeks for lesions to appear in contrast to the culture which takes
10-15 days for the development of the promastigotes (Evans, 1989).
Previous investigations by Satti et al (1958) also demonstrated that the grey
monkey (Cercopithecus sebaezis ) and the red monkey (Cercopithecns ruler)
has successfully became infected with L. donovani parasites. The method
however, was criticized for being time consuming, laborious and unsuitable
for routine diagnosis.
1.12.2. Immunological diagnosis:
Immunodiagnosis is of paramount importance in the diagnosis,
follow up and control of infections .It can be divided into two parts: one
which detects and measures antibodies in the serum and the other which can
demonstrate or detect the cell mediated response (Bray, 1985; Lakshams,
1985). Immune-diagnosis is needed for rapid and reliable detection of early
cases especially in the field when the parasite load is scanty and may not be
revealed in smears. However its main problem is cross reactions. At present
several immunological techniques based on cellular or humoral responses
are widely used for the diagnosis of VL.
1.12.2.1. Leishmanin skin test (L.S.T or Montenegro test):
This test is a delayed type hypersensitivity reaction to Leishmania
anologous to tuberculin and lepromin tests (Cheesbrough, 1975). The
antigen consists of a suspension of killed promastigotes. The test is
performed by intradermal injection of 0.1 ml phenolized antigen into one
forearm and 0.1 ml phenol saline into the other forearm (Bray, 1976). The
test is usually read after 48-72 hours (Zuckerman, 1975 Bray, 1976) A
14
positive reaction is indicated by a raised induration more than 5mm

accompanied by erythema (Manson & Bahar, 1987). The test is not of
diagnostic value in VL, but can help in epidemiological surveys of past and
subclinical infection in endemic areas as well as in the exclusion of active
VL (Manson & Bahar, 1987). Zijistra et al (1993) and El Hassan et al (1993)
showed that a positive LST indicates that a treated VL patient has developed
cell mediated immunity to Leishmania. LST is positive in active and past
infection of cutaneous leishmaniasis for life (Manson & Bahar, 1987).
1.12.2.2. Non Specific Tests:
The nonspecific reactions depend upon the increase of the
immunoglobulin seen in Kalazar. It can be demonstrated by flocculation or
precipitation (Zuckerman, 1975). The best examples are:
1.12.2.2a Formal gel test (Napier aldehyde test):
This test is performed by mixing 1 ml of serum with 2 drops of 30%
formalin. The reaction is read after 20 minutes. A positive reaction is
indicated by whitening and jelly appearance which indicate hyper
globulinaemia (Ghose, 1980). The test is positive after 1-2 months of the
infection and becomes negative after 6 months of cure. It is simple easy to
perform and cheaper therefore it is suitable for sero-epidemiological survey
(Jalil et al., 1992). It has a diagnostic value in chronic cases of kala- azar
(Sen Gupta et al., 1969).
The main disadvantage is the cross reactivity with other diseases that
cause
hyperglobulinaemia
e.g
leprosy,
TB,
trypansomiasis
and
schistosomiasis.
1.12.2.2b Antimony (chopra test) or urea stibamine test:
This test is used in India. The serum is diluted 1 in 10 with distilled
water and placed in a narrow bore test tube then 4% urea stibamine solutions
15
added and mixed gently. A positive reaction is indicated by flocculent

precipitate after 10-15 minutes. It is heavy in strong positive reaction
(Napier, 1946; Manson & Bahar, 1987).
1.12.3. Specific tests:
Various tests have been applied to detect specific anti leishmanial
antibodies. These include DAT, ELISA, IFAT, CFT (Bray, 1985). Recently
DAT, IFAT, ELISA and WB were considered as important diagnostic tools.
1.12.3.1. Complement fixation test (CFT):
Complement fixing antibodies appear in 95% of cases from the third
week of infection and disappear within 6 months of cure (Manson & Bahar,
1987). The antigen is prepared from L. donovani promastigotes (Manson &
Bahar, 1987). A titer of 1/10 is suggestive and a titer of 1/40 is diagnostic
(Manson & Bahar, 1987). The problem with CFT is that it can give false
positive results with tuberculosis, leprosy and less commonly with malaria.
Persistence of a low titer is a clue to relapse.
1.12.3.2. Indirect haemagglutination test (IHT):
The haemagglutination test follows the same course as CFT (Manson
& Bahar, 1987) .The antigen is in a soluble form prepared from
promastigotes adsorbed into tanned sheep erythrocytes and latex particles. It
is a sensitive and specific test (Manson & Bahr, 1987). A titer of 1/200 and
over is diagnostic (Bray, 1976).
1.12.3.3. Indirect fluorescent antibody test (IFAT):
This is a sensitive and specific test in diagnosing VL (Hommel,
1978; Al-Alousi et al., 1980; Manson & Bahr, 1987). It has a sensitivity of
75% and a specificity of 93% as compared to ELISA (Ashford et al., 1993).
The antigen is prepared either from promastigotes fixed with acetone
or I, form a mastigotes isolated from an infected animal. It is particularly
16
important in screening and detecting early cases of kala-azar (Manson &

Bahr, 1987) as it becomes positive early in the disease and disappears within
6 months of cure. Persistence of antibodies for more than 2 years is
indicative of relapse and is also a measure of the response to treatment
(Manson & Bahar, 1987).
The disadvantage of the test is its cross-reaction with T.cruzi (Shaw &
Volter, 1964; Bray & lanson, 1965; Madersen & Thomas, 1985; Manson &
Bahar, 1987) malaria, schistosomiasis and leprosy as well as typhoid and
larva migrans infestation (Manson & Bahr, 1987). Another disadvantage is
that the test needs a fluorescent microscope and it is not suitable for large
scale epidemiological studies.
1.12.3.4. Enzyme linked immunosorbent assay (ELISA):
The use of enzyme linked assay with an antigen or an antibody was
first described by Engvall and Perlman (1971, 1972).They assessed the
antibodies present in rabbit sera by using disposable polystyrene tubes and
alkaline phosphate as a conjugate and when compared with RIA they
showed that ELISA was sensitive. Since that time ELISA has been widely
adopted
in
the
serodiagnosis of parasitic
diseases like
malaria,
trypanosomiasis, giardiasis, schistosomiasis, ect... (Voler et al, 1980).

The principle of the test is that the antigen is adsorbed to a solid
phase (polystyrene surface) by physical adsorption to which is added the
serum and the enzyme labeled anti-immunoglobulin that retains both the
immunological and the enzyme activity. The intensity of the colour
produced when the substrate specific for the enzyme is broken is
proportional to the amount of the specific antibody present in the serum
(Voler et al., 1980).
17
1.12.3.5. Immunoblotting (Western Blot WB):

The performance of Leishmania soluble antigens for VL diagnosis was
improved through electrophoretic separation of individual reactive epitopes
as in immunoblotting techniques (Jaffe & Zaiis, 1988). Incorporation of
purified polypeptides of L.infantum in such a technique provided in addition
to the high potential for VL diagnosis also the means to study the evolution
of antibody responses throughout the course of infection. The optimal
sensitivity of immunoblotting techniques and in particular the Western Blot
(WB) was further demonstrated in patients with VL/HIV co-infection.
Nevertheless Hoerauf et al (1992) demonstrated the capability of the
immunoblot technique to differentiate kala-azar from lymphoproliferative
and autoimmune diseases that could not be differentiated by ELISA and
IFAT. However the technical and financial factors involved constituted
significant obstacles for the applicability of immunoblotting techniques at
the central laboratories of various VL endemic areas.
1.12.3.6. Direct agglutination test (DAT):
The direct agglutination test is a simple and feasible technique that
has high sensitivity and specificity (Harith et al., 1986, 1988, Hommel, et
al., 1997; Meredith et al., 1995). Because of these advantages it is one of the
most widely used immunological tests that have been applied by several
investigators in diagnosis and epidemiological studies. In this test the
antigen preparation consists of the whole organism (promastigotes) and the
serological response (mainly IgG) to surface borne antigens is measured.
Nicloe and Manceaux in 1909 were the first' to describe agglutination in the
serum of patient with Leishmania while the first diagnostic application in the
serum of patient with Leishmania of the agglutination test was performed by
Row in 1931 (cited by Hommel et al., 1997). Increased specificity of the test
18
was obtained by trypsinization of antigen (Allain & Kagan, 1975). The DAT
was modified by Harith et al (1986) who described formalin-fixed
trypsinized promastigotes stained with coomassie brillant blue and used as
an antigen in a reusable V-well microtitre plates. The sensitivity and
specificity of the DAT were further improved by testing the serum samples
in serum diluent containing gelatin instead of fetal calf serum and 0.1 ml 2mercaptoethanol (Harith et al., 1988). Cleaving agents other than trypsin e.g
pronase lipase pancreatin or 2-mercaptoethanol were evaluated for further
improvement of the test (Harith et al., 1995).
The test was recommended to be used widely in seroepidemiological surveys .EL Harith et al (1996) showed that the DAT was
valuable in differentiating PKDL from other skin conditions like leprosy.
According to Hommel et al (1997) the possible cross-reactivity with
other pathogens including T brncei, T. cruzi and Mycobacteria Tuberculosis
are the critical problem in the DAT. Andrade et al (1987) showed that L.
donvani DAT antigen could be used for the diagnosis of VL caused by L.
chagasi in Brazil. Harith et al (1988) found cross- reactivity with serum
samples from patients with African trypansomaisis and toxoplasmosis.
These cross reactions were eliminated by the addition of 2- mercapto
ethanol to the serum diluents. The decline of anti-leishmanial antibodies
level in VL patients following treatment has been a subject for intensive
research in the course of DAT evaluation. Hailu (1990) found that
significant level of anti leishmanial antibodies may be present up to 7 years
after treatment. Like as most serological tests DAT may not differentiate
between active disease, sub clinical and pervious infections and this is a
major draw back to the test (Hailu, 1990; Hommet et al., 1997; Zijistra et al.,
1991). Another major draw back to DAT is the limited stability of the
19
aqueous antigen (Harith et al., 1988). Of due to that, a DAT based on stable
- freeze -dried antigen has been developed. High sensitivity (92%) and
specificity (99.7%) of the freeze dried antigen were shown to be identical to
that of aqueous antigen (Oskam et al., 1999). The freeze -dried antigen can
be stored at ambient temperature. Due to these factors the test became one of
the best available diagnostic tools for use in the field.
1.12.3.7. Latex agglutination test (LAT):
In
search
for
quick
simple
serodiagnostic
test,
many
researcherssensitized the latex particle with Leishmania antigens. De Korte

et al 1990) performed LAT sensitized by L. infantnm soluble antigen for the
detection of anti-Z,. infantum antibodies in canine VL reservoir in southern
France but they obtained a lower sensitivity for LAT in comparison to DAT.
Moody & El Safi (1996) performed the LAT (using L. donovani soluble
antigen) in sera from Sudanese VL confirmed cases (n= 60), VL
unconfirmed cases (n=48) but with positive DAT result and sera from
healthy non endemic controls (n=75) they reported a sensitivity of 88% and
specificity of 100%. They concluded that the LAT is a simple and practical
serological technique for the diagnosis of VL particularly at dispensary level
but they recommended further evaluation of LAT using fresh sera.
1.12.3.8. Latex agglutination test (Katex) for detection of urinary
antigen:
Katex is a new latex agglutination test for the detection of leishmanial
antigen (Ag) in the urine of patients with VL (Attar et al., 2001; Hommel et
al., 2001; Rijal et al., 2003). The detected Ag which is a heat-stable,
precipitates with acetone and ethanol but not Tri acidic acid, is sensitive to
periodate and acid hydrolysis but not to Pronase E, Lipase,or neuraminidase.
The Ag is a low molecular weight glycol conjugate that can be extracted by
20
phenol water, the molecular weight of urinary Ag is 5-20 KDA was detected
in the urine of patients from Nepal, Sudan ,Brazil, Yemen and Spain and
experimentally infected animals (Sarkari et al., 2002) . No low molecular
weight antigen was detected in the urine of patients with malaria,
schistosomiasis, or diseases such as typhoid and brucellosis (Sakari et al.,
2002).The antigen is detectable in both the promastigote and amastigote
stages of the parasite. Monoclonal antibodies (mAbs) against Leishmania
glycoconjugates strongly react with this molecule, so that the detected
antigen is highly specific and diagnostic for VL.
The test is based on the adsorption of anti-leishmanial antibody to
latex particles; this reagent agglutinates within two minutes when mixed
with urine from an infected host. To obtain adequate specificity urine can be
boiled before performing the test. As antigen detection tests the Katex would
in principle provide better means for diagnosis since antigen level are
expected to broadly correlate with the parasite load. Antigen detection
systems are also ideal alternative to the antibody detection systems in
immuno compromised patients. Katex is simple test and easy to perform. It
does not require specialized equipment and easy to interpret. The test is
100% specific and 80% sensitive with parasitologically confirmed cases
from Brazil, Sudan, Yemen and Nepal (Attar et al., 2001).
1.12.4. DNA based detection methods:
In recent years several molecular biological techniques have been
developed for the sensitive detection and identification of pathogens
(Denissereno et al., 2003) .The main approaches to nucleic acid based
detection are: (1) by hybridization using DNA probes and (2) amplification
techniques including NASBA, RT.PCR for the detection of RNA and the
PCR for detection of DNA (Osman et al., 1995).
21
A group of Sudanese researchers used PCR for detection of reservoir

hosts headed by EL Hassan et al (1993) detected Leishmania DNA in
Arvicanthns niloticiis.
The availability of DNA based diagnostic methods has allowed more
sensitive, rapid detection and characterization of microorganisms .The
association of hybridization with the PCR has solved relatively limited
sensitivity of hybridization and has been used with efficacy for the diagnosis
of genetic and infectious_ diseases including protozoan parasites (Ashford et
al., 1995).
Scharfer et al (1995) examined the sensitivity of PCR for detection of
parasite DNA before and after diagnosis and treatment. They found that
PCR detects sub clinical VL cases; i.e. have detectable circulating DNA in
their blood .They showed that L. donovani DNA can be detected in the
blood despite negative serological tests, and absence splenomegaly .In
addition, they found that parasite DNA can be found in blood dried on filter
paper long before a parasitological diagnosis of VL is made of helps to
provide them to center involved in clinical trials and parasite
characterization (WHO, 1996).
1.13. Treatment:
Pentavalent antimonial drugs have been used in the treatment of VL
since 1916 (Manson & Bahr, 1987). They are the first line of VL treatment.
They are available in form of sodium antimony gluconate (Pentostam) and
meglumine antimonate (Glucantime) at the rate of 85mg/ml pentostam
available in vials (50ml) which contain 600mg. It can be given
intramuscularly or intravenously at a daily dose of 20mg/kg body weight for
a bout 30 days. Antimony compounds act against Leishmania parasites by
inhibiting purine nuclear and macro cellular synthesis (Bracyson, 1987).
22
Renal function has to be assessed before and during drug treatment because
60-80% of the drug'is renally excreted. It is also cardiotoxic and can produce
fatal arrythmias in high dosage especially during the second week after
commencing treatment (Chowdhury et al., 1998). Most of the patients
respond well to antimonial compounds .Other drugs used include
pentamidine but it has side-effects. Amphotericin B kills the parasite intra
and extracellularlly but is nephrotoxic .The new formulation of
Amphotericin with lipid (Ambisome) fortunately devoid of the toxicity of
Amphotericin B (Hoiumeur et al., 1998) and can be used for VL patients
who are not responding to the treatment of ka azar patients and they enhance
intracellular killing of the parasites. INF. has been used for the treatment of
Kala -azar patients and it also enhanced intracellular killing of Leishmania
and when used in combination with antimonial compounds in refractory VL
cases gave satisfactory results (Murray et al., 1988). All the current drugs for
kala-azar have draw backs .They are administered by injection or infusion
and require the patients to be hospitalized. Miltefosine is expected to be the
first oral treatment of kala- azar in Bihar-India (TDR News, 1999).
1.2. HIV /AIDS:
Human immunodeficiency virus (HIV) types derived from primates
(Lentiviruses) are the etiologic agents of AIDS (Geo & Butel, 1998). The
illness was described for the first time in 1981 by Luc Montagnier of France
and Robert Gallo of the United States. AIDS etiologic agent HIV-1 was
isolated by the end of 1983. Since then AIDS has become a worldwide
epidemic expanding in scope and magnitude.
About 54,862,417 persons were estimated in November 2003 to be
infected worldwide, 30% of them were in South Africa (Kahn et al., 1998).
Once they contract the disease, individuals remain infected for life if left
23
untreated. The vast majority of HIV infected individuals develop fatal

opportunistic infections such as tuberculosis as a result of HIV-induced
deficiencies in the immune system. AIDS is one of the most important
public health problems world-wide at the start of the 21st century (How et
al., 1998).
1.2.1. Virus morphology & structure:
The virion of the HIV is spherical 80-100m in diameter, of
cylinderic core. The genome is single strand RNA, linear, positive sense 910 kilobases in length, diploid and contains up to six additional replication
genes. The mature virion has three morphologic components:
-An outer envelope made up of lipid bilayer membrane containing
virus specific glycoprotein spikes.
-An internal protein capsid, within the capsid, a nucleocapsid and two
virally encoded enzymes (Reverse transcriptase and integrase).
In addition, unique cellular tRNAs are associated with each segment
of the viral genome.
The Lentivirinae subfamily to which HIV belongs is characterized
by a distinctive nucleocapsid core viewed in electron microscope as a bar or
truncated, cone-shaped nucleocapsid.
HIV has three major genes coding for structural proteins. These are
gag, pol and env. The gag-derived proteins make up the viral capsid p 24,
i.e. a 24 kilodaltan protein, the nucleocapsid protein (p17) and a matrix
protein.
The pol gene a code for the virus enzymatically active proteins most
important of which is reverse transcriptase, which performs the unique
reverse transcription of the viral RNA into double stranded DNA.
24
Also pol encodes a specific viral protease. This enzyme cleaves gag
and gag-pol derived protein into functional pieces. The third structural gene,
env, stands for "envelope". The proteins derived from env are a surface (gp
120) and a transmembrane proteinTM (gp 41) (Geo & Butel, 2001).
The TM (gp41) env product contains both a transmembrane domain
that anchors the glycoprotein in the viral envelope and a fusion domain that
facilitates viral penetration into target cells. The divergence in the envelope
of HIV complicates efforts to develop an-effective vaccine for AIDS (Volk
et al., 1996).
1.2.2. Target cells and mechanism of infection:
HIV primarily infects cells with CD4 cell surface receptor molecules,
using them to gain entry. HIV use CD4 molecule as receptor, which is
expressed on macrophages and T lymphocyte. HIV may use galactosyl
cermide as receptor in place of CD4 in neural cells. Second important
receptor is necessary for HIV-1 to gain entry to cells is chemokine receptors,
chemokines are soluble factor with chemo- attractant and cytokine
properties. CCR5, the receptor for chemokines RANTES, MIP.1, and
MIP.1B, is the receptor for macrophage-tropic strains of HIV-1. In contrast,
CXCR4, the receptor for chemokine SDF-1, is the necessary co receptor for
lymphocyte-tropic strains of HIV-1 (Buseyne et al., 1998).
The presumed mechanism is that HIV needs to bind to both a T

cell CD4 receptor and to a co-receptor. The viruses first bind to CD4 and
then to the second receptor, this interaction causes conformational change in
the viral envelope activating the gp41 fusion peptide and triggering
membrane fusion. There is evidence that individuals who possess
homozygous deletion in CCR5 may be protected from infection by HIV-1.
25
Genetic variation in the chemokine co-receptor molecule results in less

effective HIV binding and thus slows HIV replication.
Studies show that a homozygous deletion (delta-32) in the gene
coding for the chemokine receptor CCR5 significantly lowers, but does not
eliminate completely, the risk of HIV infection. (Liu et al., 1996; Huang et
al., 1996; Samson et al., 1996; Dean et al., 1996; Theodorou et al., 1997;
Biti et al., 1997). A second genetic variant (CCR2-64I) in the gene coding
for the CCR2 receptor is not protective against HIV infection, but is
associated with slower progression to AIDS by 2 to 3 years in heterozygous
or homozygous individuals. It occurs in roughly 2% of African Americans,
Caucasians, Asians, and Hispanics (Smith et al., 1997; Kostrikis et al.,
1998). A third genetic variant (SDF1-3'A) affects the regulatory region of a
gene coding for the chemokine SDF-1, which is the ligand (the normal
binding molecule) for chemokine receptor CXCR4 (Winkler et al., 1998).
1.2.3. Replication Cycle:
The replication cycle of HIV-1 include the following:
A. Virus entry:
The first step is adherence of HIV by gp 120 to host cells, (Capon &
Ward, 1991) that contain in their surface, the CD4 membrane antigen as
receptor, which is found on T.lymphocyte (Bhat et al., 1993; Long et al.,
1994). These cells have specific chemokine receptors, (Alkhatib et al., 1996)
known as co-receptor, (Deng et al., 1996) such as CCR5 or HIV-1R5, which
is present in the early stages of the disease. (Alkhatib et al., 1996; Deng et
al., 1996; Dragic et al., 1996), and CXCR4 or HIV-1X4 which is seen
during late stages of the disease (Chose et al., 1996; Doranze et al., 1996 ).
The second steps after attachment to host cells surface is penetration of HIV-
26
1virion into host cells. The gp41 is responsible for this (Stein et al., 1987;
McClure et al., 1988, 1990).
B. Replication: (Reverse transcription &Integration)
After internalization, the HIV virion is uncoated and its RNA is
copied and generates a single stranded DNA copy (Katz & Shalka, 1990).
This reaction is mediated by the Reverse Transcriptase, an RNA-and DNAdependent DNA polymerase and RNase H, which degrades the RNA
component of RNA-DNA hybrid molecules (Katz & Shalka., 1990). Then
the second DNA strain is formed, also mediated by Reverse Transcriptase,
which produce large amount of DNA (Shih et al., 1988). The recently
formed double stranded DNA penetrates the nucleus as part of the pre
integration complex reaction. The nuclear viral complexes serve as the
machines that integrate viral DNA into host cell chromosomal DNA to form
provirus. This step is dependent on the activity of the viral Integrate protein
(Pryciak & Varmus, 1992).
C. Proviral DNA:
Following the integration of the recently synthesized viral DNA into
the host DNA, this proviral DNA start to encode viral protein formation.
Initially, viral mRNA is transcribed by proviral DNA and this mRNA
mediated by host cell RNA polymerase, initiates synthesis of large poly
proteins encodes the regulatory and structural viral proteins (Jacks et al.,
1988; Parkin et al., 1992; Chamorro et al., 1992).
D. Budding:
After formation of HIV structure, the virion assembles into the
cytoplasm. HIV is released from the cell by budding. It acquires its lipid
membranes from the host cell membranes after this budding, the protease
enzyme cleaves the virion poly protein leading to maturation of the
27
infections viral particle. In the productive growth cycle the host cell is
destroyed (Earl et al., 1991).
1.2.4. Pathogenesis:
The typical course of untreated HIV infection spans about a decade.
The stages include the primary infection, dissemination of virus to lymphoid
organs, clinical latency, elevated HIV expression, clinical disease and death.
The duration between primary infection and progression to clinical disease
averages about 10 years. Through untreated cases death usually occurs
within 2 years after the onset of clinical symptoms.
Following primary infection, there is a 4-11 days period between
mucosal infection and initial viraemia; viraemia is detectable for about 8-12
weeks, virus is widely disseminated troughout the body during this time and
the lymphoid organs become seeded.
An acute mononucleosis-like syndrome develops in many patients
(50-75%) 3-6 weeks after primary infection. There is significant drop in
numbers of circulating CD4 T cell at this early time. An immune response to
HIV occurs 1 week to 3 months after infection, plasma vireamia drops and
levels of CD4 cells rebound. However the immune response is unable to
clear the infection completely and HIV infected cells persist in the lymph
nodes.
This period of clinical latency may last for as long as 10 years.
During this time there is a high level of ongoing viral replication. It's
estimated that 10 billion HIV particles are produced and destroyed each day.
The half life of the virus in plasma is about 6 hours and the virus life cycle
(from the time of infection of a cell to the production of new progeny that
infect the next cell) averages 2 6 days.
28
CD4 T lymphocytes are the major targets responsible for virus

production; appear to have similar turnover rates. Once infected, the half life
of CD4 T lymphocyte is about 1 6 days. Because of this viral proliferation
and the inherent error rate of the HIV reverse transcriptase, it is estimated
that every nucleotide of the HIV genome probably mutates on a daily bases.
HIV found in patients with late stage disease is usually much more
virulent and cytopathic than the strains of virus found in early infection.
Often a shift from monocyte or macrophage tropic (M-tropic) strains
accompanies progression to AIDS.
The cardinal feature of HIV infection is depletion of T helper,
inducer lymphocytes, as a result of the tropism of HIV for this population of
lymphocytes, which express the CD4 phenotypic marker on their surface.
The CD4 molecule is the major receptor for HIV; it has a high affinity for
the viral envelope. The HIV co-receptor on lymphocytes is the CXCR4
chemokine receptor. Early in infection primary HIV isolates are M-tropic.
However, all strains of HIV infect primary CD4 T cells (but not
immortalized T cell lines in vitro). As the infection progresses the dominant
M-tropic viruses are replaced by T-tropic viruses. Laboratory adaptation of
these primary isolates in immortalized T cell lines, results in loss of ability
to infect monocytes and macrophages.
The consequences of CD4 T cell dysfunction caused by HIV
infection are devastating because the CD4 T-lymphocyte plays a critical role
in the human immune response. It is responsible directly or indirectly for
induction of a wide array of lymphoid and non-lymphoid cell functions.
These effects include activation of macrophages, induction of functions of
cytotoxic T cells, Natural killer cells, B cells and secretion of variety of
29
soluble factors that induce growth and differentiation of lymphoid cells and
affect haematopoietic cells (Geo, 1998).
Monocytes and macrophages play a major role in the dissemination
and pathogenesis of HIV infection. Certain subsets of monocytes express the
CD4 surface antigen and therefore bind to the envelope of HIV. The HIV coreceptor on monocyte and macrophage is the CCR5 chemokine receptor.
Infected pulmonary alveolar macrophages may play a role in the
interstitial pnemuonitis seen in certain patients with AIDS.
Macrophage tropic strains of HIV predominate early after infection,
and these strains are responsible for initial infections even when the
transmitting source contains both M-tropic and T-tropic viruses.
It's believed that monocytes and macrophages serve as major
reservoirs for HIV in the body. Unlike the CD4 T.cell, the monocyte is
relatively refractory to the cytopathic effects of HIV, so that the virus can
not only survive in this cell but can be transported to various organs in the
body such as lungs and brain (Volk et al., 1996).
Lymphoid organs play a central role in HIV infection.
Lymphocytes in the peripheral blood represent only about 2% of the total
lymphocyte pool, the remainder being located chiefly in lymphoid organs
where specific immune responses are generated.
Throughout the course of untreated infection, even during the stage
of clinical latency, HIV is actively replicating in lymphoid tissues where it is
ideal for the establishment and spread of HIV infection. Cytokines are
released activating a large pool of CD4 T cells that are highly susceptible to
HIV infection. As the late stage of HIV disease progresses, the architecture
of the lymphnodes becomes disrupted.
30
Neureologic abnormalities are common in AIDS and occur to

varying degrees in 40-90% of patients. These include HIV encephalopathy,
peripheral neuropathies and most serious AIDS dementia complex. Both
direct
and
indirect
pathogenic
mechanisms
might
explain
the
neuropsychiatric manifestations of HIV.

Virus may enter the brain through infected monocyte and release
cytokines that are toxic to neurons as well as chemotactic factors that lead to
infiltration of the brain with inflammatory cells. HIV has been found in a
limited number of neurons, oligodendrocytes and astrocytes.
HIV infected persons develop both humoral and cell mediated
response against HIV related antigens. Antibodies to a number of viral
antigens develop soon after infection, but the response pattern against
specific viral antigens changes over time as patient progress to AIDS.
Antibodies to envelope glycoproteins (gp 41, gp 120, gp 160) are
maintained but those directed against the core protein (p24) decline. The
decline of Anti p24 may herald the beginning of clinical signs and other
immunologic markers of progression.
Most infected individuals make neutralizing antibodies against
HIV. The neutralizing antibodies can be measured in vitro by inhibiting HIV
infection of susceptible lymphocyte cell lines.
Cytotoxic T- lymphocytes recognize env, pol and gag genes
products, this reactivity is mediated by major histocompatiability complex
restricted to CD3 and CD8 lymphocytes. The env specific reactivity occurs in
nearly all infected people and decreases with progression of disease. Natural
killer cell activity has also been detected against HIV-1 gp 120. It's not
known which of these host responses is important in providing protection
against HIV infection. A problem confronting AIDS vaccine research is that
31
the correlates of the protective immunity are not known, including the
relative importance of humoural and cell-mediated immune response (Geo et
al., 1998).
1.2.5. Transmission:
HIV is transmitted during sexual contact (including genital-oral
sex), through parenteral exposure to a contaminated blood or blood products
and from mother to child acquiring infection through breast-feeding, during
birth and during prenatal period is probable. The presence of other sexually
transmitted diseases such as syphilis, gonorrhea, or chancroid increases the
risk of sexual HIV transmission as much as a hundred folds, because the
inflammation and sores facilitate transferring of HIV across mucosal
barriers. Since the first description of AIDS, promiscuous homosexual
activity has been recognized as a major risk factor for acquisition of the
disease.
Furthermore health care workers usually get infection by HIV
following a needle stick with contaminated blood.
There has been considerable concern that in rare circumstances
other types of transmission may occur such as through "casual" contact with
HIV infected persons or insect vectors, but there is no evidence of virus
transmission under these casual conditions (Volk et al., 1996).
1.2.6. Peripheral Blood in AIDS:
Cytopenias are commonly seen in association with HIV infection.
Anemia, thrombocytopenia, neutropenia, lymphocytopenia, monocytopenia,
or combinations of any or all of these can occur in over 90% of patients with
AIDS. The microenvironment of the marrow may also be altered by HIV
infection of stromal cells including fibroblasts, endothelial cells, reticular
cells, macrophages, osteoclasts, and steatocytes, resulting in dysregulation of
32
hematopoietic cell growth with reduced hematopoiesis.( Moses et al., 1998)

Anemia is present in over half of patients early in the course of AIDS and in
nearly all AIDS patients late in the course. The anemia is often
normochromic and normocytic, typical of anemia of chronic disease, and
iron stores are increased by measurement of serum ferritin. Though CD34
stem cells poorly express CD4 receptors and, hence, are relatively resistant
to HIV infection, mononuclear cells are infected and produce cytokines such
as TGF, TNF, and IL-1 that suppress hematopoiesis. Cytopenias can be
potentiated by drug therapy including zidovudine (ZDV), ganciclovir,
amphotericin B, or trimethoprim-sulfamethoxazole and may require dose
reduction or cessation of therapy. Though a positive direct antiglobulin test
may be present in up to 43% of HIV-infected persons, hemolytic anemia is
uncommon.( Volberding et al., 2003) anemias in AIDS occasionally may
results from use of drugs that act as folate antagonists (trimethoprimsulfamethoxazole).
Thrombocytopenia is commonly seen in about a third of AIDS
cases, is rarely severe, and may result from peripheral consumption
(splenomegaly, immune complexes) or from decreased marrow platelet
production. Thrombocytopenia may also appear in some HIV-infected
persons prior to development of clinical AIDS, and most cases are due to
platelet consumption.
Lymphopenia is present in about a third of AIDS cases due to the
decrease in T4 lymphocytes.
Neutrophilia may indicate bacterial sepsis. Bone marrow failure
leading to death in patients with AIDS is very uncommon.( Jacobson et al
.,1997; Moses et al.,1998;Volberding et al., 2003; Meynard et al.,1997).
33
Neutropenia that accompanies HIV infection can increase the risk

for infection or worse the course of infection. Neutropenia can be common
in patients with AIDS and is a risk factor for both bacterial and fungal
infections. The most common cause for neutropenia is drug therapy.
Neutropenia can result from involvement of bone marrow by opportunistic
infections,
from
pharmacologic
therapies
such
as
trimethoprim-
sulfamethoxazole, and from direct effects of HIV through accelerated

apoptosis. Both chemotaxis and phagocytic functions of neutrophils also
appear to be impaired.
Thrombosis is more likely to occur when the CD4 count is less than
200/l. ( Volberding et al., 2003; Copur et al., 2002). The antiphospholipid
syndrome has been reported in association with HIV infection ( Leder et al.,
2001).
1.2.7. Acquired immunodeficiency syndrome (AIDS):
AIDS is a group of clinical syndrome caused by HIV, characterized
by profound immune suppression with diverse clinical features, including
opportunistic infection, malignancies and central nervous system. (Osmond
et al., 1991)
Classification
The CDC classification of HIV disease was first put forth as a
categorization of HIV related symptoms into four groups and was explicitly
for "public health purposes" and not "intended as a staging system,"
although it was frequently treated as if it were a staging system in the AIDS
literature(CDC, 1986). Staging is disease classification that aims primarily to
make groupings that have different prognosis and can be used in guiding
treatment decisions. Stages attempt to classify disease in a progressive
sequence from least to most severe, each higher stage having a poorer
34
prognosis or different medical management than the preceding stage. The

current CDC classification system from the revision in 1993 combines three
categories of the CD4 count with three symptom categories and are closer to
a staging system. This description of its intended use is close to the use of a
staging system (CDC, 1993).
CD4T-lymphocyte categories:
Category 1: > 500 cells/l (or CD4% > 28%)
Category 2: 200-499 cells/ l (or CD4% 14% - 28%)
Category 3: < 200 cells/ l (or CD4% < 14%)
Categories of clinical conditions:
Category A
A
symptomatic
HIV
infection,
persistent
generalized
lymphadenopathy acute (primary) HIV infection with accompanying illness

or history of acute HIV infection in an adolescent (>13 years) with
documented HIV infections. Conditions listed in categories B and C have
not occurred.
Category B
Consists of symptomatic conditions in an HIV infected adolescent or
adult that are not included among conditions listed in clinical Category C
and that meet at least one of the following criteria:
A\ The conditions are attributed to HIV infection or are indicative of a defect
in cell mediated immunity.
B\ the conditions are considered by physicians to have a clinical course or to
require management that is complicated by HIV infection.
35
Category C
Includes the clinical conditions listed in the 1993 AIDS surveillance
case definition. For classification purposes, once a Category C condition has
occurred, the person will remain in Category C (Dennis, 1998).
1.2.8. Clinical manifestation :
The clinical manifestations of HIV disease include five stages,
primary infection, and early, middle-, advanced-, and late-stage HIV disease.
These stages of HIV infection goes on the primary infection, dissemination
of virus to lymphoid organ, clinical latency, elevated HIV expression,
clinical disease, and death. There is much individual variation in the clinical
manifestations of individual patients in the same disease stage and in the rate
of progression through the stages.
The duration between primary infection and progression to
clinical disease in untreated individuals averages about 10 years (Bacchetti

& Moss, 1989). The estimate varies with the age at which infection occurs
and is significantly shorter in infants and in older adults and varies even
between infection at age 20 and infection at age 40. Death usually occurs
within 2 years after the onset of clinical symptoms (Rosenberge et al.,
1994).
1.2.8.1. Primary infection:
Often symptoms less, but when present appear to result from
immunopathic, lymphocytopathic, and neuropathic. Some times
symptoms present with an infection mononucleosis-like illness, during
2 weeks to 3 months after exposure. Reported manifestations include
fevers, fatigue, malaise, arthralgias, myalgias, lymphadenopathy,
splenomegaly, anorexia, nausea and vomiting, diarrhea, pharyngitis,
36
headache
and
retroorbital
pain,
meningitis
and
encephalitis,
neuropathy, myelopathy, maculopapular rash, and mucocutaneous

ulceration. Lymphadenopathy onset typically occurs in the second
week of the syndrome and may be generalized or preferentially affect
the occipital, axillary, and cervical groups, fevers, rash, sore throat,
night sweats, malaise, Lymphadenopathy, diarrhea, and relative and
absolute lymphocytosis in the peripheral blood. There may be mouth
and genital ulcers, a profound transient decrease in CD4 lymphocyte.
(Schacker et al., 1998; Keet et al., 1993; Schechter et al., 1990; Pantaleo
et al., 1997; Niu et al., 1993; Henrard et al., 1997).
1.2.8.2. Early and Middle stage:

The early and middle stage of HIV disease followed the primary
infection after at least three-month. In which the level of detectable
virus in peripheral blood drop dramatically and antibodies rise
(Phillips, 1996). This period of clinical latency may last for as long as
10 years, during which the patients may show, depressed count of CD4
lymphocyte (from normal to 200 to 300cells\l). Through the
subsequent early and middle stages of HIV disease, the level of virus
detectable in the peripheral blood often remains low (Pantaleo et al.,
1998).
Clinical manifestations are usually minimal or no symptoms,
other individuals, however have subjective systemic symptoms, such as
fatigue and a few have fevers. Many patients have painless stable
lymphadenopathy, which in some cases actually regresses as HIV
disease advances. Allergy to skin testing becomes increasingly
37
probable as the CD4count falls beyond 400 cells/l, with obvious

implications for tuberculosis screening ( Pantaleo et al., 1998).
1.2.8.3. Advanced HIV disease:
Untreated patients with manifestation of advanced HIV diseases
typically have CD4 counts below 200 cells/ l, increased plasma HIV RNA
levels, and clinical manifestation indicative of sever immunocompromise,
and in addition opportunistic infections commonly occur. In the absence of
prophylaxis, Pneumocystis Carinii Pneumonia (PCP), is the most common
such infection. In-patients with HIV dementia complex, manifestations
typically become profound. Systemic symptoms including fever, fatigue, and
weight loss are prominent, often without identifiable secondary causes.
Cryptosporidium diarrhea is a common problem with limited treatment
options, and the probability that it will resolve or remit decrease HIV disease
progress. HIV plasma RNA levels typically are high (Ioachim, 1990).
1.2.8.4. Late-stage HIV disease:
As the disease advances further and the CD4 count drops below 50
cells\mm, additional opportunistic infection as well as central nervous
system occur commonly, and in homosexual males, existing Kaposi
Sarcoma may become extensive, and cause disfigurement and clinically
significant edema. Central nervous system toxoplasmosis, Cryptococcal
Meningitis, cytomegalovirus (CMV) disease, and Mycobacterium Avium
Complex (MAC) disease is frequent. Infection such as MAC, CMV,
Strongyloides Stercoralis, Herpes Zoster or Tuberculosis, which normally
colonize superficially or are limited to an organ system or a local anatomic
region, may invade tissue or disseminate widely. Simultaneous clinically
significant infection by more than one pathogen is common. Secondary
38
symptoms become increasingly problematic, including anorexia, nausea,

vomiting, diarrhea, mal-absorption, muscle wasting, and weakness.
Death eventually results from extensive disease of vital organs, most
commonly the lungs and presumably from effects of circulating toxins,
electrolyte abnormalities hematopoietic and circulatory failure and
autonomic nervous system damages (Nguyen et al., 1995).
1.2.9. Laboratory diagnosis of HIV:
Evidence of HIV can be detected by virus isolation, serological
determination of antiviral antibodies including screening methods, and
confirmatory supplemental assay, and measurement of viral nucleic acid or
antigens. Also other measurements for monitoring disease progression in
HIV-1 infected individuals, such as determination of the CD4 are helpful.
HIV-1 and HIV-2 are both detected by their antibodies, antigens and
genomes.
HIV-1 ELISA technique detects antibodies to both HIV-1 and HIV-2,
confirmation of the ELISA screening test is accomplished with specific
western blot test. Confirmation can also be done by the use of
immunoflouresence tests.
The western blot identifies antibodies specific for several HIV
antigens. Most often antibodies to HIV P24 and either gp 41 or gp 160
confirm HIV infection.
The western blot method is based on the electrophoresis separation of
major proteins of an infectious agent in a two- dimensional agarose gel
matrix. A suspension of the organism against which antibodies are being
produced is mechanically or chemically disturbed and the solubilized
antigen suspension is placed at an end of a polyacrylamide (polymer) gel.
Under influence of electrical current, the proteins migrate through the gel.
39
Most bacteria or viruses contain several major proteins that can be

recognized based on their position in the gel after electrophoresis. Smaller
proteins travel faster and migrate further in the lanes of the gel than do the
larger proteins. The protein bands are transferred from the gel to a
nitrocellulose or other type of thin membrane and the membrane is treated to
immobilize the proteins. The membrane is then cut into many thin strips,
each carrying the pattern of protein bands. When patient serum is layered
over the strip, antibodies present can be used to determine whether the
patients are infected with the agent or not. Antibodies against microbes with
numerous cross-reacting antibodies, such as T. pallidum, B. burgdorferi,
herpes simplex virus types 1 and 2 and HIV, are identified more specifically
using this technology than by methods that test for only one general
antibody type (Volk et al., 1996).
A lot of membrane diffusion assays are used for first screening but are
of less sensitivity and specificity than ELISA. HIV-1 p24 antigen testing is
used to detect acutely infected patients before appearance of antibodies.
PCR testing for HIV is useful for the newborn population who may have
maternal HIV antibodies confounding interpretation of serological test and
for all patients who may not produce detectable antibodies for months
following primary infection. The reverse transcriptase PCR test is used to
quantities virus in blood. Results of quantitation are used to manage antiretroviral therapy. Also NASBA technique is used for detection and
quantitation of the virus particles.
Blood for transfusion is screened for antibodies indicative for HIV 1
and 2 as well as HTLV.l and 2 infections. In addition HIV antigen p24 test is
performed to detect donors who may be recently infected and have not
40
produced HIV antibodies. Also latex agglutination can be used for initial
screening; CD4/CD8 ratio correlates infection of HIV (How et a., 1998).
PCR is the method that duplicates short DNA segment thousands to a
million folds. DNA fragments that can be identified with a specific probe,
but are too few in number in the original specimen to be detected, can be
duplicated (amplified) using PCR, this provides the probe with enough target
to readily identify the presence of a specific virus.
1.2.10. T.Lymphocytes:
CD4 and CD8 molecules are T cell surface glycoproteins that are
expressed on mutually exclusive subsets of mature T cells with distinct
patterns of MHC restriction. CD4 and CD8 serve as accessory molecules by
facilitating interaction of T cells with APCs or CTL target cells respectively.
The function of CD4 and CD8 are intricately associated with TCR
function and therefore accessory molecules are often called coreceptoers.
Approximatly 65% per cent of peripheral B- positive T cells express CD4,
and 35 % express CD8 (Howcroft et al., 1993).
1.2.10.1. CD4 cells:
CD4 cells are the major targets for HIV. Also known as helper Tcells, are a type of lymphocyte, which is a white blood cell that plays an
important role in the immune system. Lymphocytes control the body's
ability to recognize and fight infections and cancers. CD4 lymphocytes help
to identify, attack, and destroy specific bacteria, fungi, and other germs that
infect the body. In addition, they regulate the production of antibodies, and
cytokines. CD4 cells are produced in bone marrow and differentiated in
thymus, then migrates to spleen and lymph nodes, they circulate throughout
the body in the bloodstream. HIV binds to the surface of CD4 cells, enters
them, and either reproduces immediately, killing them in the process, or
41
remains in a resting state, reproducing when the cell becomes active (Lang et
al., 1989).
The number of CD4 cells in a blood sample, which is called CD4
count, ranges from 500 to 1600 cells/l. The CD4 cell count is a laboratory
marker of the strength of the immune system. CD4 T lymphocyte subset
count is one of the best measurements for monitoring disease progression in
HIV infected individuals (De Wolf,. 1988; Stites., 1989; Stein,. 1992).
The plasma CD4 lymphocyte count (CD4 count, T-helper cell count)
has been used since the beginning of the HIV epidemic to indicate disease
stage, although it has some limitations (Hoover et al., 1992; Malone et al.,
1990). Individual determinations may vary significantly. Identifiable causes
of variation include time of day, short-term changes in health, and
experience of the clinical laboratory performing the test. Nevertheless, over
many months, the decline in CD4 cells reflects progression of untreated

HIV disease, and prior to 1996, used CD4 counts to indicate disease
stage. The CD4 count alone does not always reflect the clinical status of
an HIV-infected individual; untreated individuals with similar CD4
counts may have very different functional status, frequency of
opportunistic infections, and constitutional symptoms and signs.
Further, the CD4 cell numbers reflect only one aspect of
immunocompetence. Treatment with potent antiretroviral therapy often
results in return of CD4 counts to high levels that probably mask other
immune system defects, thus somewhat complicating staging of
disease. To understand a patient's disease stage in the era of potent
antiretroviral therapy, the clinician should consider both the most
recent CD4count and the patient's lowest past count ("nadir CD4
42
count"). Even in patients who have responded to antiretroviral therapy

with markedly increased CD4counts, however, subsequent decline of
over time indicates continuing progression.
1.2.10.2. Function of CD4:
Two important function in the activation of CD4 cells:
1- CD4 serve as cell-cell adhesion molecule, by virtue of it is specific
affinity for class 11 MHC molecules. The invariant CD4 molecule bind
via it is two N-terminal Ig-like domains to non polymorphic B2 domain
of the class 11 MHC molecule.
2- CD4molecule may transduce signals or facilitate TCR complex
mediated signal transduction upon binding class 11 MHC molecule
there by promoting the subsequent functional responses of class
restricted T cells
1.2.10.3. Function of CD8:
The CD8 molecule is thought to have the same two general roles as
CD4 namely in cell to cell adhesion and signal transudation. First CD8 serve
as cell to cell adhesion molecules by binding to nonpolymorpgic
immunoglobulin-like 3 domain of class 1 MHC molecule there by
stabilizing the interaction of class 1 MHC-restricted T cell (usually a CTL)
with a target cell bearing class 1 MHC-associated antigen. Second the CD8
molecules may tranduce signal or may facilitate TCR: CD3-mediated signal
transduction upon binding class 1MHC molecules, there by promoting
subsequent function responses of class 1-restricted T cells.
1.2.11. Methods for CD4 &CD8 lymphocyte enumeration:
Accurate and reliable measures of CD4 T. lymphocytes are essential
for the assessment of the immune system of HIV -infected person (Turner et
43
al., 1994; Hoover et al., 1992). The pathogenesis of AIDS is largely

attributable to the decrease in T.lympocytes that bears the CD4 receptors
(DeWolf et al., 1988; Smith, 1990). Progressive depletion of CD4
T.lymphocytes is associated with an increased likelihood of clinical
complication (Hanson et al., 1995; Stein et al, 1992). Consequently, the
Public Health Service (PHS) has recommended that CD4 T.lymphocyte
levels be monitored every 3-6 month in all HIV infected persons (CDC,
1992).The measurement of CD4 T. cells levels has been used to establish
decision points for initiating pneumocystis carinii pneumonia prophylaxis
(CDC., 1995) and antiretroviral therapy (National Institutes of health., 1990)
and to monitor the efficacy of treatment (Goldman et al., 1996- De Gruttola
et al., 1994). CD4 T.lymphocyte levels are also used as prognostic indicator
in patient who have HIV disease (Fahey et al., 1990; CDC, 1994) and
recently have been included as one of the criteria for initiating prophylaxis
for several opportunistic infections that are squeal of HIV infection (CDC,
1995; CDC, 1993).More over, CD4 T.lymphocytes levels are a criterion for
categorizing HIV related clinical conditions by CDCs classification
system for HIV infection and surveillance case definition for AIDS among
adults and adolescents(CDC, 1992).
Other observations made in the era prior to the advent of combination
therapy also suggest it is likely that nearly all HIV-infected persons will
eventually lose CD4 lymphocytes and progress to AIDS in the absence of
effective treatment. For example, in about 10 years of follow-up of 288 men
HIV-seropositive at baseline in the San Francisco General Hospital Study
(1983-1993), only one (0.3%) maintained a CD4 lymphocyte count above
700/l throughout follow-up and nearly all showed some worsening of other
laboratory values predictive of AIDS, such as anti-p24 antibody levels, .B-2
44
microglobulin, or neopterin. Most HIV-positive persons, even with nearnormal CD4 lymphocyte counts, show functional lymphocyte abnormalities
that suggest their long-term immune functioning will be impaired. There are
no clearly documented instances of persons who appear to have cleared a
longstanding HIV infection, although there is a report of transient infection
in an infant (Berdberg & Raden et al., 1995).
CD4T.lymphocytes can be counted by different techniques either
automated or manually.
1.2.11.1. Automated Methods:
1.2.11.1.1. Flow cytometry:
Flowcytometry is the measerment (-metry) of cellular (cyto-)
properties as they are moving in a fluid stream (flow), past a stationary set of
detectors. Latest development goes a step further and allows cells of
different subtypes to be stored and collected for further analysis. It is capable
of rapid, quantitative multi-parameter analysis of heterogeneous cell
populations on a cell-by-cell basis (single cell analysis). Performing
flowcytometry experiments generally involve three distinct, interdependent
phases. First is the pre-flowcytometry phase, which involves staining of cells
with fluorescent reagents. Second is the flowcytometry phase, which involve
processing
the stained cells using flowcytometry instrumentation and
collecting data(acquiring) for one or more measurements(which are called

parameters) made on each individual. Finally, the analysis phase involves
analyzing the collected data.
Flowcytometry offers several technical options to obtain absolute CD4
T cell counts. In double-platform assays the flowcytometer is used to deliver
the percentage of CD4T cells, which is then combined with parameters from
a hematological analyzer to obtain the absolute CD4 count. In single
45
plateform assays the flowcytometer is used on its own to provide the

percentage of CD4 T cells and/or absolute CD4 T.cell counts. Two technical
alternatives are presently available for single-plateform approaches: the use
of volumetric instruments and the use of mirobeads.
Flowcytometry is currently the accepted standard method for
determination of absolute counts of CD4 and CD8 T.lymphocytes (Landay et
al., 1990; CDC, 1992b), the techniques is expensive and require
sophisticated equipments as well as trained personnel to perform it .In
addition, the lack of ready access to technical support and quality assurance
programs has limited the use of flowcytometry techniques in resourceconstrained contries. On the other hand, manual non-flowcytmetric
techniques such as Dynabeads and Coulter methods, which produce results,
comparable to those of flowcytometry have their own limitations. They are
labor- intensive, and only a few (10 to 15) CD4 T.lymphoyte counts can be
performed by an analyst per day (Landay et al., 1990).
1.2.11.1.2. Dedicated cytometer:
Dedicated cytometers are systems that are designed to exclusively
perform absolute CD4 and CD8 T-cell measurements without the need for a
haematology analyzer. These instruments are of simpler operation and
cheaper when compared to traditional bench-top flow cytometers. However,
they dont offer flexibility in assay and reagent choice can have high running
costs and have medium sample throughout capacity. The FACS count
(Becton Dickinson immunocytometry systeme, USA), a system based on
flowcytometeric principles; at present the most widely used dedicated
cytometer, and has been exclusively validated. Other dedicated instruments,
based on flow-and non-flow principles, have recently become available
while others may soon be commercialized.
46
A Study was done by Dieye et al. (2003) to evaluate a simple and

affordable volumetric flow cytometer (Cyflow) for direct volumetric CD4T
cell counting in Dakar using the FACSCount and the TruCount on
FACSCAN as gold standard. They found that, direct volumetric CD4 T cell
measurements on the Cyflow correlated very well with the CD4 counts
obtained by FACSCount (R2 = 0.953) and TruCount analysis on FACSCAN
(R2 = 0.961). In addition, bead-based absolute CD4 measurements on the
Cyflow correlated highly with those obtained on FACSCount (R= 0.994).
Averages of CD4 count obtained by the different platforms were 464, 450
and, 455 on the Cyflow, the FACSCount and the TruCount on FACSCAN,
respectively.
Other study was done by in Siriraj Hospital (Thailand), to evaluate two
recently developed inexpensive volumetric single-platform (SP) FCM
technologies from Partec CyFlow and Guava Technologies. Evaluation of
CyFlowgreen FCM and Guava Easy CD4/CD8 system for enumerating
CD4and CD8 T-cells was performed by comparing with the standard SP
bead-based
two
color
FACSCountTM
system,
SP
bead-based
TriTEST/TruCOUNTTM tube using FACSCaliburTM FCM and the

standard dual-platform method by using FACScanTM FCM.
The CD4T-cell counts obtained from CyFlowgreen FCM showed
excellent correlation R=0.98 and 0.98, R2=0.97 and 0.97, P<0.0001 with
mean biases of -36.2 cells/L (limit of agreement: -149.7 to +77.3 cells/L)
and -16.1 cells/.L (limit of agreement: -113.9 to +81.6 cells/L) when
compared
with
standard
TriTEST/TruCOUNTTM
system
and
FACSCountTM system, respectively. The correlations of CD8 T.cell counts

were also high R=0.98, R2=0.96, mean bias of -151.3 cells/.L (limit of
agreement: -450.3 to +147.6 cells/.L); and R=0.96, R2=0.93, mean bias of 47
115.2 cells/.L (limit of agreement: -368.3 to +137.8 cells/.L) when compared

with
TriTEST/TruCOUNTTM
system
and
FACSCountTM
system,
respectively. The CD4T-cell counts obtained from Guava Easy CD4/CD8

system showed high correlation R=0.97 and 0.97, R2=0.95 and 0.94,
P<0.0001 with mean biases of +13.1 cells/.L (limit of agreement: -117.9 to
+144.1 cells/L) and +33.2 cells/.L (limit of agreement: -101.8 to +168.3
cells/.L) when compared with standard TriTEST/TruCOUNTTM system and
FACSCountTM system, respectively.
The correlations of CD8 T-cell counts were also good R=0.96,
R2=0.92, mean bias of -79.5 cells/.L (limit of agreement: -455.7 to +296.8
cells/.L); and R=0.94, R2=0.88, mean bias of 52.3 cells/.L (limit of
agreement:
-370.7
to
+266.2
cells/.L)
when
compared
with
TriTEST/TruCOUNTTM system and FACSCountTM system, respectively.

Also Fryland et al., (2004) was evaluated Cyflow Counter by
comparing it with FacsCount in Malawi. The CD4 count range in blood
samples was 2 - 1789 cells/l. The correlation coefficient for the Cyflow
Counter was 0.92 (0.89- 0.95). The mean difference in CD4 count values
with FacsCount was 8.68 (-18.8 to 1.4). Intra-run variation was less than
3%.
In a study done by Pattanapanyasat et al., (2004) in Thai, CyFlow-green
was compared with the two reference standard methods of the 3-color
system using a FACScan or FACSCalibur FCM and the 2-color FACSCount
system (Becton-Dickinson), both of which use microbeads for determining
absolute counts.
A high correlation of absolute CD4 counts was shown when the SP
CyFlow-green was compared with the bead-based three-color TRUCount
and FACSCount (R2=0.96, mean bias -56.2 cells/l, with 95% confidence
48
interval [CI] -162.1 to 49.6; and R2=0.97, mean bias -32.9 cells/l, with
95% CI -188.5 to 52.9, respectively). The correlation of CD4 counts by the
two beadbased systems was high (R2=0.98), mean bias +25.5 cells/l, with
95% CI -40.2 to +91.3). The SP CyFlow-green yielded CD4 counts that were
41.5 and 18.0 cells/l lower than TRUCount and the FACSCount systems in
the 100-300 CD4 cells/l range (Thai national guidelines call for initiating
antiretroviral drug therapy at CD4 count <200 cells/L).
1.2.11.2. Manual Method:
Currently available manual assays rely on microscope (optical or
fluorescence). Additionally they require the use of other small equipment
such as centrifuge and magnets. These tests are designed to operate with low
sample throughput in resource-limited laboratories but their running costs
are not always lower when compared to the automated methods.
1.2.11. 2.1. TRAx CD4 (T cell Diagnosis. Cambridge. MA. USA):
This is a sandwich enzyme immunoassay method for determination
of total CD4 protein in a whole blood specimen, which has been treated with
a lysing reagent to solubilize the cell membrane and releasing the CD4
molecules as well as other markers. In the last step the absorbance is read at
490 nm using an ordinary ELISA reader. Unknown values for specimens
were interpolated in cells/l from a standard curve constructed using the
standard calibrators, which were run together with the test samples
(Lyamuya et al., 1996).
1.2.11.2.2 Dynabeads T4-T8 Quantitative method:

This is an immunomagnetic cell isolation method which uses
Dynabeads magnetic particles coated with antibodies to the CD4 and CD8
antigens to capture and isolate CD4and CD8 T lymphocytes from whole
49
blood. The isolated cells are lysed and the nuclei are counted directly either
in automated cell counter or by microscopy (fluorescent or light) after
staining. The results were expressed as cell per microliter whole blood. This
method utilizes simple technology, gives absolute CD4 and CD8 T
lymphocyte counts. However the method permits processing of few samples
at a time and the specimens must be analyzed within 24 hours of collection.
Also on lyses, the cell nuclei must be counted within one hour. In addition
manual counting is laborious and to some extent subjective, especially when
the nuclei are fragmented. Counting of samples with more than 1000cell/l
was difficult because of crowding and overlapping of some nuclei.
1.2.11.2.3. Immunocytochemistry:
Immunohistochemistry is the marriage of immunologic and
histochemical techniques, allowing phenotypic markers to be detected and
interpreted within a morphologic context. Immunohistochemical (IHC)
staining techniques allow for the visualization of antigens via the sequential
application of a specific antibody to the antigen (primary antibody), a
secondary antibody to the primary antibody and an enzyme complex with a
chromogenic substrate with interposed washing steps. The enzymatic
activation of the chromgen results in a visible reaction product at the antigen
site. The specimen may then be counterstained and cover slipped. Results
are interpreted using a light microscope and aid in the differential diagnosis
of pathophysiological processes, which may or may not be associated with a
particular antigen.
The birth of immunohistochemistry is attributable to Coons and
colleagues, who, in 1941 introduced fluorescent-labeled antibodies (Coons
et al., 1994).
50
The development of enzyme-labeled antibodies that were reactive

in fixed, paraffin-embedded tissue provided the basis for modern
immunohistochemistry (Distfano et al., 19973; Taylor & Burn., 1974).
Subsequent modifications of techniques, including the peroxidaseantiperoxidase method (Stenberger et al., 1970) and avidin-biotin-peroxidase
method, (Hsu et al., 1981) as well as the development and commercial
availability of a wide range of polyclonal and monoclonal antibodies have
combined to make immunohistochemistry an essential element of
contemporary pathology practice.
The most widespread use of immunohistochemistry in pathology is
to supplement morphologic criteria in determining the appropriate
classification of an undifferentiated neoplasm. For this type of
application the immunohistochemistry results are used to aid in the
differential diagnosis suggested by the clinical setting and histology of the
lesion. In the detection of infectious agents or identification of physiologic
substances in aberrant locations, immunohistochemical assays more directly
determine the diagnosis. In other applications, such as the detection of
prognostic markers including hormone receptors, oncogene products, or
proliferation markers, immunohistochemistry is used as an analytical assay.
The addition of analytical assays has served to alert the pathology
community to the need for more stringent controls in all aspects of
immunohistochemistry.
1.2.11.2.3.1. Indirect immunohistochemistry:
Indirect immunohistochemical methods exploit the natural capacity of
immunoglobulin to act as antigen. In this approach, the primary antibody is
raised in one species, for example, the rabbit, and is not conjugated.
Immunoglobulins from this species are then used as an immunogen in a
51
second species, for example, the goat, resulting in antibodies recognizing

immunoglobulin in the primary serum (in this example, goat anti-rabbit).
Immunoglobulin from the secondary serum is then conjugated to a detection
system as described earlier for the direct system. The complete assay
involves incubation with unlabeled primary antibody, washing, incubation
with secondary antibody, followed by washing and detection (Swanson,
1994).
The labeled secondary antibody can allow for signal amplification
compared with the direct method, and this permits the primary antibody to
be used at a higher dilution. Sensitivity is generally increased compared with
direct detection. The labeled secondary antibody can be used to detect any
primary antibody that has been raised in the species it recognizes. This
versatility allows for commercial development and standardization of these
detection reagents. Background staining associated with polyvalent
secondary antibodies can be a problem with this technique (Swanson, 1994).
A recently developed variation of this technique uses a large
dextran backbone to covalently link secondary antibody and enzyme
molecules. The complex includes up to 100 enzyme molecules and 20
secondary antibody molecules. Improved sensitivity, attributed to increased
localization of enzyme activity, has been reported (Sabattini et al., 1998).
1.2.11.2.3.1a. Antibody Bridge Techniques:
The antibody bridge technique (three-step method) is a further
modification of the indirect method. In addition to the primary and
secondary antibodies used in the indirect method, a tertiary labeled antibody
is used in the antibody bridge technique. The primary and tertiary antibodies
must be from the same or closely related species, and the secondary or
bridge antibody is directed against that species, hence binding both primary
52
and tertiary antibodies as a bridge. (In this application the secondary

antibody is unlabeled.) In a variation on the same theme, the tertiary
antibody can be an unlabeled antienzyme antibody. Addition of the enzyme
(usually horse radish peroxidase) as the fourth step results in an
immunologic reaction between enzyme (e.g., horseradish peroxidase) and
anti enzyme (anti-horseradish peroxidase) for detection.
Preformed enzyme anti enzyme complexes such as PAP
(Sternberger et al., 1970), APAAP, and GAG, are available. This
combination of tertiary antibody and enzyme into one complex that is linked
to the primary antibody by an unlabeled bridge antibody reduces the fourstep technique to a three-step technique.
Reproducibility and specificity are enhanced with the use of PAP,
which is a small, stable complex from the same or closely related species
that shares antigenic determinants against which the bridge antibody is
directed.
The efficacy of these techniques depends on the development of
high-affinity bridging antibodies, tertiary antibodies, and antienzyme
antibodies.
1.2.11.2.3.1b. Affinity Labeling Methods:
Avidin-Biotin Methods:
The extremely high affinity of avidin for biotin has been used as an
alternative to relying on the antigenic nature of immunoglobulins to provide
links between antigen localization and detection reagents. Avidin is a highmolecular-weight protein (mol. wt. 67,000) found in egg white that has very
high affinity for biotin, a low-molecular-weight water-soluble vitamin. The
avidin-biotin disassociation constant approaches the strength of a covalent
bond (Kd 10. 15), and the chemistry of utilizing these molecules as
53
reagents for affinity purification has been well characterized (Bayer &
Wilchek., 1980). One molecule covalently labeled with avidin will bind to
another molecule covalently labeled with biotin, assuming labels are
introduced in a fashion that does not sterically limit the interaction of the
affinity labels.
Because avidin molecules have four binding sites for biotin, the
geometry and proportions may be arranged to allow for multimolecular
complex formation between avidin-and biotin-labeled reagents. Although
biotin can be attached to any component of the immunohistochemical
reactionprimary, secondary, or histochemical enzymethe use of a
biotinylated secondary antibody is employed most frequently because of the
great flexibility in the approach for detection. A simple layered approach
may be used.
After localization of the biotinylated secondary antibody, detection
can be achieved with unlabeled avidin followed by biotinylated label as the
fourth step, known as the bridged avidin-biotin method. The biotinylated
label is most commonly a histochemical enzyme. The third and fourth steps
of the bridged avidin-biotin method could be combined though complex
formation between avidin and biotinylated enzyme, in the same way that the
PAP complex eliminated one step. This was first described for biotinylated
peroxidase6 and is known as the avidin-biotin complex (ABC) method.
Because of the polyvalent biotin-binding capacity of avidin, mixing
of the biotinylated enzyme and avidin can be done in a ratio that allows for
multiple enzyme molecules to be complexed to avidin while the overall
complex still retains the excess biotin binding sites required for interaction
with the biotinylated secondary antibody.
54
The significant advantage of the three-step ABC is its great

sensitivity, which has been attributed to the localization of several molecules
of peroxidase at the antigenic site. The avidin-biotin peroxidaseantiperoxidase (ABPAP) complex involves sequential use of the PAP and
ABC techniques in an attempt to further increase the number of enzyme
molecules at an antigen site. Signal amplification by these techniques
reaches a limit because of steric effects. Binding of large complexes is less
efficient, and enzymes buried within layers of other molecules may not be as
accessible to substrates required to generate the colored products. Although
first described for peroxidase, alkaline phosphatase can be biotinylated and
complexed with avidin in a similar fashion.
Avidin reagents have been made by covalent reaction to a
histochemical enzyme. For horseradish peroxidase, the resulting conjugate
can include two or three enzymes bound to an avidin molecule. The use of
conjugates is referred to as labeled avidin-biotin (LAB).The conjugates
require no mixing before use, and the sensitivity of complex versus that of
the conjugate is similar. Well-standardized commercial reagents are
available for either approach. Whereas the previous discussion has centered
on avidin, a very similar protein, streptavidin, is available. It is produced by
Streptomyces avidinii and has nearly identical biotin-binding characteristics.
Unlike avidin, streptavidin has a neutral isoelectric charge because it lacks
the carbohydrate side chains of avidin. In some applications, substituting
streptavidin for avidin may contribute to lower background.
1.2.11.2.3.2. Detection Systems:
As implied in the previous description of assay formats, antibody
alone cannot be visualized. Each assay format uses a different technique for
localizing a reporter molecule or detection system to specifically bound
55
primary antibody. These detection systems include nonenzymatic systems as

well as systems using enzyme to generate a signal.
1.2.11.2.3.3. Histochemical Enzymes and Substrates:
Histochemical enzymes are enzymes that have been used as
visualization tools for the localization of targets in tissues or cells. To be
useful in such a capacity, the enzyme must have a substrate system that
generates a product that can be visualized (i.e., a chromogen) and has
minimal diffusion from the site of production. This latter characteristic is
referred to as the substantivity of the chromogen. The final sensitivity of
detection is determined by the combined efficiency of the enzyme-substrate
system in generating signal. The enzyme-specific activity, substrate
concentration and purity, time, and temperature of reaction will all impact
results. In practice, these variables have been minimized because of the
commercial availability of these key reagents.
1.2.11.2.3.3a. Horseradish Peroxidase:
Horseradish peroxidase is the most commonly used histochemical
enzyme. It is a relatively small protein (mol. wt. 40,000) that uses peroxide
as a substrate and is inactivated by substrate excess. The enzyme substrate
reaction is rapid and enzyme activity is quenched within 15 to 30 minutes.
Color development occurs using an electron donor as histochemically
demonstrable material.
There are several different histochemical systems for this enzyme.
The two most commonly used are DAB and AEC, either of which yields
comparable results (Tubbs et al., 1979), although less diffusion from site of
liberation is noted for DAB. Peroxidase is most active at neutral pH and is
reversibly inhibited by azide, cyanide, and sulfide.
56
DAB results in a mahogany brown reaction product that does not

fade and is insoluble in organic solvents. The contrast with a light
hematoxylin counterstain is good, and standard mounting media can be used.
Intensification of the DAB reaction product, changing the color to black, can
be accomplished by addition of heavy metals (e.g., nickelchloride,
cobaltchloride, copper chloride, imidazole or osmium tetroxide) (Elias,
1990). DAB is a chemical carcinogen, and the powdered form must be
handled in a hood using mask and gloves. Liquid forms, reducing the hazard
of handling the reagent, are now commercially available.
AEC gives a red reaction product that is soluble in alcohol and can
fade with long-term storage. The contrast with a light hematoxylin
counterstain is good, but nonalcoholic forms (i.e., Mayers hematoxylin)
must be used to avoid removal of signal. Aqueous mounting media must be
used.
AEC was first introduced because of a reportedly lower carcinogenic
potential than DAB. The reagent is not free of hazard and should still be
handled with care for laboratory workers and the environment.
Other less frequently used chromogens include 4-chloro-1-naphthol,
benzidine, alpha-naphthol pyronin, p-phenylenediamine pyrocatechol
(Hanker-Yates reagent), and tetramethyl benzidine (Trojanowsky et al.,
1983). 4-Chloro-1naphthol gives a blue end product, but it tends to diffuse
from the site of precipitation. Successful use of the Hanker-Yates reagent for
immunohistochemistry on renal biopsies is reported (Sheibani et al., 1981).
1.2.11.2.3.3b. Alkaline Phosphatase:
Alkaline phosphatase is the other most commonly used histochemical
enzyme. It is much larger than horseradish peroxidase (M.W 140,000), and
its size can influence geometry of complex formation and reagent
57
penetration. The enzyme is most active at alkaline pH in the presence of

magnesium and is inactivated by acid. There is no enzyme inactivation
associated with the reaction with substrate, so color development can be
extended to result in more signal (within limits).
A variety of substrates are available. The resulting chromogens are
variously colored; red, blue, or black products can be formed. The substrates
are not carcinogenic. The final sensitivity depends on the substrate system.
The chromogens are not stable in organic reagents and require protection
from organic mounting medium or the use of aqueous based systems. Choice
of counterstain is dictated by the color of the chromogen.
Chromogens for use with alkaline phosphatase include fast dyes (Fast
Red TR, Fast Blue BB) in conjunction with naphthol substrates or NBTBCIP (McGadey, 1970). The NBT-BCIP substrate results in a dark blueblack product that is very substantive and yields the highest sensitivity of
any colorimetric detection system.
1.2.11.2.3.4. Double-or Multiple-Staining Techniques:
This topic has been thoroughly reviewed by van der Loos and
associates (van der Loos, 1993), who provide practical suggestions on when
and how to apply double-staining methods, including selection of the best
methodologic combinations. Whereas immunohistochemistry for different
antigens performed on adjacent serial sections usually suffices for routine
purposes, occasionally the need arises to visualize more than one antigen in
cells. Double or multiple staining procedures can be performed either
sequentially or simultaneously and may involve use of more than one
enzyme-chromogen combination, resulting in different colored reaction
products. Many permutations are described (Van Rooijen, 1980).
58
A sequential procedure is used when both primary antibodies are

raised in the same species. Elution of the first primary antibody or adequate
development of the first reaction product before proceeding with the second
reaction may be necessary (Elias, 1990).
When the two primary antibodies are raised in different species, a
simultaneous procedure using different enzyme-chromogen combinations
can be performed. The optimal end result in a case containing the antigens of
interest will be two highly contrasting visually different signals.
Combinations of immunogold - silver staining with horseradish
peroxidase and alkaline phosphatase results in successful triple staining.
(Krenacs, 1989).
1.2.12. Treatment and control:
Numbers of different antiretroviral agent have been developed. Of
these, the RT inhibitor AZT has been the most widely used. Major toxicities
of AZT include neutropenia, anaemia and myopathy. Several other
nucleotide analogues that act as chain termination in the synthesis of the
DNA provirus. Such as DDI didanosine, Zalcitabine (ddc) and stavudine
(d4T), but they toxicities. Non competitive inhibitor of the RT includes
nevirapine and delavirdine. In general, all TR inhibitors are limited by their
low potency, their side effects and the development of resistance. Protease
inhibitors such as saquinavir have the advantage of being able to inhibit HIV
replication in cells that are chronically infected (Butera & Folks, 1992). AZT
delays the development of opportunistic infection and prolongs survival.
How early to start AZT remains controversial. Most clinical experts
however, agree on recommending AZT when the CD4 cell count drops
below 500.The development of clinically significant AZT resistance, the
development of opportunistic infection, and progressive weight loss and
59
constitutional symptoms, seem to comprise a good indication for need to

change treatment. In USA, recommendations have been issued for the use of
AZT in HIV infected pregnant women to reduce prenatal transmission.
Currently there is no effective vaccine available and limited clinical trials are
conducted to test candidate vaccines (CDC, 1994b).
1.3. Visceral Leishmaniasis in Sudan:
VL has been reported in Sudan since the beginning of 20th century
(Zijlstra & Elhassan, 2001). The first case reported in Sudan was described
by Neave in 1904. The patient was a boy aged 8-9 years from Bahr-El Gazal
and diagnosed at Omdurman Teaching Hospital. The patient was suffering
from diarrhoea and splenomegaly and that was the second case reported in
Africa after the first case which was reported in Tunis 1903 by Laveran. The
second case reported in Sudan was an Egyptian soldier who had contracted
the disease in Singa town and died in Cairo in 1907 (Cummins, 1908). As a
result of a continuous reporting of cases of VL, two governmental
commissions were set in the period between 1908-1911 to investigate the
disease in the Eastern and the Blue Nile provinces. One commission was
headed by Bousfield (1908-1910) a pathologist of the Welcome Research
Laboratory. The other one was by two pathologists, Archibal and Marshal.
(1911-1913).
The commission was closed in October 1913 and it was concluded
that VL was endemic in the Eastern province particularly Mafaza, Singa,
Kassala, Galabat, Gadarif, Sofi and Tomat (Satti, 1958). It was reported that
dogs had been the naturally infected reservoir (Marchal, 191la).
Since then, researchers continued investigating different aspects of
VL between 1931 and 1959, which revealed valuable information in a series
of papers under the heading: "Studies in Leishmaniasis in Anglo- Egyptian
60
Sudan". They studied different aspects of the disease involving vector,

transmission and clinical presentation as well as treatment (Henderson,
1937; Kirk & Satti, 1940a; Kirk & Lewis, 1940, 1951; Kirk, 1944, 1956;
Kirk & Satti, 1947).
VL occurs in sporadic small outbreaks and sometimes attaining an
epidemic form in the Upper Nile province and South Fung District of the
Bule Nile province (Stephanson, 1940; Satti, 1958, 1963a) .A major
epidemic took place in 1988 among displaced southerners from Bentiu,
where a lot of people died (El Hassan et al., 1993). The WHO estimated that
at least 400.000 people were killed by the disease and it had been well
known by natives in Sudan by different name e.g. Seimaih, Dobal, Elsaid
fever (Thomson, 1911).
In Sudan, the disease was found to be endemic in the Eastern
Region: Kassala, Gadarif, Hawata, Rahad and Dinder River up to River
Atbara (Kirk, 1934; E1 Safi et al., 2002; Zijalstra & El Hassan, 2001). In the
center it encroaches on the Blue Nile State and to the south in the Upper Nile
province and Equatoria State (Kapoeta). Different foci were detected in
Darfur, near the Nuba Mountains (Kirk & Lewis, 1955).
The disease had been reported in Khartoum province by Shamsedin
(1969) and later by Kadaru (1995), who reported two cases from El Alafon
who had never left their home. Small outbreaks were reported in ElKhogalab village where it affected 22 school childem (Hamza et al., 1976)
and Eidumish, in the White Nile (El Safi, 1996) as well as Wad zaki near
Eldweim in the White Nile State (Kadaru, 1995). The disease attained
seasonal variations, with prepondrance between October and February
(Henderson, 1937; Kirk, 1939). There was increased incidence of
transmission during the rainy season which was supposed to be due to
61
humidity and low temperature (Manson & Bahar., 1987). It most commonly
affects young adults and was common among children in Bentiu in the
South. (Henderson, 1937) Males are more affected than females (Kirk,
1939). It is general a disease of low socio-economic classes where
malnutrition acts as a provoking factor (Kirk, 1959; Yousif & WHO, 2001).
For a considerable time, workers in the field of leishmaniasis in the
Sudan investigated the possible reservoir hosts (Kirk, 1956; Satti, 1958;
Hoogostraal et al., 1963). They observed that small outbreaks had occurred
among military personnel who had been stationed in remote uninhabited
lands. In 1963 Hoogostraal and his group demonstrated natural Leishmania
infection in the grass rats Arvican niloticus luctuosus, the rodent, Rattus
Rattus and Accomys species which co-incited with the distribution of P.
orientalis in Malakal. Accordingly, they concluded that Nile grass rats were
the possible reservoir hosts of VL.
Sixel (1987) found massive Leishmania infection in a Jakal, which
was suggested as a possible reservoir in southern Sudan. Recently a
preliminary report in two villages (Bandigues & Um Salala), in Eastern
Sudan by Mukhtar, et al (2000) described the detection of L. donvani
antibodies by DAT and ELISA in animal's sera such as sheep, cows, dogs,
camels and donkeys. In regular episemiological studies, which were carried
out by Elsafi, et al., (1997-2000) in Barbar el Fugara, a village in Gedarif
State (eastern Sudan), they found the parasite in lymph node culture of dogs,
rodents and donkeys.
In Sudan the pioneer work of Kirk and his colleagues on the vector
showed that P. orientalis was the main vector of VL in Sudan (Kirk &
Lewis, 1947). It is widely distributed in the endemic area of VL, and is
found in Kassala, Blue Nile, Darfur, Melut, Malakal and Kapoeta (Kirk &
62
Lewis, 1955). P. orientalis was found to be naturally infected with

leishmania (El Naiem, 1996). The flies rest during the day in the cavities of
trees in wet seasons and in cracks in the soil during dry seasons
(Cheesbrough, 1995). Biting commences immediately after darkness and
increases about sunrise (Hoogostraal & Dieleuin, 1962).
PKDL is most commonly reported in India (WHO, 1996). It is
known to occur in the Sudan during or shortly following treatment (Kirk and
Satti, 1940 and Satti 1963a). It can even occur without past history of VL
(EL-Hassan et al., 1992) or as puncture rash (Kirk & Satti., 1940) or as
nodular depigmented areas (Kirk & Macdonalds, 1940). It affects most
commonly the face, limbs, extensor surfaces of the arm and trunk.
1.4. HIV/AIDS in Sudan:
Years of civil war and limited epidemiological data make it difficult
to generalize about the status of HIV/AIDS in the Sudan. It is generally
agreed that the country is in the early stages of a generalized HIV/AIDS
epidemic, with an almost exclusively heterosexual transmission pattern, and
indications of higher infection rates in the South than in the North. Internally
displaced persons, refugees, prison inmates and members of the armed
forces are some of the groups believed to be particularly vulnerable to HIV
infection. In addition, high risk groups such as commercial sex workers are
known to exist in the country, and studies to further understand their risk
profile are planned. From a regional perspective, the country is very
important to the treatment scale up efforts in the WHO Eastern
Mediterranean Region (EMRO), because 73% of people requiring ART in
the region have been estimated to reside in Sudan (WHO, 2006).
The most reliable available indication of the extent of the epidemic is
the 2002 Situation Analysis study conducted in the government-controlled
63
parts of the country (11 out of 16 states in the north and 3 in the south). The
study yielded HIV prevalence rates ranging from 0.5% for soldiers, 1% for
antenatal care attendees, truck drivers, and Internally Displaced Persons
(IDPs), 2.5% among female tea sellers, to 4.4 % among female sex workers.
More recently, results of limited sentinel surveillance testing conducted
during 2004 by SNAP yielded prevalence rates of 0.95% (18/1900) among
pregnant women, 1.9% (9/465) among symptomatic STD patients, and 2.3%
(33/1436) among TB patients.
It needs to be clarified that there have been two prominently cited
estimates of the HIV sero-prevalence for the general population (1.6% as
well as 2.6%), in various documents discussing HIV/AIDS in Sudan.
However the 1.6% figure appears to have been derived from aggregation of
the overall HIV positive rate among all the samples from the various groups
tested during the epidemiological component of the 2002 Situation Analysis
study (SNAP, 2002). But the methodology employed by the study would
suggest that it was designed to determine sero-prevalence for the various
groups studied, other than the general population prevalence from the
overall positive rate among the samples collected. The estimate of 2.6 % was
based on a UNAIDS/ WHO modeling for the whole country (2003).
The limited available data from Southern Sudan suggest relatively
higher infection rates, compared to the north. Studies have yielded general
population prevalence rates ranging from 2.7 in Yei (2003) to 7% in Yambio
(2000). In the city of Juba, (located in the south but administered by the
government), 10% of female tea sellers (a group some of whose members
are believed to engage in casual / commercial sex), were found to be HIV
positive in 2002. These higher rates of infection would suggest that IDPs
hailing from the affected areas may have higher HIV rates than their host
64
communities, this being made even more likely by the disruptions to family
cohesion and sexuality norms that being an IDP could cause. Unfortunately
this assumption could also reinforce a perception of relative safety and
inaction for communities in the North of Sudan. It therefore worth pointing
out that despite citation of the report of one survey yielding of a 5% HIV
positive rate among IDPs in Khartoum, this level of infection is not
supported by other data. For instance, the 2002 situation analysis study
yielded an IDP HIV prevalence of 1%, the same as that obtained for
pregnant women in all the states, and results of sentinel testing among IDP
antenatal mothers in Khartoum, done by SNAP and CARE during 2004,
yielded a positive rate of 1.6% (11/700), very similar to the 1.5% (5/400) for
general population pregnant women in the Red Sea state, for the same period
(SNAP, 2004).
HIV/AIDS prevalence, based on scarce epidemiological and
behavioral information, is estimated by UNAIDS to be around 2.3% in the
adult population. Rates of HIV infection have been estimated by Sudan
National AIDS Control Programme (SNAP) to be at 1.6 % nationwide. For
Southern Sudan, estimates vary from 1% to 7.2% with alarming rates among
certain population (SNAP, 2006).
1.5. Overview of Leishmania/HIV co-infection:
Leishmania/HIV co-infection is emerging as an extremely serious,
new disease and it is increasingly frequent. There are important clinical,
diagnostic, chemotherapeutic, epidemiological and economic implications of
this trend. Although people are often bitten by sandflies infected with
Leishmania protozoa, most do not develop the disease. However, among
persons who are immunosuppressed (e.g. as a result of advanced HIV
infections,
immunosuppressive
treatment
65
for
organ
transplants,
haematological malignancy, auto-immune diseases), cases quickly evolve to

a
full
clinical
presentation
of
severe
leishmaniasis.
AIDS and VL are locked in a vicious circle of mutual reinforcement.

On the one hand, VL quickly accelerates the onset of AIDS (with
opportunistic diseases such as tuberculosis or pneumonia) and shortens the
life expectancy of HIV-infected people. On the other hand, HIV spurs the
spread of VL. AIDS increases the risk of VL by 100-1000 times in endemic
areas. This duo of diseases produces cumulative deficiency of the immune
response since Leishmania parasites and HIV destroy the same cells,
exponentially increasing disease severity and consequences. VL is
considered a major contributor to a fatal outcome in co-infected patients.
Lately, however, use of tri therapy, where it is available, has improved the
Prognosis for Leishmania/HIV cases. Leishmaniasis can be transmitted
directly person to person through the sharing of needles, as is often the case
among intravenous drug users. This group is the main population at risk for
co-infection.
Although cases of co-infection have so far been reported in 33
countries worldwide, most of the cases have been notified in south western
Europe (Desjeux, 2001) where up to 70% of all adult cases of VL are related
to HIV/AIDS and up to 9% of all AIDS cases suffer from newly acquired or
reactivated VL (Desjeux & UNAIDS, 1998).
Leishmania/HIV co-infections impose specific difficulties in terms of
diagnosis and treatment. The usual clinical features (fever, weight loss, liver
and spleen enlargement, inflammation of the lymph nodes) are not always
present. The clinical diagnosis can also be made difficult by associated
diseases
such
cytomegalovirus
as
cryptosporidium,
infection
or
66
disseminated
mycobacterial
cryptococcosis,
infection.
The serological diagnosis is falsely negative in 42.6% of co-infected

patients. HIV-positive patients have difficulty in producing antibodies
against new infectious agents, especially at a late stage or during relapses.
Consequently, there is a need to use two or more serological tests and
antigens freshly prepared in the laboratory to increase sensitivity.
Although multiple localizations are frequent (blood, skin, digestive
tract, lungs, central nervous system), parasitological diagnosis can be
difficult and has to be repeated to orient the treatment. Bone marrow aspirate
(BMA) remains the safest and most sensitive technique, but spleen aspirate
and liver biopsy are also used. When BMA cannot be performed, the search
for
Leishmania
can
be
conducted
in peripheral blood samples.
Treatment for co-infected patients is aimed at clinical and

parasitological cures and prevention of relapses. Unfortunately, in such
patient treatment failure, relapses due to drug resistance and drug toxicity
are very common. In south-western Europe, follow-up studies using
pentavalent antimonials, the same first-line drug used to treat classic
leishmaniasis, show a positive response in 83% of cases. However, 52% of
patients relapse within a period of one month to three years, with the number
of
relapses
ranging
from
one
to
four.
The main alternative drugs include pentamidine, amphotericin B and

amphotericin B encapsulated in liposomes. This encapsulation reduces the
occurrence of side-effects, but relapses still occur and the drug remains
extremely expensive.
Leishmania/HIV co-infections can lead to epidemiological changes
which modify the traditional patterns of zoonotic VL. Co-infected patients
harbour a high number of Leishmania in their blood so there is also a risk of
them becoming reservoirs of the disease (that is, infective for the sandfly
67
vector) as in anthroponotic foci in Bangladesh, India, Nepal and East Africa.

Consequently,
there
is
an
increased
risk
of
future
epidemics.
Experimentally, sandflies can be infected through a blood meal

containing a very small quantity of blood from co-infected patients. The
quantity may be less than the content of a needle. As 71.1% of co-infected
patients in south-western Europe are intravenous drug users, transmission of
Leishmania has occurred through the sharing of syringes in this population
group.
1.5.1. Global Leishmania/HIV co-infection:
Cases of Leishmania /HIV co-infections are being reported more
frequently in various parts of the world. It is anticipated that the number of
Leishmania /HIV co-infections will continue to rise in the coming years and
there are indications that cases are no longer restricted to endemic areas.
The overlapping geographical distribution of VL and AIDS is increasing due
to two main factors: the spread of the AIDS pandemic in suburban and rural
areas of the world, and the simultaneous spread of VL from rural to
suburban area.
Leishmania/HIV co-infections are considered a real threat, especially
in south-western Europe. Of the first 1 700 cases of co-infection which have
been reported to the World Health Organization (WHO) from 33 countries
worldwide up to 1998, 1 440 cases were from the region: Spain (835); Italy
(229); France (259); and Portugal (117). Of 965 cases retrospectively
analyzed, 83.2% were males, 85.7% were young adults (20-40 years old)
and 71.1% were intravenous drugs users.
Most co-infections in the Americas are reported in Brazil, where the
incidence of AIDS has risen from 0.8 cases per 100 000 inhabitants in 1986
to 10.5 cases per 100 000 inhabitants in 1997. As HIV transmission has
68
spread into rural areas, VL has simultaneously become more urbanized-especially in north-eastern Brazil--increasing the risk of overlapping
infection.
1.5.2. Leishmania /HIV Co infection in Africa:
The number of cases of Leishmania/HIV co-infection is expected to
rise in Africa owing to the simultaneous spread of the two infectious
diseases and their increasingly overlapping geographical distribution,
complicated by mass migration, displacement, civil unrest, and war.
In general, the reported cases of Leishmania/HIV co-infection in
Africa are a very modest estimation and would substantially increase if
active surveillance were implemented throughout the continent. Ethiopia has
a well-organized system of detection, management and reporting of coinfection. Kenya and Sudan began surveillance in 1998 and Morocco has
also established a surveillance centre.
In East Africa, cases of Leishmania/HIV co-infections have been
reported in Djibouti (10), Ethiopia (74), Kenya (15), Malawi (1) and Sudan
(3). West Africa has no official surveillance system yet, but several cases
have been reported: Cameroon (1), Guinea Bissau (1), Mali (4) and Senegal
(2). In North Africa, cases have been reported in Algeria (20) and Morocco
(4).
1.5.3. Leishmania /HIV Co infection in Sudan:
In view of the fact that both VL and HIV infections are increasing in
the Sudan particularly in eastern and southern states where both infections
overlap, VL casers co-infected with HIV may be found (Elsafi et al 19981999).
In Sudan 1990-1998 only 3cases of leishmania and HIV co-infection
were reported (Poredes et al 1997). The numbers are expected to rise owing
69
to factors such as increasing mass migration, displacement, civil unrest, war

and sex work.
Elsafi et al (1997) have confirmed the prevalence of leishmania/HIV
co-infection in Sudan. 6.5% of VL cases have been found to be co-infected
with HIV. Most (75%) HIV co-infected cases come from eastern and
southern Sudan. This is in accordance with the fact that these are the two
most affected areas in the country with regards to both VL (MSF-Holland
Report, 1996-1999) and HIV infection (SNAP Report 1997-1998).
Generally HIV co-infected VL cases presented with clinical features
typical of VL although splenomegaly was found only in 75% of the cases
(Elsafi et al 1998-1999).
In clinicoepidemiological and serological studies of Leishmania /HIV
Co- infection in Sudan, which carried out by Elsafi et al (1999-2005) to
determine the prevalence of HIV infection among patients with VL and to
investigate the clinical, epidemiological and serological features of
coinfected cases in the Sudan, three series of confirmed VL cases( total 150
patients were studied, 5%(3/60), 9.4(5/53) and 8.1(3/37) of the VL in the
three studied series, respectively, were positive for HIV. In addition, HIV
infection was confirmed in 5 serologicaly confirmed VL cases brining the
total of co-infected cases to 15. Two other co-infected patients (one
parasitologicaly and one serologicaly confirmed) had been detected prior to
the study period. The 17 co-infected cases included 14 males and 3 females.
Their age ranged rom 15 to 51 years; 9 were married and 8 were single.
Three of them presented with relapse and the rest had newly acquired VL.
They included 4 farmers, 4 labourers, one soldier, one student, two house
wifes, one merchant and the others were civil servants. They belonged to
different tribes including Nuba, Nuier, Barno, Tama, Masalit, Hawsa, Dago
70
and Nubian. Most of the cases came from eastern and southern Sudan, others
came from western, central and northen parts of the country and two cases
came from Ethiopia. The results of risk factors for HIV infection in the
second series showed that two cases reported illegal sexual contact, one had
blood transfusion, and one had STD. None of them was intravenous drug
user and all of them were resident in or had a history of travel to a VL
endemic area. All the cases complained of fever and fatigue. Other
symptoms included loss of weight (88%), abdominal pain (71%), couh
(59%), anorexia (59%), diarhoea (35%) and epistaxis (29%). The clinical
signs included pallor (82%), splenomegaly (76%), hepatomegaly (65%),
lymhpadenopathy (59%), chest signs (35%), oral candidiasis (29%), skin
lesions (24%) and jaundice (18%). Laboratory result showed that all the oinfected patients were anemic; three of them had hypochromic microcytic
anemia. The TWBCs count was< 2000 in two cases, between 2000-5000 in
two cases and >5000 in one case. A comparison of series of clinical and
epidemiological variables between HIV+ve (5) and HIV ve (48) confirmed
VL cases showed that the absence of splenomegaly (P= 0.002),
lymphadenopathy (P= 0.024), the presence of epistaxis (P= 0.018), and
abdominal pain (P= 0.018) were significantly more observed in co-infected
cases. However, no significant difference relating to possible risk factors
was observed between the two groups.
In addition, the performance of serological tests (IFAT, ELISA, WB)
and Katex in the diagnosis of VL in 12 parasitologically and/ or
serologically (DAT) confirmed patients co-infected with HIV was assessed.
Parasitological confirmation was obtained in 50% (6/12) of the cases. The
DAT was positive in 67%, IFAT in 91% and both the ELISA and WB were
positive in all cases. With the exeption of one smear negative ptient, the
71
Katex was positive in all (7) the cases; the test was strongly positive in 5
patients.
72
II. Justification & Objectives

2.1. Justification:
VL is a major health problem and has been reported in Sudan since
the beginning of 20th century (Zijlstra & Elhassan, 2001).
The first case of HIV infection in Sudan was reported in 1987 in a
hemophilic boy. Since then infection increased and AIDS become a serious
health problem (Hashim et al., 1998)
An emerging health problem is the increasing occurrence of VL/HIV
co-infection. The diagnosis of VL in co-infected patients is difficult, because
they often lack usual clinical features of VL and due to their immune
compromised status do not produce antibodies, consequently, serological
tests may be falsely negative.
In view of the fact that both VL and HIV infections are increasing in
the Sudan, VL cases co infected with HIV may be found.
As the disease is a reticulo-endotheliosis and the parasite sequestrates
in many visceral organs such as bone marrow, liver, spleen and lymph nodes
so, the infiltration of these organs may affect their functions. Since bone
marrow is considered the main site for blood cells production so, the study
of full blood count parameters may support the parasitological and
serological tests for diagnosis of VL.
Leishmania parasites escape from the humoral immune response by
hiding of amastigotes inside the host macrophages, circulating antibodies
have no effect on the infection and may even be harmful. Immunity is
entirely cell-mediated, so the study of immunological state by assaying for
leishmania-specific cell- mediated immunity is more helpful in diagnosis of
disease.
73
Flowcytometry is the standard method for accurate and automated

measurement of CD4 T.lymphocytes. The technique is expensive and
requires sophisticated equipment as well as trained personnel to perform it
.In addition, the lack of ready access to technical support and quality
assurance programs have limited the use of flowcytometry techniques in
resource-constrained
countries.
On
the
other
hand,
manual
non-
flowcytmetric techniques such as Dynabeads and Coulter methods, which

produce results, comparable to those of flowcytometry have their own
limitations and not available here. So we introduced asimple and cheap
manual slide method by extending the immunoenzyme single stain method
to more applicable double staining techniques for enumeration of CD4
&CD8, T cells, which is suitable at poor setting without a need for advanced
instruments.
2.2. Objectives:
The aims of the present study are to:
- Determine the haematological parameters of VL, VL co-infected with
HIV and HIV patients.
-
Characterize T-cell subsets of these groups.
- Extend the immunoenzyme single stain method to more applicable

double stain techniques for enumeration of T.cell subsets.
- Compare this method with flowcytometry.
74
III. Material & Methods

3.1: Study area:
3.1a. VL cases:
The present study was conducted in a health center in Tabarak-Allah
village in Gedarif state-Eastern Sudan, an area recognized as endemic for
VL. The village is located about 100 Km from Gedarif town, it is surrounded
by other villages including Barbar, Birkat Nowrin and Mashroua Firsan. The
center is specialized in Kala-azar diagnosis and treatment and is under the
responsibility of Minisry of Health, Gedarif state. It has a laboratory
equipped with one light Microscope (Olympus CH2), Refrigerator,
Centrifuge, Water bath, Geimsa stains and all necessary glassware. The
laboratory is capabale of carrying important routine investigations for most
of the important endemic diseases such as malaria, typhoid, leishmaniasis
and tubercuosis. Its staff consist of one technician, two laboratory assistants
and a laboratory attendant. The medical staff consist of one doctor, two
general medical assistants, several nurses and one specialist in nutrition.
An others group of VL patients were recruited from Al- Azaza centre
in Singa state, an area also recognized as endemic for VL.
3.1b. HIV positive individuals:
HIV seropositive individual were recruited from the VCT Centre
(Odurman Teacing Hospital). This centre provides medical care for HIV
seropositive individuals who are are refered for antiretroviral therapy.
The Centre is capabale of carrying Voluntary Councelling and Testing
(VCT) for HIV. In addition the centre also it offers antiretroviral treatment
when indicated.
The laboratory is equipped with a refrigerator, ELISA machine for
HIV testing, and a flowcytometry machine for CD4 count.
75
3.2: Study Design:

This study is a descriptive study. An eligible clinical suspect was duly
informed about the objectives and procedures of the study. In case he/she
agrees to participate, a consent form is signed, before the patient is enrolled
in the study.
A cross sectional study was done to determine the hematological
parameters and T.cell subsets in known seropositive HIV individuals.
3.3: Study subjects:
This study
was conducted on three groups of patients: VL suspect,
VL cases that are found to be co-infected with HIV and known HIV
seropositive individual.
3.3.1: VL suspect cases:
VL clinically suspects who attended the Tabarak-Allah and Al-azaza
Health centres during period of April 2005-April 2007.
The criteria for clinical selection were based on the presence of fever
for two weeks or more, splenomegaly and/or lymphadenopathy after the
exclusion of malaria.
3.3.2. HIV seropositive individuals:
All HIV seropositive individuals who had been refered to VCT Centre
(Omdurman Teaching Hospital) between Jule and Augst, 2006 were enrolled
in this study.
3.3.3. VL/ HIV co-infected cases:
This group was identified by HIV testing of parasitologically and/or
serologically confirmed VL cases. All confirmed VL cases that were also
positive for HIV were included in the study.
76
3.4: Procedures:
3.4.1. VL cases:
3.4.1.1. Data Collection:
A coded enrollment number was given for each enrolled patient. The
personal, epidemiological and clinical data of each VL suspect were
collected using a standard form. Each patient was examined by the clinician
in charge of the health centre and the clinical information was recorded in
the form.
3.4.1.2. Collection of Samples:
From each enrolled VL suspect, the following samples were collected:
lymph node and/or bone marrow aspirates, venous blood and urine, for
parasitological,
serological,
hematological
and
immunological
tests
respectively. Each blood sample was divided into two parts: one part was
collected in EDTA anti coagulant containers for full blood count parameters,
the rest of sample was drawn in plain containers, left over night at R.T and
centrifuged at 3200 rpm for three minutes. Sera were collected in 1.5 ml
eppendorf tubes and frozen at (-20c) for performance of DAT and HIV
screening. Urine samples were collected from all VL suspects in sterile
containers at the time of diagnosis for the detection of leishmania urinary
antigen.
3.4.1.3. Parasitological study:
The diagnosis of lymph node puncture/or bone marrow aspirate of VL
suspects was carried out using direct microscopy examination (Geimsa
stained smear). Specimens (n/520) from inguinal lymph node of VL suspects
were collected. Slides were cleaned with gauze and labeled with patient
name and laboratory number. The patient was rested on his/ her back with
the legs stretched out. The skin over the inguinal gland was disinfected with
77
70% alcohol and then allowed to dry. The gland was held between thumb
and index finger of the left hand. A 21 G sterile needle was inserted in the
center of the gland at aright angle to the skin, and the gland was squeezed
with the left hand while the needle rotated. The fluid then came up through
the needle, needle was rapidly drawn. The piston of the syringe was drawn
back and the needle containing lymph node aspirate was attached. After the
fluid was discharged on the slide it was quickly mixed, left to dry, fixed with
methanol and stained with Geimsa. It was then examined for amastigotes
under the microscope using X100 oil immersion (Evans, 1989; WHO, 1996).
3.4.1.4. Serological Tests:
The DAT was performed for the demonstration of specific anti
leishmania antibodies. In addition, the urine latex agglutination test for the
detection of leishmania antigen was carried out.
3.4.1.4.1. Direct agglutination Test:
The testing of sera for DAT was performed as described by Harith et
al, 1995 at Khartoum University, Faculty of Medicind.
A. Performance of DAT:
All the collected sera from the parasitologicaly confirmed VL patients

were stested with the DAT. Two fold serial dilution of serum in 0.9 %(
wt/vol) NaCl with 2%( wt/vol) gelatin and 80% (wt/vol)2ME and
urea(0.02%wt/vol) were added to Leishmania antigen using microtitre plates
with 96 V shape wells. A DAT titer of 1: 3200 was reported as a positive
result (Harith et al., 1995).
B. Reading DAT plates:
The plate was placed on a white background and the titer was
estimated compared to a negative control. The titer was the highest dilution
78
that showed visible clumping and aggregation in comparison to the negative

control (plate).
3.4.1.4.2. Latex Agglutination Test (KATEX):
A Katex Kit (Kolon Biological, UK) was prepared according to Attar,
et al (2001); the Kit contains Latex beads that have been coated with IgG
anti leishmania antibodies.
A. Principle:
The principle of test is based on agglutination of Latex beads that

have been coated with IgG in presence of leishmania urinary antigen.
B. Performance:
The test was performed according to Attar et al (2001.).

Sufficient sample tubes were labeled for each patient to be tested,
250l to 1000l of urine sample were transferred into sample tubes, then put
in a rack, immersed in boiling water and heated for 5 minutes. It was,
allowed to cool to ambient temperature before applying the test.
All reagents were brought to room temperature and the bottle of latex
was shaken well before testing, 50l of the treated urine sample were placed
in the reaction zone on the glass slide, then one drop of the well mixed latex
was added. Both were mixed until completely homogeneous and covering
the whole surface of the reaction zone. The glass slide was rotated and
rocked consistently for 2 minutes in both clock wise and anti clock wise
directions to ensure complete mixing. Then the degree of agglutination was
recorded under a good stream of daylight. Apositive and negative control
was included in each patch.
C. Interpretation of results:
Agglutination was interpreted using the cross system as follow:

++++ Majority of the latex agglutinates and moves to the reaction area.
79
+++ Agglutination resembles chalk dust scattering onto surface.

++
Clear agglutinated particles were seen against the background of
latex.
+
Agglutination was just vissible as compared to the negative control.

No vissible agglutination.
All the first three grades of agglutination were regarded as positive.
3.4.1.5: HIV Testing:

3.4.1.5a VL cases:
Sera of all parasitologically/or serologicallyconfirmed VL patients were
screened for HIV by ELISA (Microlisa HIV). All reactive samples were
confirmed using another kit (enzygnost kits) at National Health Laboratory.
Microwell ELISA is a highly sensitive enzyme immunoassay for the
detection of antibodies to HIV-1(including subgroup o&c) and HIV-2 in
human serum or plasma. The test was performed in accordance with the
manufactures instructions.
Briefly, all controls and samples were diluted in the ratio of 1:11 using
sample diluents, and incubated at 37C for 30 minutes. The wells were
washed five times with working wash solution and were reacted with 100l
of conjugate for 30 minutes at 37C. After a final wash as described
previously, then 100l of working substrate was added to each well and
incubated at R.t for 30min in dark. Then 50l of stop solution was added.
After incubation for 30 minutes at room temperature, then the absorbance
was measured at 450nm in Elisa reader after blanking A-1 well.
Negative and positive serum control samples were included for each
patch. The cutoff value was calculated as follow:
Cut off value=NCX+PCX 6.
80
Test specimens with absorbance value greater than or equal to the cut
off value were reactive by Microlisa-HIV and vice versa.
3.4.1.5b. HIV seropositive individual:
The enrolled HIV seropositive individuals already were known
previously diagnosed as seropositive for HIV, but the laboratory diagnosis of
HIV in Omdurman VCT Centre as follow; rapid test as first line tests,
ELISA, and confirmation by WB.
3.4.1.6. Hematological parameters:
3.4.1.6a. VL cases:
Parameters of full blood count (Hb, TWBCs, Platelets, differential
white blood cells count, peripheral blood picture and ESR), were carried out
immediately after collection of blood samples for VL and VL/HIV coinfected patients using manual techniques at field.
Hb Concentration:
In glass test tube, 20 l of whole blood were added to 4 ml of diluting

fluid, the suspension was mixed and incubated at R.T for at least 5 min. The
diluting fluid (Drabkins solution) lyses the RBCs membrane, the Hb
becomes free and combined with cyanide compounds to form brown color,
the absorbance of test was read along with Hb STD at 540 nm using
spectrophotemeter. The Hb concentration was calculated from:
Hb g/dl: Abs of Test X Con of STD/Abs of STD.
TWBCs count:
In glass test tube, 20 l of whole blood were added to 380 l of

diluting fluid, the suspension was mixed and incubated at R.T for at least 5
min. The diluting fluid (2% G.A.A) lyses the blood cells membrane, the
nucleus of WBCs remains intact and stains deep violet-black. The Neubauer
counting chamber (haemocytometer) was filled by Pasteur pipette. The
81
WBCs were counted under light microscope using X40. TWBCs were
calculated from:
TWBCs/ l: N x D.F/Volume (depth x area counted).
Platelets count:
Platelets count was performed from well stained-thin blood film as

follows: platelets counted in 20 fields using X100 immersion oil, mean of
platelets in 20 fields was calculated, and then multiply by 20,000 to give
platelets / l.
Differential WBCs & peripheral blood cells morphology:
Many thin films were prepared from fresh blood. The slides were air
dried, two thin blood films were fixed in absolute methanol and preserved
for performing of CD4, CD8 count by double immunocytochemical staining
method. For differential WBCs count, smear was covered by leishmans
stain for 2-3 min in staining rack, and then double volume of buffer with
neutral PH was added and mixed well with stain, left for 7 min, then washed
carefully by D.W. The film was examined under light microscope, and the
different WBCs count was done and reported as percentage. The absolute
lymphocyte count was calculated from:
Absolute lymphocyte count/ l = % of lymphocyte X TWBCs/ l.
Comment on peripheral blood cells morphology was also carried.
ESR:
1 ml of whole blood was drawn into Westergen tube using plastic

syringe with bulb, tube was hanged in ESR rack for one hour at R.T, plasma
remained at the tope of tube and RBCs settled at the bottom . The point
between plasma and backed cells was read and recorded as mm/h.
82
3.4.1.6b. HIV seropositive individuals:

Parameters of full blood count were performed for all included HIV
positive individuals using automated blood cells counter (System KX.21) at
Khartoum Teaching Hospital.
3.5. HIV individual:
3.5.1. Data Collection:
With informed consent for all enrolled HIV patients, standardized
queistionaire with clinical and epidemiological data was filled out.
3.5.2. Collection of Samples:
EDTA-anti-coagulated venous whole Blood specimens (n/30) from
HIV seropostive patients were collected and tested immediately after
collection.
3.5.3. CD4 & CD8 differential count:
3.5.3a. VL and VL/HIV co-infected patients:
CD4, CD8 differential count using double immunocytochemical staining
technigues was performed for 10 confirmed VL patients (5 co-infected with
HIV). From each patient thin blood films were prepared from EDTA
anticoaugulated blood immediately after collection and were fixed in
absolute Methanol prior to immunoenzymatic staining.
Double Immunocytochemical staining Techniques:
A. Principle:
Immunocytochemical staining techniques allow for the visualization

of antigens via the sequential application of a specific antibody to the
antigen (primary antibody), a secondary antibody to the primary antibody
and an enzyme complex with a chromogenic substrate with interposed
washing steps. The enzymatic activation of the chromgen results in a visible
reaction product at the antigen site.
83
B.Method:
All thin blood films were stained firstly with peroxidase kit and followed by
alkaline phosphatase kit.
First staining:
1. Endogenous peroxidase was neutralized using Peroxidase Block for 5 min

2. Incubated with Protein Block for 5 min,
3. Incubated with CD4 primary antibody for 15 min,
4. Incubated with Biotinylated Secondary Antibody for 15 min,
5. Incubated with Streptavidin-HRP for 15 min,
6. DAB working solution was added for 5 min,
7. Slide was washed in TBS for 2 min after each step.
Second staining:
1. Endogenous alkaline phosphatase activity was neutralized,

2. Slide was incubated for 15 min with diluted AP-ABC Blocking Serum,
3. Incubated with CD8 primary antibody for15 min,
4. Incubated with diluted AP-ABC Biotinylated Antibody for 15 min
5. Incubated with pre-prepared AP-ABC Reagent for 15 min,
6. Incubated with Fast Red substrate,
7. Counterstained with Hematoxylin,
8. Slide was washed in TBS for 2 min after each step,
9. Dehydrate, clear and mount sections.
C. Interpretation of result:
Absolute
lymphocytes
count
was
performed
before
double
immunoenzymatic staining using automated cell counter. After staining of

fixed blood smear as mentioned above, the membrane surfaces of CD4, CD8
T.lymphocytes were stained with different chromagen colors; CD4, CD8
Tcells take the brown color and the red color respectively while
84
B.lymphocytes remained unstained; at least 50 lymphocytes were counted

under light microscope using X40 and the percentage of each lymphocytes
sub-type were calculated. Consequently the absolute number of each T.cell
subsets / l was calculated from formula:
% of T cell subsets X Absolute lymphocyte count.
3.5.3b. HIV seropositive individuals:
Flow cytometry:
A. Principle:
Flowcytometry
experiments
generally
involve
three
distinct,
interdependent phases. First is the pre-flowcytometry phase, which involves

staining of cells with fluorescent reagents. Second is the flowcytometry
phase, which involve processing
the stained cells using flowcytometry
instrumentation
data(acquiring)
and
collecting
for
one
or
more
measurements(which are called parameters) made on each individual.

Finally, the analysis phase involves analyzing the collected data.
This technique was performed at VCT Centre (Um durman Teaching
Hospital) using Partec Flow Cytometry instrument (CyFlow Counter) for
all HIV seropositive individuals.
B. Method:
20 Ml of CD4 mAb labeled-PE was added to 20 l of EDTA

anticoagulated whole blood in Patrec test tube, mixed gently, incubated for
15 min at R.T (protected from light). Then 800 l of no lyses buffer was
added, mixed on vortex gently and then directly measured in the Patrec
device.
85
3.6. Data Analysis:

Data were collected in master sheet paper and analysed by computer
using Microsoft office excel (2007) and statistical paired t. test then
presented in figures and tables.
86
IV. RESULTS
4.1. VL patients:
During this study, 160 patients with active VL out of 520 VL suspects'
cases were admitted to Tabarak Allah kalazar center in Gadarif State and Alazaza center in Singa State. There were 96(60%) males and 64(40%)
females, their ages ranged from 2-60 years (mean of ages was 14 years), 125
patients (78.1%) were less than 16 years old and 35 patients (21.9%) were
more than 16 years old. All patients presented with ahistory of fever, usually
for two weeks or more, splenomegaly and lymphadenopathy. Other
symptoms included abdominal distention (65%), weight loss (85%),
anorexia (55%), epistaxis (45%), diarrhea (40%), and cough (60%), (Table:
1).
4.1.1. Hematological findings:All patients were anemic (100%); Most of them with normocytic
anemia (85.4%), and with elevated ESR (100%). Most patients were
leukopenic (80%), thrompocytopenic (95%), with relative lmyphocytosis
(70%) and neutropenia (55%) (Table: 2).
4.1.2. Prevalence of HIV among VL:
6(3.5%); 5out of 160 parasitologicaly and serologicaly confirmed VL
and 1 out of 10 serologicaly confirmed VL cases were co-infected with HIV,
the prevalence of HIV among adult VL patients (age of more than 16 years)
was 8.5% (3/35), (figure: 11), (figure: 12).
4.1.3. CD4, CD8:
CD4, CD8 count were done for five confirmed VL patients during
active disease using double immunoenzymatic staining method, results
indicated that CD4 numbers were < 250 cell/ l in all five patients(100%)
(Table: 13).
87
Table (1): Clinical Features of VL patients.

Symptoms/Signs
Fever
Loss of Weight
Abdominal
Distention
Cough
Anorexia
Epistaxis
Diarrhea
Vomiting
Splenomegaly
Hepatomegaly
Lymphadenopathy
Pallor
Jaundice
percentage
100 %
85%
65 %
65 %
55%
45 %
40 %
15%
100 %
45 %
100 %
80 %
5%
Table (2): Hematological parameters of VL patients.

Parameter
Number of patients
Hb less than 11g/dl
160 (100%)
TWBCs less than 3X103/l
128 (80%)
Platelets less than 140X103/l
152 (95%)
Lymphocytes more than 40%
112 (70%)
Neutrophiles less than 45%
89 (55%)
ESR more than 45mm/h
50/50 (100%)
88
Table (3): WBCs Morphology in VL patient.

WBCs Morphology
Number of Patients
Shift to left
Shift to right
Normal
40 (25%)
10 (6%)
110 (69%)
Table (4): Poikilocytes in VL patients.

RBCs Morphology
Number of Patients
Target cells
Erythroblastosis
54 (34 %)
24 (15%)
89
Fig(1): Frequency of Age in VL patients
90
80
70
60
50
Number of patients
Frequency
40
30
20
10
0
1_10
11_20
21_30
31_40
41_50
51-60
Age
Fig(2): Number of VL patients
100
90
80
70
60
Frequency
Number 50
40
30
20
10
0
Male
Female
Sex
90
Fig(3): Frequency of TWBCs in patients with VL
90
80
70
60
50
Numbe of patients
Frequency
40
30
20
10
0
1100-2000
2100-3000
3100-4000
4100-5000
5100-6000
TWBCS/ l
Fig(4): Frequency of Hb among VL patients.
90
80
70
60
50
Number of patients
Frequency
40
30
20
10
0
3.6-5
5.1-6.5
6.6-8
Hb g/dl
91
8.1-9.5
9.6-11
Fig(5): Frequency OF Platelets among VL patients
70
60
50
40
Number of patients
Frequency
30
20
10
0
60-79
80-99
120-139
100-119
Platelets x103
140-159
160-179
/l
Fig(6): Frquency of ESR in VL patients
16
14
12
10
Number of patients
8
Frquency
6
0
45-64
65-84
85-104
105-124
ESR mm/h
92
125-144
145-164
Fig(7): % of netrophiles in differential WBCs among VL patients
50
45
40
35
30
Number of patients
25
Frequency
20
15
10
5
0
14-26
27-39
40-52
53-65
66-79
% of Neutrophiles
Fig(8): % of Lymphocytes in differential WBCs among VL patients
60
50
40
Number of patients
30
Frequency
20
10
0
16-29
30-43
44-57
% of lymphocytes
93
58-71
72-85
Fig(9): % of Monocytes in dufferential WBCs among VL patients
70
60
50
40
Number of patients
Frequency
30
20
10
0
1_4
5_8
9_12
13_16
17_20
% of Monocytes
Fig(10): % of Eosinophiles in differential WBCs among VL patients
100
90
80
70
60
Number of patients
50
Frequency
40
30
20
10
0
0_1
2_3
4_5
% of Eosinophiles
94
6_7
8_9
4.2. HIV/VL co-infected patients:

Six (3.5%) patients with age range from 5- 49 years (mean 25 years),
all males, were co-infected with HIV.
4.2.1. Haematological findings of HIV/VL co-infected patients:
The hematological findings were anemia (100%) of normocytic
normohromic (60%), microcytic hypochromic (40%), leucopenia (100%),
thrompocytopenia (100%), relative lymphocytosis (100%), and neutropenia
(100%) (Table: 5).
4.2.2. CD4 & CD8 count:
CD4 & CD8 T.cells differential count was done for 5 co-infected
patients during active VL disease, CD4 numbers were < 200 cell/ l in all
five patients (100%) (Table: 6,13).
95
Table (5): Hematological parameters of HIV/VL co-infected patients.
Pt
Sex
No.
Age
Hb
/Year /gdL
WBCs
Plt
Lymph
Neutro
Mixed
/ l
103/l
49
8.0
1000
95
65%
26
9%
33
7.0
2700
60
58%
31
11%
6.5
2600
80
60%
35
5%
9.5
3000
90
55%
38
7%
30
9.0
1700
75
62%
30
8%
Plt: Platelets, Lymph: Lymphocytes, Neutro: Neutrophils, Mixed;monocytes

and eosinophils.
Table (6): T.cells subsets of HIV/VL co-infected patients.
Pt No.
CD4/ l
CD8/ l
165
310
188
355
180
340
195
370
172
325
96
Fig(11): Prevalence of HIV among VL patients.
3.1%
Fig(12):
Prevalen
ce of
HIV
among
adults
VL
patients.
8.5%
97
4.3. HIV seropositive individuals:

A total of 30 previously diagnosed HIV seropositive individuals
refered to VCT Centre at Um dorman Teaching Hospital for ART were
included in this study. 20 (66.7%) of them were males, and 10 (33.3%) were
females. Their ages ranged between 2 and 45 years, mean of age was 28
years.
4.3.1. Hematological findings:The most common hematological changes were: anemia (50%), most of
them with normocytic anemia (80%), leukocytosis (33.3%), lymphocytosis
(30%), and eosinophilia & monocytosis (40%) (Table: 5).
4.3.2. CD4:
CD4 T.cells counts were done for 30 HIV seropositive individual
during ART using flowytometry, 20(66.6%) of patients had CD4 count < 200
cell/ l, 6(20%) had CD4 between 200- 500 cell/ l, and 4(13.4%) had CD4
> 500 cell/ l (Table: 8).
98
Table (7): Hematological parameters of HIV seropositive individuals.

parameter
Number of patients
Hb < 11g/dl
15 (50%)
TWBCs < 3X103/l
5 (16.7%)
TWBCs > 6X103/l
10 (33.3)
Platelets < 140X103/l
4 (13.3%)
Lymphocytes > 40%
9 (30%)
Lymphocytes < 20%
5 (16.7%)
Neutrophiles > 70%
3 (10%)
Neutrophiles < 45%
7 (23.3%)
Mixed: Monocytes & Eosinophils > 15
12 (40%)
Table (8): CD4 count in HIV seropositive individuals.

CD4
cell/ l
Number of patients
percentage
< 200 cell/ l
20/30
66.6%
200-500 cell/ l
6/30
20%
> 500 cell/ l
4/30
13.4%
99
Fig(13): Frequency of age among AIDS patients
12
10
Number of patients
6
Frequency
0
2_11
12_21
22_31
Age/year
100
32_41
42_51
Fig(14): Number of HIV patients
20
18
16
14
12
Frequency of age
Number of patients 10
8
6
4
2
0
Male
Female
Sex
Fig(15): Frequency of HB among AIDS payients
10
9
8
7
6
Number of patients
5
Frequency
4
3
2
1
0
5_6.9
7_8.9
9_10.9
11_12.9
Hb g/dl
101
13_14.9
15_16.9
Fig(16): Frequency of PCV among AIDS patients
12
10
Number of patients
6
Frequency
0
20_26
27_33
34_40
PCV%
41_47
48_54
Fig(17): Frequency of TWBCs among AIDS patients
12
10
Number of patients
6
Frequency
0
1-2.9
3_4.9
5-6.9
7_8.9
TWBCs cell/ l
102
9_10.9
11_12.9
Fig(18): Frequency of platelets among AIDS patients
5
Number of patients
Frequency
0
20_119
120_219
220_319
Plateletsx103a cell/ l
320_419
42-_519
Fig(19): Frequency of Lymophcyte in differential WBCs among AIDS patients
5
Number of patients
Frequency
0
16_19
20_29
30_39
40_49
Lymphocytes%
103
50_59
60_69
Fig(20): Frequency of Neutrophils in differential WBCs among AIDS patients
12
10
Number of patients
6
Frequency
0
22_35
36_49
50_63
Neutrophils %
64_77
78_91
Fig(21): Frequency MXD cells in differential WBCS among AIDS patients
12
10
Number of patients
6
Frequency
0
3_8
9_14
15_20
MXD %
104
21_26
27_32
Fig(22): Frequency of CD4 Cells among AIDS patients
18
16
14
12
10
Number of patients
Frequency
0
0_199
200_399
400_599
CD4 cell /l
105
600_799
800_999
Table (9): Perecentage of Males and Females among the three groups of
patients.
Group
Males
96/160(60%)
Females
64/160(40%)
6/6(100%)
20/30(67.7%)
10/30(33.3%)
VL
VL/HIV
HIV
Table (10): Range and mean of ages among the three groups of patients.
Group
Mean/years
14
Range/years
2 60
25
5 49
28
2 45
VL
VL/HIV
HIV
106
Table (11): Mean of different hematological parameters among the

Parameter
VL patients
HIV patients
8.5
VL/HIV coinfected
patients
8
Hb g/dL
TWBCs X103l
2.5
2.2
5.6
PlateletsX103 l
95
80
294
Neurophils %
40
34
53
Lymphocytes %
52
60
34
Mixed;Monocytes
& Eosinophils %
14
11
Table (12): Perecentage of different types of anemias among the three

groups of patients.
Anemia
VL patients
VL/HIV coinfecetd
patients
HIV patients
50%
60%
80%
10%
40%
20%
35.4%
4.6%
Normocytic
Microcytic
Dimorphic*
Sickle
* Dimorphic anemia either normocytic with microcytic or normocytic with
macrocytic anemia.
107
Table (13): Mean of CD4 & CD8 count among the three groups of
patients.
parameters
VL patients
VL/HIV Coinfecetd
patients
HIV patients
Absolute
Lymphocytes/l
CD4 T.cells
1300
1000
2000
210
180
255
CD8 T.cells
490
340
530
CD4/CD8 Ratio
1/2.3
1/1.9
1/2.1
108
4.4. Double immunoenzymatic staining method:

Sensitivity and specificity of this staining protocol were determined
by comparison with control single-staining of adjacent smear. Sensitivity of
the staining sequence (first/second) was assessed by dividing the total
number of cells stained in the staining sequence (first/second) by the number
of cells stained with the same primary antibody in single-staining
experiment (Waiser, J et al, 2002). In this study sensitivity of the first and
second staining sequence was about 100% for this applied method.
Specificity of the second staining sequence was calculated by
employing the following algorithm: (1-(number of false doublepositive
cells in double staining of non-co localized antigens divided by the number
of positive cells in single staining experiments, stained by the same primary
antibody, which was used in the first sequence of the staining (Waiser, J et
al, 2002). Specificity of the second staining sequence was 100%.
4.5. Comparison between Flowcytometry & double immunoenzymatic:
CD4 counts were done for five known HIV patients by both
flowcytometry (Cytoflow) and manual method (double immunoenzymatic
staining). Results were analyzed using statistical paired t.test, which
indicated that there is significance variation between two methods (P <0.05).
(Table: 14).
109
Table (14): Comparison between flowcytometry and double

immunocytochemical for enumeration of CD4:
Hypothesis:
H0= no significant variation in CD4 count by both methods.
H1= there is significant variation in CD4 count by both methods.
Paired t.test: d/S/n, d= diference in parameter between two method, S=
variance (x-x')2 /n-1, n=number of samples.
No. of
Double -
Flowcyto-
samples
immuno
metry
'd
d-'d
(d-'d)2
200
255
55
-3
130
188
58
-6
36
150
199
49
+3
210
255
45
+7
49
180
233
53
-1
'd=260
(d-'d)2
d=260/5=
=104
52
S= (d-'d)2/n-1= 104 /5-1= 26.
Paired t.test: t= 'd/S/n=260/ 26/5=10/5= 4.54.
The calculated t. value= 4.54, the tabulated t. value= 1.38.
4.54 > 1.38, that mean (P < 0.05), we reject the H0 and accept the H1, so
there is significant variation in CD4 by flowcytometry and double
immunocytochemical staining method.
110
Fig(23): Comparison between mean of CD4count by flowcytometry and double immunocytochemical

methods.
111
PBP of HIV: A: Microcytic Hypochromic. B: Normocytic Normochromic with rupture lymphocyte. C:

Shift to left Neutrophil. D: Target cells. E: Late Normoblast. F: Normocytic Normochromic with normal
lymphocyte.
112
PBP of VL: A: Hypersegmented Neutrophil with Target cells. B: Microcytic Hypochromic (X40). C:
Sickle cell with late Normoblast. D: Normocytic Normochromic with shift to left. E: Prominent Target
cells. F: Macrocytosis with polychromatic cell.
113
Double immunocytochemical staining of T. cells subsets: A: Positive Tissue control (CD4 in Tonsil,
Brown). B: Negative Tissue control (B. cell in blood smear of CLL). C: CD8 in Test Blood smear (Red)
X40. D: CD4 in Test Blood smear (Brown) X40. E: CD8 in Test Blood smears (Red) X100. F: CD4 in Test
Blood smear (Brown) X100.
114
A: CD8 (Red) and CD4 (Brown) in Test Blood smear, X40. B: Leishmania amastigotes in May Grund
woal Geimsa stain in B.M smear, X100.
Partec Flow Cytometry - CyFlow Counter CD4 Counter - Cell Counter.
115
V. Disscussion
VL:
VL is a major health problem and has been reported in Sudan since
the beginning of 20th century (Zijlstra & Elhassan, 2001).
A number of 160 patients with VL were recruited from two kalazar
centers in Gedarif and Singa states indicated that this disease is endemic in
eastern Sudan. In our study, both young and older children were equally
affected with females being less affected.
The clinical features were similar to that reported in previous studies
done here. Fever, splenomegaly and lymphadenopathy were found in all
patients, other symptoms were present but less common.
Investigations of full blood count parameters showed that; anemia,
leukopenia and thrompocytopenia were pronounced in the majority of VL
patients, anemia was found in all patients; normocytic normochromic was
predominant, ESR was elevated, lymphocytosis and neutropenia were more
common. Our results agreed with another study done by Zijistra & ELHassan, (2001), who found anemia in all patients, leucopenia and
thrompocytopenia in the majority of patients. Similar results were reported
by Nassir et al., (2001) in Suadi Arabia. Sickle cell anemia was found,
which is explained by the heterogeneity of our study group that included
most of the different tribes which migrated from western Sudan.
However, the high percentage of anemia could be explained by many
other contributing factors mainly due to deficiency of hematinics, malaria
and chronic diseases. Leukopenia, thrompocytopenia and neutropenia are
116
most probably related to the delayed presentation which is accompanied by

gross splenomegaly. Target cells and erythroblastosis were explained by
liver disease and bone marrow compensation respectively. Relative
lymphocytosis could be due to cell mediated immune response.
Differential T. cells counts showed that CD4 Tcell count were low,
indicating the depressed cellular immunity during active VL, this could be
explained by deficiency of the immune response since leishmania parasites
behave as HIV in destroying same immune cells and our result agreed with
another study done in Spain by Juan et al., (1998).
Leishmania/HIV co-infection:
The present study confirmes that Leishmania/HIV co-infection is
emerging as an extremely serious, new disease and it is increasingly
frequent.The number of cases of Leishmania/HIV co-infection is expected to
rise in Sudan owing to the simultaneous spread of the two infectious
diseases and their increasingly overlapping geographical distribution,
complicated by mass migration, displacement, civil unrest, and war.
Six (3.5%) of our patients were co-infected with HIV and VL.This is
lower than reported in others studies (Elsafi et al 1997). This is explained by
the fact that most of our VL patients were less than 16 years of age, while
the highest prevalence of HIV infection is in those of more than 16 yearsof
age.
Parameters of full blood count, Tcell subset counts were affected in
co-infected cases more seveerly than in VL alone.This could be explained by
cummulative deficiency of the immune response since leishmania parasites
and HIV destroy the same cells, our result agreed with another study done
in Spain by Juan et al., (1998).
117
HIV/AIDS:
Thirty HIV seropositive individual from different states were
included when they refered for ART. 20 out of 30 patients were males, twice
the number of females recruited. This high number of males is due to fact
that most of individuals who seek HIV testing for travel requirement are
males, furthermore, males present themselves to clinics more than females
due to cultural reasons.
It was noticeable that most of HIV positive individuals were within
the age range 20-40 years. This age group is the sexually active group. This
finding is strengthened by the fact that majority of studied HIV seropositive
individuals were infected by sexual contact. Previous studies in Sudan and
sub-Saharan Africa indicated the sexual contact is a common mode of
transmission of HIV (McCarthy et al., 1989).
Parameters of full blood count were done for all HIV seropositive
individuals. Anemia, thrompocytopenia, leukocytosis, lymphocytosis,
eosinohpilia and monocytosis were found but variable. These results agreed
with an other study done by Volberding et al., (2003).
Anemia could have resulted from the use of anti retroviral drugs that
act as folate antagonists. Thrompocytopenia may be explained by peripheral
consumption (splenomegaly, immune complexes) or from decreased marrow
platelet production. Leukocytosis, eosinophila and monocytosis indicated the
secondary bacterial and opportunistic protozoal infections respectively.
In this study CD4 count was done for all HIV patients during ART
using flowcytometer. Our results indicated that, CD4 counts were < 200
cell/l in the majority of patients, normal and subnormal in some patients,
118
who are clinically improved. Similar findings were reported in other studies
done in Sudan by Magzoub et al., (2002) and in other countries by Baker et
al., (1998).
Double immunocytochemical method & Flowcytometry:
Absolute CD4 T lmphocyte counts are the most widely used
surrogate markers for determining disease progression (Fahey et al., 1990) a,
patient staging (Hoover et al., 1992) and for therapeutic monitoring of
patients with HIV infection (Gazzard, 1992; Masur et al., 1989). The cost
and expertise needed to enumerate CD4 counts can be quite prohibitive, and
many developing nations currently lack the monetary funds which would
enable them to offer this test as a means to evaluate patient disease
progression (WHO, 1992).
Flowcytometry is the standard method for accurate and automated
measurement of CD4 cells. The technique is expensive and requires
sophisticated equipment as well as trained personnel to perform it .In
addition, the lack of ready access to technical support and quality assurance
programs has limited the use of flowcytometry techniques in resourceconstrained countries. On the other hand, manual non-flowcytmetric
techniques such as Dynabeads and Coulter methods, which produce results,
comparable to those of flowcytometry have their own limitations (Landay et
al., 1990), and are not available in Sudan.
In this study, we introduced asimple and cheap manual slide method
by extending the immunoenzyme single stain method to more applicable
double staining techniques for enumeration of T. cell subsets in peripheral
blood. This is suitable at poor setting with no need for advanced instrument.
The method was comparable to flowcytometry in its diagnostic
reproducibility; 10-15 sample can be analyzed by analyst per run of 3 hours.
119
The method has many advantages; small volumes of blood is

required (50l), no multiple steps of blood handling; so less hazardous,
slides can be fixed for along time prior to staining; encourages to be used in
research purposes, more specific, sensitive, can offer absolute count for CD4,
CD8 T.cells, CD4 /CD8 ratio and even absolute B.lymphoctes count, after
staining slides also can be mounted and preserved for teaching purposes. It
can be used at field area with no need for advanced instrument, more
cheaply than flowcytometry (reagents cost per test is 7$ in comparison of
12-15$ for automation) regardless of instrument cost and maintanace. As it
depends on marker detection, others peripheral mononuclear cells that carry
CD4 such as monocytes can be differentiated morphologically from
lymphocytes, so it guarantees the specificity more than flowcytometry.
Depending on TWBCs count, the method can be oriented, so different
procedures can be used for preparation of smear; whole blood, buffy coat
and mononuclear cells layer. The latter preparation is useful in concentrating
the WBCs of late cases who are usually leucopenic. In this study we
compared
the
double
immunocytochemical
staining
method
with
flowcuyometry in small number of samples due to limitation of fund and

availability of samples.
Due to technical reasons, double immunocytochemical staining
method has been only rarely applied in the past. The use of indirect
immunohistochemistry for double-staining bears the risk of cross reactions
between the two staining sequences (Waiser, J et al, 2002). Here we
introduced the standardized protocol for double immunocytochemical
staining after we assessed different protocols for enumeration of T. cell
subsets in peripheral blood smear.
120
According to our knowledge no literature has been available on

application of this technique as quantitative method for enumeration of
T.cell subsets in peripheral blood.
121
VI. Conclusions & Recommendations.

6.1. Conclusions:
1- Pancytopenia is pronounced among VL patients, and is more
supportive for diagnosis of the disease.
2- Hematological changes are variable among AIDS patients, depending
on many contributing factors; including viremia, stage of diease,
period of treatment and co-infection with other diseases.
3- Cellular immunity is depressed among the VL, HIV, and VL / HIV
co-infected patients; with decreased in CD4 number and reversed
CD4/CD8 ratio.
4- VL / HIV co-infection is an emerging health problem in the country
which may be associated with a potential risk for more epidemics.
5- The incidence of HIV is increasing in Sudan, becoming a serious
health problem. The age groups 20-40 years are more affected, males
are affected twice more than females and the common mode of
transmission is sexual contact.
6- It is possible to apply double immunocytochemical method for
staining, differentiation, and enumeration of T.cell subsets in
peripheral blood smear.
7- Double immunoenzyme is more specific, sensitive if it is applied with
standardized operating procedures, and suitable for research and
diagnosis in poor country.
6.2. Recommendations:
1- Investigation of full blood count parameters should be carried along
with microscopic diagnosis for VL suspects.
122
2- VL/HIV co-infection is expected to rise in the coming years, further

studies are recommended to assess the magnitude of the problem at
both hospital and community levels.
3- Survey of large samples to determine the prevalence of leishmaniasis
among confirmed HIV patients.
4- Survey of random samples to determine the current prevalence of
HIV in the country.
5- CD4 count should be used to monitor the progress of HIV infection.
6- Deveolpment of effective health education program.
7- Devolpmentof effective counseling.
8- Availability of anti retroviral drugs.
9- Further study containing large samples is required to compare
between double immunocytochemical and flowcytometry for
enumeration of T.cell subsets.
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Appendix
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Haematological Findings in Kala Azar, HIV, and Kalaazar

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Haematological Findings in Kala Azar, HIV, and Kalaazar

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HaematologicalfindingsinKalaazar,HIV,andKala

A Thesis Submitted for Fulfillment of Master Degree in Medica Laboratory

I dedicate my efforts, my work, my love and my live.

(%30)9 > %45 (%23.3) 7 < %70

All parasitologicaly and serologicaly confirmed VL patients were screened

VL/HIV co-infected patients. Relative lymphocytosis and neutropenia were

Clinical Features of VL patients

Hematological parameters of VL patients.

WBCs Morphology in VL patient.

Hematological parameters of HIV/VL co-infected

T.cell subsets of HIV/VL co-infected patients.

Hematological parameters of HIV seropositive

CD4 count in HIV seropositive individuals.

Percentages of Males and Females among the three

Range and mean of ages among the three groups of

Mean of different hematological parameters among the

three groups of patients.

Percentages of different types of anemia among the

Mean of CD4 & CD8 count among the three groups of

Comparison between flowcytometry and double

immunocytochemical for enumeration of CD4.

Frequency of age in VL patients.

Frequency of WBCs in VL patients.

Frequency of Hb among VL patients.

Frequency of Platelets in VL patients.

Frequency of ESR among VL patients.

Percentage of Neutropils in WBCs differential among

Percentage of Lymphocytes in WBCs differential

Percentage of Monocytes in WBCs differential among

Percentage of Eosinohils in WBCs differential among

Prevalence of HIV among VL patients.

Prevalence of HIV among adults VL patients.

Frequency of age among AIDS patients.

Number of AIDS patients.

Frequency of Hb among AIDS patients.

Frequency of PCV among AIDS paints.

Frequency of TWBCs among AIDS patients.

Frequency of Platelets among AIDS patients.

Percentage of Lymphocytes in WBCs differential

among AIDS patients.

Percentage of Neutrophils in WBCs differential among

Percentage of MXD in WBCs differential among AIDS

Frequency of CD4 among AIDS patients.

Comparison between mean of CD4 number by two

GAG: Glucose oxidase Anti-Glucose oxidase

RNA: Ribo Nucleic Acid

CHABTER I: Introduction & literature review.

1.1.1. Leishmanasis Overview

1.1.2. Historical background of VL

1.1.5. Life Cycle

1.1.6. Reservoir hosts

1.1.7. Vectors and transmission

1.1.9. Pathology and pathogenicity of Kala-azar

1.1.10. Clinical Presentation

1.1.11. Hematological Features of VL

1.1.12.1. Parasitological diagnosis

1.1.12.1.1. Direct microscopy

1.1.12.1.2. In vitro culture of visceral Leishmania

1.1.12.1.3. Animal inoculation

1.1.12.2. Immunological diagnosis

1.1.12.2.1. Leishmanin skin test

1.1.12.2.2. Non Specific Tests

1.1.12.2.2a Formal gel test