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Indian J Pediatr

DOI 10.1007/s12098-010-0252-5

SYMPOSIUM ON INFECTIONS OLD AND NEW

Newer Methods for Microbiologic Diagnosis
of Pediatric Infections
Camilla Rodrigues & Anjali Shetty

Received: 24 September 2010 / Accepted: 27 September 2010
# Dr. K C Chaudhuri Foundation 2010

Abstract There are several advantages in making a Nocardia spp, Cryptosporidia [1]) are used by the labora-
specific diagnosis of an infectious disease. In addition to tory to identify organisms.
better patient care with appropriate therapy, once the
diagnosis is established considerable financial expense is
spared and preventive measures can be initiated. The Culture
microbiology laboratory is vital in the isolation and
identification of organisms. It is also possible by finding Blood Culture This is indicated in all infections where
an antigen, metabolic product or nucleic acid unique to that bacteremia is the hallmark e.g. in infective endocarditis,
specific organism that a diagnosis can be made and where ideally three blood sample should be drawn at
susceptibility testing can also be performed. least half an hour apart. If cultures are persistently
negative, supplemental media for nutritionally dependent
Keywords Pediatric . Infections . Diagnosis streptococci can be used. Blood culture is also helpful in
acute febrile illnesses such as typhoid, meningitis,
bacterial pneumonia, osteomyelitis and in fever of
Identification of Infectious Agents unknown origin [2].
Although recovery of bacteria from blood during
To identify microorganisms five different approaches may episodes of sepsis is not difficult, certain concepts need
be adopted. to be understood. Adequate skin preparation prior to
venepuncture is of utmost importance. In addition, a
Microscopy minimum of 5–10 ml of blood should be cultured. The
culture medium should include an anticoagulant and
Direct Microscopy Examination of coverslip preparations ideally an anticomplement and antiphagocytic substance
helps in the immediate diagnosis of cholera, amoebiasis, to reduce the intracellular killing of microorganisms by
giardiasis and helminthic infestations. neutrophils following injection of the blood into the
Various stains such as Gram stain, Acid fast stain (for culture medium.
Mycobacterium tuberculosis, Mycobacterium leprae,
Pediatric Bacterial Blood Cultures The recommended
C. Rodrigues : A. Shetty blood volume to be collected are approximately 1% of
P.D. Hinduja Hospital and Medical Research Centre,
patients total blood volume e.g. for a child weighing 18–30
Mumbai 400 050, India
lbs the total blood volume which should be collected for
A. Shetty (*) two cultures will be 10 ml [3].
Department of Microbiology, There is data to substantiate that two or three blood
5th floor, Lalita Gridhar Building, P.D. Hinduja Hospital
cultures are adequate for detecting common pathogens. The
and Medical Research Centre,
Mumbai, India recovery in the first culture is 80%, 88% in two cultures
e-mail: anjalishettyuk@yahoo.co.uk and 99% in three cultures [2].

Susceptibility Testing There are fully automated instruments available today for rapid and accurate identification as well as susceptibility Automated Blood Culture Systems testing by Minimum Inhibitory Concentration (MIC). CMV. as non-respiratory specimens. Improved and rapid isolation of organisms in (d) Phoenix (BD Diagnostics system) and the (e) Omnilog bacteremias as enteric fever. and reproducible. The BACTEC and interprets results without operator intervention. 18–24 days. have shown that the bottles need to be incubated for only Other systems include (c) Sensititer (TREK Diagnostics). sensitive. tuberculosis complex from smear negative clinical ELISA. Three such systems. Western blot. agglutination. If this is not possible and the organism Conventional Tests—Agar Based Methods Culture of is either fastidious or hazardous to grow or if takes long to mycobacteria is a much more sensitive test than smear cultivate. It is based on oxygen quenching radioimmunoassay (RIA) in detecting small quantities of of mycobacteria with fluorescent dye [7]. The indicator in this technique is an enzyme which BACTEC in monitoring mycobacterial growth showed catalyzes a chemical reaction in a substrate. Various that an inoculum of 200 viable M. Multiple studies after short incubation periods with an accuracy of 93%. nately the slow doubling time of M. The end could be detected in 12–13 days while as few as 20 product of the enzyme reaction can be quantitatively viable bacilli could be detected if one waited for 14– measured spectrophotometrically. 5 days. which are difficult to obtain such as tissue biopsies and body ELISA The ELISA assay has been used extensively to fluids. The lower cost of equipment. simple. immunofluorescence. etc.tuberculosis bacilli enzyme-substrate combinations have been used. (b)The 9240/9120/9050 (BD Diagnostic Systems). inexpensive. This detects either the examination and allows for biochemical identification of specific or nonspecific immune responses of the host or the the species considering enhancing the specificity.J) usually takes viruses e. serology is resorted to. The basis of identification system reactivity is usually based on The continuous monitoring blood culture systems are pH or enzyme profiles but can also be carbon sourced and modular with a single computer controlling the incubator visual detection of growth. Time for detection of M. Lowenstein Jensen medium (L. teria. antigen. specimens is 13–15 days. infective endocarditis. allows for an unequivocal diagnosis to be established. Different studies of the sensitivity of TB. There are culture method for smear positive respiratory as well various types of serologial reactions such as precipitation. rubella. Enzyme immunoassay (EIA) has proven to be (2) Mycobacterial Growth Indicator Tube (MGIT) 960: a significant alternative to the traditional methods in viral The test employs a new state of the art fluorescent immunology in terms not only of sensitivity and specificity technology that enables result towards positivity as but also of safety. Indian J Pediatr Timing The timing of collection of the blood culture does Automated Organism Identification and Antimicrobial not need to coincide with the fever spike. sepsis (Biolog) System [8]. presence of the antigen in the host [9]. the use of L.g. or by 17 days [5. fluorometrically. complement fixation. Unfortu. HIV. hepatitis (A–G). They include the (a) Walk-away units [4]. specific.Culture vials are incubated in individual cells and system (Dade Microscan) based on fluorescence automat- monitored at intervals of 10–15 min for evidence of ically adds reagents to conventional panels when required microbial growth. herpes simplex virus. specific and rapid same antigen is predominantly IgG driven. Serology TB Culture Techniques Isolation of the offending agent is the mainstay in the diagnosis of disease. In specimens. other means. longevity of test .tuberculosis makes Specific IgG and IgM antibodies can be detected using culture on egg/agar based solid media slow and time immunological techniques against an increasing range of consuming. It has proven to be as sensitive as rapidly as 7–10 days.J. IgM antibodies are indicative of recent infection. measles. 6]. the BacT/alert Vitek 2 system (Biomerieux) is an integrated modular (Biomerieux) and the TREK ESP culture systems (TREK system that detects bacterial growth and metabolic changes Diagnostic systems) are available in India. media and BACTEC TB 460 detect either antigen or antibody and has been described as may be justified to maximize isolation of mycobac. The primary immune response is IgM initially and this is Automated Liquid Culture Methods followed by IgG. The secondary response on re-exposure to the (1) BACTEC TB – 460 is a sensitive. Epstein Barr virus.

After day 9. This antigen is a marker of the soluble polysaccharide antigens of Neisseria early acute active infection and early diagnosis of meningitidis. The stability of the kit in high Today assays are available for rapid detection of bacterial environmental temperature and humidity of tropics antigens in various body fluids. they can be used in the initial patients with low levels of parasitemia [11].C. capnocytophaga spps. Current data suggests that galactomannan (b) Cryptococcus neoformans production is proportional to the fungal load in Latex agglutination and enzyme linked immunoas. and a low avidity. stool (b) Dengue and nasopharyngeal aspirates etc.Y.B. complications such as dengue haemorrhagic fever or In neonatal meningitis the latex agglutination dengue shock syndrome.C. The turn around time is pLDH (Parasite Lactate dehydrogenase faster in these assays—ranging from 10 min to a couple of Hrp II (histidine rich protein [12]) hours. As they cannot quanti- when smear is negative. Antigen detection can be performed from various Parasite aldolase samples as cerebrospinal fluid (CSF). Also rapid antigen have decreased sensitivity at lower levels of para- methods have the advantage that since they are antigen sitemia yielding false negative results in non immune based detection systems. a recent one. A high avidity denotes a past certain rheumatological disorders. Haemophilus influenzae tybe b and dengue allows for earlier implementation of supportive Streptococcus pneumoniae in patients of acute pyo. (TORCH). Test must be evaluated in Rubella. it is 67%.W135. IgG avidity testing matococcus have been shown to produce a cross is a method to distinguish past infection from recent reacting polysaccharide antigen. ELISA in detecting antigen. A number of modifications to the techniques methods have a sensitivity of approximately 93% and have expanded sensitivity and speed. Specific IgG and IgM antibodies can be detected using immunological techniques against an increasing range of Serum viruses eg. Dengue Enzyme Immuno Assay (EIA) NS1 anti- gen: Detection of the NS1 antigen is valuable as it CSF allows for detection of infection prior to seroconver- sion and can be detected in serum from day 1 after (a) Latex agglutination techniques are used to demonstrate onset of fever up to day 9.E). prior antibiotics have been tate. tissues [14]. tested.A rising titer is difficult to extrapolate when Trichosporon beigelli. The decision to offer galactomannan say are the preferred tests for detection of Cryptococcal testing depends on resources. urine. measles. They can be used for diagnosis as well as EIA very attractive to a large number of microbiology therapeutic monitoring [9]. RDTs initiated and culture is negative. Cross reactivity across the flavivirus group >98% but the sensitivity varies with each bacterium may occur. Epstein Barr virus. 10]. institutional index of . therapy and monitoring [13]. Cytomegalovirus and Herpes simplex infections conjunction with other clinical and diagnostic findings. they are not useful for prognostication. Hepatitis (A. spp. and N (c) Aspergillus: meningitidis B/E-coli K1 it is 71% and 65% respec. These are also useful should be taken into account. and sto- readings are taken as Optical Densities. Detection of Microbial Antigens/Products These qualitative tests are rapid. simple. easy to perform and interpret.D. Galactomannan is a heteropolysacchride present This assay can also be used for serum after appropriate in the cell wall of most Aspergillus and Penicillium treatment with EDTA. For N meninigitidis A. These techniques are specificity from 93% to 100% and are equivalent to being extensively utilized in diagnosis of Toxoplasma.This reduces the risk of genic meningitis [9. antigen assays philus influenzae and group B streptococci. These tests offer good specificity detected. Treatment with pro- infection and is based on the premise that newer antibodies nase B reduces the false positive in serum tests in do not bind avidly enough. and decreased health hazards has made the use of antigen.Indian J Pediatr reagents. stages of the illness and one does not have to wait for There are three types of antigens used specific antibody production. The latex agglutination laboratories. For S pneumoniae it is 88% and for Haemo. serum. infection. Enzyme ImmunoAssay (EIA) for Galactomannan tively. once NS1 method can be used for detection of E-Coli and Group antibodies are produced this antigen is no longer B streptococci. (a) Plasmodium species Rapid Diagnostic Tests (RDTs) for malaria have an ability to detect >100 parasites/micro litre of blood. HIV.

needless antibiotics can be avoided. homogenous ICT systems are also available incorpo- ity is around 90% but can be lower in infants due to rating monoclonal antibodies to specific viral anti- ingestion of dietary galactomannans. Nitrocellulose membranes pick up specific results may be seen in patients on piperacillin proteins of these two viruses. Virus (RSV) in patient specimens and culture confir- mation. The cut off of 60 pg/ml is under consideration. Environmental contamination Throat/Nasopharyngeal Samples/Sputum/BAL may compromise specificity. Rotavi- antifungal therapy as otherwise sensitivity is dimin. The common features of glucan assays niques are used widely in clinical laboratories.3-ß-D glucan to activate the coagulation techniques also provide data for other parasitic cascade within amoebocytes derived from the haemolymph infections. False positive results are seen in easily using lateral flow assays [15]. These is the ability of 1. children under 5 years. Adenovirus is an important This test is best performed before the initiation of cause of diarrhea in children under 2 years. Ready to use performed to enhance sensitivity. tions are used in an ELISA format detection in patients and in the CSF of patients with meningitis [9]. Respira- Urine tory Viral Screen DFA Kits utilizes fluorescein-labeled monoclonal antibodies to detect and identify in patient In patients with community-acquired pneumonia. numbers of oocysts and relatively simple preparation cystis jirovecii. In sensitivity is approx 81% with a specificity of 94%. Sandwich ELISA techniques have enhanced the usually over 95%. but the sensitivity is often excellent and ml). It is also advised that twice-weekly tests be ed by latex agglutination tests [9]. This assay has better sensitivity than M ity surpasses that of Gram stain. (exceptions being Cryptococcus spp. adenovirus. haemodialysis. The clinical specific. It is also secreted in the urine revolutionising its function. In most assays the tazobactam. chromatographic (ICT) assay for the detection of the S (b) Mycoplasma pneumoniae antigen—Respiratory secre- pneumoniae antigen in urine of patients with pneumonia. (ii) Similarly for detection of Legionella pneumophila Advances in molecular medicine have provided the serogroup I antigen in urine. Antigen produced by the parasites in the of horseshoe crabs [14]. (b) Cryptosporidial antigen Beta D Glucan 1. an in vitro rapid ICT assay is research and diagnostic laboratory with tools that are available with Binax NOW. Non-invasive. gauze. The clinical sensitivity pneumoniae antibody as it is antigen detection and the and specificity is 50–80% and 90% respectively. The advantage is that if these sensitivity to 1 ng/ml. sis are exciting new developments and seem to be the . pneumoniae (Binax). assays flag positive. cardiopulmonary bypass and patients on immunoglobulin products. Indian J Pediatr invasive aspergillosis and the hospital case mix. and and processing. Molecular methods of diagno- and the clinical sensitivity and specificity are >95% [9]. acid-fast staining tech- the Zygomycetes). In Stool addition galactomannan detection appears to have prognostic value as a high unremitting level of (a) Rota virus and adenovirus galactomannan despite antifungal therapy is associated Rotavirus is a leading cause of gastroenteritis in with an unfavourable outcome. influenza B. Their clinical usefulness depends on the geographic prevalence of the specific serogroup Molecular Methods detected. and to screen for parainfluenza 1.3-ß-D glucan antigen-ß-D glucan is Stool samples in human disease provide for high present in the cell wall of most fungi including Pneumo. and Respiratory Syncytial aetiological agent. The EIA assay utilizes serum for gut can be detected from faecal specimens quickly and antigen detection. False positive gens. children the specificity is lower due to carriage of S.2 and 3 S pneumoniae The Binax NOW test is a rapid imuno- following amplification in culture [16]. (a) Respiratory viruses: Viruses can be detected directly from patient speci- mens and by culture isolation/confirmation. The early latex agglutination tests were specificity of rotavirus is slighter less than that of specific but not very sensitive (detection limit 25 ng/ adenovirus. antigen specimens. rus and adenovirus antigens can also be demonstrat- ished. and to screen for the presence of influenza testing is useful in patients on treatment to establish the A. Urinary suffering with pneumonia and upper respiratory tract antigens of pneumococci are detected and their sensitiv- infections [17].

Nucleic acid probes form a useful adjunct to automat- ed cultures for confirmatory identification. Hepatitis C and HIV). The risk of environmental contamination is also locate specific DNA or RNA sequence(s) in tissues or very real. Real time tions. 92% and 88% respectively in patients with meningitis and RNA/RNA duplex.Indian J Pediatr answer for organisms that growth characteristics that are Transcription Mediated Amplication (TMA) TMA uses a slow (mycobacteria). In situ hybridization is used to detect and the sample. These methods and thermostable DNA polymerase generates copies include DNA probes. M. Various in house vitro method for amplifying specific DNA sequence. the required sequence. repetitive sequences. Bronchoalveolar lavage specimens or tissue. plasma. intracellulaire. elucidate and study mutations as it allows for detection Today. ribosomal nucleic acid sequence from any source. cumbersome and some- template and in the presence of nucleotides.tuberculosis complex. However in paucibacillary Molecular diagnostics for tuberculosis have evolved states like tubercular meningitis and pleural effusion because the long turn around time for culture methods. a) Direct detection by nucleic acid amplification directly (b) Nucleic acid amplification from clinical samples allows for initiation of treatment Polymerase chain reaction (PCR): PCR is an in if the clinical picture is consistent.gordonae. RNA etc.. PCR based sequencing and by alternate heating and cooling for denaturation. PCR based sequencing is used to used for real time monitoring of PCR amplicon production. Other methods include PCR-SSCP analysis. (a) Nucleic acid probes—Nucleic acid hybridization is a Molecular testing for fungi is still a long way away from powerful and widely used technique which exploits being a test routinely being used on neutropenic patients on the ability of complementary sequence in single-strand cancer chemotherapy. strategies. The lack of standardization of (ss) DNA or RNA to pair with each other to form a technical issues has been a barrier.kansasi and M. M. Commercial FDA approved systems include matically generates millions or billions of exact copies Amplicor (Roche Diagnostics) and AMTD (GenProbe) thereby making genetic analysis of tiny samples a b) Molecular identification of Mycobacterial species from relatively simple process. pneumoniae and H influenzae with a sensitivity of 100%. The optimal sample for duplex. base pair mismatch assay and other septicaemia [19]. difficult (viruses. c) Molecular methods for detection of drug resistance Genotypic methods for drug resistance in TB assess Real Time PCR This technology offers rapid PCR with the genetic determinants of resistance rather than the simultaneous amplicon detection. primers times ambiguous biochemical methods. identification of mycobacteria from culture and c) Molec- avium. The single strand nucleic acid probes used for Aspergillus PCR is still unclear. M. amplified. As these tests move from the bench to the bedside it is vital that clinicians have a working NASBA Nucleic Acid Sequence Based Amplification is knowledge of the principles and more importantly develop also like TMA as isothermal amplification wherein RNA is a framework for their limitations. blood. a dramatic reduction in the time required for the of both previously recognized and unrecognized muta- assay performance is a significant advance [18]. PCR assays include amplification of genes encoding Starting with extremely minute amounts of a particular mycobacterial antigens. This technique is commercially available a) Direct detection form clinical samples b) Molecular for the identification of M. dideoxy fingerprinting. S. Serial testing by PCR would be needed during the chromosomes by making use of radioactive or fluo. annealing and extension. The target DNA acts as a culture is replacing the slow. The fied sequence or the region of interest on the gene to DNA extraction process is laborious and may contaminate be identified. PCR-heteroduplex analysis. ular methods for detection of drug resistance [21]. The advantage of this methods that can detect low pathogen numbers as in technology being that dead cells have no transcription meningitis and are attaining importance in monitoring machinery hence only viable cells are picked up and response to treatment (Viral load assays for Hepatitis B. microarrays. They also are highly sensitive for reverse transcriptase [20]. whole hybridization should be complementary to the ampli. Unfortunately it is not readily applicable in routine multiplex PCR has been used to detect N. serum. chlamydia) or species sequence of ribosomal RNA (rRNA) as the target fastidious (mycoplasma). practice. A fluorescent signal is resistance phenotype.meningitidis. transcribed into cDNA and then RNA copies are synthe- sized using RNA polymerase. neutropenic phase which would be a substantial cost to the rescent labelled DNA/RNA probes complementary to patient. . the number of bacilli is too low to be picked up by this These methods encompass three main categories namely technology. PCR enzy.

In: Koneman’s color atlas and textbook of specimens that have a low bacillary load. In a separate meta-analysis Lifesciences. 1975. Associated laboratory infra 1985. Chap 2 6th Ed. Welsher HM. Blood cultures: principles and techniques. evaluation of any test for tuberculosis meningitis is the lack of a definitive reference standard. The large scale use loop. An in house Reverse pleuritis. real time formats as GeneXpert are global use of antibiotics with consequent antimicrobial being currently evaluated directly from samples. Lippincott Williams and several studies that have shown a significantly lower Wilkins. niques for the rapid detection of drug resistance in Since specimens such as cerebrospinal fluid (CSF) and tuberculosis: The Line Probe Assay (INNO-LiPA Rif TB pleural fluid often have low bacillary load (i. Henry is paramount.62 Line Blot Hybridization assay to simultaneously speciate for pleural fluid specimens tested by commercial NAA tests. 1.15:615–20. 8. comprehensive and performed in a real world setting out of the molecular lab up to date microbiology diagnostics. Allen SD. Easier to perform. Molecular beacon assays are Thus significant developments have occurred in the past based on a stem and loop structure with the probe in the two decades in the diagnostic arena.35:364–8. Lim ISL. In: Koneman’s color atlas and In tuberculosis. J Clin Microbiol. Anargyros P. Belgium) for the detection of resis. drug resistance should be by a new method of blood culture collection. isoniazid and streptomycin has also been NAA for TBM was a low sensitivity of 0. Atlanta: Centers for Disease Control. constrained settings as ours. 4. Data from diagnostic microbiology. and can be stress the importance of rapid. for every “home-brew” and commercial assay. A negative PCR never eliminates TB as a diagnostic selective media. Am Rev Resp Dis. p. Germany) for the simultaneous detection of assessing the accuracy of NAA tests for tuberculous resistance to isoniazid and rifampicin. Pediatr Infect Dis J. a major concern with the clinical samples [22]. hybridization of DNA obtained from clinical samples to high density oligonucleotides immobilized on a solid support. References Despite the clear advantages that molecular methods offer for drug susceptibility testing in terms of turn around time. Weaver AL. Astill DSJ. 2006.50:91–8. nucleic acid amplification (NAA) tests are capable BACTEC and Lowenstein Jensen media for culture of mycobac- teria from clinical specimens. continue to apply traditional microbiology with newer have been proposed as genotypic methods for detecting molecular testing to the understanding and application of drug resistance in M. wherever the genetic basis of resistance NK. Washington JA. of amplifying a single copy of the target genomic 7. Procop GW. and quality control issues Clin Proc. p. Introduction to microbiology. 5. O’Marcaigh AS. Chap 6. None of the available Real-time PCR Techniques The main advantages of real.e. Koneman EW. In 6. the summary estimate of sensitivity was only 0. 211–302. Tigertt WD. compared two commercially available solid-phase hybridization tech. Real-time PCR techniques have been usefulness of molecular testing is well recognised and has applied to M. 1990. 67–105. Kissling P. Public health mycobacteriology—a guide for the cost implications have to be borne in mind for resource the level III laboratory. Allen SD. Middlebrook G. 1996. molecular tests can be considered “gold” standards. Procop GW. Lippincott the arbitrariness of TB latent infection disease and may Williams and Wilkins. et al. mycobacteria and detect resistance to all known mutations Similarly in another meta analysis the diagnosis accuracy of for rifampicin. also known as biochips or DNA chips.98 [23]. 6th Ed. it is possible that NAA tests can amplify tance to RIF and the GenoType MTBDR assay (Hain the target DNA or RNA. Innogenetics. these tests may have only modest sensitivity in Enterobacteriaceae. Comparison of sequence. et al.28:1288–91. a sensitivity of 95% with a specificity of 99 to 100%. Washington C. Kaditis AG. directly to clinical samples. 2006. support this argument. such as miniaturized glass slides. The however. they tend to Assay. Lastly. In practice. . Mayo structure including proper design. 1977. J Clin Microbiol. Koneman EW. with smear-positive specimens.58 and high developed that can be performed directly on smear positive specificity of 0. Indian J Pediatr Solid-phase Hybridization Techniques There are currently sensitivity in smear-negative sputum specimens.tuberculosis strains and. to avoid cross contamination and amplicon cloud generation 3. 1997.tuberculosis. 2. Washington C. Physicians must Microarrays. Automatable radio- have a useful but limited place in evaluating persons for metric detection of growth of Mycobacterium tuberculosis in TB. confirmed by well standardized phenotypic methods. Yield of positive blood cultures in pediatric oncology patients is not fully characterized. Kubica GP. Comparison of improved theory. results of molecular tests demonstrate textbook of diagnostic microbiology.115:1066–9. Reggiardo Z. accurate. time PCR techniques are the speed of the test and a lower In Herpes Simplex Virus Encephalitis however the risk of contamination. Validation data should be available to detail mycobacterial growth inducator tube (MGIT) with radiometric both the analytical and clinical specificity and sensitivity and solid culture for recovery of acid fast bacilli. smear-negative). However. et al. Rhodes KH. Pfyffer GE. more recently. possibility and a positive result does not confirm it. These resistance in pathogens and opportunistic microorganisms assays have huge potential as they are rapid. Kent PT. They are based on the clinical medicine.