Diagnostic Microbiology for Pediatric Infections

Diagnostic Microbiology for Pediatric Infections

James Versalovic

Since the late nineteenth century, diagnostic microbiology has contributed to our understanding of infectious agents, their relevance to human pathology, and the practice of medicine. The development of staining techniques for the direct visualization of microbial pathogens by light microscopy made rapid assessment of infections possible. Microbial culture techniques permitted the isolation and complete identification of bacterial and fungal pathogens. Morphologic examination by microscopy in the laboratory has been effectively coupled to biochemical testing, immunoassays including agglutination tests, and DNA probe testing for culture confirmation. Specific viral pathogens and obligate intracellular bacteria may require culture in specialized mammalian cell lines. Advances such as rapid shell-vial culture methods and direct detection by nucleic acid amplification have reduced the need for conventional viral culture techniques. Culture-independent approaches such as serologic testing, direct antigen detection, and molecular diagnostics complement culture-based strategies by facilitating the characterization of fastidious or unculturable pathogens. Finally, molecular methods have added the dimension of
quantitative or viral load testing and genotyping for mutation detection.


Methods Details
Antigen detection Immunochromatography; direct immunofluorescence (DFA); latex agglutination; rapid enzyme immunoassay
Microscopic smears Fungal (potassium hydroxide or Calcofluor); Gram stain; routine or modified acid-fast bacillus (AFB)
Nucleic acid (DNA/RNA) amplification Branched DNA (bDNA) detection; hybrid capture; nucleic acid sequence–based amplification (NASBA); polymerase chain reaction (PCR); reverse transcription-polymerase chain reaction (RT-PCR); strand-displacement amplification (SDA); transcription-mediated amplification (TMA)
Serology (antibody testing) Immunochromatography (card-based assays); rapid enzyme immunoassays; immunofluorescence
Culture Antimicrobial susceptibility testing; biochemical testing; immunoassays (agglutination); DNA probe confirmation
Histology Hematoxylin and eosin (H&E); immunohistochemistry; special stains (acid-fast bacillus [AFB], Brown-Brenn, Brown-Hopps, Gomori methenamine silver [fungal], Warthin-Starry silver)
Serology Direct or indirect immunofluorescence (DFA or IFA); enzyme immunoassays (e.g., ELISAs)

This chapter is divided into two sections. The first section describes various methodologic approaches and how different methods may be applied for the diagnosis of pediatric infections (Table 146.1). The second section describes classes of infections and how the laboratory may address specific diagnostic concerns relative to different types of infections (Tables 146.2 and 146.3).


Direct Visualization

In the late nineteenth century, Christian Gram developed a stain (later known as the Gram stain) as a tool for the direct visualization of bacterial pathogens. The direct Gram stain remains a primary method for the rapid assessment of blood cultures
and sterile body fluids. The lower limit of detection of the Gram stain is approximately 105 organisms/mL, requiring a relatively high organism load for direct visualization. Following primary assessment by Gram stain, routine cultures are monitored for growth, and positive cultures are evaluated by Gram stain in combination with biochemical, DNA probe, and immunologic tests for identification.


Possible Infection Primary Test(s)* Secondary Test(s)
Acute pharyngitis Rapid Streptococcus pyogenes (GAS) antigen testing Direct antigen testing for respiratory viruses (enzyme immunoassays or immunofluorescence)
Bronchitis or bronchiolitis Direct antigen testing for respiratory viruses (enzyme immunoassays or immunofluorescence) Bordetella pertussis PCR; Chlamydia pneumoniae or Mycoplasma pneumoniae serologies or PCR; respiratory Gram stain and culture; respiratory virus culture; RT-PCR for human metapneumovirus
Colitis or gastroenteritis Clostridium difficile enterotoxin testing; O&P; rotavirus antigen immunoassays or stool electron microscopy; stool culture (including Campylobacter) Antigen (Cryptosporidium, Giardia) immunoassays; EHEC stool Shiga toxin antigen testing or PCR; Yersinia stool culture; RT-PCR for noroviruses.
Encephalitis Herpes simplex virus PCR of CSF M. pneumoniae IgM serology; M. pneumoniae PCR of CSF; West Nile virus CSF/serum IgM or RT-PCR; PCR of CSF for EBV, CMV, VZV.
Meningitis CSF Gram stain and culture; enterovirus RT-PCR Blood culture; Neisseria meningitidis PCR of CSF
Neonatal sepsis Blood culture; CSF Gram stain and culture Enterovirus RT-PCR; Streptococcus agalactiae rapid antigen or PCR
Osteomyelitis Blood culture Bone biopsy culture
Pneumonia Blood culture; sputum (respiratory) Gram stain and culture Legionella culture; L. pneumophila urinary antigen; Streptococcus pneumoniae urinary antigen; M. pneumoniae IgM serology or PCR.
Septic arthritis Blood culture; synovial fluid Gram stain and culture  
Urinary tract infection (cystitis or pyelonephritis) Urinalysis; urine culture Blood culture
CSF, cerebrospinal fluid; EHEC, enterohemorrhagic Escherichia coli; GAS, group A streptococcus; IgM, immunoglobulin M; O&P, ova and parasite examination; PCR, polymerase chain reaction; RT, reverse transcriptase.
* Primary: indicates testing recommended as routine laboratory or point-of-care evaluation.
Secondary: indicates testing by special request or as adjunctive or confirmatory studies.


Organism Primary Secondary
Bordetella pertussis PCR of nasopharyngeal aspirates or swabs Culture; DFA
Coagulase-negative staphylococci Culture
Chlamydia pneumoniae PCR or serology
Escherichia coli (enteric bacteria) Culture EHEC Shiga toxin antigen or PCR testing
Enterovirus RT-PCR Viral culture
Herpes simplex virus PCR or viral culture; DFA of skin lesions Serology
Legionella pneumophila Culture or urinary antigen testing Antigen testing (DFA)
Moraxella catarrhalis Culture
Mycoplasma pneumoniae PCR or serology
Neisseria meningitidis Culture or PCR Antigen testing
Respiratory viruses (influenza A and B, parainfluenza 1 to 4, respiratory syncytial virus) Direct antigen testing, enzyme immunoassays, or immunofluorescence Viral culture
Rotavirus Stool antigen testing Electron microscopy
Streptococcus agalactiae Culture PCR or antigen testing
Staphylococcus aureus Culture
Streptococcus pneumoniae Culture Urinary antigen
Streptococcus pyogenes Culture; rapid antigen testing Serology; DNA probe hybridization or PCR
DFA, direct immunofluorescence; EHEC, enterohemorrhagic Escherichia coli; PCR, polymerase chain reaction; RT, reverse transcription.

Alternative special stains have been developed for the visualization of mycobacterial, fungal, and parasitic pathogens. The acid-fast and modified acid-fast stains provide important tools for the assessment of infections with mycobacteria and filamentous bacteria. Fluorochroming methods using fluorescence microscopy (e.g., auramine-rhodamine staining) represent the standard approach for primary screening of mycobacteria in respiratory specimens owing to superior sensitivity, as compared with traditional acid-fast stains (e.g., Kinyoun). Modified acid-fast techniques rely on the application of dilute acids to detect partially acid-fast pathogens such as Nocardia directly in clinical specimens or cultures.

Fungi may be visualized directly by potassium hydroxide (KOH) or improved fluorochroming techniques with fungus-specific fluorophores such as calcofluor white. Calcofluor white staining techniques in the laboratory (similar to other fluorochroming methods) provide greater sensitivity for the primary detection of fungal pathogens and are recommended for laboratory-based evaluation. Diagnostic parasitology continues to rely heavily on the application of various staining techniques for the direct visualization of pathogens. The ova and parasite (O&P) examination of stool specimens by light microscopy is a routine approach for the evaluation of patients with gastrointestinal infections.

Antigen Detection

In the 1970s, key advances in immunology such as the development of monoclonal antibodies fueled rapid advances in the formulation of pathogen-specific immunoassays for diagnostic microbiology. Cerebrospinal fluid (CSF) bacterial antigen detection has been used for the detection of Escherichia coli K1, Haemophilus influenzae serotype b, Neisseria meningitidis (serogroups A, B, C, Y, W135), group B streptococcus, and Streptococcus pneumoniae. Unfortunately, CSF bacterial antigen detection is not clinically useful in most circumstances, especially with the advent of vaccination for H. influenzae serotype b. In a comprehensive study by Perkins and associates that included 478 CSF specimens, all true-positive samples by latex agglutination were positive by Gram stain. Rapid bacterial antigen detection by latex agglutination yielded false-positive results in 54% of the antigen-positive samples. The additional false-positive results resulted in additional costs and prolonged hospitalizations.

Antigens may be effectively concentrated in the genitourinary tract, thus establishing urine as a specimen of choice for bacterial antigen detection. Urinary antigen detection is a reliable approach for the diagnosis of Legionella pneumophila serogroup 1 infection and should be performed in parallel with culture for the assessment of Legionella infections. The respiratory pathogen, S. pneumoniae, may be detected by urinary antigen testing as an adjunctive approach. Disseminated fungal infections such as blastomycosis or histoplasmosis may be reliably detected by commercially available serum or urinary antigen testing.

Stool antigen detection represents a convenient strategy for the diagnosis of gastrointestinal infections. Screening for protozoal pathogens in stool by O&P examination is tedious and time consuming. In a study from Alberta, reported by Kabani and colleagues, with 2,652 stool specimens from 1,532 children, the O&P examination rarely uncovered enteric parasites in hospitalized children (4%) as compared with children in the pediatric gastroenterology clinic (13%). Reliable commercial antigen detection assays are available for Giardia lamblia and Cryptosporidium parvum that may exceed O&P tests with respect to sensitivity. The gastric bacterial pathogen, Helicobacter pylori, may be diagnosed and monitored following treatment by enzyme immunoassay-based stool antigen detection with excellent specificity and sensitivity, exceeding 95% in children. Antibiotic-associated colitis may be diagnosed by stool-based detection of the common antigen or enterotoxins of Clostridium difficile. A study by Markowitz and associates highlighted the necessity to test for C. difficile enterotoxins A and B as a combination for the diagnosis of C. difficile infections in children. The enterotoxin A assay missed 41.5% of C. difficile infections, and the enterotoxin B assay failed to
detect 34.9% of these infections. Rotaviruses may be detected by widespread immunoassay-based testing of stool specimens in the diagnostic evaluation of viral gastroenteritis.

Antibody Detection

Antibody detection is especially useful for the diagnosis of infections with unculturable or fastidious pathogens. Purified or recombinant antigen preparations are used in commercial assays for detection of pathogen-specific immunoglobulin M (IgM), IgG, or total antibodies in sera or CSF. The combination of nonspecific and specific serologic testing for two-step screening and confirmatory testing represents an established strategy for the diagnosis of spirochetal infections. For example, primary screening for syphilis by nonspecific rapid plasma reagin (RPR) testing is followed by confirmatory specific serologic findings if the screening test is positive. A screening test with greater sensitivity and reduced specificity is followed by a confirmatory test with greater specificity. A similar approach is used routinely for the diagnosis of Lyme disease (Borrelia burgdorferi infection) in which less specific enzyme-linked immunosorbent assay (ELISA)-based screening is employed before confirmatory immunoblot-based IgM or IgG (serum or CSF) testing.

Antibody detection is a prominent strategy in clinical virology because of the difficulty in culturing many viruses in established cell lines in the laboratory. The diagnosis of Epstein-Barr virus (EBV) infection by antibody detection is a two-step process akin to syphilis testing. Children suspected of infectious mononucleosis may be screened by heterophile testing (i.e., monospot) and confirmed with EBV-specific serologic testing. The diagnostic algorithms for hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) infection are similar in that primary ELISA-based screening is followed by more specific immunoblot assays. By contrast, hepatitis B virus (HBV) infections are evaluated with combination panels of antigen and antibody tests. Serologic testing for antibodies to Toxoplasma gondii is an important strategy for the diagnosis of toxoplasmosis. Serum IgM and IgG testing is performed routinely for the assessment of possible vertical transmission in newborns as well as disseminated infections in immunocompromised children.

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Jul 24, 2016 | Posted by in ORTHOPEDIC | Comments Off on Diagnostic Microbiology for Pediatric Infections

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