Chapter 1. Laboratory Diagnosis of Bacterial, Parasitic and Fungal Diseases

The appropriate use of tests for infectious diseases of children is critical to determining the correct diagnosis. The keys to successful testing are collection and transport of an appropriate specimen to the laboratory and correct performance of the right test in the laboratory. Communication between the clinician and laboratory staff is important in diagnostic testing, particularly if a rare or fastidious organism is suspected. General guidelines for specimen collection and transport are included in this chapter, but it is important to seek guidance from the laboratory which will be performing the test, as practice varies between laboratories.

Because bacteria and fungi are living organisms, they can proliferate or die during transportation of the specimen to the laboratory. Survival of the pathogen is required for culture, however, growth of organisms during transport is undesirable, if the quantity of bacteria is important in making a diagnosis (e.g., in urine culture) or if overgrowth by flora makes detection of a pathogen less likely (e.g., in stool culture). The time for transport to the laboratory should be minimized to reduce death or excessive growth of organisms. When transport time exceeds 1–2 hours, as in the outpatient office where transport times of up to 24 hours are sometimes required, use of specialized transport media is essential for most specimens.

If it is practical to obtain them, body fluids, tissues, and purulent material are generally preferred to specimens collected on a swab. The quantity of material, which can be collected on a swab, is small and bacteria may remain trapped on the swab, where they cannot be detected. Throat and genital specimens for bacterial culture are an exception to this rule and adequate specimens can be collected from these sites using swabs. If swabs must be submitted, submit one swab for each stain or culture ordered.

Many commercial transport systems include a swab and transport media in a tube. These systems work well for most medically important bacteria and fungi. Organisms, which do not survive well during transport (even with transport media), include Neisseria spp., Streptococcus pneumoniae, Haemophilus influenzae, Campylobacter spp., and obligate anaerobic bacteria. If these organisms are suspected, transport time to the laboratory should be minimized (<6–12 h) or media should be inoculated and incubated immediately after specimen collection or nonculture methods of detection should be used. Submission of swabs without transport media to the microbiology laboratory should be avoided, except for throat cultures for group A streptococci (Streptococcus pyogenes), which survives well for 24 hours on either dry swabs or in commercial transport media.

Obligate anaerobic bacteria are killed by the presence of oxygen, so special transport systems are used for specimens from infections that are likely to include these organisms. These include deep abscesses, fasciitis, and infections which have spread from sites heavily colonized by anaerobic bacteria, such as the oropharynx and intestine. Specimens from the oropharynx, intestine, and vagina, which are normally colonized by obligate anaerobic bacteria, generally should not be submitted for anaerobic culture, because the growth of these organisms is expected and susceptibility testing is not typically performed on anaerobic bacteria. In addition, superficial skin and wound infections are unlikely to include obligate anaerobic bacteria and so anaerobic culture is generally not useful for these infections. Unless the specimen will reach the laboratory very quickly (minutes for small specimens and up to 2 hours for larger specimens), a commercial anaerobic transport tube, jar or bag should be used to protect the viability of the bacteria. Fluids, which must be transported without an anaerobic transport system, should be sent in a capped syringe from which all the air has been purged.

Specimens for yeast culture can be transported as described for bacterial culture. The following comments apply to specimens in which a hyphael fungus (i.e., mold) is suspected, although if yeast cells are present, they will also remain viable. Tissue, fluids (respiratory, urine, sterile body fluids), hair, or nail specimens for fungal culture can generally be transported in a clean, dry container without transport media. If the specimen will not reach the laboratory within 2 hours, specimens from normally sterile sites can be kept at 37°C, while those from body sites with bacterial flora can be kept at 4°C.

Laboratory Methods for Detection and Identification of Bacteria

Detection and identification of bacteria can be performed by several methods, including microscopic examination of stained specimens, culture, antigen detection, and nucleic acid amplification tests (NAAT), which include polymerase chain reaction (PCR) and transcription mediated amplification.

The Gram stain remains a valuable tool for rapid detection and preliminary identification of bacteria. Gram-positive bacteria appear dark blue or purple because they have a thick cell wall composed of peptidoglycan, which retains crystal violet and iodine during destaining with alcohol. In contrast, Gram-negative bacteria have a thin layer of peptidoglycan surrounded by an outer membrane, and the alcohol rinse removes the crystal violet and iodine. After a counter stain with safranin, gram-negative bacteria appear pink. The Gram stain also reveals the shape and arrangement of bacteria. The morphology of commonly isolated bacteria is summarized in Table 1–1. Yeast usually stain Gram-positive and they are easily differentiated from bacteria by their greater size.

Stains for mycobacteria include stains with carbol fuchsin and auromine-O. These stains can be routinely performed on respiratory specimens and tissue, and might be performed on other specimens at the discretion of the physician and laboratory. The modified acid-fast stain is a less stringent stain than the acid-fast stain and is useful in detection and identification of Nocardia, Rhodococcus, Tsukamurella, and Gordona, all of which are positive by modified acid-fast stain, but negative by regular acid-fast stain.

Culture and biochemical tests are the mainstay of detection and identification of most bacteria. Most aerobic and facultative anaerobic bacterial pathogens will grow rapidly in routine culture, however, some species require the use of special media and so the laboratory should be informed if these are suspected. Infections with some organisms, which require special culture, are better detected by serology, NAAT or antigen tests and these are noted in the tables later in this chapter.

Detection of mycobacteria in culture usually requires specific media, although rapid-growing mycobacteria (e.g., Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium abscessus) may be detected in routine bacterial culture. Mycobacterium tuberculosis often takes several weeks to grow in culture, although the time for growth is significantly reduced by using liquid culture media. Identification of mycobacteria is also time consuming, although nucleic acid probes can now be used to identify most mycobacteria from culture quickly and accurately.

Antigen detection assays use antibodies specific for bacterial proteins or carbohydrates to test for bacteria. Rapid antigen tests for S. pyogenes (group A Streptococcus) are highly specific (>95%). These tests have sensitivities of approximately 70–85%, though the sensitivity is considerably lower (approximately 50%) when performed by nonlaboratory personnel.1,2 Because of the moderate sensitivities of these tests, specimens with negative results by antigen tests for S. pyogenes should be submitted for culture.1 The Binax NOW® test for S. pneumoniae antigen in urine is very sensitive (100%) for pneumococcal disease in children, however, it is also positive in asymptomatic children colonized with S. pneumoniae and so it has a specificity of only 50–60%.3 Antigen tests performed on CSF are discussed below, in the section on meningitis.

NAAT, such as PCR, are very sensitive because they amplify nucleic acid from the pathogen in logarithmic fashion, doubling the number of DNA or RNA molecules several times. As a result, NAAT can be more sensitive than culture, particularly for fastidious organisms. The number of U.S. Food and Drug Administration (FDA)-approved NAAT is still small, however, many large laboratories have validated non-FDA-approved tests using either commercial kits or “home-brew” assays. NAAT for Chlamydia trachomatis, Neisseria gonorrhoeae, Bordetella pertussis, and M. tuberculosis are discussed later in this chapter.

There are FDA-approved PCR tests for S. agalactiae (group B Streptococcus) in vaginal/rectal samples for screening pregnant women4 and for methicillin-resistant S. aureus in nasal swabs, for infection control screens.5 The results of these assays compare well to those obtained with conventional culture and PCR results may be available more quickly.

Antibiotic Susceptibility Testing

Several methods are used for determination of antibiotic susceptibility. The minimum inhibitory concentration (MIC) is the concentration of antibiotic, which inhibits visible growth of bacteria. The MIC can be determined by several methods, including culturing bacteria in a titration of antibiotics in agar or broth, or by using automated devices. The minimum bactericidal concentration (MBC) is the concentration of an antibiotic, which kills 99.9% of bacteria. In practice, the MIC is easily determined and predicts the susceptibility of bacteria to antibiotics, while determination of the MBC is technically cumbersome and rarely adds information beyond that from the MIC or disk-diffusion testing, and so MBC testing is seldom done. One circumstance in which determining the MBC may be useful is in selection of an aminoglycoside for treatment of endocarditis with enterobacteriaceae (enteric gram-negative rods).6

Disk-diffusion susceptibility testing is performed by coating an agar plate with a known concentration of bacteria, and then placing paper disks impregnated with antibiotics onto the plate. The antibiotic diffuses out of the paper disk and a gradient of antibiotic forms around the disk. After 16–24 hours of incubation, the diameter of the zone of growth inhibition around the disk is measured. The zone of growth inhibition around the disk is inversely proportional to the MIC, and the relationship between these values is the basis for interpretation of disk-diffusion testing results.

The interpretation of MIC values or disk diffusion zones as “susceptible,” “intermediate,” or “resistant” is performed according to guidelines which are published by the Clinical and Laboratory Standards Institute (CLSI) in the United States.7 An organism is considered susceptible, if it is inhibited by concentrations of antibiotic which will be achieved at the relevant body site with the recommended dosage, and it is considered resistant if it is not. An interpretation of intermediate means that failure of antibiotic therapy is more likely than if the organism is susceptible, but that drugs which are normally concentrated at the site of infection (e.g., penicillin in urine) or drugs given at higher doses than usual (e.g., penicillin for intermediate S. pneumoniae in meningitis) may be effective. The intermediate range also includes a buffer zone for technical variation in the test. The absorption, metabolism, and excretion of an antibiotic determine whether it will reach an effective concentration at the site of infection and so laboratories selectively report susceptibility testing based on the body site from which bacteria are isolated. For example, susceptibility results with nitrofurantoin are only reported for bacteria isolated from urine, because nitrofurantoin is concentrated in urine while reaching only subtherapeutic levels in serum and tissue. Furthermore, the interpretation of antibiotic susceptibility may vary depending upon the site infected. For example, interpretation of the MICs of S. pneumoniae with cefotaxime and ceftriaxone depends on whether the patient has meningitis or infection only outside the central nervous system because the levels of these drugs in the central nervous system and the rest of the body differ.8

Laboratory testing can, in a few cases, be used to predict that there is a significant risk that an organism, which is apparently susceptible to an antibiotic, is likely to develop resistance to that antibiotic. An increased MIC to naladixic acid in Salmonella indicates that the bacterium has mutations which make it more likely to become fully resistant to fluoroquinolones, and so treatment of these organisms with fluoroquinolones in extra-intestinal infections may be ineffective.9 An increased chance of developing resistance to clindamycin in Staphylococcus and β-hemolytic Streptococci can be detected by testing for erythromycin-inducible clindamycin resistance.10 This is done by placing clindamycin and erythromycin disks close together in disk-diffusion susceptibility testing and looking for a flattening of the zone of growth inhibition around the clindamycin disk (the “D-test”). Organisms with a positive D-test are reported as clindamycin resistant, although the laboratory may report that clindamycin may still be effective in some patients.

The only test for antibiotic synergy, which is routinely performed, is a screen for synergy of the aminoglycosides gentamicin and streptomycin with the cell wall synthesis inhibitors penicillin, ampicillin and vancomycin against Enterococcus. This is done by simply testing the growth of the Enterococcus isolate with a high level of the aminoglycoside alone. Although synergy testing is available for antibiotic resistant gram-negative organisms from patients with cystic fibrosis, there is no evidence that use of these results leads to improved patient outcomes.11

Laboratory Methods for Detection and Identification of Fungi

“Fungi” include both molds and yeasts. The distinction between molds and yeasts is based on their shape, rather than true biological relationships between the fungi. Molds grow primarily as hyphae (elongated structures that form a colony which looks fuzzy) and spore-forming structures. Aspergillus, the Zygomycetes (e.g., Mucor and Rhizopus) and the dermatophytes (e.g., Trichphyton) are all molds. Yeast grow predominantly as round or oval forms and divide by budding. Colonies of yeast can look smooth or rough, but not fuzzy. Yeast include Candida spp. and Cryptococcus neoformans. Dimorphic fungi grow as yeast at body temperature (including in tissue), but as molds at 25–30°C and these include Histoplasma capsulatum, Coccidiodes immitis, Blastomyces dermatitidis, Sporothrix schenkii, Paracoccidioides brasiliensis, and Penicillium marneffei.

Microscopic examination of specimens for fungi requires specific stains because Gram stain may stain these organisms poorly. Treatment of specimens with KOH makes most host tissues clear, but fungi remain visible. Calcofluor white stain, which can be combined with KOH, binds to fungal cell walls and fluoresces under ultraviolet light, making it easy to detect fungal structures. Geimsa or Wright's stains are useful for detecting H. capsulatum in blood or bone marrow smears. Gomori methenamine silver stain is used to stain fungi such as Pneumocystis jiroveci (formerly P. carinii) in fixed tissue

Several different media are available for fungal culture and the choice of appropriate media depends on the specimen type and suspected fungus. It is therefore important that the specimen type (body site) be specified. Malassezia require addition of lipids to the media to grow, and so it is also important to tell the laboratory if Malassezia is suspected. Malassezia cause catheter-related infections in children receiving lipid-rich parenteral nutrition and also cause tinea versicolor, and less commonly, folliculitis, seborrheic dermatitis, and intravascular catheter-associated sepsis.12

Identification of fungi, particularly molds and dimorphic fungi, is primarily based on the macroscopic and microscopic morphology of the organism. It may take several days to weeks for a mold to develop the distinctive morphology required for identification. Yeast can usually be identified more quickly. The germ-tube assay is a test for identification of Candida albicans, which can be completed within a few hours after isolation of the organism in culture. Biochemical and morphological identification of other yeast can usually be accomplished in less than a week.

Several antigen tests for fungal infections are available. The galactomannan assay detects a cell wall structure from Aspergillus and Penicillium. Studies in children, although large, include only small numbers of patients with invasive Aspergillus. The test has been very sensitive (100%) in the small number of children with confirmed or probable invasive Aspergillus.13 In adults, the sensitivity of the galactomannan test for invasive Aspergillus ranges from 29% to 100%, with higher values generally occurring in profoundly immunocompromised patients.14 The specificity of galactomannan in children at risk of invasive Aspergillus as a result of hematological disease is 89.9–97.5%.13,15 The specificity of the assay is lower in neonates and children than in adults, perhaps because of translocation of the antigen across the pediatric gut.14 False-positive results with galactomannan occur in patients treated with piperacillin-tazobactam and the assay may be positive due to infection with other fungi, including C. neoformans and Geotrichum capitatum.15,16

The β-1, 3-D glucan assay detects an antigen produced by many fungi, including Candida, Aspergillus, Fusarium, Trichosporon, and others. Infections with Cryptococcus neoformans and the Zygomycetes (Rhizopus, Mucor, Rhizomucor, Cunninghamella and Absidia) cannot be detected by the (1,3)-β-d-glucan tests. In the United States, a (1,3)-β-d-glucan test is marketed as Fungitell (Associates of Cape Cod). There are not adequate studies of the use of the Fungitell assay for detection of invasive fungal infections in children. The reference range for adults may not be appropriate for children, in whom the normal levels are (1,3)-β-d-glucan are slightly higher.17 In adults, the (1,3)-β-d-glucan assay is 64.4% sensitive and 81.1% specific for invasive fungal disease when a single specimen is used.18 If specimens are collected twice weekly in neutropenic adults with acute myelogenous leukemia or myelodysplastic syndrome, the sensitivity of the test is 100% in those with proven or probable invasive fungal infection if a single abnormally high value is counted as positive, and a positive value occurs a median of 10 days before a clinical diagnosis.19 (1,3)-β-d-glucan is common in the environment and in medical devices or solutions, and positive results have been reported as a result of dialysis, administration of immunoglobulin or surgical gauze and bandages.20

Antifungal Susceptibility Testing

Susceptibility testing for fungi is still advancing and only limited tests are available. The CLSI guideline for yeast includes Candida and C. neoformans. Interpretations of susceptibility tests are available for fluconazole, itraconazole and flucytosine.21 There are no guidelines for interpretation of testing amphotericin B susceptibility, although organisms with an MIC greater than 1 μg/mL are probably resistant. The guidelines for molds include methods for testing the MIC with amphotericin, flucytosine, fluconazole, ketoconazole, and itraconazole; however, there are no interpretive standards for determining whether an isolate is susceptible.

It is important to use one or more preservatives for transport of stool for parasite examination because some parasites rapidly become undetectable. Many laboratories request that the stool be sent in two separate preservatives: 10% buffered formalin for preparation of a stool concentrate and a preservative with polyvinyl alcohol for preparation of a permanent stained slide. Commercial kits with these preservatives are available and these have convenient tight-fitting screw caps and “fill to” lines. Preservatives, which can be used for both the concentrate and permanent stained slide, are available but the laboratory should be consulted before these are used.

Pinworms (Enterobius vermicularis) and their eggs are not readily found in stool because the female worms exit the anus and lay their eggs on the adjacent skin. To collect the eggs for diagnosis, the sticky side of cellulose (clear) tape can be applied repeatedly to different areas of the peri anal skin, preferably first thing in the morning (before passing stool). The tape is then applied to a microscope slide, with the sticky side against the glass and submitted to the laboratory. Opaque or frosted tape should not be used. Commercial collection kits with sticky transparent paddles are available and simpler to use than tape.

If blood-borne parasites are suspected, blood anticoagulated with EDTA (purple top tube) should be submitted for preparation of blood smears.

Routine testing for intestinal parasites includes microscopic examination of a wet concentrate and a permanent stained slide of stool or, for E. vermicularis, tape-preparation specimens. Routine examination of stool includes a concentrated wet preparation for detection of helminths, protozoan cysts, coccidia and microsporidia, and a permanent stain-smear for protozoa. A few intestinal parasites require special stains to be detected. If these parasites are suspected, it is important to tell the microbiology laboratory so that the right methods will be used. These parasites, along with the method used for detection and the recommended specimen, are listed in Table 1–2.

Blood-borne parasites include Plasmodium (malaria), Babesia, Trypanosoma, and several species of filaria. These pathogens can be detected by microscopic examination of Giemsa-stained blood smears. The use of thick blood films increases the sensitivity for Plasmodium and Babesia, however, thin smears should also be made because the morphology of the parasites is better preserved in this preparation so that species identification can be made. The Binax NOW® ICT malaria test is an FDA-approved rapid immunochromatographic test which differentiates P. falciparum from the other species of Plasmodium. It is sensitive for P. falciparum (96%) and P. vivax (87%), but less sensitive for P. ovale and P. malariae (both 62%) and has an overall specificity of 99%.22

Blood

Blood culture bottles specifically intended for pediatric use may provide some advantage over use of adult blood culture bottles in children, although the data supporting this are limited.23 Pediatric blood culture bottles contain a smaller volume of media than those intended for use in adults and also have a lower concentration of sodium polyanethol sulfonate (SPS), which is an anticoagulant also inhibits the antibacterial effects of blood. SPS has been suggested to inhibit growth of some bacteria (e.g., Neisseria meningitidis), however, the antibacterial effect of SPS does not appear to be significant in practice.24

The concentration of bacteria in the blood of a septic child can be quite low, so the chance of detecting a bacterial pathogen is significantly increased by culturing a larger volume of blood. The concentration of bacteria in blood is less than 1 colony-forming units (viable bacteria) per mL of blood in 23.1% of children, and less than 10 colony-forming units per mL of blood in 60.3% of children with culture-confirmed bacteremia.25 The sensitivity of blood culture in children increases when higher volumes of blood are cultured (e.g., sensitivity increases by approximately 20% when the volume is increased from 2 to 6 mL.)26

Because limited blood volume is available from children and because the yield of aerobic culture is greater than of anaerobic culture from children, anaerobic culture should be performed only in children with risk factors for sepsis with obligate anaerobes.27,28 Widely used automated continuous-monitoring pediatric blood culture systems reliably detect facultative but not obligate anaerobes. Although data are limited, risk factors for sepsis with obligate anaerobic bacteria in children may include decubitis ulcers, abdominal processes (pain, breakdown of the anatomic barrier of the intestinal tract), and neutropenia.27,28 Obligate anaerobic bacteria are also associated with deep abscesses and infections of the head and neck which extend from the oropharynx, and so anaerobic culture may also be useful in children with such infections.

Several bacteria and fungi in blood require special culture conditions or alternative methods of detection (Table 1–3).29,30 Yeast, including Candida, can be detected in conventional blood culture bottles.

Cerebrospinal Fluid

Cerebrospinal fluid (CSF) culture and Gram stain and blood culture should be routinely sent if bacterial meningitis is suspected. CSF should be transported to the microbiology laboratory at room temperature within 1 hour of specimen collection. Routine Gram stain and culture will detect most common and uncommon causes of meningitis in children. Gram stain will detect approximately half of cases of bacterial meningitis, and false-positive CSF Gram stain results, although uncommon, may be caused by observer misinterpretation, reagent contamination, or the use of an occluded lumbar needle which leads to contamination of the specimen with skin flora. Tests for bacterial antigens in CSF should not be performed as these are insensitive and nonspecific and do not add information to that from Gram stain and culture results.31 Even when bacterial antigens are detected in CSF, the result rarely affects patient care because the Gram stain is nearly always positive in these patients as well.32,33 Obligate anaerobic bacteria are an uncommon cause of meningitis in children. Anaerobic culture of CSF should be considered when there are other infections which may give anaerobes access to the meninges or blood (e.g., sinusitis, chronic ear infections, or gastrointestinal disease) or when the anatomic protection of the meninges is compromised (e.g., owing to ventricular shunt or skull fractures).34

Tests for fungi in CSF should be sent in selected patients. Immunocompromised patients, particularly those with HIV infection or prolonged corticosteroid treatment, are at risk for meningitis caused by C. neoformans. Tests for cryptococcal antigen in CSF and blood can be done quickly and are sensitive and specific.35 In adults, tests for cryptococcal antigen on CSF are more than 95% sensitive for cryptococcal meningitis regardless of HIV status.36 Serum tests for cryptococcal antigen to diagnose cryptococcal meningitis are approximately 75% sensitive in adults without HIV and 95% sensitive in adults with HIV.36 False-negative tests for cryptococcal antigen can be avoided by pronase treatment of serum specimens and by titration of serum and CSF specimens to overcome the inhibitory prozone effect of high levels of antigen.35,37 India ink stains will detect less than half of cases of cryptococcal meningitis and should not be done unless cryptococcal antigen tests are not available.38H. capsulatum and C. immitis may also be detected by antigen tests and these results will often be available more quickly than culture results. It is reasonable to do fungal culture in addition to antigen assays if fungi are suspected, but it is important to note that fungi often take weeks to grow in culture.

Stool

Routine stool culture usually includes Salmonella, Shigella, and Campylobacter. If other pathogens, such as enterohemorrhagic E. coli (EHEC or E. coli O157:H7), Y. enterocolitica or Vibrio are suspected, culture for these organisms should be specifically requested. Excreted stool is preferable to swab specimens; swab specimens should only be collected from infants or patients who are unable to produce a specimen. If a stool specimen cannot be transported to the laboratory in less than an hour, it should either be transported at 4°C, or with transport media to preserve the bacteria. Enteric pathogens, which require special culture conditions, are shown in Table 1–4.

EHEC is among the most common bacterial causes of diarrhea in children.39 EHEC can be detected either by culture or by immunoassay for the shiga-like toxin which they produce. Some laboratories only test for EHEC if the stool is visibly bloody, so it is important to communicate with the laboratory if EHEC is suspected. Approximately half of EHEC are of the serotype O157:H7, and these can be detected using MacConkey's agar containing sorbitol, which these organisms do not ferment.40 Most laboratories in the United States use sorbitol containing media for detection of EHEC. Assays for shiga-like toxin, which is produced by all serotypes of EHEC, will detect significantly more cases of EHEC infection, however, this test is used in relatively few laboratories.39,40

Clostridium difficile-associated diarrhea is best diagnosed by assays for the toxins produced by C. difficile because culture has poor specificity for symptomatic infection. Diagnosis of C. difficile associated diarrhea in young children is difficult because up to 30% of asymptomatic children younger than age 1 year are colonized by C. difficile which can lead to positive toxin assays in asymptomatic children or those with another likely cause of diarrhea.39,41 Positive toxin results in younger children should be interpreted carefully in the context of the patient's history and complete testing for other pathogens.

Respiratory Specimens

If a patient is old enough to produce a sputum sample (typically older than 5 years), sputum should be submitted for Gram stain and bacterial culture. Bacteria that commonly cause pneumonia, including streptococci, staphylococci and H. influenzae, can be grown in routine respiratory culture. In children too young or too ill to produce an adequate sputum sample, a sample collection by more invasive means such as tracheal aspiration or bronchoalveolar lavage may be necessary in some circumstances (e.g., child with chronic granulomatous disease). Regardless of whether a sputum sample is submitted, blood culture should be obtained in patients suspected of having bacterial pneumonia. As discussed above, the Binax NOW® test for S. pneumoniae antigen is sensitive, but not specific for invasive pneumococcal disease in children.

There are few studies on interpretation of respiratory Gram stains in children. In adults, a high number of polymorphonuclear leukocytes and low number of epithelial cells on Gram stain suggests that a respiratory specimen is from the lower respiratory tract and that bacterial growth is likely to be significant. A study which evaluated the utility of Gram stain in endotracheal aspirates from mechanically ventilated children found that the absence of bacteria on Gram stain suggests that culture is unlikely to detect a pathogen.42 Respiratory pathogens, which are not detected by routine culture, are listed in Table 1–5 and comments on some of these follow.

The appropriate specimen for diagnosis of pulmonary tuberculosis depends on the child's age and ability to produce sputum. If sputum can be produced, three sputum samples collected on separate days should be submitted for stain and culture for acid-fast bacteria. If sputum cannot be obtained, gastric aspirate specimens should be collected. The sensitivity of gastric aspirate culture can be increased by collecting the specimen first thing in the morning (before the patient eats), neutralization of the stomach acid by adding sodium bicarbonate or sodium carbonate to the specimen, and collection of three specimens on separate days before the initiation of therapy.43 Even with these steps, the sensitivity of culture of gastric aspirates for M. tuberculosis is, at best, approximately 50%.43,44 It is controversial whether gastric aspirate specimens should be stained for acid-fast bacilli, because oral acid-fast organisms can be detected in aspirates from patients who do not have pulmonary tuberculosis. In populations with a high prevalence of pulmonary tuberculosis, staining of gastric aspirates works well, but positive results should be interpreted with caution in patients at a low risk of pulmonary tuberculosis.44

Two NAATs have been approved by the FDA, for detection of M. tuberculosis in respiratory specimens. These tests are sensitive and specific on respiratory specimens in which acid-fast bacilli are detectable on stain (i.e., “smear-positive” specimens).45 If acid-fast bacilli are not detectable by stain, these tests are specific, but not very sensitive and so a positive result is highly predictive of tuberculosis, but a negative result should not be used to rule out tuberculosis.46 NAAT for M. tuberculosis should not be used alone, but they are a useful addition to stains for acid-fast bacilli and culture.

B. pertussis can be detected by culture, PCR or serology. Direct immunofluorescent assays for B. pertussis are not sensitive or specific and should not be used if other tests are available. If the patient has been symptomatic less than 2 weeks, culture or PCR of a nasopharyngeal swab, aspirate or wash specimen is very sensitive and typically detects two-fold to three-fold more infections than does culture.47,48 Dacron or rayon swabs with synthetic shafts are preferred because calcium alginate swabs and wooden shafts inhibit PCR. Most PCR tests detect a B. pertussis genetic sequence (IS481) that is also present in Bordetella holmseii, which is occasionally found in human samples and can lead to false-positive PCR for B. pertussis. The high sensitivity of PCR must be weighed against the potential for false-positive results, which have led to costly pseudo-outbreaks of pertussis.49 PCR for pertussis can be made more sensitive by amplifying other DNA sequences, such as the pertussis toxin gene promoter.47 There is no FDA approved serology test for antibodies to B. pertussis, however, some public health laboratories offer this test.

Infection with Mycoplasma pneumoniae is best detected by NAAT of respiratory specimens or by serological testing. There are no FDA-approved NAAT for M. pneumoniae, however, many assays have been validated by reference laboratories and have sensitivity of approximately 60% and specificity of more than 95%.50–52 The sensitivity of paired IgM samples for M. pneumoniae ranges widely, from 32% to 84% and specificity is typically approximately 90%, but also varies between assays.53 Culture is difficult and slower than NAAT or serology.

Sexually Transmitted Infections (Including Perinatal Transmission)

Diagnosis of sexually transmitted infections in adolescents can be done by the same methods used in adults. In younger children, bacteria associated with sexually transmitted infection can be acquired from the mother during delivery or as a result of sexual abuse. The body sites affected and the diagnostic tests can therefore differ in children and adults, because of the routes of transmission and social, legal and psychological consequences of the diagnosis. The collection of genital specimens from a prepubertal girl should be done only by experienced practitioners, as it can be painful when performed incorrectly.

Neisseria gonorrhoeae can be detected by Gram stain, culture or NAAT. Gram stain of a urethral specimen in a symptomatic adolescent male is a sensitive and specific test for N. gonorrhoeae infection, however, it should not be used in females or asymptomatic males. Culture for N. gonorrhoeae can be performed using urethral, cervical, vaginal, anal/rectal, conjuctival or pharyngeal swabs. The organism is labile and every effort should be made to culture specimens correctly. Culture conditions and alternative tests for genital pathogens are in Table 1–6. Culture is a reasonably sensitive test for N. gonorrhoeae (80–86%) and it is the gold standard for specificity. Molecular tests, including NAAT, for N. gonorrhoeae and Chlamydia trachomatis are discussed together below, as these are usually performed together on a single specimen.

C. trachomatis can be detected by immunoassays (ELISA or immunofluorescent staining), culture, and molecular tests. Immunoassays can give same day results, but the poor sensitivity and specificity of these tests has made them less popular than NAAT. C. trachomatis culture is performed by incubating the specimen with mammalian cells, which support replication of the bacteria, and then staining the cells by immunofluorescence with antibodies specific for C. trachomatis 2 or 3 days later. The advantages of culture are the high (gold-standard) specificity of the test and acceptability of multiple specimen sources, including urethral, cervical, vaginal, anal/rectal, conjuctival or pharyngeal swabs. Unfortunately, the sensitivity of culture for genital infection with C. trachomatis is only 52.3% (female) to 58.9% (male), which is significantly lower than that of NAAT.54 As a result, culture is used primarily for conjunctival and pharyngeal sites, from which NAAT usually cannot be done, and when sexual abuse is suspected, since the very high specificity makes a false-positive result unlikely.

There are several molecular assays for C. trachomatis and N. gonorrhoeae. PACE-2 (Gen-Probe Incorporated) is a nonamplified molecular probe test for C. trachomatis and N. gonorrhoeae. The sensitivity of PACE-2 is approximately 70% for C. trachomatis and 80% for N. gonorrhoeae and it is highly specific (>98%) for both organisms.55–57 Three NAAT are available for C. trachomatis and N. gonorrhoeae: AMPLICOR (Roche Diagnostic Systems), BDProbeTec (Becton, Dickinson and Company) and APTIMA Combo 2 Assay (Gen-Probe Incorporated). An advantage of NAAT over other tests for C. trachomatis and N. gonorrhoeae is that urine can be tested, in addition to urethral and cervical specimens. Other specimens (e.g., conjunctival and pharyngeal) are not usually acceptable for NAAT. Most studies of NAAT for diagnosis of these infections are performed in adults and adolescents and data in prepubertal children are quite limited. It is difficult to compare the performance of the available NAAT because of the use of different and problematic gold-standards, but it is clear that NAAT are the most sensitive tests for both C. trachomatis and N. gonorrhoeae.57 Most studies of adults find that the NAAT are more than 90% sensitive for both organisms and that the specificities are more than 97%.58,59 Use of urine from females may be somewhat less sensitive for both organisms (approximately 80–85%) than other acceptable specimens.58,59

The selection of tests for diagnosis of C. trachomatis and N. gonorrhoeae in children who may have been sexually abused is complex. Detection of these bacteria requires a sensitive test (e.g., the NAATs) while the significant legal, social and psychological consequences of a false-positive test requires a very specific test (e.g., culture). See Table 1–7 for a summary of the tests for bacteria and parasites recommended by the Centers for Disease Control and Prevention at initial visit and 2 weeks later for children in cases of suspected sexual abuse.60 The Centers for Disease Control and Prevention does not recommend use of NAAT for C. trachomatis or N. gonorrhoeae if culture is available, however, this is an area of controversy.60–62 Results of a NAAT might not be considered evidence of infection in legal proceedings as a result of the imperfect specificity of these tests, and this can affect the decision to use a NAAT in suspected sexual abuse.60

Syphilis can be diagnosed by microscopic detection of Treponema pallidum or by serology.60 If lesions (e.g., chancre) are present, treponemes can be detected by dark-field microscopy or immunofluorescent stain. More commonly, the diagnosis is made by serology in the absence of primary lesions. Nontreponemal serological tests (RPR and VDRL) detect antibodies against a lipid antigen, cardiolipin, rather than against the bacteria. The treponemal serological tests (FTA-ABS and TP-PA) detect antibodies specific for T. pallidum. Nontreponemal tests are used as screening tests for syphilis (e.g., in sexually active adolescents) and for following the course of disease, as the titers of the nontreponemal tests fall with successful treatment. Treponemal tests are used to confirm the nontreponemal results, but the treponemal tests usually stay positive for the life of the individual even after successful treatment.

The risk of congenital syphilis should be determined by a nontreponemal antibody test of the pregnant woman in the first trimester of pregnancy and, if the risk of syphilis is high, at delivery.60 Testing the mother is preferred to testing the child, as a low level of maternal antibody may not be detectable in the newborn. However, once the diagnosis of syphilis is made in the mother, follow-up testing in the newborn should include the same quantitative nontreponemal test as was used in the mother. A higher titer in the newborn (four-fold or greater than the titer in the mother) is highly suggestive of congenital syphilis.60 Physical manifestations of congenital syphilis and anatomic pathology of the placenta and newborn are also important in determining the risk of congenital syphilis and current guidelines should be consulted.60

Trichomonas vaginalis can be detected by wet-mount of vaginal secretions in adolescents, although this test is only 50–70% sensitive.60 More sensitive tests include culture (the current gold standard) as well as a DNA probe test, Affirm VP (Becton, Dickinson and Company), which is 90.5% sensitive and 99.8% specific for T. vaginalis and also separately detects Candida and Gardenerella vaginalis.63 A rapid antigen detection test, OSOM Trichomonas Rapid Test (Genzyme Diagnostics) is also available, and is 82% sensitive.64 Infection with T. vaginalis in suspected sexual abuse should be diagnosed by culture because the performance of the nonculture tests has not been adequately assessed in prepubertal children.60

Urine

Urine culture is the gold standard for diagnosis of urinary tract infection (UTI), but culture results require a minimum of 24 hours and so rapid screening tests are also valuable. Screening tests for UTI include biochemical tests by rapid dipstick methods (nitrite and leukocyte esterase) and microscopic examination of the urine for bacteria and leukocytes. A meta-analysis of studies of screening tests for UTI in children found that detection of any bacteria by Gram stain was the best screening test for UTI, with an estimated sensitivity of 93% and specificity of 95%.65 Dipstick assays, which are less technically demanding than Gram stain, also performed well as a screen for UTI. If either leukocyte esterase or nitrite is positive (trace or greater), the dipstick has an estimated sensitivity of 88% and if both results are negative, the dipstick has an estimated specificity of 96%. In the meta-analysis, microscopic examination of urine for leukocytes was inferior to other tests as a screening assay for UTI. But microscopy for urine leukocytes may have some value, as the number of leukocytes in the urine is the best predictor of sepsis associated with UTI in children younger than 90 days.66 When tested by dipstick methods, urine collected by bag has sensitivity comparable to that of urine collected by catheter, however, the specificity of urine collected by bag is significantly lower than that collected by catheter.67

Culture of urine is important because use of the screening tests alone will miss a significant number of UTI. Collection of specimens from children who are not toilet trained is very important to make an accurate diagnosis. Bag urine specimens are not acceptable for culture as they are frequently contaminated with normal skin and genital flora.68 Specimens properly collected by suprapubic aspiration should be sterile and growth of any number of gram-negative rods or a few thousand gram-positive cocci per mL very likely indicates a UTI.69 Pure growth of more than 10,000 pure colonies of a uropathogen collected by transurethral catheterization is likely to indicate true infection and more than 100,000 pure colonies of a uropathogen from midstream urine is also likely to indicate true infection. The presence of small numbers of urogenital flora (e.g., Lactobacillus) will usually be ignored by the laboratory, but if urogenital flora are present in quantities roughly equal to a uropathogen, the laboratory will report the culture as mixed and collection of a new culture is needed.