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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
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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
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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.
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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.
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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
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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.
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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.
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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
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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.
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Respiratory Specimens
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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.
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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.
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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
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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.
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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.
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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.
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Sexually Transmitted Infections (Including Perinatal Transmission)
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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.
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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.
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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.
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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
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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
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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.
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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
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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
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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
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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.