MICROBIOLOGIC TESTS/DIARRHEAL DISEASE
| PARTNERING WITH THE CLINICAL MICROBIOLOGY LABORATORY IN PEDIATRIC TESTING —
Nancy E. Cornish, MD, Director of Microbiology, Methodist Hospital, Children’s Hospital, and The Pathology
Center, Omaha, NE
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| What laboratory needs from clinician: specimen appropriately collected, stored, and labeled and representative
of stage of illness (collect as soon as possible after onset of disease); obtain sufficient amount for positive culture
before starting antibiotics or antivirals; avoid contamination with normal bacterial flora; send to laboratory as fast
as possible (viruses require cold transport); label (Joint Commission on Accreditation of Healthcare Organizations
[JCAHO] requires 2 methods of patient identification); if possible, let laboratory know tentative diagnosis; consult
laboratory staff about special considerations or problems
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| Culture only infected sites: contamination of plate or specimen can result in unnecessary treatment, follow-up,
and expense; collect specimen as soon as possible after onset of illness (ability to recover pathogen decreases as patient
starts to recover)
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| Send specimen (not swab): send same type of specimen to microbiology laboratory as would be sent to surgical
pathology; send as much as possible (ie, send all collected); swabs do not collect Mycobacteria, fungi, or anaerobes;
exceptions—throat, vaginal, and cervical swabs standard for those sites
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| Tabletop tips: best specimens collected with metal; send fluid in original container or syringe; label accurately
and completely; limitations of swabs—1 in 100 bacteria absorbed (only 3 make it to culture); anaerobes die on
swabs, but survive in tissue and fluid; swabs only hold 150 µL fluid; culture for bacteria requires 6 agar plates, 1
thioglycolate (THIO) broth, and 1 Gram stain; if swab yields only 3 bacteria, poor chance for successful culture;
each plate selective (one grows gram-negative organisms; one gram-positives; 3 organisms easily placed together
on ineffective medium); if effective bacterial culture requires 6 plates, THIO broth, and Gram stain, probability of
success further reduced if mycobacterial and fungal culture also desired (yield poor if 3 cultures sought from single
swab); mycobacterial cultures require ≥1.5 mL of tissue or fluid to inoculate 1 broth and 2 media tubes, and
acid-fast stain; fungal culture requires 1 mL of fluid or tissue to inoculate 2 to 3 media tubes and plates, and fungal
stain (single swab inadequate)
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| Mycobacterial and fungal cultures: collect tissue for culture and histologic examination (permits correlation of
results); fungus in culture not necessarily infection (may be colonizer or contaminant); diagnosis of invasive fungal
infection requires histologic, radiologic, and clinical correlation; fungal spores also colonize nose and respiratory
tract; environmental Mycobacteria can colonize bronchoscope and fluids in laboratory (even tap water), but also
can cause infection; finally, problem may be tumor, not infection (eg, necrotic tumor misdiagnosed as infection)
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| Labeling of specimens: identify specific anatomic site; describe location and nature of lesion precisely; document
date and hour of collection (makes delay in transport readily apparent)
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| Normal microbial flora in humans (overview): normal flora beneficial and necessary; average human has
1013 mammalian cells and 1014 bacteria; oral cavity colonized by 500 to 700 types of bacteria and yeast; 500 to
1000 types of bacteria can colonize colon; laboratory studies designed to detect organisms that grow within 24 to
48 hr (fastest growing and most predominant organisms; many others missed)
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| Anaerobes: note—“what is normal [flora] for human not in hospital is different from what is normal [flora] for human
in hospital; normal flora (sites)—skin and upper respiratory, gastrointestinal (GI), and genitourinary (GU)
tracts; key facts—anaerobes die on swabs; send tissue or fluids (collected via sterile process) from appropriate site;
send to laboratory in syringe with cap or sterile sealed container (viability maintained few hours in tissue or fluid);
if sample collected from abscess where anaerobes likely prevalent, use anaerobic transport vial (maintains viability
of all organisms, including aerobes, 1-3 days)
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| Interpreting Gram stains: squamous epithelial cells suggest contamination by normal flora; segmented white
blood cells indicate severe inflammation; positive stain requires ≥105 bacteria/mL; sensitivity for detecting bacteria
≈50% (positive result helps guide empiric antibiotic therapy; negative result less reliable)
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Specimen Collection: Tips for Selected Sites
| Abscesses: cleanse skin with alcohol to remove oil and dirt; apply iodine or aqueous iodophor (wait 30 sec; bacteria
die by drying); aspirate pus through disinfected uninvolved intact skin
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| Areas of cellulitis: cleanse skin as described above; disinfectants water-based (reason for first removing oil and
dirt); usually, diagnosis not made from cellulitis (treat empirically); for specific diagnosis—aspirate advancing
margin of erythema (consider injecting nonbacteriostatic saline subcutaneously); skin punch biopsy better approach;
blood cultures make diagnosis 4% of time (soft tissue aspirate, 10%; punch biopsy, 20%)
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| Skin nodules or ulceronodular lesions
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 | Organisms: direct inoculation—Francisella tularensis; Mycobacterium marinum (sources fish tanks and swimming
pools); Sporothrix schenckii (sources rosebushes and dirt); hematogenous dissemination—Mycobacterium (causing,
eg, tuberculosis)
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| Specimen collection: “gooey” surface likely to harbor organisms other than those causing infection; remove oils
and dirt and perform iodine preparation; obtain tissue biopsy or remove nodule surgically; send tissue for histopathology
and 3 cultures—acid-fast bacillus (AFB), fungal, and routine bacterial cultures
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| Nose cultures: not predictive of etiologic agents of sinus, middle ear, or lower respiratory tract infections; cultures
should be pathogen-directed (to detect, eg, methicillin-resistant Staphylococcus aureus [MRSA] or group A streptococci)
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| Ear cultures: bypass normal flora of ear canal (aspirate middle ear); most patients respond to empiric therapy (no
culture needed); if patient with ear tubes or complicated anatomy fails to respond to therapy, give laboratory surgically
obtained specimen; growth of unusual environmental pathogens with unusual susceptibility patterns may be
due to previous treatment with multiple antibiotics
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| Tests for influenza (overview): rapid kit test—enzyme immunoassay (EIA) takes ≈30 min; can be performed in
office; immunofluorescence test—direct fluorescent antibody (DFA) takes ≈2 hr; respiratory viral culture—in
study, 50% of positive cultures reported within 24 hr (remaining 50% within 48 hr; most other viruses in panel,
within 48 hr; negative cultures within 5 days); nucleic acid amplification—for viral diagnosis; polymerase chain reaction
(PCR) or reverse transcriptase (RT) PCR; not available routinely outside public health laboratory; rapid,
sensitive, and specific
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Recommendations from Food and Drug Administration (FDA) and Centers for Disease Control and Prevention
(CDC) for Rapid Kit Tests for Influenza
| FDA alert (2004): interpret results with caution; tests do not perform well in clinical practice, despite effectiveness
in trials; false negatives likely (false positives also occur; both vary by patient age, specimen type, and disease
prevalence); in recent data, tests unsatisfactory for clinical diagnosis of avian influenza; interpretation requires—
clinical experience, further laboratory testing, and surveillance information about circulating strains and local levels
of viral activity; optimum specimens—nasopharyngeal aspirates obtained within 3 days of onset of symptoms
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| Patient age and sensitivity of rapid tests: children have higher levels of virus in respiratory tract and shed virus
longer than adults; study by Fader—rapid test compared to viral culture (during peak season, using nasopharyngeal
aspirate); 84% of children <5 yr of age had positive test result (in midsummer, problem of false positives); in 6- to
20-yr-old age group, ≈50% positive (>50 yr of age, 33% positive); if less than optimal specimen used, sensitivity
reduced further
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| When viral activity low: probability of false-positive results high; confirm positive results by DFA, viral culture,
or PCR; during peak season—false negatives more likely and not reliable for making diagnosis; consider confirmation
by DFA, viral culture, or PCR
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| Strategies for safe use of rapid tests: confirm positive results at times of low prevalence and at beginning of
season; know local prevalence of influenza and which subtypes circulating
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| Diagnostic options for outpatients: when influenza circulating, use classic symptoms (abrupt onset of fever and
cough); test one member of family with definitive testing (if positive, treat other family members empirically if
they become ill), or test youngest ill member of family (during influenza season, 84% chance of positive test result
in patients <5 yr of age)
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| Hospitalized patients: if rapid test negative, confirm with DFA or viral culture; if positive, have laboratory save
culture isolate for further testing if needed; if positive during peak of season, have laboratory save sample for further
testing; if positive out of season, confirm with DFA or viral culture and save sample; in studies by Barenfanger
(2000) and Woo (1997), diagnosis of viral infection decreases use of antibiotics and additional testing,
and shortens hospital stays
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| Rotavirus testing: EIA antigen detection test available (high rate of false positives when disease prevalence low;
false negatives occur when disease prevalence high); in Nebraska, peak occurs in March; during summer, laboratory
performs test if physician insists (if result positive, send for confirmation testing); overreliance on positive test
result during summer can mislead clinicians and delay appropriate therapy and treatment
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| DIARRHEAL DISEASE IN THE UNITED STATES —Ellen R. Cooper, MD, Associate Professor of Pediatrics,
Boston University School of Medicine, and Boston Medical Center, Division of Infectious Disease, Boston,
MA
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| Etiology: Rotavirus most common pathogen; Caliciviruses (Norovirus and Sapporo virus) transmitted by fecal-oral
route and aerosolized by vomiting; rate of viral disease peaks in winter, especially in younger children; travel increases
risk for exposure to bacterial infection
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| Seattle study (2006): 1626 children who presented to emergency department with diarrhea; mean age 2.5 yr; median
duration of diarrhea 3 days (range 1-360 days); in 15%, blood in or on stool; pathogens recovered from stool—
bacteria in 7.3% of patients (viruses, 33%; Clostridium difficile, 6.7%); only 47% of patients had pathogen identified;
most common bacterial pathogens—Salmonella and Shiga toxin–producing Escherichia coli (E coli O157:H7
most common)
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| Factors that increase likelihood of bacterial infection: onset during summer (viruses more common in winter);
age >3 yr; history of travel; visible or microscopic blood; >10 stools in previous 24 hr; greater abdominal pain
or tenderness; only 48% of those with positive bacterial culture had leukocytes in stool; bacteria most likely identified
from stool-cup specimen (less likely from diaper; least likely from swab); older the child, the more likely infection
bacterial; 76% of positive bacterial cultures suspected clinically (of suspected cases, only 35% negative)
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| E coli–associated diarrhea: classification—enterohemorrhagic E coli (EHEC); enteropathogenic; enterotoxigenic
(ETEC; traveler’s diarrhea); enteroinvasive; enteroaggregative; EHEC—causes colitis; associated with
hemolytic uremic syndrome (HUS) in all ages and postdiarrheal thrombocytopenia purpura in adults; comparison
of infectious doses—E coli (10-100 organisms) vs Salmonella (105 -108 organisms)
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E coli O157:H7
| Diagnosis: organism ferments sorbitol slowly and can be screened on sorbitol MacConkey agar; negative colonies
translucent, but can be confirmed with antisera; rate of stool isolation greater in first 6 days after onset of diarrhea
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| Typical HUS: usually occurs after prodrome of bloody diarrhea; renal failure probably secondary to predilection of
Shiga toxin for renal circulation; rare cases after urinary tract infection (UTI) and pneumonia due to Shiga-producing
E coli or Shigella; EHEC causes at least 70% of HUS (of those, 80% due to E coli O157:H7; exception Australia);
HUS after Shigella infection more severe; sources of E coli O157:H7 infection—undercooked hamburger;
petting zoos and country fairs; unpasteurized apple cider; waterborne illness; contaminated radish sprouts (difficult
to wash); person-to-person transmission; triad of typical HUS—1) microangiopathic hemolytic anemia; 2) thrombocytopenia;
3) acute renal failure
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| Treatment of EHEC: antibiotics and antimotility drugs increase risk for HUS; supportive care key (risk for acute
renal syndrome increases with severity of dehydration); rehydrate carefully because of fluid shifts and electrolyte
imbalances; early dialysis does not affect long-term risk for renal failure
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| Risk factors for long-term complications: WBC >20,000/µL on presentation; anuria >8 days or oliguria >15
days; if >50% of glomeruli damaged, long-term renal damage more likely
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| Prevention of E coli O157:H7: screening of meat not successful; 5-day period of hay feeding prior to slaughter
may dramatically lower number of organisms in intestinal tract (E coli resistant to acid); pasteurization of apple cider
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Traveler’s Diarrhea
| Diagnosis: ETEC cause of traveler’s diarrhea in all age groups (unlike EHEC, infection requires enormous inoculum);
more common in areas where sanitation poor; clinical syndrome—results from ingestion of contaminated
food or water (usually lasts ≤3-4 days); toxic gastroenteritis caused by ingestion of preformed toxins; fever low or
absent; watery stools with cramps; 30% to 50% of travelers develop diarrhea during 1- to 2-wk stay in high-risk
area; bacterial pathogens—ETEC most common; Campylobacter common (especially in Asia); nontyphoidal Salmonella
infrequent; Shigella relatively common (infectious dose low); Vibrio parahemolytics and V cholerae associated
with undercooked seafood
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| Treatment: trimethoprim-sulfamethoxazole (TMP-SMZ) and doxycycline no longer ideal; fluoroquinolones used in
adults; macrolides first-line treatment in children; rifaximin approved for patients ≥12 yr of age (200 mg tid); antimotility
agents have role, especially in combination with antibiotics; prophylaxis—bismuth subsalicylate (BSS; use with
caution in children <3 yr of age due to risk for side effects)
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Suggested Reading
Barenfanger J et al: Clinical and financial benefits of rapid detection of respiratory viruses: an outcomes study.
J Clin Microbiol 38:2824, 2000; De Jong MD et al: Fatal avian influenza A (H5N1) in a child presenting with diarrhea
followed by coma. N Engl J Med 352:686, 2005; Denno DM et al: Etiology of diarrhea in pediatric outpatient
settings. Pediatr Infect Dis J 24:142, 2005; Fader RC: Comparison of the Binax NOW Flu A enzyme
immunochromatographic assay and R-Mix shell vial culture for the 2003-2004 influenza season. J Clin Microbiol
43:6133, 2005; Farquhar D: E coli, antibiotics and hemolytic-uremic syndrome in children. CMAJ 163:438,
2000; Hickner JM et al: Principles of appropriate antibiotic use for acute rhinosinusitis in adults: background.
Ann Intern Med 134:498, 2001; Price EH et al: Etiology of diarrhea in pediatric outpatient settings. Pediatr Infect
Dis J 24:661, 2005; Quach C et al: QuickVue influenza test for rapid detection of influenza A and B viruses in a
pediatric population. Clin Diagn Lab Immunol 9:925, 2002; Van Walraven C, Naylor CD: Do we know what
inappropriate laboratory utilization is? A systematic review of laboratory clinical audits. JAMA 12:550, 1998;
Varman M et al: Characterization and mechanisms of resistant group B streptococcal isolates obtained at a community
hospital. Eur J Clin Microbiol Infect Dis 24:431, 2005; Wald ER et al: Acute maxillary sinusitis in children.
N Engl J Med 304:749, 1981.
Resources
www.fda.gov
www.thepathologycenter.org
Educational Objectives
| The goals of this program are to improve accuracy in microbiologic testing and to improve diagnosis and treatment of
diarrhea. After hearing and assimilating this program, the clinician will be better able to:
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| 1. Provide appropriately collected and labeled specimens for microbiologic analysis.
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| 2. Interpret microbiologic test results.
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| 3. Employ strategies for safe use of rapid kit tests for influenza.
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| 4. Identify infectious causes of diarrheal disease in children.
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| 5. Choose appropriate therapy for managing diarrhea in children.
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Faculty Disclosure
In adherence to ACCME Standards for Commercial Support, Audio-Digest requires all faculty members to disclose
relevant financial relationships within the past 12 months that might create any personal conflicts of interest. Any
identified conflicts were resolved to ensure that this educational activity promotes quality in health care and not a proprietary
business or commercial interest. For this program, the faculty reported nothing to disclose.
Acknowledgements
Dr. Cornish was recorded at the Stephen A. Chartrand, MD, Memorial Seminar, Practical Pediatrics 2007 Update,
presented February 16, 2007, in Omaha, NE, and cosponsored by Creighton University School of Medicine, Department
of Pediatrics, Continuing Medical Education Division, the Children’s Hospital, Ambassador Pediatrics, and
Children’s Home Healthcare. Dr. Cooper was recorded at Current Concepts in Pediatric Infectious Disease, presented
November 3-4, 2006, in Cambridge, MA, by Children’s Hospital Boston and Boston Medical Center. The Audio-Digest
Foundation thanks Drs. Cornish and Cooper and the sponsors for their cooperation in the production of this program.
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