TABLE 9.

Testing platforms for bacteria

CategoryOrganismReference(s)SpecimenaConventional test methodbAssay
Laboratory developedASRKitTargetInstrumentChemistrySensitivity vs. conventional methodTurnaround time vs. conventional methodComments
General bacteriaGroup A streptococcus (Streptococcus pyogenes) 499 Throat swabsAntigen detection/cultureYes (Roche)c pts1 geneLightCyclerDual FRET hybridization probesGreaterFasterReal-time PCR assay required half the personnel
Group B streptococcus (Streptococcus agalactiae) 30, 211, 212Anal, vaginal or combined anal/vaginal swabsCultureYes cfb geneLightCyclerDual FRET hybridization probesEqualFasterSusceptibility testing still required in patients allergic to penicillin
AbstractgAnal, vaginal or combined anal/vaginal swabsCultureYes (I.D.I.)d cfb geneLightCycler and SmartCyclerDual FRET hybridization probes (LightCycler), molecular beacons (SmartCycler)EqualFasterSusceptibility testing still required in patients allergic to penicillin
Shiga (Vero) toxin-producing Escherichia coli 387 FecesEIA, conventional PCR assayYes stx1, stx2, stx2eLightCyclerDual FRET hybridization probesGreater (EIA or conventional PCR)Faster (conventional PCR)Differentiation of stx genes by melting curve analysis
28 FecesCultureYes stx1, stx2SmartCyclerMolecular beaconsGreaterFasterMultiple assay
Slow-growing or poorly culturable bacteria Bartonella henselae 559 Blood (endocarditis)Culture, conventional PCR assayYes ribC geneLightCyclerSYBR Green IGreater (culture or conventional PCR)FasterSerology may still be used in PCR-negative cases
Bordetella pertussis 406 IsolatesCultureYesIS481 (B. pertussis)LightCyclerDual FRET hybridization probesEqualNAiAssay for IS481 also detects but cannot differentiate Bordetella holmesii from Bordetella pertussis
7 Nasopharyngeal swabsCultureYesIS481 (B. pertussis)LightCyclerDual FRET hybridization probesGreaterFasterAssay for IS481 also detects but cannot differentiate Bordetella holmesii from Bordetella pertussis
63 Nasopharyngeal swabsCultureYesIS481 (B. pertussis)ABI Prism 7700TaqMan probesGreaterFasterAssay for IS481 also detects but cannot differentiate Bordetella holmesii from Bordetella pertussis
Bordetella pertussis and Bordetella parapertussis 405 Nasopharyngeal, throat or perinasal swabs, BALCultureYesIS481 (B. pertussis), IS1001 (B. parapertussis)LightCyclerDual FRET hybridization probesGreaterFasterAssay for IS481 also detects but cannot differentiate Bordetella holmesii from Bordetella pertussis
71 Nasopharyngeal swabs or aspirates, sputa, throat swabs, bronchial washings, pleural fluid, tracheal aspiratesDFA, cultureYesIS481 (B. pertussis), IS1001 (B. parapertussis)LightCyclerDual FRET hybridization probesGreater (DFA or culture)FasterAssay for IS481 also detects but cannot differentiate Bordetella holmesii from Bordetella pertussis
233 Nasopharyngeal swabsCultureYesIS481 (B. pertussis), IS1001 (B. parapertussis)LightCyclerDual FRET hybridization probesGreaterFasterAssay for IS481 also detects but cannot differentiate Bordetella holmesii from Bordetella pertussis
450 Nasopharyngeal swabsDFA, culture, conventional PCR assayYesYeseIS481 (B. pertussis), IS1001 (B. parapertussis)LightCyclerDual FRET hybridization probesGreater (DFA, culture, conventional PCR)Faster (DFA, culture, conventional PCR)Assay for IS481 also detects but cannot differentiate Bordetella holmesii from Bordetella pertussis
234 Nasal, pharyngeal or nasopharyngeal swabs, nasopharyngeal aspiratesCultureYesIS481 (B. pertussis) IS1001 (B. parapertussis)ABI Prism 7700TaqMan ProbesGreaterFasterAssay for IS481 also detects but cannot differentiate Bordetella holmesii from Bordetella pertussis
233 Nasopharyngeal swabsCultureYesIS481 (B. pertussis) IS1001 (B. parapertussis)ABI Prism 7700TaqMan ProbesGreaterFasterAssay for IS481 also detects but cannot differentiate Bordetella holmesii from Bordetella pertussis
486 Nasal swabs, sputa and naso-pharyngeal aspiratesCulture, conventional PCR assayYesIS481 (B. pertussis), IS1001 (B. parapertussis)iCyclerMolecular beaconsGreater (culture, conventional PCR)Faster (culture, conventional PCR)Assay for IS481 also detects but cannot differentiate Bordetella holmesii from Bordetella pertussis
Tropheryma whipplei 126 Lymph node, duodenal biopsy or cardiac valveConventional test methodYes16S-23S rRNA gene intergenic spacer region and rpoB geneLightCyclerSYBR Green IEqualNA
Agents of community-aquired pheumonia Chlamydophila pneumoniae 19 Sputa, BAL fluidFour conventional PCR assaysYes ompA ABI Prism 7700TaqMan probesSee commentsFasterReal-time PCR assay produced most consistent positive results when replicate testing of samples performed
494 Peripheral blood mononuclear cells, oropharyngeal swabsCulture, two nested conventional PCR assaysYes ompA (two domains, VD2 and VD4)ABI Prism 7700TaqMan probesSee commentsFasterVD4 real-time assay detected most positives
536 Sputa, nasopharyngeal secretions, throat swabs, bronchial aspirations, BAL fluid, pleural fluidConventional PCR assayYes pst1 genomic fragmentABI Prism 7700TaqMan probesGreaterFaster
242 Sputa, nasopharyngeal, throat specimensConventional PCR assayYes ompA geneABI Prism 7700TaqMan probesGreaterFaster
407 BAL fluid, bronchial secretions, tracheal secretions, gargle water, and throat swabsCulture, serologyYes16S rRNA geneLightCyclerDual FRET hybridization probesEqualFaster
328 Nasopharyngeal aspiratesConventional PCR assayYes pmp4 geneLightCyclerDual FRET hybridization probesEqualFaster
Legionella pneumophila 536 Sputa, nasopharyngeal secretions, throat swabs, bronchial aspirations, BAL fluid, pleural fluidConventional PCR assayYes16S rRNA geneABI Prism 7700TaqMan probesEqualFaster
535 BAL fluid, tracheal secretions, bronchial secretionsDFA, culture of urinary antigenYes16S rRNA geneLightCyclerDual FRET hybridization probesGreaterFasterResults compared to combination of results for DFA, culture, urinary antigen
547 Sputa, BAL fluid, endotracheal aspiratesDFA, cultureYes mip geneLightCyclerDual FRET hybridization probesEqual (culture), greater (DFA)Faster
Legionella pneumophila and Legionella spp. 175 SputaDFA, culture of urinary antigenYes23S-5S spacer regionABI Prism 7700TaqMan probesSlightly less sensitiveFasterConsidering only positive result for DFA, culture or urinary antigen real-time PCR was 94% sensitive, equal to culture
396 Sputa, BAL fluidCulture, conventional PCR assayYes16S rRNA geneLightCyclerSYBR Green IEqual (culture, conventional PCR)Faster (culture, conventional PCR)
391 Induced sputa, BAL fluidCultureYes16S rRNA geneLightCyclerDual FRET hybridization probesGreaterFaster
408 BAL fluidCultureYes16S rRNA geneLightCyclerDual FRET hybridization probesEqualFaster
167 BAL fluid, open lung biopsyDFA, culture for BAL, DFA, culture, Warthin starry stain, in situ hybridizationYes mip gene and 5S geneLightCyclerDual FRET hybridization probesEqual (culture for BAL), greater (DFA for BAL), greater (DFA, Warthin starry stain for open lung biopsy), less (culture and in vitro hybridization for open lung biopsy)Faster (real-time PCR faster than all methods)
Mycoplasma pneumoniae 536 Sputa, nasopharyngeal secretions, throat swabs, bronchial aspirations, BAL fluid, pleural fluidConventional PCR assayYes16S rRNA geneABI Prism 7700TaqMan probesEqualFaster
163 Nasopharyngeal secretions, pharyngeal swabs and bloodSerology conventional PCR assayYes16S rRNA geneABI Prism 7700TaqMan probesSlightly less sensitive (serology, conventional PCR)Faster (serology, conventional PCR)95% sensitivity
501 Sputa, throat washings, throat swabsConventional PCR assayYesPI geneLightCyclerDual FRET hybridization probesEqualFaster
484 Throat swabsSerology, conventional PCR assayYesPhHV sequencesiCyclerMolecular beaconsGreater (serology), equal (conventional PCR)Faster
Streptococcus pneumoniae 148 Nasopharyngeal swabsCultureYes ply geneABI Prism 7700TaqMan probesSee commentsFasterNumber of organisms detected by real-time PCR correlated with numbers
detected by semiqualitative culture. It remains unclear whether these results
    correlate with pneumococcal lower respiratory tract disease
Agents of meningitis Neisseria meningitidis 77 CSF, plasma, serum, whole bloodCultureYes ctrA geneABI Prism 7700TaqMan probesLess (sensitivity = 80%)FasterAdditional cases detected in culture-negative samples
155 CSF, plasma, serumCultureYes ctrA gene, IS1106, siaD geneABI Prism 7700TaqMan probesSlightly less (91-93%)FasterAdditional cases detected in culture-negative samples
318 CSFCultureYes16S rRNA gene, sacC, siaD, porA genesLightCyclerDual FRET hybridization probesEqualFaster
Haemophilus influenzae 77 CSF, plasma, serum, whole bloodCultureYes bexA geneABI Prism 7700TaqMan probesEqualFasterAdditional cases detected in culture-negative cases
Streptococcus pneumoniae 77 CSF, plasma, serum, whole bloodCultureYes ply geneABI Prism 7700TaqMan probesSlightly less (sensitivity = 92%)FasterAdditional cases detected in culture-negative cases
Bacteria associated with tick-borne diseases Borrelia burgdorferi sensu stricto 299 Skin biopsies (erythema migrans)CultureYes recA geneLightCyclerSYBR Green INot providedFasterMelting curve analysis used to identify different Borrelia spp.
Borrelia garinii and Borrelia afzelii 380 Bacterial isolatesCultureYes recA geneLightCyclerSYBR Green IEqualFasterMelting curve analysis used to identify different Borrelia spp.
Ehrlichia chaffeensis 288 Blood, tissueN.A.Yes16S rRNA geneiCyclerTaqMan probesSee commentsFasterPrevious “positive” samples were all identified by real-time PCR
Potential agents of bioterrorism Bacillus anthracis 29, 119Bacterial isolatesCultureRochef capB, pagA genesLightCyclerDual FRET hybridization probesEqualFasterAutoclaving of bacteria before real-time PCR test did not affect sensitivity of assay
350 Spiked nasal swab specimensCultureYes rpoB, lef genesLightCyclerDual FRET hybridization probesEqualFaster
98 Spiked swabsCultureYes rpoB geneLightCyclerDual FRET hybridization probesEqualFaster
371 Bacterial isolatesCultureYes rpoB, pagA, capC genesLightCyclerDual FRET hybridization probesEqualFaster
389 Bacterial isolatesCultureYes rpoB geneLightCyclerDual FRET hybridization probesEqualFaster
254 Bacterial isolates capC geneLightCyclerSYBR GoldEqualFaster
111 Bacterial isolatesCultureYes rpoB, pag, capC genesABI Prism 7000, 7700TaqMan probesEqualFaster
181 Bacterial isolatesCultureYesUndisclosed chromosomal and pXO2, pXO7 targetsABI Prism 7700, LightCycler, SmartCyclerTaqMan probesEqualFaster
Yersinia pestis 493 Bacterial isolatesCulture16S rRNA, pla, capI, ymt genesLightCyclerDual FRET hybridization probesEqualFaster
306 Bacterial isolatesCultureUndisclosed targetLightCycler and RAPIDhTaqMan probesEqualFaster
Bacterial antibiotic resistance genesMRSA 409 Bacterial isolatesCulture-based susceptibility testingYes mecA geneLightCyclerDual FRET hybridization probesEqualFaster
448 Blood cultures which showed gram-positive cocci in clustersCulture-based susceptibility testingYes mecA and sa442 genesLightCyclerDual FRET hybridization probesEqualFaster
472 Blood cultures which showed gram-positive cocci in clustersCulture-based susceptibility testingYes mecA and sa442 genesLightCyclerSYBR Green IEqualFaster
123 MRSA selective broth (oxacillin) inoculated with specimens from wounds, abscesses, anterior nares, perineum, urine, catheter insertion sites, skin and soft tissues, sputa, tracheaCulture-based susceptibility testingYes nuc geneLightCyclerDual FRET hybridization probesSlight decrease (sensitivity = 93.3%)Faster (selective broth and real-time PCR required ∼24 h vs. longer periods for conventional culture)
150 Bacterial isolatesCulture-based susceptibility testingYes mecA geneLightCyclerDual FRET hybridization probesEqualFaster
219 Bacterial isolatesConventional PCR assayYes mecA geneABI Prism 7700TaqMan probesEqualFaster
130 Nasal or inguinal swabsCulture-based susceptibility testingYes mecA and femA genesABI Prism 7700TaqMan probesEqualFasterPrior to real-time PCR, Staphylococcus aureus cells are selectively removed from swabs by a unique immunocapture technique
VRE 361 Bacterial isolatesConventional PCR assayYes vanA and vanB genesLightCyclerDual FRET hybridization probesEqualFaster
362 Rectal swabs with and without enrichment brothYes vanA and vanB genesLightCyclerDual FRET hybridization probesGreaterFaster
451 CultureYes (Roche) vanA and vanB genesLightCyclerDual FRET hybridization probesGreaterFaster
Extended spectrum beta-lactamases 394 Bacterial isolatesCulture-based susceptibility testingYes bla SHV genesLightCyclerDual FRET hybridization probesGreaterFaster
Penicillin resistance in Streptococcus pneumoniae 214 CSFCulture-based susceptibility testingYes pbp2b LightCyclerSYBR Green IGreaterFasterDetection of php2b gene associated with susceptibility
Penicillin resistance in Neisseria meningitides 460 Bacterial isolatesDNA (sequencing of penA gene)Yes penA gene codon 566 mutationLightCyclerDual FRET hybridization probesEqualFaster penA gene codon 566 mutation associated with penicillin resistance detected by melting curve analysis
Fluoroquinolone resistance in Staphylococcus aureus 248 Bacterial isolatesCulture-based susceptibility testingYes grlA gene mutationsSmartCyclerMolecular beaconsEqualFaster
Fluoroquinolone resistance in Yersinia pestis 275 Bacterial isolatesDNA sequencing of gyrA geneYes gyrA gene mutationsLightCyclerDual FRET hybridization probesEqualFasterMutations detected by melting curve analysis
Obligately anaerobic bacteria Clostridium difficile 27 FecesCytotoxicity assayYes tcdA and tcdB genesSmartCyclerMolecular beaconsSlightly less (sensitivity = 97%)Faster
  • a BAI, bronchoalveolar lavage; CSF, cerebrospinal fluid.

  • b EIA, enzyme immunoassay; DFA, direct fluorescent antibody; N.A., not applicable.

  • c LightCycler Strep-A assay (Roche Diagnostics Corporation, Indianapolis, IN).

  • d IDI-Strep B Assay (Infectio Diagnostics, Inc., Sainte-Foy, Quebec, Canada) for use with SmartCycler (Cepheid, Sunnyvale, CA).

  • e Prototype assay evaluated for this study now available as ASR: LightCycler Bordetella IS481/1001 assay (Roche Diagnostics Corporation).

  • f LightCycler Bacillus anthracis detection kit (Roche Applied Science, Indianapolis, IN).

  • g C. Ménard, F. J. Picard, J. Frenett, M. Gagnon, D. B. Ke, M. Ouellette, P. H. Roy, and M. G. Bergeron, Am. Soc. Microbiol. 100th Annu. Meet., abstr. C-193, 2000.

  • h RAPID refers to “ruggedized” advanced pathogen identification device, which is a field-deployable real-time PCR assay platform (Idaho Technology, Salt Lake City, Utah).

  • i NA, not applicable.