Persing, David H.

Recent analysis of the gene encoding the beta subunit of Mycobacterium tuberculosis RNA polymerase (rpoB) has demonstrated a small region that harbors the mutations most frequently associated with rifampin resistance. Earlier reports have described a high degree of sequence conservation of rpoB among mycobacteria other than M. tuberculosis and other GC-rich bacteria that can lead to false-positive amplification when applied directly to clinical specimens. We developed reagents for PCR amplification that are based on signature nucleotides discovered by comparative sequence analysis of the rpoB genes of organisms phylogenetically related to M. tuberculosis. The specificities of the reagents were challenged with 20 isolates of multiple-drug-resistant M. tuberculosis and more than 20 species of mycobacteria other than M. tuberculosis and other GC-rich organisms. A single-tube heminested PCR protocol was devised to obtain sensitivity equal to those of an IS6110-based PCR assay and culture in spiked sputum experiments. The assay correctly identified 21 of 24 (87.5%) culture-positive specimens, 13 of which were acid-fast smear-negative, in a panel of 51 clinical specimens. Three specimens that were false-positive initially were negative upon repeat testing when the assay was modified to eliminate the potential for aerosol carryover of the first-round amplification product during the open-tube addition of the second set of reaction reagents. This assay is the most sensitive and specific test to date for the direct detection of M. tuberculosis rpoB in clinical specimens. This rapid PCR-based assay can be used for the simultaneous identification of M. tuberculosis and its rifampin susceptibility genotype.

Specific diagnostic test results generated by polymerase chain reaction (PCR) depend upon control of amplicon contamination in the clinical laboratory. We compared photochemical (isopsoralen [IP]) and enzymatic (uracil N-glycosylase [UNG]) methods for their ability to prevent carryover of amplicons generated from genomic targets of five viruses. PCR products (amplicons) (herpes simplex virus, 342 bp; cytomegalovirus, 250 bp; Epstein-Barr virus, 240 bp) exposed to UV light in the presence of various concentrations of IP compound 10 (IP-10) resulted in apparent increased molecular sizes of the products, as indicated by migration patterns after gel electrophoresis, and were predictive of inactivation by the agent. For amplicons of < or = 100 bp, IP-10-induced electrophoretic shifts were related to the guanidine-cytidine (G + C) content of the PCR product; no apparent shift and no inactivation were observed for a 92-bp herpes simplex virus amplicon (G + C content, 65%), whereas the 100-bp human papillomavirus product (G + C content, 42%) showed a concentration-dependent shift (25 to 100 micrograms/ml) in electrophoretic migration and was partially inactivated. UNG effectively controlled amplicon carryover for target DNA of > or = 240 bp; however, this treatment did not inactivate the two amplicons of < or = 100 bp, regardless of the G + C content of the product. Larger products were inactivated efficiently by both methods, regardless of their G + C contents. We concluded that both IP and UNG effectively inactivated PCR amplicons but not short amplicons of < or = 100 bp. We recommend that with the adoption of PCR technology in clinical laboratories, primers should be designed to produce amplicons of at least 240 to 350 bp (depending on G + C content) and that at least one effective method of controlling carryover contamination should be incorporated into each PCR protocol.

A multiplex PCR assay for detection of the staphylococcal mecA gene (the structural gene for penicillin-binding protein 2a) was compared with agar dilution and disk diffusion susceptibility test methods for identifying methicillin resistance. The multiplex PCR assay combined two primer sets (mecA and 16S rRNA) in a single reaction. A total of 500 staphylococcal isolates (228 isolates of Staphylococcus aureus and 272 isolates of coagulase-negative staphylococci) from clinical specimens were studied. For S. aureus, 40 of 40 mecA-positive isolates and 4 of 188 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 100 and 98%, respectively). In 3 of 4 discordant isolates, resistance was due to hyperproduction of beta-lactamase. For coagulase-negative staphylococci, 148 of 159 mecA-positive isolates and 0 of 113 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 93 and 100%, respectively). Twenty-six isolates were categorized as indeterminate because of the absence of a detectable 16S rRNA product. Four of these 26 isolates contained mecA when retested. The assay is designed to be incorporated into the work flow of the clinical microbiology laboratory and allows for the identification of intrinsic resistance in a timely and reliable manner.

Persistent infection with Borrelia burgdorferi in the presence of a vigorous host immune response has been demonstrated in humans and in animal models of Lyme disease. Long-term persistence of B. burgdorferi was documented recently in our studies of BALB/c and C3H mice infected with cloned and uncloned strains of B. burgdorferi. From mice inoculated with the cloned strain, 11 isolates were recovered from the skin, bladder, and blood after 1 year of infection. Analysis of the genes encoding the major outer surface proteins (OspA and OspB) by restriction digestion and DNA sequencing showed no evidence of point mutations or other small genetic alterations after 1 year. Genomic macrorestriction analysis of whole B. burgdorferi showed no loss or gross alterations of the plasmids encoding OspA, OspB, or OspC. However, in two isolates, loss of a 38-kb plasmid encoding outer surface protein D was noted. Our studies suggest that loss or alteration of the genes encoding OspA and OspB is not a common occurrence during persistent spirochetal infection and that other possible mechanisms, including invasion of immunologically privileged sites, should be actively explored.

ABSTRACT Evaluation of drug resistance by human immunodeficiency virus (HIV) genotyping has proven to be useful for the selection of drug combinations with maximum antiretroviral activity. We compared three genotyping methods for identification of mutations known to confer drug resistance in the reverse transcriptase (RT) and protease genes of HIV type 1 (HIV-1). The HIV-GenotypR method (GenotypR; Specialty Laboratories, Inc., Santa Monica, Calif.) with the ABI 377 DNA sequencer (Applied Biosystems Inc.), the HIV PRT GeneChip assay (GeneChip; Affymetrix, Santa Clara, Calif.), and the HIV-1 RT Line Probe Assay (LiPA; Innogenetics, Alpharetta, Ga.) were used to genotype plasma samples from HIV-infected patients attending the University of Wisconsin Hospitals and Clinics and the Mayo Clinic. At the time of analysis, patients were failing combination therapy ( n = 18) or were treatment naive ( n = 6). Forty codons of the RT and protease genes were analyzed by GenotypR and GeneChip for resistance-associated mutations. LiPA analyzed seven RT codons for mutations. Each sample was genotyped by all three assays, and each assay was subjected to pairwise comparisons. At least 92% of the codons tested (by the three assays) in paired comparisons were concordant. GenotypR and GeneChip demonstrated 96.6% concordance over the 40 codons tested. GenotypR identified slightly more mutations than GeneChip and LiPA; GeneChip identified all primary mutations that corresponded to failing treatment regimens. Each assay identified at least 84% of the mutations identified by the other assays. Mutations that were discordant between the assays mainly comprised secondary mutations and natural polymorphisms. The assays had better concordance for mutations that corresponded to current failing regimens, present in the more predominant viral quasispecies. In the treatment-naive patients, GenotypR, GeneChip, and LiPA mainly identified wild-type virus. Only the LiPA identified K70R, a possible transmitted zidovudine resistance mutation, in the RT gene of a treatment-naive patient. We conclude that although discrepancies in results exist between assays, each assay showed a similar capacity to identify potentially clinically relevant mutations related to patient treatment regimens.

ABSTRACT Identification of coryneform bacteria to the species level is important in certain circumstances for differentiating contamination and/or colonization from infection, which influences decisions regarding clinical intervention. However, methods currently used in clinical microbiology laboratories for the species identification of coryneform bacteria are often inadequate. We evaluated the MicroSeq 500 16S bacterial sequencing kit (Perkin-Elmer Biosystems, Foster City, Calif.), which is designed to sequence the first 527 bp of the 16S rRNA gene for bacterial identification, by using 52 coryneform gram-positive bacilli from clinical specimens isolated from January through June 1993 at the Mayo Clinic. Compared to conventional and supplemented phenotypic methods, MicroSeq provided concordant results for identification to the genus level for all isolates. At the species level, MicroSeq provided concordant results for 27 of 42 (64.3%) Corynebacterium isolates and 5 of 6 (83.3%) Corynebacterium -related isolates, respectively. Within the Corynebacterium genus, MicroSeq gave identical species-level identifications for the clinically significant Corynebacterium diphtheriae (4 of 4) and Corynebacterium jeikeium (8 of 8), but it identified only 50.0% (15 of 30) of other species ( P < 0.01). Four isolates from the genera Arthrobacter , Brevibacterium , and Microbacterium , which could not be identified to the species level by conventional methods, were assigned a species-level identification by MicroSeq. The total elapsed time for running a MicroSeq identification was 15.5 to 18.5 h. These data demonstrate that the MicroSeq 500 16S bacterial sequencing kit provides a potentially powerful method for the definitive identification of clinical coryneform bacterium isolates.

ABSTRACT The epidemiologic relatedness of methicillin-resistant Staphylococcus aureus (MRSA) isolates is currently determined by analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis (PFGE). We have evaluated an alternative typing system (MicroSeq StaphTrack Kit; Perkin-Elmer Biosystems) based on the sequence analysis of the chromosomally encoded polymorphic repeat X region of the S. aureus protein A ( spa ) gene. A total of 69 clinical MRSA isolates were divided into 18 groups according to the number and nucleotide sequences of the spa repeats. Molecular typing results obtained both by spa sequencing and from the PFGE patterns were concordant except for one group, which contained 20 isolates recovered over a 2-year period from hospitalized patients at the Mayo Clinic. Although the spa typing patterns were indistinguishable for those isolates, PFGE analysis yielded seven related but distinguishable patterns. Further coagulase gene sequence analysis subtyped those 20 strains into four groups which followed distinct temporal and geographic distributions. During a 2-year epidemic period there were up to 7 fragment changes in PFGE patterns among epidemiologically related isolates, suggesting that PFGE may be unsuitable for long-term typing of strains involved in epidemics. Although more limited than PFGE in discriminatory power, spa sequencing analysis could be used as a screening method for typing of MRSA strains because of the shorter turnaround time, ease of use, and the inherent advantages of sequence analysis, storage, and sharing of information.

ABSTRACT Ninety-nine clinical staphylococcal isolates (58 coagulase-negative Staphylococcus spp. [CoNS] and 41 Staphylococcus aureus isolates) were evaluated for susceptibility to oxacillin. The following susceptibility testing methods, media, and incubation conditions were studied: agar dilution by using Mueller-Hinton (MH) medium (Difco) supplemented with either 0, 2, or 4% NaCl and incubation at 30 or 35°C in ambient air for 24 or 48 h; disk diffusion by using commercially prepared MH medium (Difco) and MH II agar (BBL) and incubation at 35°C in ambient air for 24 or 48 h; and agar screen (spot or swab inoculation) by using commercially prepared agar (Remel) or MH agar (Difco) prepared in-house, each containing 4% NaCl and 6 μg of oxacillin/ml (0.6-μg/ml oxacillin was also studied with MH agar prepared in-house for the agar swab method and CoNS isolates) and incubation at 35°C in ambient air for 24 or 48 h for swab inoculation and at 30 or 35°C in ambient air for 24 or 48 h for spot inoculation. The results for these methods were compared to the results for mecA gene detection by a PCR method. Given the ability to support growth and the results for susceptibility testing (the breakpoint for susceptible isolates was ≤2 μg/ml), the best methods for CoNS isolates were (i) agar dilution by using MH medium supplemented with 4% NaCl and incubation at 35°C for 48 h (no growth failures were noted, and sensitivity was 97.6%) and (ii) agar screen (swab inoculation) by using MH medium prepared in-house supplemented with 4% NaCl and containing 0.6 μg oxacillin/ml and incubation at 35°C for 48 h (one isolate that did not carry the mecA gene did not grow, and the sensitivity was 100%). All but one (agar dilution without added NaCl and incubation at 30°C for 48 h) of the methods tested revealed all oxacillin-resistant S. aureus isolates, and no growth failures occurred with any method. If the breakpoint for susceptibility was lowered to ≤1 μg/ml for agar dilution methods, more CoNS isolates with oxacillin resistance related to the mecA gene were detected when 0 or 2% NaCl agar supplementation was used. Only one CoNS isolate with mecA gene-associated resistance was not detected by using agar dilution and MH medium supplemented with 4% NaCl with incubation for 48 h. When the breakpoint for susceptibility was decreased 10-fold (from 6.0 to 0.6 μg of oxacillin per ml) for the agar swab screen method, fully 100% of the CoNS isolates that carried the mecA gene were identified.

ABSTRACT Consistent with other members of the family Flaviviridae , hepatitis C virus (HCV) demonstrates a high degree of sequence variation throughout the coding regions of its genome. However, there is a high degree of sequence conservation found within the 5′ untranslated region (UTR) of the genome, making this region a target of choice for most nucleic acid amplification-based detection assays. In this study, the Amplicor HCV test, a commercially available assay which detects the 5′UTR, was used for the detection of HCV RNA in 669 serum samples obtained from a cohort of liver transplantation patients. Amplification products obtained from the HCV-positive cases were subjected to direct sequencing and genotyping based upon seven phylogenetically informative regions within the 5′UTR. Of the 669 specimens, 416 (62.2%) tested positive for the presence of HCV RNA. Of these, 372 (89.4%) specimens were successfully classified into 11 HCV genotypes and subtypes after computer-assisted analysis of the sequence data. Forty-four (10.6%) of the HCV RNA-positive specimens were not classifiable, the majority corresponding to low-titer specimens as determined by the Chiron Quantiplex HCV RNA 2.0 assay. Additional comparative studies targeting the NS-5 region of the viral genome generally confirmed the accuracy and sensitivity of the 5′UTR-based classifications, with the exception of the misclassification of a small number of type 1a cases as type 1b. We conclude that although the high sequence conservation within the 5′UTR results in the misclassification of a small number of HCV subtypes, the overall gains of efficiency, the shorter turnaround time, the inclusion of contamination control measures, and the low rate of test failure compared to that of methods based on the NS-5 gene together constitute significant advantages over other techniques.