The use of ELISA and nonradioactive DNA hybridization assays for the detection of human cytomegalovirus.

A hybridization assay using a biotinylated DNA probe was compared to both ELISA and direct isolation methods for detecting human cytomegalovirus (HCMV). The biotin labeled HCMV AD 169 HindIII-O-DNA fragment was used in a dot-blot assay to screen for the presence of HCMV in 186 urine specimens obtained from kidney transplant patients. The biotinylated HCMV HindIII-O probe could detect 3 log10 TCID50 units of HCMV. Urine specimens were also examined for the presence of HCMV by either ELISA or direct isolation of virus in tissue culture. The HindIII-O fragment detected 12 of 20 culture positive samples (sensitivity, 60%). There were 5 samples which were probe positive and cell culture negative (specificity, 97%). The ELISA assay also detected 12 of 20 culture positive samples (sensitivity, 60%). Eight samples were ELISA positive, cell culture negative (specificity, 95%). Seven specimens were positive by all three criteria. Five specimens which were both ELISA positive and probe positive were cell culture negative. The ELISA positive, probe positive, culture negative specimens originated from patients who gave a culture positive specimen within 10 days of the original sample. The combination of probe and ELISA assays detected 16 of the 20 culture positive specimens (sensitivity, 80%). The combined use of biotinylated DNA probes and ELISA allows the detection of HCMV in urine specimens with good sensitivity and specificity.

Tuberculosis screening in the federal prison system: an opportunity to treat and prevent tuberculosis in foreign-born populations.

OBJECTIVES: Despite recent success in tuberculosis (TB) control efforts in the United States, marked declines in TB case rates have not been observed in foreign-born populations. Because foreign-born populations are becoming more important for targeted national TB control efforts, the Federal Bureau of Prisons (BOP) reviewed inmate medical data to evaluate risk factors associated with Mycobacterium tuberculosis infection and active TB disease. To improve screening strategies, BOP implemented chest radiograph screening for all inmates entering a federal detention center in San Diego, California. METHODS: Tuberculin skin test (TST) data from an approximated intake cohort of inmates entering the system from February 1 to June 30, 1999, were analyzed to assess risk factors for M. tuberculosis infection among inmates entering federal prisons. The most recent case reports of inmates diagnosed with TB disease were reviewed. All inmates entering a San Diego detention facility from July 1 to December 31, 1998, were screened for TB by symptom review, TST, and chest radiographs. RESULTS: System-wide, foreign-born inmates were 5.9 times more likely to have a positive TST than US-born inmates, and accounted for 60% of recently diagnosed TB cases. Chest radiograph screening of all inmates entering the San Diego facility reduced exposure time to active TB cases by 75%, but TB incidence remained unchanged. CONCLUSIONS: The high prevalence of M. tuberculosis infection and TB disease among foreign-born inmates entering the federal prison system presents a strategic opportunity to provide preventive therapy to a high-risk population and to identify contagious cases that might elude traditional public health efforts. Universal chest radiograph screening was no more sensitive than TST for detecting active TB cases among newly incarcerated foreign-born inmates with a high prevalence of TB infection, but the screening reduced potential TB exposures through rapid identification of contagious cases.

Detection of enterotoxigenic Escherichia coli after polymerase chain reaction amplification with a thermostable DNA polymerase.

The direct identification of enterotoxigenic Escherichia coli from clinical specimens was examined by using the polymerase chain reaction (PCR) for amplifying the heat-labile toxin (LT) gene. Two synthetic primers, each of which was 20 bases in length, were used with the thermostable DNA polymerase from Thermus aquaticus to amplify the LT gene. The amplified PCR products were detected by either gel electrophoresis or hybridization to a 24-base synthetic oligonucleotide probe conjugated to alkaline phosphatase. The PCR method detected LT-positive bacteria but did not react with E. coli producing the heat-stable toxin, enteroinvasive E. coli, Salmonella typhi, Salmonella typhimurium, or Shigella dysenteriae. By the PCR method, a single bacterium could be detected following 30 cycles of amplification. The T. aquaticus DNA polymerase was inhibited by more than 10(3) organisms in the amplification reaction mixture. A group of 40 clinical specimens consisting of 16 LT bioassay-positive and 24 LT bioassay-negative stool specimens were tested by PCR for the presence of toxigenic E. coli. The total DNA from 100 microliters of stool specimen was extracted and partially purified with a commercially available ion-exchange column. All 16 of the bioassay-positive stool specimens were positive by PCR. In addition, one stool specimen which was bioassay negative for LT but positive for LT in a previous hybridization assay with a different LT probe was also positive by PCR. This may indicate that the LT gene is present but either is not expressed or is expressed below detectable levels. Amplification of specific DNA sequences by PCR provides a highly sensitive and specific tool for the detection of pathogenic microorganisms directly from clinical specimens without the need for prior isolation. This technique may find wide application in the detection of other organisms in addition to enterotoxigenic E.coli.

Detection of human rotavirus by using an alkaline phosphatase-conjugated synthetic DNA probe in comparison with enzyme-linked immunoassay and polyacrylamide gel analysis.

An alkaline phosphatase-conjugated synthetic oligodeoxyribonucleotide probe was compared with polyacrylamide gel electrophoresis (PAGE) detection of rotavirus RNA as well as an enzyme-linked immunosorbent assay (ELISA) for the detection of rotavirus in stools from young children with gastroenteritis. The synthetic probe did not cross-react with bacterial causative agents of diarrheal disease. Extraction of viral RNA from stool samples with a phenol-chloroform mixture was suitable for most samples. In some cases fecal pigments interfered with the reaction of the probe with viral RNA. The use of ion-exchange chromatography to further purify viral RNA removed contaminating pigments and increased the sensitivity of the probe assay. Of 260 stool specimens, 77 (30%) were positive for rotavirus when tested by PAGE analysis of rotavirus RNA. The synthetic probe identified 71 rotavirus specimens when RNA obtained by phenol-chloroform extraction followed by chromatographic purification was used (sensitivity, 91.0%; specificity, 96.7%). The ELISA results also agreed well with the electrophoretic analysis (sensitivity, 98.7%; specificity 94%) and the probe assay (sensitivity, 90%; specificity, 100%). Discordant results between the ELISA and the probe assay were examined further by electron microscopy and PAGE analysis of viral RNA. The positive and negative predictive values of the probe assay in comparison with PAGE were 92.2 and 96.1%, respectively. Rotaviruses showing both long and short RNA electrophoretic patterns were detected by the probe. The probe assay coupled with chromatographic purification of rotavirus RNA is an effective method for detecting rotavirus and compares favorably with PAGE analysis and ELISA.

Molecular cloning and nucleic acid sequencing of Chlamydia trachomatis 16S rRNA genes from patient samples lacking the cryptic plasmid.

We have examined the relationship between Chlamydia trachomatis found in clinical samples in which the cryptic plasmid was absent and known serovars of C. trachomatis. PCR and RNase protection assays were used to compare 12 C. trachomatis serovars and a plasmidless L2 serovar strain with the reactivity of clinical specimens taken from patients with pelvic inflammatory disease (PID) containing the C. trachomatis 16S rRNA gene and 16S rRNA but lacking plasmid DNA. Serovars D, E, H and I were unreactive in either or both of the PCR and RNase protection assays. The plasmidless L2 strain had reactivities indistinguishable from the nucleic acids found in the PID clinical specimens. Serovar D, the plasmidless L2 strain, and nucleic acids from two of the PID specimens were further compared by amplifying, cloning and sequencing the 16S rRNA genes detected in these samples. The sequences of the 16S rRNA genes detected in the PID clinical samples and the 16S rRNA gene of the plasmidless C. trachomatis variant were indistinguishable from previously reported sequences of the C. trachomatis 16S rRNA. Serovar D showed five base changes over the same region. We conclude that although these clinical samples lack the C. trachomatis cryptic plasmid, they do contain C. trachomatis nucleic acid.

Identification of enterotoxigenic Escherichia coli using alkaline phosphatase-labeled synthetic oligodeoxyribonucleotide probes.

Alkaline phosphatase-conjugated synthetic oligodeoxyribonucleic acid probes were used to detect enterotoxigenic Escherichia coli strains containing either the heat stable or heat labile toxin genes. Both of the synthetic probes detected as little as 5 ng of purified plasmid DNA bearing the appropriate toxin gene. In addition, both probes could detect 5 X 10(6) toxigenic bacteria by colony hybridisation. No cross reactivity was observed between probes. When 197 clinical isolates of Escherichia coli were examined for toxigenicity using bioassays, 13 heat stable and 17 heat labile toxin strains were identified. Of the 13 heat stable toxin strains, 12 were positive using the heat stable toxin synthetic probe (sensitivity, 92%; specificity, 98%) while 16 of 17 bioassay heat labile toxin positive samples were identified using the heat labile toxin synthetic probe (sensitivity, 94%; specificity, 97%). Alkaline phosphatase-conjugated synthetic probes with high sensitivity and specificity should provide a rapid means of identifying toxigenic Escherichia coli.

Polymerase chain reaction assay for detection of human cytomegalovirus.

Direct detection of human cytomegalovirus (HCMV) from clinical specimens was examined by using the polymerase chain reaction (PCR) for amplifying HCMV DNA. The efficiency of the amplification reaction was examined by using three different buffers and concentrations of deoxynucleotide triphosphates. The PCR assay was most efficient with a reaction mixture containing 17 mM ammonium sulfate, 67 mM Tris hydrochloride (pH 8.5), 7 mM MgCl2, 10 mM 2-mercaptoethanol, 170 micrograms of bovine serum albumin per ml, and each deoxynucleotide triphosphate at a final concentration of 1.5 mM. After 35 cycles of amplification, 0.15 fg of a plasmid containing the cloned target gene (corresponding to approximately six gene copies) was detected. The PCR assay correctly identified all of 24 clinical isolates of HCMV. Virus in urine specimens could be disrupted by heating at 93 degrees C for 30 min. The viral DNA was amplified directly from 5 microliters of preheated urine, with no further treatment before amplification. We tested the PCR assay on urine specimens from patients who had undergone renal transplantation that had been screened for the presence of HCMV by enzyme-linked immunosorbent assay, hybridization assay, and direct virus isolation. Specimens that were positive by one or more of these assays were screened by PCR. HCMV was consistently detected by PCR in all specimens that were positive by at least one other test. No cross-reactivity to other herpesviruses or MRC-5 cellular DNA was observed.

Detection of toxigenic Escherichia coli using biotin-labelled DNA probes following enzymatic amplification of the heat labile toxin gene.

Several types of DNA probes labelled with biotin were compared for their sensitivity to detect the heat labile toxin (LT) gene in toxigenic Escherichia coli. In addition, a procedure was developed for enzymatically amplifying LT gene sequences in toxigenic E. coli. Probes were labelled with biotinylated nucleotides by either nick translation; 3' tailing; primer extension of probe DNA cloned into bacteriophage M13; sandwich hybridization; or oligolabelling of isolated DNA fragments. A single stranded probe consisting of a DNA fragment from the LT gene cloned into the bacteriophage M13mp18 and detected by hybridization to oligolabelled biotinylated M13mp18 RF DNA in a sandwich hybridization was able to detect as little as 10 pg of toxin gene DNA. Cloned LT gene DNA was serially diluted and amplified enzymatically using synthetic oligonucleotide primers. Amplified DNA was detected using biotin-labelled M13-based probes. As little as 1 fg of LT DNA could be amplified to detectable levels by this method. Experiments with LT+ bacteria resulted in the detection of as few as 1000 bacteria. The combination of enzymatic amplification coupled with M13-based DNA probes provides a highly sensitive tool for detecting pathogenic microorganisms.

Use of an alkaline phosphatase-labeled synthetic oligonucleotide probe for detection of Campylobacter jejuni and Campylobacter coli.

A commercially available synthetic nucleic acid probe (SNAP) conjugated to alkaline phosphatase was compared with standard culture techniques for detecting Campylobacter species. The SNAP was able to detect either 5 ng of C. jejuni DNA or 10(5) CFU of bacteria. The SNAP could also detect DNA extracted from 10(5) CFU in mock-infected stool samples. The SNAP detected C. jejuni and C. coli but showed no reactivity with C. laridis, C. fetus subsp. fetus, C. fetus subsp. venerealis, C. fennelliae, "C. upsaliensis," C. cinaedii, C. fecalis, C. hyointestinalis, C. mucosalis, or Helicobacter (Campylobacter) pylori. The SNAP also showed no cross-reactivity with other enteric pathogens. When applied to pure cultures, the SNAP detected 55 clinical isolates of C. jejuni and 11 clinical isolates of C. coli, with an accuracy of 100%. When applied directly to clinical specimens, the SNAP detected Campylobacter spp. in 19 of 23 culture-positive stool specimens (sensitivity, 82.6%; specificity, 100%). Pure cultures of the Campylobacter strains isolated from the four probe-negative, culture-positive stool specimens gave positive reactions with the SNAP. While the SNAP had excellent sensitivity and specificity for isolated bacterial colony isolates, the main limitation to the Campylobacter probe detection kit may be the sensitivity limit on direct detection of Campylobacter organisms in stools.

Direct detection of human cytomegalovirus in urine specimens from renal transplant patients following polymerase chain reaction amplification.

A polymerase chain reaction (PCR) assay was used to amplify human cytomegalovirus (HCMV) directly from urine specimens taken from renal transplant patients. In serial urine samples from patients who had at least one specimen positive for HCMV; the PCR assay consistently detected the presence of HCMV DNA sequences, whereas virus detection by other tests such as enzyme-linked immunosorbent assay (ELISA), nonradioactive DNA hybridization assay, and virus isolation were variable. Of 37 specimens positive by PCR, 36 were positive by either ELISA, hybridization assay, or virus isolation. Infectious virus was detected in 13 of the 37 PCR-positive urines. HCMV DNA was detected by PCR in all samples that were positive for HCMV by either hybridization assay or virus isolation. The viral genome copy number was determined by PCR assay for several urine samples that were positive by virus isolation but negative for HCMV by ELISA or hybridization assay. Viral genome copy number estimates indicated the presence of HCMV at very low levels in these urines verifying the fidelity of the virus isolation procedures. The consistency of the PCR assay makes it an ideal method for detection of infection and monitoring antiviral drug therapy in patients infected with HCMV.

The human cytomegalovirus immediate early enhancer-promoter is responsive to activation by the adenovirus-5 13S E1A gene.

The stimulatory effects of the 13S adenovirus E1A gene product on the human cytomegalovirus (HCMV) major immediate early (IE) enhancer were examined. Chimeric plasmids containing cloned portions of the HCMV major IE enhancer-promoter positioned upstream of the chloramphenicol acetyltransferase gene (cat) were cotransfected into HeLa cells with the plasmid p13S-wt which contained a cDNA encoding the adenovirus 13S E1A gene product. CAT expression from chimeric plasmids containing at least one copy of the HCMV 19 base pair (bp) repetitive motif was stimulated 10-fold in the presence of p13S-wt. The 19-bp motif contains a potential binding site for the cellular transcription factor ATF/CREB. Deletion analysis indicated that the ATF/CREB site was crucial for E1A-mediated stimulation. Insertion of a synthetic oligonucleotide homologous to a 19-bp motif and containing an ATF/CREB binding site into an HCMV chimera lacking ATF/CREB motifs conferred E1A responsivity on HCMV promoter-mediated CAT expression whereas insertion of a similar oligonucleotide containing a change of two bases in the sequence of the ATF/CREB site did not. Measurement of CAT-specific RNA verified the results of the CAT enzyme experiments. The ATF/CREB motif may be a target for stimulation of HCMV gene expression through either viral or cellular transcription factors.

Molecular epidemiology of human rotavirus infections based on genome segment variations in viral strains.

Molecular epidemiology of rotavirus infections in 621 hospitalized children was investigated by analysis of migration patterns of viral genomic ribonucleic acid (RNA) segments by electrophoresis in polyacrylamide gels. Based on migration patterns of RNA segments of 184 rotavirus strains, seven different electropherotypes were identified: 146 (79.3%) strains were "long," and 38 (20.7%) were "short" electropherotypes; 61% belonged to a single dominant "long" electropherotype, which persisted throughout the 15-month period of study, whereas six other cocirculating types appeared at varying intervals. Electrophoretic migration patterns of RNA from viral isolates of two patients suggested mixed infections with different rotaviruses. There was a lack of correlation between the electrophoretic migration of segments 10 and 11 and serologically defined subgroup specificity in three of the rotavirus strains. Rotavirus infections and different electropherotypes were observed throughout the year.

Territorial limits and functional anatomy of the simian virus 40 replication origin.

The region at and near the simian virus 40 (SV40) DNA replication origin contains a series of palindromes, a 17-base pair (bp) A + T-rich sequence, three copies of a 21-bp repeat, and two copies of a 72-bp repeat. We have constructed a series of recombinant plasmids containing sequential deletions at the region of SV40 DNA replication origin starting from the end near the repeats. These deletions were introduced by using in vitro and in vivo techniques. The relative replication efficiency of these recombinant plasmids were directly assayed in COS-1 monkey kidney cells capable of providing the tumor antigen necessary for the replication of these molecules. Recombinants lacking both copies of the 72-bp repeat did not exhibit any reduction in replication efficiency. Recombinants lacking the 21-bp repeats showed decreased replication efficiency; the reduction in replication efficiency was proportional to the number of copies of the 21-bp repeat deleted in these recombinants. A recombinant retaining the palindromes at the region of SV40 DNA replication but lacking the A + T-rich sequence and the repeats failed to replicate. Based on these results, the SV40 DNA replication origin is subdivided into two regions, and their boundaries are defined. One of these two regions is a core region containing the 17-bp, 15-bp, and 27-bp palindromes and, quite likely, the 17-bp A + T-rich sequence which are necessary for replication. The other is an auxiliary region that consists of the 21-bp repeats and has a dose-dependent enhancement effect on replication efficiency.

Determination of hepatitis C virus genotypes in the United States by cleavase fragment length polymorphism analysis.

We describe the application of a new DNA-scanning method, which has been termed Cleavase Fragment Length Polymorphism (CFLP; Third Wave Technologies, Inc., Madison, Wis.), for the determination of the genotype of hepatitis C virus (HCV). CFLP analysis results in the generation of structural fingerprints that allow discrimination of different DNA sequences. We analyzed 251-bp cDNA products generated by reverse transcription-PCR of the well-conserved 5'-noncoding region of HCV. We determined the genotypes of 87 samples by DNA sequencing and found isolates representing 98% of the types typically encountered in the United States, i.e., types 1a, 1b, 2a/c, 2b, 3a, and 4. Blinded CFLP analysis of these samples was 100% concordant with DNA sequencing results, such that closely related genotypes yielded patterns with strong familial resemblance whereas more divergent sequences yielded patterns with pronounced dissimilarities. In each case, the aggregate pattern was indicative of genotypic grouping, while finer changes suggested subgenotypic differences. We also assessed the reproducibility of CFLP analysis in HCV genotyping by analyzing three distinct isolates belonging to a single subtype. These three isolates yielded indistinguishable CFLP patterns, as did replicate analysis of a single isolate. This study demonstrates the suitability of this technology for HCV genotyping and suggests that it may provide a low-cost, high-throughput alternative to DNA sequencing or other, more costly or cumbersome genotyping approaches.

Detection and differentiation of picornaviruses in clinical samples following genomic amplification.

A polymerase chain reaction (PCR) assay was used to detect and differentiate picornaviruses (PVs), using primers homologous to the 5' non-coding and VP2 regions of the PV genome. The PCR resulted in a 530 bp PCR product for human rhinoviruses (HRVs) and a 650 bp product for polioviruses, coxsackieviruses (CV) or echoviruses. The PCR assay could detect as little as 1 p.f.u. of virus in either cerebrospinal fluid (CSF) or stool, using ethidium bromide-stained gels. Standard strains of poliovirus, CV, echovirus and HRV were detected, with the exception of echovirus type 22. In contrast, heterologous viruses, such as herpes simplex virus, human cytomegalovirus, adenovirus, influenza virus and rotavirus, as well as human and monkey cell DNA, were not amplified. In nasal swabs taken from patients with respiratory infections, the PCR detected 27 of 28 HRV isolation-positive specimens. All specimens from which viruses other than HRVs were isolated were negative by PCR. The PCR definitively identified poliovirus and CVs from the CSF or stool of patients with aseptic meningitis, as well as CV in the pericardial fluid of a patient who had suffered a myocardial infarction. Specimens taken from patients with similar pathologies, and from which heterologous viruses were isolated, were uniformly negative by PCR.

Q-beta replicase-amplified assay for detection of Mycobacterium tuberculosis directly from clinical specimens.

We report the results of a study conducted to evaluate the performance of manual Q-Beta replicase-amplified Mycobacterium tuberculosis complex assay compared with that of culture for detecting M. tuberculosis directly from digested sputum pellets. A total of 261 specimens submitted to three tuberculosis testing laboratories were analyzed. Culture and acid-fast bacillus smear results were provided by the tuberculosis testing laboratories. Of these 261 specimens, 34 (13% prevalence rate) were positive for M. tuberculosis by culture. The samples were digested and decontaminated by the testing laboratories by using their standard digestion and decontamination procedures. An aliquot of the digested and decontaminated pellet was sent to GENE-TRAK. The digested and decontaminated pellet was neutralized by washing it with 0.067 M phosphate buffer (pH 6.8), and the bacteria present in the washed pellet were heat inactivated at 100 degrees C for 15 min. The samples were combined with sample processing buffer containing GuSCN and were treated for 6 min in the GENE-TRAK Sample Processing Instrument to release the nucleic acids. The release rRNA was analyzed in a manual Q-Beta replicase assay format which incorporates elements of sandwich hybridization, reversible target capture, and Q-beta replicase signal amplification technologies. In comparison with culture, the overall assay sensitivity and specificity were 97.1 and 96.5%, respectively. The positive predictive value was 80.5%, and the negative predictive value was 99.5%. After analysis of discrepant results, the assay sensitivity and specificity were 97.3 and 97.8, respectively, and the prevalence rate was 14%. The positive predictive value and the negative predictive value were 87.8 and 99.5%, respectively. The Q-Beta replicase assay is rapid sensitive, semiquantitative, and specific for the direct detection of M. tuberculosis from clinical specimens.

Detection of Mycobacterium tuberculosis directly from spiked human sputum by Q-beta replicase-amplified assay.

We report on a rapid, sensitive, Q-Beta replicase-amplified nucleic acid hybridization assay for the detection of Mycobacterium tuberculosis directly from spiked human sputum. Specimens were processed by either an N-acetyl-L-cysteine-NaOH or a 2% NaOH digestion-decontamination method and then washed to neutralize the pH of the cell pellet. The washed sputum pellets were heated at 100 degrees C to inactivate the M. tuberculosis organisms. The heat-inactivated samples were mechanically lysed at 5,000 rpm for 6 min in the GENE-TRAK Sample Processing Instrument in the presence of zirconium oxide beads and a buffer containing guanidine thiocyanate. The released nucleic acid was subjected to the GENE-TRAK Q-Beta replicase-amplified, dual-capture assay. The assay sensitivity was 10(3) purified rRNA targets or 1 CFU of M. tuberculosis spiked into M. tuberculosis-negative human sputum. There was a low level of noise because of the limitations of performing a signal amplification assay in an open system. High levels of other mycobacterial rRNA (approximately 10(7) organisms), including rRNAs of Mycobacterium avium and Mycobacterium gordonae, did not interfere with the sensitivity of the assay.

Comparison of amplified Q beta replicase and PCR assays for detection of Mycobacterium tuberculosis.

Because of the long time required to isolate Mycobacterium tuberculosis in culture, there is an acute need for simple rapid methods for direct detection of M. tuberculosis from human sputum specimens. We have developed and characterized quantitative manual Q beta replicase and PCR assays for M. tuberculosis. The Q beta replicase assay was based on reversible target capture of M. tuberculosis 23S rRNA followed by amplification of a replicatable detector probe with Q beta replicase. For PCR assays, primers generating a 370-bp amplification product from the IS6110 insertion element were used in combination with a control plasmid containing an internal deletion in the IS6110 amplicon. Serial dilutions of M. tuberculosis were spiked into sputum and subjected to digestion and decontamination with N-acetyl-L-cysteine and NaOH. Assay conditions were optimized for hybridization and sample processing chemistries in order to maximize sample utilization. Following assay optimization, the sensitivities of the Q beta replicase and PCR assays of spiked sputum samples were 0.5 and 5.0 CFU per assay reaction, respectively. The effects of sputum matrix on each assay were examined by testing 20 patient sputum samples which had been cultured for M. tuberculosis. The culture-positive samples included smear-positive and smear-negative samples. The results of the Q beta replicase assay were not inhibited by sputum and were in 100% agreement with those of culture, including detection of 10 culture-positive specimens. However, using an internal control plasmid coamplified with each PCR as an indicator, we detected PCR inhibition in 9 of 20 samples tested. Decreasing the amount of sample assayed in the PCR 24-fold alleviated the inhibitory effects in all but two specimens, one of which was culture positive. The decreased sample utilization also resulted in a false-negative result with a third specimen which was culture positive for M. tuberculosis. Quantitative smear results and QB replicase assay estimates of the number of organisms present in these specimens were in close agreement. The QB replicase assay performed well in comparison with both culture and PCR and should offer a rapid means for detecting and controlling infection due to M. tuberculosis.

Infection with a plasmid-free variant Chlamydia related to Chlamydia trachomatis identified by using multiple assays for nucleic acid detection.

Clinical samples in transport media from 40 patients exhibiting pathologies potentially caused by Chlamydia trachomatis infection were analyzed for chlamydial nucleic acid, and the results were compared with those of culture. Chlamydial culture was performed by a shell vial centrifugation method with HeLa 229 host cells. Polymerase chain reaction (PCR) assays were used to detect either regions on a 7.5-kb plasmid characteristic of C. trachomatis (plasmid-PCR) or a segment of the 16S rRNA genes (rRNA-PCR). All PCR results were confirmed by hybridization with probes for the specific amplified products in either a Southern or a dot blot format. An RNase protection (RNP) assay was used to detect genus-specific chlamydial 16S rRNA directly from the clinical samples. The PCR assays detected C. trachomatis but not other bacteria, including Chlamydia spp. C. trachomatis was isolated from six samples which were positive by the rDNA-PCR and plasmid-PCR assays. Five of the culture-positive specimens were positive by the RNP assay. Twenty-two samples were negative by all criteria. Surprisingly, nine samples were positive by rRNA-PCR and RNP assays only. Nucleic acid sequencing of the rRNA-PCR-amplified products indicated a close relationship between the variants and C. trachomatis. The data may indicate an unrecognized process in C. trachomatis infection or that these patients were infected by a variant strain of C. trachomatis which lacks the C. trachomatis-specific plasmid.