M. D. Cave

Association between Mycobacterium tuberculosis Beijing/W Lineage Strain Infection and Extrathoracic Tuberculosis: Insights from Epidemiologic and Clinical Characterization of the Three Principal Genetic Groups of M. tuberculosis Clinical Isolates

ABSTRACT Clinical strains of Mycobacterium tuberculosis can be divided into three principal genetic groups based on the single-nucleotide polymorphisms at the katG gene codon 463 and the gyrA gene codon 95. One subgroup of genetic group 1, the Beijing/W lineage, has been widely studied because of its worldwide distribution and association with outbreaks. In order to increase our understanding of the clinical and epidemiological relevance of the genetic grouping of M. tuberculosis clinical strains and the Beijing/W lineage, we investigated the genetic grouping of 679 clinical isolates of M. tuberculosis, representing 96.3% of culture-confirmed tuberculosis cases diagnosed in Arkansas between January 1996 and December 2000 using PCR and DNA sequencing. We assessed the associations of infections by different genetic groups of M. tuberculosis strains and infection by the Beijing/W lineage strains with the clinical and epidemiological characteristics of the patients using chi-square tests and multivariate logistic regression analysis. Of the 679 study isolates, 676 fell into one of the three principal genetic groups, with 63 (9.3%) in group 1, 438 (64.8%) in group 2, and 175 (25.9%) in group 3. After adjusting for potential confounding of age, gender, race/ethnicity, human immunodeficiency virus serostatus, and plcD genotype in a multivariate logistic regression model, patients infected by the Beijing/W lineage isolates were nearly three times as likely as patients infected with the non-Beijing/W lineage isolates to have an extrathoracic involvement (odds ratio [95% confidence interval], 2.85 [1.33, 6.12]). Thus, the Beijing/W lineage strains may have some special biological features that facilitate the development of extrathoracic tuberculosis.

Detection of Viable Mycobacterium tuberculosis by Reverse Transcriptase-Strand Displacement Amplification of mRNA

Numerous assays have been described for the detection of DNA and rRNA sequences that are specific for the Mycobacterium tuberculosis complex. Although beneficial to initial diagnosis, such assays have proven unsuitable for monitoring therapeutic efficacy owing to the persistence of these nucleic acid targets long after conversion of smears and cultures to negative. However, prokaryotic mRNA has a typical half-life of only a few minutes and we have previously shown that the presence of mRNA is a good indicator of bacterial viability. The purpose of the present study was to develop a novel reverse transcriptase-strand displacement amplification system for the detection of M. tuberculosis alpha-antigen (85B protein) mRNA and to demonstrate the use of this assay in assessing chemotherapeutic efficacy in patients with pulmonary tuberculosis. The assay was applied to sequential, noninduced sputum specimens collected from four patients: 10 of 11 samples (91%) collected prior to the start of therapy were positive for alpha-antigen mRNA, compared with 1 of 8 (13%), 2 of 8 (25%), 2 of 8 (25%), and 0 of 8 collected on days 2, 4, 7, and 14 of treatment, respectively. In contrast, 39 of 44 samples (89%) collected on or before day 14 were positive for alpha-antigen DNA. The loss of detectable mRNA corresponded to a rapid drop over the first 4 days of treatment in the number of viable organisms present in each sputum sample, equivalent to a mean fall of 0.43 log10 CFU/ml/day. Analysis of mRNA is a potentially useful method for monitoring therapeutic efficacy and for rapid in vitro determination of drug susceptibility.

Differences in the prevalence of IS6110 insertion sites in Mycobacterium tuberculosis strains: low and high copy number of IS6110

SETTING Mycobacterium tuberculosis (M. tuberculosis) isolates from various parts of the USA which have few copies of the insertion sequence IS6110. OBJECTIVES To characterize the sites of insertion of IS6110 among M. tuberculosis isolates that have one to six copies of the insertion sequence. DESIGN The mixed-linker polymerase chain reaction (ML-PCR) procedure was used to amplify the terminal repeats on the ends of IS6110 and adjacent flanking sequences. From the ML-PCR products, sequences flanking 14 copies of IS6110 in strains containing less than seven copies of the insertion were determined. Sequence information from the flanking deoxyribonucleic acid was used to construct flanking primers that can be used to indicate the presence of IS6110 at a particular site when paired with outbound IS6110 primers in a PCR. Over 200 strains of diverse origin were screened for the insertion of IS6110 at several distinct sites using this procedure. RESULTS The direct repeat (DR) locus has been described as a highly preferred site for insertion of IS6110 in strains of M. tuberculosis. Another highly preferred site of insertion of IS6100, DK1, is herein described. Insertions at DK1 are highly prevalent in M. tuberculosis strains harboring two to six copies of IS6110. The prevalence of insertions at this site decreases in strains with more than six copies of IS6110, even though the sequence itself is present in strains lacking a copy of IS6110 at this site. CONCLUSION In addition to the DR locus there are other conserved sites of insertion among M. tuberculosis strains. The data further suggest a separate lineage for the high copy and the low copy strains, and a possible sequential insertion of IS6110 in strains of M. tuberculosis with less than seven copies.

Population-Based Study of Deletions in Five Different Genomic Regions of Mycobacterium tuberculosis and Possible Clinical Relevance of the Deletions

ABSTRACT Regions of difference (RDs) have been described in clinical isolates of Mycobacterium tuberculosis, but the potential epidemiological and clinical relevance of the genotypes of these RDs remains to be investigated. We screened a population-based sample of 648 isolates for the deletion of five RDs, designated RD105, RD181, RD142, RD150, and RD239, using microarray-based hybridization, PCR, and DNA sequencing and assessed the associations between the RD deletions and the clinical characteristics of the patients using chi-square analysis and multivariate logistic regression model. Of the 648 isolates, 18 (2.8%) had the RD239 deletion and 39 (6.0%) had the RD105 deletion. The deletions of RD142, RD150, and RD181 subdivided the isolates with the RD105 deletion into four groups comprising a group with concurrent deletions of RD105, RD181, and RD142 (n = 13); a group with concurrent deletions of RD105, RD181, and RD150 (n = 5); a group with concurrent deletions of RD105 and RD181 (n = 13); and a group with a deletion of RD105 only (n = 8). Extrathoracic tuberculosis is statistically significantly associated with infection with the isolates with concurrent deletions of RD105, RD181, and RD142 (adjusted odds ratio [OR] = 3.05; 95% confidence interval [CI] = 1.58, 5.90) and the isolates with concurrent deletions of RD105, RD181, and RD150 (adjusted OR = 11.09; 95% CI = 4.27, 28.80), after controlling for the previously identified risk factors for extrathoracic tuberculosis (human immunodeficiency virus serostatus, race, gender, and the genotype of the plcD gene). These two combinations of RD deletions have the potential for predicting the clinical presentation of M. tuberculosis infection in the human host.

Distribution of Insertion- and Deletion-Associated Genetic Polymorphisms among Four Mycobacterium tuberculosis Phospholipase C Genes and Associations with Extrathoracic Tuberculosis: a Population-Based Study

ABSTRACT The Mycobacterium tuberculosis genome contains four phospholipase C (PLC)-encoding genes, designated plcA, plcB, plcC, and plcD, respectively. Each of the four genes contributes to the overall PLC activity of M. tuberculosis. PLC is hypothesized to contribute to M. tuberculosis virulence. Infection of M. tuberculosis strains carrying a truncated plcD gene is associated with the occurrence of extrathoracic tuberculosis. However, whether the other three plc genes are also associated with extrathoracic tuberculosis remains to be assessed. We investigated the insertion- and deletion-associated genetic diversity in all four plc genes among 682 epidemiologically and clinically well-characterized M. tuberculosis clinical isolates using PCR, DNA sequencing, and Southern hybridization. Two hundred sixty-six (39%) of the 682 isolates had an interruption in at least one of the four plc genes, most often associated with an IS6110 insertion. The plcD gene interruption was the most common: it was observed in 233 (34%) of the isolates, compared to 4.7%, 4.1%, and 5.9% for plcA, plcB, and plcC gene interruption, respectively. The association between the plc gene genotypes and disease presentation was adjusted for clustering using generalized estimating equations for both bivariate and multivariate analyses. After controlling for the genotypes of the plcABC genes and the host-related risk factors, interruption in the plcD gene remained significantly associated with extrathoracic tuberculosis (odds ratio, 3.27; 95% confidence interval, 1.32 to 8.14). The data suggest that the plcD gene might play a more important role in the pathogenesis of thoracic TB than it does in the pathogenesis of extrathoracic TB.

Epidemiologic Import of Tuberculosis Cases Whose Isolates Have Similar but Not Identical IS6110 Restriction Fragment Length Polymorphism Patterns

ABSTRACT Isolates of Mycobacterium tuberculosis from patients with epidemiologic links frequently demonstrate identical IS6110 restriction fragment length polymorphism (RFLP) patterns (i.e., RFLP clustering) because they are infected with the same strain. Uncertainty arises with isolates that differ from one another by a few IS6110 hybridizing bands. During the period from 1 January 1996 to 31 December 1999, isolates from 585 tuberculosis (TB) cases were analyzed by RFLP, representing 98.2% of the 596 culture-positive TB cases reported in Arkansas during the study period. Of the 585 cases for which RFLP was available, 419 (71.6%) had an RFLP pattern with more than five copies of IS6110. Of the total 74 clusters, 48 comprised isolates with more than five copies of IS6110 and included 164 cases. Sixty-nine isolates with more than five copies of IS6110 comprising 16 clusters and 60 unique isolates were found to be similar to at least 1 other isolate (differing from it by one or two hybridizing bands). Among the 129 cases whose isolates were similar to other clustered or unique isolates, 16 cases were discovered with epidemiologic links: 14 (15.2%) were among the 92 cases with IS6110 RFLP patterns similar to those in clusters, and 2 (5.2%) were among the 37 unique cases that were similar to another unique case. The isolates from the epidemiologically linked patients shared common spoligotypes; all except one case shared common polymorphic GC-rich sequence (PGRS) patterns. Of the 129 patients whose isolates differed from another by one or two hybridizing IS6110 bands, 101 (78.3%) shared common spoligotypes and 87 (67.4%) shared common PGRS RFLP patterns.

Secondary typing of Mycobacterium tuberculosis isolates with matching IS6110 fingerprints from different geographic regions of the United States.

ABSTRACT Fifty-nine isolates of Mycobacterium tuberculosisobtained from different states in the United States and representing 25 interstate clusters were investigated. These clusters were identified by computer-assisted analysis of DNA fingerprints submitted during 1996 and 1997 by different laboratories participating in the CDC National Genotyping and Surveillance Network. Isolates were fingerprinted with the IS6110 right-hand probe (IS6110-3′), the IS6110 left-hand probe (IS6110-5′), and the probe pTBN12, containing the polymorphic GC-rich sequence (PGRS). Spoligotyping based on the polymorphism in the 36-bp direct-repeat locus was also performed. As a control, 43 M. tuberculosisisolates in 17 clusters obtained from patients in Arkansas during the study period were analyzed. Of the 25 interstate clusters, 19 were confirmed as correctly clustered when all the isolates were analyzed on the same gel using the IS6110-3′ probe. Of the 19 true IS6110-3′ clusters, 10 (53%) were subdivided by one or more secondary typing methods. Clustering of the control group was virtually identical by all methods. Of the three different secondary typing methods, spoligotyping was the least discriminating. IS6110-5′ fingerprinting was as discriminating as PGRS fingerprinting. The data indicate that the IS6110-5′ probe not only is a useful secondary typing method but also probably would prove to be a more useful primary typing method for a genotyping network which involves isolates from different geographic regions.