Thomas C. Victor

Exogenous Reinfection as a Cause of Recurrent Tuberculosis after Curative Treatment

BACKGROUND For decades it has been assumed that postprimary tuberculosis is usually caused by reactivation of endogenous infection rather than by a new, exogenous infection. METHODS We performed DNA fingerprinting with restriction-fragment-length polymorphism analysis on pairs of isolates of Mycobacterium tuberculosis from 16 compliant patients who had a relapse of pulmonary tuberculosis after curative treatment of postprimary tuberculosis. The patients lived in areas of South Africa where tuberculosis is endemic. Medical records were reviewed for clinical data. RESULTS For 12 of the 16 patients, the restriction-fragment-length polymorphism banding patterns for the isolates obtained after the relapse were different from those for the isolates from the initial tuberculous disease. This finding indicates that reinfection was the cause of the recurrence of tuberculosis after curative treatment. Two patients had reinfections with a multidrug-resistant strain. All 15 patients who were tested for the human immunodeficiency virus were seronegative. CONCLUSIONS Exogenous reinfection appears to be a major cause of postprimary tuberculosis after a previous cure in an area with a high incidence of this disease. This finding emphasizes the importance of achieving cures and of preventing anyone with infectious tuberculosis from exposing others to the disease.

Genomic analysis identifies targets of convergent positive selection in drug-resistant Mycobacterium tuberculosis.

M. tuberculosis is evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly sequenced and 7 previously sequenced M. tuberculosis whole genomes, we identified genome-wide signatures of positive selection specific to the 47 drug-resistant strains. By searching for convergent evolution—the independent fixation of mutations in the same nucleotide position or gene—we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode components in cell wall biosynthesis, transcriptional regulation and DNA repair pathways. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.

Analysis for a Limited Number of Gene Codons Can Predict Drug Resistance of Mycobacterium tuberculosis in a High-Incidence Community

ABSTRACT Correct and rapid diagnosis is essential in the management of multidrug-resistant tuberculosis (MDR-TB). In this population-based study of 61 patients with drug-resistant tuberculosis, we evaluated the frequency of mutations and compared the performance of genotypic (mutation analysis by dot blot hybridization) and phenotypic (indirect proportion method) drug resistance tests. Three selected codons (rpoB531, rpoB526, and katG315) allowed identification of 90% of MDR-TB cases. Ninety percent of rifampin, streptomycin, and ethambutol resistance and 75% of isoniazid resistance were detected by screening for six codons: rpoB531, rpoB526, rrs-513, rpsL43, embB306, and katG315. The performance (reproducibility, sensitivity, and specificity) of the genotypic method was superior to that of the routine phenotypic method, with the exception of sensitivity for isoniazid resistance. A commercialized molecular genetic test for a limited number of target loci might be a good alternative for a drug resistance screening test in the context of an MDR “DOTS-plus” strategy.

Emergence and Spread of Extensively and Totally Drug-Resistant Tuberculosis, South Africa

Factors driving the increase in drug-resistant tuberculosis (TB) in the Eastern Cape Province, South Africa, are not understood. A convenience sample of 309 drug-susceptible and 342 multidrug-resistant (MDR) TB isolates, collected July 2008–July 2009, were characterized by spoligotyping, DNA fingerprinting, insertion site mapping, and targeted DNA sequencing. Analysis of molecular-based data showed diverse genetic backgrounds among drug-sensitive and MDR TB sensu stricto isolates in contrast to restricted genetic backgrounds among pre–extensively drug-resistant (pre-XDR) TB and XDR TB isolates. Second-line drug resistance was significantly associated with the atypical Beijing genotype. DNA fingerprinting and sequencing demonstrated that the pre-XDR and XDR atypical Beijing isolates evolved from a common progenitor; 85% and 92%, respectively, were clustered, indicating transmission. Ninety-three percent of atypical XDR Beijing isolates had mutations that confer resistance to 10 anti-TB drugs, and some isolates also were resistant to para-aminosalicylic acid. These findings suggest the emergence of totally drug-resistant TB.

Rifampicin Reduces Susceptibility to Ofloxacin in Rifampicin-resistant Mycobacterium tuberculosis through Efflux

RATIONALE Central dogma suggests that rifampicin resistance in Mycobacterium tuberculosis develops solely through rpoB gene mutations. OBJECTIVE To determine whether rifampicin induces efflux pumps activation in rifampicin resistant M. tuberculosis strains thereby defining rifampicin resistance levels and reducing ofloxacin susceptibility. METHODS Rifampicin and/or ofloxacin minimum inhibitory concentrations (MICs) were determined in rifampicin resistant strains by culture in BACTEC 12B medium. Verapamil and reserpine were included to determine their effect on rifampicin and ofloxacin susceptibility. RT-qPCR was applied to assess expression of efflux pump/transporter genes after rifampicin exposure. To determine whether verapamil could restore susceptibility to first-line drugs, BALB/c mice were infected with a MDR-TB strain and treated with first-line drugs with/without verapamil. MEASUREMENTS AND MAIN FINDINGS Rifampicin MICs varied independently of rpoB mutation and genetic background. Addition reserpine and verapamil significantly restored rifampicin susceptibility (p = 0.0000). RT-qPCR demonstrated that rifampicin induced differential expression of efflux/transporter genes in MDR-TB isolates. Incubation of rifampicin mono-resistant strains in rifampicin (2 μg/ml) for 7 days induced ofloxacin resistance (MIC > 2 μg/ml) in strains with an rpoB531 mutation. Ofloxacin susceptibility was restored by exposure to efflux pump inhibitors. Studies in BALB/c mice showed that verapamil in combination with first-line drugs significantly reduced pulmonary CFUs after 1 and 2 months treatment (p < 0.05). CONCLUSION Exposure of rifampicin resistant M. tuberculosis strains to rifampicin can potentially compromise the efficacy of the second-line treatment regimens containing ofloxacin, thereby emphasising the need for rapid diagnostics to guide treatment. Efflux pump inhibitors have the potential to improve the efficacy of anti-tuberculosis drug treatment.

Emergence of Increased Resistance and Extensively Drug-Resistant Tuberculosis Despite Treatment Adherence, South Africa

Improved infection control, rapid diagnostic tools, enhanced screening strategies, and pharmacokinetic studies are needed.

Transmission of a Multidrug-Resistant Mycobacterium tuberculosis Strain Resembling “Strain W” among Noninstitutionalized, Human Immunodeficiency Virus—Seronegative Patients

Since 1990, several outbreaks of multidrug-resistant tuberculosis (MDR-TB) have been described among institutionalized patients infected with human immunodeficiency virus (HIV). We describe a community MDR-TB outbreak among HIV-seronegative patients in Cape Town, South Africa. Isolates were characterized by restriction fragment length polymorphism (RFLP) analysis and dot-blot hybridization analysis of mutations conferring resistance for isoniazid, rifampin, streptomycin, and ethambutol. All isolates were identical on RFLP analysis. In 2 patients, RFLP analysis showed exogenous reinfection during or after treatment for drug-susceptible TB. Mutation analysis confirmed the genotypic identity of the isolates. The infecting strain was genotypically related to strain W, which is responsible for the majority of MDR-TB outbreaks in New York City. Transmission of MDR-TB is thus not limited to HIV-seropositive patients in an institutional setting but occurs within a community.

Isoniazid-Induced Transient High-Level Resistance in Mycobacterium tuberculosis

ABSTRACT An American Type Culture Collection reference strain and eight clinical strains of Mycobacterium tuberculosis, all of which were susceptible to isoniazid (INH) (mean MIC, 0.06 mg/liter) and negative for the Ser315Thr katG mutation, were left in their BACTEC 12B vials (for use with the BACTEC 460-TB method) containing 0.1 mg of INH per liter for periods of up to 28 days after the completion of the antibiotic susceptibility test. Each eventually grew to levels compatible with those of INH-resistant strains. Successive passages in INH-containing BACTEC 12B vials and onto solid media showed that the resistance noted above was maintained. Successive passages of these M. tuberculosis strains in which INH resistance had been induced into BACTEC 12B vials or solid media containing stepwise increases in INH concentrations eventually yielded organisms resistant to 20 mg of INH per liter. Transfer of cells in which INH resistance had been induced to drug-free medium followed by repeated passages in that medium eventually yielded organisms whose susceptibility to INH was identical to that of the original parent strains. The cycle of induced INH resistance could be repeated with these now INH-susceptible cells. The use of M. tuberculosis identification probes and IS6110-based restriction fragment length polymorphism analyses of cultures throughout the induction of INH resistance and the reversal of resistance in drug-free medium eliminated the possibility that the culture was contaminated or that the initial specimen had a mixed type of infection. Induced high-level resistance to INH (20 mg/liter) could be reduced 100-fold with a subinhibitory concentration of reserpine but not with verapamil. These results collectively suggest that high-level resistance to INH can be induced in INH-susceptible M. tuberculosis strains by the induction of a reserpine-sensitive efflux mechanism.

gyrA mutations and phenotypic susceptibility levels to ofloxacin and moxifloxacin in clinical isolates of Mycobacterium tuberculosis

OBJECTIVES To compare mutations in the quinolone resistance-determining region of the gyrA gene and flanking sequences with the MICs of ofloxacin and moxifloxacin for Mycobacterium tuberculosis. METHODS The presence of mutations in 177 drug-resistant M. tuberculosis isolates was determined by DNA sequencing and the MICs quantified by MGIT 960. RESULTS Single nucleotide polymorphisms were detected at codons 94 (n = 30), 90 (n = 12), 91 (n = 3), 89 (n = 1), 88 (n = 1) and 80 (n = 1). Four isolates with double mutations D94G plus A90V (n = 2) and D94G plus D94N (n = 2) reflect mixed populations. Agreement between genotypic and phenotypic susceptibility was high (≥97%) for both drugs. Mutant isolates had an MIC(50) of 8.0 mg/L and an MIC(90) of >10 mg/L for ofloxacin compared with an MIC(50) and MIC(90) of 2.0 mg/L for moxifloxacin. Codons 94 and 88 were linked to higher levels of fluoroquinolone resistance compared with codons 90, 91 and 89. The MIC distributions for the wild-type isolates ranged from ≤0.5 to 2.0 mg/L for ofloxacin and from ≤0.125 to 0.25 mg/L for moxifloxacin. However, 96% of the isolates with genetic alterations had MICs ≤2.0 mg/L for moxifloxacin, which is within its achievable serum levels. CONCLUSIONS This study provides quantitative evidence that the addition of moxifloxacin to extensively drug-resistant tuberculosis (XDR-TB) regimens based on a clinical breakpoint of 2.0 mg/L has merit. The use of moxifloxacin in the treatment of multidrug-resistant tuberculosis may prevent the acquisition of additional mutations and development of XDR-TB.

The non-clonality of drug resistance in Beijing-genotype isolates of Mycobacterium tuberculosis from the Western Cape of South Africa.

BackgroundThe Beijing genotype of M. tuberculosis is a virulent strain that is disseminating worldwide and has a strong association with drug resistance. In the Western Cape of South Africa, epidemiological studies have identified the R220 cluster of the Beijing genotype as a major contributor to a recent outbreak of drug-resistant tuberculosis. Although the outbreak is considered to be due to clonal transmission, the relationship among drug resistant isolates has not yet been established.ResultsTo better understand the evolution of drug resistance among these strains, 14 drug-resistant clinical isolates of the Beijing genotype were sequenced by whole-genome sequencing, including eight from R220 and six from a more ancestral Beijing cluster, R86, for comparison. While each cluster shares a distinct resistance mutation for isoniazid, mapping of other drug-resistance mutations onto a phylogenetic tree constructed from single nucleotide polymorphisms shows that resistance mutations to many drugs have arisen multiple times independently within each cluster of isolates. Thus, drug resistance among these isolates appears to be acquired, not clonally derived. This observation suggests that, although the Beijing genotype as a whole might have selective advantages enabling its rapid dissemination, the XDR isolates are relatively less fit and do not propagate well. Although it has been hypothesized that the increased frequency of drug resistance in some Beijing lineages might be caused by a mutator phenotype, no significant shift in synonymous substitution patterns is observed in the genomes.ConclusionWhile MDR-TB is spreading by transmission in the Western Cape, our data suggests that further drug resistance (i.e. XDR-TB) at this stage is acquired.