Older age at first tuberculosis diagnosis is associated with tuberculosis recurrence in HIV-negative persons.

SETTING: Tuberculosis (TB) clinic in Durban, South Africa. OBJECTIVE: To assess the factors associated with TB recurrence among human immunodeficiency virus (HIV) negative adults and children. DESIGN: We conducted a retrospective longitudinal study from January 2000 to December 2012. We defined recurrence as a TB episode occurring within the study period after treatment completion or cure of a previous episode. We used a multivariable Poisson regression model to assess the factors associated with the number of recurrences among HIV-negative patients. RESULTS: Among 17 941 patients with known HIV status, 3653 (20%) were HIV-negative; of these, 235 (6%) had one recurrence, 21 (1%) had two recurrences and 4 (0.1%) had three recurrences. The median follow-up time from the end of treatment for the first episode was 3.0 years (interquartile range 1.9-4.2). Age at the first TB episode was significantly associated with the number of TB recurrences: younger patients had the lowest rate of recurrence, with a steady increase in rates until age 40 years, after which rates stabilized. CONCLUSIONS: TB recurrence rates among HIV-negative patients were higher at increased age at the first TB episode. Further translational studies are needed to clarify the factors that drive multiple TB recurrences in older age, including impaired immunity, the results of which have implications for TB vaccine development.

Isoniazid-monoresistant tuberculosis is associated with poor treatment outcomes in Durban, South Africa.

SETTING: A large tuberculosis (TB) clinic in Durban, South Africa. OBJECTIVE: To determine the association between isoniazid (INH) monoresistant TB and treatment outcomes. DESIGN: We performed a retrospective longitudinal study of patients seen from 2000 to 2012 to compare episodes of INH-monoresistant TB with those of drug-susceptible TB using logistic regression with robust standard errors. INH-monoresistant TB was treated with modified regimens. RESULTS: Among 18 058 TB patients, there were 19 979 TB episodes for which drug susceptibility testing was performed. Of these, 557 were INH-monoresistant and 16 311 were drug-susceptible. Loss to follow-up, transfer, and human immunodeficiency virus (HIV) co-infection (41% had known HIV status) were similar between groups. INH-monoresistant episodes were more likely to result in treatment failure (4.1% vs. 0.6%, P < 0.001) and death (3.2% vs. 1.8%, P = 0.01) than drug-susceptible episodes. After adjustment for age, sex, race, retreatment status, and disease site, INH-monoresistant episodes were more likely to have resulted in treatment failure (OR 6.84, 95%CI 4.29-10.89, P < 0.001) and death (OR 1.81, 95%CI 1.11-2.95, P = 0.02). CONCLUSION: INH monoresistance was associated with worse clinical outcomes than drug-susceptible TB. Our findings support the need for rapid diagnostic tests for INH resistance and improved treatment regimens for INH-monoresistant TB.

High-throughput method for detecting genomic-deletion polymorphisms.

DNA microarrays have been successfully used with different microorganisms, including Mycobacterium tuberculosis, to detect genomic deletions relative to a reference strain. However, the cost and complexity of the microarray system are obstacles to its widespread use in large-scale studies. In order to evaluate the extent and role of large sequence polymorphisms (LSPs) or insertion-deletion events in bacterial populations, we developed a technique, termed deligotyping, which hybridizes multiplex-PCR products to membrane-bound, highly specific oligonucleotide probes. The approach has the benefits of being low cost and capable of simultaneously interrogating more than 40 bacterial strains for the presence of 43 genomic regions. The deletions represented on the membrane were selected from previous comparative genomic studies and ongoing microarray experiments. Highly specific probes for these deletions were designed and attached to a membrane for hybridization with strain-derived targets. The targets were generated by multiplex PCR, allowing simultaneous amplifications of 43 different genomic loci in a single reaction. To validate our approach, 100 strains that had been analyzed with a high-density microarray were analyzed. The membrane accurately detected the deletions identified by the microarray approach, with a sensitivity of 99.9% and a specificity of 98.0%. The deligotyping technique allows the rapid and reliable screening of large numbers of M. tuberculosis isolates for LSPs. This technique can be used to provide insights into the epidemiology, genomic evolution, and population structure of M. tuberculosis and can be adapted for the study of other organisms.

Molecular epidemiology of tuberculosis.

Despite the almost 50 yrs since the introduction of curative antituberculosis drugs, Mycobacterium tuberculosis continues to exert an enormous toll on world health, and tuberculosis remains the world's leading cause of death due to a single infectious agent. This has stimulated research efforts into finding new tools to tackle the continuing tuberculosis pandemic. One of the few successes to date has been the development of a new discipline, molecular epidemiology. This has added a further dimension to the classical epidemiology of tuberculosis and enhanced understanding of how M. tuberculosis continues to be successfully transmitted within populations. In the process, inadequacies in tuberculosis control programmes have been identified, helping accumulate resources for their improvement. Other technologies, based on knowledge of the complete genome sequence of M. tuberculosis, which will provide newer tools for probing the epidemiology of tuberculosis, are now emerging. In spite of these advances, tuberculosis continues to remain a devastating infectious disease, disproportionately impacting on the world's poorest countries. The future challenge for molecular epidemiology is to provide better understanding of the transmission dynamics of tuberculosis in these settings and to stimulate the implementation of control measures on a more global scale.

Inactivation of the Mycobacterium tuberculosis Nramp orthologue (mntH) does not affect virulence in a mouse model of tuberculosis.

Mycobacterium tuberculosis is an intracellular pathogen which can survive and multiply within the phagosomal compartment of the macrophage, and in doing so has to withstand the various macrophage defense mechanisms, which include limitation of iron and other metals. Analysis of the complete genome sequence of M. tuberculosis revealed an extensive array of cation transporters, including mntH, an orthologue of the eukaryotic Nramp (natural resistance-associated macrophage protein) gene, that encodes a proton-dependent divalent metal transporter. To assess the effect of this transporter on intracellular survival and pathogenesis, an mntH knock-out mutant of M. tuberculosis H37Rv was created and assayed in bone marrow-derived macrophages and in a murine model of tuberculosis. In neither of these systems was any loss of fitness associated with inactivation of mntH, demonstrating that Nramp orthologues are not important determinants of mycobacterial virulence.

A new evolutionary scenario for the Mycobacterium tuberculosis complex.

The distribution of 20 variable regions resulting from insertion-deletion events in the genomes of the tubercle bacilli has been evaluated in a total of 100 strains of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, and Mycobacterium bovis. This approach showed that the majority of these polymorphisms did not occur independently in the different strains of the M. tuberculosis complex but, rather, resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis specific deletion (TbD1), M. tuberculosis strains can be divided into ancestral and "modern" strains, the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters. Furthermore, successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum, M. microti, and M. bovis that diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the often-presented hypothesis that M. tuberculosis, the etiological agent of human tuberculosis evolved from M. bovis, the agent of bovine disease. M. canettii and ancestral M. tuberculosis strains lack none of these deleted regions, and, therefore, seem to be direct descendants of tubercle bacilli that existed before the M. africanum-->M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could well have been a human pathogen already.

The evolution of mycobacterial pathogenicity: clues from comparative genomics.

Comparative genomics, and related technologies, are helping to unravel the molecular basis of the pathogenesis, host range, evolution and phenotypic differences of the slow-growing mycobacteria. In the highly conserved Mycobacterium tuberculosis complex, where single-nucleotide polymorphisms are rare, insertion and deletion events (InDels) are the principal source of genome plasticity. InDels result from recombinational or insertion sequence (IS)-mediated events, expansion of repetitive DNA sequences, or replication errors based on repetitive motifs that remove blocks of genes or contract coding sequences. Comparative genomic analyses also suggest that loss of genes is part of the ongoing evolution of the slow-growing mycobacterial pathogens and might also explain how the vaccine strain BCG became attenuated.

Regulation of catalase-peroxidase (KatG) expression, isoniazid sensitivity and virulence by furA of Mycobacterium tuberculosis.

Mycobacterium tuberculosis has two genes for ferric uptake regulator orthologues, one of which, furA, is situated immediately upstream of katG encoding catalase-peroxidase, a major virulence factor that also activates the prodrug isoniazid. This association suggested that furA might regulate katG and other genes involved in pathogenesis. Transcript mapping showed katG to be expressed from a strong promoter, with consensus -10 and -35 elements, preceding furA. No promoter activity was demonstrated downstream of the furA start codon, using different gene reporter systems, indicating that furA and katG are co-transcribed from a common regulatory region. The respective roles of these two genes in the isoniazid susceptibility and virulence of M. tuberculosis were assessed by combinatorial complementation of a Delta(furA-katG) strain that is heavily attenuated in a mouse model of tuberculosis. In the absence of furA, katG was upregulated, cells became hypersensitive to isoniazid, and full virulence was restored, indicating that furA regulates the transcription of both genes. When furA alone was introduced into the Delta(furA-katG) mutant, survival in mouse lungs was moderately increased, suggesting that FurA could regulate genes, other than katG, that are involved in pathogenesis. These do not include the oxidative stress genes ahpC and sodA, or those for siderophore production.

Comparative genomics uncovers large tandem chromosomal duplications in Mycobacterium bovis BCG Pasteur.

On direct comparison of minimal sets of ordered clones from bacterial artificial chromosome (BAC) libraries representing the complete genomes of Mycobacterium tuberculosis H37Rv and the vaccine strain, Mycobacterium bovis BCG Pasteur, two major rearrangements were identified in the genome of M. bovis BCG Pasteur. These were shown to correspond to two tandem duplications, DU1 and DU2, of 29 668 bp and 36 161 bp, respectively. While DU1 resulted from a single duplication event, DU2 apparently arose from duplication of a 100 kb genomic segment that subsequently incurred an internal deletion of 64 kb. Several lines of evidence suggest that DU2 may continue to expand, since two copies were detected in a subpopulation of BCG Pasteur cells. BCG strains harbouring DU1 and DU2 are diploid for at least 58 genes and contain two copies of oriC, the chromosomal origin of replication. These findings indicate that these genomic regions of the BCG genome are still dynamic. Although the role of DU1 and DU2 in the attenuation and/or altered immunogenicity of BCG is yet unknown, knowledge of their existence will facilitate quality control of BCG vaccine lots and may help in monitoring the efficacy of the world's most widely used vaccine.

The rabbit study: ritonavir and saquinavir in combination in saquinavir-experienced and previously untreated patients.

Thirteen protease inhibitor-naive patients with HIV-1 infection, and 12 patients with a median of 58 months prior treatment with saquinavir (SQV) monotherapy, were treated with SQV (400 mg twice daily) and ritonavir (RIT, 500 mg twice daily) in a study designed to assess the effect of prior treatment with SQV monotherapy on the antiretroviral activity of RIT-SQV combination therapy. Median baseline viral load and CD4+ cell counts were 155,000 and 262,000 copies/ml and 333 and 225 cells/mm3 in the naive and experienced groups, respectively. Mean viral load changes at 24 weeks were -1.63 and -0.27 log copies/ml in the naive and SQV-experienced groups, respectively (intent-to-treat analysis). Baseline genotype by point mutation assay and sequencing in the SQV-experienced group was highly predictive of virological response. Eight of 11 SQV-experienced patients had evidence of phenotypic resistance to RIT at baseline, despite previous treatment with SQV only. There was strong correlation between phenotypic resistance to RIT and the presence of the L90M mutation. We conclude that prolonged prior treatment with saquinavir monotherapy may produce cross-resistance to ritonavir and reduce the subsequent response to ritonavir-saquinavir in combination. In this study, both phenotypic resistance to ritonavir and presence of the L90M mutation predicted the viral load response to ritonavir-saquinavir.

The in vivo effects of combination antiretroviral drug therapy on peripheral blood CD34+ cell colony-forming units from HIV type 1-infected patients.

This study investigated the effects of a combination antiretroviral drug regimen (indinavir and two nucleoside analogs or ritonavir and saquinavir) on the levels of CD34+ colony-forming units (CFU-Cs) in the peripheral blood of HIV-1+ patients. Ten patients who were receiving combination antiretroviral drug therapy were studied and their peripheral blood CD34+ CFU-Cs were measured prior to, 1 month after, and 4 to 6 months after the commencement of therapy. The levels of CD4+ T cells increased significantly in these patients (paired t test, p = 0.0027) and plasma viral load became undetectable in all but one patient studied. Measurements of the CFU-Cs showed that their levels tended to increase on the commencement of therapy, and these levels became significantly higher than baseline by 4-6 months (paired t test, p = 0.0293). Analysis of the different colony phenotype demonstrated that the main contributor to this increase consisted of burst-forming unit erythroid (BFU-E) cells. These data also demonstrated that there was an inverse correlation between the rise in CFU-Cs at 4-6 months compared with CD4+ cell, CD8+ cell, and neutrophil counts, and hemoglobin concentration, at baseline. The demonstrated increase in the levels of CD34+ CFU-Cs suggests that HIV-1 may have an inhibitory effect on these cells in vivo, and that this inhibition may be abrogated by suppression of viral replication.