Ruth Stavrum

Pyrazinamide Resistance among South African Multidrug-Resistant Mycobacterium tuberculosis Isolates

ABSTRACT Pyrazinamide is important in tuberculosis treatment, as it is bactericidal to semidormant mycobacteria not killed by other antituberculosis drugs. Pyrazinamide is also one of the cornerstone drugs retained in the treatment of multidrug-resistant tuberculosis (MDR-TB). However, due to technical difficulties, routine drug susceptibility testing of Mycobacterium tuberculosis for pyrazinamide is, in many laboratories, not performed. The objective of our study was to generate information on pyrazinamide susceptibility among South African MDR and susceptible M. tuberculosis isolates from pulmonary tuberculosis patients. Seventy-one MDR and 59 fully susceptible M. tuberculosis isolates collected during the national surveillance study (2001 to 2002, by the Medical Research Council, South Africa) were examined for pyrazinamide susceptibility by the radiometric Bactec 460 TB system, pyrazinamidase activity (by Waynes assay), and sequencing of the pncA gene. The frequency of pyrazinamide resistance (by the Bactec system) among the MDR M. tuberculosis isolates was 37 of 71 (52.1%) and 6 of 59 (10.2%) among fully sensitive isolates. A total of 25 unique mutations in the pncA gene were detected. The majority of these were point mutations that resulted in amino acid substitutions. Twenty-eight isolates had identical mutations in the pncA gene, but could be differentiated from each other by a combination of the spoligotype patterns and 12 mycobacterial interspersed repetitive-unit loci. A high proportion of South African MDR M. tuberculosis isolates were resistant to pyrazinamide, suggesting an evaluation of its role in patients treated previously for tuberculosis as well as its role in the treatment of MDR-TB.

High Diversity of Mycobacterium tuberculosis Genotypes in South Africa and Preponderance of Mixed Infections among ST53 Isolates

ABSTRACT The reemergence of tuberculosis (TB) has become a major health problem worldwide, especially in Asia and Africa. Failure to combat this disease due to nonadherence or inappropriate drug regimens has selected for the emergence of multiple-drug-resistant (MDR) TB. The development of new molecular genotyping techniques has revealed the presence of mixed Mycobacterium tuberculosis infections, which may accelerate the emergence of drug-resistant strains. There are some studies describing the local distribution of circulating strains in South Africa, but to date, reports describing the frequency and distribution of M. tuberculosis genotypes, and specifically MDR genotypes, across the different provinces are limited. Thus, 252 isolates (of which 109 were MDR) from eight of the nine provinces of South Africa were analyzed by spoligotyping. Spoligotyping showed 10 different lineages, and ST53 (11.1%) and ST1 (10.3%) were the most frequent genotypes. Of the 75 different spoligopatterns observed, 20 (7.9%) were previously unreported. Analysis of the mycobacterial interspersed repetitive units of variable-number tandem repeats of the ST53 and ST1 isolates revealed that ∼54% of the ST53 isolates were of mixed M. tuberculosis subpopulations. Drug resistance (defined as resistance to at least isoniazid and/or rifampin) could only be linked to a history of previous anti-TB treatment (adjusted odds ratio, 4.0; 95% confidence interval, 2.27 to 7.10; P = <0.0001). This study describes a high diversity of circulating genotypes in South Africa in addition to a high frequency of mixed M. tuberculosis subpopulations among the ST53 isolates. MDR TB in South Africa could not be attributed to the spread of any single lineage.

Isoniazid and rifampicin resistance-associated mutations in Mycobacterium tuberculosis isolates from Yangon, Myanmar: implications for rapid molecular testing

OBJECTIVES To evaluate the frequency and nature of mutations in genes associated with resistance to rifampicin and isoniazid in Mycobacterium tuberculosis isolates collected from Yangon, Myanmar. METHODS Ninety-six isoniazid-resistant M. tuberculosis isolates, including 29 multidrug-resistant isolates, were analysed for mutations in the rpoB, katG, inhA, oxyR and ahpC genes. RESULTS Mutations in the rpoB gene were detected in 25 (86.2%) of the 29 rifampicin-resistant isolates. Of the 96 isoniazid-resistant isolates, 61 (63.5%) had mutations in codon 315 of the catalase-peroxidase-encoding gene (katG). Mutations in codon 315 were observed at a higher frequency in the multidrug-resistant isolates than in the isoniazid-resistant isolates (86.2% versus 53.7%, respectively, P = 0.003). Mutations in the oxyR-ahpC promoter region and in the inhA gene were observed in 14.6% and 2.1% of the isolates, respectively. Genotyping performed on the 96 M. tuberculosis isolates revealed a total of 94 different genotyping patterns. A distinct genotypic pattern was found in 92 isolates, whereas 4 isolates belonged to two clusters with identical genotypes, suggesting that the majority of the isolates were not from an outbreak of a single drug-resistant clone. CONCLUSIONS This study provides the first molecular characterization of isoniazid- and rifampicin-resistant M. tuberculosis isolates from Myanmar and gives information on the molecular basis for rifampicin and isoniazid drug resistance in M. tuberculosis. The study generates useful information for the development of potential rapid molecular drug susceptibility tests.

Identification of biomarkers for Mycobacterium tuberculosis infection and disease in BCG-vaccinated young children in Southern India.

Pediatric tuberculosis (TB) often goes undiagnosed because of the lack of reliable diagnostic methods. With the aim of assessing biomarker(s) that can aid in the diagnosis of TB infection and disease, we investigated 746 Indian children with suspected TB. Whole-blood mRNA from 210 children was examined by dual-color Reverse-Transcriptase Multiple Ligation-dependent Probe-Amplification for the expression of 45 genes and a Bio-Plex assay for the expression of cytokines/chemokines in QuantiFERON supernatants. The study shows that transcription of SEC14L1, GUSB, BPI, CCR7 and TGFβ-1 (all P⩽0.05) was downregulated in TB disease compared with uninfected controls, while transcription of RAB33A was downregulated in TB disease compared with both latent TB (P<0.05) and controls (P<0.01). The transcription of CD4, TGFβ-1 (P<0.01) and the expression of IL-2 (P<0.01) and IL-13 (P<0.05) was upregulated in latent TB compared with that in controls. Using the Least Absolute Shrinkage and Selection Operator (lasso) model, RAB33A alone discriminated between TB disease and latent TB (area under the curve (AUC) 77.5%), whereas a combination of RAB33A, CXCL10, SEC14L1, FOXP3 and TNFRSF1A was effective in discriminating between TB disease and controls (AUC 91.7%). A combination of 11 biomarkers predicted latent TB with moderate discriminatory power (AUC 72.2%). In conclusion, RAB33A is a potential biomarker for TB disease, whereas CD4, TGFβ-1 and IL-2, IL-13 may identify latent TB in children.

Genomic Diversity among Beijing and non-Beijing Mycobacterium tuberculosis Isolates from Myanmar

Background The Beijing family of Mycobacterium tuberculosis is dominant in countries in East Asia. Genomic polymorphisms are a source of diversity within the M. tuberculosis genome and may account for the variation of virulence among M. tuberculosis isolates. Till date there are no studies that have examined the genomic composition of M. tuberculosis isolates from the high TB-burden country, Myanmar. Methodology/Principle Findings Twenty-two M. tuberculosis isolates from Myanmar were screened on whole-genome arrays containing genes from M. tuberculosis H37Rv, M. tuberculosis CDC1551 and M. bovis AF22197. Screening identified 198 deletions or extra regions in the clinical isolates compared to H37Rv. Twenty-two regions differentiated between Beijing and non-Beijing isolates and were verified by PCR on an additional 40 isolates. Six regions (Rv0071-0074 [RD105], Rv1572-1576c [RD149], Rv1585c-1587c [RD149], MT1798-Rv1755c [RD152], Rv1761c [RD152] and Rv0279c) were deleted in Beijing isolates, of which 4 (Rv1572-1576c, Rv1585c-1587c, MT1798-Rv1755c and Rv1761c) were variably deleted among ST42 isolates, indicating a closer relationship between the Beijing and ST42 lineages. The TbD1 region, Mb1582-Mb1583 was deleted in Beijing and ST42 isolates. One M. bovis gene of unknown function, Mb3184c was present in all isolates, except 11 of 13 ST42 isolates. The CDC1551 gene, MT1360 coding for a putative adenylate cyclase, was present in all Beijing and ST42 isolates (except 1). The pks15/1 gene, coding for a putative virulence factor, was intact in all Beijing and non-Beijing isolates, except in ST42 and ST53 isolates. Conclusion This study describes previously unreported deletions/extra regions in Beijing and non-Beijing M. tuberculosis isolates. The modern and highly frequent ST42 lineage showed a closer relationship to the hypervirulent Beijing lineage than to the ancient non-Beijing lineages. The pks15/1 gene was disrupted only in modern non-Beijing isolates. This is the first report of an in-depth analysis on the genomic diversity of M. tuberculosis isolates from Myanmar.

Modulation of Transcriptional and Inflammatory Responses in Murine Macrophages by the Mycobacterium tuberculosis Mammalian Cell Entry (Mce) 1 Complex

The outcome of many infections depends on the initial interactions between agent and host. Aiming at elucidating the effect of the M. tuberculosis Mce1 protein complex on host transcriptional and immunological responses to infection with M. tuberculosis, RNA from murine macrophages at 15, 30, 60 min, 4 and 10 hrs post-infection with M. tuberculosis H37Rv or Δ-mce1 H37Rv was analyzed by whole-genome microarrays and RT-QPCR. Immunological responses were measured using a 23-plex cytokine assay. Compared to uninfected controls, 524 versus 64 genes were up-regulated by 15 min post H37Rv- and Δ-mce1 H37Rv-infection, respectively. By 15 min post-H37Rv infection, a decline of 17 cytokines combined with up-regulation of Ccl24 (26.5-fold), Clec4a2 (23.2-fold) and Pparγ (10.5-fold) indicated an anti-inflammatory response initiated by IL-13. Down-regulation of Il13ra1 combined with up-regulation of Il12b (30.2-fold), suggested switch to a pro-inflammatory response by 4 hrs post H37Rv-infection. Whereas no significant change in cytokine concentration or transcription was observed during the first hour post Δ-mce1 H37Rv-infection, a significant decline of IL-1b, IL-9, IL-13, Eotaxin and GM-CSF combined with increased transcription of Il12b (25.1-fold) and Inb1 (17.9-fold) by 4 hrs, indicated a pro-inflammatory response. The balance between pro-and anti-inflammatory responses during the early stages of infection may have significant bearing on outcome.

Mycobacterium tuberculosis Mce1 protein complex initiates rapid induction of transcription of genes involved in substrate trafficking.

The mammalian cell entry (Mce)1 protein complex has an important role during the initial phase of a Mycobacterium tuberculosis (M. tuberculosis) infection. Murine macrophages were infected with M. tuberculosis H37Rv or Δ-mce1 H37Rv, and total RNA was isolated from the host cells at 15, 30 and 60 min, and 4 and 10 h post-infection. With the aim of studying the role for the Mce1 protein complex on host gene expression, the RNA was hybridized onto 44 K whole-genome microarrays. Selected genes were verified by reverse-transcriptase quantitative PCR (RT-QPCR). ‘Transport’ was the most overrepresented biological process during the first hour post H37Rv infection. Five genes (Abca1 (21.0-fold), Slc16a10 (3.1-fold), Slc6a12 (17.9-fold), Slc6a8 (2.3-fold) and Nr1h3, (5.5-fold)) involved in substrate trafficking were verified by RT-QPCR to be upregulated by >2-fold 1 h post H37Rv infection. By 1 h post Δ-mce1 H37Rv infection, only Abca1 and Slc6a12 were upregulated by >2-fold. A number of other genes, which may be directly involved in substrate trafficking or share the same transcription, were found to have expression profiles similar to the genes involved in substrate trafficking. The Mce1 protein complex has a significant role in the transcriptional activation of genes involved in substrate trafficking during the initial phase of an M. tuberculosis infection.

Concordant or discordant results by the tuberculin skin test and the quantiFERON-TB test in children reflect immune biomarker profiles

The tuberculin skin test (TST) and QuantiFERON-TB-Gold-In-tube (QFTGIT) are adjunctive tests used in the diagnosis of pediatric tuberculosis (TB). Neither test can rule out TB; however, a positive test usually triggers preventive treatment in TB contacts aged <5 years. TST and QFTGIT can give divergent results and it is unclear how discordant results should be interpreted in terms of TB risk and preventive treatment. To understand the immune processes underlying concordant or discordant TST and QFTGIT results, we analyzed immune responses in children from Palamaner Taluk in India (a TB-endemic region with routine neonatal BCG vaccination) who were referred to a TB case verification ward on suspicion of TB. Two hundred and ten children aged <3 years were classified according to their TST and QFTGIT results, and their immune responses analyzed by dual-colour-Reverse-Transcriptase-Multiple-Ligation-dependent-Probe-Amplification, using a panel of 45 genes and a 10-plex antigen-specific enzyme-linked immunosorbent assay. We show that immune biomarkers FPR1, TNFRSF1A and interferon (IFN)-γ are upregulated (all P<0.05) in concordant test-positive children, whereas BPI is downregulated (P<0.05). In contrast, SEC14L1 (P=0.034) and Interferon gamma-induced protein 10 (IP-10) (P=0.001) are differentially expressed between the TST+QFTGIT− /TST−QFTGIT+ groups. Known TB exposure was more frequent in concordant positive children and results were consistent with elevated expression of genes associated with inflammatory responses. Children with discordant test results displayed a mixed profile with activation of both pro- and anti-inflammatory markers. TST and/or QFTGIT positivity appears to reflect distinct but overlapping aspects of host immunity.

Increased level of acute phase reactants in patients infected with modern Mycobacterium tuberculosis genotypes in Mwanza, Tanzania

BackgroundThere is increasing evidence to suggest that different Mycobacterium tuberculosis lineages cause variations in the clinical presentation of tuberculosis (TB). Certain M. tuberculosis genotypes/lineages have been shown to be more likely to cause active TB in human populations from a distinct genetic ancestry. This study describes the genetic biodiversity of M. tuberculosis genotypes in Mwanza city, Tanzania and the clinical presentation of the disease caused by isolates of different lineages.MethodsTwo-hundred-fifty-two isolates from pulmonary TB patients in Mwanza, Tanzania were characterized by spoligotyping, and 45 isolates were further characterized by mycobacterium interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR). The patients’ level of the acute phase reactants AGP, CRP and neutrophil counts, in addition to BMI, were measured and compared to the M. tuberculosis lineage of the infectious agent for each patient.ResultsThe most frequent genotype was ST59 (48 out of 248 [19.4%]), belonging to the Euro-American lineage LAM11_ZWE, followed by ST21 (CAS_KILI lineage [44 out of 248 [17.7%]). A low degree of diversity (15.7% [39 different ST’s out of 248 isolates]) of genotypes, in addition to a high level of mixed M. tuberculosis sub-populations among isolates with an unreported spoligotype pattern (10 out of 20 isolates [50.0%]) and isolates belonging to the ST53 lineage (13 out of 25 [52%]) was observed. Isolates of the ‘modern’ (TbD1-) Euro-American lineage induced higher levels of α1-acid glycoprotein (β = 0.4, P = 0.02; 95% CI [0.06-0.66]) and neutrophil counts (β = 0.9, P = 0.02; 95% CI [0.12-1.64]) and had lower BMI score (β = -1.0, P = 0.04; 95% CI[-1.89 – (-0.03)]). LAM11_ZWE (‘modern’) isolates induced higher levels of CRP (β = 24.4, P = 0.05; 95% CI[0.24-48.63]) and neutrophil counts (β = 0.9, P = 0.03; 95% CI[0.09-1.70]).ConclusionThe low diversity of genotypes may be explained by an evolutionary advantage of the most common lineages over other lineages combined with optimal conditions for transmission, such as overcrowding and inadequate ventilation. The induction of higher levels of acute phase reactants in patients infected by ‘modern’ lineage isolates compared to ‘ancient’ lineages may suggest increased virulence among ‘modern’ lineage isolates.

Effect of Non-tuberculous Mycobacteria on Host Biomarkers Potentially Relevant for Tuberculosis Management

Background Non-tuberculous mycobacteria (NTM) are different from Mycobacterium tuberculosis (MTB) both in their ubiquitous environmental distribution and in their reduced capacity to cause disease. While often neglected in favour of other infectious diseases, NTM may interfere with important aspects of TB control and management, namely the efficacy of new anti-tuberculosis (TB) vaccines; the immuno-diagnostic Tuberculin skin test (TST) and QuantiFERON TB Gold In Tube assay (QFTGIT); and immune biomarkers explored for their diagnostic and/or predictive potential. Our objective was therefore to explore host immune biomarkers in children who had NTM isolated from respiratory and/or gastric specimens. Methodology and Principle Findings The present study was nested within a prospective cohort study of BCG-vaccinated neonates in Southern India. In this setting, immune biomarkers from peripheral blood were analyzed in 210 children aged <3 years evaluated for TB using dual-colour-Reverse-Transcriptase-Multiple-Ligation-dependent-Probe-Amplification (dcRT-MLPA) and Bio-Plex assays. The children were classified based on clinical examination, chest X-rays and mycobacterial culture reports as either: 1) TB disease, 2) NTM present and 3) controls. The study shows a down-regulation of RAB33A (p<0.001) and up-regulation of TGFβ1, IL-2 and IL-6 (all p<0.05) in children with TB disease, and that RAB33A, TGFBR2 and IL-10 (all p<0.05) were differentially expressed in children with NTM present when compared to children that were culture negative for MTB and NTM (controls). Conclusions and Significance Carriage of NTM may reduce the specificity of future diagnostic and predictive immune biomarkers relevant to TB management.