Andrea L. Gibson

Tuberculosis Is Associated with a Down-Modulatory Lung Immune Response That Impairs Th1-Type Immunity

Immune mediators associated with human tuberculosis (TB) remain poorly defined. This study quantified levels of lung immune mediator gene expression at the time of diagnosis and during anti-TB treatment using cells obtained by induced sputum. Upon comparison to patients with other infectious lung diseases and volunteers, active pulmonary TB cases expressed significantly higher levels of mediators that counteract Th1-type and innate immunity. Despite the concomitant heightened levels of Th1-type mediators, immune activation may be rendered ineffectual by high levels of intracellular (SOCS and IRAK-M) and extracellular (IL-10 and TGF-βRII, IL-1Rn, and IDO) immune suppressive mediators. These modulators are a direct response to Mycobacterium tuberculosis as, by day 30 of anti-TB treatment, many suppressive factors declined to that of controls whereas most Th1-type and innate immune mediators rose above pretreatment levels. Challenge of human immune cells with M. tuberculosis in vitro up-regulated these immune modulators as well. The observed low levels of NO synthase-2 produced by alveolar macrophages at TB diagnosis, along with the heightened amounts of suppressive mediators, support the conclusion that M. tuberculosis actively promotes down-modulatory mediators to counteract Th1-type and innate immunity as an immunopathological strategy. Our data highlight the potential application of immune mediators as surrogate markers for TB diagnosis or treatment response.

Discovery of a Novel Mycobacterium tuberculosis Lineage That Is a Major Cause of Tuberculosis in Rio de Janeiro, Brazil

ABSTRACT The current study evaluated Mycobacterium tuberculosis isolates from Rio de Janeiro, Brazil, for genomic deletions. One locus in our panel of PCR targets failed to amplify in ∼30% of strains. A single novel long sequence polymorphism (>26.3 kb) was characterized and designated RDRio. Homologous recombination between two similar protein-coding genes is proposed as the mechanism for deleting or modifying 10 genes, including two potentially immunogenic PPE proteins. The flanking regions of the RDRio locus were identical in all strains bearing the deletion. Genetic testing by principal genetic group, spoligotyping, variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR), and IS6110-based restriction fragment length polymorphism analysis cumulatively support the idea that RDRio strains are derived from a common ancestor belonging solely to the Latin American-Mediterranean spoligotype family. The RDRio lineage is therefore the predominant clade causing tuberculosis (TB) in Rio de Janeiro and, as indicated by genotypic clustering in MIRU-VNTR analysis, the most significant source of recent transmission. Limited retrospective reviews of bacteriological and patient records showed a lack of association with multidrug resistance or specific risk factors for TB. However, trends in the data did suggest that RDRio strains may cause a form of TB with a distinct clinical presentation. Overall, the high prevalence of this genotype may be related to enhanced virulence, transmissibility, and/or specific adaptation to a Euro-Latin American host population. The identification of RDRio strains outside of Brazil points to the ongoing intercontinental dissemination of this important genotype. Further studies are needed to determine the differential strain-specific features, pathobiology, and worldwide prevalence of RDRioM. tuberculosis.

Molecular Epidemiology of Disease Due to Mycobacterium bovis in Humans in the United Kingdom

ABSTRACT Mycobacterium bovis is the causative agent of bovine tuberculosis, with a wide host range. Fifty human M. bovis isolates were typed using spoligotyping and variable number tandem repeats (VNTR). Fifteen of these spoligotypes have not yet been recorded in cattle. The predominant spoligotype in humans and cattle was subdivided by VNTR.

Application of Sensitive and Specific Molecular Methods To Uncover Global Dissemination of the Major RDRio Sublineage of the Latin American-Mediterranean Mycobacterium tuberculosis Spoligotype Family

ABSTRACT The Latin American-Mediterranean (LAM) family of Mycobacterium tuberculosis is believed to be the cause of ∼15% of tuberculosis cases worldwide. Previously, we defined a prevalent sublineage of the LAM family in Brazil by a single characteristic genomic deletion designated RDRio. Using the Brazilian strains, we pinpoint an Ag85C103 single nucleotide polymorphism (SNP) (screened by restriction fragment length polymorphism [RFLP] analysis) that correctly identified all LAM family strains. Importantly, all RDRio strains concomitantly possessed the RD174 deletion. These genetic signatures, along with a newly developed multiplex PCR for rapid differentiation between “wild-type” and RDRio strains, were then used to analyze an international collection of M. tuberculosis strains. RDRioM. tuberculosis was identified from four continents involving 11 countries. Phylogenetic analysis of the IS6110-RFLP patterns from representative RDRio and LAM strains from Brazil, along with all representative clusters from a South African database, confirmed their genetic relatedness and transcontinental transmission. The Ag85C103 SNP RFLP, as compared to results obtained using a PCR method targeting a LAM-restricted IS6110 element, correctly identified 99.8% of LAM spoligotype strains. Together, these tests were more accurate than spoligotyping at categorizing strains with indefinable spoligotypes and segregated true LAM strains from those with convergent spoligotypes. The fact that RDRio strains were identified worldwide highlights the importance of this LAM family sublineage and suggests that this strain is a global threat that should be specifically targeted by public health resources. Our provision of simple and robust molecular methods will assist the evaluation of the LAM family and the RDRio sublineage.

High Rates of Clustering of Strains Causing Tuberculosis in Harare, Zimbabwe: a Molecular Epidemiological Study

ABSTRACT We examined the pattern of tuberculosis (TB) transmission (i.e., reactivation versus recent transmission) and the impact of human immunodeficiency virus (HIV) infection in Harare, Zimbabwe. Consecutive adult smear-positive pulmonary TB patients presenting to an urban hospital in Harare were enrolled. A detailed epidemiological questionnaire was completed, and tests for HIV type 1 and CD4 cell counts were performed for each patient. Molecular fingerprinting of the genomic DNA recovered from cultures of sputum was performed by two molecular typing methods: spacer oligonucleotide typing (spoligotyping) and analysis of variable number of tandem DNA repeats (VNTRs). A cluster was defined as isolates from two or more patients that shared the same spoligotype pattern or the same VNTR pattern, or both. DNA suitable for typing was recovered from 224 patients. The prevalence of HIV infection was 79%. Of 187 patient isolates (78.6%) typed by both spoligotyping and analysis of VNTRs, 147 were identified as part of a cluster by both methods. By spoligotyping alone, 84.1% of patient isolates were grouped into 20 clusters. The cluster size was generally <8 patient isolates, although three large clusters comprised 68, 25, and 23 patient isolates. A total of 89.4% of the patient isolates grouped into 12 clusters defined by analysis of VNTRs, with 2 large clusters consisting of 127 and 13 patient isolates, respectively. Thirty-six percent of patient isolates with a shared spoligotype and 17% with a shared VNTR pattern were geographically linked within Harare, but they were not linked on the basis of the patients home district. In a multivariate analysis, there were no independent predictors of clustering, including HIV infection status. Comparison with the International Spoligotype database (Pasteur Institute, Pointe à Pitre, Guadeloupe) demonstrated that our three largest spoligotype clusters are well recognized and ubiquitous in Africa. In this epidemiologically well characterized urban population with a high prevalence of HIV infection, we identified a very high level of strain clustering, indicating substantial ongoing recent TB transmission. Geographic linkage could be detected in a proportion of these clusters. A small group of actively circulating strains accounted for most of the cases of TB transmission.

Mycobacterium bovis infection, United Kingdom.

We describe the first documented spillover of bovine tuberculosis from animals into the human population of the United Kingdom since the resurgence of the disease in cattle in the country. This finding suggests that there may be a small risk for transmission to humans, making continued vigilance particularly necessary.

RDRio Mycobacterium tuberculosis infection is associated with a higher frequency of cavitary pulmonary disease.

ABSTRACT Molecular genotyping has shown Mycobacterium tuberculosis lineages to be geographically restricted and associated with distinct ethnic populations. Whether tuberculosis (TB) caused by some M. tuberculosis lineages can present with a differential clinical spectrum is controversial because of very limited clinical data. We recently reported on the discovery of RDRioM. tuberculosis, a Latin American-Mediterranean sublineage that is the predominant cause of TB in Rio de Janeiro, Brazil. To investigate the clinical attributes of TB caused by RDRio strains, we studied a cohort of TB cases from Belo Horizonte, Brazil, in which clinical information recorded on a standardized questionnaire was collected at the time of microbiological testing. These patients were referred for culture and drug susceptibility testing because of the clinical suspicion of “complicated” TB, as demonstrated by high rates of multidrug resistance (12%) and cavitary TB (80%). We performed spoligotyping and RDRio genotyping on the M. tuberculosis strains and analyzed the clinical data from these patients. RDRioM. tuberculosis accounted for 37% of the total TB burden. Multivariate analysis found a significant association between TB caused by RDRio strains and pulmonary cavitation and residence in Belo Horizonte. Since cavitary TB is associated with higher sputum bacillary load, our findings support the hypothesis that RDRioM. tuberculosis is associated with a more “severe” disease as a strategy to increase transmission. Future studies are needed to confirm these observations and to better define the contribution of RDRioM. tuberculosis to the global TB epidemic.

Progression Toward an Improved DNA Amplification-Based Typing Technique in the Study of Mycobacterium tuberculosis Epidemiology

ABSTRACT While high-copy-number IS6110-based restriction fragment length polymorphism (HCN-RFLP) is the gold standard for typing most Mycobacterium tuberculosis strains, the time taken for culturing and low throughput make it impractical for large-scale prospective typing of large numbers of isolates. The development of a new method, mycobacterial interspersed repetitive units (MIRU), a variation of the original variable-number tandem repeat (VNTR) technique, may provide a viable alternative. Panels based on the original 12-loci MIRU (12MIRU), a combination of 12MIRU and remaining ETR loci (15MIRU-VNTR), and an extended panel with an additional 10 novel regions (25VNTR) were used to study three populations with varying degrees of epidemiological data. MIRU discrimination increased with panel size and the addition of spoligotyping. Combining these two techniques enabled a reduction in the panel size from 25 to 14 loci without a significant loss in discrimination. However, 25VNTR alone or in combination with spoligotyping still possessed weaker discrimination than RFLP for high-copy-number isolates.

Distinct clinical and epidemiological features of tuberculosis in New York City caused by the RD Rio Mycobacterium tuberculosis sublineage

BACKGROUND Genetic tracking of Mycobacterium tuberculosis is a cornerstone of tuberculosis (TB) control programs. The RD(Rio) M. tuberculosis sublineage was previously associated with TB in Brazil. We investigated 3847 M. tuberculosis isolates and registry data from New York City (NYC) (2001-2005) to: (1) affirm the position of RD(Rio) strains within the M. tuberculosis phylogenetic structure, (2) determine its prevalence, and (3) define transmission, demographic, and clinical characteristics associated with RD(Rio) TB. METHODS Isolates classified as RD(Rio) or non-RD(Rio) M. tuberculosis by multiplex PCR were further classified as clustered (≥2 isolates) or unique based primarily upon IS6110-RFLP patterns and lineage-specific cluster proportions were calculated. The secondary case rate of RD(Rio) was compared with other prevalent M. tuberculosis lineages. Genotype data were merged with the data from the NYC TB Registry to assess demographic and clinical characteristics. RESULTS RD(Rio) strains were found to: (1) be restricted to the Latin American-Mediterranean family, (2) cause approximately 8% of TB cases in NYC, and (3) be associated with heightened transmission as shown by: (i) a higher cluster proportion compared to other prevalent lineages, (ii) a higher secondary case rate, and (iii) cases in children. Furthermore, RD(Rio) strains were significantly associated with US-born Black or Hispanic race, birth in Latin American and Caribbean countries, and isoniazid resistance. CONCLUSIONS The RD(Rio) genotype is a single M. tuberculosis strain population that is emerging in NYC. The findings suggest that expanded RD(Rio) case and exposure identification could be of benefit due to its association with heightened transmission.

Can 15-Locus Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Analysis Provide Insight into the Evolution of Mycobacterium tuberculosis?

ABSTRACT The phylogeny and evolution of the bacterium Mycobacterium tuberculosis is still poorly understood despite the application of a variety of molecular techniques. We analyzed 469 M. tuberculosis and 49 Mycobacterium bovis isolates to evaluate if the mycobacterial interspersed repetitive units-variable-number tandem repeats (MIRU-VNTR) commonly used for epidemiological studies can define the phylogeny of the M. tuberculosis complex. This population was characterized by previously identified silent single-nucleotide polymorphisms (sSNPs) or by a macroarray based on these sSNPs that was developed in this study. MIRU-VNTR phylogenetic codes capable of differentiating between phylogenetic lineages were identified. Overall, there was 90.9% concordance between the lineages of isolates as defined by the MIRU-VNTR and sSNP analyses. The MIRU-VNTR phylogenetic code was unique to M. bovis and was not observed in any M. tuberculosis isolates. The codes were able to differentiate between different M. tuberculosis strain families such as Beijing, Delhi, and East African-Indian. Discrepant isolates with similar but not identical MIRU-VNTR codes often displayed a stepwise trend suggestive of bidirectional evolution. A lineage-specific panel of MIRU-VNTR can be used to subdivide each lineage for epidemiological purposes. MIRU-VNTR is a valuable tool for phylogenetic studies and could define an evolutionarily uncharacterized population of M. tuberculosis complex organisms.