IS6110-based Restriction Fragment Length Polymorphism Research Articles

TB transmission is associated with prolonged stay in a low socio-economic, high burdened TB and HIV community in Cape Town, South Africa

BackgroundWhile several studies have assessed the associations between biological factors and tuberculosis (TB) transmission, our understanding of the associations between TB transmission and social and economic factors remains incomplete. We aimed to explore associations between community TB transmission and socio-economic factors within a high TB-HIV burdened setting.MethodsWe conducted a cross-sectional molecular epidemiology study among adult patients attending a routine TB clinic. Demographic and clinical data were extracted from TB registers and clinical folders; social and economic data were collected using interviewer-administered questionnaires; Mycobacterium tuberculosis isolates were genotyped and classified as clustered/non-clustered using IS6110-based Restriction Fragment Length Polymorphism. Composite “social” and “economic” scores were generated from social and economic data. Data were analyzed using StataCorp version 15.0 software. Stratified, bivariable analyses were performed using chi-squared. Wilcoxon signed rank tests; univariable and multivariable logistic regression models were developed to explore associations in the social, economic, traditional and composite TB risk factors with TB transmission.ResultsOf the 505 patient Mtb strains, 348(69%) cases were classified as clustered and 157(31%) were non-clustered. Clustered cases were more likely to have lived longer in the study community, (odds ratio [OR] = 1.05, 95% Confidence interval [C.I]:1.02–1.09, p = 0.006); in the same house (OR = 1.04, C.I: 0.99–1.08, p = 0.06); and had increased household crowding conditions (i.e fewer rooms used for sleeping, OR = 0.45, C.I:0.21–0.95, p = 0.04). Although a higher proportion of clustered cases had a low economic score, no statistically significant association was found between clustering and either the economic score (p = 0.13) or social score (p = 0.26).ConclusionsWe report a novel association between Mtb transmission and prolonged stay within a high burdened community. Transmission was also associated with fewer rooms for sleeping in a household. Increased social interaction and prolonged residence in a high burdened community are important factors linked to Mtb transmission, possibly due to increased probability of higher effective contact rates. The possible importance of degrees of poverty within low socio-economic setting warrants further study.

Genotypic Characterisation of Mycobacterium tuberculosis Isolates from Tuberculous Meningitis Patients at a Tertiary Neurocare Centre in Southern India

Aims: Specific genotypes of Mycobacterium tuberculosis (MTB) have been reported to cause outbreaks of pulmonary tuberculosis (TB) in geographical areas that are endemic to TB. However, since there is little epidemiological evidence on the association of particular genotypes that cause tuberculous meningitis (TBM), we sought to investigate the association of specific MTB strains with infection of the central nervous system (CNS). Materials and Methods: We carried out a genetic characterisation of 89 MTB isolates from TBM patients at a Southern Indian tertiary neurocare centre and compared the genotypes with strains of pulmonary TB isolated from Indian immigrants in New York City. We applied the standard methods of genotyping of MTB, namely, IS6110-based restriction fragment length polymorphism and spoligotyping for strain identification, along with principal genetic grouping and single-nucleotide polymorphism cluster analysis. Results: The analysis revealed a high-level of diversity amongst the strain population. The genotypes of the isolates from TBM patients paralleled the pulmonary TB strain population recovered from the Indian immigrants in NYC. Conclusions: We conclude that there is no apparent association between genotypes of MTB and propensity to infect CNS tissue.

Molecular Strain Typing of Mycobacterium tuberculosis: a Review of Frequently Used Methods.

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, remains one of the most serious global health problems. Molecular typing of M. tuberculosis has been used for various epidemiologic purposes as well as for clinical management. Currently, many techniques are available to type M. tuberculosis. Choosing the most appropriate technique in accordance with the existing laboratory conditions and the specific features of the geographic region is important. Insertion sequence IS6110-based restriction fragment length polymorphism (RFLP) analysis is considered the gold standard for the molecular epidemiologic investigations of tuberculosis. However, other polymerase chain reaction-based methods such as spacer oligonucleotide typing (spoligotyping), which detects 43 spacer sequence-interspersing direct repeats (DRs) in the genomic DR region; mycobacterial interspersed repetitive units–variable number tandem repeats, (MIRU-VNTR), which determines the number and size of tandem repetitive DNA sequences; repetitive-sequence-based PCR (rep-PCR), which provides high-throughput genotypic fingerprinting of multiple Mycobacterium species; and the recently developed genome-based whole genome sequencing methods demonstrate similar discriminatory power and greater convenience. This review focuses on techniques frequently used for the molecular typing of M. tuberculosis and discusses their general aspects and applications.

Epidemiologic Correlates of Pyrazinamide-Resistant Mycobacterium tuberculosis in New York City.

Pyrazinamide (PZA) has important sterilizing activity in tuberculosis (TB) chemotherapy. We describe trends, risk factors, and molecular epidemiology associated with PZA-resistant (PZA(r)) Mycobacterium tuberculosis in New York City (NYC). From 2001 to 2008, all incident culture-positive TB cases reported by the NYC Department of Health and Mental Hygiene (DOHMH) were genotyped by IS6110-based restriction fragment length polymorphism and spoligotype. Multidrug-resistant (MDR) isolates underwent DNA sequencing of resistance-determining regions of pncA, rpoB, katG, and fabG1. Demographic and clinical information were extracted from the NYC DOHMH TB registry. During this period, PZA(r) doubled (1.6% to 3.6%) overall, accounting for 44% (70/159) of the MDR population and 1.4% (75/5511) of the non-MDR population. Molecular genotyping revealed strong microbial phylogenetic associations with PZA(r). Clustered isolates and those from acid-fast bacillus (AFB) smear-positive cases had 2.7 (95% confidence interval [CI] = 1.71 to 4.36) and 2.0 (95% CI = 1.19 to 3.43) times higher odds of being PZA(r), respectively, indicating a strong likelihood of recent transmission. Among the MDR population, PZA(r) was acquired somewhat more frequently via primary transmission than by independent pathways. Our molecular analysis also revealed that several historic M. tuberculosis strains responsible for MDR TB outbreaks in the early 1990s were continuing to circulate in NYC. We conclude that the increasing incidence of PZA(r), with clear microbial risk factors, underscores the importance of routine PZA drug susceptibility testing and M. tuberculosis genotyping for the identification, control, and prevention of increasingly resistant organisms.

Transmission of tuberculosis in a South African community with a high prevalence of HIV infection.

In settings of high tuberculosis transmission, little is known of the interaction between human immunodeficiency virus (HIV) positive and HIV-negative tuberculosis disease and of the impact of antiretroviral treatment (ART) programs on tuberculosis transmission dynamics. Mycobacterium tuberculosis isolates were collected from patients with tuberculosis who resided in a South African township with a high burden of tuberculosis and HIV infection. Demographic and clinical data were extracted from clinic records. Isolates underwent IS6110-based restriction fragment length polymorphism analysis. Patients with unique (nonclustered) M. tuberculosis genotypes and cluster index cases (ie, the first tuberculosis case in a cluster) were defined as having tuberculosis due to reactivation of latent M. tuberculosis infection. Secondary cases in clusters were defined as having tuberculosis due to recent M. tuberculosis infection. Overall, 311 M. tuberculosis genotypes were identified among 718 isolates from 710 patients; 224 (31%) isolates were unique strains, and 478 (67%) occurred in 87 clusters. Cluster index cases were significantly more likely than other tuberculosis cases to be HIV negative. HIV-positive patients were more likely to be secondary cases (P = .001), including patients receiving ART (P = .004). Only 8% of cases of adult-adult transmission of tuberculosis occurred on shared residential plots. Recent infection accounted for the majority of tuberculosis cases, particularly among HIV-positive patients, including patients receiving ART. HIV-negative patients may be disproportionally responsible for ongoing transmission.

Factors affecting tuberculosis strain success over 10 years in a high TB- and HIV-burdened community

Factors associated with Mycobacterium tuberculosis (Mtb) strain success over time in high burdened communities are unknown. Mtb isolates collected over 10 years from sputum-positive tuberculosis (TB) patients resident in the study site underwent IS6110-based restriction fragment length polymorphism analysis. Clinical, demographic and social data were extracted from clinic records and interviewer-administered questionnaires. Strains were defined as persistently successful, transiently successful or unsuccessful based on the average number of cases per year and their continued presence over time. Genotyping data were available on 789 TB cases. Of the 311 distinct Mtb strains (≥6 bands) identified, 247 were categorized as unsuccessful strains, 12 transiently successful and 10 persistently successful strains. Strain success was not associated with age, gender, antiretroviral use or social factors. Persistently successful strains were less likely to be drug-resistant compared with transiently successful strains [odds ratio (OR): 0.13; 95% confidence interval (CI): 0.04 - 0.5]. Persistently successful strains were positively associated with host HIV-infection compared with unsuccessful strains, but this finding was not robust in sensitivity analyses. Pathogen characteristics appear to play a greater role in Mtb strain success compared with social or host factors. This study supports the need for further investigations into the role of pathogen characteristics in strain success.

Nosocomial transmission of extensively drug-resistant tuberculosis in a rural hospital in South Africa.

Extensively drug-resistant tuberculosis (XDR-tuberculosis) is a global public health threat, but few data exist elucidating factors driving this epidemic. The initial XDR-tuberculosis report from South Africa suggested transmission is an important factor, but detailed epidemiologic and molecular analyses were not available for further characterization. We performed a retrospective, observational study among XDR-tuberculosis patients to identify hospital-associated epidemiologic links. We used spoligotyping, IS6110-based restriction fragment-length polymorphism analysis, and sequencing of resistance-determining regions to identify clusters. Social network analysis was used to construct transmission networks among genotypically clustered patients. Among 148 XDR-tuberculosis patients, 98% were infected with human immunodeficiency virus (HIV), and 59% had smear-positive tuberculosis. Nearly all (93%) were hospitalized while infectious with XDR-tuberculosis (median duration, 15 days; interquartile range: 10-25 days). Genotyping identified a predominant cluster comprising 96% of isolates. Epidemiologic links were identified for 82% of patients; social network analysis demonstrated multiple generations of transmission across a highly interconnected network. The XDR-tuberculosis epidemic in Tugela Ferry, South Africa, has been highly clonal. However, the epidemic is not the result of a point-source outbreak; rather, a high degree of interconnectedness allowed multiple generations of nosocomial transmission. Similar to the outbreaks of multidrug-resistant tuberculosis in the 1990s, poor infection control, delayed diagnosis, and a high HIV prevalence facilitated transmission. Important lessons from those outbreaks must be applied to stem further expansion of this epidemic.

English

There is scarcity of data on molecular epidemiology of tuberculosis (TB) in many of the high burden countries of sub-Saharan Africa including Nigeria. This may be due to inadequate diagnostic and research capacities. There is a need to build capacity on molecular epidemiologic techniques in order to enhance TB diagnosis, treatment and prevention strategies. Some of the more popular molecular typing methods being used globally include IS6110-based restriction fragment length polymorphism (RFLP),polymerase chain reaction (PCR) -based methods such as spacer oligonucleotide typing or spoligotyping, mycobacterial interspersed repetitive units-multilocus variable number tandem repeats (MIRU-VNTR), single –nucleotide polymorphisms and large –sequence polymorphism analysis. Among other things, molecular technique(s) to be adopted for use in a particular setting depend on the state of the infrastructure including laboratory facilities, presence of skilled personnel and cost. In Nigeria however, availability of PCR-based diagnostic assay –MTBDR plus assay (HAIN- Lifesciences, Germany) in some TB reference laboratories may serve as a springboard for introduction of advanced TB molecular epidemiologic tools in the country. Key words: Molecular epidemiologic tools, tuberculosis, Nigeria.

Factors associated with recently transmitted Mycobacterium tuberculosis strain MS0006 in Hinds County, Mississippi.

The objective of this study was to investigate risk factors associated with tuberculosis (TB) transmission that was caused by Mycobacterium tuberculosis strain MS0006 from 2004 to 2009 in Hinds County, Mississippi. DNA fingerprinting using spoligotyping, mycobacterial interspersed repetitive unit, and IS6110-based restriction fragment length polymorphism of culture-confirmed cases of TB was performed. Clinical and demographic factors associated with strain MS0006 were analyzed by univariate and multivariate analysis. Of the 144 cases of TB diagnosed during the study period, 117 were culture positive with fingerprints available. There were 48 different strains, of which 6 clustered strains were distributed among 74 patients. The MS0006 strain accounted for 46.2% of all culture-confirmed cases. Risk factors for having the MS0006 strain in a univariate analysis included homelessness, HIV co-infection, sputum smear negativity, tuberculin skin test negativity, and noninjectable drug use. Multivariate analysis identified homelessness (odds ratio 7.88, 95% confidence interval 2.90-21.35) and African American race (odds ratio 5.80, 95% confidence interval 1.37-24.55) as independent predictors of having TB caused by the MS0006 strain of M. tuberculosis. Our findings suggest that a majority of recently transmitted TB in the studied county was caused by the MS0006 strain. African American race and homelessness were significant risk factors for inclusion in the cluster. Molecular epidemiology techniques continue to provide in-depth analysis of disease transmission and play a vital role in effective contact tracing and interruption of ongoing transmission.

Systematic Survey of Clonal Complexity in Tuberculosis at a Populational Level and Detailed Characterization of the Isolates Involved

Clonally complex infections by Mycobacterium tuberculosis are progressively more accepted. Studies of their dimension in epidemiological scenarios where the infective pressure is not high are scarce. Our study systematically searched for clonally complex infections (mixed infections by more than one strain and simultaneous presence of clonal variants) by applying mycobacterial interspersed repetitive-unit (MIRU)-variable-number tandem-repeat (VNTR) analysis to M. tuberculosis isolates from two population-based samples of respiratory (703 cases) and respiratory-extrapulmonary (R+E) tuberculosis (TB) cases (71 cases) in a context of moderate TB incidence. Clonally complex infections were found in 11 (1.6%) of the respiratory TB cases and in 10 (14.1%) of those with R+E TB. Among the 21 cases with clonally complex TB, 9 were infected by 2 independent strains and the remaining 12 showed the simultaneous presence of 2 to 3 clonal variants. For the 10 R+E TB cases with clonally complex infections, compartmentalization (different compositions of strains/clonal variants in independent infected sites) was found in 9 of them. All the strains/clonal variants were also genotyped by IS6110-based restriction fragment length polymorphism analysis, which split two MIRU-defined clonal variants, although in general, it showed a lower discriminatory power to identify the clonal heterogeneity revealed by MIRU-VNTR analysis. The comparative analysis of IS6110 insertion sites between coinfecting clonal variants showed differences in the genes coding for a cutinase, a PPE family protein, and two conserved hypothetical proteins. Diagnostic delay, existence of previous TB, risk for overexposure, and clustered/orphan status of the involved strains were analyzed to propose possible explanations for the cases with clonally complex infections. Our study characterizes in detail all the clonally complex infections by M. tuberculosis found in a systematic survey and contributes to the characterization that these phenomena can be found to an extent higher than expected, even in an unselected population-based sample lacking high infective pressure.

Population-based investigation of fluoroquinolones resistant tuberculosis in rural eastern China

Empirical use of fluoroquinolones (FQ) to treat a variety of bacterial infections may inadvertently select for FQ-resistant strains of Mycobacterium tuberculosis(MTB), especially in rural China where the use of FQ in treating infections has not been standardized. Here we determine the prevalence and describe the transmission of FQ-resistant MTB in two rural counties in eastern China through a combination of conventional epidemiology with IS6110-based restriction fragment length polymorphism(RFLP) analysis and DNA sequencing of drug-resistance determining regions. Phenotypic FQ resistance was detected in 31 of 351(8.8%) isolates. FQ resistance was equally distributed between patient-isolates deemed drug resistant and drug-susceptible, but mostly observed in those with treatment history of respiratory infection. Mutations in gyrA were found in 54.8% of FQ resistant isolates, and one isolate with a gyrB mutation. Despite predominating in entire bacilli population(69.2%), Beijing family strain had similar proportion of FQ resistance to the other(10.3% vs. 4.7%, p = 0.060). IS6110RFLP identified 2 clusters(4 isolates) among FQ resistant isolates and 3 clusters composed of both 4 FQ resistant isolates and 6 FQ susceptible isolates. Our results indicate that FQ-resistant MTB has emerged among the circulating bacillary population in rural eastern China. The relatively low level of clustering among FQ-resistant strains suggests most are acquired de novo, likely due to widespread FQ use.

Molecular Detection of Mixed Infections of Mycobacterium tuberculosis Strains in Sputum Samples from Patients in Karonga District, Malawi

The occurrence of mixed infections of Mycobacterium tuberculosis is no longer disputed. However, their frequency, and the impact they may have on our understanding of tuberculosis (TB) pathogenesis and epidemiology, remains undetermined. Most previous studies of frequency applied genotyping techniques to cultured M. tuberculosis isolates and found mixed infections to be rare. PCR-based techniques may be more sensitive for detecting multiple M. tuberculosis strains and can be applied to sputum. To date, one study in South Africa has used a PCR approach and suggested that mixed infection could be common. We investigated mixed infections in northern Malawi using two lineage-specific PCR assays targeting the Latin American-Mediterranean (LAM) and Beijing lineages. Compared with spoligotyping, the specificity and sensitivity of both assays was 100%. From 160 culture-positive sputa, mixed LAM and non-LAM strains were detected in 4 sputa belonging to 2 (2.8%) patients. Both patients were HIV positive, with no history of TB. Cultured isolates from both patients showed only LAM by PCR and spoligotyping. In a set of 377 cultured isolates, 4 were mixed LAM and non-LAM. Only one showed evidence of more than one M. tuberculosis strain using IS6110-based restriction fragment length polymorphism (IS6110-RFLP) and spoligotyping analyses. Corresponding sputa for the 4 isolates were unavailable. Mixed Beijing and non-Beijing strains were not detected in this study. Mixed infections appear to be rare in our setting and are unlikely to affect findings based on DNA fingerprinting data. Molecular methods, which avoid the selective nature of culture and target distinct strains, are well suited to detection of mixed infections.

Mycobacterium tuberculosis Spoligotypes in Monterrey, Mexico

Although tuberculosis is still a public health problem in Mexico, there is little information about the genetic characteristics of the isolates. In the present study, we analyzed by spoligotyping 180 Mycobacterium tuberculosis clinical isolates from the urban area of Monterrey, Mexico, including drug-susceptible and drug-resistant isolates. The spoligotype patterns were compared with those in the international SITVIT2 spoligotyping database. Four isolates presented spoligotype patterns not found in the database (orphan types); the rest were distributed among 44 spoligo international types (SITs). SIT53 (clade T1) and SIT119 (clade X1) were predominant and included 43 (23.8%) and 28 (15.5%) of the isolates, respectively. In order to determine if there was a dominant spoligotype in the group of multidrug-resistant isolates, 37 of them were analyzed by IS6110-based restriction fragment length polymorphism assays, and scarce clustering of strains with more than five bands was observed. Fourteen isolates of this multidrug-resistant group presented four bands or less and were distributed in four SITs: SIT53 (n = 8), SIT92 (n = 3), SIT70 (n = 2), and SIT3038 (n = 1). When the molecular detection of mutations in the katG and rpoB genes were analyzed in these isolates with low copy numbers of IS6110, only two isolates shared the same IS6110, spoligotyping, and mutations patterns. When the distribution of the spoligotypes was analyzed by age cohort, SIT119 was predominantly found in patients 0 to 20 years old, especially in males, accounting for up to 40% of the isolates. In contrast, SIT53 was more prevalent in older females. This analysis demonstrates the variability of M. tuberculosis isolates in Monterrey and the partial dominance of SIT53 and SIT119 in that area of Mexico.

Molecular Epidemiology ofMycobacterium tuberculosisin a South African Community with High HIV Prevalence

To explore the relationship between human immunodeficiency virus (HIV) and Mycobacterium tuberculosis genotypes, we performed IS6110-based restriction fragment-length polymorphism analysis on M. tuberculosis culture specimens from patients with smear-positive tuberculosis in a periurban community in South Africa from 2001 through 2005. Among 151 isolates, 95 strains were identified within 26 families, with 54% clustering. HIV status was associated with W-Beijing strains (P = .009) but not with clustering per se. The high frequency of clustering suggests ongoing transmission in both HIV-negative and HIV-positive individuals in this community. The strong association between W-Beijing and HIV infection may have important implications for tuberculosis control.

Molecular Analysis of Isoniazid Resistance in Different Genotypes ofMycobacterium tuberculosisIsolates from Iran

Considering the significant increase of isoniazid (INH) resistance in Iranian Mycobacterium tuberculosis isolates in the last few years and to investigate the prevalence and diagnostic potential of the most commonly reported mutations associated with INH resistance in Iran, we analyzed parts of the katG gene and fabG1-inhA and oxyR-ahpC regulatory regions in a sample of 48 INH-resistant and 25 INH-sensitive isolates. Mutations in the katG 315, fabG1-inhA, and oxyR-ahpC regulatory regions were detected in 58.3%, 18.7%, and 39.6% of isolates, respectively. The R463L polymorphism in the katG gene and the ahpC46A were detected with high frequency in both INH-resistant and -sensitive isolates. Spoligotyping and IS6110-based restriction fragment length polymorphism patterns revealed that most of the isolates containing ahpC46A and katG 463Leu polymorphism belonged to the Central Asian (CAS) super family. The tight relationship between ahpC46A and katG 463Leu polymorphisms and the CAS super family highlights the importance of the CAS super family in INH resistance in Iran. In conclusion, mutations at katG codon 315 or the fabG1-inhA regulatory region were identified in 77.0% of the INH-resistant isolates and in none of the INH-sensitive strains, and are highly predictive of isonizid resistance in Iranian isolates.

Predominance of Mycobacterium tuberculosis EAI and Beijing Lineages in Yangon, Myanmar

Isolates of the Mycobacterium tuberculosis Beijing lineage are associated with high rates of transmission, hypervirulence and drug resistance. The Beijing lineage has been shown to dominate the tuberculosis (TB) epidemic in East Asia; however, the diversity and frequency of M. tuberculosis genotypes from Myanmar are unknown. We present the first comprehensive study describing the M. tuberculosis isolates circulating in Yangon, Myanmar. Thus, 310 isolates from pulmonary TB patients from Yangon, Myanmar, were genotyped by spoligotyping and IS6110-based restriction fragment length polymorphism analysis (IS6110 RFLP). The most frequent lineages observed were the East African-Indian (EAI; 48.4%; n = 150) and Beijing (31.9%; n = 99) lineages. Isolates belonging to the most frequent shared types (STs), ST1 (n = 98; Beijing), ST292 (n = 28; EAI), and ST89 (n = 11; EAI), had >or=75% similarity in their IS6110 patterns. Five of 11 Beijing isolates comprising five clusters with identical IS6110 RFLP patterns could be discriminated by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. Of the 150 EAI isolates, 40 isolates (26.7%) had only one IS6110 copy, and 17 of these isolates could be discriminated by MIRU-VNTR analysis. The findings from this study suggest that although there is a predominance of the ancient EAI lineage in Yangon, the TB epidemic in Yangon is driven by clonal expansion of the ST1 genotype. The Beijing lineage isolates (21.4%) were more likely (P = 0.009) than EAI lineage isolates to be multidrug resistant (MDR) (1.3%; odds ratio, 3.2, adjusted for the patients' history of exposure to anti-TB drugs), suggesting that the spread of MDR Beijing isolates is a major problem in Yangon.

Factors Associated with Differences between Conventional Contact Tracing and Molecular Epidemiology in Study of Tuberculosis Transmission and Analysis in the City of Barcelona, Spain

The aim of this study was to analyze the factors associated with conventional contact tracing (CCT) and molecular epidemiology (ME) methods in assessing tuberculosis (TB) transmission, comparing the populations studied and the epidemiological links established by both methods. Data were obtained from TB case and CCT registries, and ME was performed using IS6110-based restriction fragment length polymorphism (RFLP) analysis and mycobacterial interspersed repetitive unit 12 (MIRU12) typing as a secondary typing method. During two years (2003 and 2004), 892 cases of TB were reported, of which 687 (77%) were confirmed by culture. RFLP analysis was performed with 463 (67.4%) of the 687 isolated strains, and MIRU12 types in 75 strains were evaluated; 280 strains (60.5%) had a unique RFLP pattern, and 183 (39.5%) shared patterns, grouping into 65 clusters. CCT of 613 (68.7%) of 892 cases detected 44 clusters involving 101 patients. The results of both CCT and ME methods yielded 96 clusters involving 255 patients. The household link was the one most frequently identified by CCT (corresponding to 80.7% of the cases clustered by this method), whereas nonhousehold and unknown links were associated with 94.1% of the strains clustered by ME. When both methods were used in 351 cases (39.3%), they showed the same results in 214 cases (61%). Of the remainder, 106 (30.2%) were clustered only by ME, 19 (5.5%) were clustered only by CCT, and 12 (3.4%) were clustered by both methods but into different clusters. Patients with factors potentially associated with social problems were less frequently studied by CCT (P = 0.002), whereas patients of <15 years of age, most with negative cultures, were less frequently studied by ME (P = 0.005). Significant differences in the populations studied by ME versus CCT were observed, possibly explaining the scarce correlation found between the results of these methods. Moreover, ME allowed the detection of nonhousehold contact relationships, whereas CCT was more useful for tracing transmission chains involving patients of <15 years of age. In conclusion, the two methods are complementary, suggesting the need to improve the methodology of contact study protocols.

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

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 approximately 30% of strains. A single novel long sequence polymorphism (>26.3 kb) was characterized and designated RD(Rio). 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 RD(Rio) 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 RD(Rio) strains are derived from a common ancestor belonging solely to the Latin American-Mediterranean spoligotype family. The RD(Rio) 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 RD(Rio) 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 RD(Rio) 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 RD(Rio) M. tuberculosis.

Endogenous reactivation and true treatment failure as causes of recurrent tuberculosis in a high incidence setting with a low HIV infection

To determine the relative frequencies of reinfection vs. reactivation or treatment failure in patients from a high tuberculosis incidence setting with a low prevalence of HIV infection. We performed DNA fingerprinting on serial isolates from one and multiple TB episodes from 97 retreatment patients; 35 patients had been previously cured, whereas 62 had not. DNA fingerprinting patterns of recurrence Mycobacterium tuberculosis isolates of 5 of the 35 previously cured patients did not match with those of the corresponding initial isolates, indicating reinfection. We did not document reinfection during treatment. Isolates from each of the remaining 30 previously cured patients had identical DNA fingerprinting results, indicating reactivation. DNA fingerprinting patterns of isolates from the 62 patients with persistently positive sputum smears were identical, suggesting treatment failure. These findings suggest that reinfection is not a common cause of relapse and treatment failure in this rural predominantly HIV-free population despite the high incidence of TB.

P1835 Genotypic characterisation of Mycobacterium tuberculosis isolates from Bogotá, Colombia based on IS6110–based restriction fragment length polymorphism

P1835 Genotypic characterisation of Mycobacterium tuberculosis isolates from Bogotá, Colombia based on IS6110–based restriction fragment length polymorphism