Madalene Richardson

Proportion of tuberculosis transmission that takes place in households in a high-incidence area

The prevalence of infection among household contacts of people with tuberculosis is high. This information frequently guides active case finding. We analysed DNA fingerprints of Mycobacterium tuberculosis from 765 tuberculosis patients in Ravensmead and Uitsig, adjacent suburbs of Cape Town, South Africa. In 129 households in which DNA fingerprints were available for more than one patient, we identified 313 patients, of whom 145 (46%) had a fingerprint pattern matching that of another member of the household. The proportion of transmission in the community that took place in the household was 19%, and therefore, in this high-incidence area, tuberculosis transmission occurs mainly outside the household.

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.

Microevolution of the direct repeat region of Mycobacterium tuberculosis: Implications for interpretation of spoligotyping data

ABSTRACT The direct repeat (DR) region has been determined to be an important chromosomal domain for studying the evolution of Mycobacterium tuberculosis. Despite this, very little is known about microevolutionary events associated with clonal expansion and how such events influence the interpretation of both restriction fragment length polymorphism (RFLP) and spoligotype data. This study examined the structure of the DR region in three independently evolving lineages of M. tuberculosis with a combination of DR-RFLP, spoligotyping, and partial DNA sequencing. The results show that the duplication of direct variable repeat (DVR) sequences and single-nucleotide polymorphisms is rare; conversely, the deletion of DVR sequences and IS6110-mediated mutation is observed frequently. Deletion of either single or contiguous DVR sequences was observed. The deletion of adjacent DVR sequences occurred in a dependent manner rather than as an accumulation of independent events. Insertion of IS6110 into either the direct repeat or spacer sequences influenced the spoligotype pattern, resulting in apparent deletion of DVR sequences. Homologous recombination between adjacent IS6110 elements led to extensive deletion in the DR region, again demonstrating a dependent evolutionary mechanism. Different isolates from the same strain family and isolates from different strain families were observed to converge to the same spoligotype pattern. In conclusion, the binary data of the spoligotype are unable to provide sufficient information to accurately establish genotypic relationships between certain clinical isolates of M. tuberculosis. This has important implications for molecular epidemiologic strain tracking and for the application of spoligotype data to phylogenetic analysis of M. tuberculosis isolates.

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.

Impact of social interactions in the community on the transmission of tuberculosis in a high incidence area

BACKGROUND Tuberculosis (TB) is transmitted by close contact with an infectious person. It is assumed that close contact occurs amongst household members and that contact outside the house is “casual” and does not play a major role in the transmission of TB. METHODS This study was conducted in an impoverished area with a high incidence of TB and a low HIV seropositive prevalence. Thirty three households with 84 TB patients were identified between February 1993 and April 1996 and the transmission of TB was studied by combiningMycobacterium tuberculosis fingerprinting with in depth sociological interviews. RESULTS Forty two strain genotypes were identified in the 84 patients. In 15 households all the patients had identical strains, in nine households all the patients had different strains, and in nine households some patients had identical strains and one had a different strain. In 26 houses at least one patient had a strain which formed part of a larger community cluster and in 12 of these households the patient(s) had contact with a community member who had the identical strain. In 58% of the cases the contact took place while drinking in social groups. CONCLUSION In high incidence areas contact outside the household may be important for the transmission of TB. This contact often takes place during recreation which, in the case of this study of impoverished people, consisted of drinking in social groups. Social interaction patterns should be studied and understood for effective implementation of control strategies.

Multiple Mycobacterium tuberculosis Strains in Early Cultures from Patients in a High-Incidence Community Setting

ABSTRACT In an ongoing molecular epidemiology study, human immunodeficiency virus-negative patients with first-time pulmonary tuberculosis from a high-incidence community were enrolled. Mycobacterium tuberculosis strains were identified by restriction fragment length polymorphism analysis with two fingerprinting probes. Of 131 patients, 3 (2.3%) were shown to have a mixture of strains in one or two of their serial cultures. This study further investigated these cases with disease caused by multiple M. tuberculosis strains in the context of the molecular epidemiology of the study setting.

Calculation of the Stability of the IS6110 Banding Pattern in Patients with Persistent Mycobacterium tuberculosis Disease

ABSTRACT The interpretation of molecular epidemiologic data of Mycobacterium tuberculosis infection is dependent on the understanding of the stability and evolutionary characteristics of the DNA fingerprinting marker used to classify clinical isolates. This study investigated the stability of the IS6110 banding pattern in serial tuberculosis isolates collected from patients resident in an area with a high incidence of tuberculosis. Evolutionary changes were observed in 4% of the strains, and a half-life (t1/2) of 8.74 years was calculated, assuming a constant rate of change over time. This rate may be composed of a high rate of change seen during the early disease phase (t1/2 = 0.57 years) and a low rate of change seen in the late disease phase (t1/2 = 10.69 years). The early rate probably reflects change occurring during active growth prior to therapy, while the low late rate may reflect change occurring during or after treatment. We demonstrate that the calculation of these rates is strongly influenced by the time interval between onset of disease and sputum sampling. These calculations are further complicated by partial replacement of the original strain population, resulting in the sporadic appearance of clonal variants in sputum specimens. Therefore, the true extent of genetic diversity may be underestimated within each host, thereby influencing molecular epidemiological data used to establish transmission chains.

Evolution of the IS6110-Based Restriction Fragment Length Polymorphism Pattern during the Transmission of Mycobacterium tuberculosis

ABSTRACT Interpretation of the molecular epidemiological data of Mycobacterium tuberculosis is dependent on the validity of the assumptions that have been made. It is assumed that the IS6110 banding pattern is sufficiently stable to define epidemiological events representing ongoing transmission. However, molecular epidemiological data also support the observation that the IS6110 banding pattern may change over time. Factors affecting this rate may include the nature and duration of disease in a host and the opportunity to experience different host environments during the transmission cycle. To estimate the rate of IS6110 change occurring during the process of transmission, M. tuberculosis isolates from epidemiologically linked patients were genotypically characterized by restriction fragment length polymorphism (RFLP) analysis. The identification of IS6110 banding pattern changes during ongoing transmission suggested that a rate could be estimated. IS6110 change was significantly associated with strains with >5 IS6110 elements (P = 0.013) and was not observed in low-copy-number isolates. The minimum rate of appearance of variant strains was calculated to be 0.14 variant cases per source-case per year. This data suggest that clustering of isolates based on identical RFLP patterns is expected to underestimate transmission in patients infected with high-copy-number isolates. A model based on the rate of appearance of both variant and invariant strains demonstrates that the genotypically defined population structure may change by 18.6% during the study period of approximately 6.5 years. The implications for the use of RFLP data for epidemiologic study are discussed.

Mapping of IS6110 flanking regions in clinical isolates of Mycobacterium tuberculosis demonstrates genome plasticity.

Southern hybridization was used in combination with IS6110 insertion‐locus‐specific probes in a comparative study to determine the structure of chromosomal domains flanking IS6110 elements in clinical isolates of Mycobacterium tuberculosis. The resulting restriction fragment length polymorphism (RFLP) data demonstrated three mutational mechanisms responsible for the polymorphisms observed: IS6110 insertion, chromosomal mutation and deletion. The frequency of IS6110 insertion within many of the chromosomal regions demonstrates that preferential integration regions are common in M. tuberculosis. Mapping the IS6110 insertion positions and chromosomal deletions in relation to the M. tuberculosis H37Rv and M. bovis BCG genome sequences reveals numerous disruptions of predicted open reading frames (ORFs). A phylogenetic tree, based on the mutational data, showed a number of independently evolving lineages of M. tuberculosis, while analysis of the mutational events occurring at each branch point suggests both divergent and convergent evolution. A significant positive correlation was demonstrated between the mutation rate and the frequency of occurrence of different isolates in families of strains, suggesting that evolution may impact on strain ‘fitness’ or that strain proliferation may increase the chance of mutation. We conclude that the genome of clinical isolates of M. tuberculosis continues to evolve.

Clonal Expansion of a Globally Disseminated Lineage of Mycobacterium tuberculosis with Low IS6110 Copy Numbers

ABSTRACT Knowledge of the clonal expansion of Mycobacterium tuberculosis and accurate identification of predominant evolutionary lineages in this species remain limited, especially with regard to low-IS6110-copy-number strains. In this study, 170 M. tuberculosis isolates with ≤6 IS6110 insertions identified in Cape Town, South Africa, were characterized by principal genetic grouping, restriction fragment length polymorphism analysis, spoligotyping, IS6110 insertion site mapping, and variable-number tandem repeat (VNTR) typing. These analyses indicated that all but one of the isolates analyzed were members of principal genetic group 2 and of the same low-IS6110-copy-number lineage. The remaining isolate was a member of principal genetic group 1 and a different low-IS6110-copy-number lineage. Phylogenetic reconstruction suggests clonal expansion through sequential acquisition of additional IS6110 copies, expansion and contraction of VNTR sequences, and the deletion of specific direct-variable-repeat sequences. Furthermore, comparison of the genotypic data of 91 representative low-IS6110-copy-number isolates from Cape Town, other southern African regions, Europe, and the United States suggests that certain low-IS6110-copy-number strain spoligotypes and IS6110 fingerprints were acquired in the distant past. These clones have subsequently become widely disseminated and now play an important role in the global tuberculosis epidemic.