IS6110 DNA Fingerprinting Research Articles

Whole genome sequencing provides additional insights into recurrent tuberculosis classified as endogenous reactivation by IS6110 DNA fingerprinting

Recurrent tuberculosis (TB) after successful TB treatment occurs due to endogenous reactivation (relapse) or exogenous reinfection. We revisited the conclusions of relapse in a high TB incidence setting that were drawn on the basis of IS6110 restriction fragment length polymorphism (RFLP) analysis in a large retrospective cohort study in suburban Cape Town, South Africa. Using whole genome sequencing (WGS), we undertook pair-wise genome comparison of Mycobacterium tuberculosis strains cultured from diagnostic sputum samples collected at the index and recurrent TB episode for 25 recurrent TB cases who had been classified as relapse based on identical DNA fingerprint patterns in the earlier study. We found that paired strain genome sequences were identical or showed minimal variant differences in 22 of 25 recurrent TB cases, consistent with relapse. One showed 20 variant differences, suggestive of exogenous reinfection. Two of the 25 had mixed infections, each with the index episode strain detected as the dominant strain at recurrence in one of these patients, the minority strain harboured drug-resistance conferring mutations (rpoB, katG). In conclusion, our study highlights the additional value of WGS for investigating recurrent TB in settings with high infection pressure and closely related circulating strains, where the extent of re- and mixed infection may be underestimated.

Integration of molecular typing results into tuberculosis surveillance in Germany-A pilot study.

An integrated molecular surveillance for tuberculosis (TB) improves the understanding of ongoing TB transmission by combining molecular typing and epidemiological data. However, the implementation of an integrated molecular surveillance for TB is complex and requires thoughtful consideration of feasibility, demand, public health benefits and legal issues. We aimed to pilot the integration of molecular typing results between 2008 and 2010 in the German Federal State of Baden-Württemberg (population 10.88 Million) as preparation for a nationwide implementation. Culture positive TB cases were typed by IS6110 DNA fingerprinting and results were integrated into routine notification data. Demographic and clinical characteristics of cases and clusters were described and new epidemiological links detected after integrating typing data were calculated. Furthermore, a cross-sectional survey was performed among local public health offices to evaluate their perception and experiences. Overall, typing results were available for 83% of notified culture positive TB cases, out of which 25% were clustered. Age <15 years (OR = 4.96, 95% CI: 1.69–14.55) and being born in Germany (OR = 2.01, 95% CI: 1.44–2.80) were associated with clustering. At cluster level, molecular typing information allowed the identification of previously unknown epidemiological links in 11% of the clusters. In 59% of the clusters it was not possible to identify any epidemiological link. Clusters extending over different counties were less likely to have epidemiological links identified among their cases (OR = 11.53, 95% CI: 3.48–98.23). The majority of local public health offices found molecular typing useful for their work. Our study illustrates the feasibility of integrating typing data into the German TB notification system and depicts its added public health value as complementary strategy in TB surveillance, especially to uncover transmission events among geographically separated TB patients. It also emphasizes that special efforts are required to strengthen the communication between local public health offices in different counties to enhance TB control.

The Evolution of Strain Typing in the Mycobacterium tuberculosis Complex.

Tuberculosis (TB) is a contagious disease with a complex epidemiology. Therefore, molecular typing (genotyping) of Mycobacterium tuberculosis complex (MTBC) strains is of primary importance to effectively guide outbreak investigations, define transmission dynamics and assist global epidemiological surveillance of the disease. Large-scale genotyping is also needed to get better insights into the biological diversity and the evolution of the pathogen. Thanks to its shorter turnaround and simple numerical nomenclature system, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) typing, based on 24 standardized plus 4 hypervariable loci, optionally combined with spoligotyping, has replaced IS6110 DNA fingerprinting over the last decade as a gold standard among classical strain typing methods for many applications. With the continuous progress and decreasing costs of next-generation sequencing (NGS) technologies, typing based on whole genome sequencing (WGS) is now increasingly performed for near complete exploitation of the available genetic information. However, some important challenges remain such as the lack of standardization of WGS analysis pipelines, the need of databases for sharing WGS data at a global level, and a better understanding of the relevant genomic distances for defining clusters of recent TB transmission in different epidemiological contexts. This chapter provides an overview of the evolution of genotyping methods over the last three decades, which culminated with the development of WGS-based methods. It addresses the relative advantages and limitations of these techniques, indicates current challenges and potential directions for facilitating standardization of WGS-based typing, and provides suggestions on what method to use depending on the specific research question.

Rapid and simple identification of Beijing genotype strain of Mycobacterium tuberculosis using a loop-mediated isothermal amplification assay.

Beijing genotype strains of Mycobacterium tuberculosis are geographically widespread and pose a notorious public health problem, these strains causing outbreaks of multidrug-resistant tuberculosis (TB); some studies have reported an association with drug resistance. Because the prevalence of Beijing strain has a substantial impact on TB control programs, the availability of a rapid and reliable method for detecting these strains is important for epidemiological monitoring of their circulation. The main methods currently used to identify Beijing genotype strains are IS6110 DNA fingerprinting, spoligotyping and PCR to detect specific deletions such as region of difference (RD)207. More recently, multiplex PCR assay using a Beijing-specific single nucleotide polymorphism (SNP) has been developed for detecting Beijing lineage strains. However, these methods are time-consuming and technically demanding. In the present study, a loop-mediated isothermal amplification (LAMP) assay that allows specific identification of Beijing genotype strain was developed. This Beijing genotype strain-identifying LAMP assay was performed 214 clinical isolates and the results compared with those of conventional PCR that targeted RD207 and Rv0679c-targreting multiplex PCR for Beijing lineage identification. LAMP assay showed 100% sensitivity and specificity compared with RD207-PCR. Furthermore, the sensitivity and specificity were 99.3% and 100%, respectively, compared with Rv0679c-multiplex PCR. This LAMP assay could be used routinely in local laboratories to monitor the prevalence of the Beijing genotype strain and thereby used to help control the spread of these potentially highly virulent and drug resistant strains.

Molecular Epidemiological Interpretation of the Epidemic of Extensively Drug-Resistant Tuberculosis in South Africa

We show that the interpretation of molecular epidemiological data for extensively drug-resistant tuberculosis (XDR-TB) is dependent on the number of different markers used to define transmission. Using spoligotyping, IS6110 DNA fingerprinting, and DNA sequence data, we show that XDR-TB in South Africa (2006 to 2008) was predominantly driven by the acquisition of second-line drug resistance.

Evaluation of Mycobacterium tuberculosis Typing Methods in a 4-Year Study in Schleswig-Holstein, Northern Germany

In order to evaluate the discriminatory power of different methods for genotyping of Mycobacterium tuberculosis complex (MTBC) isolates, we compared the performance of (i) IS6110 DNA fingerprint typing, (ii) spoligotyping, and (iii) 24-loci mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing in a long-term study on the epidemiology of tuberculosis (TB) in Schleswig-Holstein, the northernmost federal state of Germany. In total, we analyzed 277 MTBC isolates collected from patients between the years 2006 and 2010. The collection comprised a broad spectrum of 13 different genotypes, among which strains of the Haarlem genotype (31%) were most prominent, followed by strains belonging to the Delhi and Beijing lineages (7% and 6%, respectively). On the basis of IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping analyses, 211 isolates had unique patterns (76%) and 66 isolates (24%) were in 20 clusters. MIRU-VNTR combined with spoligotyping analyses revealed 202 isolates with unique patterns (73%) and 75 isolates in 18 clusters (27%). Overall, there was 93.1% concordance between the typing results obtained; 198 strains were identified as unique, and 60 isolates were clustered by both typing combinations (including all 31 isolates with confirmed epidemiological links). Of the remaining 19 isolates with discrepant results, 15 were falsely clustered by MIRU-VNTR (six Beijing genotype strains) and four were clustered by IS6110 RFLP (low IS6110 copy number) only. In conclusion, in the study population investigated, a minority of isolates, especially of the Beijing genotype, clustered by standard 24-loci MIRU-VNTR and without an obvious epidemiological link may require second-line typing by IS6110 RFLP or hypervariable MIRU-VNTR loci.

Using a Label-free Proteomics Method to Identify Differentially Abundant Proteins in Closely Related Hypo- and Hypervirulent Clinical Mycobacterium tuberculosis Beijing Isolates

Although the genome of the Mycobacterium tuberculosis H37Rv laboratory strain has been available for over 10 years, it is only recently that genomic information from clinical isolates has been used to generate the hypothesis of virulence differences between different strains. In addition, the relationship between strains displaying differing virulence in an epidemiological setting and their behavior in animal models has received little attention. The potential causes for variation in virulence between strains, as determined by differential protein expression, have similarly been a neglected area of investigation. In this study, we used a label-free quantitative proteomics approach to estimate differences in protein abundance between two closely related Beijing genotypes that have been shown to be hyper- and hypovirulent on the basis of both epidemiological and mouse model studies. We were able to identify a total of 1668 proteins from both samples, and protein abundance calculations revealed that 48 proteins were over-represented in the hypovirulent isolate, whereas 53 were over-represented in the hypervirulent. Functional classification of these results shows that molecules of cell wall organization and DNA transcription regulatory proteins may have a critical influence in defining the level of virulence. The reduction in the presence of ESAT-6, other Esx-like proteins, and FbpD (MPT51) in the hypervirulent strain indicates that changes in the repertoire of highly immunogenic proteins can be a defensive process undertaken by the virulent cell. In addition, most of the previously well characterized gene targets related to virulence were found to be similarly expressed in our model. Our data support the use of proteomics as a complementary tool for genomic comparisons to understand the biology of M. tuberculosis virulence.

Transmission dynamics of pulmonary tuberculosis between autochthonous and immigrant sub-populations

BackgroundThe overall incidence of tuberculosis (TB) in Western Europe has been declining since the 19th Century. However, immigrant sub-groups from high-prevalence countries are slowing down this trend. The aim of this study was to describe how immigration influences TB transmission in Germany. For that we prospectively investigated the dynamics of TB transmission between TB high-prevalence immigrant and TB low-prevalence local populations with molecular epidemiological methods and conventional contact investigations. Besides, we assessed transmission in relation to social mixing using an innovative tool that measures the integration of immigrants into the local social environment.MethodsA prospective study of confirmed culture positive cases of pulmonary TB and their contacts was carried out in a German federal state from 2003 to 2005. Data for the study included: 1) case data routinely collected by the local public health staff and transmitted to the state health office and the national surveillance centre, 2) a study questionnaire designed to capture social interactions of relevance for TB transmission and 3) molecular genotyping data (IS6110 DNA fingerprint and spoligotyping). The proportion of German cases caused by foreign-born cases, and vice versa, was estimated and an integration index was computed using a selected set of questions from the study questionnaire.ResultsA total of 749 cases of culture-positive pulmonary tuberculosis voluntarily enrolled in the study, representing 57.8% of all registered cases diagnosed over the study period. Data that included study questionnaire and DNA fingerprinting were available for 41% (n = 308) of the study participants. Forty-seven clusters, defined as a least two cases infected by the same TB strains, were identified by molecular methods and included 132 (17%) of the study participants. Epidemiological links were identified for 28% of the clusters by conventional epidemiological data. In mixed clusters, defined as clusters including German and foreign-born individuals, the probability of cases to be caused by foreign-born cases was estimated at 18.3%. We observed a trend to mixed clusters with increasing time spent by immigrants in the host country. This group also presented comparatively higher integration indexes than immigrants in immigrant-only clusters.ConclusionOur results confirm the findings of other studies that there is no significant TB transmission from TB high-prevalence immigrant to TB low-prevalence autochthonous population. This may be explained by the good performance of tuberculosis screening programmes for certain groups arriving in Germany from high- prevalence countries, by a low degree of mixing of immigrants with the local population or by a combination of both.

Mycobacterium tuberculosis Transmission Is Not Related to Household Genotype in a Setting of High Endemicity

Among the different strains of Mycobacterium tuberculosis, Beijing has been identified as an emerging genotype. Enhanced transmissibility provides a potential mechanism for genotype selection. This study evaluated whether the Beijing genotype is more readily transmitted than other prevalent genotypes to children in contact with an adult tuberculosis (TB) index case in the child's household. We conducted a prospective, community-based study at two primary health care clinics in Cape Town, South Africa, from January 2003 through December 2004. Bacteriologically confirmed new adult pulmonary TB cases were genotyped by IS6110 DNA fingerprinting; household contacts less than 5 years were traced and screened for M. tuberculosis infection and/or disease. A total of 187 adult index cases were identified from 174 households with children aged less than 5 years. Of 261 child contacts aged 0 to 5 years, 219 (83.9%) were completely evaluated and the isolate from the index case was successfully genotyped. M. tuberculosis infection (induration of >or=10 mm by Mantoux tuberculin skin test) was documented in 118/219 (53.9%) children; 34 (15.5%) had radiographic signs suggestive of active TB. There was no significant difference in the ratio of infected children among those exposed to the Beijing genotype (51/89; 57.3%) and those exposed to non-Beijing genotypes (55/115; 47.8%) (odds ratio, 1.5; 95% confidence interval, 0.8 to 2.7). Genotyping was successful for six children diagnosed with active TB; the isolates from only two children had IS6110 fingerprints that were identical to the IS6110 fingerprint of the isolate from the presumed index case. We found no significant association between the M. tuberculosis genotype and transmissibility within the household. However, undocumented M. tuberculosis exposure may have been a major confounding factor in this setting with a high burden of TB.

Mixed infection and clonal representativeness of a single sputum sample in tuberculosis patients from a penitentiary hospital in Georgia

BackgroundStudies on recurrent tuberculosis (TB), TB molecular epidemiology and drug susceptibility testing rely on the analysis of one Mycobacterium tuberculosis isolate from a single sputum sample collected at different disease episodes. This scheme rests on the postulate that a culture of one sputum sample is homogeneous and representative of the total bacillary population in a patient.MethodsWe systematically analysed several pre-treatment isolates from each of 199 smear-positive male adult inmates admitted to a prison TB hospital by standard IS6110 DNA fingerprinting, followed by PCR typing based on multiple loci containing variable number of tandem repeats (VNTRs) on a subset of isolates. Drug susceptibility testing (DST) was performed on all isolates for isoniazid, rifampicin, streptomycin and ethambutol.ResultsWe found mixed infection in 26 (13.1%) cases. In contrast, analysis of a single pre-treatment isolate per patient would have led to missed mixed infections in all or 14 of these 26 cases by using only standard DNA fingerprinting or the PCR multilocus-based method, respectively. Differences in DST among isolates from the same patient were observed in 10 cases, of which 6 were from patients with mixed infection.ConclusionThese results suggest that the actual heterogeneity of the bacillary population in patients, especially in high TB incidence settings, may be frequently underestimated using current analytical schemes. These findings have therefore important implications for correct interpretation and evaluation of molecular epidemiology data and in treatment evaluations.

Rapid Detection of Mycobacterium tuberculosis Beijing Genotype Strains by Real-Time PCR

Mycobacterium tuberculosis strains of the Beijing genotype were first identified in China and neighboring countries and have attracted special attention due to their global emergence and association with drug resistance. To further analyze the spread and special characteristics of Beijing genotype strains, accurate, rapid and sensitive methods that overcome the drawbacks of the classical methods such as IS6110 DNA fingerprinting or spoligotyping for the identification of strains of this genotype are needed. Based on the nucleotide sequences of M. tuberculosis SAWC0780 and H37Rv, primers and fluorogenic 5' nuclease (TaqMan) probes for real-time PCR assays specific for Beijing and non-Beijing strains, respectively, were designed. The detection limits for the real-time PCR assays were about 5 and 10 copies of chromosomal DNA, respectively. In mixtures of Beijing and non-Beijing DNA, a multiplex assay was able to detect (i) one copy of Beijing DNA in approximately 1,000 copies of non-Beijing DNA and (ii) one copy of non-Beijing DNA in approximately 2,000 copies of Beijing DNA. In a blinded analysis of a collection of 103 multidrug-resistant strains isolated in Germany in 2001, all 62 Beijing and all 41 non-Beijing strains were correctly identified. In conclusion, the real-time assay allows for the rapid and specific detection of Beijing and non-Beijing strains. The major advantages of this test in comparison to other methods used for the identification of Beijing strains are its simplicity and sensitivity and the fact that amplification and detection occur within one reaction tube.

Analysis of DNA fingerprints of Mycobacterium Tuberculosis Isolates from Patients Registered at Health Center in Gyeonggi Province in 2004

Background : IS6110 DNA fingerprint is a very useful tool for investigating the transmission of tuberculosis. The aim of this study was to identify the epidemiological situations within a given area (one province). Methods : The 681 Mycbobacterium tuberculosis isolates from patients, who were registered at health centers in Gyeonggi Province from May to December in 2004, were subjected to IS6110 DNA fingerprinting. Patients belonging to clusters were interviewed by health-workers to determine their previous contacts or household TB history. Results : The number of IS6110 copies of the 681 isolates showed diverse fingerprint patterns from 0 to 21 of which the most prevalent copy number was 10 from 120 isolates (17.6%). Thirty-three isolates (4.8%) belonged to the K strain, and 128 isolates (18.8%) belonged to the K family. There were 180 (26.4%) isolates belonged belonging to fifty clusters, of which two clusters were within household transmission. Forty-three (23.9%) out of 180 patients resided in an area under the same health center control. The rate of clusters in those aged 60-70 was higher than in any other age group ( 95% CI of RR : 1.072 ~ 1.988). Conclusion : This is the first report of an epidemiological survey based on a whole province using a DNA fingerprinting technique for M. tuberculosis. These results will be helpful in developing a program or policies to prevent the transmission of TB.

Reinfection and Mixed Infection Cause Changing Mycobacterium tuberculosis Drug-Resistance Patterns

Multiple infections with different strains of Mycobacterium tuberculosis may occur in settings where the infection pressure is high. The relevance of mixed infections for the patient, clinician, and control program remains unclear. This study aimed to describe reinfection and mixed infection as underlying mechanisms of changing drug-susceptibility patterns in serial sputum cultures. Serial M. tuberculosis sputum cultures from patients diagnosed with multi-drug-resistant (MDR) tuberculosis were evaluated by phenotypic drug-susceptibility testing and mutation detection methods. Genotypic analysis was done by IS6110 DNA fingerprinting and a novel strain-specific polymerase chain reaction amplification method. DNA fingerprinting analysis of serial sputum cultures from 48 patients with MDR tuberculosis attributed 10 cases to reinfection and 1 case to mixed infection. In contrast, strain-specific polymerase chain reaction amplification analysis in 9 of the 11 cases demonstrated mixed infection in 5 cases, reinfection in 3 cases, and laboratory contamination in 1 case. Analysis of clinical data suggests that first-line therapy can select for a resistant subpopulation, whereas poor adherence or second-line therapy resulted in the reemergence of the drug-susceptible subpopulations. We have shown that, in some patients with MDR tuberculosis, mixed infection may be responsible for observations attributed to reinfection by DNA fingerprinting. We conclude that treatment and adherence determines which strain is dominant. We hypothesize that treatment with second-line drugs may lead to reemergence of the drug-susceptible strain in patients with mixed infection.

Targeted Hybridization of IS 6110 Fingerprints Identifies the W-Beijing Mycobacterium tuberculosis Strains among Clinical Isolates

Targeted IS6110-based RFLP genotyping can be applied to rapidly identify specific groups of biomedically/epidemiologically relevant Mycobacterium tuberculosis clinical isolates. One such group is the W-Beijing strain family (also known as Beijing/W), implicated in significant nosocomial and community outbreaks worldwide. Using previously defined criteria, we developed a simple and accurate method to identify members of the W-Beijing family, based on rehybridization of Southern blot membranes used previously in routine IS6110 DNA fingerprint analysis. The hybridization probe constructed ("W-Beijing polyprobe") contains the PCR-amplified fragments specific for three M. tuberculosis chromosomal loci used for the identification of W-Beijing strains. The targets include the dnaA-dnaN and NTF regions and the direct repeat locus. A total of 526 selected clinical isolates (representative of 253 different IS6110-defined strain types) were analyzed using the W-Beijing polyprobe. A total of 148 isolates from this collection were found to be members of the W-Beijing phylogenetic lineage, comprising 106 strains from the W-Beijing family (46 clusters) and 42 related isolates. Rehybridization results were confirmed by computer-assisted analysis. The sensitivity and specificity of this method were estimated at 98.7% and 99.7%, respectively. This study demonstrates that the W-Beijing polyprobe can accurately and reliably discriminate members of the W-Beijing phylogenetic lineage and the W-Beijing family of M. tuberculosis strains.

Molecular Genotyping of Mycobacterium tuberculosis Isolates from Turkey

To survey the circulating strains of Mycobacterium tuberculosis in Turkey, all clinical isolates (381 patients) recovered in laboratories in six cities during one-month periods were collected and typed by IS6110-DNA fingerprinting and spoligotyping. Drug susceptibilities were also determined. About 23% of the isolates were resistant to one or more drugs and about 4% were multidrug resistant (i.e., resistant to at least isoniazid and rifampin). IS6110-DNA fingerprints and spoligotypes were obtained from 368 and 374 strains, respectively. Of the 374 isolates spoligotyped, 87 (23%) displayed unique spoligotypes and 287 (77%) displayed one of 34 spoligotypes (2-77 isolates per pattern). The clustered spoligotypes included ones that matched spoligotypes of the T (37% of isolates), LAM (20%), Haarlem (8%) and Beijing (2%) families. Of the 368 isolates IS6110-typed, 232 (63%) displayed unique IS6110-fingerprint patterns and 136 (37%) displayed one of 35 patterns (2-34 isolates per pattern). When IS6110 fingerprinting and spoligotyping information were combined for the 381 isolates tested, 273 isolates (72%) displayed unique genotypes and 108 isolates (28%) displayed one of 34 genotypes (2-24 isolates per genotype). In summary, many different strains are circulating in Turkey with no single strain appearing to be dominant as has been observed in other areas of the world with high tuberculosis incidence.

Laboratory Cross-Contamination Of Mycobacterium Tuberculosis During Preparation Of Smears

Introduction Materials And Methods Results Discussion References Abstract A possible cross-contamination of Mycobacterium tuberculosis at the Epidemiology and Hygiene Center of Havana (EHCH) occurred in 1999. Nine M. tuberculosis isolates from two acid -fast bacilli (AFB) smear-positive sputa and seven AFB smear-negative specimens were analyzed by IS6110 DNA fingerprinting. All isolates had identical fingerprints indicating a laboratory cross-contamination. No breaking rules were registered on the specimen transportation, sample labeling neither during sputum processing for culturing. Additional investigations in the peripheral laboratory (ML-HC2) showed that the recent entrance of new personnel in conjunction with severe difficulties in preparing smears for microscopy might have provoked the cross-contamination incident. The use of only one metal wire inoculation loop for smearing all series of sputa, a sand flask without alcohol and a faulty burner for loop sterilization, indicating the cross-contamination most likely occurred in ML-LC2. Multiple actions taken in the ML-LC2 have allowed that none laboratory contamination event has been reported after this.A possible cross-contamination of Mycobacterium tuberculosis at the Epidemiology and Hygiene Center of Havana (EHCH) occurred in 1999. Nine M. tuberculosis isolates from two acid -fast bacilli (AFB) smear-positive sputa and seven AFB smear-negative specimens were analyzed by IS6110 DNA fingerprinting. All isolates had identical fingerprints indicating a laboratory cross-contamination. No breaking rules were registered on the specimen transportation, sample labeling neither during sputum processing for culturing. Additional investigations in the peripheral laboratory (ML-HC2) showed that the recent entrance of new personnel in conjunction with severe difficulties in preparing smears for microscopy might have provoked the cross-contamination incident. The use of only one metal wire inoculation loop for smearing all series of sputa, a sand flask without alcohol and a faulty burner for loop sterilization, indicating the cross-contamination most likely occurred in ML-LC2. Multiple actions taken in the ML-LC2 have allowed that none laboratory contamination event has been reported after this.

Conventional and Molecular Epidemiology of Tuberculosis in Homeless Patients in Budapest, Hungary

In Hungary the incidence of tuberculosis among the homeless population was 676 per 100,000 in 2002. Sixty-nine percent (140 patients) of all homeless tuberculosis patients were notified in Budapest (the capital). Therefore, a retrospective study that included 66 homeless tuberculosis patients notified in Budapest in 2002 was conducted to determine the rate of recent transmission of the disease and medical risk factors and to identify transmission pathways by means of conventional and molecular epidemiologic methods. IS6110 DNA fingerprinting revealed that 71.2% of the isolates could be clustered. Thirty-four (51.5%) patients belonged to five major clusters (size, from 4 to 11 individuals), and 13 (19.7%) belonged to six smaller clusters. Additional analysis of patient records found that 2 (18%) of the 11 patients in cluster A, 3 (37.5%) of the 8 patients in cluster B, and 2 (33%) of the 6 patients in cluster C were residents of the same three homeless shelters during the diagnosis of tuberculosis. Review of the database of the National Tuberculosis Surveillance Center (NTSC) revealed that 21.2% of the cases have not been reported to the NTSC. These findings indicate that the screening and treatment of tuberculosis among the homeless need to be strengthened and also warrant the review of environmental control steps in public shelters. Improvement of adherence of clinicians to surveillance reporting regulations is also necessary.

Use of genetic distance as a measure of ongoing transmission of Mycobacterium tuberculosis.

The stability of the genotypic marker IS6110, used to define the epidemiology of Mycobacterium tuberculosis, is one of the most important factors influencing the interpretation of DNA fingerprint data. We propose that evolved strains should be considered together with clustered strains to represent chains of ongoing transmission. For the present study we used a large set of fingerprint data for strains collected between 1992 and 1998 from residents of a community with a high incidence of tuberculosis in Cape Town, South Africa. Interstrain genetic distances were calculated by counting the banding pattern mismatches in the IS6110 DNA fingerprints of different isolates. These data demonstrate that the propensity to change by one or two bands is independent of the IS6110 copy number. Hence, the genetic distance between pairs of isolates can be simply expressed as the number of differences in the banding patterns. From this foundation, a data set which identifies newly evolved strains has been generated. Inclusion of these evolved strains into various molecular epidemiological calculations significantly increased the estimate of ongoing transmission in this study setting. The indication is that nearly all cases of tuberculosis in this community are due to ongoing transmission. This has important implications for tuberculosis control, as it indicates that the control measures used at present are unable to reduce the level of transmission. This technique may also be applicable to the study of low-incidence tuberculosis outbreaks as well as the analysis of epidemiological data from other disease epidemics.

IS6110 restriction fragment length polymorphism of Mycobacterium tuberculosis isolated from patients with pulmonary tuberculosis in Campinas, Brazil: evidence of intercontinental distribution of strains.

Tuberculosis (TB) is a major concern in developing countries. In Brazil, few genotyping studies have been conducted to verify the number of IS6110 copies present in local prevalent strains of Mycobacterium tuberculosis, the distribution and clustering of strains. IS6110 DNA fingerprinting was performed on a sample of M. tuberculosis isolates from patients with AFB smear-positive pulmonary TB, at a hospital in Brazil. The IS6110 profiles were analyzed and compared to a M. tuberculosis database of the Houston Tuberculosis Initiative, Houston, US. Seventy-six fingerprints were obtained from 98 patients. All M. tuberculosis strains had an IS6110 copy number between 5-21 allowing for differentiation of the isolates. Human immunodeficiency virus infection was confirmed in nearly half the patients of whom data was available. Fifty-eight strains had unique patterns, while 17 strains were grouped in 7 clusters (2 to 6 strains). When compared to the HTI database, 6 strains matched isolates from El Paso, Ciudad de Juarez, Houston, and New York. Recently acquired infections were documented in 19% of cases. The community transmission of infection is intense, since some clustered strains were recovered during the four-year study period. The intercontinental dissemination of M. tuberculosis strains is suspected by demonstration of identical fingerprints in a distant country.

Mycobacterium tuberculosisTransmission between Cluster Members with Similar Fingerprint Patterns

Molecular epidemiologic studies provide evidence of transmission of Mycobacterium tuberculosis within clusters of patients whose isolates share identical IS6110-DNA fingerprint patterns. However, M. tuberculosis transmission among patients whose isolates have similar but not identical DNA fingerprint patterns (i.e., differing by a single band) has not been well documented. We used DNA fingerprinting, combined with conventional epidemiology, to show unsuspected patterns of tuberculosis transmission associated with three public bars in the same city. Among clustered TB cases, DNA fingerprinting analysis of isolates with similar and identical fingerprints helped us discover epidemiologic links missed during routine tuberculosis contact investigations.