P. C. Hopewell

Impact of bacterial genetics on the transmission of isoniazid-resistant Mycobacterium tuberculosis.

Understanding the ecology of drug-resistant pathogens is essential for devising rational programs to preserve the effective lifespan of antimicrobial agents and to abrogate epidemics of drug-resistant organisms. Mathematical models predict that strain fitness is an important determinant of multidrug-resistant Mycobacterium tuberculosis transmission, but the effects of strain diversity have been largely overlooked. Here we compared the impact of resistance mutations on the transmission of isoniazid-resistant M. tuberculosis in San Francisco during a 9-y period. Strains with a KatG S315T or inhA promoter mutation were more likely to spread than strains with other mutations. The impact of these mutations on the transmission of isoniazid-resistant strains was comparable to the effect of other clinical determinants of transmission. Associations were apparent between specific drug resistance mutations and the main M. tuberculosis lineages. Our results show that in addition to host and environmental factors, strain genetic diversity can influence the transmission dynamics of drug-resistant bacteria.

A Molecular Epidemiologic Analysis of Tuberculosis Trends in San Francisco, 1991–1997

During the late 1980s and early 1990s, when tuberculosis case rates were increasing in many parts of the United States, it became apparent that considerable transmission of Mycobacterium tuberculosis was occurring and that the infection often progressed rapidly to tuberculosis (1-4). Transmission of M. tuberculosis followed by rapid progression to clinical disease was directly shown by many instances of institutional transmission and was indirectly shown by population-based studies using genotyping of the organism to infer epidemiologic links among patients with tuberculosis (5-14). Case rates are again decreasing in many urban areas and in the United States as a whole (15, 16). In New York City, the reduction in the tuberculosis case rate has been attributed mainly to the use of directly observed therapy, but other control measures have also been instituted (15). Many of the control interventions focused on preventing transmission of M. tuberculosis in high-risk groups and in such settings as hospitals, prisons, and shelters. Prevention of M. tuberculosis transmission would decrease the rate of tuberculosis cases that result from recent infection followed by a short latency period and rapid progression to clinical disease. Other measures, such as increased use of isoniazid preventive therapy, would decrease tuberculosis cases caused by reactivation of latent infection. In San Francisco, California, we identified a high proportion of clustered cases by using both conventional and molecular epidemiologic methods (9, 12); cases were considered to be clustered if the isolates had identical DNA fingerprints. These findings indicated that considerable transmission of M. tuberculosis was occurring within the population. On the basis of these observations, tuberculosis control measures directed toward reducing transmission of M. tuberculosis in high-risk groups were instituted beginning in early 1991. We hypothesized that these measures would reduce the frequency of tuberculosis resulting from recent transmission (clustering) as well as decrease the overall incidence of the disease. In this observational study, we describe the trends in tuberculosis incidence and the molecular epidemiologic pattern of the disease in San Francisco from 1 January 1991 through 31 December 1997. Methods Study Sample Tuberculosis was diagnosed in 2051 persons in San Francisco between 1 January 1991 and 31 December 1997. Of these cases, 1761 (86%) were confirmed by isolation of M. tuberculosis. Deoxyribonucleic acid fingerprinting using IS6110-based restriction fragment length polymorphism analysis was done on isolates from 1548 (88%) of the 1761 culture-positive cases. Six or more IS6110 hybridizing bands were found in 1238 (80%) of the tested isolates, and the remaining 310 isolates had five or fewer IS6110 bands. We excluded isolates from 25 cases (1.6% of cases for which DNA fingerprinting was done) that met criteria for laboratory cross-contamination. Polymorphic guanine-cytosine-rich sequence (PGRS)-based restriction fragment length polymorphism analysis was done on 257 of the 310 (83%) non-laboratory contaminant isolates with five or fewer IS6110 bands. Thus, 1495 of 1761 patients (85% of all culture-positive cases) had isolates with complete DNA fingerprinting data. No significant year-to-year differences were seen in the percentage of isolates for which IS6110-based and PGRS-based DNA fingerprinting was done. At the time of diagnosis, tuberculosis control personnel entered standard demographic information into the Centers for Disease Control and Preventions Report of a Verified Case of Tuberculosis form. Annual case rates of tuberculosis in San Francisco were calculated per 100 000 persons by using 1990 U.S. Census data on the population of San Francisco residents according to age, sex, place of birth, and race or ethnicity (17). The prevalence of HIV infection in San Francisco was obtained by using data from the 1997 Consensus Report on HIV Prevalence and Incidence in San Francisco (18). DNA Fingerprinting Methods IS6110-based DNA fingerprinting was performed by using the internationally standardized method, and the resulting patterns were compared by using BioImage Whole Band Analyzer software (version 3.3, BioImage Corp., Ann Arbor, Michigan) (19, 20). Isolates that had identical IS6110 fingerprints that contained five or fewer hybridizing bands were further analyzed with PGRS-based fingerprinting (21-24). Genomic DNA was digested with Sma1 and was probed by using a consensus sequence of the PGRS (22). The resulting patterns were examined visually and were defined as matching if the number, relative intensity, and molecular weights of the bands were identical. Definition of Laboratory Cross-Contamination Laboratory cross-contamination was considered to be the cause of a positive culture when all of the following criteria were met: 1) A patient had only one positive culture and that specimen tested negative on microscopic examination for acid-fast bacilli; 2) the specimen had been processed in the laboratory within 28 days of a specimen from another patient who had a positive result on microscopic examination for acid-fast bacilli; and 3) the microscopically positive specimen had the same DNA fingerprint as the putative contaminated specimen (25). Definition of Clustering Clustering was defined as the occurrence, within 1 year, of two or more isolates from different patients that contained 1) six or more IS6110 bands in an identical pattern or 2) five or fewer IS6110 bands that matched identically and had an identical PGRS pattern. A 1-year period for defining clustering was used because we were especially interested in assessing transmission of M. tuberculosis that resulted in rapid progression to disease. Clustered cases were identified as follows. For every initial isolate of M. tuberculosis from an incident case of tuberculosis, the database of IS6110 fingerprints was searched to identify any matching patterns within the previous year. If a match was found in the previous year, the case was counted as being in a cluster. For example, if the fingerprint from a case diagnosed in March matched the fingerprint from a case diagnosed the previous August, the case diagnosed in March was counted as clustered. Because no match preceding the first case identified with a particular fingerprint (index cases) will be found, these cases were counted as not clustered. Data from the 1990 U.S. Census from California (17) and the 1997 Consensus Report on HIV Prevalence and Incidence in San Francisco (18) were used to calculate annual rates of clustered cases per 100 000 persons for each year from 1992 to 1997, both for the population as a whole and according to place of birth (United States or outside of the United States) and presence of HIV infection. Because we did not begin performing comprehensive DNA fingerprinting until 1 January 1991, we could not compare isolates from patients in whom tuberculosis was diagnosed in the first 1-year period (1 January 1991 through 31 December 1991) with isolates from previous cases. Thus, the comparison of strains began with isolates obtained after 1 January 1992. Of the 1495 cases with isolates that had complete DNA fingerprinting data, 225 were from patients whose diagnosis was established between 1 January 1991 and 31 December 1991, and 1270 were from patients whose diagnosis was established between 1 January 1992 and 31 December 1997. These 1270 patients made up the final study sample for the analysis of clustering over time. Intensification of Tuberculosis Control Measures Tuberculosis control measures were intensified in five areas beginning in 1991, partly on the basis of findings from our earlier molecular epidemiologic studies (12, 26). 1. Improved contact investigations. Disease control investigators were trained to improve communication with specific populations at risk, including homeless persons and persons with a history of substance abuse. In addition, the assessment of what constitutes significant contact was broadened. The median number of contacts per new case increased from 1 in 1991 to 4 in 1995 and was 3 in 1996 and 1997 (P<0.001; chi-square test). The percentage of cases with no contacts identified decreased from 25% in 1990 to 3% in 1997 (Table 1). The total number of contacts identified each year increased between 1991 (approximately 1100) and 1992 and continued to range from approximately 2000 to 3000 from 1993 through 1997. The number of contacts infected with M. tuberculosis identified through these investigations increased from 498 in 1991 to 956 in 1997, and approximately 80% of the contacts completed a course of isoniazid preventive therapy in each of these years. The number of cases of active tuberculosis found through contact investigations ranged from 19 to 25 per year during the study period. Table 1. Data on Contacts of Tuberculosis Cases and Number of Case-Patients with Tuberculosis Receiving Directly Observed Therapy per Year in San Francisco, California, 1991-1997 2. Expanded use of directly observed therapy. Overall, 28% (541 of 1958) of all patients with incident cases received directly observed therapy as part of their treatment during the study period (Table 1). The proportion of patients receiving directly observed therapy increased from 22% in 1991 to a peak of 35% in 1996; in 1997, the percentage was 22%. The percentage of all patients who had documented negative sputum cultures within 3 months of the initiation of therapy increased slightly, from 65% in 1990 to 68% in 1997. 3. Development of an HIV-related tuberculosis prevention program. Persons seen at any of six city-based HIV primary care and methadone maintenance clinics underwent tuberculin skin testing and were evaluated clinically for tuberculosis. Depending on HIV status, tuberculin reactors were treated with a 12- or 6-month course of isoniazid preventive therapy after exclusion of active tubercu

Influence of M. tuberculosis Lineage Variability within a Clinical Trial for Pulmonary Tuberculosis

Recent studies suggest that M. tuberculosis lineage and host genetics interact to impact how active tuberculosis presents clinically. We determined the phylogenetic lineages of M. tuberculosis isolates from participants enrolled in the Tuberculosis Trials Consortium Study 28, conducted in Brazil, Canada, South Africa, Spain, Uganda and the United States, and secondarily explored the relationship between lineage, clinical presentation and response to treatment. Large sequence polymorphisms and single nucleotide polymorphisms were analyzed to determine lineage and sublineage of isolates. Of 306 isolates genotyped, 246 (80.4%) belonged to the Euro-American lineage, with sublineage 724 predominating at African sites (99/192, 51.5%), and the Euro-American strains other than 724 predominating at non-African sites (89/114, 78.1%). Uneven distribution of lineages across regions limited our ability to discern significant associations, nonetheless, in univariate analyses, Euro-American sublineage 724 was associated with more severe disease at baseline, and along with the East Asian lineage was associated with lower bacteriologic conversion after 8 weeks of treatment. Disease presentation and response to drug treatment varied by lineage, but these associations were no longer statistically significant after adjustment for other variables associated with week-8 culture status.

Pathways and costs of care for patients with tuberculosis symptoms in rural Uganda

SETTING Latent tuberculous infection (LTBI) remains a significant source of new active tuberculosis cases. Recent guidelines encourage primary care physicians to prescribe LTBI treatment; however, there have been no investigations into the impact on treatment completion. OBJECTIVE To estimate LTBI treatment initiation and completion rates by primary care physicians. DESIGN A historical cohort study was conducted with Quebec residents dispensed isoniazid (INH) between 1 January 1998 and 31 December 2005. Information was obtained from administrative health insurance data. Regression modeling was used to estimate the association of completion rates with prescribing physician specialty, after adjustment for initial health status and other patient characteristics. RESULTS A total of 14,753 people were dispensed INH for LTBI treatment. Primary care physicians initiated 3863 (26%) treatments. This proportion decreased from 28.7% in 1998 to 21.1% in 2005. Patients initiated on treatment by primary care physicians were less likely to complete treatment (OR 0.79, 95%CI 0.72-0.86). Only 5977 (40.5%) patients completed treatment; the average treatment duration of the primary care physician group was 11 days less (P < 0.0001). CONCLUSION Primary care physicians initiated a substantial number of LTBI treatments, but less than half of patients completed treatment regardless of the physician specialty. Primary care physicians should be supported to enhance treatment completion.

Sublineages of lineage 4 (Euro-American) Mycobacterium tuberculosis differ in genotypic clustering.

SETTING Mycobacterium tuberculosis is classified into six phylogenetic lineages, each of which can be divided into sublineages. Sublineages of the same lineage have phenotypic differences, including their capacity to cause disease (pathogenicity). OBJECTIVE 1) To test the hypothesis that different sublineages of lineage 4, which causes most of the tuberculosis (TB) in the United States, have varying ability to cause secondary cases as determined by genotypic clustering, a proxy for pathogenicity; and 2) to determine if spoligotype and mycobacterial interspersed repetitive units (MIRU) typing could infer sublineage. DESIGN We included TB cases caused by lineage 4 strains from our community-based study in San Francisco. Sublineage was determined by regions of difference. Genotypic clustering was determined by insertion sequence 6110 and polymorphic guanine-cytosine-rich sequence. Associations between sublineages and patient characteristics were evaluated with adjusted and unadjusted analyses. RESULTS The most frequent sublineage was H37Rv-like. In the adjusted analysis, sublineage 183 was associated with clustering and homelessness. We found that strains from different sublineages had convergent spoligotype and MIRU types. CONCLUSIONS Sublineage 183 is associated with genotypic clustering, evidence of its being more able to cause secondary cases than the other lineage 4 sublineages. This finding suggests that bacterial factors contribute to the pathogenesis of TB. Spoligotype and MIRU type cannot be used to infer sublineage.

Reduced sensitivity of the QuantiFERON(®) test in diabetic patients with smear-negative tuberculosis.

SETTING Immunosuppressive conditions have been associated with low sensitivity of interferon-gamma release assays (IGRAs) and the tuberculin skin test (TST) for the diagnosis of tuberculosis (TB). However, no systematic analysis of patient and bacterial characteristics has been performed before. OBJECTIVE To determine the sensitivity and the risk factors for false-negative QuantiFERON(®)-TB (QFT) assay and TST in TB patients. DESIGN We performed a retrospective analysis of data collected in a community-based study of TB in San Francisco, CA, USA. We included 300 TB patients who underwent QFT and TST. RESULTS The risk factors for false-negative QFT were human immunodeficiency virus infection and the use of QuantiFERON(®)-TB Gold. In patients with sputum smear-negative TB, diabetes mellitus (DM) was associated with false-negative QFT (OR 2.85, 95%CI 1.02-7.97, P = 0.045). TST sensitivity was higher than QFT sensitivity in DM patients (OR 9.46, 95%CI 2.53-35.3). CONCLUSIONS In San Francisco, QFT sensitivity was lower than that of TST, especially in patients with DM. Stratified analysis by sputum smear results showed that this association was specific to smear-negative TB. In contrast, TST was not affected by the presence of DM.

Interplay of strain and race/ethnicity in the innate immune response to M. tuberculosis

Background The roles of host and pathogen factors in determining innate immune responses to M. tuberculosis are not fully understood. In this study, we examined host macrophage immune responses of 3 race/ethnic groups to 3 genetically and geographically diverse M. tuberculosis lineages. Methods Monocyte-derived macrophages from healthy Filipinos, Chinese and non-Hispanic White study participants (approximately 45 individuals/group) were challenged with M. tuberculosis whole cell lysates of clinical strains Beijing HN878 (lineage 2), Manila T31 (lineage 1), CDC1551 (lineage 4), the reference strain H37Rv (lineage 4), as well as with Toll-like receptor 2 agonist lipoteichoic acid (TLR2/LTA) and TLR4 agonist lipopolysaccharide (TLR4/LPS). Following overnight incubation, multiplex assays for nine cytokines: IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, IFNγ, TNFα, and GM-CSF, were batch applied to supernatants. Results Filipino macrophages produced less IL-1, IL-6, and more IL-8, compared to macrophages from Chinese and Whites. Race/ethnicity had only subtle effects or no impact on the levels of IL-10, IL-12p70, TNFα and GM-CSF. In response to the Toll-like receptor 2 agonist lipoteichoic acid (TLR2/LTA), Filipino macrophages again had lower IL-1 and IL-6 responses and a higher IL-8 response, compared to Chinese and Whites. The TLR2/LTA-stimulated Filipino macrophages also produced lower amounts of IL-10, TNFα and GM-CSF. Race/ethnicity had no impact on IL-12p70 levels released in response to TLR2/LTA. The responses to TLR4 agonist lipopolysaccharide (TLR4/LPS) were similar to the TLR2/LTA responses, for IL-1, IL-6, IL-8, and IL-10. However, TLR4/LPS triggered the release of less IL-12p70 from Filipino macrophages, and less TNFα from White macrophages. Conclusions Both host race/ethnicity and pathogen strain influence the innate immune response. Such variation may have implications for the development of new tools across TB therapeutics, immunodiagnostics and vaccines.

Clinical and bacteriological characteristics associated with clustering of multidrug-resistant tuberculosis.

SETTING The impact of the genetic characteristics of Mycobacterium tuberculosis on the clustering of multidrug-resistant tuberculosis (MDR-TB) has not been analyzed together with clinical and demographic characteristics. OBJECTIVE To determine factors associated with genotypic clustering of MDR-TB in a community-based study. DESIGN We measured the proportion of clustered cases among MDR-TB patients and determined the impact of clinical and demographic characteristics and that of three M. tuberculosis genetic characteristics: lineage, drug resistance-associated mutations, and rpoA and rpoC compensatory mutations. RESULTS Of 174 patients from California and Texas included in the study, the number infected by East-Asian, Euro-American, Indo-Oceanic and East-African-Indian M. tuberculosis lineages were respectively 70 (40.2%), 69 (39.7%), 33 (19.0%) and 2 (1.1%). The most common mutations associated with isoniazid and rifampin resistance were respectively katG S315T and rpoB S531L. Potential compensatory mutations in rpoA and rpoC were found in 35 isolates (20.1%). Hispanic ethnicity (OR 26.50, 95%CI 3.73-386.80), infection with an East-Asian M. tuberculosis lineage (OR 30.00, 95%CI 4.20-462.40) and rpoB mutation S531L (OR 4.03, 95%CI 1.05-23.10) were independent factors associated with genotypic clustering. CONCLUSION Among the bacterial factors studied, East-Asian lineage and rpoB S531L mutation were independently associated with genotypic clustering, suggesting that bacterial factors have an impact on the ability of M. tuberculosis to cause secondary cases.

Impact of Euro-American sublineages of Mycobacterium tuberculosis on new infections among named contacts

BACKGROUND The impact of demographic, clinical, and bacterial factors on new infection by Euro-American lineage Mycobacterium tuberculosis among contacts of patients with tuberculosis (TB) has not been evaluated. OBJECTIVE To describe the risk factors for new infection by Euro-American M. tuberculosis sublineages in San Francisco, California. DESIGN We included contacts of patients with TB due to Euro-American M. tuberculosis. Sublineages were determined by large-sequence polymorphisms. We used tuberculin skin testing or QuantiFERON®-TB Gold In-Tube to identify contacts with new infection. Regression models with generalized estimating equations were used to determine the risk factors for new infection. RESULTS We included 1488 contacts from 134 patients with TB. There were 79 (5.3%) contacts with new infection. In adjusted analyses, contacts of patients with TB due to region of difference 219 M. tuberculosis sublineage were less likely to have new infection (OR 0.23, 95%CI 0.06-0.84) than those with other sublineages. Other risk factors for new infection were contacts exposed to more than one patient with TB, contacts exposed for 30 days, or contacts with a history of smoking or excessive alcohol consumption. CONCLUSIONS In addition to well-known exposure and clinical characteristics, bacterial characteristics independently contribute to the transmissibility of TB in San Francisco.