Molecular characterisation of multidrug-resistant Mycobacterium tuberculosis isolates from a high-burden tuberculosis state in Brazil.

Tuberculosis (TB) is the leading cause of death among infectious diseases worldwide. Among the estimated cases of drug-resistant TB, approximately 60% occur in the BRICS countries (Brazil, Russia, India, China and South Africa). Among Brazilian states, primary and acquired multidrug-resistant TB (MDR-TB) rates were the highest in Rio Grande do Sul (RS). This study aimed to perform molecular characterisation of MDR-TB in the State of RS, a high-burden Brazilian state. We performed molecular characterisation of MDR-TB cases in RS, defined by drug susceptibility testing, using 131 Mycobacterium tuberculosis (M.tb) DNA samples from the Central Laboratory. We carried out MIRU-VNTR 24loci, spoligotyping, sequencing of the katG, inhA and rpoB genes and RDRio sublineage identification. The most frequent families found were LAM (65.6%) and Haarlem (22.1%). RDRio deletion was observed in 42 (32%) of the M.tb isolates. Among MDR-TB cases, eight (6.1%) did not present mutations in the studied genes. In 116 (88.5%) M.tb isolates, we found mutations associated with rifampicin (RIF) resistance in rpoB gene, and in 112 isolates (85.5%), we observed mutations related to isoniazid resistance in katG and inhA genes. An insertion of 12 nucleotides (CCAGAACAACCC) at the 516 codon in the rpoB gene, possibly responsible for a decreased interaction of RIF and RNA polymerase, was found in 19/131 of the isolates, belonging mostly to LAM and Haarlem families. These results enable a better understanding of the dynamics of transmission and evolution of MDR-TB in the region.

Outcomes from patients with presumed drug resistant tuberculosis in five reference centers in Brazil.

BACKGROUND: The implementation of rapid drug susceptibility testing (DST) is a current global priority for TB control. However, data are scarce on patient-relevant outcomes for presumptive diagnosis of drug-resistant tuberculosis (pDR-TB) evaluated under field conditions in high burden countries. METHODS: Observational study of pDR-TB patients referred by primary and secondary health units. TB reference centers addressing DR-TB in five cities in Brazil. Patients age 18 years and older were eligible if pDR-TB, culture positive results for Mycobacterium tuberculosis and, if no prior DST results from another laboratory were used by a physician to start anti-TB treatment. The outcome measures were median time from triage to initiating appropriate anti-TB treatment, empirical treatment and, the treatment outcomes. RESULTS: Between February,16th, 2011 and February, 15th, 2012, among 175 pDR TB cases, 110 (63.0%) confirmed TB cases with DST results were enrolled. Among study participants, 72 (65.5%) were male and 62 (56.4%) aged 26 to 45 years. At triage, empirical treatment was given to 106 (96.0%) subjects. Among those, 85 were treated with first line drugs and 21 with second line. Median time for DST results was 69.5 [interquartile - IQR: 35.7-111.0] days and, for initiating appropriate anti-TB treatment, the median time was 1.0 (IQR: 0-41.2) days. Among 95 patients that were followed-up during the first 6 month period, 24 (25.3%; IC: 17.5%-34.9%) changed or initiated the treatment after DST results: 16/29 MDRTB, 5/21 DR-TB and 3/45 DS-TB cases. Comparing the treatment outcome to DS-TB cases, MDRTB had higher proportions changing or initiating treatment after DST results (p = 0.01) and favorable outcomes (p = 0.07). CONCLUSIONS: This study shows a high rate of empirical treatment and long delay for DST results. Strategies to speed up the detection and early treatment of drug resistant TB should be prioritized.

Factors associated with non-completion of latent tuberculosis infection treatment in Rio de Janeiro, Brazil: A non-matched case control study.

INTRODUCTION: There are scarce data on the routine latent tuberculosis infection treatment (LTBIT) and factors associated with a non-completion in high tuberculosis burden countries. Therefore, in this study we aimed to evaluate the factors associated with non-completion of LTBIT. MATERIALS AND METHODS: This was a non-matched case control study conducted at a University Hospital in Rio de Janeiro, Brazil. A total of 114 cases and 404 controls were enrolled between January/1999 and December/2009. Cases were close contacts who did not complete the LTBIT and controls were the contacts that completed it. Multivariate analysis was used to investigate risk factors associated with non-completion of LTBIT among contacts in two different periods of recruitment. RESULTS: Factors associated with non-completion LTBIT included: drug use (OR 23.33, 95% CI 1.83-296.1), TB treatment default by the index case (OR 16.97, 95% CI 3.63-79.24) and drug intolerance. TB disease rates after two years of follow up varied from 0.4% to 1.9%. The number necessary to treat to prevent one TB case among contacts was 116. CONCLUSIONS: Non-completion treatment by the index case and illicit drug use were associated with not completing latent tuberculosis infection treatment and no tuberculosis disease was identified among those who completed latent tuberculosis infection treatment.

Challenges and perspectives for improved management of HIV/Mycobacterium tuberculosis co-infection.

HIV and Mycobacterium tuberculosis (MTB) are two widespread and highly successful microbes whose synergy in pathogenesis has created a significant threat for human health globally. In acknowledgement of this fact, the European Union (EU) has funded a multinational support action, the European Network for global cooperation in the field of AIDS and TB (EUCO-Net), that brings together experts from Europe and those regions that bear the highest burden of HIV/MTB co-infection. Here, we summarise the main outcome of the EUCO-Net project derived from an expert group meeting that took place in Stellenbosch (South Africa) (AIDS/TB Workshop on Research Challenges and Opportunities for Future Collaboration) and the subsequent discussions, and propose priority areas for research and concerted actions that will have impact on future EU calls.

Contribution of Xpert MTB/RIF to clinical diagnosis in adolescents with tuberculosis in Rio de Janeiro, Brazil.

SETTING: Rio de Janeiro, RJ, Brazil, a high tuberculosis (TB) burden city.OBJECTIVE: To compare the sociodemographics, clinical characteristics, care process indicators (CPIs) and treatment outcomes among adolescents with pulmonary TB (PTB) and those with PTB + extrapulmonary TB (EPTB), who underwent testing with Xpert® and sputum culture.DESIGN: This was a retrospective study of data from three national databases from 2014 to 2016 of adolescents (aged 10-18 years) residing and notified in Rio de Janeiro City. Three groups were identified according to their Xpert and culture results: Group 1, Xpert- and culture-positive; Group 2, Xpert-positive and culture-negative; and Group 3, Xpert- and culture-negative. Study CPIs were as follows: the time between 'sample collection and Xpert result release', 'sample collection and treatment initiation' and 'notification and treatment outcome'.RESULTS: Of 258 adolescents included in the study, 223 (86.4%) were in Group 1, 20 (7.8%) in Group 2 and 15 (5.8%) in Group 3. Groups 1 and 2 had a similar profile. Compared to Group 1, Group 3 had a higher proportion of HIV-positive cases (21.4% vs. 3.0%, P = 0.016), adolescents with a hospital diagnosis (53.3% vs. 7.6%, P < 0.001), and PTB + EPTB cases (20% vs. 0.4%; P < 0.001). There were no statistically significant differences in CPIs or treatment outcomes.CONCLUSION: The clinical diagnosis was decisive in more critical or complex patients, despite Xpert-negative results.

Development of multiplex assay for rapid characterization of Mycobacterium tuberculosis.

We have developed a multiplex assay, based on multiplex ligation-dependent probe amplification (MLPA), that allows simultaneous detection of multiple drug resistance mutations and genotype-specific mutations at any location in the Mycobacterium tuberculosis genome. The assay was validated on a reference panel of well-characterized strains, and the results show that M. tuberculosis can be accurately characterized by our assay. Eighteen discriminatory markers identifying drug resistance (rpoB, katG, inhA, embB), members of the M. tuberculosis complex (16S rRNA, IS6110, TbD1), the principal genotypic group (katG, gyrA), and Haarlem and Beijing strains (ogt, mutT2, mutT4) were targeted. A sequence specificity of 100% was reached for 16 of the 18 selected genetic targets. In addition, a panel of 47 clinical M. tuberculosis isolates was tested by MLPA in order to determine the correlation between phenotypic drug resistance and MLPA and between spoligotyping and MLPA. Again, all mutations present in these isolates that were targeted by the 16 functional probes were identified. Resistance-associated mutations were detected by MLPA in 71% of the identified rifampin-resistant strains and in 80% of the phenotypically isoniazid-resistant strains. Furthermore, there was a perfect correlation between MLPA results and spoligotypes. When MLPA is used on confirmed M. tuberculosis clinical specimens, it can be a useful and informative instrument to aid in the detection of drug resistance, especially in laboratories where drug susceptibility testing is not common practice and where the rates of multidrug-resistant and extensively drug resistant tuberculosis are high. The flexibility and specificity of MLPA, along with the ability to simultaneously genotype and detect drug resistance mutations, make MLPA a promising tool for pathogen characterization.

Prevalence of pyrazinamide resistance and Wayne assay performance analysis in a tuberculosis cohort in Lima, Peru.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB) regimens often contain pyrazinamide (PZA) even if susceptibility to the drug has not been confirmed. This gap is due to the limited availability and reliability of PZA susceptibility testing. OBJECTIVES: To estimate the prevalence of PZA resistance using the Wayne assay among TB patients in Lima, Peru, to describe characteristics associated with PZA resistance and to compare the performance of Wayne with that of BACTEC™ MGIT™ 960. METHODS: PZA susceptibility using the Wayne assay was tested in patients diagnosed with culture-positive pulmonary TB from September 2009 to August 2012. Factors associated with PZA resistance were evaluated. We compared the performance of the Wayne assay to that of MGIT 960 in a convenience sample. RESULTS: The prevalence of PZA resistance was 6.6% (95%CI 5.8-7.5) among 3277 patients, and 47.7% (95%CI 42.7-52.6) among a subset of 405 MDR-TB patients. In multivariable analysis, MDR-TB (OR 86.0, 95%CI 54.0-136.9) and Latin American-Mediterranean lineage (OR 3.40, 95%CI 2.33-4.96) were associated with PZA resistance. The Wayne assay was in agreement with MGIT 960 in 83.9% of samples (κ 0.66, 95%CI 0.56-0.76). CONCLUSION: PZA resistance was detected using the Wayne assay in nearly half of MDR-TB patients in Lima. This test can inform the selection and composition of regimens, especially those dependent on additional resistance.

A multicenter evaluation of tuberculin skin test positivity and conversion among health care workers in Brazilian hospitals.

SETTING: Four general Brazilian hospitals. OBJECTIVE: To assess the occupational risk of Mycobacterium tuberculosis (TB) in participating hospitals. DESIGN: In phase one of this longitudinal study, a cross-sectional survey documented baseline tuberculin skin test (TST) positivity rates. In phase two, TST conversion rates were evaluated in participants with an initial negative two-step TST. TST conversion data were analyzed to determine risk factors for TB infection using an increase of > or = 10 mm compared to baseline TST. RESULTS: The initial TST positivity rate was 63.1%; the follow-up TST conversion rate was 10.7 per 1000 person-months (p-m). Hospital of employment, recent bacille Calmette-Guerin (BCG) vaccination, nosocomial TB exposure, and employment as a nurse were independent risk factors for TST conversion. Hospitals without TB infection control measures had higher conversion rates than those with control measures (16.0 vs. 7.8/ 1000 p-m, P < 0.001). CONCLUSIONS: This study indicates an important occupational risk of infection in health care settings with a high TB incidence. Longitudinal TST studies are a valuable tool to assess the occupational risk of TB, even in BCG-vaccinated populations, and should be used to direct limited resources for infection control.

Latent tuberculosis infection among undergraduate medical students in Rio de Janeiro State, Brazil.

SETTING: Five medical schools in three cities with different tuberculosis (TB) incidence rates in Rio de Janeiro State, Brazil. OBJECTIVE: To estimate prevalence of and associated factors for latent tuberculosis infection (LTBI) among medical students. DESIGN: A cross-sectional survey was conducted among undergraduate students in pre-clinical, early and late clinical years from schools in cities with low (28/100,000), intermediate (63/100,000) and high (114/100,000) TB incidence rates. Information on socio-demographic profile, previous BCG vaccination, potential TB exposure, co-morbidity and use of respiratory protective masks was obtained. A tuberculin skin test (TST) was performed using the Mantoux technique by an experienced professional. A positive TST, defined as induration > or = 10 mm, was considered LTBI. RESULTS: LTBI prevalence was 6.9% (95%CI 5.4-8.6). In multivariate analysis, male sex (adjusted odds ratio [aOR] 1.8; 95% CI 1.1-3.0), late clinical years (aOR 1.9; 95% CI 1.01-3.5), intermediate TB incidence (aOR 4.3; 95% CI 1.3-14.6) and high TB incidence in the city of medical school (aOR 5.1; 95% CI 1.6-16.8) were significantly associated with LTBI. CONCLUSIONS: The higher prevalence of LTBI in late clinical years suggests that medical students are at increased risk for nosocomial Mycobacterium tuberculosis infection. The implementation of a TB control program may be necessary in medical schools, particularly in cities with higher TB incidence.

HIV infection in 567 active pulmonary tuberculosis patients in Brazil.

We studied 567 patients with active pulmonary tuberculosis (APT) in Rio de Janeiro, Brazil, by using a standardized questionnaire and by testing blood for HIV antibodies. The rate of HIV infection was 3.9% in 1987, 4.8% in 1988, and 5.2% in 1989, and did not differ by sex. It was highest (7.4%) in the 15- to 39-year age group. There was no difference between patients infected and not infected by HIV with regard to education, income, housing, or employment. Among all patients with definite HIV risk behavior, the HIV infection rate was 23.3%, rising to 31.2% among homo/bisexual men and 36.4% among intravenous drug users, and the rate was 6.5% for blood-transfusion recipients. Among patients who denied risk behavior, the rate was 1.2%. Generalized lymphadenopathy and oral candidiasis occurred with greater frequency among HIV-infected patients (p < 0.0001). Applying the World Health Organization 1985 clinical criteria and revised case definition for AIDS, we found, respectively, sensitivities of 34% and 76.9% and specificities of 31% and 26.3%; in the Rio de Janeiro environment, these clinical criteria without HIV serology should not be adopted for tuberculosis patients. For chest radiographs, a significant association was found between HIV infection and the occurrence of atypical images (p = 0.0001), and hilar and/or mediastinal adenopathy (p = 0.0002) and absence of cavities (p = 0.0003). A PPD (purified protein derivative) skin test induration of < 5 mm was identified in 53% of the HIV-positive cases and in 31.3% of the HIV-negative cases. Only 11.5% of HIV-infected APT patients met the Centers for Disease Control 1987 AIDS criteria.

Pulmonary function changes in the acute stage of histoplasmosis, with follow-up. An analysis of eight cases.

We have studied eight patients, six children and two adults, during a microepidemic of soil- and patient-proven histoplasmosis. Pulmonary function tests were performed between the 15th and 23rd days after the onset of symptoms, and repeated between the fifth and sixth, the ninth and tenth and the 15th and 22nd months afterward. Initial abnormalities were mild in seven cases and severe in one. There was a restrictive pattern in three cases and an obstructive pattern in two. The fraction of CO extraction was reduced in five cases and the diffusing capacity for CO was reduced in five of six cases so tested. Hypoxemia was present in three cases. On follow-up, the obstructive defect had disappeared by the sixth month, the restrictive pattern by the tenth month, and the diffusing defect still remained in three cases by the end of follow-up. Hypoxemia remained only in the severe case.

Retreatment tuberculosis cases. Factors associated with drug resistance and adverse outcomes.

STUDY OBJECTIVE: Risk factors associated with treatment failure and multidrug-resistant tuberculosis (MDR-TB) were examined among HIV-seronegative patients who were previously treated for tuberculosis (TB). DESIGN: Prospective, cohort study of patients referred to the study hospital for retreatment of TB between March 1986 and March 1990. PATIENTS: The patients belonged to three groups, according to outcomes following their previous treatment: 37 patients who abandoned treatment or suffered relapse after completion of therapy (group A), 91 patients who failed to respond to the first-line drug regimen (group B), and 78 patients who failed to respond to the second-line drug regimen (group C). RESULTS: Patients with Mycobacterium tuberculosis strains resistant to rifampin and isoniazid were found in 2 (6%) in group A, 29 (33%) in group B, and 49 (65%) in group C. Cure was achieved in 77% in group A, 54% in group B, and 36% in group C. Death occurred in none of the patients in group A, 8% in group B, and 24% in group C. In a multivariate logistic regression analysis, unfavorable response (failure to sterilize sputum culture, death, and abandonment) was significantly associated with infection with a multidrug-resistant M tuberculosis strain (p = 0.0002), cavitary disease (p = 0.0029), or irregular use of medications (p < 0.0001). CONCLUSIONS: These observations show that a previous treatment outcome and current clinical and epidemiologic histories can be used to predict the development of MDR-TB and adverse outcomes in patients undergoing retreatment for TB. Such information may be useful for identifying appropriate patient candidates for programs such as directly observed therapy.

Health sector reform in Brazil: impact on tuberculosis control.

This paper comments on the reform process of the health sector policies that took place after 1986 in Brazil, and its negative impact on the National Tuberculosis Control Programme (NTP). Decentralisation was followed by a slow transition from a vertical programme to an integrated programme. In 1990, the NTP was dismantled due to fiscal constraints, and in 1992, the NTP component was reorganised, with national and regional coordinators and subsequent increased support to state programmes. In 1996, the health sector reform continued its process, but this consisted mainly of cuts in health budgets and rapid decentralisation from federal level to unprepared states and municipalities, leading to the weakening of local tuberculosis control programmes. Only recently has government commitment been secured, with a new National Plan on Tuberculosis Control which includes the World Health Organization strategy for TB control--the implementation of the DOTS strategy (directly-observed treatment, short-course)--and efforts are being concentrated in 5500 municipalities. The programme has a centralised administration which supports decentralised implementation through out-patient clinics, and resources will be focused on local service delivery.

Defaulting from anti-tuberculosis treatment in a teaching hospital in Rio de Janeiro, Brazil.

SETTING: Few studies have investigated factors associated with defaulting from anti-tuberculosis (TB) therapy in hospital settings. OBJECTIVE: To identify the factors associated with defaulting from treatment among TB in-patients in Rio de Janeiro city, Brazil. DESIGN: Case-control study. METHODS: All study participants initiated anti-tuberculosis treatment in a teaching hospital. A defaulting case was defined as a person who did not return for anti-tuberculosis medications after 60 days. Cases and controls were interviewed by a trained health care worker using a standardized form. RESULTS: From 1 January to 31 December 1997, 228 TB cases were registered. After a review of the medical records, 39 were excluded. Household visits were performed in 189 patients; 46 subjects were identified as cases and 117 as controls. Defaulting from anti-tuberculosis treatment was observed in 66 cases (28.9%) before and in 46 (20.2%) after a home visit. After multivariate analysis, the strongest predictors of defaulting from treatment were: 1) returning card not provided (OR 0.099; 95%CI 0.008-1.2; P = 0.07), 2) not feeling comfortable with a doctor (OR 0.16; 95%CI 0.33-0.015; P = 0.001), and 3) blood pressure not measured (OR 0.072; 95%CI 0.036-0.79; P = 0.024). CONCLUSIONS: In this hospital, the factors associated with defaulting from anti-tuberculosis treatment highlight the necessity for a structured TB Control Program. It is expected that the implementation of such a program, pursuing specific approaches, should enhance completion of anti-tuberculosis treatment and cure.

Medical students at risk of nosocomial transmission of Mycobacterium tuberculosis.

SETTING: University and teaching hospital in Rio de Janeiro, Brazil, a city with a high prevalence of tuberculosis (TB). OBJECTIVE: To determine whether medical students are at increased risk of nosocomial transmission of Mycobacterium tuberculosis relative to other university students. DESIGN: A cross-sectional study of medical and chemical engineering students in different levels of their training programmes. Information about socio-demographic characteristics, BCG vaccination history, and potential exposures to TB were obtained using a standardised questionnaire. Tuberculin skin testing (TST) was used to determine the prevalence of infection with TB. RESULTS: Medical students have an increasing prevalence of TST positivity as they advance in their training programme to increasing levels of study (4.6%, 7.8%, 16.2%, respectively, P < 0.001), but chemical engineering students do not (4.2%, 4.3%, 4.4%, respectively, P = 0.913). The risks are greatest during the years of clinical training, when medical students have increased contact with patients. CONCLUSIONS: Medical students in this setting may be at increased risk of M. tuberculosis infection, relative to chemical engineering students. A programme of routine tuberculin skin testing is needed, combined with interventions to reduce the risk of nosocomial transmission in the workplace.

Mycobacterium tuberculosis lipid antigens: use of multi-antigen based enzyme immunoassay for free and complex dissociated antibodies.

OBJECTIVE: To test the sensitivity and specificity of four lipid antigens of Mycobacterium tuberculosis: BDA-TDA, DAT, SL-I, and PIMs, adsorbed in the same microplate well, to detect reactive IgG by enzyme-immunoassay (EIA) from plain serum (MA-EIA) and dissociated immune complexes (ICMA-EIA). DESIGN: IgG antibodies against four antigens, placed in the same microplate well, were evaluated in serum from 155 tuberculous (TB) cases non-infected with the human immunodeficiency virus (HIV): 78 patients with positive bacilloscopy and culture, 33 patients with positive culture and 44 patients diagnosed by clinical and radiological criteria; and from 211 HIV negative control subjects: 32 patients with other pulmonary diseases, 100 healthy people and 79 close contacts. RESULTS: MA-EIA had an overall sensitivity and specificity of 61% (94/155) and 95% (200/211), respectively. We further examined whether the dissociation of immune complexes increases the number of positive reactions in those initially found to be seronegative (SN). The subset of 112 (76 controls and 36 TB) MA-EIA SN samples tested using ICMA-EIA yielded an overall sensitivity and specificity of 83% and 100%. The ICMA-EIA results improved the overall sensitivity from 61 to 80% without changing specificity. CONCLUSION: These preliminary results suggest that MA-EIA followed by ICMA-EIA, for SN samples, might serve as a fast, cheap, and easy method for the diagnosis of TB in less than 48 hours.

Association between an early humoral response to Mycobacterium tuberculosis antigens and later development of tuberculosis in human immunodeficiency virus-infected individuals.

OBJECTIVE: To evaluate the association between an early humoral response to Mycobacterium tuberculosis antigens and the later development of tuberculosis (TB) disease in human immunodeficiency virus (HIV)-infected individuals. METHODS: Using an ELISA test, IgG antibodies against 4 M. tuberculosis antigens--purified protein derivative (PPD); 2,3 diacyl trehalose (DAT); a lipooligosaccharide (LOS) and a trehalose dicarboxylic acid bis N,N-dioctadecylamide (BDA.TDA)--were measured in sera from 25 HIV-infected tuberculous patients and 52 HIV-infected persons without TB. RESULTS: With the DAT and LOS antigens, a positive result in sera obtained in the 12 months preceding the onset of TB was significantly associated with later development of TB. Using the BDA.TDA antigen, the same association was observed in sera collected during the 6 months before the diagnosis of TB. No significant association was found with the PPD antigen. CONCLUSIONS: These results suggest that specific antibody markers may be useful to evaluate the risk of active TB in HIV-infected individuals, and a helpful indicator for preventive treatment.

Mycobacterium tuberculosis typing: usefulness of DRE-PCR to confirm cross-contamination in the mycobacteriology laboratory of a general reference hospital for AIDS.

In this study two molecular typing methods, a simple double repetitive element PCR-based assay and the standardized restriction fragment length polymorphism (RFLP), were used to confirm cross-contamination in the mycobacteriology laboratory. Clinical specimens from 12 patients, submitted for acid-fast bacilli stain smear and processed for culture in Lowenstein-Jensen on the same day, resulted in positive bacterioscopy (+++) and confluent growth only for one of the patients. The specimens from all the other patients but two were smear-negative and culture-positive, with one or two colonies. None of them had clinical symptoms and radiological findings for active tuberculosis (TB). The suspicion of false-positive cultures arose when a health care worker who had had a PPD skin test conversion, claimed to be healthy and had no TB symptoms, was found to have a positive sputum culture. DRE-PCR demonstrated that all nine cultures typed belonged to one cluster, further confirmed by RFLP. Although DRE-PCR has been found to be poorly reproducible, it has enough discriminatory power to be useful for rapid epidemiological investigation in selected settings.