Risk factors associated with cluster size of Mycobacterium tuberculosis (Mtb) of different RFLP lineages in Brazil.

BACKGROUND: Tuberculosis (TB) transmission is influenced by patient-related risk, environment and bacteriological factors. We determined the risk factors associated with cluster size of IS6110 RFLP based genotypes of Mycobacterium tuberculosis (Mtb) isolates from Vitoria, Espirito Santo, Brazil. METHODS: Cross-sectional study of new TB cases identified in the metropolitan area of Vitoria, Brazil between 2000 and 2010. Mtb isolates were genotyped by the IS6110 RFLP, spoligotyping and RDRio. The isolates were classified according to genotype cluster sizes by three genotyping methods and associated patient epidemiologic characteristics. Regression Model was performed to identify factors associated with cluster size. RESULTS: Among 959 Mtb isolates, 461 (48%) cases had an isolate that belonged to an RFLP cluster, and six clusters with ten or more isolates were identified. Of the isolates spoligotyped, 448 (52%) were classified as LAM and 412 (48%) as non-LAM. Our regression model found that 6-9 isolates/RFLP cluster were more likely belong to the LAM family, having the RDRio genotype and to be smear-positive (adjusted OR = 1.17, 95% CI 1.08-1.26; adjusted OR = 1.25, 95% CI 1.14-1.37; crude OR = 2.68, 95% IC 1.13-6.34; respectively) and living in a Serra city neighborhood decrease the risk of being in the 6-9 isolates/RFLP cluster (adjusted OR = 0.29, 95% CI, 0.10-0.84), than in the others groups. Individuals aged 21 to 30, 31 to 40 and > 50 years were less likely of belonging the 2-5 isolates/RFLP cluster than unique patterns compared to individuals < 20 years of age (adjusted OR = 0.49, 95% CI 0.28-0.85, OR = 0.43 95% CI 0.24-0.77and OR = 0. 49, 95% CI 0.26-0.91), respectively. The extrapulmonary disease was less likely to occur in those infected with strains in the 2-5 isolates/cluster group (adjustment OR = 0.45, 95% CI 0.24-0.85) than unique patterns. CONCLUSIONS: We found that a large proportion of new TB infections in Vitoria is caused by prevalent Mtb genotypes belonging to the LAM family and RDRio genotypes. Such information demonstrates that some genotypes are more likely to cause recent transmission. Targeting interventions such as screening in specific areas and social risk groups, should be a priority for reducing transmission.

Genotypic and Spatial Analysis of Mycobacterium tuberculosis Transmission in a High-Incidence Urban Setting.

BACKGROUND: Genotyping Mycobacterium tuberculosis isolates allows study of dynamics of tuberculosis transmission, while geoprocessing allows spatial analysis of clinical and epidemiological data. Here, genotyping data and spatial analysis were combined to characterize tuberculosis transmission in Vitória, Brazil, to identify distinct neighborhoods and risk factors associated with recent tuberculosis transmission. METHODS: From 2003 to 2007, 503 isolates were genotyped by IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The analysis included kernel density estimation, K-function analysis, and a t test distance analysis. Mycobacterium tuberculosis isolates belonging to identical RFLP patterns (clusters) were considered to represent recent tuberculosis infection (cases). RESULTS: Of 503 genotyped isolates, 242 (48%) were categorized into 70 distinct clusters belonging to 12 RFLP families. The proportion of recent transmission was 34.2%. Kernel density maps indicated 3 areas of intense concentration of cases. K-function analysis of the largest RFLP clusters and families showed they co-localized in space. The distance analysis confirmed these results and demonstrated that unique strain patterns (controls) randomly distributed in space. A logit model identified young age, positive smear test, and lower Index of Quality of Urban Municipality as risk factors for recent transmission. The predicted probabilities for each neighborhood were mapped and identified neighborhoods with high risk for recent transmission. CONCLUSIONS: Spatial and genotypic clustering of M. tuberculosis isolates revealed ongoing active transmission of tuberculosis caused by a small subset of strains in specific neighborhoods of the city. Such information provides an opportunity to target tuberculosis transmission control, such as through rigorous and more focused contact investigation programs.

Use of in-house PCR for identification of Mycobacterium tuberculosis in BACTEC broth cultures of respiratory specimens.

We evaluated the ability of a PCR assay to identify Mycobacterium tuberculosis complex (MTBC) from positive BACTEC 12B broth cultures. A total of 107 sputum samples were processed and inoculated into Ogawa slants and BACTEC 12B vials. At a growth index (GI) > or=30, 1.0 ml of the 12B broth was removed, stored, and assayed with PCR. Molecular results were compared to those obtained by phenotypic identification methods, including the BACTEC NAP method. The average times required to perform PCR and NAP were compared. Of the 107 broth cultures evaluated, 90 were NAP positive, while 91 were PCR positive for MTBC. Of particular interest were three contaminated BACTEC 12B broth cultures yielding microorganisms other than acid-fast bacilli growth with a MTBC that were successfully identified by PCR, resulting in a mean time of 14 days to identify MTBC before NAP identification. These results suggest that PCR could be used as an alternative to the NAP test for the rapid identification of MTBC in BACTEC 12B cultures, particularly in those that contained both MTBC and nontuberculous mycobacteria.

Evaluation of a commercial test based on ligase chain reaction for direct detection of Mycobacterium tuberculosis in respiratory specimens.

A ligase chain reaction DNA amplification method for direct detection of Mycobacterium tuberculosis (Abbott LCx MTB) in respiratory specimens was evaluated. Results from LCx MTB Assay were compared with those from acid fast bacilli smear, culture, and final clinical diagnosis for each patient. A total of 297 respiratory specimens (sputum and bronchial lavage) from 193 patients were tested. The sensitivity, specificity, positive predictive value and negative predictive value of LC vs culture were 92.7%, 93%, 67.8% and 98.7%, respectively. When compared to the clinical final diagnosis, the sensitivity, specificity, PPV and NPV for LCx were 88.9%, 96.8%, 86.5% and 97.4%, respectively. The sensitivity of LCx MTB assay was 75% for smear-negative, culture positive samples. The results indicate that LCx MTB assay is a rapid, simple and valuable technique as a complementary tool for the diagnosis of tuberculosis.

Extrapulmonary tuberculosis: Mycobacterium tuberculosis strains and host risk factors in a large urban setting in Brazil.

BACKGROUND: Factors related to the development of extrapulmonary forms of tuberculosis (EPTB) are still poorly understood, particularly in high-endemic countries like Brazil. The objective of the paper is to determine host and Mycobacterium tuberculosis (MTB) strain-related factors associated with the development of EPTB in Espírito Santo state, Brazil. METHODS AND FINDINGS: We conducted a retrospective laboratory-based surveillance study of new tuberculosis (TB) cases diagnosed in Espírito Santo state, Brazil between 1998 and 2007. We genotyped 612 isolates of MTB from 606 TB patients using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) typing and compared sociodemographic and clinical characteristics of patients with pulmonary TB (PTB) and EPTB. Among 606 patients, 464 (77%) had PTB, 79 (13%) had EPTB, 51 (8%) had both, and 12 (2%) had miliary TB. The IS6110 RFLP analysis demonstrated that 250 (41%) isolates belonged to clustered RFLP patterns, 27 (11%) of which were from EPTB. We identified 73 clusters including 35 (48%) composed of 2 isolates each. By spoligotyping, 506 (83%) MTB isolates fell into known patterns and 106 (17%) fell into patterns with no family assignment; 297 (48%) isolates belonged to the Latin-American Mediterranean family. Higher school level (4-7 years OR: 0.16 95% CI 0.34-0.73 and > 8 years of education, OR 0.06 95% CI 0.009-0.50) white ethnicity (OR: 2.54 95% CI 1.03-6.25) and HIV infection (OR: 16.83 95% CI 5.23-54.18) were associated with EPTB. No specific strain lineage or percentage of clustering was associated with EPTB. CONCLUSIONS: These results demonstrate that risk factors for EPTB are related more to host than to MTB strain lineage characteristics.

Cavitary disease and quantitative sputum bacillary load in cases of pulmonary tuberculosis.

We examined sputum bacterial loads in adults with newly diagnosed tuberculosis using quantitative culture and time-until-positive (DTP) culture in BACTEC 460. Patients with cavitary disease had higher CFU levels than those without cavities and shorter DTPs. Within radiographic strata of moderately and far advanced tuberculosis, higher CFU counts were associated with cavitary disease.

Use of in-house PCR for identification of Mycobacterium tuberculosis in BACTEC broth cultures of respiratory specimens

We evaluated the ability of a PCR assay to identify Mycobacterium tuberculosis complex (MTBC) from positive BACTEC® 12B broth cultures. A total of 107 sputum samples were processed and inoculated into Ogawa slants and BACTEC® 12B vials. At a growth index (GI) > 30, 1.0 ml of the 12B broth was removed, stored, and assayed with PCR. Molecular results were compared to those obtained by phenotypic identification methods, including the BACTEC® NAP method. The average times required to perform PCR and NAP were compared. Of the 107 broth cultures evaluated, 90 were NAP positive, while 91 were PCR positive for MTBC. Of particular interest were three contaminated BACTEC® 12B broth cultures yielding microorganisms other than acid-fast bacilli growth with a MTBC that were successfully identified by PCR, resulting in a mean time of 14 days to identify MTBC before NAP identification. These results suggest that PCR could be used as an alternative to the NAP test for the rapid identification of MTBC in BACTEC® 12B cultures, particularly in those that contained both MTBC and nontuberculous mycobacteria.

Evaluation of a commercial test based on ligase chain reaction for direct detection of Mycobacterium tuberculosis in respiratory specimens

O método de amplificação de DNA baseado na reação em cadeia da ligase (Abbott LCx MTB) foi avaliado para detecção do Mycobacterium tuberculosis em espécimes pulmonares. Os resultados do LCx MTB foram comparados aos resultados de baciloscopia, cultura e diagnóstico clínico para cada paciente. Um total de 297 espécimes (escarro e lavado broncoalveolar) de 189 pacientes foram testadas. Os valores de sensibilidade, especificidade, valor preditivo positivo e valor preditivo negativo do LCX vs cultura foram 92,7%, 93%, 67,8% e 98,7%, respectivamente. Quando comparados ao diagnóstico clínico, os valores de sensibilidade, especificidade, VPP e VPN para o LCx foram 88,9%, 96,8%, 86,5% e 97,4%, respectivamente. A sensibilidade do LCx MTB foi de 75% para as amostras com baciloscopia negativa e cultura positiva. Os resultados indicam que o teste LCx MTB é simples, rápido, eficiente e pode ser utilizado como um recurso complementar para o diagnóstico da tuberculose.