Macrophage migration inhibitory factor - 794 CATT5-8 microsatellite polymorphism and susceptibility of tuberculosis.

PURPOSE: The establishment of candidate genetic determinants associated with tuberculosis (TB) is a challenge, considering the divergent frequencies among populations. The objective of this study was to evaluate the association between MIF - 794 CATT 5-8 polymorphism and susceptibility to TB. METHODS: Case-control study. Patients > 18 years, with pulmonary TB were included. The control group consisted of blood donors and household contacts, not relatives, healthy and > 18 years. MIF - 794 CATT 5-8 were genotyped using sequencing of PCR and capillary electrophoresis. RESULTS: 126 patients and 119 controls were included. The genotype 5/5 was more frequent among cases (15.1%) than in controls (5.9%) (p = 0.019). Cases had more frequently the allele 5 (29.4%) as compared with controls (19.3%) (p = 0.010). Prevalence of 7/X + 8/X genotypes was not different between cases and controls (p = 0.821). There was no difference between patients with alleles 7 and 8 and those with alleles 5 and 6 (p = 0.608). CONCLUSIONS: The genotype 5/5 and the allele 5 of MIF - 794 CATT 5-8 were more frequent among TB patients than in controls.

Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides.

BACKGROUND: Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. OBJECTIVES: To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in M. tuberculosis resistance to Isoniazid (INH) directly in Ziehl-Neelsen (ZN) stained slides. METHODS: Were analysed 55 independent DNA samples extracted from clinical isolates of M. tuberculosis by sequencing. For application in TB diagnosis resistance, 59 ZN-stained slides were used. The sensitivity, specificity and Kappa index, with a 95% confidence interval (CI95%), were determined. FINDINGS: The agreement between the tests was, for the katG target, the Kappa index of 0.89 (CI95%: 0.7-1.0). The sensitivity and specificity were 97.6% (CI95%: 87.7-99.9) and 91.7% (CI95%: 61.5-99.5), respectively. For inhA, the Kappa index was 0.92 (CI95%: 0.8-1.0), the sensitivity and specificity were 94.4% (CI95%: 72.7-99.8) and 97.3% (CI95%: 85.8-99.9), respectively. The use of ZN-stained slides for drug-resistant TB detection showed significant results when compared to other standard tests for drug resistance. MAIN CONCLUSIONS: qPCR genotyping proved to be an efficient method to detect genes that confer M. tuberculosis resistance to INH. Thus, qPCR genotyping may be an alternative instead of sequencing.

Canine visceral leishmaniasis diagnosis: a comparative performance of serological and molecular tests in symptomatic and asymptomatic dogs.

Although serological assays have been widely used for the diagnosis of canine visceral leishmaniasis (CVL), they present different performances depending on the clinical profile of the dogs. This study evaluated the accuracy of serological tests, immunochromatographic (Dual Path Platform: DPP®) and enzyme-linked immunosorbent (ELISA EIE®), for CVL in relation to the detection of Leishmania DNA through real-time polymerase chain reaction (real-time PCR) in samples from symptomatic and asymptomatic dogs from a non-endemic area in the state of Rio Grande do Sul, Southern Brazil. Serum from 140 dogs (39 symptomatic and 101 asymptomatic) was tested by DPP and ELISA followed by real-time PCR. From a total of 140 samples evaluated, Leishmania DNA was detected by real-time PCR in 41.4% (58/140). Moreover, 67.2% of samples positive in real-time PCR were positive in both DPP and ELISA (39/58), showing moderate agreement between methods. In the symptomatic group, one sample non-reactive in both serological assays was positive in real-time PCR, whereas in the asymptomatic group, 17.8% non-reactive or undetermined samples in serological assays were positive in the molecular method. Leishmania DNA was not detected in 17.9% reactive samples by serological assays from the symptomatic group, and in 3.9% from asymptomatic dogs. Real-time PCR demonstrated greater homogeneity between symptomatic and asymptomatic groups compared with DPP and ELISA. The molecular method can help to establish the correct CVL diagnosis, particularly in asymptomatic dogs, avoiding undesirable euthanasia.

Macrophage migration inhibitory factor -173 G>C single nucleotide polymorphism and its association with active pulmonary tuberculosis.

PURPOSE: The establishment of candidate genes associated with susceptibility to TB is a challenge especially due to divergent frequencies among different populations. The objective of this study was to evaluate the association between macrophage migration inhibitory factor (MIF) -173 G>C single nucleotide polymorphism (SNP) and susceptibility to pulmonary TB in a population of southern Brazil. METHODS: Case-control study. Patients > 18 years old, diagnosed with pulmonary TB were included. The control group consisted of blood donors and household contacts, not relatives, healthy and > 18 years old. MIF -173 G>C SNPs were genotyped using real-time PCR using a TaqMan SNP Genotyping assay. RESULTS: 174 patients and 166 controls were included. There were no statistically significant differences between cases and controls regarding genotype prevalence (p>0.05). Comparing patients with normal genotype (GG) with those with at least one C allele, there was also no statistically significant difference (p = 0.135). Also, there was no statistically significant difference comparing the homozygous for the mutation (CC) with the other patients (GG and CG) (p = 0.864). CONCLUSIONS: We did not find association between MIF -173 G>C polymorphism and susceptibility to pulmonary TB.

Improvement of Mycobacterium tuberculosis detection in sputum using DNA extracted by sonication.

Tuberculosis (TB) is one of the infectious diseases with high mortality in the world. DNA amplification techniques have been used to overcome barriers to the diagnosis of this disease. However, the success of these methodologies is highly dependent on the DNA obtained from the sample. This study was carried out to verify whether the DNA extracted by sonication (in house method) could yield suitable DNA for amplification by real-time PCR (qPCR). Sixty sputum samples were submitted to DNA extraction using sonication compared to a commercial method (Detect-TB kit, Labtest/MG-Brazil). All DNA samples were amplified by qPCR for IS6110 region (IS6110-qPCR/SYBR Green assay). Out of 60 samples, 40 were positive for TB; of these, all had positive results when extracted by sonication (100%) and 80% when extracted by the commercial method. The limit of detection (LOD) of Mycobacterium tuberculosis (H37Rv strain) by qPCR was 14 CFU/mL when the DNA was extracted by sonication, compared to countless colonies when extracted by commercial kit. In conclusion, the sonication protocol (without purification step) proved to be a simple, fast, and suitable method for obtaining DNA for use in qPCR from sputum samples.

Geospatial intelligence and health analitycs: Its application and utility in a city with high tuberculosis incidence in Brazil.

BACKGROUND: Geospatial Intelligence and Health Analysis have been used to identify tuberculosis (TB) hotspots and to better understand their relationship to social and economic factors. The purpose of this study was to use geospatial intelligence to assess the distribution of TB and its correlations with Human Development Index (HDI) in a city with high TB incidence in Brazil. METHODS: We conducted an ecological study, using National System of Information on Noticeable Disease (SINAN) to identify TB cases. Geocoding was performed using QGIS 2.0 software and Google Maps API 3.0. We applied geospatial intelligence to detect where in the city clustering of TB cases occurred, and assessed the association of an area's HDI (each one of the components - longevity, education, and income) with TB spatial distribution. RESULTS: During the study period (2011-2013), there were 737 TB cases. TB cases showed heterogeneity across the 29 neighborhoods. The neighborhoods with HDI-income lower than the mean had higher TB incidence (p = 0.036). CONCLUSIONS: We found several hotspots of TB across the 29 neighborhoods, and an inverse association between HDI-income and TB incidence. These findings provide useful information and may help to guide TB control programs.

Improvement of Mycobacterium tuberculosis detection in sputum using DNA extracted by sonication

Abstract Tuberculosis (TB) is one of the infectious diseases with high mortality in the world. DNA amplification techniques have been used to overcome barriers to the diagnosis of this disease. However, the success of these methodologies is highly dependent on the DNA obtained from the sample. This study was carried out to verify whether the DNA extracted by sonication (in house method) could yield suitable DNA for amplification by real-time PCR (qPCR). Sixty sputum samples were submitted to DNA extraction using sonication compared to a commercial method (Detect-TB kit, Labtest/MG-Brazil). All DNA samples were amplified by qPCR for IS6110 region (IS6110-qPCR/SYBR Green assay). Out of 60 samples, 40 were positive for TB; of these, all had positive results when extracted by sonication (100%) and 80% when extracted by the commercial method. The limit of detection (LOD) of Mycobacterium tuberculosis (H37Rv strain) by qPCR was 14CFU/mL when the DNA was extracted by sonication, compared to countless colonies when extracted by commercial kit. In conclusion, the sonication protocol (without purification step) proved to be a simple, fast, and suitable method for obtaining DNA for use in qPCR from sputum samples.

Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides

BACKGROUND Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. OBJECTIVES To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in M. tuberculosis resistance to Isoniazid (INH) directly in Ziehl-Neelsen (ZN) stained slides. METHODS Were analysed 55 independent DNA samples extracted from clinical isolates of M. tuberculosis by sequencing. For application in TB diagnosis resistance, 59 ZN-stained slides were used. The sensitivity, specificity and Kappa index, with a 95% confidence interval (CI95%), were determined. FINDINGS The agreement between the tests was, for the katG target, the Kappa index of 0.89 (CI95%: 0.7-1.0). The sensitivity and specificity were 97.6% (CI95%: 87.7-99.9) and 91.7% (CI95%: 61.5-99.5), respectively. For inhA, the Kappa index was 0.92 (CI95%: 0.8-1.0), the sensitivity and specificity were 94.4% (CI95%: 72.7-99.8) and 97.3% (CI95%: 85.8-99.9), respectively. The use of ZN-stained slides for drug-resistant TB detection showed significant results when compared to other standard tests for drug resistance. MAIN CONCLUSIONS qPCR genotyping proved to be an efficient method to detect genes that confer M. tuberculosis resistance to INH. Thus, qPCR genotyping may be an alternative instead of sequencing.