Genetic diversity of Mycobacterium tuberculosis isoniazid monoresistant and multidrug-resistant in Rio Grande do Sul, a tuberculosis high-burden state in Brazil.

Tuberculosis (TB) remains a major public health problem in the world and Brazil is among the countries with the highest incidence and prevalence rates, and Rio Grande do Sul, a Brazilian state, occupy a prominent position. Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) further aggravates this scenario, making it more difficult to treat and control the disease. Isoniazid monoresistance (IMR) may increase the risk of progression to MDR-TB and treatment failure. However, most drug resistance molecular tests only focus on detecting rifampicin (RIF) resistance.In the present study, we characterized a total of 63 drug resistant isolates of M. tuberculosis (35 MDR, 26 IMR and two isolates monoresistant to rifampicin [RMR]) of the Rio Grande do Sul state by MIRU-VNTR (24 loci), spoligotyping, presence of RDRio, fbpC103, pks15/1 and sequencing of the katG, rpoB and inhA genes. We observed a higher proportion of the LAM family 30/63 (47.61%). In IMR, mutations were found in the katG gene (98% at codon 315) in 72.5%, and mutations in the promoter region of the inhA gene in 6.25% of the isolates. In MDR-TB and RMR-TB isolates, 92.1% had mutations in the rpoB gene (57% at codon 531). The presence of a 12 bp insertion between codons 516 and 517 of the rpoB gene in MDR-TB isolates was found in five isolates. In conclusion, we observed that the highest frequency of IMR-TB and MDR-TB strains belong to the LAM and Haarlem genotypes in Rio Grande do Sul state. A significant number of isolates previously characterized as Mycobacterium pinnipedi2 through spoligotyping were found to belong to the M. tuberculosis LAM family. This was responsible for a number of significant cases and the molecular profile of this strain and the pattern of mutations related to drug resistance were analyzed. These findings may contribute to a better understanding about the spread of M. tuberculosis resistant in southern of Brazil.

Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus.

BACKGROUND: To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. OBJECTIVE: To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). METHOD: 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. RESULTS: With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. CONCLUSION: Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.

Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus

BACKGROUND To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. OBJECTIVE To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). METHOD 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. RESULTS With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. CONCLUSION Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.

Association between cytokine gene polymorphisms and outcome of hepatitis B virus infection in southern Brazil.

A number of studies have demonstrated associations between cytokine gene polymorphisms and outcome of hepatitis B virus (HBV) infection. However, no general consensus has been reached, possibly due to differences between ethnic groups. In this study, 345 individuals living in southern Brazil, including 196 chronic HBV carriers and 149 subjects who had spontaneously recovered from acute infection, were enrolled to evaluate the influence of cytokine gene polymorphisms on the outcome of HBV infection. Most participants were of European descent. Genotyping of IL2-330 G/T, IL4-589C/T, IL6-174 G/C, IL10-592C/A, IL10-1082 A/G, IL17A-197 G/A, IL17A-692 T/C, TNF-α-238 G/A, and TNF-α-308 G/A single nucleotide polymorphisms was performed by using the minisequencing (single base extension) method. By multivariable analysis, a statistically significant association was found between genotypic profile AA + GA in TNF-α-308 and chronic HBV infection (OR, 1.82; 95%CI, 1.01-3.27; P = 0.046). In southern Brazil, the carriers of the -308A allele in the TNF-α gene promoter have a moderately higher risk of becoming chronic carriers in case of HBV infection. In addition, patients with chronic active hepatitis B (n = 60) exhibited a decreased frequency (3.3%) of the TNF-238A allele when compared to that (14.8%) found among asymptomatic HBV carriers (n = 136), suggesting that this could be a protective factor against liver injury (OR, 0.17; 95%CI, 0.04-0.076; P = 0.023). J. Med. Virol. 88:1759-1766, 2016. © 2016 Wiley Periodicals, Inc.

Single Nucleotide Polymorphisms in IL17A and IL6 Are Associated with Decreased Risk for Pulmonary Tuberculosis in Southern Brazilian Population.

In Mycobacterium tuberculosis (MTB) infection, the complex interaction of host immune system and the mycobacteria is associated with levels of cytokines production that play a major role in determining the outcome of the disease. Several single-nucleotide polymorphisms (SNPs) in cytokine genes have been associated with tuberculosis (TB) outcome. The aim of this study was to evaluate the association between previously reported SNPs IL2-330 T>G (rs2069762); IL4-590 C>T (rs2243250); IL6-174 G>C (rs1800795); IL10-592 A>C (rs1800872); IL10-1082 G>A (rs1800896); IL17A -692 C>T (rs8193036); IL17A -197 G>A (rs2275913); TNF -238 G>A (rs361525); TNF -308 G>A (rs1800629) and IFNG +874 T>A (rs2430561) and pulmonary TB (PTB) susceptibility. We conducted a case-control study in individuals from Southern Brazil who were recruited between February 2012 and October 2013 in a high incidence TB city. We performed a multiplex genotyping assay in 191 patients with PTB and 175 healthy subjects. Our results suggest a decreased risk for PTB development associated with the IL17A -197A allele (OR = 0.29; p = 0.04), AA genotype (OR = 0.12; p = 0.04) and A carrier (AG/AA) (OR = 0.29; p = 0.004) and IL6 -174C carrier (CC/CG) (OR = 0.46; p = 0.04). We could not properly analyze IL17A -692 C>T (rs8193036) and IFNG +874T>A due to genotypic inconsistencies and found no evidence of association for the IL2, IL4, IL10 and TNF polymorphisms and PTB. In conclusion, our results show a protective effect of IL17 and IL6 polymorphisms on PTB outcome in Southern Brazilian population.

Identification of the third/extra allele for forensic application in cases with TPOX tri-allelic pattern.

Genotyping of polymorphic short tandem repeats (STRs) loci is widely used in forensic DNA analysis. STR loci eventually present tri-allelic pattern as a genotyping irregularity and, in that situation, the doubt about the tri-allele locus frequency calculation can reduce the analysis strength. In the TPOX human STR locus, tri-allelic genotypes have been reported with a widely varied frequency among human populations. We investigate whether there is a single extra allele (the third allele) in the TPOX tri-allelic pattern, what it is, and where it is, aiming to understand its genomic anatomy and to propose the knowledge of this TPOX extra allele from genetic profile, thus preserving the two standard TPOX alleles in forensic analyses. We looked for TPOX tri-allelic subjects in 75,113 Brazilian families. Considering only the parental generation (mother+father) we had 150,226 unrelated subjects evaluated. From this total, we found 88 unrelated subjects with tri-allelic pattern in the TPOX locus (0.06%; 88/150,226). Seventy three of these 88 subjects (73/88; 83%) had the Clayton's original Type 2 tri-allelic pattern (three peaks of even intensity). The remaining 17% (15/88) show a new Type 2 derived category with heterozygote peak imbalance (one double dose peak plus one regular sized peak). In this paper we present detailed data from 66 trios (mother+father+child) with true biological relationships. In 39 of these families (39/66; 59%) the extra TPOX allele was transmitted either from the mother or from the father to the child. Evidences indicated the allele 10 as the extra TPOX allele, and it is on the X chromosome. The present data, which support the previous Lane hypothesis, improve the knowledge about tri-allelic pattern of TPOX CODIS' locus allowing the use of TPOX profile in forensic analyses even when with tri-allelic pattern. This evaluation is now available for different forensic applications.

Genetic data and de novo mutation rates in father-son pairs of 23 Y-STR loci in Southern Brazil population.

We evaluated haplotype and allele frequencies, as well as statistical forensic parameters, for 23 Y-chromosome short tandem repeats (STRs) loci of the PowerPlex®Y23 system (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, Y-GATA-H4, DYS481, DYS533, DYS549, DYS570, DYS576, DYS643) in a sample of 150 apparently healthy males, resident in South Brazil. A total of 150 different haplotypes were identified. The highest gene diversity (GD) was observed for the single locus marker DYS570 (GD = 0.7888) and for a two-locus system DYS385 (GD = 0.9009). We also examined 150 father-son pairs by the same system, and a total of 13 mutations were identified in the 3450 father-son allelic transfers, with an overall mutation rate across the 23 loci of 3.768 × 10(-3) (95% CI: 3.542 × 10(-3) to 3.944 × 10(-3)). In all cases there was only one locus mutated with gain/loss of repeats in the son (5 one-repeat gains, and 7 one-repeat and 1 two-repeat losses); we observed no instances of mutations involving a non-integral number of repeats.

Investigation of paternity with alleged father deceased or missing: analysis of success at the end of the report.

In this work we present a retrospective study of 858 cases of paternity investigation performed in Rio Grande do Sul, Southern Brazil, from 2007 to 2012, where the alleged father was deceased or missing. These cases represent 3.3% (858/26187) of paternity tests performed in that period. Considering the analysis of 17 DNA short tandem repeat loci, we present here the proportion of cases with conclusive results according to the number of relatives of the unavailable alleged father investigated and their kinship. The results show 81.0% (695/858) of cases with conclusive results and their characteristics.

Genetic diagnosis in recently transfused patients.

Analysis of recently transfused patients is usually postponed to avoid spurious results because of contamination with donor's cells. However, little is known about the extent of this influence in routine molecular diagnostic tests. To elucidate this question, we tested a mix of blood samples from 2 α-1-antitrypsin-deficient patients diagnosed as Pi*Z homozygous with 1 normal donor at 1:1, 1:10, 1:20, and 1:30 proportions. Human identification panel and Pi*Z allele detection were used to establish the detection limit of a blood mixture. Mixtures of 1:1 and 1:10 were easily detected with both techniques, whereas for 1:30, it was necessary to change the equipment settings to identify the mixture. Moreover, the heterozygous pattern observed for the mixtures on Pi*Z genotyping was weaker at this level of mixture. We further evaluated the degree of mixture detectable in 20 transfused patients who received 1 blood unit (concentrate of irradiated or nonirradiated red blood cells) using the human identification panel. Two days after the transfusion, the presence of the donor's markers was not detected, suggesting that after this time point the levels of admixture are below 1:30. The methods applied in the present study showed adequate sensitivity to identify alleles of the so-called "smaller population" of cells up to 3%, approximately. The same result was obtained in a "diagnostic situation," in which the blood mixture was submitted to a PCR-RFLP protocol to detect a mutation.

Mutation rate estimates for 13 STR loci in a large population from Rio Grande do Sul, Southern Brazil.

Short tandem repeat (STR) polymorphisms have been extensively used in forensic genetics analysis. Knowledge about the locus-specific mutation rates of STRs improves forensic probability calculations and interpretations of diversity data. To incorporate single-locus diversity information into autosomal STR mutation rate estimations, 13 STR loci were studied during 2007-2009 in 10,959 paternity investigation cases from Rio Grande do Sul, the southernmost state of Brazil, covering an overall number of 284,934 allelic transfers. A total of 355 mutations were identified; 348 repeats were gains or losses of one step, three were gains or losses of two steps, and four were gains or losses of not stepwise mutation. The mutation rates ranged from 4.6 × 10(-5) to 2.3 × 10(-3), and the overall mutation rate estimate was 1.2 × 10(-3). The average of the paternal mutation rate (1.8 × 10(-3)) was five times higher than the maternal rate (0.36 × 10(-3)). The observed mutational features for STRs have important consequences for forensic applications, including the definition of criteria for exclusion in paternity testing and the interpretation of DNA profiles in identification analysis.

Population genetic analyses of the AmpFlSTR® NGM™ in Brazil.

Population data of 15 short tandem repeat loci of the AmpFlSTR® next generation multiplex (NGM)™ were obtained from a sample of 835 individuals. The loci are the ten short tandem repeats (STRs) in the SGM Plus® Kit plus the EDNAP- and ENSFI-recommended STRs D10S1248, D22S1045, D2S441, D1S1656, and D12S391. Allele frequency and other forensically relevant statistics data were generated for the NGM loci into five current country macroregions of Brazil (North, Northeast, Central West, Southeast, and South). All the analyzed loci meet Hardy-Weinberg equilibrium expectations and no linkage disequilibrium in all pairs of loci. The observed and expected heterozygosity, power of discrimination, polymorphic information content, and the other population-genetic indices were calculated. The overall power of discrimination was greater than 0.99999999999999999996 and the combined power of exclusion was greater than 0.9999998 in all Brazilian populations. Comparative analysis between populations from different Brazilian macroregions as well as between Brazil and Caucasian, African Americans, and Hispanic US populations are presented.

Allele frequencies of 15 STRs in a representative sample of the Brazilian population.

Allele frequencies for 15 short tandem repeat (STR) loci were obtained from a sample of 12,030 individuals undergoing paternity testing. This sample includes individuals from all States in Brazil, combined according to the current country division into five regions (North, Northeast, Central West, Southeast, and South). The most polymorphic loci were D2S1338 and D18S51. All the analysed loci meet Hardy-Weinberg equilibrium expectations. Combined power of discrimination and combined power of exclusion for the 15 tested STR loci were 0.999999999999990 and 0.9999992, respectively. Comparative analysis between populations from different Brazilian macroregions as well as between Brazil and other relevant populations are presented.

15 STR loci frequencies in the population from Paraná, Southern Brazil.

Allele frequencies for 15 short tandem repeats (STR) loci were obtained from a sample of 4076 unrelated individuals undergoing paternity testing. The population is from Paraná, Southern Brazil. The loci are the most commonly used in forensic and paternity testing, being analyzed by the AmpFlSTR((R)) Identifiler (Applied Biosystems) commercial kit. The most polymorphic loci were D2S1338 and D18S51. Excepting the D13S317, all loci were in Hardy-Weinberg equilibrium. Comparative analyses between our population data and other populations are presented.

Population genetic data for 11 STR loci, including SE33, in Southern Brazil.

Allele frequencies for 11 STR autosomal loci (D3S1358, vWA, D16S539, D2S1338, D8S1179, SE33, D19S433, TH01, FGA, D21S11, D18S51) were analysed in unrelated individuals undergoing paternity testing in Rio Grande do Sul State, Southern Brazil. The most polimorphic locus was SE33. The distributions of the genotypes in the evaluated loci are in Hardy-Weinberg equilibrium after Bonferroni's correction.

15 STR loci frequencies with mutation rates in the population from Rio Grande do Sul, Southern Brazil.

Allele frequencies for 15 short tandem repeats (STR) loci were obtained from a sample of 2038 individuals undergoing paternity testing. The population is from Rio Grande do Sul, Brazil. The loci are the most commonly used in forensic and paternity testing, being analysed by the AmpFlSTR Identifiler (Applied Biosystems) commercial kit. The most polymorphic locus was D2S1338. Mutation rates were ascertained from this population sample. All the loci analysed reached the Hardy-Weinberg equilibrium. The results of genetic distance are consistent with the European-derived origins of Rio Grande do Sul population.