The Role of GeneXpert® for Tuberculosis Diagnostics in Brazil: An Examination from a Historical and Epidemiological Perspective.

The rapid molecular test (RMT) performed on the GeneXpert® system is widely used as a control strategy and surveillance technique for tuberculosis (TB). In the region of the Americas, TB incidence is slowly increasing owing to an upward trend in Brazil, which is among the high TB-burden countries (HBCs), ranking in the 19th position. In this context, we aimed to (i) describe the implementation and history of RMT-TB (Xpert® MTB/RIF and Xpert® MTB/RIF Ultra) in Brazil; (ii) to evaluate the national RMT laboratory distribution, TB, and resistance to RIF detection by RMT; and (iii) to correlate these data with Brazilian TB incidence. The quantitative data of Xpert® MTB/RIF and Xpert® MTB/RIF Ultra assays performed in the pulmonary TB investigation from 2014 to 2020 were provided by the Brazilian Ministry of Health. A spatial visualization using ArcGIS software was performed. The Southeast region constituted about half of the RMT laboratories-from 39.4% to 45.9% of the total value over the five regions. Regarding the federal units, the São Paulo state alone represented from 20.2% to 34.1% (5.0 to 8.5 times the value) of RMT laboratories over the years observed. There were significant differences (p < 0.0001) in the frequency of RMT laboratories between all years of the historical series. There was an unequal distribution of RMT laboratories between Brazilian regions and federal units. This alerts us for the surveillance of rapid molecular detection of TB in different parts of the country, with the possibility of improving the distribution of tests in areas of higher incidence in order to achieve the level of disease control recommended by national and worldwide authorities.

Spatial Analysis of Tuberculosis Patient Flow in a Neglected Region of Northern Brazil.

Tuberculosis (TB) is still considered a priority due to its high incidence rate in Brazil. In this context, we aimed to evaluate the flow of care between the municipalities of patients diagnosed with TB through notification forms of the Information System for Diseases and Notifications (SINAN) in a neglected region of Northern Brazil, Ilha do Marajó, state of Para. For this, we performed a descriptive, retrospective study on data obtained from the National Register of Health Establishments and SINAN from 2013 to 2018. We used Pearson's Chi-square and G Test with p-value < 0.05 for descriptive statistics and spatial analysis technique on flow network analysis. Of the 749 cases, 16.5% were notified in another municipality that was not the patient's residence. Regarding diagnostic methods, a positive bacterioscopy was adopted for 56% of the patients; culture was not performed for 82% of cases. Histopathological examination was not performed in 90% of the individuals. Rapid molecular test (RMT) was performed in only six (5%) cases. The region needs greater attention focused on diagnostic tests, suggesting that the introduction of RMT and culture by Ogawa-Kudoh could improve the region's health network to minimise patient displacement and thus avoid the increase in the transmission chain of TB.

Genomic Diversity of the Rarely Observed Genotype of the Mycobacterium tuberculosis Central Asian (CAS) Lineage 3 from North Brazil.

Mycobacterium tuberculosis (Mtb) Central Asian Strain (CAS) Lineage 3 (L3) genotype is predominantly found in East-Africa, Central-Asia, Western-Asia, and South-Asia; however, a new spoligotyping CAS/SIT2545 was found in northern regions of Brazil. We aimed to characterize and describe the genetic diversity and perform a phylogenetic assessment of this novel genotype. We performed 24-MIRU-VNTR loci and Whole-genome sequencing (WGS) of six Brazilian isolates previously spoligotyped. The libraries were prepared using a Nextera-XT kit and sequenced in a NextSeq 550 Illumina instrument. We performed lineage assignment and genomic characterization. From publicly available genomes of Mtb L3 and other lineages, we created a robust dataset to run the MTBSeq pipeline and perform a phylogenetic analysis. MIRU-VNTR and WGS confirmed CAS/SIT2545 belongs to L3. Out of 1691 genomes, 1350 (79.83%) passed in quality control (genomic coverage > 95%). Strain 431 differed in 52 single nucleotide variants (SNV), confirming it does not belong to the same transmission chain. The eight genomes from a global dataset clustered closer to Brazilian strains differed in >52 SNVs. We hypothesized L3 and L1 were introduced in Brazilian Northern in the same historical event; however, there is a need for additional studies exploring the genetic diversity of Mtb Brazilian Northern.

Molecular detection of mycobacterium tuberculosis complex species in triatomines found in urban households and Peridomiciles in a city with high prevalence of tuberculosis in Northeastern Brazil.

Background: Triatomine insects, native to northeastern Brazil, have been found in the urban peridomicile. The city of Sobral has a high number of tuberculosis cases and several triatomine species. This study investigates the presence of mycobacteria, particularly Mycobacterium tuberculosis complex (MTBC) species, in triatomines captured in the urban perimeter of Sobral. Methods: We analyzed 167 triatomines captured in urban households and peridomiciles of Sobral. Mycobacteria were identified by the PRA-hsp65 method followed by partial sequencing of the hsp65 and rpoB genes. The sequences confirmed as MTBC were also typed by mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) and spoligotyping. Results: Triatoma brasiliensis (38.6%), Triatoma pseudomaculata (32.9%), Panstrongylus lutzi (24.3%) were the most frequently identified. In 51.1% (70/167) of them, species of the Mycobacteriaceae family were detected by PRA-hsp65; of these, 31.4% (22/70) were identified as belonging to MTBC species. Nine (12.9%) of the triatomine samples were confirmed by sequencing as belonging to MTBC species. MIRU-VNTR genotyping suggests that the presence of different MTBC sublines in the triatomines should be investigated. Conclusion: This is the first report of MTBC lineages in triatomine insects. These results indicate the migration and adaptation of these insects in an urban setting.

Whole-Genome Sequencing of Alcaligenes faecalis HZ01, with Potential to Inhibit Nontuberculous Mycobacterial Growth.

Alcaligenes faecalis is a Gram-negative rod that is ubiquitous in the environment and is an opportunistic human pathogen. Here, we report the whole-genome sequencing analysis of A. faecalis HZ01, which presents mycobacterial growth inhibitory activity and was isolated from a contaminated culture of Mycobacterium chubuense ATCC 27278.

Molecular epidemiology of Mycobacterium tuberculosis in Brazil before the whole genome sequencing era: a literature review.

Molecular-typing can help in unraveling epidemiological scenarios and improvement for disease control strategies. A literature review of Mycobacterium tuberculosis transmission in Brazil through genotyping on 56 studies published from 1996-2019 was performed. The clustering rate for mycobacterial interspersed repetitive units - variable tandem repeats (MIRU-VNTR) of 1,613 isolates were: 73%, 33% and 28% based on 12, 15 and 24-loci, respectively; while for RFLP-IS6110 were: 84% among prison population in Rio de Janeiro, 69% among multidrug-resistant isolates in Rio Grande do Sul, and 56.2% in general population in São Paulo. These findings could improve tuberculosis (TB) surveillance and set up a solid basis to build a database of Mycobacterium genomes.

Evaluation of drug susceptibility profile of Mycobacterium tuberculosis Lineage 1 from Brazil based on whole genome sequencing and phenotypic methods.

BACKGROUND: The evaluation of procedures for drug susceptibility prediction of Mycobacterium tuberculosis based on genomic data against the conventional reference method test based on culture is realistic considering the scenario of growing number of tools proposals based on whole-genome sequences (WGS). OBJECTIVES: This study aimed to evaluate drug susceptibility testing (DST) outcome based on WGS tools and the phenotypic methods performed on isolates of M. tuberculosis Lineage 1 from the state of Pará, Brazil, generally associated with low levels of drug resistance. METHODOLOGY: Culture based DST was performed using the Proportion Method in Löwenstein-Jensen medium on 71 isolates that had been submitted to WGS. We analysed the seven main genome sequence-based tools for resistance and lineage prediction applied to M. tuberculosis and for comparison evaluation we have used the Kappa concordance test. FINDINGS: When comparing the WGS-based tools against the DST, we observed the highest level of agreement using TB-profiler. Among the tools, TB-profiler, KvarQ and Mykrobe were those which identified the largest number of TB-MDR cases. Comparing the four most sensitive tools regarding resistance prediction, agreement was observed for 43 genomes. MAIN CONCLUSIONS: Drug resistance profiling using next-generation sequencing offers rapid assessment of resistance-associated mutations, therefore facilitating rapid access to effective treatment.

Molecular epidemiology of mycobacteria among herds in Marajó Island, Brazil, reveals strains genetically related and potential zoonotic risk of clinical relevance.

Mycobacterium bovis is the main causative agent of bovine tuberculosis (bTB) being among the animal-adapted Mycobacterium tuberculosis complex. Herds can also be infected with non-tuberculous mycobacteria (NTM) causing a negative effect on the economy and on animal and human health through zoonotic infections. Molecular tools are required for mycobacteria identification; thus, it is laborious to determine the epidemiological information of mycobacteria among herds. We aimed to describe the mycobacterial pathogens associated with cases of suspected bTB lesions in cattle/buffaloes slaughtered for consumption and to investigate bTB transmission. We evaluated 74 lesion samples from 48 animals (27 bovine/21 buffaloes) from 16 mapped farms. Positives samples from nested-PCR were cultured in Lowenstein-Jensen (LJ), 2% pyruvate (LJ + P), and 2% glycerol (LJ + G) media, followed by Ziehl-Neelsen (ZN) staining technique and partial gene sequencing (hsp65, rpoB, and 16S-rRNA). Spoligotyping and 24-MIRU-VNTR were performed. The LJ + P increased the chance of obtaining bacilli. The respiratory tract and the oral cavity were the most important infection route. In addition, the calcified part of the lesions suggested chronic bTB. Spoligotypes of M. bovis (SIT986/SB0885) differed from others found in South America, and the MIRU-VNTR 24 loci suggested that bTB was associated to highly related strains. The NTM species found are of clinical importance in humans.

Analysis of potential household transmission events of tuberculosis in the city of Belem, Brazil.

Tuberculosis (TB) is an infectious disease with a higher risk for infection and disease among household contacts (HHC). Here, we report a molecular epidemiology-based approach to study disease transmission and the genetic characteristics of Mycobacterium tuberculosis (Mtb) strains among HHC in the city of Belem, the capital of the state of Para in north Brazil. The study included 63 TB patients belonging to 26 HHC groups (HHC1 to HHC26). Spoligotyping and 24-loci Mycobacterial Interspersed Repetitive Unit - Variable Number of Tandem Repeat (MIRU-VNTR) revealed indistinguishable bacterial genotypes among 26 patients in 14 (53.8%) HHC groups. Drug susceptibility testing (DST) revealed that 45 (71.4%) of the Mtb isolates were multidrug resistant. The major cluster composed of isolates from five HHCs and on three of these, whole genome sequencing (WGS) was performed confirming their high genetic similarity. These results pinpoint the need for improved vigilance for TB control in households in the city of Belém. When comparing WGS versus phenotypic resistance detection methods as DST and Minimum Inhibitory Concentration (MIC) our data suggest that depending on the colonies selection, results may present variation.