Non-tuberculous mycobacteria hybridisation profiles in the GenoType MTBDRplus assay: experience from a diagnostic routine of a high-throughput laboratory.

Introduction. Disease caused by non-tuberculous mycobacteria (NTM) is an emergent problem. Because NTM pulmonary disease and tuberculosis (TB) have similar clinical presentations, many cases of NTM may be misdiagnosed as TB before laboratory identification of the NTM species.Hypothesis/Gap Statement. Clinical laboratories should always perform differentiation between Mycobacterium tuberculosis complex (MTBC) and NTM to guide patients' correct treatment.Aim. To describe the characteristics and to identify mycobacterial isolates presumptively classified as MTBC by macroscopic characteristics in culture media that tested negative in GenoType MTBDRplus.Methodology. All cultures from February 2019 to December 2021 showing MTBC macroscopic characteristics were processed by GenoType MTBDRplus. MTBC-negative cultures underwent species identification by immunochromatography, line probe assays and PRA-hsp65. Patients' data were obtained from Brazilian surveillance systems.Results. Only 479 (3.1%) of 15 696 isolates presumptively identified as MTBC were not confirmed by GenoType MTBDRplus and were then subjected to identification. A total of 344 isolates were shown to be NTM, of which 309 (64.5%) and 35 (7.3%) were identified to the species and genus levels, respectively. Of the 204 NTM isolates with MTBC characteristics, the most frequent species were M. fortuitum (n=52, 25.5%), M. abscessus complex (MABC; n=27, 13.2%) and M. avium complex (MAC; n=26, 12.7%). Regarding the GenoType MTBDRplus results from NTM isolates, there were diverse hybridisation profiles with rpoB gene's different wild-type (WT) probes. Seventy-six (16.1%) of the 473 patients were classified as having NTM disease, the most frequent being MAC (n=15, 19.7%), MABC (n=13, 17.1%), M. kansasii (n=10, 13.2%) and M. fortuitum (n=6, 7.9%).Conclusion. Because the signs and symptoms of pulmonary TB are similar to those of pulmonary mycobacteriosis and treatment regimens for TB and NTM are different, identifying the disease-causing species is paramount to indicate the correct management. Thus, in the laboratory routine, when an isolate presumptively classified as MTBC is MTBC-negative, it is still essential to perform subsequent identification.

A descriptive study on isoniazid resistance-associated mutations, clustering and treatment outcomes of drug-resistant tuberculosis in a high burden country.

PURPOSE: To describe katG and inhA mutations, clinical characteristics, treatment outcomes and clustering of drug-resistant tuberculosis (TB) in the State of São Paulo, southeast Brazil. METHODS: Mycobacterium tuberculosis isolates from patients diagnosed with drug-resistant TB were screened for mutations in katG and inhA genes by line probe assay and Sanger sequencing, and typed by IS6110-restriction fragment-length polymorphism for clustering assessment. Clinical, epidemiological and demographic data were obtained from surveillance information systems for TB. RESULTS: Among the 298 isolates studied, 127 (42.6%) were isoniazid-monoresistant, 36 (12.1%) polydrug-resistant, 93 (31.2%) MDR, 16 (5.4%) pre-extensively drug-resistant (pre-XDR), 9 (3%) extensively drug-resistant (XDR) and 17 (5.7%) susceptible after isoniazid retesting. The frequency of katG 315 mutations alone was higher in MDR isolates, while inhA promoter mutations alone were more common in isoniazid-monoresistant isolates. Twenty-six isolates phenotypically resistant to isoniazid had no mutations either in katG or inhA genes. The isolates with inhA mutations were found more frequently in clusters (75%) when compared to the isolates with katG 315 mutations (59.8%, p = 0.04). In our population, being 35-64 years old, presenting MDR-, pre-XDR- or XDR-TB and being a retreatment case were associated with unfavourable TB treatment outcomes. CONCLUSION: We found that katG and inhA mutations were not equally distributed between isoniazid-monoresistant and MDR isolates. In our population, clustering was higher for isolates with inhA mutations. Finally, unfavourable TB outcomes were associated with specific factors.

Rapid response of a public health reference laboratory to the COVID-19 pandemic.

Introduction. Brazil was one of the most affected countries by the COVID-19 pandemic. Instituto Adolfo Lutz (IAL) is the reference laboratory for COVID-19 in São Paulo, the most populous state in Brazil. In April 2020, a secondary diagnostic pole named IAL-2 was created to enhance IAL's capacity for COVID-19 diagnosis.Hypothesis/Gap Statement. Public health laboratories must be prepared to rapidly respond to emerging epidemics or pandemics.Aim. To describe the design of IAL-2 and correlate the results of RT-qPCR tests for COVID-19 with secondary data on suspected cases of SARS-CoV-2 infection in the São Paulo state.Methodology. This is a retrospective study based on the analysis of secondary data from patients suspected of infection by SARS-CoV-2 whose clinical samples were submitted to real-time PCR after reverse transcription (RT-qPCR) at IAL-2, between 1 April 2020 and 8 March 2022. RT-qPCR Ct results of the different kits used were also analysed.Results. IAL-2 was implemented in April 2020, just over a month after the detection of the first COVID-19 case in Brazil. The laboratory performed 304,250 RT-qPCR tests during the study period, of which 98 319 (32.3 %) were positive, 205827 (67.7 %) negative, and 104 (0.03 %) inconclusive for SARS-CoV-2. RT-qPCR Ct values≤30 for E/N genes of SARS-CoV-2 were presented by 79.7 % of all the samples included in the study.Conclusion. IAL was able to rapidly implement a new laboratory structure to support the processing of an enormous number of samples for diagnosis of COVID-19, outlining strategies to carry out work with quality, using different RT-qPCR protocols.

New insights on tuberculosis transmission dynamics and drug susceptibility profiles among the prison population in Southern Brazil based on whole-genome sequencing.

BACKGROUND: The rate of tuberculosis (TB) infection among the prison population (PP) in Brazil is 28 times higher than that in the general population, and prison environment favors the spread of TB. OBJECTIVE: To describe TB transmission dynamics and drug resistance profiles in PP using whole-genome sequencing (WGS). METHODS: This was a retrospective study of Mycobacterium tuberculosis cultivated from people incarcerated in 55 prisons between 2016 and 2019; only one isolate per prisoner was included. Information about movement from one prison to another was tracked. Clinical information was collected, and WGS was performed on isolates obtained at the time of TB diagnosis. RESULTS: Among 134 prisoners included in the study, we detected 16 clusters with a total of 58 (43%) cases of M. tuberculosis. Clusters ranged from two to seven isolates with five or fewer single nucleotide polymorphism (SNP) differences, suggesting a recent transmission. Six (4.4%) isolates were resistant to at least one anti-TB drug. Two of these clustered together and showed resistance to rifampicin, isoniazid, and fluoroquinolones, with 100% concordance between WGS and phenotypic drug-susceptibility testing. Prisoners with clustered isolates had a high amount of movement between prisons (two to eight moves) during the study period. CONCLUSIONS: WGS demonstrated the recent transmission of TB within prisons in Brazil. The high movement among prisoners seems to be related to the transmission of the same M. tuberculosis strain within the prison system. Screening for TB before and after the movement of prisoners using rapid molecular tests could play a role in reducing transmission.

GenoType MTBDRsl for detection of second-line drugs and ethambutol resistance in multidrug-resistant Mycobacterium tuberculosis isolates at a high-throughput laboratory.

We assessed the performance of MTBDRsl for detection of resistance to fluoroquinolones, aminoglycosides/cyclic peptides, and ethambutol compared to BACTEC MGIT 960 by subjecting simultaneously to both tests 385 phenotypically multidrug-resistant-Mycobacterium tuberculosis isolates from Sao Paulo, Brazil. Discordances were resolved by Sanger sequencing. MTBDRsl correctly detected 99.7% of the multidrug-resistant isolates, 87.8% of the pre-XDR, and 73.9% of the XDR. The assay showed sensitivity of 86.4%, 100%, 85.2% and 76.4% for fluoroquinolones, amikacin/kanamycin, capreomycin and ethambutol, respectively. Specificity was 100% for fluoroquinolones and aminoglycosides/cyclic peptides, and 93.6% for ethambutol. Most fluoroquinolone-discordances were due to mutations in genome regions not targeted by the MTBDRsl v. 1.0: gyrA_H70R and gyrB_R446C, D461N, D449V, and N488D. Capreomycin-resistant isolates with wild-type rrs results on MTBDRsl presented tlyA mutations. MTBDRsl presented good performance for detecting resistance to second-line drugs and ethambutol in clinical isolates. In our setting, multidrug-resistant. isolates presented mutations not targeted by the molecular assay.

New insights on tuberculosis transmission dynamics and drug susceptibility profiles among the prison population in Southern Brazil based on whole-genome sequencing

ABSTRACT Background: The rate of tuberculosis (TB) infection among the prison population (PP) in Brazil is 28 times higher than that in the general population, and prison environment favors the spread of TB. Objective: To describe TB transmission dynamics and drug resistance profiles in PP using whole-genome sequencing (WGS). Methods: This was a retrospective study of Mycobacterium tuberculosis cultivated from people incarcerated in 55 prisons between 2016 and 2019; only one isolate per prisoner was included. Information about movement from one prison to another was tracked. Clinical information was collected, and WGS was performed on isolates obtained at the time of TB diagnosis. Results: Among 134 prisoners included in the study, we detected 16 clusters with a total of 58 (43%) cases of M. tuberculosis. Clusters ranged from two to seven isolates with five or fewer single nucleotide polymorphism (SNP) differences, suggesting a recent transmission. Six (4.4%) isolates were resistant to at least one anti-TB drug. Two of these clustered together and showed resistance to rifampicin, isoniazid, and fluoroquinolones, with 100% concordance between WGS and phenotypic drug-susceptibility testing. Prisoners with clustered isolates had a high amount of movement between prisons (two to eight moves) during the study period. Conclusions: WGS demonstrated the recent transmission of TB within prisons in Brazil. The high movement among prisoners seems to be related to the transmission of the same M. tuberculosis strain within the prison system. Screening for TB before and after the movement of prisoners using rapid molecular tests could play a role in reducing transmission.

GenoType MTBDRsl for detection of second-line drugs and ethambutol resistance in multidrug-resistant Mycobacterium tuberculosis isolates at a high-throughput laboratory

We assessed the performance of MTBDRsl for detection of resistance to fluoroquinolones, aminoglycosides/cyclic peptides, and ethambutol compared to BACTEC MGIT 960 by subjecting simultaneously to both tests 385 phenotypically multidrug-resistant-Mycobacterium tuberculosis isolates from Sao Paulo, Brazil. Discordances were resolved by Sanger sequencing. MTBDRsl correctly detected 99.7% of the multidrug-resistant isolates, 87.8% of the pre-XDR, and 73.9% of the XDR. The assay showed sensitivity of 86.4%, 100%, 85.2% and 76.4% for fluoroquinolones, amikacin/kanamycin, capreomycin and ethambutol, respectively. Specificity was 100% for fluoroquinolones and aminoglycosides/cyclic peptides, and 93.6% for ethambutol. Most fluoroquinolone-discordances were due to mutations in genome regions not targeted by the MTBDRsl v. 1.0: gyrA_H70R and gyrB_R446C, D461N, D449V, and N488D. Capreomycin-resistant isolates with wild-type rrs results on MTBDRsl presented tlyA mutations. MTBDRsl presented good performance for detecting resistance to second-line drugs and ethambutol in clinical isolates. In our setting, multidrug-resistant. isolates presented mutations not targeted by the molecular assay.

Correlating genetic mutations with isoniazid phenotypic levels of resistance in Mycobacterium tuberculosis isolates from patients with drug-resistant tuberculosis in a high burden setting.

We analysed mutations in katG, inhA and rpoB genes, and isoniazid phenotypic resistance levels in Mycobacterium tuberculosis isolates from drug-resistant TB patients from São Paulo state, Brazil. Isolates resistant to the critical concentration of isoniazid in MGIT (0.1 µg/mL) were screened for mutations in katG 315 codon, inhA promoter region and rpoB RRDR by MTBDRplus assay and subjected to determination of isoniazid resistance levels by MGIT 960. Discordances were resolved by Sanger sequencing. Among the 203 isolates studied, 109 (54%) were isoniazid-monoresistant, 47 (23%) MDR, 29 (14%) polydrug-resistant, 12 (6%) pre-XDR and 6 (3%) XDR. MTBDRplus detected isoniazid mutations in 75% (153/203) of the isolates. Sequencing of the entire katG and inhA genes revealed mutations in 18/50 wild-type isolates by MTBDRplus (10 with novel mutations), resulting in a total of 32/203 (16%) isolates with no mutations detected. 81/83 (98%) isolates with katG 315 mutations alone had intermediate resistance. Of the 66 isolates with inhA C-15T mutation alone, 51 (77%) showed low-level, 14 (21%) intermediate and 1 (2%) high-level resistance. 5/6 (83%) isolates with mutations in both katG and inhA had high-level resistance. Inferred mutations corresponded to 22% (16/73) of all mutations found in rpoB. Mutations detected in katG regions other than codon 315 in this study might be potential new isoniazid resistance markers and could explain phenotypic resistance in some isolates without katG and inhA classic mutations. In our setting, 16% of isoniazid-resistant isolates, some with high-level resistance, presented no mutations either in katG or inhA.

Transmission of Mycobacterium tuberculosis presenting unusually high discordance between genotypic and phenotypic resistance to rifampicin in an endemic tuberculosis setting.

BACKGROUND: Since the implementation of the Xpert MTB/RIF in Sao Paulo, Brazil, numerous Mycobacterium tuberculosis isolates presenting "rifampicin-resistant genotype with rifampicin-susceptible phenotype" were observed. OBJECTIVE: To evaluate the prevalence, rpoB mutations and transmission of M. tuberculosis resistant to rifampicin on Xpert MTB/RIF but susceptible on BACTEC MGIT system, in Sao Paulo state. METHODS: Patients' isolates with this pattern of rifampicin discordance, collected from 2014 to 2017, had their rpoB predominant rifampicin-resistance-determining region sequenced and were genotyped by IS6110 restriction fragment-length polymorphism. FINDINGS: The prevalence of rifampicin-discordant M. tuberculosis with genotypic resistance was 55.1% (156/283). Among the sequenced and genotyped isolates, 75.5% (111/147) were in clusters, largely associated with the type of rpoB mutation. Most isolates (98.6%; 72/73) harbouring the predominant mutation, His445Asn, were pooled into the two largest clusters, SP2ga (42/72; 58.3%) and SP5o (12/72; 16.7%). Ranking second, isolates carrying the silent mutation Phe433Phe were mostly (92.3%; 24/26) gathered into four groups of the family SP25. CONCLUSION: These findings suggest that this unusual high rifampicin discrepancy proportion was greatly influenced by few actively circulating clusters. Further studies on many of the rpoB mutations identified in our setting are needed to elucidate their association with phenotypic rifampicin resistance.

Phenotypic characterization of Neisseria meningitidis strains isolated from invasive meningococcal disease in Brazil from 2002 to 2017.

INTRODUCTION: Invasive meningococcal disease (IMD) has a high rate of fatality and may cause severe clinical sequelae. Over the years, the epidemiology of IMD has changed significantly in various regions of the world, and laboratory surveillance of this disease is important for mapping epidemiologic changes. AIM: To perform phenotypic characterization of Neisseria meningitidis strains isolated from invasive disease in Brazil from 2002 to 2017, as a complementation of the data obtained in the period of 1990-2001. METHODOLOGY: In total, 8,689 isolates sent to Adolfo Lutz Institute confirmed as N. meningitidis by conventional methods were serogrouped by slide agglutination against MenA, MenB, MenC, MenE, MenW, MenX, MenY and MenZ; serotyped and serosubtyped by a whole-cell dot-blotting assay with monoclonal antibodies. RESULTS: The isolates were sent from all regions of Brazil, and the southeast region was responsible for the largest number of isolates (57.2 %). Overall, the total sample (n=8,689) was represented by serogroups C (n=4,729; 54.4 %), B (n=3,313; 38.1 %), W (n=423; 4.9 %), Y (n=203; 2.3 %), X (n=5; 0.1 %) and others (n=16; 0.2 %). A shift in the prevalence of serogroups was observed in 2006, when serogroup C became the most prevalent (65.5 %), surpassing the serogroup B (21.9 %). The main isolated phenotypes were C:23:P1.14-6; B:4,7:P1.19,15; W:2a:P1.5 and W:2a:P1.5,2. CONCLUSION: The data show an important change in the distribution of meningococcal serogroups, serotypes and subtypes occurring during 2002-2017. A continuous laboratory-based surveillance provides robust information to implement appropriate strategies to IMD control.

Transmission and prevalence of drug-resistant tuberculosis in a Brazilian setting under a directly observed therapy short-course strategy.

INTRODUCTION: We aimed to estimate the prevalence and transmission of drug-resistant tuberculosis in a high-burden Brazilian setting under directly observed therapy short-course strategy. METHODS: Isolates of culture-confirmed pulmonary tuberculosis patients from Guarulhos, Brazil, diagnosed in October 2007-2011 were subjected to drug susceptibility and IS6110-restriction fragment length polymorphism testing. RESULTS: The overall resistance prevalence was 11.5% and the multi-drug resistance rate was 4.2%. Twenty-six (43.3%) of 60 drug-resistant isolates were clustered. Epidemiological relationships were identified in 11 (42.3%) patients; 30.8% of the cases were transmitted in households. CONCLUSIONS: Drug-resistant tuberculosis was relatively low and transmitted in households and the community.

Frequency of first and second-line drug resistance-associated mutations among resistant Mycobacterium tuberculosis clinical isolates from São Paulo, Brazil.

BACKGROUND Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the number of new cases of multidrug resistant TB (MDR-TB), pre extensively drug-resistant TB (pre-XDR-TB) and extensively drug-resistant TB (XDR-TB) has increased considerably worldwide. OBJECTIVES Herein, using 156 M. tuberculosis isolates from 106 patients previously classified as MDR or pre-XDR or XDR isolates, we investigated the genetic mutation profiles associated with phenotypic resistances in patients with MDR-TB, pre-XDR-TB and XDR-TB, treatment outcomes and resistance evolution. METHODS Molecular analyses were performed by partial sequencing of the rpoB, katG, gyrA, gyrB, rrs genes and analysis of the fabG-inhA promoter region. Clinical, epidemiologic and demographic data were obtained from the TB Notification database system of São Paulo (TB-WEB) and the Information System for Special Tuberculosis Treatments (SITE-TB). FINDINGS Drug resistance was attributed to previously known mutations and a novel Asp449Val mutation in gyrB was observed in four isolates from the same patient. Ten patients had more than one isolate evaluated and eight of these patients displayed resistance progression. MAIN CONCLUSIONS The present study is the first to report the frequency of mutations related to second-line drug resistance in MDR-TB, pre-XDR-TB and XDR-TB isolates. The results could lead to the improvement of available technologies for the rapid detection of drug resistant TB.

Transmission and prevalence of drug-resistant tuberculosis in a Brazilian setting under a directly observed therapy short-course strategy

Abstract INTRODUCTION: We aimed to estimate the prevalence and transmission of drug-resistant tuberculosis in a high-burden Brazilian setting under directly observed therapy short-course strategy. METHODS: Isolates of culture-confirmed pulmonary tuberculosis patients from Guarulhos, Brazil, diagnosed in October 2007-2011 were subjected to drug susceptibility and IS6110-restriction fragment length polymorphism testing. RESULTS: The overall resistance prevalence was 11.5% and the multi-drug resistance rate was 4.2%. Twenty-six (43.3%) of 60 drug-resistant isolates were clustered. Epidemiological relationships were identified in 11 (42.3%) patients; 30.8% of the cases were transmitted in households. CONCLUSIONS: Drug-resistant tuberculosis was relatively low and transmitted in households and the community.

Determinação do nível de resistência a isoniazida em cepas de Mycobacterium tuberculosis e correlação com mutações nos genes que conferem resistência a este fármaco: katG e inhA / Determination of the level of resistance to isoniazid in strains of Mycobacterium tuberculosis and correlation with mutations in genes that confer resistance to this drug: katG and inhA

A tuberculose (TB) resistente aos fármacos é um problema mundial. As mutações mais frequentes associadas à resistência à isoniazida em Mycobacterium tuberculosis ocorrem no códon 315 do gene katG, causando níveis moderados a altos de resistência, e na região promotora do gene inhA, associadas a baixos níveis de resistência. Mutações no gene rpoB associam-se à resistência à rifampicina. Este estudo analisou mutações nos genes katG, inhA e rpoB e níveis fenotípicos de resistência à isoniazida em isolados de M. tuberculosis de pacientes com TB resistente do estado de São Paulo, no período de outubro/2008 a março/2009 e no ano de 2016. Dos 374 isolados analisados, 165 (44%) eram monorresistentes à isoniazida, 47 (13%) polirresistentes (resistentes à isoniazida+fármaco(s) de 1ª linha exceto rifampicina), 122 (33%) multirresistentes (MR), 24 (6%) pré-extensivamente resistentes (pré-XDR) e 16 (4%) XDR. Entre os isolados com a mutação Ser315Thr no katG, 99,4% apresentaram resistência intermediária ou alta à isoniazida, enquanto 73,7% dos isolados com a mutação C-15T no inhA apresentaram baixa resistência. Dos isolados com mutações no katG e inhA, 66,6% mostraram resistência alta e 32,4% resistência intermediária. Observou-se que 58,8% dos isolados tipados estavam em cluster e 43,7% deles pertenciam...(AU)

Drug-resistant tuberculosis (TB) is a problem worldwide. The most frequent mutations associated with isoniazid resistance in Mycobacterium tuberculosis isolates occur in codon 315 of the katG gene, which have been associated with moderate- to high-levels of resistance, and in the promoter region of the inhA gene, causing low-level isoniazid resistance. In its turn, the rpoB gene is associated with resistance to rifampicin. This study analyzed mutations in katG, inhA and rpoB genes and phenotypic levels of isoniazid resistance in M. tuberculosis isolates from drug-resistant TB patients from the state of São Paulo, in the period of October 2008 to March 2009 and during the year of 2016. Of the 374 M. tuberculosis isolates analyzed 165 (44%) were monoresistant to isoniazid, 47 (13%) polydrug-resistant (resistant to isoniazid+1st line drug(s) except rifampicin), 122 (33%) multidrug-resistant (MDR), 24 (6%) pre-extensively drug-resistant (pre-XDR) and 16 (4%) XDR. Among isolates with the katG Ser315Thr mutation, 99.4% had intermediate or high-level isoniazid resistance, while 73.7% of the isolates with the inhA C15T mutation had low-level resistance. Among isolates with mutations in katG and inhA, 66.6% had high-level resistance and 32.4% had intermediate resistance. It was observed that 58.8% of the isolates submitted to typing were clustered, and 43.7% of them belonged to the five most prevalent clusters: SP5, SP2c, SP12, SP5ac and SP1i. Isolates with katG 315 mutations were more frequently clustered when compared to isolates with other katG mutations or with no mutations (p= 0.002 and 0.01, respectively). Isolates with the inhA C-15T mutation were more frequently clustered when compared to isolates with katG 315 mutations (p= 0.006). Statistically significant differences were found between cure rates of isoniazidmonoresistant TB patients and MDR-TB patients (63.4% vs 47.1%, p= 0.02), as well as between isoniazid-monoresistant TB patients and XDR-TB patients (63.4% vs 12.5%, p= 0.01). Patients with polydrug-resistant TB also had higher cure rates than patients with MDR-TB (70% vs 47.1%, p= 0.02) and XDR-TB (12.5%, p= 0.004). The present study showed that katG mutations are associated with higher levels of isoniazid resistance and inhA mutations are found in isolates with lower resistance levels. In addition, drugresistant TB was transmitted among patients from the state of São Paulo, whose cure rates were relatively low. (AU)

Detection of Drug Resistant Mycobacterium Tuberculosis Strains Using Kit SIRE Nitratase®: a Multicenter Study

Abstract (1) Background: The Commercial Kit SIRE Nitratase® PlastLabor, is a drug susceptibility test kit used to detect Mycobacterium tuberculosis resistance to first-line TB treatment drugs. The present study aimed at evaluating its performance in a multicenter study. (2) Methods: To determine its accuracy, the proportion methods in Lowenstein Jensen medium or the BACTECTMMGITTM960 system was used as a gold standard. (3) Results: The study revealed that the respective accuracies of the kit with 190 M. tuberculosis clinical isolates, using the proportion methods in Lowenstein Jensen medium or BACTECTMMGITTM960 system as a gold standard, were 93.9% and 94.6%, 96.9% and 94.6%, 98.0% and 97.8%, and 98.0% and 98.9%, for streptomycin, isoniazid, rifampicin, and ethambutol, respectively. (4) Conclusion: Thus, the kit can rapidly screen resistance to streptomycin, isoniazid, rifampicin, and ethambutol. Additionally, it does not require sophisticated equipment; hence, it can be easily used in the laboratories of low and middle income countries.

Speeding up the diagnosis of multidrug-resistant tuberculosis in a high-burden region with the use of a commercial line probe assay.

OBJECTIVE: To evaluate the rapid diagnosis of multidrug-resistant tuberculosis, by using a commercial line probe assay for rifampicin and isoniazid detection (LPA-plus), in the routine workflow of a tuberculosis reference laboratory. METHODS: The LPA-plus was prospectively evaluated on 341 isolates concurrently submitted to the automated liquid drug susceptibility testing system. RESULTS: Among 303 phenotypically valid results, none was genotypically rifampicin false-susceptible (13/13; 100% sensitivity). Two rifampicin-susceptible isolates harboured rpoB mutations (288/290; 99.3% specificity) which, however, were non-resistance-conferring mutations. LPA-plus missed three isoniazid-resistant isolates (23/26; 88.5% sensitivity) and detected all isoniazid-susceptible isolates (277/277; 100% specificity). Among the 38 (11%) invalid phenotypic results, LPA-plus identified 31 rifampicin- and isoniazid-susceptible isolates, one isoniazid-resistant and six as non-Mycobacterium tuberculosis complex. CONCLUSIONS: LPA-plus showed excellent agreement (≥91%) and accuracy (≥99%). Implementing LPA-plus in our setting can speed up the diagnosis of multidrug-resistant tuberculosis, yield a significantly higher number of valid results than phenotypic drug susceptibility testing and provide further information on the drug-resistance level.

Speeding up the diagnosis of multidrug-resistant tuberculosis in a high-burden region with the use of a commercial line probe assay / Agilizando o diagnóstico da tuberculose multirresistente em uma região endêmica com o uso de um teste comercial de sondas em linha

ABSTRACT Objective: To evaluate the rapid diagnosis of multidrug-resistant tuberculosis, by using a commercial line probe assay for rifampicin and isoniazid detection (LPA-plus), in the routine workflow of a tuberculosis reference laboratory. Methods: The LPA-plus was prospectively evaluated on 341 isolates concurrently submitted to the automated liquid drug susceptibility testing system. Results: Among 303 phenotypically valid results, none was genotypically rifampicin false-susceptible (13/13; 100% sensitivity). Two rifampicin-susceptible isolates harboured rpoB mutations (288/290; 99.3% specificity) which, however, were non-resistance-conferring mutations. LPA-plus missed three isoniazid-resistant isolates (23/26; 88.5% sensitivity) and detected all isoniazid-susceptible isolates (277/277; 100% specificity). Among the 38 (11%) invalid phenotypic results, LPA-plus identified 31 rifampicin- and isoniazid-susceptible isolates, one isoniazid-resistant and six as non-Mycobacterium tuberculosis complex. Conclusions: LPA-plus showed excellent agreement (≥91%) and accuracy (≥99%). Implementing LPA-plus in our setting can speed up the diagnosis of multidrug-resistant tuberculosis, yield a significantly higher number of valid results than phenotypic drug susceptibility testing and provide further information on the drug-resistance level.

RESUMO Objetivo: Avaliar o diagnóstico rápido de tuberculose multirresistente, utilizando um teste comercial de sondas em linha (LPA-plus), na rotina de um laboratório de referência de tuberculose. Métodos: O teste LPA-plus foi avaliado prospectivamente em 341 isolados simultaneamente submetidos ao teste de suscetibilidade aos antimicrobianos em meio líquido, pelo sistema automatizado. Resultados: Entre os 303 resultados fenotipicamente válidos, nenhum foi genotipicamente falso suscetível à rifampicina (13/13; 100% de sensibilidade). Dois isolados sensíveis à rifampicina apresentavam mutações no gene rpoB (288/290; especificidade de 99,3%), as quais, no entanto, não são associadas à resistência a rifampicina. O LPA-plus não identificou resistência à isoniazida em três isolados fenotipicamente resistentes (23/26; 88,5% de sensibilidade) e detectou todos os isolados sensíveis à isoniazida (277/277; especificidade de 100%). Entre os 38 (11%) resultados fenotípicos inválidos, o LPA-plus identificou 31 isolados sensíveis à rifampicina e à isoniazida, um resistente à isoniazida e seis como micobactérias não tuberculosas. Conclusões: O LPA-plus mostrou excelente concordância (≥91%) e acurácia (≥99%). Sua implementação pode acelerar o diagnóstico da tuberculose multirresistente, produzir número significativamente maior de resultados válidos do que o teste fenotípico de suscetibilidade aos antimicrobianos e fornecer informações adicionais sobre o nível de resistência aos fármacos.

Surveillance of antimicrobial resistance in Neisseria meningitidis strains isolated from invasive cases in Brazil from 2009 to 2016.

PURPOSE: To describe the antimicrobial resistance profile of Neisseria meningitidis isolates causing invasive disease in Brazil from 2009 to 2016. METHODOLOGY: Among 3548 N. meningitidis isolates received, 2888 (81.4 %) were analysed for antimicrobial resistance using the broth microdilution technique, as recommended by the Clinical and Laboratory Standards Institute. Isolates were tested for ciprofloxacin, chloramphenicol, ceftriaxone, penicillin G, ampicillin and rifampin. RESULTS: All the isolates tested were susceptible to ceftriaxone, while 953 (33.0 %), 1307 (45.3 %) and 2 (0.07 %) isolates were penicillin G-, ampicillin- and rifampin-intermediate, respectively. Resistance to rifampin, ciprofloxacin and chloramphenicol was shown by three isolates (0.1 %), two isolates (0.07 %) and one (0.03 %) isolate, respectively. Although no isolates were resistant to penicillin G in the period of 2009-2016, our results show an upward trend in minimum inhibitory concentrations (MICs) for this drug as of 2010 (P<0.001). There was no significant difference between different gender and age groups of patients for reduced susceptibility to penicillin G. There was a higher frequency of isolates with reduced susceptibility to penicillin G in the South and Southeast regions (P<0.001). This reduced susceptibility was also associated with serotype 19 inside serogroup B (P<0.001). CONCLUSION: Despite the decrease in susceptibility to penicillin G and ampicillin observed from 2010, the overall resistance of N. meningitidis isolates to the antimicrobials tested remained uncommon and sporadic, confirming their efficacy for chemoprophylaxis or treatment of invasive meningococcal disease (IMD) in Brazil. Continued surveillance of N. meningitidis antimicrobial susceptibility profiles is important in order to monitor variations in resistance either geographically, over time or in association with emergent clones.

Growth characteristics of liquid cultures increase the reliability of presumptive identification of Mycobacterium tuberculosis complex.

We evaluated the microscopic and macroscopic characteristics of mycobacteria growth indicator tube (MGIT) cultures for the presumptive identification of the Mycobacterium tuberculosis complex (MTBC) and assessed the reliability of this strategy for correctly directing isolates to drug susceptibility testing (DST) or species identification. A total of 1526 isolates of mycobacteria received at the Instituto Adolfo Lutz were prospectively subjected to presumptive identification by the observation of growth characteristics along with cord formation detection via microscopy. The presumptive identification showed a sensitivity, specificity and accuracy of 98.8, 92.5 and 97.9 %, respectively. Macroscopic analysis of MTBC isolates that would have been erroneously classified as non-tuberculous mycobacteria based solely on microscopic morphology enabled us to direct them rapidly to DST, representing a substantial gain to patients. In conclusion, the growth characteristics of mycobacteria in MGIT, when considered along with cord formation, increased the reliability of the presumptive identification, which has a great impact on the laboratory budget and turnaround times.

Emergence of resistance to ciprofloxacin in Neisseria meningitidis in Brazil.

To prevent secondary invasive meningococcal disease (IMD) cases and outbreaks, antimicrobial prophylaxis of high-risk contacts is indicated. This study reports two ciprofloxacin-resistant Neisseria meningitidis strains in Brazil. The 3523 N. meningitidis isolates collected throughout Brazil from 2009 to 2016 were evaluated for antimicrobial resistance. Meningococcal isolates showing minimal inhibitory concentrations, MICs≥0.125µg ml-1 to ciprofloxacin, were analysed to determine the presence of mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC genes. Two ciprofloxacin-resistant N. meningitidis isolates were found, both presenting a single mutation in the quinolone resistance-determining region of the gyrA gene. These results confirmed that ciprofloxacin is still a first-line drug for chemoprophylaxis. However, we highlight the importance of continued surveillance to monitor the trends of N. meningitidis susceptibility profiles to the antimicrobials recommended for chemoprophylaxis and IMD treatment.