Impact of Xpert MTB/RIF implementation in tuberculosis case detection and control in Brazil: a nationwide intervention time-series analysis (2011-2022).

Background: Since 2014, Brazil has gradually implemented the Xpert MTB/RIF (Xpert) test to enhance early tuberculosis (TB) and drug-resistant (DR-TB) detection and control, yet its nationwide impact remains underexplored. Our study conducts an intervention time-series analysis (ITSA) to evaluate how the Xpert's implementation has improved TB and DR-TB detection nationwide. Methods: 1,061,776 cases from Brazil's National TB Registry (2011-2022) were reviewed and ITSA (2011-2019) was used to gauge the impact of the Xpert's adoption on TB and DR-TB notification. Granger Causality and dynamic regression modelling determined if incorporating Xpert testing as an external regressor enhanced forecasting accuracy for Brazil's future TB trends. Findings: Xpert implementation resulted in a 9.7% increase in TB notification and substantial improvements in DR-TB (63.6%) and drug-susceptible TB (92.1%) detection compared to expected notifications if it had not been implemented. Xpert testing counts also presented a time-dependent relationship with DR-TB detection post-implementation, and improved predictions in forecasting models, which depicted a potential increase in TB and DR-TB detection in the next six years. Interpretation: This study underscores the critical role of Xpert's adoption in boosting TB and DR-TB detection in Brazil, reinforcing the case for its widespread use in disease control. Improvements in prediction accuracy resulting from integrating Xpert data are crucial for allocating resources and reducing the incidence of TB. By acknowledging Xpert's role in both disease control and improving predictions, we advocate for its expanded use and further research into advanced molecular diagnostics for effective TB and DR-TB control. Funding: FIOCRUZ.

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.

Pooling of sputum samples to increase tuberculosis diagnostic capacity in Brazil during the COVID-19 pandemic.

OBJECTIVES: We assessed whether combining (pooling) four individual's samples and testing with Xpert Ultra has the same accuracy as testing samples individually as a more efficient testing method. METHODS: We conducted a cross-sectional study of individuals with presumptive tuberculosis attending primary health care or general hospital facilities in Alagoas, Brazil. The sputum samples of four consecutive individuals were pooled and the pool and individual samples were tested with Xpert Ultra. The agreement of the tests was compared using kappa statistics. We estimated the sensitivity and specificity of pooling using the individual test as the reference standard and potential cartridge savings. RESULTS: A total of 396 participants were tested. A total of 95 (24.0%) individual samples were Mycobacterium tuberculosis (MTB)-positive, 300 (75.8%) "MTB not detected", including 20 "MTB trace", and one reported an error. A total of 99 pools of four samples were tested, of which 62 (62.6%) had MTB detected and 37 (37.4%) MTB not detected, including six (6.1%) with MTB trace. The agreement between individual and pooled testing was 96.0%. Pooling had a sensitivity of 95.0% (95% confidence interval 86.9-99%), specificity of 97.1% (95% confidence interval 85.1-99.9%), and kappa of 0.913. The method saved 12.4% of cartridge costs. CONCLUSION: The pooled testing of specimens had a high level of agreement with individual testing. The pooling of samples for testing improves the efficiency of testing, potentially enabling the screening and testing of larger numbers of individuals more cost-effectively.

Linkage to care and treatment outcomes for patients diagnosed with drug-susceptible tuberculosis using Xpert MTB/RIF assay in Thaba-Tseka district in Lesotho.

Objectives: To evaluate linkage to care and treatment outcomes of patients with tuberculosis (TB) confirmed by Xpert MTB/RIF assay in Thaba-Tseka district, Lesotho. Design: This was a retrospective cohort study of adult patients diagnosed with drug-susceptible TB using the Xpert MTB/RIF assay at two laboratories in Thaba-Tseka district from January 2016 to December 2020. Results: Six hundred and fifty-five eligible participants were identified for inclusion in this study. Their median age was 40 [interquartile range (IQR) 32-54] years, and 468 (71.45%) were male. Evidence of linkage to care was found for 459 (70.08%) participants, but there was no documentation on treatment initiation for 196 (29.92%) participants. The median time to treatment initiation was 0 days (same-day initiation) (IQR 0-4) and the treatment success rate was 86%. Treatment success was associated with negative sputum smear results after 2, 5 and 6 months (χ2, P<0.001). The overall mortality rate was 10%, with no trend of mortality reduction. Conclusion: There is a need to address the issue of linkage to care of patients diagnosed with TB in Thaba-Tseka district. Efforts should be made to reduce TB mortality in line with the World Health Organization's 'End TB strategy' target.

Prueba Xpert® MTB/RIF para el diagnóstico de Tuberculosis en el Instituto Médico La Floresta

La necesidad de un diagnóstico rápido, sensible y específico para tuberculosis es apremiante; se estiman 4 000 muertes cada día y aproximadamente 28 000 personas se contagian de esta enfermedad. La prueba Xpert®MTB/RIF consiste en un ensayo de diagnóstico in vitro de reacción en cadena de la polimerasa (PCR) en tiempo real automatizada, semicuantitativa, que en tan solo dos horas, permite detectar el ácido desoxirribonucleico (ADN) del complejo Mycobacterium tuberculosis (MTB) y mutaciones asociadas a la resistencia a rifampicina. Objetivo: Describir la experiencia del ensayo Xpert®MTB/ RIF y su valor diagnóstico en Dpto. de Microbiología del Instituto Médico La Floresta, desde abril 2012 hasta abril 2021. Métodos: Estudio descriptivo de muestras respiratorias y de otras procedencias, de pacientes con sospecha de tuberculosis, ensayadas por PCR a través del Xpert®MTB/RIF. Adicionalmente se realizó Ziehl-Neelsen y cultivo por el método de Ogawa Kudoh modificado, dependiendo del volumen de muestra recibida. Resultados: De 618 PCR realizadas, 58 muestras fueron positivas (9,4 %), 56 correspondieron a adultos y 2 a niños, 81 % se clasificaron como tuberculosis pulmonar y 19 % como extrapulmonar. A 37 se les realizó cultivo, de las cuales 10 no desarrollaron crecimiento, y a 33 adicionalmente Ziehl-Neelsen, de las cuales 12 presentaron baciloscopias negativas. En cinco de las muestras se detectó resistencia a rifampicina. Conclusiones: A través de la prueba Xpert®MTB/RIF fue posible detectar MTB en aquellas con baciloscopias negativas, en las que no desarrollaron crecimiento en el cultivo y permitió la rápida instauración de tratamiento en la tuberculosis multirresistente.

The rapid, sensitive, and specific diagnosis for tuberculosis is urgent; 4 000 deaths are estimated every day and approximately 28 000 people are infected with this disease. The Xpert®MTB/RIF test consists of an automated, semi-quantitative real-time polymerase chain reaction (PCR) in vitro diagnostic assay that, in just two hours, allows the detection of Mycobacterium tuberculosis complex (MTB) DNA and mutations associated with resistance to rifampicin. Objective: To describe the experience of the Xpert®MTB/RIF assay and its diagnostic value in the Department of Microbiology of the La Floresta Medical Institute, from April 2012 to April 2021. Methods: Descriptive study of respiratory samples and other sources, from patients with suspected of tuberculosis, assayed by PCR through Xpert®MTB/RIF. Additionally, Ziehl-Neelsen and culture were performed by the modified Ogawa Kudoh method, depending on the volume of sample received. Results: Of 618 PCR performed, 58 samples were positive (9.4 %), 56 corresponded to adults and 2 to children, 81 % were classified as pulmonary tuberculosis and 19 % as extrapulmonary. Culture was performed on 37, of which 10 did not develop growth, and on 33 additionally Ziehl-Neelsen, of which 12 presented negative bacilloscopy. Rifampicin resistance was detected in five of the samples. Conclusions: Through the Xpert®MTB/RIF test, it was possible to detect MTB in those with negative bacilloscopy, in which they did not develop growth in the culture and allowed the rapid establishment of treatment in multidrug-resistant tuberculosis.

Contribución del uso de XPERT MTB/RIF y su costo-efectividad en el diagnóstico de tuberculosis pulmonar y la resistencia a rifampicina: una comparación con métodos diagnósticos no moleculares / Contribution of the use of xpert MTB/RIF and its cost-effectiveness in the diagnosis of pulmonary tuberculosis and resistance to rifampicin: a comparison with non molecular diagnostic methods

Resumen Introducción: La tuberculosis es un problema de salud pública; su control requiere diagnóstico temprano y tratamiento oportuno. Xpert MTB/RIF® es una tecno logía diagnóstica basada en PCR en tiempo real, detecta el Complejo Mycobacterium tuberculosis y la susceptibilidad a rifampicina. Objetivo: Determinar la contribución del Xpert MTB/RIF y su costo-efectividad en la detección de tuberculosis y la resistencia a rifampicina en muestras respirato rias al compararlo con métodos de diagnóstico no moleculares Materiales y Métodos: Se analizaron 1.574 muestras de pacientes con sospecha de tuberculosis pulmonar que fueron procesadas para microscopía con coloración fluorescente de auramina-rodamina, Xpert MTB/RIF y cultivo en BACTEC MGIT 960. Los resultados obtenidos se compararon entre los métodos no moleculares y los moleculares para la detección de M. tuberculosis y susceptibilidad a rifampicina y se realizó un análisis comparativo de costos y costo efectividad. Resultados: 19,2% de las muestras fueron positivas por alguna de las técnicas usadas. Xpert MTB/RIF detectó M. tuberculosis en 90,4% del total de muestras positivas con un índice Kappa de 0,77 (IC95%: 0,74-0,82) comparado con el cultivo. La resistencia a rifampicina por Xpert fue 8,1%, sensibilidad 94,1% (IC95%: 73,0-99,0%), especificidad 98,4% (IC95%: 95,5-99,5%) y Kappa de 0,88 (IC95%: 0,76-1,00). La razón incremental de costo efectividad (RICE) fue menor en Xpert MTB/RIF comparada con el cultivo. Conclusión: Xpert MTB/RIF es una prueba eficiente y costo efectiva en la detección de casos de M. tuberculosis en muestras pulmonares comparado con los mé todos de diagnóstico basados en cultivo, sin embargo y a diferencia del Xpert MTB/RIF, estos pueden aportar en el diagnóstico con el aislamiento de especies de micobacterias no tuberculosas y la susceptibilidad a isoniazida y otros medicamentos.

Abstract Introduction: Tuberculosis is a public health problem its control requires early diagnosis and timely treatment. Xpert MTB/RIF is a real-time PCR based diagnostic technology, detects the Mycobacterium tuberculosis complex and rifampicin resistance. Objective: To determine the contribution of Xpert MTB/RIF and its cost-effectiveness in the detection of potential positive cases for tuberculosis and resistance to rifampicin in respiratory samples comparatively with diagnostic non molecular methods Materials and Methods: From 2013 to 2015, 1.574 clinical samples of patients with suspected pulmonary tuberculosis were evaluated by smear microscopy using auramina-rodamina stain, Xpert and culture in liquid medium BACTEC MGIT 960®. Results: 19,2% of the samples were positive for any of the methods used, Xpert detected M. tuberculosis in 90,4% of the positive samples and the concordance between Xpert and cultures had a Kappa index of 0,71 (IC95%: 0,62-0,72). Xpert identified resistance to rifampicin in 8,1% of the clinical samples studied with a sensitivity 94.1% (IC95%: 73,0-99,0%), specificity 98,4% (IC95%: 95,5-99,5%) and Kappa index 0,88 (IC95%: 0,76-1,00). Xpert had an incremental cost effectiveness ratio lower than culture (RICE). Conclusion: Xpert MTB/Rif is efficient diagnostic technique and comparable with culture in cost effectiveness for pulmonary tuberculosis diagnosis. However, culture based methods, in contrast to Xpert, may allow the isolation and identification of non tuberculosis mycobacterial species and the possibility to perform susceptibility for other antituberculous drugs.

Caracterización clínica y microbiológica de pacientes con trazas en Xpert MTB/RIF Ultra / Clinical and microbiological characterization of patients with Xpert MTB/RIF Ultra traces

INTRODUCCIÓN: El Xpert MTB/RIF Ultra (Ultra) ha mejorado dramáticamente el diagnóstico de la tuberculosis (TBC). Con él ha nacido la categoría de trazas, que es la menor carga bacilar detectable por este examen. OBJETIVO: Describir las características clínicas de los pacientes con presencia de trazas en el Ultra y evaluar la confirmación de la TBC como diagnóstico clínico. MATERIALES Y MÉTODOS: Estudio descriptivo de serie de casos. Se extrajo la información de fichas clínicas de pacientes con positividad a trazas. Se confrontaron datos clínicos, microbiológicos e histopatológicos. RESULTADOS: Se analizaron 21 pacientes. La edad promedio fue de 52 años. Todos los casos presentaron baciloscopias negativas. Cuatro cultivos en medio líquido MGIT fueron positivos, dos en pleura parietal, uno en líquido pleural y otro en expectoración. En pleura parietal, tres casos presentaron granulomas con necrosis caseosa y un granuloma esbozos de necrosis. En tejido pulmonar se observaron dos casos con granulomas con esbozos de necrosis y dos con granulomas no necrotizantes. Tres pacientes tenían el antecedente de TBC previa, se interpretó la positividad de trazas en ellos como falsos positivos. Finalmente se diagnosticaron 13 casos como TBC activa, donde cinco de ellos fueron TBC pleurales. La mayor concordancia clínica, microbiológica e histopatológica fue en muestras de líquido y tejido pleural. DISCUSIÓN: Se debe interpretar con cautela los hallazgos de esta prueba en muestras de vía aérea; el análisis multidisciplinario (clínica, imágenes, microbiología, histología) es crucial en las decisiones de nuestras conductas clínicas futuras. El hallazgo de trazas en pleura tiene, a nuestro parecer, un alto valor diagnóstico en el estudio de la tuberculosis en esta localización.

INTRODUCTION: Xpert MTB/RIF Ultra has dramatically changed the diagnosis of tuberculosis. A new category called traces appeared, which is the smallest amount of bacillar load detectable. OBJECTIVE: Describe the clinical characteristics of patients that present traces in Xpert MTB/RIF Ultra test, and to evaluate the confirmation of tuberculosis as clinical diagnosis. METHODS: We perform a descriptive case series study. Information was recovered from clinical records of patients with positive test for traces. Clinical, histopathological and microbiological results were confronted. RESULTS: Twenty one patients were analyzed. The mean age was 52 years-old. All cases had negative smear microscopy and four MGIT cultures were positive, two in pleural fluid and another in sputum. In parietal pleura, three cases presented granulomas with caseous necrosis, and one showed granuloma with very little necrosis. In pleural tissue we observed two cases of granulomas with traces of necrosis and two with non-necrotizing granulomas. Three patients had history of previous tuberculosis and positive traces, the test was interpreted as a false positive result. Finally, active tuberculosis was diagnosed in 13 cases, and five of them were pleural tuberculosis. The highest clinical, microbiological and histopathological agreement was in fluid and pleural tissue samples. DISCUSSION: The findings of Xpert MTB/RIF Ultra in airway samples must be interpreted carefully. Multi-disciplinary analysis is crucial in future clinical decisions. The finding of traces in pleura has, in our opinion, a high diagnostic value in the study of tuberculosis in this location.

Viability assessment of Mycobacterium tuberculosis complex in OMNIgene • SPUTUM reagent using the BACTEC MGIT 960 System and Xpert MTB/RIF assay.

The World Health Organization advocates that sputum specimens submitted to tuberculosis (TB) diagnostic should be processed within 48 h after collection and be stored under cooling. We aimed to assess the performance of OMNIgene • SPUTUM reagent in maintaining viable specimens of Mycobacterium tuberculosis complex (MTBC) during transportation of sputum samples without refrigeration, in comparison to the standard protocol of the National TB Control Program. Sputum samples obtained in southeastern Brazil (June 2017 to July 2018) from 100 sequential patients with positive acid-fast bacillus smear microscopy were divided into two portions. Portion 1 continued to be cooled (standard protocol, STA), but portion 2 was added to OMNIgene • SPUTUM reagent (alternative protocol, OMS) until concomitant further processing. Both portions of all samples were cultured using MGIT and tested by Xpert MTB/RIF assay. Growth of MTBC in the first 42 days was detected in 96% of the cultures under the STA and 88% under the OMS. Intervals between processing and detecting MTBC growth in the two portions significantly differed (p = 0.0001). Portions under the two protocols showed similar results in the MTBC detection by Xpert assay and culture contamination by non-MTBC. The OMNIgene reagent liquefies and decontaminates sputum leading to a decrease in processing time. Although there was a small delay in mycobacterial growth, the OMNIgene reagent can be useful in specimens transported from collection sites over a long distance to centralized testing centers, maintaining viable MTBC for at least 8 days at room temperature.

Examination of respiratory specimens improves microbiological diagnosis of patients with presumptive extrapulmonary tuberculosis.

OBJECTIVES: Bacteriological confirmation of extrapulmonary tuberculosis (EPTB) is challenging for several reasons: the paucibacillary nature of the sample; scarce resources, mainly in middle and low-income countries; the need for hospitalization; and unfavorable outcomes. We evaluated the diagnostic role of respiratory specimen examination prospectively in a cohort of patients with presumptive EPTB. METHODS: From July 2018 to January 2019, in a tuberculosis (TB)/HIV reference hospital, a cohort of 157 patients with presumed EPTB was evaluated. Xpert® MTB/RIF Ultra or a culture-positive result was considered for bacteriologically confirmed TB. RESULTS: Out of 157 patients with presumptive EPTB, 97 (62%) provided extrapulmonary and respiratory specimens and 60 (38%) extrapulmonary specimens only. Of the 60 patients with extrapulmonary samples, 5 (8%) were positive. Of those with respiratory and extrapulmonary samples, 27 (28%) were positive: 10 in both the respiratory and extrapulmonary samples, 6 in the extrapulmonary sample only, and 11 in the respiratory sample only. A respiratory specimen examination increased by 6-fold the chance of bacteriological confirmation of TB (odds ratio = 5.97 [1.11-47.17]). CONCLUSION: We conclude that respiratory samples should be examined in patients with presumptive EPTB.

Should treatment of low-level rifampicin mono-resistant tuberculosis be different?

BACKGROUND: Rifampicin resistant tuberculosis (RR-TB) was frequently detected in Suriname after the introduction of Xpert MTB/RIF in 2012. Subsequent phenotypic drug-susceptibility testing (DST) was not conclusive at that moment, while RR-TB patients treated with first-line tuberculostatics had good treatment outcome. In our study, we analysed this interesting observation. METHODS: We collected demographic and clinical characteristics and treatment outcome of TB patients from May 2012-December 2018 and performed a univariate and multivariate analysis to assess possible associations with resistance to rifampicin. Secondly, we conducted whole genome sequencing on all available Mycobacterium tuberculosis isolates that had a rifampicin resistance in the Xpert MTB/RIF test and performed phenotypic DST on selected isolates. FINDINGS: RR-TB was detected in 59 (9.6%) patients confirmed by Xpert. These patients were treated with rifampicin-containing regimens in most (88%) of the cases. In all 32 samples examined, a D435Y mutation in the rpoB gene was identified; only one isolate revealed an additional isoniazid mutation. Phenotypic DST indicated low-level rifampicin resistance. In multivariate analysis, the Creole ethnicity was a factor associated with rifampicin resistance (aOR 3.5; 95%CI 1.9-6.4). The treatment success rate for patients with RR-TB (78.0%) was comparable to the treatment outcome in non-RR-TB patients 77.8%. INTERPRETATION: This study confirms a low-level rifampicin mono-resistance in TB patients of Suriname. These patients could benefit from a first-line regimen with high dose rifampicin (or rifabutin), rather than from the lengthy treatment regimens for rifampicin-resistant and multi-drug resistant TB, a concept of stratified medicine also advocated for the treatment of TB. FUNDING: None.

Cost-effectiveness of Xpert®MTB/RIF in the diagnosis of tuberculosis: pragmatic study

Abstract INTRODUCTION: The intensification of research and innovation with the creation of networks of rapid and effective molecular tests as strategies for the end of tuberculosis are essential to avoid late diagnosis and for the eradication of the disease. We aimed to evaluate the cost-effectiveness of Xpert®MTB/RIF (Xpert) in the diagnosis of drug-resistant tuberculosis in reference units, in scenarios with and without subsidies, and the respective cost adjustment for today. METHODS: The analyses were performed considering as criterion of effectiveness, negative culture or clinical improvement in the sixth month of follow-up. The comparison was performed using two diagnostic strategies for the drug susceptibility test (DST), BactecTMMGITTM960 System, versus Xpert. The cost effectiveness and incremental cost-effectiveness ratio (ICER) were calculated and dollar-corrected for American inflation (US$ 1.00 = R$ 5,29). RESULTS: Subsidized Xpert had the lowest cost of US$ 33.48 (R$67,52) and the highest incremental average efficiency (13.57), thus being a dominated analysis. After the inflation was calculated, the mean cost was DST-MGIT=US$ 74.85 (R$ 396,73) and Xpert = US$ 37.33 (R$197,86) with subsidies. CONCLUSIONS: The Xpert in the diagnosis of TB-DR in these reference units was cost-effective with subsidies. In the absence of a subsidy, Xpert in TB-DR is not characterized as cost effective. This factor reveals the vulnerability of countries dependent on international organizations' subsidy policies.

Rendimiento del GeneXpert en el diagnóstico de tuberculosis pulmonar y extrapulmonar en la edad pediátrica / GeneXpert for the diagnosis of pulmonary and extrapulmonary tuberculosis in pediatric patients / GeneXpert para o diagnóstico da tuberculose pulmonar e extrapulmonar em pacientes pediátricos

resumen está disponible en el texto completo

Summary: Introduction: the World Health Organization (WHO) recommends molecular biology techniques, such as Xpert MTB/RIF, for the diagnosis of tuberculosis (TB) and for the detection of Rifampicin resistance. In Uruguay, the Xpert MTB/RIF has been used since 2014, and no research papers have yet assessed its performance. A Cochrane review recommends the assessment of the Xpert diagnostic accuracy in difficult to diagnose groups, such as, children, people living with HIV and with extrapulmonary tuberculosis. Objectives: describe cases of TB in children of under 15 years of age in Uruguay during 2018 and 2019 and describe the influence of the various diagnostic tests on the bacteriological confirmation of the disease. Evaluate the performance of the Xpert MTB/RIF for the diagnosis of TB in respiratory and non- respiratory samples using the culture as a reference standard. Compare the performance of GeneXpert with smear microscopy for TB diagnosis. Material and methods: analytical, retrospective study of children of under 15 years of age in Uruguay between January 2018 and June 2019, based on data obtained from the PNC-TB information system. Clinical-epidemiological characteristics of the TB cases were described. Definitions were taken from CHLA-EP, as per WHO recommendations. All respiratory and non-respiratory samples received by the National Reference Laboratory in Tuberculosis of the CHLA-EP from 1/1/2018 to 6/30/2019, entered in the IT system ("TB soft") were analyzed; they belonged to patients with clinical suspicion of TB, studied as contacts, or to TB risk groups (patients with immunodeficiency or at risk of immunosuppression, mainly). All samples underwent smear microscopy and/or Xpert MTB/RIF (according to the CHLA-EP protocol) and culture. The detection of Rifampicin resistance in the Xpert was compared with first- line drug sensitivity tests using molecular methods made from the cultures. The sensitivity, specificity, PPV and NPV of GeneXpert and ZN microscopy were calculated using Mycobacterium tuberculosis culture as gold standard. We calculated the Xpert positive and negative likelihood ratio (LR). Results: 67 patients under 15 years of age were diagnosed with TB, and 46% cases were bacteriologically confirmed. A total of 1670 samples were analyzed; 82% respiratory and 17% non-respiratory. A total of 32 samples showed a positive culture for M. tuberculosis (14 respiratory and 18 non- respiratory). One rifampicin resistance sample was detected in the Xpert that was not confirmed in the culture. The sensitivity of Xpert for all samples was 80%; the specificity 99,5%; PPV 80%; NPV 99,5%. In the case of smear microscopy for all samples: S 44,4%, specificity 99,4%, PPV 70,6%; NPV 98,2%. Respiratory samples: Xpert S 100%; E 99,4%; PPV 66,7%; NPV 100%. Bacilloscopy: S 72,7%; E 99,6%; PPV 72,7%; NPV 99,6%. Non-respiratory samples: Xpert S: 66,7%; E 100%; PPV 100%; NPV 97,9%. Bacilloscopy: S 25%; E 98,8%; PPV 66,7%; NPV 93,2%. The LR + of the Xpert for all samples was 160 and the LR - 0,2. Conclusions: TB in children under 15 remains difficult to diagnose. Bacteriological confirmation was attempted in 88% of TB cases, and almost 50% showed positive results by some bacteriological technique. The Xpert showed a good sensitivity and specificity profile in both respiratory and non-respiratory samples, similar to those reported in international papers. The main contribution in relation to smear microscopy is the greater sensitivity for the diagnosis of TB in children under 15 years of age. The Xpert is very useful for TB diagnosis when it is positive, although it does not ensure we can rule out the disease in case of negative results.

Resumo: Introdução: a Organização Mundial da Saúde (OMS) recomenda técnicas de biologia molecular, como o Xpert MTB / RIF para o diagnóstico de tuberculose (TB) e para a detecção de resistência à Rifampicina. No Uruguai, o Xpert MTB / RIF é usado desde 2014, e o seu desempenho ainda não tem sido avaliado. Uma revisão recente da Cochrane promove que pesquisas futuras devem avaliar a precisão diagnóstica do Xpert, em grupos difíceis de diagnosticar, como crianças, pessoas vivendo com HIV e pessoas com tuberculose extrapulmonar. Objetivos: descrever os casos de tuberculose em crianças menores de 15 anos no Uruguai nos anos 2018-2019 e a contribuição dos diferentes testes de diagnóstico na confirmação bacteriológica da doença. Avaliar o desempenho do Xpert MTB / RIF para o diagnóstico de TB em amostras respiratórias e não respiratórias de pacientes menores de 15 anos, utilizando a cultura como padrão de referência. Comparar o desempenho do GeneXpert com a baciloscopia para o diagnóstico da TB. Material e métodos: estudo analítico e retrospectivo de crianças menores de 15 anos estudadas para TB no Uruguai entre janeiro de 2018 e junho de 2019, utilizando a base em dados do sistema informático PNC-TB. Descrevemos as características clínico-epidemiológicas dos casos de TB. As definições foram retiradas do CHLA-EP de acordo com as recomendações da OMS. Todas as amostras respiratórias e não respiratórias recebidas pelo Laboratório Nacional de Referência (LNR) em Tuberculose do CHLA-EP de 01/01/2018 a 30/06/2019, inseridas no sistema computacional (TB Soft), corresponderam a pacientes com suspeita clínica de TB, estudados como contatos ou na detecção de TB em grupos de risco (pacientes com imunodeficiências ou com risco de imunossupressão, principalmente). As amostras foram realizadas por esfregaço (baciloscopia) e/ou Xpert MTB/RIF (de acordo com o protocolo CHLA-EP) e por cultura. A detecção da resistência à Rifampicina no Xpert foi comparada com os testes de sensibilidade a drogas de primeira linha (PSD), utilizando os métodos moleculares das culturas. A sensibilidade, especificidade, PPV e NPV do Xpert e esfregaço foram calculados usando a cultura como padrão de referência. Calculamos a razão de verossimilhança positiva e negativa (LR) do Xpert. Resultados: 67 crianças menores de 15 anos foram diagnosticadas com TB, e 46% dos casos foram confirmados bacteriologicamente. 1670 amostras foram analisadas; 82% respiratórias e 17% não respiratórias. 32 amostras tiveram uma cultura positiva para M. tuberculosis (14 respiratórias e 18 não respiratórias). A sensibilidade (S) do Xpert para todas as amostras foi de 80% (IC95% 37,5-96,3), especificidade (E) 99,5% (IC95% 97,3-99,9), PPV 80% (37,5-96,3), NPV 99,5% (97,3-99,9). Baciloscopia: S de 46,1% (28,7-64,5), E 99,5% (98,7-99,8), PPV 75% (50,5-89,8), VPL 98,3% (97,2-99). Amostras respiratórias: Xpert S 100%; E 99,3% VPP 66% e VPN 100%. Baciloscopia: S 66,6%, E 99,8%, PPV 80%, NPV 99,7%. Amostras não respiratórias: Xpert S: 66,6%, E 100%, PPV 100%, NPV 97,9%; Esfregaço S: 25%, E 99,3%, PPV 80%, NPV 93%. O LR + do Xpert para todas as amostras foi de 160 e o LR - 0,2. Conclusões: a TB em crianças menores de 15 anos é ainda difícil de diagnosticar. Tentamos a confirmação bacteriológica em 88% dos casos de TB, e quase 50% deles tiveram resultados positivos utilizando alguma técnica bacteriológica. O Xpert mostrou um bom perfil de sensibilidade e especificidade em amostras respiratórias e não respiratórias, semelhante ao relatado em papers internacionais. A principal contribuição em relação à baciloscopia é a maior sensibilidade para o diagnóstico de TB em menores de 15 anos. O Xpert é muito útil para o diagnóstico de TB em caso de ser positivo, embora não permita descartar a doença em casos negativos.

Association of Xpert MTB/RIF Cycle Threshold Values with Tuberculosis Treatment Outcomes.

PURPOSE: Considering the current recommendation of the World Health Organization to replace sputum smear microscopy with Xpert MTB/RIF as an initial diagnostic test for tuberculosis (TB), and that culture takes time to provide results, the cycle threshold (CT) of the Xpert test may be the only way to assess bacillary load. The objective of this study is to evaluate the association of bacillary load, measured by the Xpert CT, with the TB treatment outcomes. METHODS: In cohort study, Xpert CT values were evaluated in cured and non-cured (failure and death) patients. Multivariate analysis was performed to evaluate if CT is independently associated with TB treatment outcomes. RESULTS: During this study period, 155 patients (84 cured and 71 non-cured) met the inclusion and were included in the analysis. The mean CT value for Xpert MTB/RIF test was 20.7 ± 5.6 in cured patients and 17.1 ± 5.6 in non-cured patients (p < 0.0001). Previous TB was more frequent in non-cured (28.2%) than in cured patients (7.1%) (p < 0.0001). Non-cured patients were younger than cured ones (37.1 ± 13.3 vs 43.6 ± 16.2; p = 0.006). HIV was more frequent in non-cured (28.2%) than in cured patients (15.5%), although this difference was not statistically significant (p = 0.054). In multivariate analysis, CT values, age, previous TB, and HIV were independently associated with non-cure. CONCLUSIONS: Lower Xpert MTB/RIF CT values were independently associated with worse treatment outcomes. The information from even a single test performed before starting treatment proved to be a relatively good predictor of TB treatment outcome.

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.

Diagnostic performance of the Xpert Carba-R™ assay directly from rectal swabs for active surveillance of carbapenemase-producing organisms in the largest Brazilian University Hospital.

BACKGROUND: The global spread of carbapenemase-producing organisms (CPO) has been considered by international health authorities as a critical public health concern. Brazil has a high CPO prevalence according to distinct publications but many routine microbiology laboratories have only phenotypic resources to evaluate this epidemiological situation, which is time-consuming and detects only carbapenem-resistant isolates missing CPO susceptible expressing a slightly decreased susceptibility. New molecular platforms can detect CPO faster but a local evaluation is essential. AIM: To evaluate the performance of CPO detection direct from rectal swabs with the Xpert Carba-R™ assay (Cepheid, Sunnyvale, CA) in the largest Brazilian University Hospital. METHODS: A prospective diagnostic accuracy study of CPO was performed with the collection of rectal swabs from patients admitted into the Intensive Care Unit (ICU) and into the Emergency Department (ED) between April and July 2016. The Xpert Carba-R™ assay results were compared with carbapenem-resistant Enterobacterales (CRE) surveillance cultures plus in-house PCR carbapenemase detection (reference method). In case of discordant results between methods, additional tests were performed. The limit of detection (LoD) for the CRE culture and the Xpert Carba-R™ assay were performed with contrived isolates of known carbapenemases genes. RESULTS: A total of 921 clinical rectal swabs were analyzed being 21% (196/921) from the ICU and 79% (725/921) from the ED. Overall, the Xpert Carba-R™ assay detected 9.9% (91/921) of CPOs being 9.5% (87/921) positive only for blaKPC and 0.4% (4/921) positive only for blaNDM. The reference method detected 9.1% (84/921) CPO being 77 (8.4%) blaKPC, 5 blaVIM (0.5%) and 2 blaNDM (0.2%). No IMP or OXA-48 like gene was detected. Overall, twelve samples, 1.3% (10 blaKPC, 2 blaNDM) were Xpert Carba-R™ positive but negative by the reference method. Five isolates (0.5%) were positive for blaVIM only by in-house PCR and confirmed to be blaVIM-2 by DNA sequencing. The Kappa value, sensitivity, specificity, positive/negative predictive values and accuracy of the Xpert Carba-R™ assay were; 0.893 (95% confidence interval [CI], 0.842-0.944), 94% (86.7-98.0), 98.6% (97.5-99.3), 86.8% (78.1-93.0), 99.4% (98.6-99.8) and 98.2% (97.3-99.1), respectively. The LoD for blaKPC of the Xpert Carba-R™ assay and the CRE cultures were 101 CFU/swab. CONCLUSION: The Xpert Carba-R™ assay is an accurate test to detect CPO directly from the rectal swabs with significant lower turnaround time (TAT) when compared to the reference method (CRE culture plus in-house PCR). Xpert Carba-R™ may, therefore, be regarded as a good and fast epidemiological tool.

Oral swab testing by Xpert® MTB/RIF Ultra for mass tuberculosis screening in prisons.

Diagnosis of pulmonary tuberculosis is usually achieved by testing sputum for presence of Mycobacterium tuberculosis by microscopy, culture or nucleic acid amplification tests. However, many individuals are unable to produce sputum, particularly early in the course of illness. Studies have reported that oral swabs, assayed by nucleic acid amplification tests, may be a suitable substitute or complement to sputum testing. To determine whether this method could be useful of case finding, in which bacillary load is often lower, we evaluated it in the setting of a mass tuberculosis screening study in prison inmates in Brazil. For this sub-study, we enrolled 128 individuals with pulmonary tuberculosis confirmed by sputum Xpert testing, and 128 controls who tested negative by sputum culture and Xpert. We collected two oral swabs by participant, prior to starting treatment. Swabs were collected from the tongue by brushing along the surface for 10 times. The sensitivity of a single oral swab was 43% (N = 55/128; 95% CI: 34-52%). Using two consecutive oral swabs the sensitivity increased to 51% (N = 66/128; 95% CI: 43-60%). The specificity was 100% (128/128). In participants with high mycobaterial load in the sputum, the combined sensitivity was 90% (N = 9/10). In the participants with medium mycobaterial load in the sputum, the combined sensitivity was 79% (N = 23/29). In the participants with low or very low mycobaterial load in the sputum, the combined sensitivity was 38% (N = 34/89). Our data suggest that oral swab sampling, assayed by Xpert, has comparable sensitivity to sputum in participants with high and medium mycobacterial load in the sputum. However, 70% (89/128) of individuals identified through our mass screening study (Carbone et al.) had detection number low or very low in their sputum. In this population, oral swab testing may not have sufficient sensitivity as currently performed. Further studies are needed to identify alternative non-sputum sampling strategies in this population.

Evaluation of Xpert MTB/RIF Ultra performance for pulmonary tuberculosis (TB) diagnosis in a city with high TB incidence in Brazil.

INTRODUCTION: Since 2018, World Health Organization (WHO) recommended the Xpert MTB/RIF Ultra use for pulmonary and extrapulmonary TB diagnosis, and suggested that Xpert Ultra should be tested in various populations, with different geographical and epidemiological settings. METHODS: Cross-sectional study with prospective data collection. Outpatients aged >18 years with respiratory symptoms suggestive of pulmonary TB were invited to participate. Sensitivity, specificity, positive and negative predictive values of the test were calculated and compared with the traditional Xpert MTB/RIF. RESULTS: During the study period, 180 patients met the inclusion and were included in the analysis. Xpert MTB/RIF Ultra test was positive in 33 patients (18.3%), and RIF resistance was detected in 1 (3.1%) patient. Considering culture as the gold standard, the sensitivity, specificity, positive predictive value, and negative predictive value of Xpert MTB/RIF Ultra were 100.0% (95% CI 85.2-100.0), 93.6% (95% CI 88.6-96.9), 69.7% (95% CI 55.8-80.7), and 100.0% (95% CI 87.2-100.0), respectively. The area under the ROC curve was 0.97 for the Xpert MTB/RIF Ultra test (95% CI 0.93 to 0.99; p < 0.0001). There was no difference statistically significant between sensitivities and specificities of Xpert MTB/RIF and Xpert MTB/RIF Ultra (p > 0.05). CONCLUSIONS: This is the first study in Brazil to evaluate the accuracy of Xpert MTB/RIF Ultra in individuals with presumptive pulmonary TB. The test showed an excellent sensitivity and a high specificity, demonstrating that it is a useful tool for pulmonary TB diagnosis.

Will more sensitive diagnostics identify tuberculosis missed by clinicians? Evaluating Xpert MTB/RIF testing in Guatemala.

OBJECTIVE: To assess the impact of introducing Xpert as a follow-on test after smear microscopy on the total number pulmonary TB notifications. METHOD: Genexpert systems were installed in six departments across Guatemala, and Xpert was indicated as a follow-on test for people with smear-negative results. We analyzed notifications to national tuberculosis (TB) programmes (NTP) and the project's laboratory data to assess coverage of the intervention and case detection yield. Changes in quarterly TB notifications were analyzed using a simple pre/post comparison and a regression model controlling for secular notification trends. Using a mix of project and NTP data we estimated the theoretical yield of the intervention if testing coverage achieved 100%. RESULTS: Over 18,000 smear-negative individuals were eligible for Xpert testing during the intervention period. Seven thousand, one hundred and ninety-three people (39.6% of those eligible) were tested on Xpert resulting in the detection of 199 people with smear-negative, Xpert positive results (2.8% positivity rate). In the year before testing began 1098 people with smear positive and 195 people with smear negative results were notified in the six intervention departments. During the intervention, smear-positive notification remained roughly stable (1090 individuals, 0.7%), but smear-negative notifications increased by 167 individuals (85.6%) to an all-time high of 362. After controlling for secular notifications trends over an eight-quarter pre-intervention period, combined pulmonary TB notifications (both smear positive and negative) were 19% higher than trend predictions. If Xpert testing coverage approached 100% of those eligible, we estimate there would have been a+41% increase in TB notifications. CONCLUSIONS: We measured a large gain in pulmonary notifications through the introduction of Xpert testing alone. This indicates a large number of people with TB in Guatemala are seeking health care and being tested, yet are not diagnosed or treated because they lack bacteriological confirmation. Wider use of more sensitive TB diagnostics and/or improvements in the number of people clinically diagnosed with TB have the potential to significantly increase TB notifications in this setting, and potentially in other settings where a low proportion of pulmonary notifications are clinically diagnosed.

Modelling the impact of chest X-ray and alternative triage approaches prior to seeking a tuberculosis diagnosis.

BACKGROUND: Tuberculosis is a major challenge to health in the developing world. Triage prior to diagnostic testing could potentially reduce the volume of tests and costs associated with using the more accurate, but costly, Xpert MTB/RIF assay. An effective methodology to predict the impact of introducing triage prior to tuberculosis diagnostic testing could be useful in helping to guide policy. METHODS: The development and use of operational modelling to project the impact on case detection and health system costs of alternative triage approaches for tuberculosis, with or without X-ray, based on data from Porto Alegre City, Brazil. RESULTS: Most of the triage approaches modelled without X-ray were predicted to provide no significant benefit. One approach based on an artificial neural network applied to patient and symptom characteristics was projected to increase case detection (82% vs. 75%) compared to microscopy, and reduce costs compared to Xpert without triage. In addition, use of X-ray before diagnostic testing for HIV-negative patients could maintain diagnostic yield of using Xpert without triage, and reduce costs. CONCLUSION: A model for the impact assessment of alternative triage approaches has been tested. The results from using the approach demonstrate its usefulness in informing policy in a typical high burden setting for tuberculosis.

Group B Streptococcus detection in pregnant women: comparison of qPCR assay, culture, and the Xpert GBS rapid test.

BACKGROUND: Group B Streptococcus (GBS) is one of the most important causative agents of neonatal sepsis. As administration of prophylactic antibiotics during labor can prevent GBS infection, routine screening for this bacterium in prenatal care before the onset of labor is recommended. However, many women present in labor without having undergone such testing during antenatal care, and the turnaround time of detection methods is insufficient for results to be obtained before delivery. METHODS: Vaginal and anorectal specimens were collected from 270 pregnant women. Each sample was tested by Xpert GBS, qPCR, and culture for GBS detection. RESULTS: The overall prevalence of maternal GBS colonization was 30.7% according to Xpert GBS, 51.1% according to qPCR, and 14.3% according to cultures. Considering the qPCR method as the reference, the Xpert GBS had a sensitivity of 53% and specificity of 93%. Positive Xpert GBS results were correlated to marital status (married or cohabitating) and with prematurity as a cause of neonatal hospitalization. Positive cultures were related with ischemic-hypoxic encephalopathy requiring therapeutic hypothermia. CONCLUSIONS: Combined enrichment/qPCR and the Xpert GBS rapid test found a high prevalence of GBS colonization. The Xpert GBS technique gives faster results and could be useful for evaluating mothers who present without antenatal GBS screening results and are at risk of preterm labor, thus allowing institution of prophylactic antibiotic therapy.