RESUMEN
The Roche cobas MTB and MTB-RIF/INH assays allow for detection of Mycobacterium tuberculosis complex (MTBC) nucleic acid and rifampicin (RIF) and isoniazid (INH) resistance-associated mutations in an automated, high-throughput workflow. In this study, we evaluated the performance of these assays, employing samples from settings of low and high tuberculosis (TB) burdens. A total of 325 frozen, leftover respiratory samples collected from treatment-naive patients with presumptive TB in Germany (n = 280) and presumptive RIF-resistant TB in Sierra Leone (n = 45) were used in this study. cobas MTB results for detection of MTBC DNA from N-acetyl-l-cysteine-sodium hydroxide (NALC-NaOH)-treated samples were compared to culture results. Predictions of RIF and INH resistance by the cobas MTB-RIF/INH assay were compared to a composite reference standard (phenotypic drug susceptibility testing and line probe assay). Whole-genome sequencing was used to resolve discordances. The overall sensitivity of cobas MTB for detection of MTBC DNA in culture-positive samples (n = 102) was 89.2% (95% confidence interval [CI], 81.7 to 93.9%). The specificity of cobas MTB was 98.6% (95% CI, 96.1 to 99.5%). Sensitivity and specificity for detection of RIF and INH resistance were 88.4% (95% CI, 75.5 to 94.9%) and 97.6% (95% CI, 87.4 to 99.6%) and 76.6% (95% CI, 62.8 to 86.4%) and 100.0% (95% CI, 90.8 to 100.0%), respectively. Discordant results for RIF and INH resistance were mainly due to uncommon mutations in samples from Sierra Leone that were not covered by the cobas MTB-RIF/INH assay. In conclusion, cobas MTB and MTB-RIF/INH assays provide accurate detection of MTBC DNA and resistance-associated mutations in respiratory samples. The influence of regional variations in the prevalence of resistance-conferring mutations requires further investigation.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Alemania , Humanos , Isoniazida/farmacología , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/genética , Rifampin/farmacología , Sensibilidad y Especificidad , Sierra Leona , Esputo , Tuberculosis Resistente a Múltiples Medicamentos/diagnósticoRESUMEN
The Ebola virus disease (EVD) outbreak in West Africa has highlighted an urgent need for point-of-care (POC) assays for the diagnosis of this devastating disease in resource-limited African countries. The diagnostic performance characteristics of a prototype Cepheid GeneXpert Ebola POC used to detect Ebola virus (EBOV) in stored serum and plasma samples collected from suspected EVD cases in Sierra Leone in 2014 and 2015 was evaluated. The GeneXpert Ebola POC is a self-contained single-cartridge automated system that targets the glycoprotein (GP) and nucleoprotein (NP) genes of EBOV and yields results within 90 min. Results from 281 patient samples were compared to the results of a TaqMan real-time reverse transcription-PCR (RT-PCR) targeting the polymerase gene and performed on two real-time PCR machines. Agreement between the three platforms was 100% at cycle threshold (CT) values of ≤34.99, but discordant results were noted between CT values of 35 and 45.The diagnostic sensitivity of the three platforms was 100% in 91 patient samples that were confirmed to be infectious by virus isolation. All three molecular platforms detected viral EBOV RNA in additional samples that did not contain viable EBOV. The analytical sensitivity of the GeneXpert Ebola POC for the detection of NP was higher, and comparable to that of polymerase gene detection, than that for the detection of GP when using a titrated laboratory stock of EBOV. There was no detectable cross-reactivity with other hemorrhagic fever viruses or arboviruses. The GeneXpert Ebola POC offers an easy to operate and sensitive diagnostic tool that can be used for the rapid screening of suspected EVD cases in treatment or in holding centers during EVD outbreaks.
Asunto(s)
Ebolavirus/genética , Fiebre Hemorrágica Ebola/diagnóstico , Fiebre Hemorrágica Ebola/virología , Técnicas de Diagnóstico Molecular , Pruebas en el Punto de Atención , Línea Celular , Ebolavirus/clasificación , Ebolavirus/aislamiento & purificación , Genes Virales , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Sierra Leona , Carga ViralRESUMEN
OBJECTIVES: We evaluated the ability of FluoroType MTBDR version 2 (FTv2; Hain Lifescience), a second-step real-time PCR assay, to simultaneously detect Mycobacterium tuberculosis complex (MTBC) DNA and mutations conferring resistance to rifampicin (RIF) and isoniazid (INH), in pulmonary and extrapulmonary samples from patients and compared them with corresponding cultures. METHODS: FTv2 MTBC was evaluated on 1815 and 432 samples from Denmark (DK) and Germany (DE), respectively. RIF and INH resistance mutations were assessed in the German samples and 110 samples from Sierra Leone and subsequently compared to phenotypic antimicrobial susceptibility testing and a composite reference DNA (CRD) based on the GenoType MTBDR line-probe assay and Sanger sequencing or whole-genome sequencing. RESULTS: Of the 584 (557 smear-negative) Danish and 277 (85 smear-negative) German sputum samples, 42 (16) and 246 (54) were culture positive, and 44 (18) and 222 (35) were FTv2 positive, providing an FTv2 sensitivity and specificity of 0.86 (0.63) and 0.98 (DK), 0.90 (0.65) and 1.00 (DE), respectively. The count, sensitivities, and specificities for all pulmonary samples were 1434, 0.79, and 0.99 (DK) and 347, 0.86, and 1.00 (DE), respectively; for extrapulmonary samples, 381, 0.33, 0.99 (DK) and 83, 0.50, and 1.00 (DE). The valid count, sensitivity, and specificity compared with CRD for detecting resistance mutations were RIF 355, 0.99, 0.96, and INH 340, 1.00, and 0.98, respectively. DISCUSSION: FTv2 reliably detects MTBC DNA in pulmonary and extrapulmonary samples and detects resistance mutations for INH and RIF resistance in inhA promoter, katG, and rpoB genes.
Asunto(s)
Isoniazida , Mutación , Mycobacterium tuberculosis , Rifampin , Tuberculosis Resistente a Múltiples Medicamentos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Humanos , Alemania , Dinamarca , Sierra Leona , Rifampin/farmacología , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Isoniazida/farmacología , Antituberculosos/farmacología , Pruebas de Sensibilidad Microbiana , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/diagnóstico , Proteínas Bacterianas/genética , Sensibilidad y Especificidad , Esputo/microbiología , ARN Polimerasas Dirigidas por ADN/genéticaRESUMEN
Multidrug-resistant tuberculosis (MDR-TB) management has become a serious global health challenge. Understanding its epidemic determinants on the regional level is crucial for developing effective control measures. We used whole genome sequencing data of 238 of Mycobacterium tuberculosis complex (MTBC) strains to determine drug resistance profiles, phylogeny, and transmission dynamics of MDR/rifampicin-resistant (RR) MTBC strains from Sierra Leone. Forty-two strains were classified as RR, 196 as MDR, 5 were resistant to bedaquiline (BDQ) and clofazimine (CFZ), but none was found to be resistant to fluoroquinolones. Sixty-one (26%) strains were resistant to all first-line drugs, three of which had additional resistance to BDQ/CFZ. The strains were classified into six major MTBC lineages (L), with strains of L4 being the most prevalent, 62% (n = 147), followed by L6 (Mycobacterium africanum) strains, (21%, n = 50). The overall clustering rate (using ≤d12 single-nucleotide polymorphism threshold) was 44%, stratified into 31 clusters ranging from 2 to 16 strains. The largest cluster (n = 16) was formed by sublineage 2.2.1 Beijing Ancestral 3 strains, which developed MDR several times. Meanwhile, 10 of the L6 strains had a primary MDR transmission. We observed a high diversity of drug resistance mutations, including borderline resistance mutations to isoniazid and rifampicin, and mutations were not detected by commercial assays. In conclusion, one in five strains investigated was resistant to all first-line drugs, three of which had evidence of BDQ/CFZ resistance. Implementation of interventions such as rapid diagnostics that prevent further resistance development and stop MDR-TB transmission chains in the country is urgently needed. IMPORTANCE: A substantial proportion of MDR-TB strains in Sierra Leone were resistant against all first line drugs; however this makes the all-oral-six-month BPaLM regimen or other 6-9 months all oral regimens still viable, mainly because there was no FQ resistance.Resistance to BDQ was detected, as well as RR, due to mutations outside of the hotspot region. While the prevalence of those resistances was low, it is still cause for concern and needs to be closely monitored.
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Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Humanos , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Rifampin/farmacología , Sierra Leona/epidemiología , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/epidemiología , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Pruebas de Sensibilidad Microbiana , Farmacorresistencia Bacteriana Múltiple/genéticaRESUMEN
Filovirus serological diagnosis and epidemiological investigations are hampered due to the unavailability of validated immunoassays. Diagnostic performance of three indirect enzyme-linked immunosorbent assays (I-ELISA) was evaluated for the detection of IgG antibody to Ebola virus (EBOV) in human sera. One I-ELISA was based on a whole EBOV antigen (WAg) and two utilized recombinant nucleocapsid (NP) and glycoproteins (GP), respectively. Validation data sets derived from individual sera collected in South Africa (SA), representing an EBOV non-endemic country, and from sera collected during an Ebola disease (EBOD) outbreak in Sierra Leone (SL), were categorized according to the compounded results of the three I-ELISAs and real time reverse-transcription polymerase chain reaction (RT-PCR). At the cut-off values selected at 95% accuracy level by the two-graph receiver operating characteristic analysis, specificity in the SA EBOV negative serum panel (n = 273) ranged from 98.17% (GP ELISA) to 99.27% (WAg ELISA). Diagnostic specificity in the SL EBOV negative panel (n = 676) was 100% by the three ELISAs. The diagnostic sensitivity in 423 RT-PCR confirmed EBOD patients was dependent on the time when the serum was collected after onset of disease. It significantly increased 2 weeks post-onset, reaching 100% sensitivity by WAg and NP and 98.1% by GP I-ELISA.
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Anticuerpos Antivirales/sangre , Ensayo de Inmunoadsorción Enzimática , Fiebre Hemorrágica Ebola/sangre , Fiebre Hemorrágica Ebola/diagnóstico , Inmunoglobulina G/sangre , Antígenos Virales/inmunología , Brotes de Enfermedades , Ebolavirus , Glicoproteínas/inmunología , Humanos , Proteínas de la Nucleocápside/inmunología , Sensibilidad y Especificidad , Sierra Leona , SudáfricaRESUMEN
We generated genome sequences from 218 cases of Ebola virus disease (EVD) in Sierra Leone (SLE) during 2014â»2015 to complement available datasets, particularly by including cases from a period of low sequence coverage during peak transmission of Ebola virus (EBOV) in the highly-affected Western Area division of SLE. The combined dataset was utilized to produce phylogenetic and phylodynamic inferences, to study sinkâ»source dynamics and virus dispersal from highly-populated transmission hotspots. We identified four districts in SLE where EBOV was introduced and transmission occurred without onward exportation to other districts. We also identified six districts that substantially contributed to the dispersal of the virus and prolonged the EVD outbreak: five of these served as major hubs, with lots of movement in and out, and one acted primarily as a source, exporting the virus to other areas of the country. Positive correlations between case numbers, inter-district transition events, and district population sizes reaffirm that population size was a driver of EBOV transmission dynamics in SLE. The data presented here confirm the role of urban hubs in virus dispersal and of a delayed laboratory response in the expansion and perpetuation of the EVD outbreak in SLE.
Asunto(s)
Ebolavirus/genética , Fiebre Hemorrágica Ebola/transmisión , Filogenia , Brotes de Enfermedades , Ebolavirus/clasificación , Genoma Viral , Fiebre Hemorrágica Ebola/epidemiología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Sierra Leona/epidemiologíaRESUMEN
BACKGROUND: In August 2014, the National Institute for Communicable Diseases (NICD) in South Africa established a modular high-biosafety field Ebola diagnostic laboratory (SA FEDL) near Freetown, Sierra Leone in response to the rapidly increasing number of Ebola virus disease (EVD) cases. METHODS AND FINDINGS: The SA FEDL operated in the Western Area of Sierra Leone, which remained a "hotspot" of the EVD epidemic for months. The FEDL was the only diagnostic capacity available to respond to the overwhelming demand for rapid EVD laboratory diagnosis for several weeks in the initial stages of the EVD crisis in the capital of Sierra Leone. Furthermore, the NICD set out to establish local capacity amongst Sierra Leonean nationals in all aspects of the FEDL functions from the outset. This led to the successful hand-over of the FEDL to the Sierra Leone Ministry of Health and Sanitation in March 2015. Between 25 August 2014 and 22 June 2016, the laboratory tested 11,250 specimens mostly from the Western Urban and Western Rural regions of Sierra Leone, of which 2,379 (21.14%) tested positive for Ebola virus RNA. CONCLUSIONS: The bio-safety standards and the portability of the SA FEDL, offered a cost-effective and practical alternative for the rapid deployment of a field-operated high biocontainment facility. The SA FEDL teams demonstrated that it is highly beneficial to train the national staff in the course of formidable disease outbreak and accomplished their full integration into all operational and diagnostic aspects of the laboratory. This initiative contributed to the international efforts in bringing the EVD outbreak under control in Sierra Leone, as well as capacitating local African scientists and technologists to respond to diagnostic needs that might be required in future outbreaks of highly contagious pathogens.