Evaluación de la técnica Xpert® MTB/RIF para la detección de Mycobacterium tuberculosis complex en muestras extra-pulmonares / Evaluation of Xpert® MTB/RIF technique for Mycobacterium tuberculosis complex detection in extra-respiratory specimens

Resumen La tuberculosis (TBC) extra-pulmonar alcanza al 26,2% de los casos totales de TBC en Chile. El cultivo es el método estándar de oro, pero es lento. La técnica Xpert® MTB/RIF permite detectar Mycobacterium tuberculosis complex (MTBc) por RPC en tiempo real en menos de 3 h, sin embargo, ha sido validada sólo para muestras respiratorias. El objetivo de este estudio fue determinar la utilidad de la prueba Xpert® MTB/RIF en la detección de MTBc en muestras extra-pulmonares en comparación con un estándar de oro combinado consistente en un cultivo de micobacterias positivo (medio sólido y líquido) y/o un método molecular validado positivo (q-RPC, Cobas® TaqMan-MTB). Se analizaron 50 muestras extra-pulmonares, de las cuales 25 fueron definidas positivas y 25 negativas para MTBc en base a estándar de oro combinado. Las 25 muestras definidas positivas tuvieron un resultado positivo por Xpert® MTB/RIF; de las 25 muestras definidas negativas, 24 tuvieron un resultado negativo y una de ellas un resultado positivo. Se obtuvo una concordancia global entre Xpert® MTB/RIF y el estándar de oro combinado de 98%. La prueba Xpert® MTB/RIF fue capaz de detectar 12 casos de TBC extra-pulmonar con baciloscopia negativa y 3 casos con cultivo negativo. El método Xpert® MTB/RIF ha demostrado tener una sensibilidad similar al q-RPC para detectar MTBc en muestras extra-pulmonares y permite reducir sustancialmente el tiempo de diagnóstico.

Extra-pulmonary tuberculosis (TB) represents the 26.2% of total TB cases in Chile. Culture is the gold standard method, but the process is extremely slow. Xpert®MTB/RIF technique detects Mycobacterium tuberculosis complex (MTBc) through real time PCR in less than 3 h. However, it has been validated only for respiratory specimens. We aimed to determine the performance of Xpert®MTB/RIF test in detecting MTBc in extra-respiratory specimens compared with a combined gold standard consisting in a positive (liquid and solid) mycobacterial culture and/or a positive validated molecular method (q-RPC, Cobas®TaqMan®-MTB). Fifty extra-respiratory specimens were analyzed, from which 25 were positive and 25 negative for MTBc based on the combined gold standard. The 25 positive specimens had a positive result by Xpert®MTB/RIF; from the 25 negative specimens, 24 had a negative result and one had a positive result. We obtained an overall concordance of 98% between Xpert®MTB/RIF and the combined gold standard. Xpert®MTB/RIF test was able to detect 12 smear-negative specimens and 3 culture-negative specimens, all of them corresponding to extra-pulmonary TB cases. Xpert®MTB/RIF showed similar sensitivity to q-RPC in detecting MTBc in extra-respiratory specimens. This procedure allowed a substantial reduction in the time of diagnosis.

Analytical and Clinical Validation of Six Commercial Middle East Respiratory Syndrome Coronavirus RNA Detection Kits Based on Real-Time Reverse-Transcription PCR

BACKGROUND: During the 2015 outbreak of Middle East Respiratory Syndrome coronavirus (MERS-CoV), six different commercial MERS-CoV RNA detection kits based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) were available in Korea. We performed analytical and clinical validations of these kits. METHODS: PowerChek (Kogene Biotech, Korea), DiaPlexQ (SolGent, Korea), Anyplex (Seegene, Korea), AccuPower (Bioneer, Korea), LightMix (Roche Molecular Diagnostics, Switzerland), and UltraFast kits (Nanobiosys, Korea) were evaluated. Limits of detection (LOD) with 95% probability values were estimated by testing 16 replicates of upstream of the envelope gene (upE) and open reading frame 1a (ORF1a) RNA transcripts. Specificity was estimated by using 28 nasopharyngeal swabs that were positive for other respiratory viruses. Clinical sensitivity was evaluated by using 18 lower respiratory specimens. The sensitivity test panel and the high inhibition panel were composed of nine specimens each, including eight and six specimens that were positive for MERS-CoV, respectively. RESULTS: The LODs for upE ranged from 21.88 to 263.03 copies/reaction, and those for ORF1a ranged from 6.92 to 128.82 copies/reaction. No cross-reactivity with other respiratory viruses was found. All six kits correctly identified 8 of 8 (100%) positive clinical specimens. Based on results from the high inhibition panel, PowerChek and AccuPower were the least sensitive to the presence of PCR inhibition. CONCLUSIONS: The overall sensitivity and specificity of all six assay systems were sufficient for diagnosing MERS-CoV infection. However, the analytical sensitivity and detection ability in specimens with PCR inhibition could be improved with the use of appropriate internal controls.

Nontuberculous Mycobacteria Isolated from Respiratory Specimens during Recent Two Years: Distribution and Clinical Significance / 대한임상미생물학회지

BACKGROUND: The isolation of nontuberculous mycobacteria (NTM) has been increasing worldwide as well as its clinical importance. The aim of this study was to investigate the distribution and clinical significance of NTM that has been isolated from respiratory specimens during a recent two-year period at a tertiary hospital. METHODS: We analyzed respiratory samples that were obtained between January 2009 and December 2010 for AFB culture. We retrospectively reviewed the electronic medical records of these patients to obtain both clinical and radiologic information. NTM pulmonary disease was defined by using the guidelines provided by the America Thoracic Society/Infectious Diseases Society of America. RESULTS: Among the 1,601 specimens that resulted in a positive AFB culture, 310 (19.4%) were NTM. In 189 patients, the most common isolate was M. avium-intracellulare complex (MAC) (127, 67.2%), which was then followed by M. abscessus (31, 16.4%), M. fortuitum (10, 5.3%), M. kansasii (9, 4.8%), and other NTM species. Of these, 93 (49.2%) patients were diagnosed with NTM pulmonary disease. MAC, M. abscessus, and M. kansasii were more virulent than the other species. None of the cases of NTM pulmonary disease were caused by M. fortuitum, M. chelonae, M. peregrinum, M. terrae complex, or M. gordonae. CONCLUSION: In Korea, the prevalence of NTM isolates is increasing, as are the cases of pulmonary disease. The pathogenic potential of NTM differs enormously by species and as a result the treatment of NTM lung disease depends on which species has caused the infection. The isolation and identification of NTM isolated from respiratory specimens are mandatory in order for clinical microbiology laboratories to make an accurate diagnosis and suggest the proper treatment of the NTM disease.

Recovery Rate and Characteristics of Nontuberculous Mycobacterial Isolates in a University Hospital in Korea / 결핵및호흡기질환

BACKGROUND: The incidence of nontuberculous mycobacterium (NTM) infections in Korea is increasing. This retrospective study was performed to examine the recovery rate of NTM from respiratory specimens as well as the isolated NTM colony characteristics, and to assess the clinical significance of a NTM isolation. METHODS: The results of the respiratory specimens requested for an acid-fast bacilli (AFB) examination during 2002 at Asan Medical Center, along with the patients clinical characteristics were analyzed. RESULTS: A total 26,820 respiratory specimens were requested for the acid-fast bacilli (AFB) smear and culture during the study period. The proportion of M. tuberculosis and NTM isolation was 5.7% and 2.2%, respectively. Among the AFB smear and culture positive specimens, 12.2% were found to be NTM. The scotochromogen showing a low colony count < 20, which appeared to be contaminants, were isolated in 31.8% of the 584 NTM isolates. Excluding the low-colony scotochromogens, the M. avium-intracellulare complex was the most common NTM isolates (42.1%), and was also the most common causative organism for NTM pulmonary diseases. 8.4% (23/275) and 17.8% (49/275) of patients with NTM isolates met the American and British Thoracic Society diagnostic criteria for NTM pulmonary disease, respectively. CONCLUSION: In case of a positive AFB-smear or culture result, the possibility of NTM being a causative organism should always be considered, even in Korea, which has an intermediate incidence of tuberculosis.