Comparative Analysis of In-House RT-qPCR Detection of SARS-CoV-2 for Resource-Constrained Settings.

We developed and standardized an efficient and cost-effective in-house RT-PCR method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated sensitivity, specificity, and other statistical parameters by different RT-qPCR methods including triplex, duplex, and simplex assays adapted from the initial World Health Organization- (WHO) recommended protocol. This protocol included the identification of the E envelope gene (E gene; specific to the Sarvecovirus genus), RdRp gene of the RNA-dependent RNA polymerase (specific for SARS-CoV-2), and RNase P gene as endogenous control. The detection limit of the E and the RdRp genes were 3.8 copies and 33.8 copies per 1 µL of RNA, respectively, in both triplex and duplex reactions. The sensitivity for the RdRp gene in the triplex and duplex RT-qPCR tests were 98.3% and 83.1%, respectively. We showed a decrease in sensitivity for the RdRp gene by 60% when the E gene acquired Ct values > 31 in the diagnostic tests. This is associated with the specific detection limit of each gene and possible interferences in the protocol. Hence, developing efficient and cost-effective methodologies that can be adapted to various health emergency scenarios is important, especially in developing countries or settings where resources are limited.

Evaluation of an in-house PCR for diagnosing scrub typhus along with a preliminary study of genotypic characterization of Orientia tsutshugamushi circulating in Chhattisgarh: A prospective clinico-microbiological study.

PURPOSE: Scrub typhus, caused by Orientia tsutsugamushi (O. tsutsugamushi) present nonspecific clinical features during manifestation of acute undifferentiated febrile illness (AUFI) to render its early diagnosis difficult. Accordingly, this study was undertaken to assess an in-house groEL PCR versus IgM ELISA for the diagnosis of scrub typhus and to genotypically characterise the randomly selected scrub typhus positive cases. METHODS: Blood samples, collected from two hundred twenty one (221) AUFI cases were subjected to groEL PCR and IgM ELISA for diagnosis of scrub typhus. Eleven randomly selected PCR positive cases were processed for DNA sequencing to determine the genetic diversity of O. tsutsugamushi in Chhattisgarh. RESULTS: Scrub typhus prevalence of 35.2% were detected among AUFI cases using both in-house groEL PCR and IgM ELISA. PCR alone showed sensitivity, specificity, positive and negative predictive values of 66.6% (CI: 55.08-76.94), 100% (CI: 90 to 100),100% (CI: 93.15 to 100) and 57.37% (CI: 44.05 to 69.96) while for IgM ELISA, these parameters were 62.8% (CI: 51.13-73.50), 100% (CI: 90 to 100), 100% (CI: 92.75 to 100) and 54.68% (CI: 41.75 to 67.18) respectively. PCR and ELISA could detect scrub typhus in 37.2% and 33.3% cases, when tested alone. groEL PCR detected the O. tsutsugamushi throughout the course of infection. Phylogenetic analysis depicted 5 of 11 positive cases belonged to Kuroki, Japan strain of O. tsutsugamushi, followed by Gilliam and Karp strain in 4 and 2 cases respectively. CONCLUSION: Scrub typhus should be considered in differential diagnosis of AUFI. groEL PCR may aid on to IgM ELISA test for optimum laboratory diagnosis of scrub typhus by its implementation especially in seronegative cases. Predominance of Kuroki-like strain followed by Gillian and Karp strains of O. tsutsugamushi in Chhattisgarh confirm variable geographical distribution of O. tsutsugamushi and provide the baseline epidemiological data which will eventually be used to help the researchers for developing better diagnostic tests and vaccine covering the predominant genotypes.

Performance of Nucleic Acid Amplification Tests in Patients with Presumptive Pulmonary Tuberculosis in Taiwan.

INTRODUCTION: Several nucleic acid amplification tests (NAATs) for detection of Mycobacterium tuberculosis (TB) complex (MTBC) are available in Taiwan; however, their performances may differ and have not been extensively evaluated. Therefore, we aimed to explore the accuracy of NAATs overall followed by comparison between platforms commonly used in Taiwan. METHODS: This study enrolled presumptive pulmonary TB patients with NAATs throughout Taiwan. The diagnostic performance of smear microscopy and NAATs was assessed using sputum culture as a reference standard. To investigate the performance of NAATs in excluding non-tuberculous mycobacteria (NTM), we quantified the false-positive proportion of NAATs in patients infected with NTM. RESULTS: Of the 4126 enrollees, 860 (20.8%) had positive NAATs. The sensitivity and specificity of NAATs were 83.2% and 96.7%, respectively, compared to 81.5% and 55.3% for smear. There was no significant difference in sensitivity between the NAATs and smear; however, the specificity of smear was significantly lower than that of the NAATs [difference 41.4%, 95% confidence interval (CI) 39.6-43.2%]. There was no significant difference in sensitivity among Roche Cobas Amplicor Mycobacterium tuberculosis assay (Amplicor), Xpert MTB/RIF assay (Xpert) and in-house polymerase chain reaction (in-house PCR) (82.2% versus 83.8% versus 82.4%); however, in-house PCR was significantly less specific than Amplicor (difference 5.3%, 95% CI 2.4-8.2%) and Xpert (difference 5.8%, 95% CI 3.1-8.5%). The sensitivity of NAATs among smear-negative cases was 33.1% (95% CI 26.0-40.3%). In-house PCR had a significantly higher false-positive rate among specimens that were culture positive for NTM than Amplicor (7.7% versus 0.3%; difference 7.4%, 95% CI 3.4-11.5%) and Xpert (7.7% versus 0.7%; difference 7.0%, 95% CI 2.9-11.0%). CONCLUSION: The NAATs overall had a relatively high sensitivity and specificity in detecting MTBC while Amplicor and Xpert performed better than in-house PCR in excluding NTM. Our findings will be useful for the development of national policy.

Leprosy in a low-incidence setting : Case report relevant to metagenomic next generation sequencing applications.

Leprosy is a disease caused by Mycobacterium leprae that results in disability. In 2000 the World Health Organization announced that leprosy had been eradicated. In nonendemic areas diagnosing leprosy is becoming a challenge for inexperienced clinicians. This case involves a male patient suffering from chronic numbness, hand deformity and recurrent erythema. Skin biopsy revealed granuloma and acid-fast staining of short-rod bacteria. Peripheral venous blood was subjected to metagenomic next generation sequencing and bioinformatics analysis, which revealed 3 unique sequence reads of M. leprae. Paraffin-embedded tissue and fresh samples scraped from skin lesions were subjected to in-house PCR targeting 16S rRNA, hsp65, rpoB, rpoT, ribF-rpsO, and mmaA. Sanger sequencing of amplicons from fresh samples and paraffin-embedded tissue verified the presence of M. leprae. For inexperienced clinicians in nonendemic areas nucleic acid amplification tests, such as in-house PCR, are helpful for diagnosing leprosy but sequence reads from metagenomic next generation sequencing may also provide evidence when interpreted cautiously.

Usefulness of in-house PCR methods for hepatitis B virus DNA detection.

The aim of the present study was to evaluate the performance of three in-house PCR techniques for HBV DNA detection and compare it with commercial quantitative methods to evaluate the usefulness of in-house methods for HBV diagnosis. Three panels of HBsAg reactive sera samples were evaluated: (i) 50 samples were examined using three methods for in-house qualitative PCR and the Cobas Amplicor HBV Monitor Assay; (ii) 87 samples were assayed using in-house semi-nested PCR and the Cobas TaqMan HBV test; (iii) 11 serial samples obtained from 2 HBV-infected individuals were assayed using the Cobas Amplicor HBV test and semi-nested PCR. In panel I, HBV DNA was detected in 44 samples using the Cobas Amplicor HBV test, 42 samples using semi-nested PCR (90% concordance with Cobas Amplicor), 22 samples using PCR for the core gene (63.6% concordance) and 29 samples using single-round PCR for the pre-S/S gene (75% concordance). In panel II, HBV DNA was quantified in 78 of the 87 HBsAg reactive samples using Cobas TaqMan but 52 samples using semi-nested PCR (67.8% concordance). HBV DNA was detected in serial samples until the 17th and 26th week after first donation using in-house semi-nested PCR and the Cobas Amplicor HBV test, respectively. In-house semi-nested PCR presented adequate concordance with commercial methods as an alternative method for HBV molecular diagnosis in low-resource settings.

Uso clínico de la reacción en cadena de la polimerasa e hibridización en el diagnóstico de tuberculosis / Clinical use of the polymerase chain reaction and hybridization in diagnosing tuberculosis

La tuberculosis (TBC) es una causa importante de morbimortalidad en nuestro país. Las herramientas de diagnóstico no son 100% eficientes y rápidas para ayudar al clínico en su decisión médica. Nuevas pruebas, como la reacción en cadena de la polimerasa (PCR), son necesarias. Evaluar la utilidad clínica de una PCR para IS6110, en muestras clínicas de pacientes con sospecha de tuberculosis pulmonar o extrapulmonar. Sesenta y una muestras de 46 pacientes del Hospital General del Este, Dr. Domingo Luciani de Caracas, Venezuela, fueron procesadas para la detección del M. Tuberculosis. Se practicó baciloscopia, cultivo en LJ y PCR e hibridación. Los pacientes se clasificaron como TBC positivos si tenían evidencia clínica o radiológica, y positivas algunas de estas pruebas: PPD, ZN, cultivo, biopsia positiva o respuesta favorable al tratamiento. Los TBC negativos con evidencia clínica o radiológica positiva y todas las pruebas negativas. La sensibilidad de la PCR sola fue del 75% y la especificidad del 61%. La PCR e hibridación juntas mostraron sensibilidad del 90% y especificidad del 57%. Los valores predictivos positivos y negativos fueron del 62% y 88%, respectivamente

Tuberculosis (TBC) remains a leading cause of morbidity and mortality in Venezuela. The diagnos tic tools used are not 100% specific, sensitive, efficient or fast enough to help clinicians take the right clinical decision. New tests, like polymerase chain reaction (PCR), are necessary. We evaluated the clinical usefulness of an IS6110 in-house PCR in clinical samples from patients suspected of having pulmonary or extrapulmonary TBC. Sixty one samples from 46 patients were processed for detection of M. tuberculosis by ZN stained smear examination, LJ medium culture, and PCR and hybridization assay. The patients were classified as TBC positive when they had clinical or radiological evidence plus any positive of the following tests: PPD, ZN, LJ culture, biopsy or anti-TBC treatment response and TBC negative when they had clinical or radiological evidence but all the applied tests used were negative. PCR sensibility was 75% and specificity was 61%, using PCR alone, but when PCR and hybridization were evaluated together the sensibility was 90% and specificity was 57%. The predictive positive and negative values were 62% and 88% respectively