METHODS: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay.
RESULTS: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6-98.2) and 100% (95% CI = 78.5-100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (κ = 0.913, P
Methods: A total of 42 patients with congenital heart defects, as confirmed by echocardiography, were recruited. Genetic molecular analysis using a fluorescence in situ hybridization (FISH) technique was conducted as part of routine 22q11.2DS screening, followed by multiplex ligation-dependent probe amplification (MLPA), which serves as a confirmatory test.
Results: Two of the 42 CHD cases (4.76%) indicated the presence of 22q11.2DS, and interestingly, both cases have conotruncal heart defects. In terms of concordance of techniques used, MLPA is superior since it can detect deletions within the 22q11.2 locus and outside of the typically deleted region (TDR) as well as duplications.
Conclusion: The incidence of 22q11.2DS among patients with CHD in the east coast of Malaysia is 0.047. MLPA is a scalable and affordable alternative molecular diagnostic method in the screening of 22q11.2DS and can be routinely applied for the diagnosis of deletion syndromes.
METHODS: In this study, mouthwash, saliva, and buccal cytobrush samples were collected from β-thalassemia major patients who had previously been characterized using DNA extracted from peripheral blood. DNA was extracted from mouthwash, saliva, and buccal cytobrush samples using the conventional inexpensive phenol-chloroform method and was measured by spectrophotometry for yield and purity. Molecular characterization of β-globin gene mutations was carried out using the amplification refractory mutation system (ARMS).
RESULTS: DNA extracted from mouthwash, saliva, and buccal cytobrush samples produced high concentration and pure DNA. The purified DNA was successfully amplified using ARMS. Results of the β-globin gene mutations using DNA from the three non-invasive samples were in 100% concordance with results from DNA extracted from peripheral blood.
CONCLUSIONS: The conventional in-house developed methods for non-invasive sample collection and DNA extraction from these samples are effective and negate the use of more expensive commercial kits. In conclusion, DNA extracted from mouthwash, saliva, and buccal cytobrush samples provided sufficiently high amounts of pure DNA suitable for molecular analysis of β-thalassemia.
METHODS: RNA was extracted from nasopharyngeal swab samples by a simple RNA extraction method.
RESULTS: Testing of 77 samples demonstrated 91.2% sensitivity (95% confidence interval [CI]: 78-98.2%) and 100% specificity (95% confidence interval: 92-100%) using UDG RT-LAMP.
CONCLUSION: This colorimetric UDG RT-LAMP is a simple-to-use, fast, and easy-to-interpret method, which could serve as an alternative for diagnosis of SARS-CoV-2 infection, especially in remote hospitals and laboratories with under-equipped medical facilities.
MATERIALS AND METHODS: Our search was limited to original papers in the English language from 2010 to 2018 using several databases including Pubmed, Scopus, Google Scholar, Iranmedex, and Scientific Information Database. A manual search of references provided in the included papers was also performed.
RESULTS: Of 101 electronically searched citations, 43 met the inclusion criteria. ELISA is commonly used for qualitative and screening detection, and WB and PCR techniques are used to confirm infection.
CONCLUSION: Among all the reported methods for detection of HTLV-1, only serological and molecular tests are used as the most common technical assays for HTLV-1. The ELISA assay, without a confirmatory test, has several limitations and affect the accuracy of the results. Owing to the prevalence of HTLV-1 and limitations of the current detection methods, further evaluation of the accuracy of these methods is needed. There are new opportunities for applying novel technological advances in microfluidics, biosensors, and lab-on-a-chip systems to perform HTLV-1 diagnostics.
OBJECTIVE: To design and evaluate a molecular diagnostic tool for detection and identification of all currently recognized and potentially future emergent CoVs from the Orthocoronavirinae subfamily.
STUDY DESIGN AND RESULTS: We designed a semi-nested, reverse transcription RT-PCR assay based upon 38 published genome sequences of human and animal CoVs. We evaluated this assay with 14 human and animal CoVs and 11 other non-CoV respiratory viruses. Through sequencing the assay's target amplicon, the assay correctly identified each of the CoVs; no cross-reactivity with 11 common respiratory viruses was observed. The limits of detection ranged from 4 to 4 × 102 copies/reaction, depending on the CoV species tested. To assess the assay's clinical performance, we tested a large panel of previously studied specimens: 192 human respiratory specimens from pneumonia patients, 5 clinical specimens from COVID-19 patients, 81 poultry oral secretion specimens, 109 pig slurry specimens, and 31 aerosol samples from a live bird market. The amplicons of all RT-PCR-positive samples were confirmed by Sanger sequencing. Our assay performed well with all tested specimens across all sample types.
CONCLUSIONS: This assay can be used for detection and identification of all previously recognized CoVs, including SARS-CoV-2, and potentially any emergent CoVs in the Orthocoronavirinae subfamily.
METHODOLOGY: A combination of nucleic acid-based assays, including in house generic polymerase chain reaction (PCR) assays for enteroviruses, flaviviruses and phleboviruses, a commercial real-time PCR assay for herpesviruses and a commercial real time multiplex PCR, enabling detection of frequently-observed viral, bacterial and fungal agents were employed for screening.
RESULTS: The microbial agent could be characterized in 10 (10%) of the 100 specimens. Viral etiology could be demonstrated in 7 (70%) specimens, which comprises Human Herpesvirus 6 (4/7), Herpes Simplex virus type1 (2/7) and Enteroviruses (1/7). In 3 specimens (30%), Streptococcus pneumoniae, Listeria monocytogenes and Staphylococcus aureus were detected via the multiplex PCR, which were also isolated in bacteriological media. All specimens with detectable viral nucleic acids, as well as unreactive specimens via nucleic acid testing remained negative in bacteriological cultures.
CONCLUSIONS: Herpes and enteroviruses were identified as the primary causative agents of central nervous system infections in children. Enterovirus testing must be included in the diagnostic work-up of relevant cases.
METHODS: This study adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis for Diagnostic Test Accuracy (PRISMA-DTA) guideline. Relevant studies in the health-related electronic databases were searched. According to the criteria set for this study, eligible studies were identified. The quality of included studies was evaluated with the use of a quality assessment checklist. A summary performance estimates such as pooled sensitivity and specificity were stratified by type of LAMP. Bivariate model for data analyses was applied. Summary receiver operating characteristics plots were created to display the results of individual studies in a receiver operating characteristics space. Meta-regression analysis was performed to investigate the sources of heterogeneity among individual studies.
RESULTS: Twenty-seven studies across 17 endemic countries were identified. The vast majority of studies were with unclear risk of bias in the selection of index test. Overall, the pooled test performances were high for Pan LAMP (sensitivity: 0.95, 95% CI 0.91 to 0.97; specificity: 0.98, 95% CI 0.95 to 0.99), Plasmodium falciparum (Pf) LAMP (sensitivity: 0.96, 95% CI 0.94 to 0.98; specificity: 0.99, 95% CI 0.96 to 1.00) or for Plasmodium vivax (Pv) LAMP from 6 studies (sensitivity: 0.98, 95% CI 0.92 to 0.99; specificity: 0.99, 95% CI 0.72 to 1.00). The area under the curve for Pan LAMP (0.99, 95% CI 0.98-1.00), Pf LAMP (0.99, 95% CI 0.97-0.99) and Pv LAMP was (1.00, 95% CI 0.98-1.00) indicated that the diagnostic performance of these tests were within the excellent accuracy range. Meta-regression analysis showed that sample size had the greatest impact on test performance, among other factors.
CONCLUSIONS: The current findings suggest that LAMP-based assays are appropriate for detecting low-level malaria parasite infections in the field and would become valuable tools for malaria control and elimination programmes. Future well-designed larger sample studies on LAMP assessment in passive and active malaria surveillances that use PCR as the reference standard and provide sufficient data to construct 2 × 2 diagnostic table are needed.