Quality of the sample-based RNA determines the real-time RT-PCR results in the laboratory diagnosis of COVID-19.

Effect of the quality of sample-based RNA on COVID-19 real-time RT-PCR results was investigated. The purity of the extracts was dependent on the extraction method (P<0.0001) and affected the test interpretations (P = 0.002). Gross RNA concentration negatively correlated with Ct values (P < 0.0001). The presence of impurities contributed to inconclusive test results.

Relationship between clinical and laboratory features with infecting dengue virus serotypes in a sample of dengue suspected adult patients from 2015-2017 in Sri Lanka.

Dengue is a major viral disease affecting the tropics. Although previous research has focused on the relationship between the infecting dengue virus (DENV) serotypes and disease severity, less work has been done on the relationship between the clinical and laboratory features and the infecting DENV serotypes in Sri Lanka. We evaluated the relationship between the clinical and laboratory features and the infecting DENV serotypes in adult patients with clinically suspected dengue admitted to the Base Hospital, Mawanella, Sri Lanka from December 2015 to March 2017. Blood samples of 200 dengue suspected patients were tested for the infecting DENV serotypes using a reverse transcription polymerase chain reaction with primers targeting the envelope region of the virus. Relationship between the infecting DENV serotypes with clinical and laboratory features was assessed using Z score and paired t tests. Of the 200 patients tested, 39 (19.5%) were positive for DENV, any of the four DENV serotypes alone or in combination. The highest number of infections was noted with DENV-2 (n=18, 46.1%). Fever (P=0.000) and rash (P=0.017) were frequently noted in DENV negative patients while bleeding (P=0.012) was more frequently noted in DENV serotype positive patients. Platelet count of <100,000 µl-1 was significantly associated with DENV serotype positivity (P=0.000). Platelet count of <100,000 µl-1 (P=0.035) and haemoglobin (Hb) of >13mgdl-1 (P=0.016) were noted in 15 of the 18 DENV-2 positive patients. Clinical and laboratory features of severe dengue with bleeding manifestations, low platelet counts and high Hb were noted in DENV-2 infections.

Diagnostic utility and validation of a newly developed real time loop mediated isothermal amplification method for the detection of SARS CoV-2 infection.

Background: Detecting SARS-CoV-2 using a simple real time molecular assay will be helpful for the mitigation efforts in low / middle income countries during the pandemic. We have developed and validated a rapid and simple real time loop mediated isothermal amplification assay (LAMP) for screening of SARS-CoV-2 infection in known infected and non-infected individuals. Methods: Six sets of primers were designed targeting the N-gene of the SARS-CoV-2 (Accession ID MN994468). LAMP reactions were performed using Warm Start 2X Master Mix and real-time PCR machine at 65 °C for 60 cycles with 15 s for each cycle. Results were read by visualizing turbidity under ultraviolet light and real time fluorescence detection through FAM channel of the real time PCR machine. We tested a total of 320 including 240 SARS CoV-2 positive (Ct values <40) and 80 SARS CoV-2 negative samples as tested by a real time RT-PCR using the newly developed LAMP assay. Results: A total of 206 out of 240 SARS CoV-2 positive samples were tested positive by the newly developed LAMP assay with a sensitivity of 86%. All 80 SARS CoV-2 negative samples were tested negative by the newly developed LAMP assay with a specificity of 100%. Conclusion: The newly developed real time LAMP assay has a sensitivity of 86% and specificity of 100% compared to the real time RT-PCR for the detection of SARS CoV-2. The new assay will be useful to screen large number of samples if adopted to minimize the time and cost.

Circulating dengue virus serotypes and vertical transmission in Aedes larvae during outbreak and inter-outbreak seasons in a high dengue risk area of Sri Lanka.

BACKGROUND: Spatial and temporal changes in the dengue incidence are associated with multiple factors, such as climate, immunity among a population against dengue viruses (DENV), circulating DENV serotypes and vertical transmission (VT) of DENV in an area at a given time. The level of VT in a specific location has epidemiological implications in terms of viral maintenance in vectors. Identification of the circulating DENV serotypes in both patients and Aedes mosquito larvae in an area may be useful for the early detection of outbreaks. We report here the results of a prospective descriptive study that was conducted to detect the levels of VT in Aedes mosquito larvae and circulating DENV serotypes in patients and Aedes mosquito larvae from December 2015 to March 2017 in an area of Sri Lanka at high risk for dengue. METHODS: A total of 200 patients with clinically suspected dengue who had been admitted to a tertiary care hospital during a dengue outbreak (3 study periods: December 2015-January 2016, June-August 2016, December 2016-January 2017) and in the inter-outbreak periods (February-May 2016 and September-November 2016) were investigated. Blood samples were drawn from the study participants to test for DENV. The houses of the study participants were visited within 7 days of admission to the hospital, and Aedes larvae were also collected within a radius of 400 m from the houses. The larvae were separately identified to species and then pooled according to each patient's identification number. Patients' sera and the Aedes larvae were tested to identify the infecting DENV serotypes using a reverse transcription PCR (RT-PCR) method. Levels of VT in Aedes mosquito larvae were also identified. RESULTS: All four DENV serotypes (DENV-1 to -4) were identified in the study area. In the early part of the study (December 2015-February 2016), DENV-3 was predominant and from April 2016 to March 2017, DENV-2 became the most predominant type. Four cases of DENV co-infections were noted during the study period in patients. Interestingly, all four DENV serotypes were detected in Aedes albopictus larvae, which was the prominent immature vectorial form identified throughout the study period in the area, showing 9.8% VT of DENV. With the exception of DENV-4, the other three DENV serotypes were identified in Aedes aegypti larvae with a VT of 8.1%. CONCLUSION: Comparatively high rates of VT of DENV was detected in Ae. albopictus and Ae. aegypti larvae. A shift in the predominant DENV serotype with simultaneous circulation of all four DENV serotypes was identified in the study area from December 2015 to March 2017.