Development and Validation of Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) as a Simple and Rapid Diagnostic Tool for SARS-CoV-2 Detection.

Since the COVID-19 pandemic outbreak in the world, many countries have searched for quick diagnostic tools to detect the virus. There are many ways to design diagnostic assays; however, each may have its limitations. A quick, sensitive, specific, and simple approach is essential for highly rapidly transmitted infections, such as SARS-CoV-2. This study aimed to develop a rapid and cost-effective diagnostic tool using a one-step Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) approach. The results were observed using the naked eye within 30-60 min using turbidity or colorimetric analysis. The sensitivity, specificity, and lowest limit of detection (LoD) for SARS-CoV-2 RNA against the RT-LAMP assay were assessed. This assay was also verified and validated against commercial quantitative RT-PCR used by health authorities in Saudi Arabia. Furthermore, a quick and direct sampling from the saliva, or buccal cavity, was applied after simple modification, using proteinase K and heating at 98 °C for 5 min to avoid routine RNA extraction. This rapid single-tube diagnostic tool detected COVID-19 with an accuracy rate of 95% for both genes (ORF1a and N) and an LoD for the ORF1a and N genes as 39 and 25 copies/reaction, respectively. It can be potentially used as a high-throughput national screening for different respiratory-based infections within the Middle East region, such as the MERS virus or major zoonotic pathogens such as Mycobacterium paratuberculosis and Brucella spp., particularly in remote and rural areas where lab equipment is limited.

Assessment of 12 qualitative RT-PCR commercial kits for the detection of SARS-CoV-2.

The emergence of the novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the late months of 2019 had the officials to declare a public health emergency leading to a global response. Public measurements rely on an accurate diagnosis of individuals infected with the virus by using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The aim of our study is to relate the fundamental clinical and analytical performance of SARS-CoV-2 (RT-PCR) commercial kits. A total of 94 clinical samples were selected. Generally, 400 µl of each respiratory specimen was subjected to extraction using ExiPrep 96 Viral RNA Kit. All kits master mix preparation, cycling protocol, thermocycler, and results interpretation were carried out according to the manufacturer's instructions of use and recommendations. The performance of the kits was comparable except for the LYRA kit as it was less sensitive (F = 67, p < .001). Overall, four kits scored a sensitivity of 100% including: BGI, IQ Real, Sansure, and RADI. For specificity, all the tested kits scored above 95%. The performance of these commercial kits by gene target showed no significant change in CT values which indicates that kits disparities are mainly linked to the oligonucleotide of the gene target. We believe that most of the commercially available RT-PCR kits included in this study can be used for routine diagnosis of patients with SARS-CoV-2. We recommend including kits with multiple targets in order to monitor the virus changes over time.

The aspartate aminotransferase/platelet count ratio index as a marker of dengue virus infection: Course of illness.

BACKGROUND: The usefulness of laboratory tests in the decision-making process with regard to early identification of dengue virus infection has not been widely reported, particularly the aspartate aminotransferase (AST)/platelet count ratio index during a patient's days of illness. The aim of this study was to examine the pattern of the ratio index over the course of illness and identify whether it is a marker of dengue virus infection in dengue patients, as well as to assess the role of other laboratory tests. METHODS: A chart review of 205 dengue patients was analyzed using available records of 845 laboratory results within different time intervals or exam dates during the course of illness. We used repeated measures mixed binary logistic regression analyses to model the dengue virus infection, defined as giving at least one positive antibody test (yes/no). RESULTS: The high risk of dengue virus infection in dengue patients was found in the male gender (adjusted OR=4.316, 95% CI: 1.285-14.498, P=0.018), in patients with a high AST/platelet count ratio index (adjusted OR=1.438, 95% CI: 1.057-1.957, P=0.021), in patients with a low MCV level (adjusted OR=0.815, 95% CI: 0.679-0.978, P=0.028), and in patients with a low ALT level (adjusted OR=0.996, 95% CI: 0.993-0.999, P=0.010). CONCLUSION: Laboratory markers, in particular the AST/platelet count ratio index, can be useful for clinicians to strengthen the decision-making process in primary care settings. Furthermore, our model revealed that low MCV and low ALT are predictors of the dengue virus infection, while being a male increases the risk of dengue virus infection. More studies are needed to evaluate the impact of the AST/platelet count ratio index on the severity of dengue fever infection during the onset of symptoms and course of treatment.

Demographic Variations of MERS-CoV Infection among Suspected and Confirmed Cases: An Epidemiological Analysis of Laboratory-Based Data from Riyadh Regional Laboratory.

Introduction. Middle East respiratory syndrome coronavirus was first recognized in September 2012 in Saudi Arabia. The clinical presentations of MERS and non-MERS SARI are often similar. Therefore, the identification of suspected cases that may have higher chances of being diagnosed as cases of MERS-CoV is essential. However, the real challenge is to flag these patients through some demographic markers. The nature of these markers has not previously been investigated in Saudi Arabia, and hence, this study aims to identify them. METHODS: It was a surveillance system-based study, for which data from a total of 23,646 suspected patients in Riyadh and Al Qassim regions were analyzed from January 2017 until December 2017 to estimate the prevalence of MERS-CoV among suspected cases and to determine potential demographic risk factors related to the confirmation of the diagnosis. RESULTS: Of 23,646 suspected cases, 119 (0.5%) were confirmed by laboratory results. These confirmed cases (67.2% of which were males) had a mean age of 43.23 years (SD ± 22.8). Around 42.2% of the confirmed cases were aged between 41 and 60 years and about 47% of confirmed cases had their suspected specimen tested in the summer. The study identified three significant and independent predictors for confirmation of the disease: an age between 41 and 60 years, male gender, and summer season admission. CONCLUSION: The study provides evidence that the MERS-CoV epidemic in the subject regions has specific characteristics that might help future plans for the prevention and management of such a contagious disease. Future studies should aim to confirm such findings in other regions of Saudi Arabia as well and explore potential preventable risk factors.

Climate factors and incidence of Middle East respiratory syndrome coronavirus.

BACKGROUND: Our understanding of climate factors and their links to the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) outbreaks is incomplete. This study aimed to estimate the monthly incidence of MERS-CoV cases and to investigate their correlation to climate factors. METHODS: The study used aggregated monthly MERS-CoV cases that reported to the Saudi Center for Disease Prevention and Control from the Riyadh Region between November 1, 2012 and December 31, 2018. Data on the meteorological situation throughout the study period was calculated based on Google reports on the Riyadh Region (24.7136°N, 46.6753°E). The Poisson regression was used to estimate the incidence rate ratio (IRR) and its 95% confidence intervals (CI) for each climate factor. RESULTS: A total of 712 MERS-CoV cases were included in the analysis (mean age 54.2±9.9 years), and more than half (404) (56.1%) MERS-CoV cases were diagnosed during a five-month period from April to August. The highest peak timing positioned in August 2015, followed by April 2014, June 2017, March 2015, and June 2016. High temperatures (IRR=1.054, 95% CI: 1.043-1.065) and a high ultraviolet index (IRR=1.401, 95% CI: 1.331-1.475) were correlated with a higher incidence of MERS-CoV cases. However, low relative humidity (IRR=0.956, 95% CI: 0.948-0.964) and low wind speed (IRR=0.945, 95% CI: 0.912-0.979) were correlated with a lower incidence of MERS-CoV cases. CONCLUSION: The novel coronavirus, MERS-CoV, is influenced by climate conditions with increasing incidence between April and August. High temperature, high ultraviolet index, low wind speed, and low relative humidity are contributors to increased MERS-CoV cases. The climate factors must be evaluated in hospitals and community settings and integrated into guidelines to serve as source of control measures to prevent and eliminate the risk of infection.

Isolation and growth characterization of novel full length and deletion mutant human MERS-CoV strains from clinical specimens collected during 2015.

Middle East respiratory syndrome (MERS) is a viral respiratory illness first reported in Saudi Arabia in September 2012 caused by the human coronavirus (CoV), MERS-CoV. Using full-genome sequencing and phylogenetic analysis, scientists have identified three clades and multiple lineages of MERS-CoV in humans and the zoonotic host, dromedary camels. In this study, we have characterized eight MERS-CoV isolates collected from patients in Saudi Arabia in 2015. We have performed full-genome sequencing on the viral isolates, and compared them to the corresponding clinical specimens. All isolates were clade B, lineages 4 and 5. Three of the isolates carry deletions located on three independent regions of the genome in the 5'UTR, ORF1a and ORF3. All novel MERS-CoV strains replicated efficiently in Vero and Huh7 cells. Viruses with deletions in the 5'UTR and ORF1a exhibited impaired viral release in Vero cells. These data emphasize the plasticity of the MERS-CoV genome during human infection.