Portable RT-PCR System: a Rapid and Scalable Diagnostic Tool for COVID-19 Testing.

Combating the ongoing coronavirus disease 2019 (COVID-19) pandemic demands accurate, rapid, and point-of-care testing with fast results to triage cases for isolation and treatment. The current testing relies on reverse transcriptase PCR (RT-PCR), which is routinely performed in well-equipped laboratories by trained professionals at specific locations. However, during busy periods, high numbers of samples queued for testing can delay the test results, impacting efforts to reduce the infection risk. Besides, the absence of well-established laboratories at remote sites and low-resourced environments can contribute to a silent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These reasons compel the need to accommodate point-of-care testing for COVID-19 that meets the ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable). This study assessed the agreement and accuracy of the portable Biomeme SARS-CoV-2 system against the gold standard tests. Nasopharyngeal and nasal swabs were used. Of the 192 samples tested using the Biomeme SARS-CoV-2 system, the results from 189 samples (98.4%) were in agreement with the reference standard-of-care RT-PCR testing for SARS-CoV-2. The portable system generated simultaneous results for nine samples in 80 min with high positive and negative percent agreements of 99.0% and 97.8%, respectively. We performed separate testing in a sealed glove box, offering complete biosafety containment. Thus, the Biomeme SARS-CoV-2 system can help decentralize COVID-19 testing and offer rapid test results for patients in remote and low-resourced settings.

Molecular characterisation and clinical outcomes of Candida auris infection: Single-centre experience in Saudi Arabia.

BACKGROUND: Candida auris is a difficult-to-diagnose multidrug-resistant yeast that can cause invasive infections with high mortality. Since emerging in 2009, this pathogen has been associated with numerous outbreaks around the world. Whole genome sequencing (WGS) is instrumental for understanding the emergence and local transmission of this pathogen. OBJECTIVES: To describe the clinical, molecular characteristics of Candida auris infection and clinical outcome in our centre. PATIENTS AND METHODS: Patients with positive cultures for Candida auris were identified in a microbiology database. Clinical characteristics and antifungal susceptibility were obtained. Isolates were sent to the US CDC for whole genome sequencing. RESULTS: Seven unique patients with eight different isolates were identified. Seven isolates were sent to the US CDC for whole genome sequencing. None of the patients had bloodstream infection. Thirty-day mortality was higher in infected patients compared with those who were colonised. Seven of the eight isolates were resistant to both fluconazole, and five were resistant to amphotericin B. WGS analysis demonstrated that the seven isolates belonged to the South Asian clade but formed two distinct subclades suggesting two independent introductions and ongoing transmission within the facility. CONCLUSIONS: Candida auris is associated with a high mortality rate in infected patients. Strict infection control measures and surveillance for asymptomatic cases are warranted to halt ongoing transmission.

Whole genome sequencing of resistance and virulence genes in multi-drug resistant Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic bacterium that causes serious hospital-acquired infections. To assess the risk of clinically isolated P. aeruginosa to human health, we analyzed the resistance and virulence mechanisms of a collection of clinical isolates. METHODS: This was a retrospective study in which P. aeruginosa isolates collected from January 1, 2018 to August 31, 2019 were analyzed using phenotypic and whole-genome sequencing (WGS) methods. The analysis included 48 clinical samples. Median patient age was 54.0 (29.5) years, and 58.3% of patients were women. Data from the microbiology laboratory database were reviewed to identify P. aeruginosa isolates. All unique isolates available for further testing were included, and related clinical data were collected. Infections were defined as hospital acquired if the index culture was obtained at least 48 h after hospitalization. RESULTS: High-risk P. aeruginosa clones, including sequence types (STs) ST235 and ST111, were identified, in addition to 12 new STs. The isolates showed varying degrees of biofilm formation ability when evaluated at room temperature, along with reduced metabolic activity, as measured by metabolic staining, suggesting their ability to evade antimicrobial therapy. Most isolates (77.1%) were multidrug resistant (MDR), with the highest resistance and susceptibility rates to beta-lactams and colistimethate sodium, respectively. CONCLUSIONS: The MDR phenotypes of the examined isolates can be explained by the high prevalence of efflux-mediated resistance- and hydrolytic enzyme-encoding genes. These isolates had high cytotoxic potential, as indicated by the detection of toxin production-related genes.

SARS-CoV-2 spike gene Sanger sequencing methodology to identify variants of concern.

The global demand for rapid identification of circulating SARS-CoV-2 variants of concern has led to a shortage of commercial kits. Therefore, this study aimed to develop and validate a rapid, cost-efficient genome sequencing protocol to identify circulating SARS-CoV-2 (variants of concern). Sets of primers flanking the SARS-CoV-2 spike gene were designed, verified and then validated using 282 nasopharyngeal positive samples for SARS-CoV-2. Protocol specificity was confirmed by comparing these results with SARS-CoV-2 whole-genome sequencing of the same samples. Out of 282 samples, 123 contained the alpha variant, 78 beta and 13 delta, which were indicted using in-house primers and next-generation sequencing; the numbers of variants found were 100% identical to the reference genome. This protocol is easily adaptable for detection of emerging variants during the pandemic.

Association of SARS-CoV-2 Nucleocapsid Protein Mutations with Patient Demographic and Clinical Characteristics during the Delta and Omicron Waves.

SARS-CoV-2 genomic mutations outside the spike protein that may increase transmissibility and disease severity have not been well characterized. This study identified mutations in the nucleocapsid protein and their possible association with patient characteristics. We analyzed 695 samples from patients with confirmed COVID-19 in Saudi Arabia between 1 April 2021, and 30 April 2022. Nucleocapsid protein mutations were identified through whole genome sequencing. 𝜒2 tests and t tests assessed associations between mutations and patient characteristics. Logistic regression estimated the risk of intensive care unit (ICU) admission or death. Of the 60 mutations identified, R203K was the most common, followed by G204R, P13L, E31del, R32del, and S33del. These mutations were associated with reduced risk of ICU admission. P13L, E31del, R32del, and S33del were also associated with reduced risk of death. By contrast, D63G, R203M, and D377Y were associated with increased risk of ICU admission. Most mutations were detected in the SR-rich region, which was associated with low risk of death. The C-tail and central linker regions were associated with increased risk of ICU admission, whereas the N-arm region was associated with reduced ICU admission risk. Consequently, mutations in the N protein must be observed, as they may exacerbate viral infection and disease severity. Additional research is needed to validate the mutations' associations with clinical outcomes.

Epidemiology and Clinical Characteristics of People with Confirmed SARS-CoV-2 Infection during the Early COVID-19 Pandemic in Saudi Arabia.

This study provides epidemiologic and clinical characteristics of 492 consecutive patients diagnosed with SARS-CoV-2 infection at King Faisal Specialist Hospital and Research Centre in Saudi Arabia between March and September 2020. Data were collected from electronic case reports. The cohort was 54% male, with 20.4% aged >60 years, 19.9% aged 31−40 years, and 17% aged 41−50 years. The median incubation period was 16 days, with upper and lower 95% quartiles of 27 and 10 days, respectively. Most patients (79.2%) were symptomatic. Variables significantly different between symptomatic and asymptomatic patients were age, blood oxygen saturation percentage, hemoglobin level, lymphocyte count, neutrophil to lymphocyte (NTL) ratio, and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) level. Asymptomatic patients were mostly younger, with lower body mass index and ALT and AST levels but higher lymphocyte counts, NTL ratio, and CD4, CD8, natural killer cell, IgG, and IgM levels. Factors associated with increased risk of mortality were age (>42 years) and comorbidities, particularly diabetes mellitus and hypertension. Patients who were not given an antiviral regimen were associated with better prognosis than patients who received an antiviral regimen (HR, 0.07; 95% CI, 0.011−0.25). These findings will help clinicians and policymakers adopt best management and treatment options for SARS-CoV-2 infection.

SARS-CoV-2 genetic diversity and variants of concern in Saudi Arabia.

INTRODUCTION: In December 2019, a new severe acute respiratory syndrome coronavirus, SARS-CoV-2, emerged in China, causing coronavirus disease 2019. The present study investigated genetic profiles and variations of SARS-CoV-2 distributed in different regions of Saudi Arabia to begin to understand the pathogenesis and transmission of SARS-CoV-2 in this country and analyzed associations of these variations with host factors. METHODOLOGY: In total, 774 SARS-CoV-2 genomic sequences obtained and annotated by the Global Initiative on Sharing All Influenza Data (GISAID) were captured and analyzed. RESULTS: The most common SARS-CoV-2 clades in Saudi Arabia were GH followed by O, GR, G, and S. Statistically significant associations were detected between clades and patient outcome. Age, as a host factor, was significantly associated with many variables, including virus geographical location, clade, and patient outcome. The most common variants detected were the NSP12_P323L mutation 94.9%, followed by the D614G mutation (76%) and the NS3_Q57H mutation (71.4%). The concerned variants B.1.1.7, B.1.351, and P.1 were not detected in our population. D614G was associated with higher morbidities than the wild-type virus, including higher rates of death and hospitalization. The NS3_Q57H mutation was the only variant associated with better patient outcome than the wild type. Risk of death was highest with the NSP12_P323L mutation (OR = 1.84; 95% CI = 0.37-9.30) and lowest with the NS3_Q57H mutation (OR = 0.43; 95% CI = 0.25-0.727). CONCLUSIONS: SARS-CoV-2 has evolved uniquely and independently in Saudi Arabia. Our findings provide evidence to begin linking the evolutionary implications to host factors and their effects on the virus severity and transmission.

Development and validation of an in-house, low-cost SARS-CoV-2 detection assay.

BACKGROUND: One major challenge for detecting the virus that causes COVID-19 is commercial SARS-CoV-2 testing kit or reagent availability. To allow every laboratory or hospital access to an in-house assay, we developed a low-cost SARS-CoV-2 detection assay protocol using in-house primers and reagents/equipment on hand in most biology or diagnostic laboratories: a SYBR Green-based RT-PCR. RNA extraction has also become a major bottleneck due to limited supplies and the required labor. Thus, we validated an alternative RNA extraction protocol. METHODS: We designed and synthesized in-house primers according to SARS-CoV-2 genome sequences retrieved from GISAID database. One hundred and ninety patient samples were collected by nasopharyngeal swab, coded, and used to develop and validate the assay protocol. RNA extraction was performed using TRI reagent-based RNA protocol to inactivate the virus; thus, testing was conducted in a conventional biosafety level 2 laboratory. RESULTS: The sensitivity and specificity of the primers were evaluated using 190 patient samples previously tested for SARS-CoV-2. The positive amplicons were sequenced to confirm the results. The assay protocol was developed, and the specificity of each RT-PCR product was confirmed using melting curve analyses. Of 190 samples, the SYBR Green-based RT-PCR assay detected SARS-CoV-2 target genes in 88 samples, with no false-positive results. These findings indicate that the sensitivity of our assay was 97.7% and specificity of 100% with those of the diagnostic laboratory that tested the same samples using a Rotor-Gene PCR cycler with an Altona Diagnostics SARS-CoV-2 kit (R2 = 0.89). CONCLUSIONS: These approaches are reliable, repeatable, specific, sensitive, simple, and low-cost tools for the detection of SARS-CoV-2 in a conventional biosafety level 2 laboratory, offering alternative approaches when commercial kits are unavailable or not affordable.

Demand management and optimization of clinical laboratory services in a tertiary referral center in Saudi Arabia.

BACKGROUND: Demand for clinical laboratory services in our insti.tution has increased by 7% each year in the past 5 years, while the amount budgeted for services has remained fixed. To address the issue, we conducted a pilot study to curb inappropriate demand by implementing a minimum retest interval (time-based restrictions on the ordering certain tests) and thus reduce costs. OBJECTIVE: Explore the impact (financial and work volume) of restricting overuse of laboratory tests that add to costs but provide no additional clinical value. DESIGN: Pilot study of means to reduce costs and workload. SETTING: Clinical laboratory that provides diagnostic support to a tertiary care center specializing in transplantation and oncology. METHODS: With the engagement of clinical colleagues, we selected 13 tests characterized by high volume, high cost, or a perception of overuse that adds no clinical value. The selection was also based on established lock-out frequencies identified in a literature review. Data was captured on test numbers before and after initiating computer-based lock-outs along with the reference laboratory cost of these tests for the first 6 months of 2016 and 2017. MAIN OUTCOME MEASURES: Alterations in testing patterns (mimimum retest intervals) and frequencies for tests. RESULTS: The number of tests ordered during the 6-month period in 2017 were reduced by an average of 6.6% versus the same period for 2016, saving 2.03 million Saudi Arabian Riyals (SAR). Given a 7% annual growth in the preceding 5 years, the volume was reduced by 13% in real terms. The percentage reduction in number of tests ranged from as little as 0.2% for PT to 70.3% for an enzyme immunoassay. Savings were 1.4 million SAR in hematology and 0.36 million SAR in microbiology over the 6-month period. CONCLUSION: Minimum retest intervals using computer-based rules are effective in supporting strategies to manage demand. LIMITATIONS: This approach may not be applicable to all laboratory tests; however, the success of this pilot study would encourage more widespread use of this approach. CONFLICT OF INTEREST: None.

The Use of Next-Generation Sequencing in the Identification of a Fastidious Pathogen: A Lesson From a Clinical Setup.

Clostridium haemolyticum is the causal agent of bacillary hemoglobinuria in cattle, goat, sheep, and ruminants. In this study, we report the first recorded human-infecting C. haemolyticum strain collected from an 18-year-old woman diagnosed with acute lymphoblastic leukemia. After failure of traditional techniques, only next-generation sequencing (NGS) technology in combination with bioinformatics, phylogenetic, and pathogenomics analyses revealed that our King Faisal Specialist Hospital and Research Center (KFSHRC) bacterial isolate belongs to C. haemolyticum species. KFSHRC isolate is composed of 1 chromosome and 4 plasmids. The total genome size is estimated to be 2.7 Mbp with a low GC content of 28.02%. Comparative pathogenomics analysis showed that C. haemolyticum KFSHRC isolate is a potential virulent pathogenic bacterium as it possesses the virulence factors necessary to establish an infection, acquire essential nutrients, resist antimicrobial agents, and tolerate hostile conditions both in the human host and in its surrounding environment. These factors are included in the main chromosome in addition to novel recombination of the plasmids, and they could be the reason for the incidence of that human infection. This work demonstrated the importance of using NGS in medical microbiology for pathogen identification. It also demonstrates the importance of sequencing more microbial samples and sharing this information in public databases to facilitate the identification of pathogenic microbes with better accuracy.