Point Prevalence Survey of Antimicrobial Use and Resistance during the COVID-19 Era among Hospitals in Saudi Arabia and the Implications.

The inappropriate prescribing of antimicrobials increases antimicrobial resistance (AMR), which poses an appreciable threat to public health, increasing morbidity and mortality. Inappropriate antimicrobial prescribing includes their prescribing in patients hospitalized with COVID-19, despite limited evidence of bacterial infections or coinfections. Knowledge of current antimicrobial utilization in Saudi Arabia is currently limited. Consequently, the objective of this study was to document current antimicrobial prescribing patterns among Saudi hospitals during the COVID-19 pandemic. This study included patients with or without COVID-19 who were admitted to five hospitals in Makkah, Saudi Arabia. Data were gathered using the Global PPS methodology and analyzed using descriptive statistics. Out of 897 hospitalized patients, 518 were treated with antibiotics (57.7%), with an average of 1.9 antibiotics per patient. There were 174 culture reports collected, representing 36.5% of all cases. The most common indication for antibiotics use was community-acquired infections, accounting for 61.4% of all cases. 'Watch' antibiotics were the most commonly prescribed antibiotics, with the cephalosporins and carbapenems representing 38.7% of all antibiotics prescribed, followed by the penicillins (23.2%). Notably, Piperacillin/Tazobactam and Azithromycin were prescribed at relatively higher rates for COVID-19 patients. These findings highlight the need for continuous efforts to optimize the rational use of antibiotics through instigating appropriate antimicrobial stewardship programs in hospitals and, as a result, reduce AMR in the country.

Impact of the SARS-CoV-2 nucleocapsid 203K/204R mutations on the inflammatory immune response in COVID-19 severity.

BACKGROUND: The excessive inflammatory responses provoked by SARS-CoV-2 infection are critical factors affecting the severity and mortality of COVID-19. Previous work found that two adjacent co-occurring mutations R203K and G204R (KR) on the nucleocapsid (N) protein correlate with increased disease severity in COVID-19 patients. However, links with the host immune response remain unclear. METHODS: Here, we grouped nasopharyngeal swab samples of COVID-19 patients into two cohorts based on the presence and absence of SARS-CoV-2 nucleocapsid KR mutations. We performed nasopharyngeal transcriptome analysis of age, gender, and ethnicity-matched COVID-19 patients infected with either SARS-CoV-2 with KR mutations in the N protein (KR patients n = 39) or with the wild-type N protein (RG patients n = 39) and compared to healthy controls (n = 34). The impact of KR mutation on immune response was further characterized experimentally by transcriptomic and proteomic profiling of virus-like-particle (VLP) incubated cells. RESULTS: We observed markedly elevated expression of proinflammatory cytokines, chemokines, and interferon-stimulated (ISGs) genes in the KR patients compared to RG patients. Using nasopharyngeal transcriptome data, we found significantly higher levels of neutrophils and neutrophil-to-lymphocyte (NLR) ratio in KR patients than in the RG patients. Furthermore, transcriptomic and proteomic profiling of VLP incubated cells confirmed a similar hyper-inflammatory response mediated by the KR variant. CONCLUSIONS: Our data demonstrate an unforeseen connection between nucleocapsid KR mutations and augmented inflammatory immune response in severe COVID-19 patients. These findings provide insights into how mutations in SARS-CoV-2 modulate host immune output and pathogenesis and may contribute to more efficient therapeutics and vaccine development.

Institutional review boards in Saudi Arabia: the first survey-based report on their functions and operations.

BACKGROUND: Institutional review boards (IRBs) are formally designated to review, approve, and monitor biomedical research. They are responsible for ensuring that researchers comply with the ethical guidelines concerning human research participants. Given that IRBs might face different obstacles that cause delays in their processes or conflicts with investigators, this study aims to report the functions, roles, resources, and review process of IRBs in Saudi Arabia. METHOD: This was a cross-sectional self-reported survey conducted from March 2021 to March 2022. The survey was sent to 53 IRB chairpersons and the administration directors (or secretary) across the country through email after receiving verbal consent. The validated survey consisted of eight aspects: (a) organizational aspects, (b) membership and educational training, (c) submission arrangements and materials, (d) minutes, (e) review procedures, (f) communicating a decision, (g) continuing review, and (h) research ethics committee (REC) resources. A total of 200 points indicated optimal IRB functions. RESULTS: Twenty-six IRBs across Saudi Arabia responded to the survey. Overall, the IRBs in this study scored a total of 150/200 of the points on the self-assessment tool. Relatively newer IRBs (established less than ten years ago) conducted meetings at least once in a month, had annual funding, had more balanced gender representation, tended to score higher than older IRBs. The organizational aspect score was the lowest among all items in the survey (14.3 score difference, p-value < 0.01). The average turnaround time for expedited research from proposal submission to final decision was 7 days, while it was 20.5 days for the full committee review. CONCLUSION: Saudi IRBs performed generally well. However, there is room for focused improvement with respect to extra resources and organizational issues that require closer evaluation and guidance from the regulatory bodies.

The Association between Colorectal Cancer and Colonoscopic Conditions in Saudi Patients: A 10-Year Cross-Sectional-Retrospective Study.

Background: In Saudi Arabia, colorectal cancer (CRC) is the most common cancer in males and the third most common cancer in females. The current gold standard for colorectal cancer diagnosis is colonoscopy. Several concerns regarding the balance of ordering colonoscopy procedures for patients presenting with signs and symptoms. There are also several concerns regarding over-ordering the procedure when unnecessary. The current study aimed to evaluate the association between colorectal cancer and colonoscopic conditions in Saudi patients. Methods: A 10-year cross-sectional study was conducted at Alnoor Specialty Hospital, Makkah, over the last ten years. Colonoscopy reports of patients were evaluated to identify the colonoscopy manifestations associated with mass, polyps, and bleeding. Results: The current study evaluated 2158 cases admitted to the hospital for colonoscopic diagnosis. Results indicated that most of the patients were males (55.4%). Additionally, results showed a significant statistical association between tumor and bleeding, polyp, and hemorrhage. Moreover, it highlighted the association between polyps and bleeding, inflammation, and diverticulosis. Conclusion: CRC screening in Saudi Arabia is comprehensive; however, there are a few areas for improvement, including standardization of colorectal cancer pathology reporting to improve the health system's quality. Also, the current study identified conditions that are significantly associated with reported colon polyps and tumors, which could aid in stratifying patients selected for screening via colonoscopy.

Knowledge, attitude, and perception of community pharmacists towards antimicrobial stewardship in Saudi Arabia: A descriptive cross-sectional study.

Background: Antimicrobial resistance is of paramount concern globally. Community pharmacists (CPs) play a vital role in supporting judicious antimicrobial use in the community as they are the key healthcare providers at a public level. This study aimed to assess the knowledge, attitude, and perception of CPs towards antimicrobial stewardship at the community level in Saudi Arabia. Methods: A self-administered questionnaire was distributed to all community pharmacies in four major cities of Saudi Arabia. A simple random sampling approach was used to select pharmacies in each chain. Results: A total of 520 CPs responded to the survey with a response rate of 98.6 %. Most of the pharmacists (n = 479, 92.1 %) accepted that antimicrobial stewardship programs are essential tools to limit injudicious usage of antimicrobials at the community level. Interestingly, very few (n = 105, 21 %) agreed to recommend antibiotics for common illnesses, including upper respiratory tract infections, cold, and flu without a valid prescription. Further, we found a significant role of Saudi health authorities, e.g., Saudi food & drug authorities and the Ministry of Health, in restricting antimicrobials sale in community pharmacies without a valid prescription. Conclusion: Our study findings revealed that CPs had good knowledge about antimicrobial stewardship in Saudi Arabia. The CPs play an active role in the optimization of antimicrobial therapy and infections caused by different microbes. Strict policies by the Saudi health care authority regarding the restricted dispensing of antimicrobials are welcomed by the CPs and thus may contribute toward lowering of antimicrobial resistance burden on the patients and Saudi health care authorities.

Patterns of Thyroid Cancer Mortality and Incidence in Saudi Arabia: A 30-Year Study.

Thyroid cancer is the most prevalent endocrine cancer among the female population in the Kingdom of Saudi Arabia (KSA) and the ninth most common in the male population in Saudi Arabia. Over the past years, an increasing incidence of thyroid cancer has been reported in Saudi Arabia. However, the etiology of thyroid cancer is still not clear. Therefore, this study aimed to estimate thyroid cancer incidence and mortality trends in Saudi Arabia from 1990 to 2019. The current study utilized the Global Burden of Disease and the Institute for Health Metrics and Evaluation databases to extract prevalence data of thyroid cancer in Saudi Arabia from 1990 to 2019. Moreover, the current project utilizes Global Burden of Disease (GBD) web-based tools to visualize these data. In total, 23,846 cases (17,220 females and 6626 males) were diagnosed with thyroid cancer in Saudi Arabia from 1990 to 2019. The incidence is higher in females than in males. Over these 30 years, women's incidence steadily increased by 15-fold versus a 22-fold increase in men. Moreover, there were 2056 deaths in total caused by thyroid cancer in KSA. The mortality rate in women steadily increased by threefold in the same period. However, the increase in mortality was higher in males (sixfold). A high percentage of YLLs was observed in males, with around 24.8% ranging from 30 to 34 and 40 to 45 years. Thyroid cancer incidence rates have increased exponentially between 1990 and 2019. The expansion of the incidence of thyroid cancer in Saudi Arabia could be due to the increased development in detection and diagnosis. The current study provided evidence of the need to increase awareness and diagnosis in the male population.

Dose optimization of ß-lactams antibiotics in pediatrics and adults: A systematic review.

Background: ß-lactams remain the cornerstone of the empirical therapy to treat various bacterial infections. This systematic review aimed to analyze the data describing the dosing regimen of ß-lactams. Methods: Systematic scientific and grey literature was performed in accordance with Preferred Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. The studies were retrieved and screened on the basis of pre-defined exclusion and inclusion criteria. The cohort studies, randomized controlled trials (RCT) and case reports that reported the dosing schedule of ß-lactams are included in this study. Results: A total of 52 studies met the inclusion criteria, of which 40 were cohort studies, 2 were case reports and 10 were RCTs. The majority of the studies (34/52) studied the pharmacokinetic (PK) parameters of a drug. A total of 20 studies proposed dosing schedule in pediatrics while 32 studies proposed dosing regimen among adults. Piperacillin (12/52) and Meropenem (11/52) were the most commonly used ß-lactams used in hospitalized patients. As per available evidence, continuous infusion is considered as the most appropriate mode of administration to optimize the safety and efficacy of the treatment and improve the clinical outcomes. Conclusion: Appropriate antibiotic therapy is challenging due to pathophysiological changes among different age groups. The optimization of pharmacokinetic/pharmacodynamic parameters is useful to support alternative dosing regimens such as an increase in dosing interval, continuous infusion, and increased bolus doses.

High Seroprevalence of SARS-CoV-2 IgG and RNA among Asymptomatic Blood Donors in Makkah Region, Saudi Arabia.

The gold-standard approach for diagnosing and confirming Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) infection is reverse transcription-polymerase chain reaction (RT-PCR). This method, however, is inefficient in detecting previous or dormant viral infections. The presence of antigen-specific antibodies is the fingerprint and cardinal sign for diagnosis and determination of exposure to infectious agents including Corona virus disease-2019 (COVID-19). This cross-sectional study examined the presence of SARS-CoV-2 spike-specific immunoglobulin G (IgG) among asymptomatic blood donors in Makkah region. A total of 4368 asymptomatic blood donors were enrolled. They were screened for spike-specific IgG using ELISA and COVID-19 RNA by real-time PCR. COVID-19 IgG was detected among 2248 subjects (51.5%) while COVID-19-RNA was detected among 473 (10.8%) subjects. The IgG frequency was significantly higher among males and non-Saudi residents (p < 0.001 each) with no significant variation in IgG positivity among blood donors with different blood groups. In addition, COVID-19 RNA frequency was significantly higher among donors below 40-years old (p = 0.047, χ2 = 3.95), and non-Saudi residents (p = 0.001, χ2 = 304.5). The COVID-19 IgG levels were significantly higher among the RNA-positive donors (p = 001), and non-Saudi residents (p = 0.041), with no variations with age or blood group (p > 0.05). This study reveals a very high prevalence of COVID-19 IgG and RNA among asymptomatic blood donors in Makkah, Saudi Arabia indicating a high exposure rate of the general population to COVID-19; particularly foreign residents. It sheds light on the spread on COVID-19 among apparently healthy individuals at the beginning of the pandemic and could help in designing various control measures to minimize viral spread.

Clinical and Serological Findings of COVID-19 Participants in the Region of Makkah, Saudi Arabia.

Makkah in Saudi Arabia hosts the largest annual religious event in the world. Despite the many strict rules enacted, including Hajj cancellation, city lockdowns, and social distancing, the region has the second highest number of new COVID-19 cases in Saudi Arabia. Public health interventions that identify, isolate, and manage new cases could slow the infection rate. While RT-PCR is the current gold standard in SARS-CoV-2 identification, it yields false positive and negative results, which mandates the use of complementary serological tests. Here, we report the utility of serological assays during the acute phase of individuals with moderate and severe clinical manifestations of SARS-CoV-2 (COVID19). Fifty participants with positive RT-PCR results for SARS-CoV-2 were enrolled in this study. Following RT-PCR diagnosis, serum samples from the same participants were analyzed using in-house ELISA (IgM, IgA, and IgG) and microneutralization test (MNT) for the presence of antibodies. Of the 50 individuals analyzed, 43 (86%) showed a neutralizing antibody titer of ≥20. Univariate analysis with neutralizing antibodies as a dependent variable and the degree of disease severity and underlying medical conditions as fixed factors revealed that patients with no previous history of non-communicable diseases and moderate clinical manifestation had the strongest neutralizing antibody response "Mean: 561.11". Participants with severe symptoms and other underlying disorders, including deceased individuals, demonstrated the lowest neutralizing antibody response. Anti-spike protein antibody responses, as measured by ELISA, showed a statistically significant correlation with neutralizing antibodies. This reinforces the speculation that serological assays complement molecular testing for diagnostics; however, patients' previous medical history (anamnesis) should be considered in interpreting serological results.

Barriers to routine checkups use among Saudis from the perspective of primary care providers: A qualitative study.

OBJECTIVES: To explore the perspectives of primary care providers (PCPs) on the low use of and barriers to routine checkups among Saudi adults. METHODS: A qualitative phenomenological study design was used. Interviews were carried out between (December 2020 and February 2021) with 19 PCPs working at 5 primary healthcare centers (PHCs) operated by the Ministry of Health (MOH) in Makkah, Saudi Arabia. Descriptive statistics were performed to characterize participants, and a directed content analysis was carried out to examine major themes. RESULTS: Primary care providers identified a number of barriers that contributed to a low uptake of routine checkup among Saudis. These barriers to routine checkups were classified into 3 main themes: patient-related barriers, provider-related barriers, and healthcare system-related barriers. Lack of knowledge of patients, crowdedness at PHCs, and busy staff at PHCs were the most frequently mentioned barriers that hamper the use of routine checkups. CONCLUSION: This study presented new insight into the low use of routine checkups by obtaining the perspective of PCPs. Although results point to potential targets for interventions to increase routine checkups, additional research is recommended with a representative sample of PCPs randomly selected from the healthcare system to inform future policy and decision making related to improving use of routine care available through the Saudi Healthcare System.

The Role of Genetics, Epigenetics, and the Environment in ASD: A Mini Review.

According to recent findings, variances in autism spectrum disorder (ASD) risk factors might be determined by several factors, including molecular genetic variants. Accumulated evidence has also revealed the important role of biological and chemical pathways in ASD aetiology. In this paper, we assess several reviews with regard to their quality of evidence and provide a brief outline of the presumed mechanisms of the genetic, epigenetic, and environmental risk factors of ASD. We also review some of the critical literature, which supports the basis of each factor in the underlying and specific risk patterns of ASD. Finally, we consider some of the implications of recent research regarding potential molecular targets for future investigations.

Epidemiological Features of COVID-19 in Makkah City: A Retrospective Data Analysis.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is continuing to circulate and change, affecting billions of people worldwide and leading to increased mortality rates, especially in the Holy City of Makkah, Kingdom of Saudi Arabia (KSA). This study was aimed at investigating the epidemiological features of COVID-19 in Makkah City, KSA. METHODS: A retrospective analysis was conducted to investigate the prevalence of COVID-19 and the association between the severity and mortality of COVID-19 with demographic factors and comorbidities. RESULTS: Among 4,742 COVID-19 patients, the incidence rate observed in males was 66.7%, and 69.7% were from Al-Noor Specialized Hospital. The highest incidence rate (25.2%) was found in the age group > 60 years old, followed by the age group 51-60 years (21.8%). Furthermore, the highest frequency was observed in patients from Saudi Arabia (36.8%), followed by patients from Myanmar (14.7%) and Bangladesh (9.4%). The overall frequency of COVID-19 severity and death was 20.3% and 11.6%, respectively. Body mass index analysis showed that 1% of the patients were underweight, while 9.2% were overweight and 4.4% were obese. In addition, 9.6% had diabetes, 6.9% had hypertension, 1.1% had heart disease, and 2.2% had other chronic diseases. CONCLUSION: The study concluded that the overall percentages of severe COVID-19 (intensive care unit) cases and deaths in Makkah City, KSA, were 20.3% and 11.6%, respectively, and there was a higher incidence in male patients. The severity of infection was shown to have a strong significant correlation with different chronic diseases, nationality, body mass index, death rate (mortality), heart disease, and length of hospital stay (P < 0.05). Despite these findings, more studies are needed to explain the underlying mechanisms that influence the overall health status of patients with specific characteristics and comorbidities.

A Systematic Review on Clinical Safety and Efficacy of Vancomycin Loading Dose in Critically Ill Patients.

BACKGROUND: The clinical significance of utilizing a vancomycin loading dose in critically ill patients remains unclear. OBJECTIVE: The main aim of this systematic review is to evaluate the clinical safety and efficacy of the vancomycin loading dose in critically ill patients. METHODS: We performed a systematic review using PRISMA guidelines. PubMed, the Web of Science, MEDLINE, Scopus, Google Scholar, the Saudi Digital Library and other databases were searched. Studies that reported clinical outcomes among patients receiving the vancomycin LD were considered eligible. Data for this study were collected using PubMed, the Web of Science, MEDLINE, Scopus, Google Scholar and the Saudi Digital Library using the following terms: "vancomycin", "safety", "efficacy" and "loading dose" combined with the Boolean operator "AND" or "OR". RESULTS: A total of 17 articles, including 2 RCTs, 11 retrospective cohorts and 4 other studies, met the inclusion/exclusion criteria out of a total 1189 studies. Patients had different clinical characteristics representing a heterogenous group, including patients in critical condition, with renal impairment, sepsis, MRSA infection and hospitalized patients for hemodialysis or in the emergency department. CONCLUSIONS: The study shows that the target therapeutic level is achieved more easily among patients receiving a weight-based LD as compared to patients received the usual dose without an increased risk of new-onset adverse drug reactions.

SARS-CoV-2 genomes from Saudi Arabia implicate nucleocapsid mutations in host response and increased viral load.

Monitoring SARS-CoV-2 spread and evolution through genome sequencing is essential in handling the COVID-19 pandemic. Here, we sequenced 892 SARS-CoV-2 genomes collected from patients in Saudi Arabia from March to August 2020. We show that two consecutive mutations (R203K/G204R) in the nucleocapsid (N) protein are associated with higher viral loads in COVID-19 patients. Our comparative biochemical analysis reveals that the mutant N protein displays enhanced viral RNA binding and differential interaction with key host proteins. We found increased interaction of GSK3A kinase simultaneously with hyper-phosphorylation of the adjacent serine site (S206) in the mutant N protein. Furthermore, the host cell transcriptome analysis suggests that the mutant N protein produces dysregulated interferon response genes. Here, we provide crucial information in linking the R203K/G204R mutations in the N protein to modulations of host-virus interactions and underline the potential of the nucleocapsid protein as a drug target during infection.

Emphasizing the link between blood types in multi-ethnic disparities and COVID-19 infection in Makkah, Saudi Arabia.

OBJECTIVES: To analyze the impact and distribution of blood groups in different ethnicities and the extent of susceptibility to infection with COVID-19 in Makkah, Saudi Arabia. METHODS: A retrospective study was performed on 4,609 COVID-19 patients from five ethnic groups to assess the impact and distribution of different blood types and susceptibility to COVID-19 infection. The study was carried out between November 2020 and June 2021 in the College of Medicine, Umm Al-Qura University in collaboration with the General Directorate of Health Affairs, Makkah, Saudi Arabia. RESULTS: Blood group (A, B, and O) distributions in 2,617 COVID-19 patients with local control populations was done. Our study found that in both Saudi and non-Saudi populations, blood groups O and A were associated with higher infection rates, whereas blood group AB was associated with lower infection rates (p=0.0001). COVID-19 seems to be associated with blood groups A, B, and AB (RR=3.23, 95% CI=2.702-3.821, p=0.0001). COVID-19 risk was lower in people with O blood group (RR=0.783, 95% CI=0.733-0.836, p=0.0001). South Asians had higher odds of COVID-19 infection when compared to Saudi cases and other ethnic groups (OR=1.12, 95 % CI: 1.074-1.24, p=0.04). CONCLUSION: We emphasize that COVID-19 infection is not proportional among ethnically related blood groups. Notably, RhD-negative protect against COVID-19, whereas A and O blood types are more susceptible. Thus, when assessing COVID-19 prognosis and vaccination priority, blood groups A and O are critical.

Dose Optimization of Colistin: A Systematic Review.

Colistin is considered a last treatment option for multi-drug and extensively resistant Gram-negative infections. We aimed to assess the available data on the dosing strategy of colistin. A systematic review was performed to identify all published studies on the dose optimization of colistin. Grey literature and electronic databases were searched. Data were collected in a specified form and the quality of the included articles was then assessed using the Newcastle-Ottawa scale for cohort studies, the Cochrane bias tool for randomized clinical trials (RCT), and the Joanna Briggs Institute (JBI) critical checklist for case reports. A total of 19 studies were included, of which 16 were cohort studies, one was a RCT, and two were case reports. A total of 18 studies proposed a dosing regimen for adults, while only one study proposed a dosing schedule for pediatric populations. As per the available evidence, a loading dose of 9 million international units (MIU) of colistin followed by a maintenance dose of 4.5 MIU every 12 h was considered the most appropriate dosing strategy to optimize the safety and efficacy of treatment and improve clinical outcomes. This review supports the administration of a loading dose followed by a maintenance dose of colistin in severe and life-threatening multi-drug Gram-negative bacterial infections.

The epidemiology and incidence of dengue in Makkah, Saudi Arabia, during 2017-2019.

OBJECTIVES: To study the epidemiology of dengue incidence and understand the dynamics of dengue transmission in Makkah, Kingdom of Saudi Arabia (KSA), between 2017-2019. METHODS: This is a cross-sectional study. Health and demographic data was obtained for all confirmed dengue cases in Makkah, KSA, in the years 2017-2019 from the Vector-Borne and Zoonotic Diseases Administration (VBZDA) in Makkah and the Makkah Regional Laboratory, KSA. In addition, entomological data about Aedes density was obtained from the VBZDA. Descriptive epidemiological methods were used to determine the occurrence and distribution of dengue cases. RESULTS: Laboratory-confirmed dengue cases were higher in 2019 as compared to 2017 and 2018, suggesting an outbreak of dengue in Makkah, KSA, in 2019. The incidence of confirmed dengue cases was 204 in 2017, 163 in 2018 and 748 in 2019. Dengue mostly affected people in the 25-44 age group, accounting for approximately half of the annual dengue cases each year. Men were at a higher dengue incidence risk when compared to women, and Saudi women had a higher risk rate for dengue cases when compared to non-Saudi women in all 3 years studied. There was no dengue related death in these 3 years. CONCLUSION: The dengue incidence increased in Makkah, KSA, in 2019 as compared to the previous 2 years, owing to heavy rainfall in 2019. Post-rainfall Vector control efforts may help contain the disease in Makkah, KSA.

Simultaneous detection and mutation surveillance of SARS-CoV-2 and multiple respiratory viruses by rapid field-deployable sequencing.

BACKGROUND: Strategies for monitoring the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are crucial for combating the pandemic. Detection and mutation surveillance of SARS-CoV-2 and other respiratory viruses require separate and complex workflows that rely on highly specialized facilities, personnel, and reagents. To date, no method can rapidly diagnose multiple viral infections and determine variants in a high-throughput manner. METHODS: We describe a method for multiplex isothermal amplification-based sequencing and real-time analysis of multiple viral genomes, termed nanopore sequencing of isothermal rapid viral amplification for near real-time analysis (NIRVANA). It can simultaneously detect SARS-CoV-2, influenza A, human adenovirus, and human coronavirus and monitor mutations for up to 96 samples in real time. FINDINGS: NIRVANA showed high sensitivity and specificity for SARS-CoV-2 in 70 clinical samples with a detection limit of 20 viral RNA copies per µL of extracted nucleic acid. It also detected the influenza A co-infection in two samples. The variant analysis results of SARS-CoV-2-positive samples mirror the epidemiology of coronavirus disease 2019 (COVID-19). Additionally, NIRVANA could simultaneously detect SARS-CoV-2 and pepper mild mottle virus (PMMoV) (an omnipresent virus and water-quality indicator) in municipal wastewater samples. CONCLUSIONS: NIRVANA provides high-confidence detection of both SARS-CoV-2 and other respiratory viruses and mutation surveillance of SARS-CoV-2 on the fly. We expect it to offer a promising solution for rapid field-deployable detection and mutational surveillance of pandemic viruses. FUNDING: M.L. is supported by KAUST Office of Sponsored Research (BAS/1/1080-01). This work is supported by KAUST Competitive Research Grant (URF/1/3412-01-01; M.L. and J.C.I.B.) and Universidad Catolica San Antonio de Murcia (J.C.I.B.). A.M.H. is supported by Saudi Ministry of Education (project 436).

Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes.

One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT-PCR kits in the market, their high cost and unavailability due to airport closures and shipment restriction became a major bottleneck that had driven the desire to produce the key components of such kits locally. Here, we provide a simple, economical, and powerful one-step qRT-PCR kit based on patent-free, specifically tailored versions of Moloney murine leukemia virus reverse transcriptase and Thermus aquaticus DNA polymerase and termed R3T (Rapid Research Response Team) one-step qRT-PCR. We also demonstrate the robustness of our enzyme production strategies and provide the optimal reaction conditions for their efficient augmentation in a one-step approach. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity to that of the Invitrogen SuperScript III Platinum One-step qRT-PCR and TaqPath one-step RT-qPCR kits. Overall, our kit has shown robust performance in both laboratory settings and the Saudi Ministry of Health-approved testing facility.

A Robust, Safe, and Scalable Magnetic Nanoparticle Workflow for RNA Extraction of Pathogens from Clinical and Wastewater Samples.

Molecular diagnosis and surveillance of pathogens such as SARS-CoV-2 depend on nucleic acid isolation. Pandemics at the scale of COVID-19 can cause a global shortage of proprietary commercial reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. An open-source method, magnetic-nanoparticle-aided viral RNA isolation from contagious samples (MAVRICS), built upon readily available reagents, and easily assembled in any basically equipped laboratory, is thus developed. The performance of MAVRICS is evaluated using validated pathogen detection assays and real-world and contrived samples. Unlike conventional methods, MAVRICS works directly in samples inactivated in phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities. MAVRICS allows wastewater biomass immobilized on membranes to be directly inactivated and lysed in TRIzol followed by RNA extraction by magnetic nanoparticles, thereby greatly reducing biohazard risk and simplifying processing procedures. Using 39 COVID-19 patient samples and two wastewater samples, it is shown that MAVRICS rivals commercial kits in detection of SARS-CoV-2, influenza viruses, and respiratory syncytial virus. Therefore, MAVRICS is safe, fast, and scalable. It is field-deployable with minimal equipment requirements and could become an enabling technology for widespread testing and wastewater monitoring of diverse pathogens.