Viral Load Samples Research Articles

Design, development, and validation of a strand-specific RT-qPCR assay to differentiate replicating versus nonreplicating Rabies virus.

The Rabies virus, a single-strand RNA virus with a negative-sense polarity, is responsible for causing encephalitis and is a zoonotic disease. If not promptly treated after infection, it has a close to 100% fatality rate. Similar to other negative-sense polarity single-strand RNA viruses, the Rabies virus requires the creation of a positive-strand RNA intermediate for replication. One approach to identify this replication activity is to detect the complementary strand of the viral RNA genome in suspected infected cells or tissues. The reported Rabies virus RT-qPCR detection methods are designed to detect total viral load in samples without distinguishing between positive- and negative-strand for RNA viruses. As such, in this study, a sensitive Taqman-based strand-specific RT-qPCR assay has been developed to quantitatively detect both the positive- and negative-strand of the Rabies virus. This method demonstrates good reproducibility across a wide dynamic range and exhibits linearity of 8 logs with a lower limit of detection of 103 copies/μL for the positive-strand and 9 logs with a lower limit of detection of 102 copies/μL for the negative-strand. Notably, it can accurately detect a specific viral RNA strand even in the presence of high levels of the opposite strand, confirming the method’s specificity. In summary, a reliable strand-specific RT-qPCR assay has been developed and validated to differentiate replicating from non-replicating Rabies virus.

B-195 Accelerating Molecular Diagnostics: Development of a novel Rapid, Integrated PCR System for Acute Respiratory Infections Detection

Abstract Background The COVID-19 pandemic and acute respiratory infections have underscored the necessity for high-throughput and timely molecular diagnostics. Existing molecular diagnostic methods are labor-intensive, a challenge particularly evident in large-scale testing. This study aims to streamline the nucleic acid testing process by integrating extraction and detection in PCR testing, thereby optimizing efficiency. Methods We developed 'EXP-160R', an integrated PCR system capable of rapid thermal cycling. This innovation employs advanced temperature control technology to markedly reduce heating, cooling, and annealing-extension times. The PCR reagent-kit (Zybio Inc.) was optimized to complement the instrument's capabilities, aiming for pathogen nucleic acid detection in acute respiratory infections within 25 minutes. The precision of in-tube temperature during rapid amplification was validated by using temperature-sensitive molecular probes in system development stage. Performance evaluation used the WHO International Standard for SARS-CoV-2 RNA to assess sensitivity and detection limits. We also analyzed 500 clinical samples from multiple Chinese hospitals, including 295 COVID-19 positives and 205 negatives, comparing the EXP-160R system's performance with an NMPA-approved PCR kit. Results The innovative system significantly improved PCR cycle times, enabling detection of 16 samples in 22 minutes and a throughput of 2400 tests per day, without compromising sensitivity or specificity. Validation confirmed a detection limit of 200 copies/mL, and the system achieved a 100% detection rate for medium and high viral load samples, with a coefficient of variation (CV) for Ct values ≤ 5%. Clinical trial comparisons with clinical standard diagnostics revealed a sensitivity of 98.05%, specificity of 100%, accuracy of 99.2%, and a Kappa coefficient of 0.984. When compared to the established PCR kit, our system showed comparable efficacy, with a sensitivity of 98.50%, specificity of 98.67%, accuracy of 98.6%, and a Kappa of 0.971. Conclusions The rapid PCR system represents a significant advancement in molecular diagnostics, offering rapid, high-throughput, and accurate detection of SARS-CoV-2. This system is particularly beneficial in urgent testing scenarios, such as airports and fever clinics. It addresses current pandemic needs and sets a new benchmark for future respiratory and infectious disease diagnostics, enhancing both efficiency and user experience.

Full genomic sequence characterization of the chikungunya virus from an imported case with serum viral concentration below culturable level

Chikungunya virus (CHIKV) infection, responsible for Chikungunya fever and occasionally severe symptoms, has emerged as an increasing global health concern following several large-scale outbreaks from Africa, Asia, Europe, and America. Over the past two decades, South and Southeast Asia regions have gradually become hot spots for outbreaks involving multiple CHIKV lineages. In China, most CHIKV infections are imported, making it crucial to trace the origins and transmission routes for effective prevention and control. In January 2024, a case of imported chikungunya fever was confirmed in Guangzhou City, Guangdong Province, China. However, the serum CHIKV viral concentration was too low for cultivation (RT-PCR detection, Ct = 32.62). Despite this, we successfully obtained the viral genome sequence directly from the whole blood sample using an optimized metatranscriptomic sequencing strategy, achieving a full-length viral genome with an average depth of 54.3X. Further analysis confirmed that the CHIKV virus belonged to the Asian lineage, traced to Timor-Leste, where an endemic CHIKV outbreak had been reported in January 2024, consistent with the patient’s travel history. Finally, we analyzed genetic evolutionary trends and amino acid site variations. This study highlights the identification of a CHIKV infection origin using direct whole-blood metatranscriptomic sequencing, a valuable method for rapidly sequencing low viral-load samples.

Evaluation of Xpert point-of-care assays for detection of HIV infection in persons using long-acting cabotegravir for pre-exposure prophylaxis.

HIV RNA assays can detect HIV infections earlier than HIV rapid tests or Ag/Ab tests in persons using CAB-LA PrEP. Earlier HIV diagnosis could allow for earlier treatment initiation and reduced risk of INSTI resistance. POC tests may help detect HIV infection before CAB-LA administration and may be more accessible than laboratory-based assays in some settings. In this study, the POC Xpert VL-XC assay detected HIV RNA in most samples from individuals who received CAB-LA PrEP and had delayed detection of HIV infection with HIV rapid tests and an Ag/Ab test. The performance of this assay was similar to laboratory-based HIV RNA assays in this cohort. The POC Xpert Qual-XC assay detects both HIV RNA and DNA, with a higher viral load cutoff for RNA detection. This assay was negative for most lower viral load samples and did not offer an advantage for HIV screening in persons using CAB-LA PrEP.

Evaluation of Abbott ID-NOW Rapid Assay with Real-Time Reverse Transcription PCR Assay for Detection of SARS-CoV-2

Rapid nucleic acid assays have been approved by FDA for managing the COVID-19 pandemic, however its analytical efficiency has not been thoroughly validated. This study evaluates the detection and identification of COVID-19 virus using Abbott ID-Now to rapidly identify cases and intervention practices in comparison to nucleic acid detection. Nasopharyngeal Swabs collected from 611 participants were tested for Abbott ID-NOW and LabGun COVID-19 ExoFast RT-PCR Kit as per manufacturer’s protocol. The results from the ID NOW™ COVID-19 assay were evaluated by comparing results with the standard RT-PCR, which served as a standard reference. The infection burden of SARS-CoV-2 in the population of UAE was 11.62%. Compared to detection using real time-based platforms, the sensitivity, specificity, positive and negative predictive values of the ID-Now were 84.51%, 99.81%, 98.36% and 98.00% respectively for COVID-19. A stratified analysis was also carried out using cycle threshold (Ct) values categorizing as Ct>33 as with low viral loads while those with Ct<33 as high. This demonstrated statistically significant (P<0.0001) decrease in sensitivity in samples (97.87% in low Ct value samples versus 58.33% in high Ct value samples). Even though the sensitivity for Abbott ID NOW™ in this study was lower, the specificity, positive predictive values and negative predictive values were significant in low viral load samples. It is easy to use and interpret, giving early information to support clinical decision-making ID-NOW could be possibly used as a point-of-care test after evaluation in epidemic and endemic settings.

Determining the rate and reasons for specimen rejection among specimens referred for HIV viral load determination through referral linkage to Debre Tabor Comprehensive Specialized Hospital, north-central, Ethiopia,2023: Retrospective study

BackgroundThe preanalytical phase encompasses the time between the clinician's test order to the sample being ready for analysis. Of all errors during the laboratory diagnostic process,70 % appeared in the pre-analytical phase. In clinical laboratories, it is crucial to ensure proper specimen collection and handling, which is essential to guarantee quality assessment, monitoring process standardization, improving performance, and ensuring patient safety. Despite this importance, no study has been conducted in the study area to investigate the rate and reasons for human immunodeficiency virus viral load sample rejection. ObjectiveTo determine the rate of human immunodeficiency virus viral load sample rejection (number of preanalytical errors) documented during the preanalytical phase and articulate possible causes for specimen rejection. Material and methodsA retrospective study was conducted at Debre Tabor Comprehensive Specialized Hospital from January 1st to January 31, 2023. During the study period, 5950 samples were extracted from the human immunodeficiency virus viral load laboratory sample tracking log books, which were sent to the hospital for viral load testing between August 2021 to November 2022. The collected data were cleaned and entered into EPI data version 4.6 before transferred it to STATA version 14.0 for analysis. Descriptive statistics such as frequencies, percentages, and cross-tabulations were used to summarize the findings. ResultsThe study found that improper sample handling was common during the preanalytical phase. According to the current study, 3.6 % of the sample was rejected at pre analytical stage. The most common reasons for specimen rejection were using inappropriate containers (64.0 %) uncentrifuged specimens (20.4 %); hemolyzed specimens (7.0 %); insufficient specimen volume (6.2 %); clotted specimens (1.9 %); and specimen labeling problems (0.5 %). ConclusionThis study found that the most common preanalytical error was using an inappropriate sample collection container, followed by uncentrifuged samples, Therefore, it is recommended that mentorship programs be developed to educate staff on the preanalytical phase of laboratory testing, specifically on sample collection, storage, and transportation for HIV viral load testing. Additionally, the quality management system of laboratory processes should be strengthened to ensure accuracy and minimize errors.

Evaluation of LN34 Pan-Lyssavirus RT-qPCR assay for rabies diagnosis in Brazil

Rabies, a fatal zoonotic viral disease affecting mammals, including humans, remains a significant global health concern, particularly in low-income countries. The disease, primarily transmitted through infected animal saliva, prompts urgent diagnosis for timely post-exposure prophylaxis (PEP). The gold standard diagnostic test, direct fluorescent antibody test (dFAT), while sensitive, suffers from limitations such as subjective interpretation and high costs. As a confirmatory technique, the LN34 Pan-Lyssavirus RT-qPCR assay has emerged as a promising tool for universal Lyssavirus detection. This study evaluated its performance using 130 rabies virus isolates representing eleven Brazilian variants and 303 clinical samples from surveillance operations. The LN34 assay demonstrated 100% sensitivity and 98% specificity compared to dFAT. Additionally, it detected all samples, including those missed by dFAT, indicating superior sensitivity. The assay's specificity was confirmed through Sanger nucleotide sequencing, with only a minimal false-positive rate. Comparative analysis revealed higher accuracy and concordance with dFAT than traditional rabies tissue culture infection tests (RTCIT). False-negative RTCIT results were attributed to low viral load or suboptimal sampling. These findings underscore the LN34 assay's utility as a confirmatory technique, enhancing rabies surveillance and control in Brazil. Its widespread adoption could significantly improve diagnostic sensitivity, crucial for effective PEP and public health interventions.

Enhanced detection of African swine fever virus in samples with low viral load using digital PCR technology

Detection of low viral load samples has long been a challenge for African swine fever (ASF) prevention and control. This study aimed to compare the detection efficacy of droplet digital PCR(ddPCR) and quantitative PCR(qPCR) for African swine fever virus (ASFV) at different viral loads, with a focus on assessing the accuracy of ddPCR in detecting low viral load samples. The results revealed that ddPCR had a detection limit of 1.97 (95% CI 1.48 – 4.12) copies/reaction and was 18.99 times more sensitive than qPCR (detection limit: 37.42, 95% CI 29.56 – 69.87 copies/reaction). In the quantification of high, medium, and low viral load samples, ddPCR showed superior stability with lower intra- (2.06% – 7.58%) and inter-assay (3.83% – 7.50%) coefficients of variation than those of qPCR (intra-assay: 8.08%–29.86%; inter-assay: 9.27%–34.58%). Bland-Altman analysis indicated acceptable consistency between ddPCR and qPCR for high and medium viral load samples; however, discrepancies were observed for low viral load samples, where two samples (2/24, 8.33%) exhibited deviations beyond the acceptable range (−46.18 copies/reaction). Moreover, ddPCR demonstrated better performance in detecting ASFV in clinical samples from asymptomatic pigs and environmental samples, with qPCR showing false negative rates of 7.69% (2/26) and 27.27% (12/44), respectively. McNemar analysis revealed significant differences between the two methods (P = 0.000) for samples with a viral load <100 copies/reaction. The results of this study demonstrate that ddPCR has better detection limits and adaptability than qPCR, allowing for a more accurate detection of ASFV in early-stage infections and low-concentration environmental samples. These findings highlight the potential of ddPCR in the prevention and control of ASF.

Characteristics associated with viral suppression among HIV-infected children aged 0-14 years in Mozambique, 2019.

The human immunodeficiency virus (HIV) is a global public health problem, disproportionally affecting sub-Saharan African countries including Mozambique. In 2019, of 150,000 estimated HIV-infected children in Mozambique, only 95,080 were on antiretroviral treatment and 73% virally suppressed. The objective of this study was to determine the characteristics associated with viral suppression in children. A cross-sectional study was carried out using records of viral load samples from children aged 0 to 14 years old who underwent viral load tests in 2019 in Mozambique. Secondary analyses were conducted on data obtained from Data Intensive Systems and Applications (DISA) of children enrolled in health facilities who had viral load tests registered. Viral suppression was defined as the presence of less than 1,000 copies/ml of blood. Multivariate logistic regression analysis was used to evaluate the characteristics associated with viral suppression. Of the 33,559 viral load sample records analyzed, 53% (17,794/33,559) were female. The average patient age was 8 (sd ± 4) years old. About 44% (14,888/33,559) of the children had a suppressed viral load, with 55% (8,258/14,888) being female and 16% (2,319/14,888) belonging to the 1-4 years old age group. Characteristics associated with viral suppression were the age groups of 5-9 years [AOR = 1.73; 95% CI 1.34-2.23; p<0.001] and 10-14 years old [AOR = 1.92; 95% CI 1.50-2.48; p<0.001] versus < 1 year. Other factors such as living in Maputo City [AOR = 1.61; 95% CI 1.26-2.05; p <0.001] versus Tete Province were also associated with viral suppression. Factors such as being male [AOR = 0.83; 95% CI 0.80-0.87; p <0.001)], living in the provinces of Niassa [AOR = 0.75; 95% CI 0.56-0.99; p <0.003], Cabo Delgado [AOR = 0.77; 95% CI 0.60-0.99; p <0.045] and Zambezia [AOR = 0.72 (95% CI: 0.56-0.92, p<0.008)] versus Tete Province, or being on ART for 2-5 years [AOR = 0.72 (95% CI: 0.61-0.85, p<0.001)] versus 11-14 years were associated with not being virally suppressed. More than half of children did not achieve viral suppression. The odds of viral suppression were highest among children aged 5-14 years and among children living in Maputo city. Further research is needed to better understand the challenges in achieving viral suppression in children.

Characterizing a visual lateral flow device for rapid SARS-CoV-2 virus protein detection: pre-clinical and system assessment.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections have affected more than 769 million individuals worldwide over the last few years. Although the pandemic is transitioning into an endemic, the COVID-19 outbreak is still a global concern. A rapid screening platform is needed for effective preventive and control measures. Herein, a visual rapid lateral flow platform for SARS-CoV-2 nucleocapsid protein detection is developed. Under optimal conditions, the system demonstrated good detection sensitivity and selectivity against tested respiratory viruses. The system provides direct visual detection with a limit of 0.7 ng of the nucleocapsid protein per mL of a sample (0.7 ng mL-1) within 15 minutes. Further, a correlation between direct visual detection and semi-quantitative analysis using a reader showed a similar detection limit (R2 = 0.9571). The repeatability and reproducibility studies highlighted the potential of the system for the rapid screening of SARS-CoV-2 infection, with variations within 5% and 10% at high and low protein concentrations, respectively. Subsequent pre-clinical validation to correlate the performance with the standard molecular approach (RT-PCR) using 170 nasopharyngeal swabs demonstrated 98% estimated sensitivity (95% CI, 89.35-99.95%) and 100% specificity (95% CI, 96.38-100%). The positive and negative predictive values were reported to be 100% and 99%, respectively, with an accuracy of 99.3%. With high viral load samples (Ct value ≤25, n = 47), the system demonstrated 100% detection sensitivity and specificity. The proposed technique provides a valuable platform for potential use in rapid screening, particularly during pandemics, where diagnostic capacity and mass screening are crucial.

Targeted solutions to increase dolutegravir coverage, viral load testing coverage, and viral suppression among children living with HIV in Togo: An analysis of routine facility data.

According to UNAIDS, Togo halved AIDS-related deaths among children ages 0-14 from 2010 to 2020. However, available data show low dolutegravir (DTG)-containing antiretroviral therapy (ART) coverage and low viral load suppression (VLS) among children living with HIV (CLHIV). We analyzed routine facility data before and after implementation of root-cause-based solutions for improving DTG coverage, viral load (VL) testing coverage, and VLS among CLHIV. We analyzed routine data for CLHIV ≤14 years from October 2019 through September 2022. We assessed proportion of CLHIV on ART receiving DTG, VL testing coverage (CLHIV on ART with documented VL test result), and VLS (CLHIV with documented VL test result of <1,000 copies among those with test result). From October 2019 to September 2020, 52% were on a DTG-containing regimen, 48% had documented VL test results, and 64% had VLS. Site-level teams conducted a root-cause analysis and designed corresponding solutions implemented beginning October 2020: line listing and contacting eligible CLHIV to start/transition to DTG-containing regimen and collect VL samples; ART adherence support; monthly DTG stock monitoring; tracking pending VL test results through laboratory focal persons; documenting VL test results; and informing caregivers within one week if CLHIV not virally suppressed. Granular data were used to prioritize technical assistance to sites with lowest DTG coverage, VL testing coverage, and VLS. From baseline (October 2019-September 2020) to endline (October 2021-September 2022), increases were observed for DTG coverage (52% to 71%), VL testing coverage (48% to 90%), and VLS (64% to 82%). Age-disaggregated data showed positive trends. Root-cause-based solutions and granular data use increased DTG coverage, resulting in increased VL testing and VLS among CLHIV. These interventions should be scaled and become the national standard of care.

HIV Type-1 Genotyping in the Context of the COVID-19 Pandemic: A Critical Tool for Identifying Antiretroviral Drug Resistance and Optimizing Treatment Strategies

Abstract Introduction/Objective The COVID-19 pandemic strained the healthcare system and the WHO recently reported increased rates of antibiotic resistance. Meanwhile, antiretroviral resistance can also lead to treatment failure and disease progression, especially in HIV patients. Here, we investigated the rates of HIV drug resistance (HIVDR) in our HIV population between pre and post-COVID-19 eras. Methods/Case Report The post-COVID-19 era began on 03/11/2020. Patients positive for HIV-1 between 01/2019- 08/2021 were included (total: 94 patients, 46 pre-COVID-19; 48 post-COVID-19). Patients’ charts were reviewed for demographics, laboratory results (including HIV-1 sequencing/genotyping (e.g., Genosure)), clinical presentation and management. Student t-test and Fisher exact test were calculated. Results (if a Case Study enter NA) Most patients were male (70%) and had a mean age of 49 years. 70% of patients had sufficient viral loads in samples reflexed to HIV genotyping. Post-COVID-19 patients harbored lower viral loads (1.8x105 vs. 3.4x105 copies/mL, p=0.05), CD4 counts (174 vs. 196 cells/mcL, p=0.7), CD8 counts (555 vs. 800 cells/mcL, p=0.04), and higher CD4/CD8 ratio (0.312 vs. 0.266, p=0.57). Overall, 20% of patients had HIVDR. Following COVID-19 pandemic, there was a 9% increase in HIVDR, predominantly seen in non-nucleotide reverse transcriptase inhibitors (NNRTI), although insignificant. Specifically, high resistance was documented for drugs Efavirenz (16%), Nevirapine (16%), and Rilpivirine (13%). We observed an increased prevalence of G190A, K101, K103, and E138 mutations conferring resistance to NNRTIs. Changes to therapy were observed in 47.8% (42/88) of total cases, with an increase of 12.5% in patients from post-COVID-19 era. Additionally, changes to therapy were observed more in patients with infectious diseases consultation (16% increase). Conclusion Increasing HIVDR was seen in our patient population post-COVID-19. Continued surveillance is critical to managing HIVDR and improving patient outcomes. The clinical utility of sequencing tests can outweigh the cost if results are appropriately acted upon which may involve the participation of other clinical teams and the antimicrobial stewardship committee.

Digital CRISPR/Cas13a-RPA based assay for rapid and sensitive detection of JC virus in renal transplant patients

JC viruses (JCVs), members of the Polyomaviruses family, are a type of circular double-stranded DNA (dsDNA) viruses and can lead to opportunistic infections. Infection of JCVs in kidney transplant patients can lead to polyomavirus-associated nephropathy. Thus, early detection and quantification of JC virus in urine or blood will effectively improve the survival rate of kidney transplant patients and reduce the mortality of patients. At present, PCR has been used as a routine method for JC virus detection in clinical practice. However, PCR cannot achieve absolute quantitative detection of viral nucleic acid. rendering it unsuitable for certain clinical applications with specific requirements. Herein, we proposed a JC virus nucleic acid testing platform, named MdCaR, which couples a microfluidic droplet device with CRISPR-Cas13a and recombinase polymerase amplification. MdCaR can quantitatively detect JC virus through digital droplet. The detection limit achieves 1 copy/mL, and the assay takes only 40 min. Twelve renal transplant patients and 20 control subjects’ specimens were analyzed. MdCaR conveyed a good performance in testing these samples, especially for detecting the low viral load samples identified as negative by qPCR. Taken altogether, MdCaR could be regarded as a promising, rapid JCV detection strategy and widely applied for diagnosis for JCV infection.

Evaluation of Rapid Molecular Viral Load Monitoring with ABBOTT m-PIMATM HIV-1/2 VL

World Health Organization recommends rapid results of Human Immunodeficiency Virus (HIV) viral load by using Point of Care (POC) testing, delivering faster decision-making to prevent transmission and suppress drug resistance and greater likelihood of starting therapy if the clinic provides care and treatment services. The m-PIMATM HIV-1/2VL cartridge is an RT-PCR POC test that detects HIV-1 and HIV-2 RNA viral load, recognizing the conserved-part 5'-UTR of the HIV genome. The standard, Roche Cobas®4800 system HIV-1, targets gag and LTR regions. This study aimed to determine the correlation, sensitivity, and specificity of Abbott m-PIMATM HIV-1/2VL compared to Roche Cobas®4800 system HIV-1. A cross-sectional study at Dharmais Hospital Laboratory. EDTA plasma obtained from 200 HIV patients, consisted of 80 high-viral-load samples (&gt;1000 copies/mL), 80 low-viral-load samples (&lt;1000 copies/mL), and 40 undetectable-viral-load samples (&lt;14.2 copies/mL). Kappa Cohen was used to measure the categorical correlation. Sensitivity and specificity were calculated with diagnostic test. Dharmais Ethic Committee released ethical clearance No.0132/KEPK/IX/2020. The categorical correlation of Abbott m-PIMATM HIV-1/2 VL in determining the group of high or low viral load samples was 0.948 (very strong correlation). The categorical correlation deciding whether the HIV viral load sample was high, low, or undetectable was 0.714 (strong correlation). The categorical sensitivity of Abbott m-PIMATM HIV-1/2 VL compared to Roche Cobas®4800 system HIV-1 in categorizing HIV viral load was 97.5%, specificity of 97.5%. Abbott m-PIMATM HIV-1/2 VL performance in determining HIV RNA viral load group samples has a strong correlation compared to Roche Cobas®4800 system HIV-1. A larger study is required.

Analysis of blood and nasal epithelial transcriptomes to identify mechanisms associated with control of SARS-CoV-2 viral load in the upper respiratory tract

The amount of SARS-CoV-2 detected in the upper respiratory tract (URT viral load) is a key driver of transmission of infection. Current evidence suggests that mechanisms constraining URT viral load are different from those controlling lower respiratory tract viral load and disease severity. Understanding such mechanisms may help to develop treatments and vaccine strategies to reduce transmission. Combining mathematical modelling of URT viral load dynamics with transcriptome analyses we aimed to identify mechanisms controlling URT viral load. COVID-19 patients were recruited in Spain during the first wave of the pandemic. RNA sequencing of peripheral blood and targeted NanoString nCounter transcriptome analysis of nasal epithelium were performed and gene expression analysed in relation to paired URT viral load samples collected within 15 days of symptom onset. Proportions of major immune cells in blood were estimated from transcriptional data using computational differential estimation. Weighted correlation network analysis (adjusted for cell proportions) and fixed transcriptional repertoire analysis were used to identify associations with URT viral load, quantified as standard deviations (z-scores) from an expected trajectory over time. Eighty-two subjects (50% female, median age 54 years (range 3-73)) with COVID-19 were recruited. Paired URT viral load samples were available for 16 blood transcriptome samples, and 17 respiratory epithelial transcriptome samples. Natural Killer (NK) cells were the only blood cell type significantly correlated with URT viral load z-scores (r=-0.62, P=0.010). Twenty-four blood gene expression modules were significantly correlated with URT viral load z-score, the most significant being a module of genes connected around IFNA14 (Interferon Alpha-14) expression (r=-0.60, P=1e-10). In fixed repertoire analysis, prostanoid-related gene expression was significantly associated with higher viral load. In nasal epithelium, only GNLY (granulysin) gene expression showed significant negative correlation with viral load. Correlations between the transcriptional host response and inter-individual variations in SARS-CoV-2 URT viral load, revealed many molecular mechanisms plausibly favouring or constraining viral replication. Existing evidence corroborates many of these mechanisms, including likely roles for NK cells, granulysin, prostanoids and interferon alpha-14. Inhibition of prostanoid production and administration of interferon alpha-14 may be attractive transmission-blocking interventions.

Comparative Performance of Cobas 6800, 8800 with Cobas Ampliprep/TaqMan for HIV Viral Load Quantification

Human immunodeficiency virus (HIV) remains a serious public health challenge, especially in Africa. Viral load (VL) is the major marker in monitoring disease prognosis. With the increased demand for HIV VL, countries are equipping fewer laboratories with higher throughput machines based on real-time polymerase chain reaction (PCR) technology. They are fully automated, faster, possess improved assay sensitivity/specificity, higher throughput, more dynamic ranges and reduced contamination rates. This study sought to verify the analytical performance of two new Cobas instruments against the Cobas Ampliprep/TaqMan (CAP/CTM), used in tracking viral suppression in Nigeria. In a cross-sectional study, aliquots of clinical HIV viral load samples were assayed on the three platforms. Values obtained with the CAP/CTM were compared with those from Cobas 6800/8800 in a routine clinical setting. Between June and August 2019, 100 plasma samples collected were analysed in parallel using both techniques. Accuracy, inter-assay precision, linearity and carry-over effect analyses were conducted. The correlation coefficients of 0.997, 0.874, and 0.878 were obtained for C8800/C6800; C8800/CAP/CTM, and C6800/CAP/CTM, respectively. The level of agreement using a Bland–Altman plot was 94.2%. The systems produced good analytical performance, comparable and correlated with CAP/CTM. Therefore, Cobas 6800 and 8800 machines are suitable and acceptable for clinical samples; and recommended for high-volume laboratories.

Ultrasensitive PCR system for HBV DNA detection: Risk stratification for occult hepatitis B virus infection in English blood donors.

Occult hepatitis B (HBV) infection (OBI), characterized by low viral loads, accounts for much of the risk of HBV transfusion-transmitted infection. With anticore antibodies (anti-HBc) screening introduced in England, the imperative to identify OBI donors has increased. We aimed to develop an ultra-sensitive PCR system and investigate risk factors for HBV DNA presence in blood donations. Seven extraction methods and three PCR assays were compared. The optimal system was sought to determine HBV DNA presence in anti-HBc-positive donations. Predictors of DNA positivity were subsequently investigated. Extraction from 5 mL of plasma increased sample representation and resulted in HBV DNA detection in low viral load samples (~0.5 IU/mL). Screening of 487 763 donations in 2022 identified two OBI donors and 2042 anti-HBc-positive donors, 412 of the latter with anti-HBs < 100 mIU/mL. Testing of 134 anti-HBc-positive donations utilizing the 5 mL extraction method identified two further HBV DNA-positive donations. Higher anti-HBc titer and anti-HBs negativity were significant predictors of DNA detectability in anti-HBc-positive donations. An ultrasensitive PCR assay identified potentially infectious donations increasing HBV DNA detection in anti-HBc-positive donors from 0.5% to 1.9%. Anti-HBc titers may further complement the risk stratification for DNA positivity in anti-HBc screening and minimize unnecessary donor deferral.

Rapid detection of monkeypox virus using a CRISPR-Cas12a mediated assay: a laboratory validation and evaluation study

The 2022 outbreak of mpox (formerly known as monkeypox) led to the spread of monkeypox virus (MPXV) in over 110 countries, demanding effective disease management and surveillance. As current diagnostics rely largely on centralised laboratory testing, our objective was to develop a simple rapid point-of-care assay to detect MPXV in clinical samples using isothermal amplification coupled with CRISPR and CRISPR-associated protein (Cas) technology. In this proof-of-concept study, we developed a portable isothermal amplification CRISPR-Cas12a-based assay for the detection of MPXV. We designed a panel of 22 primer-guide RNA sets using pangenome and gene-agnostic approaches, and subsequently shortlisted the three sets producing the strongest signals for evaluation of analytical sensitivity and specificity using a fluorescence-based readout. The set displaying 100% specificity and the lowest limit of detection (LOD) was selected for further assay validation using both a fluorescence-based and lateral-flow readout. Assay specificity was confirmed using a panel of viral and bacterial pathogens. Finally, we did a blind concordance study on genomic DNA extracted from 185 clinical samples, comparing assay results with a gold-standard quantitative PCR (qPCR) assay. We identified the optimal time to detection and analysed the performance of the assay relative to qPCR using receiver operating characteristic (ROC) curves. We also assessed the compatibility with lateral-flow strips, both visually and computationally, where strips were interpreted blinded to the fluorescence results on the basis of the presence or absence of test bands. With an optimal run duration of approximately 45 min from isothermal amplification to CRISPR-assay readout, the MPXV recombinase polymerase amplification CRISPR-Cas12a-based assay with the selected primer-guide set had an LOD of 1 copy per μL and 100% specificity against tested viral pathogens. Blinded concordance testing of 185 clinical samples resulted in 100% sensitivity (95% CI 89·3-100) and 99·3% specificity (95% CI 95·7-100) using the fluorescence readout. For optimal time to detection by fluorescence readout, we estimated the areas under the ROC curve to be 0·98 at 2 min and 0·99 at 4 min. Lateral-flow strips had 100% sensitivity (89·3-100) and 98·6% specificity (94·7-100) with both visual and computational assessment. Overall, lateral-flow results were highly concordant with fluorescence-based readouts (179 of 185 tests, 96·8% concordant), with discrepancies associated with low viral load samples. Our assay for the diagnosis of mpox displayed good performance characteristics compared with qPCR. Although optimisation of the assay will be required before deployment, its usability and versatility present a potential solution to MPXV detection in low-resource and remote settings, as well as a means of community-based, on-site testing. Victorian Medical Research Accelerator Fund and the Australian Government Department of Health.

A new approach to the epidemiological control of infectious diseases in the Russian population

The article describes a development related to the field of public health and molecular biology, in particular, medical diagnostics. We present the Isascreen-8 portable analyzer (Registration Certificate No. RZN 2022/17322, May 24, 2022) and its application for rapid diagnostics of a new coronavirus infection with the use of commercially available sets IsoAmp SARS-CoV-2 (Lytech Co. Ltd., Russia), and also our own development TB-IZATEST (Invention Application No. 2022133809, December 22, 2022) for diagnostics of tuberculosis pathogen. The approach proposed in this article can be used for rapid detection of genetic material (DNA/RNA) of Mycobacterium tuberculosis and SARS-CoV-2 in biological samples using the method of loop isothermal amplification in less than 20 minutes, and also as a universal platform for detection of genetic material of other pathogens in a biological sample. Using the proposed approach, various research tasks for epidemiological monitoring of tuberculosis and new coronavirus infection, qualitative and quantitative analysis of bacterial and viral load in samples, including evaluation of the effectiveness of the prescribed antibiotic therapy regimen, can be solved. The advantage is that this method can be used not only in diagnostic laboratories, but also in field laboratories in the most remote regions of Russia if the mandatory sanitary and epidemiological requirements are observed. With sufficient distribution, the proposed approach could help ensure epidemiological control of the prevalence of these socially significant infections.

Biomolecular Features of COVID-19 in Hodeidah, Yemen

Background: The biomolecular technique namely Real-Time Polymerase Chain Reaction (RT-PCR) is very important in confirmation of coronavirus disease 2019 (COVID-19) but in progressive infection and predication of the illness severity is not used.&#x0D; Objective: Therefore, the study aimed to determine the relationship between the viral load of COVID-19 infection and the severity of illness based on cycle threshold (Ct).&#x0D; Methodology: The research was designed in a case series study. The study included 60 patients that were confirmed by the RT-PCR test with COVID-19 and divided into two major groups. The first major group was mild and moderate cases (n:20) that were treated at home (outpatient) and the second major group was severe and critical cases (n:40) that were treated in the isolation center (inpatient).&#x0D; Results: The results showed no relationship between the viral load and severity of illness, where the minimum Ct was 14, and the maximum Ct was 36. The mean of Ct was 22 ± 7. On the other hand, the low Ct (high viral load) was reported in early detection cases. In addition , 60% (36 cases) of patients had low Ct (high viral load ; Ct ≤ 15 and ≤ 25 ) 40% of patients (24 cases) had high Ct (low viral load ; Ct ≤ 26 and ≤ 39 ) that was more than 25 Ct and less than 39 cycles . On the other mean , 95 % of first group cases (mild and moderate cases ) had high viral load based on Ct -values and 42.5 % of second group (severe and critical) had low viral load based on Ct -values. On the other hand, kinetic of viral load–based Ct where the viral load was reported in the first week as very high (low Ct 21) with a longitudinal assessment of RT-PCR test results in individuals requiring third –fourth weeks to clear COVID-19 RNA showed a significant reduction of the viral load in samples (Ct values &gt; 30). &#x0D; Conclusion: The study concluded that several factors can affect the Ct of RT-PCR (onset date, collection technique, type of swab, sampling method). Briefly, the COVID-19 RT-PCR test cannot be used as a predictor of the severity of illness.