Malaria diagnostic methods with the elimination goal in view.

Malaria control measures have been in use for years but have not completely curbed the spread of infection. Ultimately, global elimination is the goal. A major playmaker in the various approaches to reaching the goal is the issue of proper diagnosis. Various diagnostic techniques were adopted in different regions and geographical locations over the decades, and these have invariably produced diverse outcomes. In this review, we looked at the various approaches used in malaria diagnostics with a focus on methods favorably used during pre-elimination and elimination phases as well as in endemic regions. Microscopy, rapid diagnostic testing (RDT), loop-mediated isothermal amplification (LAMP), and polymerase chain reaction (PCR) are common methods applied depending on prevailing factors, each with its strengths and limitations. As the drive toward the elimination goal intensifies, the search for ideal, simple, fast, and reliable point-of-care diagnostic tools is needed more than ever before to be used in conjunction with a functional surveillance system supported by the ideal vaccine.

A method for campus-wide SARS-CoV-2 surveillance at a large public university.

The systematic screening of asymptomatic and pre-symptomatic individuals is a powerful tool for controlling community transmission of infectious disease on college campuses. Faced with a paucity of testing in the beginning of the COVID-19 pandemic, many universities developed molecular diagnostic laboratories focused on SARS-CoV-2 diagnostic testing on campus and in their broader communities. We established the UC Santa Cruz Molecular Diagnostic Lab in early April 2020 and began testing clinical samples just five weeks later. Using a clinically-validated laboratory developed test (LDT) that avoided supply chain constraints, an automated sample pooling and processing workflow, and a custom laboratory information management system (LIMS), we expanded testing from a handful of clinical samples per day to thousands per day with the testing capacity to screen our entire campus population twice per week. In this report we describe the technical, logistical, and regulatory processes that enabled our pop-up lab to scale testing and reporting capacity to thousands of tests per day.

Diagnostic accuracy of three commercially available one step RT-PCR assays for the detection of SARS-CoV-2 in resource limited settings.

BACKGROUND: COVID-19 is an ongoing public health pandemic regardless of the countless efforts made by various actors. Quality diagnostic tests are important for early detection and control. Notably, several commercially available one step RT-PCR based assays have been recommended by the WHO. Yet, their analytic and diagnostic performances have not been well documented in resource-limited settings. Hence, this study aimed to evaluate the diagnostic sensitivities and specificities of three commercially available one step reverse transcriptase-polymerase chain reaction (RT-PCR) assays in Ethiopia in clinical setting. METHODS: A cross-sectional study was conducted from April to June, 2021 on 279 respiratory swabs originating from community surveillance, contact cases and suspect cases. RNA was extracted using manual extraction method. Master-mix preparation, amplification and result interpretation was done as per the respective manufacturer. Agreements between RT-PCRs were analyzed using kappa values. Bayesian latent class models (BLCM) were fitted to obtain reliable estimates of diagnostic sensitivities, specificities of the three assays and prevalence in the absence of a true gold standard. RESULTS: Among the 279 respiratory samples, 50(18%), 59(21.2%), and 69(24.7%) were tested positive by TIB, Da An, and BGI assays, respectively. Moderate to substantial level of agreement was reported among the three assays with kappa value between 0 .55 and 0.72. Based on the BLCM relatively high specificities (95% CI) of 0.991(0.973-1.000), 0.961(0.930-0.991) and 0.916(0.875-0.952) and considerably lower sensitivities with 0.813(0.658-0.938), 0.836(0.712-0.940) and 0.810(0.687-0.920) for TIB MOLBIOL, Da An and BGI respectively were found. CONCLUSIONS: While all the three RT-PCR assays displayed comparable sensitivities, the specificities of TIB MOLBIOL and Da An were considerably higher than BGI. These results help adjust the apparent prevalence determined by the three RT-PCRs and thus support public health decisions in resource limited settings and consider alternatives as per their prioritization matrix.

Genomic surveillance of SARS-CoV-2 Spike gene by sanger sequencing.

The SARS-CoV-2 responsible for the ongoing COVID pandemic reveals particular evolutionary dynamics and an extensive polymorphism, mainly in Spike gene. Monitoring the S gene mutations is crucial for successful controlling measures and detecting variants that can evade vaccine immunity. Even after the costs reduction resulting from the pandemic, the new generation sequencing methodologies remain unavailable to a large number of scientific groups. Therefore, to support the urgent surveillance of SARS-CoV-2 S gene, this work describes a new feasible protocol for complete nucleotide sequencing of the S gene using the Sanger technique. Such a methodology could be easily adopted by any laboratory with experience in sequencing, adding to effective surveillance of SARS-CoV-2 spreading and evolution.

RNA reference materials with defined viral RNA loads of SARS-CoV-2-A useful tool towards a better PCR assay harmonization.

SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load. Consequently, there was a need to introduce standards to support estimation of SARS-CoV-2 viral load in diagnostic specimens. In a collaborative study, INSTAND established two reference materials (RMs) containing heat-inactivated SARS-CoV-2 with SARS-CoV-2 RNA loads of ~107 copies/mL (RM 1) and ~106 copies/mL (RM 2), respectively. Quantification was performed by RT-qPCR using synthetic SARS-CoV-2 RNA standards and digital PCR. Between November 2020 and February 2021, German laboratories were invited to use the two RMs to anchor their Ct values measured in routine diagnostic specimens, with the Ct values of the two RMs. A total of 305 laboratories in Germany were supplied with RM 1 and RM 2. The laboratories were requested to report their measured Ct values together with details on the PCR method they used to INSTAND. This resultant 1,109 data sets were differentiated by test system and targeted gene region. Our findings demonstrate that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that they are treated as being unique to an individual laboratory. For this reason, clinical guidance based on viral loads should not cite Ct values. The RMs described were a suitable tool to determine the specific laboratory Ct for a given viral load. Furthermore, as Ct values can also vary between runs when using the same instrument, such RMs could be used as run controls to ensure reproducibility of the quantitative measurements.

Chimeric crRNA improves CRISPR-Cas12a specificity in the N501Y mutation detection of Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2.

Many CRISPR/Cas platforms have been established for the detection of SARS-CoV-2. But the detection platform of the variants of SARS-CoV-2 is scarce because its specificity is very challenging to achieve for those with only one or a few nucleotide(s) differences. Here, we report for the first time that chimeric crRNA could be critical in enhancing the specificity of CRISPR-Cas12a detecting of N501Y, which is shared by Alpha, Beta, Gamma, and Mu variants of SARS-CoV-2 without compromising its sensitivity. This strategy could also be applied to detect other SARS-CoV-2 variants that differ only one or a few nucleotide(s) differences.

In Situ Nanocoating on Porous Pyrolyzed Paper Enables Antibiofouling and Sensitive Electrochemical Analyses in Biological Fluids.

Electrochemical detection in complex biofluids is a long-standing challenge as electrode biofouling hampers its sensing performance and commercial translation. To overcome this drawback, pyrolyzed paper as porous electrode coupled with the drop casting of an off-the-shelf polysorbate, that is, Tween 20 (T20), is described here by taking advantage of the in situ formation of a hydrophilic nanocoating (2 nm layer of T20). The latter prevents biofouling while providing the capillarity of samples through paper pores, leveraging redox reactions across both only partially fouled and fresh electrodic surfaces with increasing detection areas. The nanometric thickness of this blocking layer is also essential by not significantly impairing the electron-transfer kinetics. These phenomena behave synergistically to enhance the sensibility that further increases over long-term exposures (4 h) in biological fluids. While the state-of-the-art antibiofouling strategies compromise the sensibility, this approach leads to peak currents that are up to 12.5-fold higher than the original currents after 1 h exposure to unprocessed human plasma. Label-free impedimetric immunoassays through modular bioconjugation by directly anchoring spike protein on gold nanoparticles are also allowed, as demonstrated for the COVID-19 screening of patient sera. The scalability and simplicity of the platform combined with its unique ability to operate in biofluids with enhanced sensibility provide the generation of promising biosensing technologies toward real-world applications in point-of-care diagnostics, mass testing, and in-home monitoring of chronic diseases.

Column Agglutination Assay Using Polystyrene Microbeads for Rapid Detection of Antibodies against SARS-CoV-2.

Rapid serology platforms are essential in disease pandemics for a variety of applications, including epidemiological surveillance, contact tracing, vaccination monitoring, and primary diagnosis in resource-limited areas. Laboratory-based enzyme-linked immunosorbent assay (ELISA) platforms are inherently multistep processes that require trained personnel and are of relatively limited throughput. As an alternative, agglutination-based systems have been developed; however, they rely on donor red blood cells and are not yet available for high-throughput screening. Column agglutination tests are a mainstay of pretransfusion blood typing and can be performed at a range of scales, ranging from manual through to fully automated testing. Here, we describe a column agglutination test using colored microbeads coated with recombinant SARS-CoV-2 spike protein that agglutinates when incubated with serum samples collected from patients recently infected with SARS-CoV-2. After confirming specific agglutination, we optimized centrifugal force and time to distinguish samples from uninfected vs SARS-CoV-2-infected individuals and then showed concordant results against ELISA for 22 clinical samples, and also a set of serial bleeds from one donor at days 6-10 postinfection. Our study demonstrates the use of a simple, scalable, and rapid diagnostic platform that can be tailored to detect antibodies raised against SARS-CoV-2 and can be easily integrated with established laboratory frameworks worldwide.

A rapid and affordable point of care test for antibodies against SARS-CoV-2 based on hemagglutination and artificial intelligence interpretation.

Diagnostic tests that detect antibodies (AB) against SARS-CoV-2 for evaluation of seroprevalence and guidance of health care measures are important tools for managing the COVID-19 pandemic. Current tests have certain limitations with regard to turnaround time, costs and availability, particularly in point-of-care (POC) settings. We established a hemagglutination-based AB test that is based on bi-specific proteins which contain a dromedary-derived antibody (nanobody) binding red blood cells (RBD) and a SARS-CoV-2-derived antigen, such as the receptor-binding domain of the Spike protein (Spike-RBD). While the nanobody mediates swift binding to RBC, the antigen moiety directs instantaneous, visually apparent hemagglutination in the presence of SARS-CoV-2-specific AB generated in COVID-19 patients or vaccinated individuals. Method comparison studies with assays cleared by emergency use authorization demonstrate high specificity and sensitivity. To further increase objectivity of test interpretation, we developed an image analysis tool based on digital image acquisition (via a cell phone) and a machine learning algorithm based on defined sample-training and -validation datasets. Preliminary data, including a small clinical study, provides proof of principle for test performance in a POC setting. Together, the data support the interpretation that this AB test format, which we refer to as 'NanoSpot.ai', is suitable for POC testing, can be manufactured at very low costs and, based on its generic mode of action, can likely be adapted to a variety of other pathogens.

Validation and deployment of a direct saliva real-time RT-PCR test on pooled samples for COVID-19 surveillance testing.

A direct, real-time reverse transcriptase PCR test on pooled saliva was validated in 2,786 participants against oropharyngeal swabs. Among asymptomatic/pre-symptomatic participants, the test was found to be in 99.21% agreement and 45% more sensitive than contemporaneous oropharyngeal swabs. The test was then used for surveillance testing on 44,242 saliva samples from asymptomatic participants. Those whose saliva showed evidence of SARS-CoV-2 within 50 cycles of amplification were referred for confirmatory testing, with 87% of those tested by nasal swab within 72 hours receiving a positive diagnostic result on Abbott ID NOW or real-time PCR platforms. Median Ct values on the saliva PCR for those with a positive and negative confirmatory tests was 30.67 and 35.92 respectively, however, binary logistic regression analysis of the saliva Ct values indicates that Ct thresholds as high as 47 may be useful in a surveillance setting. Overall, data indicate that direct RT-PCR testing of pooled saliva samples is an effective method of SARS-CoV-2 surveillance.

Humoral immune response to SARS-CoV-2 in five different groups of individuals at different environmental and professional risk of infection.

It is partially unknown whether the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection persists with time. To address this issue, we detected the presence of SARS-CoV-2 antibodies in different groups of individuals previously diagnosed with COVID-19 disease (group 1 and 2), or potentially exposed to SARS-CoV-2 infection (group 3 and 4), and in a representative group of individuals with limited environmental exposure to the virus due to lockdown restrictions (group 5). The primary outcome was specific anti-SARS-CoV-2 antibodies in the different groups assessed by qualitative and quantitative analysis at baseline, 3 and 6 months follow-up. The seroconversion rate at baseline test was 95% in group 1, 61% in group 2, 40% in group 3, 17% in group 4 and 3% in group 5. Multivariate logistic regression analysis revealed male gender, close COVID-19 contact and presence of COVID-19 related symptoms strongly associated with serological positivity. The percentage of positive individuals as assessed by the qualitative and quantitative tests was superimposable. At the quantitative test, the median level of SARS-CoV-2 antibody levels measured in positive cases retested at 6-months increased significantly from baseline. The study indicates that assessing antibody response to SARS-CoV-2 through qualitative and quantitative testing is a reliable disease surveillance tool.

The evaluation of the utility of the GENECUBE HQ SARS-CoV-2 for anterior nasal samples and saliva samples with a new rapid examination protocol.

INTRODUCTION: GENECUBE® is a rapid molecular identification system, and previous studies demonstrated that GENECUBE® HQ SARS-CoV-2 showed excellent analytical performance for the detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) with nasopharyngeal samples. However, other respiratory samples have not been evaluated. METHODS: This prospective comparison between GENECUBE® HQ SARS-CoV-2 and reference real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed for the detection of SARS-CoV-2 using anterior nasal samples and saliva samples. Additionally, we evaluated a new rapid examination protocol using GENECUBE® HQ SARS-CoV-2 for the detection of SARS-CoV-2 with saliva samples. For the rapid protocol, in the preparation of saliva samples, purification and extraction processes were adjusted, and the total process time was shortened to approximately 35 minutes. RESULTS: For 359 anterior nasal samples, the total-, positive-, and negative concordance of the two assays was 99.7% (358/359), 98.1% (51/52), and 100% (307/307), respectively. For saliva samples, the total-, positive-, and negative concordance of the two assays was 99.6% (239/240), 100% (56/56), and 99.5% (183/184), respectively. With the new protocol, total-, positive-, and negative concordance of the two assays was 98.8% (237/240), 100% (56/56), and 98.4% (181/184), respectively. In all discordance cases, SARS-CoV-2 was detected by additional molecular examinations. CONCLUSION: GENECUBE® HQ SARS-CoV-2 provided high analytical performance for the detection of SARS-CoV-2 in anterior nasal samples and saliva samples.

Seroprevalence of antibodies against SARS-Cov-2 in the high impacted sub-district in Jakarta, Indonesia.

BACKGROUND: Understanding the actual prevalence of COVID-19 transmission in the community is vital for strategic responses to the pandemic. This study aims to estimate the actual infection of COVID-19 through a seroprevalence survey and to predict infection fatality rate (IFR) in Tanjung Priok, the hardest-hit sub-district by the COVID-19 in Jakarta, Indonesia. METHODS: We conducted a venous blood sampling (phlebotomy) to 3,196 individuals in Tanjung Priok between Nov 23, 2020, and Feb 19, 2021 to detect their antibodies against SARS-CoV-2. Using an enumerator-administered questionnaire, we collected data on the respondents' demographic characteristics, COVID-19 test history, COVID-19 symptoms in the last 14 days, comorbidities, and protective behaviours during the last month. We employed descriptive analysis to estimate the seroprevalence and IFR. FINDINGS: The prevalence of Antibody against SARS-CoV-2 was 28.52% (95% CI 25.44-31.81%), with the result being higher in females than males (OR 1.20; 95% CI 1.02-1.42). By the end of the data collection (February 9, 2021), the cumulative cases of COVID-19 in Tanjung Priok were reported to be experienced by 9,861 people (2.4%). Those aged 45-65 were more likely to be seropositive than 15-19 years old (OR 1.42; 95% CI 1.05-1.92). Nearly one third (31%) of the subjects who developed at least one COVID-19 symptom in the last 14 days of the data collection were seropositive. The estimated IFR was 0.08% (95% CI 0.07-0.09), with a higher figure recorded in males (0.09; 95% CI 0.08-0.10) than females (0.07; 95% CI 0.06-0.08), and oldest age group (45-65) (0.21; 95% CI 0.18-0.23) than other younger groups. CONCLUSION: An under-reporting issue was found between the estimated COVID-19 seroprevalence and the reported cumulative cases in Tanjung Priok. More efforts are required to amplify epidemiological surveillance by the provincial and local governments.

Predicting Hospital Length of Stay at Admission Using Global and Country-Specific Competing Risk Analysis of Structural, Patient, and Nutrition-Related Data from nutritionDay 2007-2015.

Hospital length of stay (LOS) is an important clinical and economic outcome and knowing its predictors could lead to better planning of resources needed during hospitalization. This analysis sought to identify structure, patient, and nutrition-related predictors of LOS available at the time of admission in the global nutritionDay dataset and to analyze variations by country for countries with n > 750. Data from 2006-2015 (n = 155,524) was utilized for descriptive and multivariable cause-specific Cox proportional hazards competing-risks analyses of total LOS from admission. Time to event analysis on 90,480 complete cases included: discharged (n = 65,509), transferred (n = 11,553), or in-hospital death (n = 3199). The median LOS was 6 days (25th and 75th percentile: 4-12). There is robust evidence that LOS is predicted by patient characteristics such as age, affected organs, and comorbidities in all three outcomes. Having lost weight in the last three months led to a longer time to discharge (Hazard Ratio (HR) 0.89; 99.9% Confidence Interval (CI) 0.85-0.93), shorter time to transfer (HR 1.40; 99.9% CI 1.24-1.57) or death (HR 2.34; 99.9% CI 1.86-2.94). The impact of having a dietician and screening patients at admission varied by country. Despite country variability in outcomes and LOS, the factors that predict LOS at admission are consistent globally.

Comparison of IL-6 measurement methods with a special emphasis on COVID-19 patients according to equipment and sample type.

BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine associated with various diseases, including coronavirus disease (COVID-19). Although IL-6 levels can be assessed using serum samples, use of the AFIAS (Boditech Med Inc.) automated immunoassay analyzer enables quick and simple measurement of IL-6 levels in both serum and whole blood specimens. This study aimed to assess the correlation between IL-6 measurements obtained from the AFIAS IL-6 assay and Elecsys IL-6 assay (Roche Diagnostics). Additionally, utilization of the AFIAS IL-6 assay was evaluated. METHODS: The IL-6 levels from 113 serum samples quantified using two assay systems were evaluated for their degree of correlation. Meanwhile, the linearity, analytical sensitivity, and precision/reproducibility of the AFIAS IL-6 assay were also assessed. RESULTS: Quantification of IL-6 with the AFIAS IL-6 and Elecsys IL-6 assays showed excellent agreement (kappa 0.802) and were found to be correlated (y = -0.2781 + 1.068x; 95% confidence interval: 1.007-1.124). AFIAS IL-6 showed good analytical performances. IL-6 levels were significantly higher in deceased patients compared to those with non-complicated disease and those who were intubated (p = 0.002 and p < 0.0001, respectively). Finally, IL-6 levels more accurately predicted poor prognosis in patients, than did C-reactive protein (area under the curve, 0.716 vs. 0.634). CONCLUSION: The overall analytical performance of the AFIAS assay was comparable to that of the Elecsys IL-6 assay. In light of the ongoing COVID-19 pandemic, the AFIAS may be an attractive tool for measuring IL-6 levels.

Routine COVID-19 testing may not be necessary for most cancer patients.

Cancer patients are at risk for severe complications or death from COVID-19 infection. Therefore, the need for routine COVID-19 testing in this population was evaluated. Between 1st August and 30th October 2020, 150 cancer patients were included. Symptoms of COVID-19 infection were evaluated. All eligible individuals went through RT-PCR and serological tests for COVID-19. At the same time, 920 non-cancer patients were recruited from a random sample of individuals who were subject to routine molecular and anti-body screening tests. Of 150 cancer patients, 7 (4.7%) were RT-PCR positive. Comorbidity made a significant difference in the RT-PCR positivity of cancer patients, 71.4% positive versus 25.8% negative (P-value = 0.02). The average age for negative and positive groups was 53.3 and 58.2 respectively (P-value = 0.01). No significant difference was observed between cancer and non-cancer patients regarding COVID-19 antibody tests. However, cancer patients were 3 times less likely to have a positive RT-PCR test result OR = 0.33 (CI: 0.15-0.73). The probability of cancer patients having a positive routine test was significantly lower than non-cancer patients, and the concept that all cancer patients should be routinely tested for COVID-19 may be incorrect. Nevertheless, there may be a subgroup of patients with comorbidities or older age who may benefit from routine COVID-19 testing. Importantly, these results could not be subjected to multivariate analysis.

Evaluation of a SARS-CoV-2 antigen-detecting rapid diagnostic test as a self-test: Diagnostic performance and usability.

To control the spread of the coronavirus disease 2019 (COVID-19) epidemics, it is necessary to have easy-to-use, reliable diagnostic tests available. The nasopharyngeal sampling method being often uncomfortable, nasal sampling could prove to be a viable alternative to the reference sampling method. We performed a multicentre, prospective validation study of the COVID-VIRO® test, using a nasal swab sampling method, in a point-of-care setting. In addition, we performed a multicentre, prospective, and usability study to validate the use of the rapid antigen nasal diagnostic test by laypersons. In March 2021, 239 asymptomatic and symptomatic patients were included in the validation study. Compared with reverse-transcription polymerase chain reaction on nasopharyngeal samples, the sensitivity and specificity of the COVID-VIRO® Antigen test combined with a nasal sampling method were evaluated as 96.88% and 100%, respectively. A total of 101 individuals were included in the usability study. Among these, 99% of the participants rated the instructions material as good, 98% of the subjects executed the test procedure well, and 98% of the participants were able to correctly interpret the test results. This study validates the relevance of COVID-VIRO® as a diagnostic tool from nasal specimens as well as its usability in the general population. COVID-VIRO® diagnostic performances and ease of use make it suitable for widespread utilization.

Comparison of SARS-CoV-2 detection with the Cobas® 6800/8800 system on gargle samples using two sample processing methods with combined oropharyngeal/nasopharyngeal swab.

BACKGROUND: Gargle samples have been proposed as a noninvasive method for detection of SARS-CoV-2 RNA. The clinical performance of gargle specimens diluted in Cobas® PCR Media and in Cobas® Omni Lysis Reagent was compared to oropharyngeal/nasopharyngeal swab (ONPS) for the detection of SARS-CoV-2 RNA. STUDY DESIGN: Participants were recruited prospectively in two COVID-19 screening clinics. In addition to the ONPS, participants gargled with 5 ml of natural spring water split in the laboratory as follows: 1 ml was added to 4.3 ml of polymerase chain reaction (PCR) media and 400 µl was added to 200 µl of lysis buffer. Testing was performed with the Cobas® SARS-CoV-2 test on the Cobas® 6800 or 8800 platforms. RESULTS: Overall, 134/647 (20.7%) participants were considered infected because the ONPS or at least one gargle test was positive. ONPS had, respectively, a sensitivity of 96.3% (95% confidence interval [CI]: 91.3-98.5); both gargle processing methods were slightly less but equally sensitive (90.3% [95% CI: 83.9-94.3]). When ONPS and gargle specimens were both positive, the mean cycle threshold (Ct ) was significantly higher for gargles, suggesting lower viral loads. CONCLUSION: Gargle specimens directly added in PCR Media provide a similar clinical sensitivity to chemical lysis, both having a slightly, not significantly, lower sensitivity to ONPS.

Evaluation of five widely used serologic assays for antibodies to SARS-CoV-2.

Reliable diagnostic technologies are pivotal to the fight against COVID-19. While real-time reverse transcription-polymerase chain reaction (rRT-PCR) remains the gold standard, commercial assays for antibodies against (SARS-CoV-2) have emerged. We sought to examine 5 widely used commercial methods. We measured antibodies against SARS-CoV-2 with assays, Abbott-IgG, Roche-IgT (total antibodies, isotype-unspecific), EUROIMMUN-IgG, EUROIMMUN-IgA, DiaSorin-IgG, in 191 serum samples from patients with rRT-PCR proven COVID-19 between days 0 and 47 after the onset of clinical symptoms and in biobank samples collected in 2018. The assays were calibrated using the manufacturers' instructions; results are in multiples of the assay specific cut-offs (Abbott, Roche, EUROIMMUN) or in arbitrary units (AU/mL, DiaSorin). The assays for IgG and IgT have approximately the same sensitivity and specificity for detecting seroconversion which starts at approximately day 3 after symptom onset, sensitivity reached 93% on day 16 and was 100% for each assay on day 20. The assay for IgA antibodies was superior in sensitivity and had a lower specificity than the others. Bivariate non-parametric correlation coefficients ranged between 0.738 and 0.991. Commercial assays for IgG or total antibodies against SARS-CoV-2 are largely equivalent for establishing seroconversion but differ at high antibody titres. Increased sensitivity to detect seroconversion is afforded by including IgA antibodies. Further international efforts to harmonise assays for antibodies against SARS-CoV-2 are urgently needed.

Cycle threshold values in RT-PCR to determine dynamics of SARS-CoV-2 viral load: An approach to reduce the isolation period for COVID-19 patients.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) has affected all inhabited continents, and India is currently experiencing a devastating second wave of coronavirus disease-2019 (COVID-19). Here, we examined the duration of clearance of SARS-CoV-2 in respiratory samples from 207 infected cases by real-time reverse-transcription polymerase chain reaction (RT-PCR). A substantial proportion of COVID-19 positive cases with cycle threshold (Ct) values more than or equal to 31 (45.7%) were subsequently tested negative for SARS-CoV-2 RNA within 7 days of initial detection of the viral load. A total of 60% of all the patients with COVID-19, irrespective of their Ct values, cleared SARS-CoV-2 RNA within 14 days of the initial detection. Longitudinal assessment of RT-PCR test results in individuals requiring 15-30 days to clear SARS-CoV-2 RNA showed a significant reduction of the viral load in samples with high or intermediate viral loads (Ct values ≤ 25 and between 26 and 30, respectively) but the follow-up group with low viral RNA (Ct values ≥ 31) exhibited a stable viral load. Together, these results suggest that COVID-19 positive cases with Ct values more than or equal to 31 require reduced duration to clear SARS-CoV-2, and thus, a shorter isolation period for this group might be considered to facilitate adequate space in the COVID Care Centres and reduce the burden on healthcare infrastructure.