DEVELOPMENT AND VALIDATION OF AN ENZYME-LINKED IMMUNOASSAY KIT FOR DIAGNOSIS AND SURVEILLANCE OF COVID-19.

There is a massive demand to identify alternative methods to detect new cases of COVID-19 as well as to investigate the epidemiology of the disease. In many countries, importation of commercial kits poses a significant impact on their testing capacity and increases the costs for the public health system. We have developed an ELISA to detect IgG antibodies against SARS-CoV-2 using a recombinant viral nucleocapsid (rN) protein expressed in E. coli. Using a total of 894 clinical samples we showed that the rN-ELISA was able to detect IgG antibodies against SARS-CoV-2 with high sensitivity (97.5%) and specificity (96.3%) when compared to a commercial antibody test. After three external validation studies, we showed that the test accuracy was higher than 90%. The rN-ELISA IgG kit constitutes a convenient and specific method for the large-scale determination of SARS-CoV-2 antibodies in human sera with high reliability.

Tracking the turnover of SARS-CoV-2 VOCs Gamma to Delta in a Brazilian state (Minas Gerais) with a high-vaccination status.

The emergence and global dissemination of Severe Acute Respiratory Syndrome virus 2 (SARS-CoV-2) variants of concern (VOCs) have been described as the main factor driving the Coronavirus Disease 2019 pandemic. In Brazil, the Gamma variant dominated the epidemiological scenario during the first period of 2021. Many Brazilian regions detected the Delta variant after its first description and documented its spread. To monitor the introduction and spread of VOC Delta, we performed Polymerase Chain Reaction (PCR) genotyping and genome sequencing in ten regional sentinel units from June to October 2021 in the State of Minas Gerais (MG). We documented the introduction and spread of Delta, comprising 70 per cent of the cases 8 weeks later. Comparing the viral loads of the Gamma and Delta dominance periods, we provide additional evidence that the latter is more transmissible. The spread and dominance of Delta did not culminate in the increase in cases and deaths, suggesting that the vaccination may have restrained the epidemic growth. Analysis of 224 novel Delta genomes revealed that Rio de Janeiro state was the primary source for disseminating this variant in the state of MG. We present the establishment of Delta, providing evidence of its enhanced transmissibility and showing that this variant shift did not aggravate the epidemiological scenario in a high immunity setting.

Development and validation of an enzyme-linked immunoassay kit for diagnosis and surveillance of COVID-19.

There is a massive demand to identify alternative methods to detect new cases of COVID-19 as well as to investigate the epidemiology of the disease. In many countries, importation of commercial kits poses a significant impact on their testing capacity and increases the costs for the public health system. We have developed an ELISA to detect IgG antibodies against SARS-CoV-2 using a recombinant viral nucleocapsid (rN) protein expressed in E. coli. Using a total of 894 clinical samples we showed that the rN-ELISA was able to detect IgG antibodies against SARS-CoV-2 with high sensitivity (97.5%) and specificity (96.3%) when compared to a commercial antibody test. After three external validation studies, we showed that the test accuracy was higher than 90%. The rN-ELISA IgG kit constitutes a convenient and specific method for the large-scale determination of SARS-CoV-2 antibodies in human sera with high reliability.

Previous Infection with SARS-CoV-2 Correlates with Increased Protective Humoral Responses after a Single Dose of an Inactivated COVID-19 Vaccine.

Previous studies have indicated that antibody responses can be robustly induced after the vaccination in individuals previously infected by SARS-CoV-2. To evaluate anti-SARS-CoV-2 humoral responses in vaccinated individuals with or without a previous history of COVID-19, we compared levels of anti-SARS-CoV-2 antibodies in the sera from 21 vaccinees, including COVID-19-recovered or -naïve individuals in different times, before and after immunization with an inactivated COVID-19 vaccine. Anti-SARS-CoV-2-specific antibodies elicited after COVID-19 and/or immunization with an inactivated vaccine were measured by ELISA and Plaque Reduction Neutralizing assays. Antibody kinetics were consistently different between the two vaccine doses for naïve individuals, contrasting with the SARS-CoV-2-recovered subjects in which we observed no additional increase in antibody levels following the second dose. Sera from SARS-CoV2-naïve individuals had no detectable neutralizing activity against lineage B.1 SARS-CoV-2 or Gamma variant five months after the second vaccine dose. Contrarily, SARS-CoV-2-recovered subjects retained considerable neutralizing activity against both viruses. We conclude that a single inactivated SARS-CoV-2 vaccine dose may be sufficient to induce protective antibody responses in individuals with previous history of SARS-CoV-2 infection.

Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern.

The coronavirus disease 2019 (COVID-19) pandemic unfolded due to the widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 ± 2.7 viral genomic copies/µL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65°C for 30 min. When compared to reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), up to cycle-threshold (Ct) value 32, RT-LAMP presented 98% [95% confidence interval (CI) = 95.3-99.5%] sensitivity and 100% (95% CI = 94.5-100%) specificity for SARS-CoV-2 RNA detection targeting E and N genes. No cross-reactivity was detected when testing other non-SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction-free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern and variants of interest, such as variants occurring in Brazil named gamma (P.1), zeta (P.2), delta (B.1.617.2), B.1.1.374, and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipment, infrastructure, and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive, and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

The use of denaturing solution as collection and transport media to improve SARS-CoV-2 RNA detection and reduce infection of laboratory personnel.

Accurate testing to detect SARS-CoV-2 RNA is key to counteract the virus spread. Nonetheless, the number of diagnostic laboratories able to perform qPCR tests is limited, particularly in developing countries. We describe the use of a virus-inactivating, denaturing solution (DS) to decrease virus infectivity in clinical specimens without affecting RNA integrity. Swab samples were collected from infected patients and from laboratory personnel using a commercially available viral transport solution and the in-house DS. Samples were tested by RT-qPCR, and exposure to infective viruses was also accessed by ELISA. The DS used did not interfere with viral genome detection and was able to maintain RNA integrity for up to 16 days at room temperature. Furthermore, virus loaded onto DS were inactivated, as attested by attempts to grow SARS-CoV-2 in cell monolayers after DS desalt filtration to remove toxic residues. The DS described here provides a strategy to maintain diagnostic accuracy and protects diagnostic laboratory personnel from accidental infection, as it has helped to protect our lab crew.

Adjusting the Cut-Off and Maximum Pool Size in RT-qPCR Pool Testing for SARS-CoV-2.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-CoV-2 RNA is an essential test to monitor the occurrence of COVID-19. A methodology is proposed for the determination of maximum pool size and adjustments of cut-off values of cycle threshold (Ct in RT-qPCR pool testing, to compensate for the dilution caused by pooling. The trade-off between pool size and test sensitivity is stated explicitly. The procedure was designed to ensure that samples that would be detectable in individual testing remain detectable in pool testing. The proposed relaxation in cut-off is dependent on the pool size, allowing a relatively tight correction to avoid loss of detection of positive samples. The methodology was evaluated in a study of pool testing of adults attending a public emergency care unit, reference for COVID-19 in Belo Horizonte, Brazil, and presenting flu-like symptoms. Even samples on the edge of detectability in individual testing were detected correctly. The proposed procedure enhances the consistency of RT-qPCR pool testing by enforcing that the scales of detectability in pool processing and in individual sample processing are compatible. This may enhance the contribution of pool testing to large-scale testing for COVID-19.