Amplified parallel antigen rapid test for point-of-care salivary detection of SARS-CoV-2 with improved sensitivity.

In the ongoing COVID-19 pandemic, simple, rapid, point-of-care tests not requiring trained personnel for primary care testing are essential. Saliva-based antigen rapid tests (ARTs) can fulfil this need, but these tests require overnight-fasted samples; without which independent studies have demonstrated sensitivities of only 11.7 to 23.1%. Herein, we report an Amplified Parallel ART (AP-ART) with sensitivity above 90%, even with non-fasted samples. The virus was captured multimodally, using both anti-spike protein antibodies and Angiotensin Converting Enzyme 2 (ACE2) protein. It also featured two parallel flow channels. The first contained spike protein binding gold nanoparticles which produced a visible red line upon encountering the virus. The second contained signal amplifying nanoparticles that complex with the former and amplify the signal without any linker. Compared to existing dual gold amplification techniques, a limit of detection of one order of magnitude lower was achieved (0.0064 ng·mL-1). AP-ART performance in detecting SARS-CoV-2 in saliva of COVID-19 patients was investigated using a case-control study (139 participants enrolled and 162 saliva samples tested). Unlike commercially available ARTs, the sensitivity of AP-ART was maintained even when non-fasting saliva was used. Compared to the gold standard reverse transcription-polymerase chain reaction testing on nasopharyngeal samples, non-fasting saliva tested on AP-ART showed a sensitivity of 97.0% (95% CI: 84.7-99.8); without amplification, the sensitivity was 72.7% (95% CI: 83.7-94.8). Thus, AP-ART has the potential to be developed for point-of-care testing, which may be particularly important in resource-limited settings, and for early diagnosis to initiate newly approved therapies to reduce COVID-19 severity.

BNT162b2 mRNA Vaccine-Induced Immune Response in Oral Fluids and Serum.

OBJECTIVES: The COVID-19 vaccine is currently being administered worldwide to address the ongoing pandemic. Although these vaccines have proven effective in preventing severe disease, the level of immunity required to prevent respiratory mucosal infection remains less well understood. Therefore, it is desirable to develop a noninvasive screening strategy such as oral fluid to monitor secreted antibodies longitudinally as potential surrogates of mucosal immunity. METHODS: We evaluated the anti-spike protein antibodies in gingival crevicular fluid (GCF) and saliva and compared them to immune responses in the blood of 50 healthy health care workers following 2 doses of intramuscular Pfizer/BioNTech-BNT162b2 vaccine. RESULTS: The antibodies to SARS-CoV-2 spike and subdomain proteins (RBD, S1, S2, and NTD) were significantly higher in serum than oral fluids but showed a greater detection rate and higher median titres in GCF than saliva. For all tested SARS-CoV-2 antigens, IgG in GCF (as opposed to saliva) showed a more significant and stronger correlation with IgG in serum. Serum-neutralising antibodies (Nab) titres also displayed a significant and stronger correlation with anti-spike protein and their subdomains in GCF than saliva. Interestingly, the time post-second dose of vaccine and sex had a similar influence on IgG in serum and GCF. However, interferon (IFN)-γ-producing T-cell responses showed no association with SARS-Cov-2 IgG antibodies in serum, GCF, or saliva and neutralisation antibodies in serum. The correlation matrix of all measured parameters grouped serum and GCF IgG parameters separately from salivary IgG parameters indicating that GCF better represents the humoural response in serum than saliva. CONCLUSIONS: Within limitations, we propose that GCF could be a less invasive alternative to serum and more appropriate than saliva to detect antibody responses by current COVID-19 vaccines if the GCF collection procedure could be standardised. Further research is needed to investigate the suitability of GCF for community immune surveillance for vaccines.

Peridomestic Aedes malayensis and Aedes albopictus are capable vectors of arboviruses in cities.

BACKGROUND: Dengue and chikungunya are global re-emerging mosquito-borne diseases. In Singapore, sustained vector control coupled with household improvements reduced domestic mosquito populations for the past 45 years, particularly the primary vector Aedes aegypti. However, while disease incidence was low for the first 30 years following vector control implementation, outbreaks have re-emerged in the past 15 years. Epidemiological observations point to the importance of peridomestic infection in areas not targeted by control programs. We investigated the role of vectors in peri-domestic areas. METHODS: We carried out entomological surveys to identify the Aedes species present in vegetated sites in highly populated areas and determine whether mosquitoes were present in open-air areas frequented by people. We compared vector competence of Aedes albopictus and Aedes malayensis with Ae. aegypti after oral infection with sympatric dengue serotype 2 and chikungunya viruses. Mosquito saliva was tested for the presence of infectious virus particles as a surrogate for transmission following oral infection. RESULTS: We identified Aedes albopictus and Aedes malayensis throughout Singapore and quantified their presence in forested and opened grassy areas. Both Ae. albopictus and Ae. malayensis can occupy sylvatic niches and were highly susceptible to both arboviruses. A majority of saliva of infected Ae. malayensis contained infectious particles for both viruses. CONCLUSIONS: Our study reveals the prevalence of competent vectors in peri-domestic areas, including Ae. malayensis for which we established the vector status. Epidemics can be driven by infection foci, which are epidemiologically enhanced in the context of low herd immunity, selective pressure on arbovirus transmission and the presence of infectious asymptomatic persons, all these conditions being present in Singapore. Learning from Singapore's vector control success that reduced domestic vector populations, but has not sustainably reduced arboviral incidence, we suggest including peri-domestic vectors in the scope of vector management.