The decline of measles antibody titers in previously vaccinated adults: a cross-sectional analysis

ABSTRACT The global reemergence of measles in 2018-2019 reinforces the relevance of high-coverage immunization to maintain the disease elimination. During an outbreak in the Sao Paulo State in 2019, several measles cases were reported in individuals who were adequately vaccinated according to the current immunization schedule recommends. This study aimed to assess measles IgG antibody seropositivity and titers in previously vaccinated adults. A cross-sectional study was conducted at CRIE-HC-FMUSP (Sao Paulo, Brazil) in 2019. It included healthy adults who had received two or more Measles-Mumps-Rubella vaccines (MMR) and excluded individuals with immunocompromising conditions. Measles IgG antibodies were measured and compared by ELISA (Euroimmun®) and chemiluminescence (LIASON®). The association of seropositivity and titers with variables of interest (age, sex, profession, previous measles, number of measles-containing vaccine doses, interval between MMR doses, and time elapsed since the last MMR dose) was analyzed. A total of 162 participants were evaluated, predominantly young (median age 30 years), women (69.8%) and healthcare professionals (61.7%). The median interval between MMR doses was 13.2 years, and the median time since the last dose was 10.4 years. The seropositivity rate was 32.7% by ELISA and 75.3% by CLIA, and a strong positive correlation was found between the tests. Multivariate analyses revealed that age and time since the last dose were independently associated with positivity. Despite being a single-center evaluation, our results suggest that measles seropositivity may be lower than expected in adequately immunized adults. Seropositivity was higher among older individuals and those with a shorter time since the last MMR vaccine dose.

Accuracy of a raw saliva-based COVID-19 RT-LAMP diagnostic assay

ABSTRACT The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic demanded rapid diagnosis to isolate new COVID-19 cases and prevent disease transmission. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) rapidly became the gold standard for diagnosis. However, due to the high cost and delay of the results, other types of diagnosis were implemented, such as COVID-19 Ag Rapid Tests and Reverse Transcription Technique followed by Loop-Mediated isothermal Amplification (RT-LAMP). In this work, we validated the use of RT-LAMP in saliva samples rather than nasopharyngeal swabs, as the collection is more comfortable. First, we selected 5 primer sets based on the limit of detection for SARS-CoV-2 RNA, then validated their sensitivity and specificity in patient samples. A total of 117 samples were analyzed by fluorometric RT-LAMP and compared with qRT-PCR results. Our results show that the use of a high-sensitive primer ORF1-a, together with a low-sensitive primer set Gene E (time to threshold of 22.9 and 36.4 minutes, respectively, using 200 copies of viral RNA), achieved sensitivity in purified RNA from saliva samples of 95.2% (95% CI 76.1-99.8) with 90.5% specificity (95% CI 69.6-98.8) (n = 42).As RNA purification increases the turnaround time, we tested the outcome of RT-LAMP utilizing raw saliva samples without purification. The test achieved a sensitivity of 81.8% (95% CI 59.7-94.8) and a specificity of 90.9% (95% CI 70.8-98.8). As a result, the accuracy of 92.9% (95% CI 80.5-98.5) in purified RNA-saliva samples was lowered to an acceptable level of 86.4% (95% CI 72.6-94.8) in raw saliva. Although mass vaccination has been implemented, new strains and low vaccination progress helped to spread COVID-19. This study shows that it is feasible to track new COVID-19 cases in a large population with the use of raw saliva as sample in RT-LAMP assay which yields accurate results and offers a less invasive test.

In vitro susceptibility of eighteen clinical isolates of human monkeypox virus to tecovirimat

BACKGROUND In 2022, an outbreak of mpox that started in European countries spread worldwide through human-to-human transmission. Cases have been mostly mild, but severe clinical presentations have been reported. In these cases, tecovirimat has been the drug of choice to treat patients with aggravated disease. OBJECTIVES Here we investigated the tecovirimat susceptibility of 18 clinical isolates of monkeypox virus (MPXV) obtained from different regions of Brazil. METHODS Different concentrations of tecovirimat were added to cell monolayers infected with each MPXV isolate. After 72 hours, cells were fixed and stained for plaque visualization, counting, and measurement. The ortholog of F13L gene from each MPXV isolate was polymerase chain reaction (PCR)-amplified, sequenced, and the predicted protein sequences were analyzed. FINDINGS The eighteen MPXV isolates generated plaques of different sizes. Although all isolates were highly sensitive to the drug, two showed different response curves and IC50 values. However, the target protein of tecovirimat, F13 (VP37), was 100% conserved in all MPXV isolates and therefore does not explain the difference in sensitivity. MAIN CONCLUSIONS Our results support screening different MPXV isolates for tecovirimat susceptibility as an important tool to better use of the restricted number of tecovirimat doses available in low-income countries to treat patients with mpox.

COVID-19 diagnosis by RT-qPCR in alternative specimens

BACKGROUND The high demand for adequate material for the gold standard reverse transcription real-time polymerase chain reaction (RT-qPCR)-based diagnosis imposed by the Coronavirus disease 2019 (COVID-19) pandemic, combined with the inherent contamination risks for healthcare workers during nasopharyngeal swab (NP) sample collection and the discomfort it causes patients, brought the need to identify alternative specimens suitable for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). OBJECTIVES The aim of this work was to compare saliva and gingival fluid swabs to NP swabs as specimens for RT-qPCR-based SARS-CoV-2 diagnosis. METHODS We compared gingival fluid swabs (n = 158) and saliva (n = 207) to the rayon-tipped NP swabs obtained from mild-symptomatic and asymptomatic subjects as specimens for RT-qPCR for SARS-CoV-2 detection. FINDINGS When compared to NP swabs, gingival fluid swabs had a concordance rate of 15.4% among positive samples, zero among inconclusive, and 100% among negative ones. For saliva samples, the concordance rate was 67.6% among positive samples, 42.9% among inconclusive, and 96.8% among negative ones. However, the concordance rate between saliva and NP swabs was higher (96.9%) within samples with lower cycle threshold (Ct) values (Ct > 10 ≤ 25). MAIN CONCLUSIONS Our data suggests that whereas gingival fluid swabs are not substitutes for NP swabs, saliva might be considered whenever NP swabs are not available or recommended.

Identification and characterisation of SARS-CoV-2 and Human alphaherpesvirus 1 from a productive coinfection in a fatal COVID-19 case

BACKGROUND During routine Coronavirus disease 2019 (COVID-19) diagnosis, an unusually high viral load was detected by reverse transcription real-time polymerase chain reaction (RT-qPCR) in a nasopharyngeal swab sample collected from a patient with respiratory and neurological symptoms who rapidly succumbed to the disease. Therefore we sought to characterise the infection. OBJECTIVES We aimed to determine and characterise the etiological agent responsible for the poor outcome. METHODS Classical virological methods, such as plaque assay and plaque reduction neutralisation test combined with amplicon-based sequencing, as well as a viral metagenomic approach, were performed to characterise the etiological agents of the infection. FINDINGS Plaque assay revealed two distinct plaque phenotypes, suggesting either the presence of two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains or a productive coinfection of two different species of virus. Amplicon-based sequencing did not support the presence of any SARS-CoV-2 genetic variants that would explain the high viral load and suggested the presence of a single SARS-CoV-2 strain. Nonetheless, the viral metagenomic analysis revealed that Coronaviridae and Herpesviridae were the predominant virus families within the sample. This finding was confirmed by a plaque reduction neutralisation test and PCR. MAIN CONCLUSIONS We characterised a productive coinfection of SARS-CoV-2 and Herpes simplex virus 1 (HSV-1) in a patient with severe symptoms that succumbed to the disease. Although we cannot establish the causal relationship between the coinfection and the severity of the clinical case, this work serves as a warning for future studies focused on the interplay between SARS-CoV-2 and HSV-1 coinfection and COVID-19 severity.

Performance of an alternative RT-PCR procedure using residual sample from the Panbio™ Ag COVID-19 test

ABSTRACT Introduction: In the current standard of care (SoC) RT-PCR method for COVID-19, the patient's swab was extracted in viral transport media (VTM). For the PanbioTM COVID-19 Ag Rapid Test, the patient swab is flushed out in extraction buffer, of which a small fraction is used for testing, leaving more than half the sample unused. This study was designed to show that RT-PCR results from the residual sample of the PanbioTM COVID-19 Ag Rapid Test (called Novel RT-PCR) are not worse than the SoC RT-PCR result. Methods: The study was performed using (1) dilution series of five patient samples, and (2) 413 patient samples comparing SOC versus Novel RT-PCR results. Results: For the dilution series samples, all tested positive by both methods. The bias between Ct values of Novel RT-PCR and SoC RT-PCR did not exceed 3.00 Ct using primers N1 and N2. A total of 413 COVID symptomatic patients seeking COVID testing were tested, of which 89 patients tested positive and 324 tested negative with SoC RT-PCR. In 324 patients who tested negative with SoC RT-PCR, 323 tested negative with Novel RT-PCR, and one (1) tested positive. Out of 89 who tested positive with SoC RT-PCR, 80 tested positive with the Novel RT-PCR, and nine patients showed a negative test result. The Overall Percent Agreement for the 413 valid patient sample pairs was 97.5 [95% CI 97 to 98]. Conclusion: The study demonstrated that the performance of the Novel RT-PCR method is acceptable compared to the SoC RT-PCR method and can be a useful tool to perform RTPCR without the need for new swab collections.