ABSTRACT
Cancer patients display immunomodulation related to malignancy and anti-cancer therapies, but how these factors impact COVID-19 remains unknown. To investigate immune responses in cancer patients with COVID-19, we undertook a prospective case-control study, enrolling hospitalized solid tumor patients with acute COVID-19, as well as age-, gender-, and comorbidity-matched COVID-19 patients without cancer as controls. Using biospecimens collected during hospitalization, we performed virologic measurements as well as in-depth immunophenotyping of cellular, antibody and cytokine responses. We enrolled 17 cancer patients (cases) admitted to Yale-New Haven Hospital between March 15 and June 30, 2020 with COVID-19, as well as 17 matched non-cancer patients (controls) admitted with COVID-19. No significant differences were observed between cases and controls based on patient characteristics (age, gender, race, co-morbidities, smoking history, days from symptom onset to COVID-19 diagnosis) or outcomes (COVID-19 severity, length of hospital stay, rate of intubation or mortality). The most common primary tumor sites were lung (4/17) and gastrointestinal (4/17);all cases had received cancer-directed therapy within 6 months of COVID-19 diagnosis, with 13/17 receiving treatment less than 1 month prior to hospitalization. Three of 17 cases had received immune checkpoint inhibitor therapies. Despite having similar SARS-CoV-2 viral RNA loads at the time of COVID-19 diagnosis when compared with controls, cancer cases had increased viral RNA abundance during hospitalization, suggesting slower clearance. Antibody responses against SARS-CoV-2 were preserved in cancer cases, with cases displaying similar levels of IgM and IgG antibodies directed against SARS-CoV-2 epitopes compared to controls. Cytokine profiling revealed higher plasma levels of CCL3, IL1A and CXCL12 in cancer cases compared to controls. Using flow cytometric immunophenotyping, we found that innate immune and non-T cell adaptive immune parameters were similar between cases and controls hospitalized with COVID-19. However, among cancer cases on conventional therapies, T cell lymphopenia was more profound, and these cases demonstrated higher levels of CD8+ exhausted (CD8+CD45RA-PD1+TIM3+ ), CD8+GranzymeB+ and CD4+CD38+HLA-DR+ and CD8+CD38+HLA-DR+ activated T cells when compared with controls;interestingly, these differences were not observed in patients who had received immune checkpoint inhibition. Thus, we found reduced viral RNA clearance and specific alterations in T cell and cytokine responses in cancer patients hospitalized with COVID-19 compared with matched controls with COVID-19. This dysregulated T cell response in cancer patients, which may reflect immune modulation due to chronic antigen stimulation as well as cancer therapies, may lead to altered virologic and clinical outcomes in this population.
ABSTRACT
Background. Streptococcus pneumoniae (pneumococcus) is a common colonizer of the upper respiratory tract and can progress to cause invasive and mucosal disease. Additionally, infection with pneumococcus can complicate respiratory viral infections (influenza, respiratory syncytial virus, etc.) by exacerbating the initial disease. Limited data exist describing the potential relationship of SARS-CoV-2 infection with pneumococcus and the role of co-infection in influencing COVID-19 severity. Methods. Inpatients and healthcare workers testing positive for SARS-CoV-2 during March-August 2020 were tested for pneumococcus through culture-enrichment of saliva followed by RT-qPCR (to identify carriage) and for inpatients only, serotype-specific urine antigen detection (UAD) assays (to identify pneumococcal pneumonia). A multinomial multivariate regression model was used to examine the relationship between pneumococcal detection and COVID-19 severity. Results. Among the 126 subjects who tested positive for SARS-CoV-2, the median age was 62 years;54.9% of subjects were male;88.89% were inpatients;23.5% had an ICU stay;and 13.5% died. Pneumococcus was detected in 17 subjects (13.5%) by any method, including 5 subjects (4.0%) by RT-qPCR and 12 subjects (13.6%) by UAD. Little to no bacterial growth was observed on 21/235 culture plates. Detection by UAD was associated with both moderate and severe COVID-19 disease while RT-qPCR detection in saliva was not associated with severity. None of the 12 individuals who were UAD-positive died. Conclusion. Pneumococcal pneumonia (as determined by UAD) continues to occur during the ongoing pandemic and may be associated with more serious COVID-19 outcomes. Detection of pneumococcal carriage may be masked by high levels of antibiotic use. Future studies should better characterize the relationship between pneumococcus and SARS-CoV-2 across all disease severity levels.
ABSTRACT
Background. Quickly detecting and isolating individuals positive for SARSCoV-2 is essential for limiting virus spread. Policy makers rely on the number of active cases to make decisions, and individuals use this information to evaluate risk should they return to public spaces. Robust testing strategies have been plagued with limited authorized diagnostic assays and high test prices, with large-scale implementation hampered by worldwide supply chain issues. Methods. Having identified its potential early in the pandemic, we simplified saliva-based COVID-19 diagnostic testing by (1) not requiring collection tubes with preservatives, (2) replacing nucleic acid extraction with a simple enzymatic and heating step, and (3) testing specimens for SARS-CoV-2 in dualplex RT-qPCR. Moreover, we validated this approach ("SalivaDirect") with reagents and instruments from multiple vendors to circumvent supply chain disruptions. Results. SalivaDirect's simplified protocol does not compromise on sensitivity. In our hospital cohort, we found a high positive agreement (94%) between saliva tested with SalivaDirect and nasopharyngeal swabs tested with a commercial RT-qPCR kit. With the National Basketball Association we tested 3,779 saliva specimens from healthy individuals and detected low rates of invalid (0.3%) and false-positive (< 0.05%) results. Using comparative assays and sample types, we also demonstrated SalivaDirect to efficiently detect SARS-CoV-2 in asymptomatic individuals. SalivaDirect is a simplified method for SARS-CoV-2 detection (A) Schematic overview of SalivaDirect workflow depicting the main steps of mixing saliva with proteinase K, heat inactivation, and dualplex qRT-PCR testing. Figure created with Biorender.com. (B) SARS-CoV-2 is stable in saliva for at least 7 days at 4C, room temperature (RT;19C), and 30C without addition of stabilizing buffers. Spiked-in saliva samples of low virus concentrations (12, 25, and 50 SARSCoV-2 copies/mL) were kept at the indicated temperature for 7 days and then tested with SalivaDirect. N1 cycle threshold (Ct) values were lower when kept for 7 days at 30C as compared to fresh specimens (Kruskal-Wallis;p = 0.03). Horizontal bars indicate the median. (C) Comparing Ct values for saliva treated with proteinase K and heat as compared to nucleic extraction yields higher N1 Ct values without extraction (Wilcoxon;p < 0.01). (D) Testing extracted nucleic acid from saliva with the N1 primer-probe set (singleplex) as compared to a multiplex assay showed stronger N1 detection in multiplex (Wilcoxon;p < 0.01). The dotted line in (B)-(D) indicates the limit of detection. Conclusion. Saliva is a valid alternative to swabs for SARS-CoV-2 screening. Importantly, SalivaDirect enables labs to utilize existing infrastructure, improving test implementation time and requiring limited investment to scale-up to meet mass testing needs. With the safe and reliable self-collection of saliva, our vision is to help provide accessible and equitable testing solutions, especially in low-resource and remote settings.
ABSTRACT
Background: Smell loss has been recognized as an important, and potentially early, sign of COVID-19. However, to date smell loss has only been assessed in retrospective, COVID+ cohorts, and largely through self-report. The objective of this study was to implement a daily standardized behavioral test of smell sensitivity in healthcare workers (HCW) to capture changes in smell sensitivity over time and to assess whether these changes occur prior to positive COVID test. Methods: The study enrolled 500 high-risk COVID-negative HCW during the COVID-19 epidemic in Connecticut, beginning March 28, 2020 (80% F, mean age 38, 58% nurses). Initially, HCW received a daily symptom questionnaire with parosmia screening questions. On April 23 we introduced the “Jiffy”, a daily at-home psychophysical test of smell sensitivity, where olfactory stimuli are sampled and rated for perceived intensity. SARS-CoV-2 infection was tested every three days by PCR of nasopharyngeal swabs or saliva Results: Of the first 500 enrolled HCW, 376 HCW (75%) completed the Jiffy 4528 times (mean 12 times/HCW). 17/500 HCW (3.4%) had a COVID+ test, of which 9/17 (53%) reported smell loss through the Jiffy or the daily symptom survey. 6/9 (67%) reported smell loss that preceded or was concurrent with a COVID+ test. 8/17 COVID+ HCW completed the Jiffy, with 5/8 (63%) reporting reductions in smell versus 42/368 (11%) COVID- HCW (OR=13, 95% CI: 2.4-85, p=.001). COVID+ HCW rated their greatest reduction in smell sensitivity as slight (40%) and severe (60%), versus slight (88%) and moderate (12%) in COVID- HCW. 16/17 COVID+ HCW completed a daily symptom survey (mean 14 times/HCW), with 8/16 (50%) ever reporting parosmia versus 90/466 (19%) of COVID- HCW (OR=4.2, 95% CI: 1.3-13, p=.007). Overall, parosmia was the first reported symptom in 3/13 (23%) COVID+ HCW who reported symptoms. Conclusion: We conducted a prospective study of smell testing in a population at high risk for COVID-19 using two parallel approaches. Our results demonstrate the feasibility of at-home smell testing for assessing parosmia during COVID-19, in some cases even prior to a positive PCR result. Given the urgent need for widespread, lowcost, non-invasive testing for COVID-19, we are now developing an easy-to-use app to distribute this survey more widely to high-risk populations. (Table Presented).