Characteristics, barriers, and career intentions of a national cohort of LGBTQ+ MD/PhD and DO/PhD trainees.

BACKGROUND: Lesbian, gay, bisexual, transgender, queer, non-binary, intersex, and/or asexual (LGBTQ+) individuals continue to suffer worse health outcomes compared to the general population. Data on LGBTQ+ individuals in medicine, particularly in medical training, remain sparse. National studies of LGBTQ+ students in MD/PhD and DO/PhD training programs have not been reported. METHODS: Trainees pursuing MD, DO, MD/PhD, and DO/PhD degrees at 32 nationally representative institutions completed a 70-item survey about their future career and anticipated challenges using an online survey tool from September 2012 to December 2014. There were 4,433 respondents to the survey. Of those, 2,837 completed the gender identity questions and 2,849 completed the sexual orientation questions. Completion of these questions was required for inclusion. Survey results were analyzed to examine differences between LGBTQ+ and non-LGBTQ+ medical and dual degree trainees. RESULTS: LGBTQ+ students were underrepresented among MD/PhD and DO/PhD trainees (8.70%) compared to the US population, though their representation was higher than among MD and DO trainees (5.20%). LGBTQ+ dual degree trainees endorsed the greatest interest in pursuing careers involving academic medicine, with varying career focuses including research, clinical duties, education, and advocacy. LGBTQ+ dual degree trainees prioritized opportunities in patient care, work-life balance, and research as the most important factors for their career selection. Importantly, a higher percentage of LGBTQ+ dual degree trainees (15.50%) identified sexual harassment as a past barrier to career advancement compared to their non-LGBTQ+ peers (8.27%). LGBTQ+ dual degree trainees were more likely to report having a mentor who advocated for them. CONCLUSIONS: LGBTQ+ physician scientist trainees remain under-represented and under-studied. It is vital that medical institutions devote more time and resources towards identifying and addressing the unique needs of this group in training. Training programs should be aware of the current and prior challenges faced by their LGBTQ+ dual degree trainees, work to overcome the unique barriers they face, highlight the strengths and unique perspectives they bring, and foster their professional growth and goals during and beyond their training.

Specific COVID-19 Symptoms Correlate with High Antibody Levels against SARS-CoV-2.

Lasting immunity will be critical for overcoming COVID-19. However, the factors associated with the development of high titers of anti-SARS-CoV-2 Abs and how long those Abs persist remain incompletely defined. In particular, an understanding of the relationship between COVID-19 symptoms and anti-SARS-CoV-2 Abs is limited. To address these unknowns, we quantified serum anti-SARS- CoV-2 Abs in clinically diverse COVID-19 convalescent human subjects 5 wk (n = 113) and 3 mo (n = 79) after symptom resolution with three methods: a novel multiplex assay to quantify IgG against four SARS-CoV-2 Ags, a new SARS-CoV-2 receptor binding domain-angiotensin converting enzyme 2 inhibition assay, and a SARS-CoV-2 neutralizing assay. We then identified clinical and demographic factors, including never-before-assessed COVID-19 symptoms, that consistently correlate with high anti-SARS-CoV-2 Ab levels. We detected anti-SARS-CoV-2 Abs in 98% of COVID-19 convalescent subjects 5 wk after symptom resolution, and Ab levels did not decline at 3 mo. Greater disease severity, older age, male sex, higher body mass index, and higher Charlson Comorbidity Index score correlated with increased anti-SARS-CoV-2 Ab levels. Moreover, we report for the first time (to our knowledge) that COVID-19 symptoms, most consistently fever, body aches, and low appetite, correlate with higher anti-SARS-CoV-2 Ab levels. Our results provide robust and new insights into the development and persistence of anti-SARS-CoV-2 Abs.

The landscape of antibody binding in SARS-CoV-2 infection.

The search for potential antibody-based diagnostics, vaccines, and therapeutics for pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has focused almost exclusively on the spike (S) and nucleocapsid (N) proteins. Coronavirus membrane (M), ORF3a, and ORF8 proteins are humoral immunogens in other coronaviruses (CoVs) but remain largely uninvestigated for SARS-CoV-2. Here, we use ultradense peptide microarray mapping to show that SARS-CoV-2 infection induces robust antibody responses to epitopes throughout the SARS-CoV-2 proteome, particularly in M, in which 1 epitope achieved excellent diagnostic accuracy. We map 79 B cell epitopes throughout the SARS-CoV-2 proteome and demonstrate that antibodies that develop in response to SARS-CoV-2 infection bind homologous peptide sequences in the 6 other known human CoVs. We also confirm reactivity against 4 of our top-ranking epitopes by enzyme-linked immunosorbent assay (ELISA). Illness severity correlated with increased reactivity to 9 SARS-CoV-2 epitopes in S, M, N, and ORF3a in our population. Our results demonstrate previously unknown, highly reactive B cell epitopes throughout the full proteome of SARS-CoV-2 and other CoV proteins.

Initial evaluation of a mobile SARS-CoV-2 RT-LAMP testing strategy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control in the United States remains hampered, in part, by testing limitations. We evaluated a simple, outdoor, mobile, colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay workflow where self-collected saliva is tested for SARS-CoV-2 RNA. From July 16 to November 19, 2020, 4,704 surveillance samples were collected from volunteers and tested for SARS-CoV-2 at 5 sites. A total of 21 samples tested positive for SARS-CoV-2 by RT-LAMP; 12 were confirmed positive by subsequent quantitative reverse-transcription polymerase chain reaction (qRT-PCR) testing, while 8 were negative for SARS-CoV-2 RNA, and 1 could not be confirmed because the donor did not consent to further molecular testing. We estimated the RT-LAMP assay's false-negative rate from July 16 to September 17, 2020 by pooling residual heat-inactivated saliva that was unambiguously negative by RT-LAMP into groups of 6 or less and testing for SARS-CoV-2 RNA by qRT-PCR. We observed a 98.8% concordance between the RT-LAMP and qRT-PCR assays, with only 5 of 421 RT-LAMP negative pools (2,493 samples) testing positive in the more sensitive qRT-PCR assay. Overall, we demonstrate a rapid testing method that can be implemented outside the traditional laboratory setting by individuals with basic molecular biology skills and can effectively identify asymptomatic individuals who would not typically meet the criteria for symptom-based testing modalities.

Fever, Diarrhea, and Severe Disease Correlate with High Persistent Antibody Levels against SARS-CoV-2.

Lasting immunity will be critical for overcoming the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, factors that drive the development of high titers of anti-SARS-CoV-2 antibodies and how long those antibodies persist remain unclear. Our objective was to comprehensively evaluate anti-SARS-CoV-2 antibodies in a clinically diverse COVID-19 convalescent cohort at defined time points to determine if anti-SARS-CoV-2 antibodies persist and to identify clinical and demographic factors that correlate with high titers. Using a novel multiplex assay to quantify IgG against four SARS-CoV-2 antigens, a receptor binding domain-angiotensin converting enzyme 2 inhibition assay, and a SARS-CoV-2 neutralization assay, we found that 98% of COVID-19 convalescent subjects had anti-SARS-CoV-2 antibodies five weeks after symptom resolution (n=113). Further, antibody levels did not decline three months after symptom resolution (n=79). As expected, greater disease severity, older age, male sex, obesity, and higher Charlson Comorbidity Index score correlated with increased anti-SARS-CoV-2 antibody levels. We demonstrated for the first time that COVID-19 symptoms, namely fever, abdominal pain, diarrhea and low appetite, correlated consistently with higher anti-SARS-CoV-2 antibody levels. Our results provide new insights into the development and persistence of anti-SARS-CoV-2 antibodies.

The landscape of antibody binding in SARS-CoV-2 infection.

The search for potential antibody-based diagnostics, vaccines, and therapeutics for pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has focused almost exclusively on the spike (S) and nucleocapsid (N) proteins. Coronavirus membrane (M), ORF3a, and ORF8 proteins are humoral immunogens in other coronaviruses (CoVs) but remain largely uninvestigated for SARS-CoV-2. Here we use ultradense peptide microarray mapping to show that SARS-CoV-2 infection induces robust antibody responses to epitopes throughout the SARS-CoV-2 proteome, particularly in M, in which one epitope achieved excellent diagnostic accuracy. We map 79 B cell epitopes throughout the SARS-CoV-2 proteome and demonstrate that antibodies that develop in response to SARS-CoV-2 infection bind homologous peptide sequences in the six other known human CoVs. We also confirm reactivity against four of our top-ranking epitopes by enzyme-linked immunosorbent assay (ELISA). Illness severity correlated with increased reactivity to nine SARS-CoV-2 epitopes in S, M, N, and ORF3a in our population. Our results demonstrate previously unknown, highly reactive B cell epitopes throughout the full proteome of SARS-CoV-2 and other CoV proteins.

Gender Can Influence Student Experiences in MD-PhD Training.

Background: Female physician-scientists have led major advances in medicine broadly and particularly in women's health. Women remain underrepresented in dual MD-PhD degree programs that train many physician-scientists despite gender parity among medical and biomedical research students. Materials and Methods: To explore how the training environment might be experienced differently for male and female students in one MD-PhD program, the authors analyzed gender differences in annual symposium speakers with exact binomial tests, student participation as question-askers at a weekly seminar with logistic regression, and number of publications with quasi-Poisson generalized linear models. They compared male and female students' perceptions of gender-based discrimination using a survey, including qualitative analysis of free text responses. The program consisted of 71 total students in the 2017-2018 and 2018-2019 academic years. Female students comprised 42.0% (81/191) of program matriculants from 1997 to 2019. Results: Male and female students were equally likely to present at the annual program symposium, but faculty (p = 0.001) and keynote (p = 0.012) presenters were more likely to be male. Compared with male counterparts, female students asked fewer seminar questions (p < 0.005) and female speakers received more questions (p = 0.03). Female students perceived less support and differed from men in reasons for asking or not asking seminar questions. Free text responses described repeated small acts of discrimination toward women with cumulative impact. Positive program changes followed presentation of findings to program leaders and students. Conclusions: The authors identified several aspects of one MD-PhD program that could discourage career or training persistence of female students. Increasing awareness of these issues was temporally related to positive programmatic changes.

Initial Evaluation of a Mobile SARS-CoV-2 RT-LAMP Testing Strategy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control in the United States remains hampered, in part, by testing limitations. We evaluated a simple, outdoor, mobile, colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay workflow where self-collected saliva is tested for SARS-CoV-2 RNA. From July 16, 2020, to November 19, 2020, surveillance samples (n = 4704) were collected from volunteers and tested for SARS-CoV-2 at 5 sites. Twenty-one samples tested positive for SARS-CoV-2 by RT-LAMP; 12 were confirmed positive by subsequent quantitative reverse-transcription polymerase chain reaction (qRT-PCR) testing, whereas 8 tested negative for SARS-CoV-2 RNA, and 1 could not be confirmed because the donor did not consent to further molecular testing. We estimated the false-negative rate of the RT-LAMP assay only from July 16, 2020, to September 17, 2020 by pooling residual heat-inactivated saliva that was unambiguously negative by RT-LAMP into groups of 6 or fewer and testing for SARS-CoV-2 RNA by qRT-PCR. We observed a 98.8% concordance between the RT-LAMP and qRT-PCR assays, with only 5 of 421 RT-LAMP-negative pools (2493 total samples) testing positive in the more-sensitive qRT-PCR assay. Overall, we demonstrate a rapid testing method that can be implemented outside the traditional laboratory setting by individuals with basic molecular biology skills and that can effectively identify asymptomatic individuals who would not typically meet the criteria for symptom-based testing modalities.

Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab samples.

SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Frequent surveillance of individuals attending work or school is currently unavailable to most people but will likely be necessary to reduce the ~50% of transmission that occurs when individuals are nonsymptomatic. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed primary samples with a limit of detection (LOD) of ~625 copies/µl, approximately 100-fold lower sensitivity than qRT-PCR. While less sensitive than extraction-based molecular methods, RT-LAMP without RNA extraction is fast and inexpensive. Here we also demonstrate that adding a lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Furthermore, purified RNA in this assay achieves a similar LOD to qRT-PCR. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 surveillance programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained.

Revealing fine-scale spatiotemporal differences in SARS-CoV-2 introduction and spread.

Evidence-based public health approaches that minimize the introduction and spread of new SARS-CoV-2 transmission clusters are urgently needed in the United States and other countries struggling with expanding epidemics. Here we analyze 247 full-genome SARS-CoV-2 sequences from two nearby communities in Wisconsin, USA, and find surprisingly distinct patterns of viral spread. Dane County had the 12th known introduction of SARS-CoV-2 in the United States, but this did not lead to descendant community spread. Instead, the Dane County outbreak was seeded by multiple later introductions, followed by limited community spread. In contrast, relatively few introductions in Milwaukee County led to extensive community spread. We present evidence for reduced viral spread in both counties following the statewide "Safer at Home" order, which went into effect 25 March 2020. Our results suggest patterns of SARS-CoV-2 transmission may vary substantially even in nearby communities. Understanding these local patterns will enable better targeting of public health interventions.

Discovery of a Novel Simian Pegivirus in Common Marmosets (Callithrix jacchus) with Lymphocytic Enterocolitis.

From 2010 to 2015, 73 common marmosets (Callithrix jacchus) housed at the Wisconsin National Primate Research Center (WNPRC) were diagnosed postmortem with lymphocytic enterocolitis. We used unbiased deep-sequencing to screen the blood of deceased enterocolitis-positive marmosets for viruses. In five out of eight common marmosets with lymphocytic enterocolitis, we discovered a novel pegivirus not present in ten matched, clinically normal controls. The novel virus, which we named Southwest bike trail virus (SOBV), is most closely related (68% nucleotide identity) to a strain of simian pegivirus A isolated from a three-striped night monkey (Aotus trivirgatus). We screened 146 living WNPRC common marmosets for SOBV, finding an overall prevalence of 34% (50/146). Over four years, 85 of these 146 animals died or were euthanized. Histological examination revealed 27 SOBV-positive marmosets from this cohort had lymphocytic enterocolitis, compared to 42 SOBV-negative marmosets, indicating no association between SOBV and disease in this cohort (p = 0.0798). We also detected SOBV in two of 33 (6%) clinically normal marmosets screened during transfer from the New England Primate Research Center, suggesting SOBV could be exerting confounding influences on comparisons of common marmoset studies from multiple colonies.

Distinct patterns of SARS-CoV-2 transmission in two nearby communities in Wisconsin, USA.

Evidence-based public health approaches that minimize the introduction and spread of new SARS-CoV-2 transmission clusters are urgently needed in the United States and other countries struggling with expanding epidemics. Here we analyze 247 full-genome SARS-CoV-2 sequences from two nearby communities in Wisconsin, USA, and find surprisingly distinct patterns of viral spread. Dane County had the 12th known introduction of SARS-CoV-2 in the United States, but this did not lead to descendant community spread. Instead, the Dane County outbreak was seeded by multiple later introductions, followed by limited community spread. In contrast, relatively few introductions in Milwaukee County led to extensive community spread. We present evidence for reduced viral spread in both counties, and limited viral transmission between counties, following the statewide Safer-at-Home public health order, which went into effect 25 March 2020. Our results suggest that early containment efforts suppressed the spread of SARS-CoV-2 within Wisconsin.

Antibody responses to Zika virus proteins in pregnant and non-pregnant macaques.

The specificity of the antibody response against Zika virus (ZIKV) is not well-characterized. This is due, in part, to the antigenic similarity between ZIKV and closely related dengue virus (DENV) serotypes. Since these and other similar viruses co-circulate, are spread by the same mosquito species, and can cause similar acute clinical syndromes, it is difficult to disentangle ZIKV-specific antibody responses from responses to closely-related arboviruses in humans. Here we use high-density peptide microarrays to profile anti-ZIKV antibody reactivity in pregnant and non-pregnant macaque monkeys with known exposure histories and compare these results to reactivity following DENV infection. We also compare cross-reactive binding of ZIKV-immune sera to the full proteomes of 28 arboviruses. We independently confirm a purported ZIKV-specific IgG antibody response targeting ZIKV nonstructural protein 2B (NS2B) that was recently reported in ZIKV-infected people and we show that antibody reactivity in pregnant animals can be detected as late as 127 days post-infection (dpi). However, we also show that these responses wane over time, sometimes rapidly, and in one case the response was elicited following DENV infection in a previously ZIKV-exposed animal. These results suggest epidemiologic studies assessing seroprevalence of ZIKV immunity using linear epitope-based strategies will remain challenging to interpret due to susceptibility to false positive results. However, the method used here demonstrates the potential for rapid profiling of proteome-wide antibody responses to a myriad of neglected diseases simultaneously and may be especially useful for distinguishing antibody reactivity among closely related pathogens.