Experiences of Health Departments on Community Engagement and Implementation of a COVID-19 Self-testing Program.

CONTEXT: Health departments (HDs) work on the front lines to ensure the health of their communities, providing a unique perspective to public health response activities. Say Yes! COVID Test (SYCT) is a US federally funded program providing free COVID-19 self-tests to communities with high COVID-19 transmission, low vaccination rates, and high social vulnerability. The collaboration with 9 HDs was key for the program distribution of 5.8 million COVID-19 self-tests between March 31 and November 30, 2021. OBJECTIVE: The objective of this study was to gather qualitative in-depth information on the experiences of HDs with the SYCT program to better understand the successes and barriers to implementing community-focused self-testing programs. DESIGN: Key informant (KI) interviews. SETTING: Online interviews conducted between November and December 2021. PARTICIPANTS: Sixteen program leads representing 9 HDs were purposefully sampled as KIs. KIs completed 60-minute structured interviews conducted by one trained facilitator and recorded. MAIN OUTCOME MEASURES: Key themes and lessons learned were identified using grounded theory. RESULTS: Based on perceptions of KIs, HDs that maximized community partnerships for test distribution were more certain that populations at a higher risk for COVID-19 were reached. Where the HD relied predominantly on direct-to-consumer distribution, KIs were less certain that communities at higher risk were served. Privacy and anonymity in testing were themes linked to higher perceived community acceptance. KIs reported that self-test demand and distribution levels increased during higher COVID-19 transmission levels. CONCLUSION: HDs that build bridges and engage with community partners and trusted leaders are better prepared to identify and link high-risk populations with health services and resources. When collaborating with trusted community organizations, KIs perceived that the SYCT program overcame barriers such as mistrust of government intervention and desire for privacy and motivated community members to utilize this resource to protect themselves against COVID-19.

Multistate Outbreak of SARS-CoV-2 Infections, Including Vaccine Breakthrough Infections, Associated with Large Public Gatherings, United States.

During July 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.617.2 variant infections, including vaccine breakthrough infections, occurred after large public gatherings in Provincetown, Massachusetts, USA, prompting a multistate investigation. Public health departments identified primary and secondary cases by using coronavirus disease surveillance data, case investigations, and contact tracing. A primary case was defined as SARS-CoV-2 detected <14 days after travel to or residence in Provincetown during July 3-17. A secondary case was defined as SARS-CoV-2 detected <14 days after close contact with a person who had a primary case but without travel to or residence in Provincetown during July 3-August 10. We identified 1,098 primary cases and 30 secondary cases associated with 26 primary cases among fully and non-fully vaccinated persons. Large gatherings can have widespread effects on SARS-CoV-2 transmission, and fully vaccinated persons should take precautions, such as masking, to prevent SARS-CoV-2 transmission, particularly during substantial or high transmission.

Multistate Outbreak of SARS-CoV-2 B.1.1.529 (Omicron) Variant Infections Among Persons in a Social Network Attending a Convention - New York City, November 18-December 20, 2021.

On December 2, 2021, the Minnesota Department of Health (MDH) notified CDC of a COVID-19 case caused by sequence-confirmed SARS-CoV-2 B.1.1.529 (Omicron) variant in a Minnesota resident (patient A), the first such case identified in the state and one of the earliest identified in the United States. Patient A had attended a large indoor convention in New York, New York with approximately 53,000 attendees from 52 U.S jurisdictions and 30 foreign countries during November 19-21, 2021, and had close contact† during 5 days with 29 fellow attendees. The convention required attendees to have received ≥1 COVID-19 vaccine dose and enforced mask-use while indoors. On November 22, these close contact attendees were directly and immediately notified by patient A of their exposure to SARS-CoV-2, and they sought testing over the next few days while quarantined or isolated. As part of the larger investigation into SARS-CoV-2 transmission at the convention, a subinvestigation was conducted during December by CDC, MDH, and respective state and local health departments to characterize the epidemiology of Omicron variant infection among this group of close contacts and determine the extent of secondary household transmission. Among 30 convention attendees that included patient A (the index patient) and the 29 other close contacts, 23 were interviewed, among whom all were fully vaccinated, including 11 (48%) who had received a booster dose; all 23 sought testing, and 16 (70%) received a positive SARS-CoV-2 test result. Fewer attendees who had received a booster dose before the convention received a positive test result (six of 11) compared with those who had not received a booster dose (10 of 12). The 16 attendees with positive test results had a total of 20 household contacts, 18 of whom sought testing after exposure; six received a positive test result for SARS-CoV-2. None of the persons with positive test results was hospitalized or died. There was limited convention-associated transmission identified outside of this cluster; the larger investigation included cases of both SARS-CoV-2 B.1.617.2 (Delta) and Omicron, and all Omicron cases were associated with this group (1). Data from this investigation reinforces the importance of COVID-19 booster doses in combination with early notification and other multicomponent prevention measures to limit transmission and prevent severe illness from Omicron and other SARS-CoV-2 variants.

Epidemiologic Findings from Case Investigations and Contact Tracing for First 200 Cases of Coronavirus Disease, Santa Clara County, California, USA.

In January 2020, Santa Clara County, California, USA, began identifying laboratory-confirmed coronavirus disease among residents. County staff conducted case and contact investigations focused on households and collected detailed case demographic, occupation, exposure, and outcome information. We describe the first 200 test-positive cases during January 31-March 20, 2020, to inform future case and contact investigations. Probable infection sources included community transmission (104 cases), known close contact with a confirmed case-patient (66 cases), and travel (30 cases). Disease patterns across race and ethnicity, occupational, and household factors suggested multiple infection risk factors. Disproportionately high percentages of case-patients from racial and ethnic subgroups worked outside the home (Hispanic [86%] and Filipino [100%]); household transmission was more common among persons from Vietnam (53%). Even with the few initial cases, detailed case and contact investigations of household contacts capturing occupational and disaggregated race and ethnicity data helped identify at-risk groups and focused solutions for disease control.

COVID-19-Associated Deaths After SARS-CoV-2 Infection During Pregnancy - Mississippi, March 1, 2020-October 6, 2021.

Pregnant and recently pregnant women are at increased risk for severe illness and death from COVID-19 compared with women who are not pregnant or were not recently pregnant (1,2). CDC recommends COVID-19 vaccination for women who are pregnant, recently pregnant, trying to become pregnant, or might become pregnant in the future.*,† This report describes 15 COVID-19-associated deaths after infection with SARS-CoV-2 (the virus that causes COVID-19) during pregnancy in Mississippi during March 1, 2020-October 6, 2021.

Community Transmission of SARS-CoV-2 Associated with a Local Bar Opening Event - Illinois, February 2021.

During February 2021, an opening event was held indoors at a rural Illinois bar that accommodates approximately 100 persons. The Illinois Department of Public Health (IDPH) and local health department staff members investigated a COVID-19 outbreak associated with this opening event. Overall, 46 COVID-19 cases were linked to the event, including cases in 26 patrons and three staff members who attended the opening event and 17 secondary cases. Four persons with cases had COVID-19-like symptoms on the same day they attended the event. Secondary cases included 12 cases in eight households with children, two on a school sports team, and three in a long-term care facility (LTCF). Transmission associated with the opening event resulted in one school closure affecting 650 children (9,100 lost person-days of school) and hospitalization of one LTCF resident with COVID-19. These findings demonstrate that opening up settings such as bars, where mask wearing and physical distancing are challenging, can increase the risk for community transmission of SARS-CoV-2, the virus that causes COVID-19. As community businesses begin to reopen, a multicomponent approach should be emphasized in settings such as bars to prevent transmission* (1). This includes enforcing consistent and correct mask use, maintaining ≥6 ft of physical distance between persons, reducing indoor bar occupancy, prioritizing outdoor seating, improving building ventilation, and promoting behaviors such as staying at home when ill, as well as implementing contact tracing in combination with isolation and quarantine when COVID-19 cases are diagnosed.

Zika virus detection in amniotic fluid and Zika-associated birth defects.

BACKGROUND: Zika virus infection during pregnancy can cause serious birth defects, which include brain and eye abnormalities. The clinical importance of detection of Zika virus RNA in amniotic fluid is unknown. OBJECTIVE: The purpose of this study was to describe patterns of Zika virus RNA testing of amniotic fluid relative to other clinical specimens and to examine the association between Zika virus detection in amniotic fluid and Zika-associated birth defects. Our null hypothesis was that Zika virus detection in amniotic fluid was not associated with Zika-associated birth defects. STUDY DESIGN: We conducted a retrospective cohort analysis of women with amniotic fluid specimens submitted to Colombia's National Institute of Health as part of national Zika virus surveillance from January 2016 to January 2017. Specimens (maternal serum, amniotic fluid, cord blood, umbilical cord tissue, and placental tissue) were tested for the presence of Zika virus RNA with the use of a singleplex or multiplex real-time reverse transcriptase-polymerase chain reaction assay. Birth defect information was abstracted from maternal prenatal and infant birth records and reviewed by expert clinicians. Chi-square and Fisher's exact tests were used to compare the frequency of Zika-associated birth defects (defined as brain abnormalities [with or without microcephaly, but excluding neural tube defects and their associated findings] or eye abnormalities) by frequency of detection of Zika virus RNA in amniotic fluid. RESULTS: Our analysis included 128 women with amniotic fluid specimens. Seventy-five women (58%) had prenatally collected amniotic fluid; 42 women (33%) had amniotic fluid collected at delivery, and 11 women (9%) had missing collection dates. Ninety-one women had both amniotic fluid and other clinical specimens submitted for testing, which allowed for comparison across specimen types. Of those 91 women, 68 had evidence of Zika virus infection based on detection of Zika virus RNA in ≥1 specimen. Testing of amniotic fluid that was collected prenatally or at delivery identified 39 of these Zika virus infections (57%; 15 [22%] infections were identified only in amniotic fluid), and 29 infections (43%) were identified in other specimen types and not amniotic fluid. Among women who were included in the analysis, 89 had pregnancy outcome information available, which allowed for the assessment of the presence of Zika-associated birth defects. Zika-associated birth defects were significantly (P<.05) more common among pregnancies with Zika virus RNA detected in amniotic fluid specimens collected prenatally (19/32 specimens; 59%) than for those with no laboratory evidence of Zika virus infection in any specimen (6/23 specimens; 26%), but the proportion was similar in pregnancies with only Zika virus RNA detected in specimens other than amniotic fluid (10/23 specimens; 43%). Although Zika-associated birth defects were more common among women with any Zika virus RNA detected in amniotic fluid specimens (ie, collected prenatally or at delivery; 21/43 specimens; 49%) than those with no laboratory evidence of Zika virus infection (6/23 specimens; 26%), this comparison did not reach statistical significance (P=.07). CONCLUSION: Testing of amniotic fluid provided additional evidence for maternal diagnosis of Zika virus infection. Zika-associated birth defects were more common among women with Zika virus RNA that was detected in prenatal amniotic fluid specimens than women with no laboratory evidence of Zika virus infection, but similar to women with Zika virus RNA detected in other, nonamniotic fluid specimen types.

Disparities in Incidence of COVID-19 Among Underrepresented Racial/Ethnic Groups in Counties Identified as Hotspots During June 5-18, 2020 - 22 States, February-June 2020.

During January 1, 2020-August 10, 2020, an estimated 5 million cases of coronavirus disease 2019 (COVID-19) were reported in the United States.* Published state and national data indicate that persons of color might be more likely to become infected with SARS-CoV-2, the virus that causes COVID-19, experience more severe COVID-19-associated illness, including that requiring hospitalization, and have higher risk for death from COVID-19 (1-5). CDC examined county-level disparities in COVID-19 cases among underrepresented racial/ethnic groups in counties identified as hotspots, which are defined using algorithmic thresholds related to the number of new cases and the changes in incidence.† Disparities were defined as difference of ≥5% between the proportion of cases and the proportion of the population or a ratio ≥1.5 for the proportion of cases to the proportion of the population for underrepresented racial/ethnic groups in each county. During June 5-18, 205 counties in 33 states were identified as hotspots; among these counties, race was reported for ≥50% of cumulative cases in 79 (38.5%) counties in 22 states; 96.2% of these counties had disparities in COVID-19 cases in one or more underrepresented racial/ethnic groups. Hispanic/Latino (Hispanic) persons were the largest group by population size (3.5 million persons) living in hotspot counties where a disproportionate number of cases among that group was identified, followed by black/African American (black) persons (2 million), American Indian/Alaska Native (AI/AN) persons (61,000), Asian persons (36,000), and Native Hawaiian/other Pacific Islander (NHPI) persons (31,000). Examining county-level data disaggregated by race/ethnicity can help identify health disparities in COVID-19 cases and inform strategies for preventing and slowing SARS-CoV-2 transmission. More complete race/ethnicity data are needed to fully inform public health decision-making. Addressing the pandemic's disproportionate incidence of COVID-19 in communities of color can reduce the community-wide impact of COVID-19 and improve health outcomes.

Current trends in translational research in leptospirosis.

PURPOSE OF REVIEW: In the past years, the importance of studying leptospirosis in a translational context has become more evident. This review addresses recent findings in the study of leptospirosis infection, focusing on those applicable to public health, or that will affect management and diagnosis of cases of leptospirosis. RECENT FINDINGS: We review here recent findings regarding translational aspects of leptospirosis research. Briefly, PCR or a combination of serology and PCR seem to have a higher sensitivity than the current gold standard (microagglutination test). More clinical trials are needed to determine the best treatment for mild and severe leptospirosis. Dendritic cells and γδ T cells seem to have an important role in the immune response to leptospirosis. Environmental assessment is emerging as a very useful tool. SUMMARY: In order to understand leptospirosis, multiple aspects need to be considered, including host, pathogen and environment. In this review, we will address newer diagnostics, current advances in immunology and treatment and the growing role of environmental assessment.

The Impact of Community-Based Testing Sites and Gift Incentives on COVID-19 Testing Uptake in Maryland, April 29 - May 9, 2021.

PURPOSE: Information on incentives for COVID-19 testing is needed to understand effective practices that encourage testing uptake. We describe characteristics of those who received an incentive after performing a rapid antigen test. DESIGN: Cross-sectional descriptive analysis of survey data. SETTING: During April 29-May 9, 2021, COVID-19 rapid antigen testing was offered in 2 Maryland cities. SAMPLE: Convenience sample of 553 adults (≥18 years) who tested and received an incentive; 93% consented to survey. MEASURES: Survey questions assessed reasons for testing, testing history, barriers, and demographics. ANALYSIS: Robust Poisson regressions were used to determine characteristic differences based on testing history and between participants who would re-test in the future without an incentive vs participants who would not. RESULTS: The most common reasons for testing were the desire to be tested (n = 280; 54%) and convenience of location (n = 146; 28%). Those motivated by an incentive to test (n = 110; 21%) were 5.83 times as likely to state they would not test again without an incentive, compared to those with other reasons for testing (95% CI: 2.67-12.72, P < .001). CRITICAL LIMITATIONS: No comparative study group. CONCLUSION: Results indicate internal motivation and convenience were prominent factors supporting testing uptake. Incentives may increase community testing participation, particularly among people who have never tested. Keywords COVID-19, pandemic, incentives, health behavior, community testing.

Expression of sialic acids and other nonulosonic acids in Leptospira.

BACKGROUND: Sialic acids are negatively charged nine carbon backbone sugars expressed on mammalian cell surfaces. Sialic acids are part of a larger family of nonulosonic acid (NulO) molecules that includes pseudaminic and legionaminic acids. Microbial expression of sialic acids and other nonulosonic acids has been shown to contribute to host-microbe interactions in a variety of contexts, including participation in colonization, immune subversion, and behaviors such as biofilm formation, autoagglutination and motility. Previous research has suggested that some spirochetes may also express these molecules. RESULTS: Here we use a combination of molecular tools to investigate the presence of NulO biosynthetic gene clusters among clinical and saprophytic isolates of the genus Leptospira. Polymerase chain reaction and Southern blotting suggested that a variety of leptospires encoded NulO biosynthetic pathways. High performance liquid chromatography and mass spectrometry analyses provided biochemical evidence that di-N-acetylated NulO molecules are expressed at relatively high levels by L. interrogans serovar Lai strain 55601, and at lower levels by L. alexanderi serovar Manhao and L. fainei serovar Hurstbridge. Endogenous expression of N-acetylneuraminic acid (Neu5Ac, the most common sialic acid) was documented in L. interrogans serovar Copenhageni strain L1-130. Neu5Ac biosynthesis is also supported by a unique gene fusion event resulting in an enzyme with an N-terminal N-acetylneuraminic acid synthase domain and a C-terminal phosphatase domain. This gene fusion suggests that L. interrogans uses a Neu5Ac biosynthetic pathway more similar to animals than to other bacteria. Analysis of the composition and phylogeny of putative NulO biosynthetic gene clusters in L. interrogans serovar Lai and serovar Copenhageni revealed that both strains have complete biosynthetic pathways for legionamimic acid synthesis, a molecule with the same stereochemistry as sialic acid. Lectin-based affinity purification of NulO-modified molecules, followed by mass spectrometric identification suggests post-translational modification of surface lipoproteins, including Loa22. CONCLUSIONS: Leptospira species encode NulO biosynthetic pathways and synthesize multiple NulO molecules including sialic acid. Additional studies are needed to clarify the exact context and functional significance of NulO expression. These findings have implications for immune evasion during systemic leptospirosis.

Change in unemployment by social vulnerability among United States counties with rapid increases in COVID-19 incidence-July 1-October 31, 2020.

OBJECTIVE: During the COVID-19 pandemic, the unemployment rate in the United States peaked at 14.8% in April 2020. We examined patterns in unemployment following this peak in counties with rapid increases in COVID-19 incidence. METHOD: We used CDC aggregate county data to identify counties with rapid increases in COVID-19 incidence (rapid riser counties) during July 1-October 31, 2020. We used a linear regression model with fixed effect to calculate the change of unemployment rate difference in these counties, stratified by the county's social vulnerability (an indicator compiled by CDC) in the two months before the rapid riser index month compared to the index month plus one month after the index month. RESULTS: Among the 585 (19% of U.S. counties) rapid riser counties identified, the unemployment rate gap between the most and least socially vulnerable counties widened by 0.40 percentage point (p<0.01) after experiencing a rapid rise in COVID-19 incidence. Driving the gap were counties with lower socioeconomic status, with a higher percentage of people in racial and ethnic minority groups, and with limited English proficiency. CONCLUSION: The widened unemployment gap after COVID-19 incidence rapid rise between the most and least socially vulnerable counties suggests that it may take longer for socially and economically disadvantaged communities to recover. Loss of income and benefits due to unemployment could hinder behaviors that prevent spread of COVID-19 (e.g., seeking healthcare) and could impede response efforts including testing and vaccination. Addressing the social needs within these vulnerable communities could help support public health response measures.

Central monitoring in a randomized, open-label, controlled phase 3 clinical trial for a treatment-shortening regimen for pulmonary tuberculosis.

INTRODUCTION: With the growing use of online study management systems and rapid availability of data, timely data review and quality assessments are necessary to ensure proper clinical trial implementation. In this report we describe central monitoring used to ensure protocol compliance and accurate data reporting, implemented during a large phase 3 clinical trial. MATERIAL AND METHODS: The Tuberculosis Trials Consortium (TBTC) Study 31/AIDS Clinical Trials Group (ACTG) study A5349 (S31) is an international, multi-site, randomized, open-label, controlled, non-inferiority phase 3 clinical trial comparing two 4-month regimens to a standard 6 month regimen for treatment of drug-susceptible tuberculosis (TB) among adolescents and adults with a sample size of 2500 participants. RESULTS: Central monitoring utilized primary study data in a five-tiered approach, including (1) real-time data checks & topic-specific intervention reports, (2) missing forms reports, (3) quality assurance metrics, (4) critical data reports and (5) protocol deviation identification, aimed to detect and resolve quality challenges. Over the course of the study, 240 data checks and reports were programed across the five tiers used. DISCUSSION: This use of primary study data to identify issues rapidly allowed the study sponsor to focus quality assurance and data cleaning activities on prioritized data, related to protocol compliance and accurate reporting of study results. Our approach enabled us to become more efficient and effective as we informed sites about deviations, resolved missing or inconsistent data, provided targeted guidance, and gained a deeper understanding of challenges experienced at clinical trial sites. TRIAL REGISTRATION: This trial was registered with ClinicalTrials.gov (Identifier: NCT02410772) on April 8, 2015.

Pregnancy, Birth, Infant, and Early Childhood Neurodevelopmental Outcomes among a Cohort of Women with Symptoms of Zika Virus Disease during Pregnancy in Three Surveillance Sites, Project Vigilancia de Embarazadas con Zika (VEZ), Colombia, 2016-2018.

Project Vigilancia de Embarazadas con Zika (VEZ), an intensified surveillance of pregnant women with symptoms of the Zika virus disease (ZVD) in Colombia, aimed to evaluate the relationship between symptoms of ZVD during pregnancy and adverse pregnancy, birth, and infant outcomes and early childhood neurodevelopmental outcomes. During May-November 2016, pregnant women in three Colombian cities who were reported with symptoms of ZVD to the national surveillance system, or with symptoms of ZVD visiting participating clinics, were enrolled in Project VEZ. Data from maternal and pediatric (up to two years of age) medical records were abstracted. Available maternal specimens were tested for the presence of the Zika virus ribonucleic acid and/or anti-Zika virus immunoglobulin antibodies. Of 1213 enrolled pregnant women with symptoms of ZVD, 1180 had a known pregnancy outcome. Results of the Zika virus laboratory testing were available for 569 (48.2%) pregnancies with a known pregnancy outcome though testing timing varied and was often distal to the timing of symptoms; 254 (21.5% of the whole cohort; 44.6% of those with testing results) were confirmed or presumptive positive for the Zika virus infection. Of pregnancies with a known outcome, 50 (4.2%) fetuses/infants had Zika-associated brain or eye defects, which included microcephaly at birth. Early childhood adverse neurodevelopmental outcomes were more common among those with Zika-associated birth defects than among those without and more common among those with laboratory evidence of a Zika virus infection compared with the full cohort. The proportion of fetuses/infants with any Zika-associated brain or eye defect was consistent with the proportion seen in other studies. Enhancements to Colombia's existing national surveillance enabled the assessment of adverse outcomes associated with ZVD in pregnancy.

Novel hypertonic saline-sodium hydroxide (HS-SH) method for decontamination and concentration of sputum samples for Mycobacterium tuberculosis microscopy and culture.

This study evaluated a new decontamination and concentration (DC) method for sputum microscopy and culture. Sputum samples from patients with suspected pulmonary tuberculosis (TB) (n=106) were tested using the proposed hypertonic saline-sodium hydroxide (HS-SH) DC method, the recommended N-acetyl-L-cysteine-sodium citrate-sodium hydroxide (NALC-NaOH) DC method and unconcentrated direct smear (Ziehl-Neelsen) techniques for the presence of mycobacteria using Löwenstein-Jensen culture and light microscopy. Of 94 valid specimens, 21 (22.3%) were positive in culture and were further characterized as Mycobacterium tuberculosis. The sensitivity for acid-fast bacilli (AFB) smears was increased from 28.6% using the direct method to 71.4% (HS-SH) and 66.7% (NALC-NaOH) using DC methods. Both concentration techniques were highly comparable for culture (kappa=0.794) and smear (kappa=0.631) for AFB. Thus the proposed HS-SH DC method improved the sensitivity of AFB microscopy compared with a routine unconcentrated direct smear; its performance was comparable to that of the NALC-NaOH DC method for AFB smears and culture, but it was methodologically simpler and less expensive, making it a promising candidate for evaluation by national TB control programmes in developing countries.

A simple and improved method for diagnosis of tuberculosis using hypertonic saline and sodium hydroxide to concentrate and decontaminate sputum.

We report the use of hypertonic saline and sodium hydroxide (HS-SH) for concentration-decontamination of sputum samples for the diagnosis of tuberculosis. This method increases detection of mycobacteria in smears compared to examination of direct smears, is comparable and reduces contamination in cultures compared to NALC-NaOH.

Whole Genome Shotgun Sequencing Shows Selection on Leptospira Regulatory Proteins During in vitro Culture Attenuation.

Leptospirosis is the most common zoonotic disease worldwide with an estimated 500,000 severe cases reported annually, and case fatality rates of 12-25%, due primarily to acute kidney and lung injuries. Despite its prevalence, the molecular mechanisms underlying leptospirosis pathogenesis remain poorly understood. To identify virulence-related genes in Leptospira interrogans, we delineated cumulative genome changes that occurred during serial in vitro passage of a highly virulent strain of L. interrogans serovar Lai into a nearly avirulent isogenic derivative. Comparison of protein coding and computationally predicted noncoding RNA (ncRNA) genes between these two polyclonal strains identified 15 nonsynonymous single nucleotide variant (nsSNV) alleles that increased in frequency and 19 that decreased, whereas no changes in allelic frequency were observed among the ncRNA genes. Some of the nsSNV alleles were in six genes shown previously to be transcriptionally upregulated during exposure to in vivo-like conditions. Five of these nsSNVs were in evolutionarily conserved positions in genes related to signal transduction and metabolism. Frequency changes of minor nsSNV alleles identified in this study likely contributed to the loss of virulence during serial in vitro culture. The identification of new virulence-associated genes should spur additional experimental inquiry into their potential role in Leptospira pathogenesis.