Last data update: Sep 16, 2024. (Total: 47680 publications since 2009)
Records 1-30 (of 150 Records) |
Query Trace: Powers A [original query] |
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Central nervous system infection in a pediatric population in West Java
Alisjahbana DH , Nurmawati S , Milanti M , Djauhari H , Ledermann JP , Antonjaya U , Dewi YP , Johar E , Wiyatno A , Sriyani IY , Alisjahbana B , Safari D , Myint KSA , Powers AM , Hakim DD . PLoS Negl Trop Dis 2023 17 (11) e0011769 Central nervous system (CNS) viral infections are critical causes of morbidity and mortality in children; however, comprehensive data on etiology is lacking in developing countries such as Indonesia. To study the etiology of CNS infections in a pediatric population, 50 children admitted to two hospitals in Bandung, West Java, during 2017-2018 were enrolled in a CNS infection study. Cerebrospinal fluid and serum specimens were tested using molecular, serological, and virus isolation platforms for a number of viral and bacteriological agents. Causal pathogens were identified in 10 out of 50 (20%) and included cytomegalovirus (n = 4), Streptococcus pneumoniae (n = 2), tuberculosis (n = 2), Salmonella serotype Typhi (n = 1) and dengue virus (n = 1). Our study highlights the importance of using a wide range of molecular and serological detection methods to identify CNS pathogens, as well as the challenges of establishing the etiology of CNS infections in pediatric populations of countries with limited laboratory capacity. |
Improved mosquito housing and saliva collection method enhances safety while facilitating longitudinal assessment of individual mosquito vector competence for arboviruses
Ledermann JP , Burns PL , Perinet LC , Powers AM , Byers NM . Vector Borne Zoonotic Dis 2023 24 (1) 55-63 Background: Assessing the potential for mosquitoes to transmit medically important arboviruses is essential for understanding their threat to human populations. Currently, vector competence studies are typically performed by collecting saliva using a glass capillary tube system which involves sacrificing the mosquito at the time of saliva collection allowing only a single data point. These techniques also require handling infected mosquitoes and glass capillaries, constituting a safety risk. Materials and Methods: To improve the efficiency and safety of assessing vector competence, a novel containment and saliva collection approach for individually housed mosquitoes was developed. The improved housing, allowing longitudinal tracking of individual mosquitoes, consists of a 12-well Corning polystyrene plate sealed with a three-dimensional printed lid that holds organdy netting firmly against the rims of the wells. Results: This method provides excellent mosquito survival for five species of mosquitoes, with at least 79% of each species tested surviving for more than 2 weeks, comparable to the carton survival rates of ≥76%. When the plate housing system was used to assess vector infection, replication of West Nile virus (WNV) in mosquito tissues was similar to traditional containment mosquito housing. Mosquito saliva was collected using either blotting paper pads or traditional glass capillaries to assay viral transmission. The blotting paper collection showed similar or better sensitivity than the capillary method; in addition, longitudinal saliva samples could be collected from individual mosquitoes housed in the 12-well plates. Conclusions: The improved housing and saliva collection technique described herein provides a safer and more informative method for determining vector competence in mosquitoes. |
Developing a prototype pathogen plan and research priorities for the alphaviruses
Powers AM , Williamson LE , Carnahan RH , Crowe JE Jr , Hyde JL , Jonsson CB , Nasar F , Weaver SC . J Infect Dis 2023 228 S414-s426 The Togaviridae family, genus, Alphavirus, includes several mosquito-borne human pathogens with the potential to spread to near pandemic proportions. Most of these are zoonotic, with spillover infections of humans and domestic animals, but a few such as chikungunya virus (CHIKV) have the ability to use humans as amplification hosts for transmission in urban settings and explosive outbreaks. Most alphaviruses cause nonspecific acute febrile illness, with pathogenesis sometimes leading to either encephalitis or arthralgic manifestations with severe and chronic morbidity and occasional mortality. The development of countermeasures, especially against CHIKV and Venezuelan equine encephalitis virus that are major threats, has included vaccines and antibody-based therapeutics that are likely to also be successful for rapid responses with other members of the family. However, further work with these prototypes and other alphavirus pathogens should target better understanding of human tropism and pathogenesis, more comprehensive identification of cellular receptors and entry, and better understanding of structural mechanisms of neutralization. |
Characterization of a monoclonal antibody specific to California serogroup orthobunyaviruses and development as a chimeric immunoglobulin M-positive control in human diagnostics
Powers JA , Boroughs KL , Mikula S , Goodman CH , Davis EH , Thrasher EM , Hughes HR , Biggerstaff BJ , Calvert AE . Microbiol Spectr 2023 11 (5) e0196623 California serogroup viruses (CSGVs) of medical importance in the United States include La Crosse virus, Jamestown Canyon virus (JCV), California encephalitis virus, and snowshoe hare virus. Current diagnosis of CSGVs relies heavily on serologic techniques for detecting immunoglobulin M (IgM), an indication of a recent CSGV infection. However, human-positive control sera reactive to viruses in the serogroup are scarce because detection of recent infections is rare. Here, we describe the development of new murine monoclonal antibodies (MAbs) reactive to CSGVs and the engineering of a human-murine chimeric antibody by combining the variable regions of the broadly CSGV cross-reactive murine MAb, 3-3B6/2-3B2 and the constant region of the human IgM. MAb 3-3B6/2-3B2 recognizes a tertiary epitope on the Gn/Gc heterodimer, and epitopes important in JCV neutralization were mapped to the Gc glycoprotein. This engineered human IgM constitutively expressed in a HEK-293 stable cell line can replace human-positive control sera in diagnostic serological techniques such as IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA). Compared to the parent murine MAbs, the human-chimeric IgM antibody had identical serological activity to CSGVs in ELISA and demonstrated equivalent reactivity compared to human immune sera in the MAC-ELISA.IMPORTANCEOrthobunyaviruses in the California serogroup cause severe neurological disease in children and adults. While these viruses are known to circulate widely in North America, their occurrence is rare. Serological testing for CSGVs is hindered by the limited availability and volumes of human-positive specimens needed as controls in serologic assays. Here, we described the development of a murine monoclonal antibody cross-reactive to CSGVs engineered to contain the variable regions of the murine antibody on the backbone of human IgM. The chimeric IgM produced from the stably expressing HEK293 cell line was evaluated for use as a surrogate human-positive control in a serologic diagnostic test. |
Attitudes toward COVID-19 illness and COVID-19 vaccination among pregnant women: a cross-sectional multicenter study during August-December 2020 (preprint)
Battarbee AN , Stockwell MS , Varner M , Newes-Adeyi G , Daugherty M , Gyamfi-Bannerman C , Tita AT , Vorwaller K , Vargas C , Subramaniam A , Reichle L , Galang RR , Powers E , Lucca-Susana M , Parks M , Chen TJ , Razzaghi H , Dawood FS . medRxiv 2021 2021.03.26.21254402 Objective Evaluate pregnant women’s attitudes toward COVID-19 illness and vaccination and identify factors associated with vaccine acceptability.Study Design Cross-sectional survey among pregnant women enrolled in a prospective COVID-19 cohort study in Salt Lake City, UT, Birmingham, AL, and New York, NY, August 9– December 10, 2020. Women were eligible if they were 18-50 years old and <28 weeks of gestation. Upon enrollment, women completed surveys regarding concerns about COVID-19 illness and likelihood of getting COVID-19 vaccine if one were available during pregnancy. Vaccine acceptability was defined as a response of “very likely” or “somewhat likely” on a 4-point Likert scale. Factors associated with vaccine acceptability were assessed with multivariable logistic regression.Results Of 939 pregnant women eligible for the main cohort study, 915 (97%) consented to participate. Among these 915 women, 39% self-identified as White, 23% Black, 33% Hispanic, and 4% Other. Sixty-two percent received an influenza vaccine last season. Seventy-two percent worried about getting sick with COVID-19. If they were to get sick, 92% worried about harm to their pregnancy and 80% about harm to themselves. Only 41% reported they would get a vaccine. Of women who were unlikely to get vaccinated, the most frequently cited concern was vaccine safety for their pregnancy (82%). Non-Hispanic Black and Hispanic women had lower odds of accepting a vaccine compared with non-Hispanic White women (adjusted odds ratios (aOR) 0.4, 95%CI 0.2–0.6 for both). Receipt of influenza vaccine during the previous season was associated with higher odds of vaccine acceptability (aOR 2.1, 95%CI 1.5-3.0).Conclusion Although most pregnant women worried about COVID-19 illness, <50% were willing to get vaccinated during pregnancy. Racial and ethnic disparities in plans to accept COVID-19 vaccine highlight the need to prioritize strategies to address perceived barriers among groups at high risk for COVID-19.Competing Interest StatementAll authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf and declare: no support from any organization for the submitted work; author CGB has an unrestricted grant from SMFM/AMAG to study prematurity.Funding StatementFunding: This study was funded by the US Centers for Disease Control and Prevention through Contract # 75D30120C08150 with Abt Associates.Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Centralized Institutional Review Board approval was obtained (IRB-AAAT1906), and informed consent was obtained from all participants. The Columbia University Medical Center IRB served as the centralized IRB for this study.All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesData will automatically be sequestered by site. |
Neutralizing antibody responses to messenger RNA coronavirus disease 2019 vaccines versus severe acute respiratory syndrome coronavirus 2 infection among pregnant women and vaccine-induced antibody transfer to infants
Dawood FS , Tita A , Stockwell MS , Newes-Adeyi G , Wielgosz K , Gyamfi-Bannerman C , Battarbee A , Reichle L , Thornburg N , Ellington S , Galang RR , Vorwaller K , Vargas CY , Morrill T , Parks M , Powers E , Gibson M , Varner M . Open Forum Infect Dis 2023 10 (5) ofad204 BACKGROUND: Early coronavirus disease 2019 (COVID-19) vaccine trials excluded pregnant women, resulting in limited data about immunogenicity and maternal-fetal antibody transfer, particularly by gestational timing of vaccination. METHODS: In this multicenter observational immunogenicity study, pregnant and nonpregnant women receiving COVID-19 vaccines were prospectively enrolled. Participants had sera collected before vaccination, at 14-28 days after each vaccine dose, at delivery (umbilical cord and peripheral), and from their infants at 3 and 6 months. Geometric mean titers (GMTs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ID(50) neutralizing antibody (nAb) against D614G-like viruses were compared by participant characteristics. RESULTS: Overall, 23 nonpregnant and 85 pregnant participants (trimester of first vaccine dose: 10 first, 47 second, 28 third) were enrolled. Ninety-three percent (76/82 with blood samples) of pregnant participants had detectable SARS-CoV-2 nAb after 2 vaccine doses, but GMTs (95% confidence intervals) were lower in pregnant participants than nonpregnant participants (1722 [1136-2612] vs 4419 [2012-9703]; P = .04). By 3 and 6 months, 28% and 74% of infants, respectively, of vaccinated participants had no detectable nAb to D614G-like viruses. Among the 71 pregnant participants without detectable nAb before vaccination, cord blood GMTs at delivery were 5-fold higher among participants vaccinated during the third versus first trimester, and cord blood nAb titers appeared inversely correlated with weeks since first vaccine dose (R(2) = 0.06, P = .06). CONCLUSIONS: Though most pregnant women develop nAb after 2 doses of mRNA COVID-19 vaccines, this analysis suggests that infant protection from maternal vaccination varies by gestational timing of vaccination and wanes. Additional prevention strategies such as caregiver vaccination may warrant consideration to optimize infant protection. |
Emergency powers and the pandemic: Reflecting on state legislative reforms and the future of public health response
Davis M , Dedon L , Hoffman S , Baker-White A , Engleman D , Sunshine G . J Emerg Manag 2023 21 (7) 19-35 The first 2 years of combatting the COVID-19 pandemic necessitated an unprecedented use of emergency powers. States responded with an equally unprecedented flurry of legislative changes to the legal underpinnings of emergency response and public health authorities. In this article, we provide a brief background on the framework and use of governors and state health officials' emergency powers. We then analyze several key themes, including both the enhancement and restriction of powers, emerging from emergency management and public health legislation introduced in state and territorial legislatures. During the 2020 and 2021 state and territorial legislative sessions, we tracked legislation related to the emergency powers of governors and state health officials. Legislators introduced hundreds of bills impacting these powers, some enhancing and others restricting emergency powers. Enhancements included increasing vaccine access and expanding the pool of eligible medical professions that could administer vaccinations, strengthening public health investigation and enforcement authority for state agencies, and preclusion of local orders by orders at the state level. Restrictions included establishing oversight mechanisms for executive actions, limits on the duration of the emergency, limiting the scope of emergency powers allowed during a declared emergency, and other restraints. By describing these legislative trends, we hope to inform governors, state health officials, policymakers, and emergency managers about how changes in the law may impact future public health and emergency response capabilities. Understanding this new legal landscape is critical to effectively preparing for future threats. © 2023 Weston Medical Publishing. All rights reserved. |
Travel history among persons infected with SARS-CoV-2 variants of concern in the United States, December 2020-February 2021.
Dunajcik A , Haire K , Thomas JD , Moriarty LF , Springer Y , Villanueva JM , MacNeil A , Silk B , Nemhauser JB , Byrkit R , Taylor M , Queen K , Tong S , Lee J , Batra D , Paden C , Henderson T , Kunkes A , Ojo M , Firestone M , Martin Webb L , Freeland M , Brown CM , Williams T , Allen K , Kauerauf J , Wilson E , Jain S , McDonald E , Silver E , Stous S , Wadford D , Radcliffe R , Marriott C , Owes JP , Bart SM , Sosa LE , Oakeson K , Wodniak N , Shaffner J , Brown Q , Westergaard R , Salinas A , Hallyburton S , Ogale Y , Offutt-Powell T , Bonner K , Tubach S , Van Houten C , Hughes V , Reeb V , Galeazzi C , Khuntia S , McGee S , Hicks JT , Dinesh Patel D , Krueger A , Hughes S , Jeanty F , Wang JC , Lee EH , Assanah-Deane T , Tompkins M , Dougherty K , Naqvi O , Donahue M , Frederick J , Abdalhamid B , Powers AM , Anderson M . PLOS Glob Public Health 2023 3 (3) e0001252 The first three SARS-CoV-2 phylogenetic lineages classified as variants of concern (VOCs) in the United States (U.S.) from December 15, 2020 to February 28, 2021, Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1) lineages, were initially detected internationally. This investigation examined available travel history of coronavirus disease 2019 (COVID-19) cases reported in the U.S. in whom laboratory testing showed one of these initial VOCs. Travel history, demographics, and health outcomes for a convenience sample of persons infected with a SARS-CoV-2 VOC from December 15, 2020 through February 28, 2021 were provided by 35 state and city health departments, and proportion reporting travel was calculated. Of 1,761 confirmed VOC cases analyzed, 1,368 had available data on travel history. Of those with data on travel history, 1,168 (85%) reported no travel preceding laboratory confirmation of SARS-CoV-2 and only 105 (8%) reported international travel during the 30 days preceding a positive SARS-CoV-2 test or symptom onset. International travel was reported by 92/1,304 (7%) of persons infected with the Alpha variant, 7/55 (22%) with Beta, and 5/9 (56%) with Gamma. Of the first three SARS-CoV-2 lineages designated as VOCs in the U.S., international travel was common only among the few Gamma cases. Most persons infected with Alpha and Beta variant reported no travel history, therefore, community transmission of these VOCs was likely common in the U.S. by March 2021. These findings underscore the importance of global surveillance using whole genome sequencing to detect and inform mitigation strategies for emerging SARS-CoV-2 VOCs. |
Yata Virus (Family Rhabdoviridae, Genus Ephemerovirus) Isolation from Mosquitoes from Uganda, the First Reported Isolation since 1969.
Perinet LC , Mutebi JP , Powers AM , Lutwama JJ , Mossel EC . Diseases 2023 11 (1) As a part of a systematic study of mosquitoes and associated viruses in Uganda, a virus was isolated from a pool of Mansonia uniformis collected in July 2017, in the Kitgum District of northern Uganda. Sequence analysis determined that the virus is Yata virus (YATAV; Ephemerovirus yata; family Rhabdoviridae). The only previous reported isolation of YATAV was in 1969 in Birao, Central African Republic, also from Ma. uniformis mosquitoes. The current sequence is over 99% identical at the nucleotide level to the original isolate, indicating a high level of YATAV genomic stability. |
Identification of mosquito proteins that differentially interact with alphavirus nonstructural protein 3, a determinant of vector specificity.
Byers NM , Burns PL , Stuchlik O , Reed MS , Ledermann JP , Pohl J , Powers AM . PLoS Negl Trop Dis 2023 17 (1) e0011028 Chikungunya virus (CHIKV) and the closely related onyong-nyong virus (ONNV) are arthritogenic arboviruses that have caused significant, often debilitating, disease in millions of people. However, despite their kinship, they are vectored by different mosquito subfamilies that diverged 180 million years ago (anopheline versus culicine subfamilies). Previous work indicated that the nonstructural protein 3 (nsP3) of these alphaviruses was partially responsible for this vector specificity. To better understand the cellular components controlling alphavirus vector specificity, a cell culture model system of the anopheline restriction of CHIKV was developed along with a protein expression strategy. Mosquito proteins that differentially interacted with CHIKV nsP3 or ONNV nsP3 were identified. Six proteins were identified that specifically bound ONNV nsP3, ten that bound CHIKV nsP3 and eight that interacted with both. In addition to identifying novel factors that may play a role in virus/vector processing, these lists included host proteins that have been previously implicated as contributing to alphavirus replication. |
Vitamin C status of US adults assessed as part of the National Health and Nutrition Examination Survey remained unchanged between 2003-2006 and 2017-2018
Powers CD , Sternberg MR , Patel SB , Pfeiffer CM , Storandt RJ , Schleicher RL . J Appl Lab Med 2023 8 (2) 272-284 BACKGROUND: We compared serum vitamin C (VIC) status of the adult (≥20 y) US population in the National Health and Nutrition Examination Survey (NHANES) 2017-2018 with combined data from 2003-2004 and 2005-2006. METHODS: VIC was measured using HPLC with electrochemical detection. Mean data were stratified by age, sex, race/Hispanic origin, income, body mass index, dietary intake, supplement use, and smoking status. Prevalence of VIC deficiency (<11.4 μmol/L) was calculated. RESULTS: In NHANES 2017-2018, the mean VIC was 8 μmol/L higher in people ≥60 y compared with those 20-59 y of age, 10 μmol/L lower in men vs women, 8 μmol/L lower in low vs high income, 11 μmol/L lower in obese vs healthy weight, and 15 μmol/L lower in smokers vs nonsmokers. Differences in mean VIC across race/Hispanic origin groups ranged from 2 to 7 μmol/L. Mean VIC was 27 μmol/L higher with vitamin C-containing supplement use and positively associated (Spearman ρ = 0.33; P < 0.0001) with increasing dietary intake. The associations between mean VIC and the investigated covariates were generally consistent and the prevalence of deficiency was not significantly different between survey periods (6.8% vs 7.0%; P = 0.83). However, a few subgroups had double the risk. We found no significant survey differences in mean VIC (51.2 vs 54.0 μmol/L; P = 0.09). CONCLUSIONS: Overall VIC status of the US adult population has remained stable since last assessed in the NHANES 2005-2006 survey. Vitamin C deficiency remained high for those with low dietary intake and who smoke. |
Exposure of Egyptian rousette bats (Rousettus aegyptiacus) and a little free-tailed bat (Chaerephon pumilus) to alphaviruses in Uganda
Kading RC , Borland EM , Mossel EC , Nakayiki T , Nalikka B , Ledermann JP , Crabtree MB , Panella NA , Nyakarahuka L , Gilbert AT , Kerbis-Peterhans JC , Towner JS , Amman BR , Sealy TK , Miller BR , Lutwama JJ , Kityo RM , Powers AM . Diseases 2022 10 (4) The reservoir for zoonotic o'nyong-nyong virus (ONNV) has remained unknown since this virus was first recognized in Uganda in 1959. Building on existing evidence for mosquito blood-feeding on various frugivorous bat species in Uganda, and seroprevalence for arboviruses among bats in Uganda, we sought to assess if serum samples collected from bats in Uganda demonstrated evidence of exposure to ONNV or the closely related zoonotic chikungunya virus (CHIKV). In total, 652 serum samples collected from six bat species were tested by plaque reduction neutralization test (PRNT) for neutralizing antibodies against ONNV and CHIKV. Forty out of 303 (13.2%) Egyptian rousettes from Maramagambo Forest and 1/13 (8%) little free-tailed bats from Banga Nakiwogo, Entebbe contained neutralizing antibodies against ONNV. In addition, 2/303 (0.7%) of these Egyptian rousettes contained neutralizing antibodies to CHIKV, and 8/303 (2.6%) contained neutralizing antibodies that were nonspecifically reactive to alphaviruses. These data support the interepidemic circulation of ONNV and CHIKV in Uganda, although Egyptian rousette bats are unlikely to serve as reservoirs for these viruses given the inconsistent occurrence of antibody-positive bats. |
When a Touch of Gold Was Used to Heal the King’s Evil
Krugman J , Chorba T . Emerg Infect Dis 2022 28 (3) 765-7 Throughout history, divine approval has been claimed by many rulers in establishing legitimacy of their monarchy and has been integral to governance in the development of many cultures. In ancient and Imperial China, a tradition of a mandate of heaven, as the will of the universe or natural law, was used to justify the position of the ruler. In the Inca Empire, the traditional ruler was considered the progeny of the sun god and in that capacity had to be accorded absolute power over the people, such as the sun itself has. European history is replete with similar traditions of monarchical claims for legitimacy. In Britain and in France, the evolution of the concept of “the divine right of kings” and the resultant philosophic traditions favoring or opposing such a concept shaped much of the history of the past millennium. Both monarchies claimed to rule by divine will, and to this day, the British Coronation service includes a sacred anointing of the new king or queen. | | Many religious traditions have had thaumaturgic (relating to supernatural powers) touch as a tradition. In Britain, reference to the monarch as having divine power in “the royal touch” dates to the 11th century, when it was believed that Edward the Confessor, last of the Anglo-Saxon kings, possessed powers to heal the sick through some form of laying on of hands. In official ceremonies in his and subsequent reigns, subjects could approach the monarch to seek the imperial touch, hoping to cure their ailments or diseases. For centuries, the disease that most readily lent itself to the occasional appearance of success in this regard was scrofula (i.e., lymphadenitis—most commonly tuberculous cervical lymphadenitis), which would manifest itself with painful and visible sores that could go into remission and even go into resolution, giving the impression of a royally induced cure. Scrofula is a term that has fallen into disuse like many other medical terms in English (e.g., catarrh, ague, quinsy, dropsy, and grippe), principally because of diagnostic advances and more precise disease characterization. However, because of the association of its spontaneous remission with the royal touch, tuberculous lymphadenitis was also called “the king's evil,” and throughout most of the past millennium, its presence in European populations was very common. |
Agreement Between Pregnant Individuals' Self-Report of Coronavirus Disease 2019 (COVID-19) Vaccination and Medical Record Documentation.
Wielgosz K , Dawood FS , Stockwell MS , Varner M , Newes-Adeyi G , Ellington S , Vargas C , Bruno AM , Powers E , Morrill T , Reichle L , Battarbee AN , Tita AT . Obstet Gynecol 2022 140 (6) 989-992 For public health research such as vaccine uptake or effectiveness assessments, self-reported coronavirus disease 2019 (COVID-19) vaccination status may be a more efficient measure than verifying vaccination status from medical records if agreement between sources is high. We assessed agreement between self-reported and medical record-documented COVID-19 vaccination status among pregnant individuals followed in a cohort during August 2020-October 2021. At end of pregnancy, participants completed questionnaires about COVID-19 vaccine receipt during pregnancy; staff verified vaccination status using medical records. Agreement was assessed between self-reported and medical record vaccination status using Cohen's kappa. There was high agreement between self-reported and medical record vaccination status (Kappa coefficient=0.94, 95% CI 0.91-0.98), suggesting that self-report may be acceptable for ascertaining COVID-19 vaccination status during pregnancy. |
Complete Genome Sequence of O'nyong Nyong Virus Isolated from a Febrile Patient in 2017 in Uganda.
Ledermann JP , Kayiwa JT , Perinet LC , Apangu T , Acayo S , Lutwama JJ , Powers AM , Mossel EC . Microbiol Resour Announc 2022 11 (12) e0069222 Despite causing numerous large outbreaks in the 20th century, few isolates of o'nyong nyong virus (ONNV) have been fully sequenced. Here, we report the complete genome sequence of an isolate of ONNV obtained from a febrile patient in northwest Uganda in 2017, designated ONNV UVRI0804. |
The role of depression in secondary HIV transmission among people who inject drugs in Vietnam: A mathematical modeling analysis
Levintow SN , Pence BW , Sripaipan T , Ha TV , Chu VA , Quan VM , Latkin CA , Go VF , Powers KA . PLoS One 2022 17 (10) e0275995 BACKGROUND: Among people who inject drugs (PWID), depression burden is high and may interfere with HIV prevention efforts. Although depression is known to affect injecting behaviors and HIV treatment, its overall impact on HIV transmission has not been quantified. Using mathematical modeling, we sought to estimate secondary HIV transmissions and identify differences by depression among PWID. METHODS: We analyzed longitudinal data from 455 PWID living with HIV in Vietnam during 2009-2013. Using a Bernoulli process model with individual-level viral load and behavioral data from baseline and 6-month follow-up visits, we estimated secondary HIV transmission events from participants to their potentially susceptible injecting partners. To evaluate differences by depression, we compared modeled transmissions per 1,000 PWID across depressive symptom categories (severe, mild, or no symptoms) in the three months before each visit. RESULTS: We estimated a median of 41.2 (2.5th, 97.5th percentiles: 33.2-49.2) secondary transmissions from all reported acts of sharing injection equipment with 833 injecting partners in the three months before baseline. Nearly half (41%) of modeled transmissions arose from fewer than 5% of participants in that period. Modeled transmissions per 1,000 PWID in that period were highest for severe depressive symptoms (100.4, 80.6-120.2) vs. mild (87.0, 68.2-109.4) or no symptoms (78.9, 63.4-94.1). Transmission estimates fell to near-zero at the 6-month visit. CONCLUSIONS: Secondary transmissions were predicted to increase with depression severity, although most arose from a small number of participants. Our findings suggest that effective depression interventions could have the important added benefit of reducing HIV transmission among PWID. |
Neurological disease associated with chikungunya in Indonesia
Myint KSA , Mawuntu AHP , Haryanto S , Imran D , Dian S , Dewi YP , Ganiem AR , Anggreani R , Iskandar MM , Bernadus JBB , Maharani K , Susanto D , Estiasari R , Dewi H , Kristiani A , Gaghiwu L , Johar E , Yudhaputri FA , Antonjaya U , Ledermann JP , van Crevel R , Hamers RL , Powers AM . Am J Trop Med Hyg 2022 107 (2) 291-5 Chikungunya virus (CHIKV) is recognized but rarely considered as a cause of central nervous system infection in endemic areas. A total of 244 patients with acute meningoencephalitis in Indonesia were retrospectively tested to identify whether any CHIKV infection was associated with neurological manifestations, especially in provinces known for CHIKV endemicity. Cerebrospinal fluid and blood specimens were tested using CHIKV-specific real-time reverse transcription polymerase chain reaction and IgM ELISA, alongside a panel of neurotropic viruses. We report four cases of suspected or confirmed CHIKV-associated neurological disease, including CHIKV RNA detection in cerebrospinal fluid (CSF) of one patient and in acute serum of another, and CHIKV IgM in CSF of three patients and in serum of a fourth. In conclusion, CHIKV should be considered as a cause of neurologic disease in endemic areas and especially during outbreaks, in addition to the more common arboviral diseases such as dengue and Japanese encephalitis viruses. |
Development of HEK-293 cell lines constitutively expressing flaviviral antigens for use in diagnostics
Powers JA , Skinner B , Davis BS , Biggerstaff BJ , Robb L , Gordon E , Calvert AE , Chang GJ . Microbiol Spectr 2022 10 (3) e0059222 Flaviviruses are important human pathogens worldwide. Diagnostic testing for these viruses is difficult because many of the pathogens require specialized biocontainment. To address this issue, we generated 39 virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells of 13 different flaviviruses, including dengue, yellow fever, Japanese encephalitis, West Nile, St. Louis encephalitis, Zika, Rocio, Ilheus, Usutu, and Powassan viruses. Antigen secretion was stable for at least 10 cell passages, as measured by enzyme-linked immunosorbent assays and immunofluorescence assays. Thirty-five cell lines (90%) had stable antigen expression over 10 passages, with three of these cell lines (7%) increasing in antigen expression and one cell line (3%) decreasing in antigen expression. Antigen secretion in the HEK-293 cell lines was higher than in previously developed COS-1 cell line counterparts. These antigens can replace current antigens derived from live or inactivated virus for safer use in diagnostic testing. IMPORTANCE Serological diagnostic testing for flaviviral infections is hindered by the need for specialized biocontainment for preparation of reagents and assay implementation. The use of previously developed COS-1 cell lines secreting noninfectious recombinant viral antigen is limited due to diminished antigen secretion over time. Here, we describe the generation of 39 flaviviral virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells representing 13 medically important flaviviruses. Antigen production was more stable and statistically higher in these newly developed cell lines than in their COS-1 cell line counterparts. The use of these cell lines for production of flaviviral antigens will expand serological diagnostic testing of flaviviruses worldwide. |
Resurgence of interest in eastern equine encephalitis virus vaccine development
Powers AM . J Med Entomol 2022 59 (1) 20-26 Eastern equine encephalitis virus (EEEV; Family Togaviridae), is an endemic pathogen first isolated in 1933 with distribution primarily in the eastern US and Canada. The virus has caused periodic outbreaks in both humans and equines along the eastern seaboard and through the southern coastal states. While the outbreaks caused by EEEV have been sporadic and varied geographically since the discovery of the virus, it has continued to expand its range moving into the Midwest states as well. Additionally, one of the largest outbreaks was recorded in 2019 prompting concerns that outbreaks were becoming larger and more frequent. Because the virus can cause serious disease and because it is transmissible by both mosquitoes and aerosol, there has been renewed interest in identifying potential options for vaccines. Currently, there are no licensed vaccines and control relies completely on the use of personal protective measures and integrated vector control which have limited effectiveness for the EEEV vectors. Several vaccine candidates are currently being developed; this review will describe the multiple options under consideration for future development and assess their relative advantages and disadvantages. |
Monoclonal antibodies to Cache Valley virus for serological diagnosis.
Skinner B , Mikula S , Davis BS , Powers JA , Hughes HR , Calvert AE . PLoS Negl Trop Dis 2022 16 (1) e0010156 Cache Valley virus (CVV) is a mosquito-borne virus in the genus Orthobunyavirus, family Peribunyaviridae. It was first isolated from a Culiseta inorata mosquito in Cache Valley, Utah in 1956 and is known to circulate widely in the Americas. While only a handful of human cases have been reported since its discovery, it is the causative agent of fetal death and severe malformations in livestock. CVV has recently emerged as a potential viral pathogen causing severe disease in humans. Currently, the only serological assay available for diagnostic testing is plaque reduction neutralization test which takes several days to perform and requires biocontainment. To expand diagnostic capacity to detect CVV infections by immunoassays, 12 hybridoma clones secreting anti-CVV murine monoclonal antibodies (MAbs) were developed. All MAbs developed were found to be non-neutralizing and specific to the nucleoprotein of CVV. Cross-reactivity experiments with related orthobunyaviruses revealed several of the MAbs reacted with Tensaw, Fort Sherman, Tlacotalpan, Maguari, Playas, and Potosi viruses. Our data shows that MAbs CVV14, CVV15, CVV17, and CVV18 have high specific reactivity as a detector in an IgM antibody capture test with human sera. |
The global epidemiology of chikungunya from 1999 to 2020: A systematic literature review to inform the development and introduction of vaccines
Bettis AA , L'Azou Jackson M , Yoon IK , Breugelmans JG , Goios A , Gubler DJ , Powers AM . PLoS Negl Trop Dis 2022 16 (1) e0010069 Chikungunya fever is an acute febrile illness that is often associated with severe polyarthralgia in humans. The disease is caused by chikungunya virus (CHIKV), a mosquito-borne alphavirus. Since its reemergence in 2004, the virus has spread throughout the tropical world and several subtropical areas affecting millions of people to become a global public health issue. Given the significant disease burden, there is a need for medical countermeasures and several vaccine candidates are in clinical development. To characterize the global epidemiology of chikungunya and inform vaccine development, we undertook a systematic literature review in MEDLINE and additional public domain sources published up to June 13, 2020 and assessed epidemiological trends from 1999 to 2020. Observational studies addressing CHIKV epidemiology were included and studies not reporting primary data were excluded. Only descriptive analyses were conducted. Of 3,883 relevant sources identified, 371 were eligible for inclusion. 46% of the included studies were published after 2016. Ninety-seven outbreak reports from 45 countries and 50 seroprevalence studies from 31 countries were retrieved, including from Africa, Asia, Oceania, the Americas, and Europe. Several countries reported multiple outbreaks, but these were sporadic and unpredictable. Substantial gaps in epidemiological knowledge were identified, specifically granular data on disease incidence and age-specific infection rates. The retrieved studies revealed a diversity of methodologies and study designs, reflecting a lack of standardized procedures used to characterize this disease. Nevertheless, available epidemiological data emphasized the challenges to conduct vaccine efficacy trials due to disease unpredictability. A better understanding of chikungunya disease dynamics with appropriate granularity and better insights into the duration of long-term population immunity is critical to assist in the planning and success of vaccine development efforts pre and post licensure. |
Comparing statistical methods for detecting and estimating waning efficacy of rotavirus vaccines in developing countries.
Haber M , Tate JE , Lopman BA , Qi W , Ainslie KEC , Parashar UD . Hum Vaccin Immunother 2021 17 (11) 1-4 INTRODUCTION: Vaccination has significantly reduced morbidity and mortality resulting from rotavirus infection worldwide. However, rotavirus vaccine efficacy (VE) appears to wane over the first 2 years since vaccination, particularly in developing countries. Statistical methods for detecting VE waning and estimating its rate have been used in a few studies, but comparisons of methods for evaluating VE waning have not yet been performed. In this work we present and compare three methods - Durham's method, Tian's method, and time-dependent covariate (TDC) method - based on generalizations of the Cox proportional hazard model. METHODS: We developed a new stochastic agent-based simulation model to generate data from a hypothetical rotavirus vaccine trial where the protective efficacy of the vaccine may vary over time. Input parameters to the simulation model were obtained from studies on rotavirus infections in four developing countries. We applied each of the methods to four simulated datasets and compared the type-1 error probabilities and the powers of the resulting statistical tests. We also compared estimated and true values of VE over time. RESULTS: Durham's method had the highest power of detecting true VE waning of the three methods. This method also provided quite accurate estimates of VE in each period and of the per-period drop in VE. CONCLUSIONS: Durham's method is somewhat more powerful than the other two Cox proportional hazards model-based methods for detecting VE waning and provides more information about the temporal behavior of VE. |
Attitudes Toward COVID-19 Illness and COVID-19 Vaccination among Pregnant Women: A Cross-Sectional Multicenter Study during August-December 2020.
Battarbee AN , Stockwell MS , Varner M , Newes-Adeyi G , Daugherty M , Gyamfi-Bannerman C , Tita AT , Vorwaller K , Vargas C , Subramaniam A , Reichle L , Galang RR , Powers E , Lucca-Susana M , Parks M , Chen TJ , Razzaghi H , Dawood FS . Am J Perinatol 2021 39 (1) 75-83 OBJECTIVE: The aim of the study was to evaluate pregnant women's attitudes toward COVID-19 illness and vaccination and identify factors associated with vaccine acceptability. STUDY DESIGN: This was a cross-sectional survey among pregnant women enrolled in a prospective COVID-19 cohort study in Salt Lake City, UT, Birmingham, AL, and New York, NY, from August 9 to December 10, 2020. Women were eligible if they were 18 to 50 years old and <28 weeks of gestation. Upon enrollment, women completed surveys regarding concerns about COVID-19 illness and likelihood of getting COVID-19 vaccine if one were available during pregnancy. Vaccine acceptability was defined as a response of "very likely" or "somewhat likely" on a 4-point Likert scale. Factors associated with vaccine acceptability were assessed with multivariable logistic regression. RESULTS: Of 939 pregnant women eligible for the main cohort study, 915 (97%) consented to participate. Among these 915 women, 39% self-identified as White, 23% Black, 33% Hispanic, and 4% Other. Sixty-two percent received an influenza vaccine last season. Seventy-two percent worried about getting sick with COVID-19. If they were to get sick, 92% worried about harm to their pregnancy and 80% about harm to themselves. Only 41% reported they would get a vaccine. Of women who were unlikely to get vaccinated, the most frequently cited concern was vaccine safety for their pregnancy (82%). Non-Hispanic Black and Hispanic women had lower odds of accepting a vaccine compared with non-Hispanic White women (adjusted odds ratios [aOR] 0.4, 95% CI 0.2-0.6 for both). Receipt of influenza vaccine during the previous season was associated with higher odds of vaccine acceptability (aOR 2.1, 95% CI 1.5-3.0). CONCLUSION: Although most pregnant women worried about COVID-19 illness, <50% were willing to get vaccinated during pregnancy. Racial and ethnic disparities in plans to accept COVID-19 vaccine highlight the need to prioritize strategies to address perceived barriers among groups at high risk for COVID-19. KEY POINTS: · Less than half of pregnant patients stated they would get a COVID-19 vaccine.. · Protecting their baby was the most common reason for acceptance and refusal of the COVID-19 vaccine.. · Patients of minority race/ethnicity and those without prior influenza vaccination were less likely to accept the COVID-19 vaccine.. |
Incidence, Clinical Characteristics, and Risk Factors of SARS-CoV-2 Infection among Pregnant Individuals in the United States.
Dawood FS , Varner M , Tita A , Newes-Adeyi G , Gyamfi-Bannerman C , Battarbee A , Bruno A , Daugherty M , Reichle L , Vorwaller K , Vargas C , Parks M , Powers E , Lucca-Susana M , Gibson M , Subramaniam A , Cheng YJ , Feng PJ , Ellington S , Galang RR , Meece J , Flygare C , Stockwell MS . Clin Infect Dis 2021 74 (12) 2218-2226 BACKGROUND: Data about the risk of SARS-CoV-2 infection among pregnant individuals are needed to inform infection prevention guidance and counseling for this population. METHODS: We prospectively followed a cohort of pregnant individuals during August 2020-March 2021 at three U.S. sites. The three primary outcomes were incidence rates of any SARS-CoV-2 infection, symptomatic infection, and asymptomatic infection, during pregnancy during periods of SARS-CoV-2 circulation. Participants self-collected weekly mid-turbinate nasal swabs for SARS-CoV-2 RT-PCR testing, completed weekly illness symptom questionnaires, and submitted additional swabs with COVID-19-like symptoms. An overall SARS-CoV-2 infection incidence rate weighted by population counts of women of reproductive age in each state was calculated. RESULTS: Among 1098 pregnant individuals followed for a mean of 10 weeks, nine percent (99/1098) had SARS-CoV-2 infections during the study. Population weighted incidence rates of SARS-CoV-2 infection were 10.0 per 1,000 (95% confidence interval [CI] 5.7-14.3) person-weeks for any infection, 5.7 per 1,000 (95% CI 1.7-9.7) for symptomatic infections, and 3.5 per 1,000 (95% CI 0-7.1) for asymptomatic infections. Among 96 participants with SARS-CoV-2 infection and symptom data, the most common symptoms were nasal congestion (72%), cough (64%), headache (59%), and change in taste or smell (54%); 28% had measured or subjective fever. The median symptom duration was 10 days (IQR6-16 days). CONCLUSION: Pregnant individuals had a 1% risk of SARS-CoV-2 infection per week. Study findings provide information about SARS-CoV-2 infection risk during pregnancy to inform counseling for pregnant individuals about infection prevention practices, including COVID-19 vaccination. |
Detection of dengue virus serotype 1 in central nervous system of a child in Bandung, West Java: A case report
Alisjahbana DH , Nurmawati S , Hakim DDL , Milanti M , Dewi YP , Johar E , Myint KSA , Lederman JP , Powers AM , Alisjahbana B , Antonjaya U . SAGE Open Med Case Reports 2021 9 2050313X211034393 Central nervous system involvement of dengue virus is increasingly reported from endemic areas. This study describes the clinical characteristics and laboratory features of a pediatric patient enrolled in a central nervous system illness study conducted in 2017–2018 to identify viral and bacterial etiologies in Indonesian children. Dengue diagnostics including molecular and serological testing were performed on an encephalitis patient who presented with both classical dengue and neurological clinical symptoms. Dengue virus serotype 1 RNA was detected in both cerebrospinal fluid and serum by serotype-specific reverse transcription polymerase chain reaction, and the E gene was successfully sequenced. Anti-dengue virus immunoglobulin M was detected in both admission and discharge sera, whereas anti-dengue virus immunoglobulin G was identified only in the discharge serum. This study describes the central nervous system complications in a case with dengue virus infection in West Java, Indonesia, and highlights the potential for dengue virus serotype 1, a serotype rarely associated with neurotropism, to cause encephalitis. © The Author(s) 2021. |
Stability of Zika virus antibodies in specimens from a retrospective serological study
Sasmono RT , Johar E , Yohan B , Ma'roef CN , Soebandrio A , Myint KS , Pronyk P , Hadinegoro SR , Soepardi EJ , Bouckenooghe A , Hawley W , Rosenberg R , Powers AM . Am J Trop Med Hyg 2021 105 (3) 853 We would like to respond to the letter by Zhang and others regarding our study published in the American Journal of Tropical Medicine and Hygiene.1 Our serum samples were all stored in annually calibrated –80°C freezers. The samples were only thawed once. In general, antibodies are known to remain stable in frozen storage over lengthy periods. There are numerous publications regarding antibody stability during storage,2–6 and we believe that all antibodies, including antibodies against Zika virus, will remain stable during storage. |
Association Between Caseload Surge and COVID-19 Survival in 558 U.S. Hospitals, March to August 2020.
Kadri SS , Sun J , Lawandi A , Strich JR , Busch LM , Keller M , Babiker A , Yek C , Malik S , Krack J , Dekker JP , Spaulding AB , Ricotta E , Powers Iii JH , Rhee C , Klompas M , Athale J , Boehmer TK , Gundlapalli AV , Bentley W , Datta SD , Danner RL , Demirkale CY , Warner S . Ann Intern Med 2021 174 (9) 1240-1251 BACKGROUND: Several U.S. hospitals had surges in COVID-19 caseload, but their effect on COVID-19 survival rates remains unclear, especially independent of temporal changes in survival. OBJECTIVE: To determine the association between hospitals' severity-weighted COVID-19 caseload and COVID-19 mortality risk and identify effect modifiers of this relationship. DESIGN: Retrospective cohort study. (ClinicalTrials.gov: NCT04688372). SETTING: 558 U.S. hospitals in the Premier Healthcare Database. PARTICIPANTS: Adult COVID-19-coded inpatients admitted from March to August 2020 with discharge dispositions by October 2020. MEASUREMENTS: Each hospital-month was stratified by percentile rank on a surge index (a severity-weighted measure of COVID-19 caseload relative to pre-COVID-19 bed capacity). The effect of surge index on risk-adjusted odds ratio (aOR) of in-hospital mortality or discharge to hospice was calculated using hierarchical modeling; interaction by surge attributes was assessed. RESULTS: Of 144 116 inpatients with COVID-19 at 558 U.S. hospitals, 78 144 (54.2%) were admitted to hospitals in the top surge index decile. Overall, 25 344 (17.6%) died; crude COVID-19 mortality decreased over time across all surge index strata. However, compared with nonsurging (<50th surge index percentile) hospital-months, aORs in the 50th to 75th, 75th to 90th, 90th to 95th, 95th to 99th, and greater than 99th percentiles were 1.11 (95% CI, 1.01 to 1.23), 1.24 (CI, 1.12 to 1.38), 1.42 (CI, 1.27 to 1.60), 1.59 (CI, 1.41 to 1.80), and 2.00 (CI, 1.69 to 2.38), respectively. The surge index was associated with mortality across ward, intensive care unit, and intubated patients. The surge-mortality relationship was stronger in June to August than in March to May (slope difference, 0.10 [CI, 0.033 to 0.16]) despite greater corticosteroid use and more judicious intubation during later and higher-surging months. Nearly 1 in 4 COVID-19 deaths (5868 [CI, 3584 to 8171]; 23.2%) was potentially attributable to hospitals strained by surging caseload. LIMITATION: Residual confounding. CONCLUSION: Despite improvements in COVID-19 survival between March and August 2020, surges in hospital COVID-19 caseload remained detrimental to survival and potentially eroded benefits gained from emerging treatments. Bolstering preventive measures and supporting surging hospitals will save many lives. PRIMARY FUNDING SOURCE: Intramural Research Program of the National Institutes of Health Clinical Center, the National Institute of Allergy and Infectious Diseases, and the National Cancer Institute. |
Spatiotemporal Heterogeneity of Zika Virus Transmission in Indonesia: Serosurveillance Data from a Pediatric Population
Sasmono RT , Johar E , Yohan B , Ma'roef CN , Pronyk P , Hadinegoro SR , Soepardi EJ , Bouckenooghe A , Hawley WA , Rosenberg R , Powers AM , Soebandrio A , Myint KSA . Am J Trop Med Hyg 2021 104 (6) 2220-3 The presence of Zika virus (ZIKV) in Indonesia has been recognized since the 1970s, but its transmission dynamics there have been poorly understood. To understand more fully the geographic distribution and burden of ZIKV disease, we performed retrospective serological tests on specimens collected from asymptomatic children age 5 to 9 years old living at 30 sites in 14 provinces. Of 870 serum samples tested, 9.2% were found to be positive for anti-ZIKV antibodies, as confirmed by plaque reduction neutralization assays. This was the same overall prevalence reported previously for 1- to 4-year-old children collected at the same sites at the same time. Together with geographic differences in seroprevalence between the age groups, these data suggest that, although ZIKV might be endemic in Indonesia, its occurrence has been focal and episodic. |
Respiratory Viral Infections and Infection Prevention Practices among Women with Acute Respiratory Illness during Delivery Hospitalizations during the 2019-2020 Influenza Season
Dawood FS , Varner M , Munoz F , Stockwell MS , Suyama J , Li DK , Tita A , Mathias L , Shakib JH , Piedra PA , Gyamfi-Bannerman C , Weissman A , Ferber J , Battarbee AN , Wesley MG , Vorwaller K , Powers E , Gibson M , Bond N , Santarcangelo P , Avadhanula V , Newes-Adeyi G , Hunt DR , Subramaniam A , Sanusi A , Boone A , Ogokeh C , Macio I , Odouli R , Thind P , Vargas CY , Almonte C , Galang R , Shapiro-Mendoza C , Campbell AP . J Infect Dis 2021 225 (1) 50-54 We conducted a cross-sectional study of pregnant women with acute respiratory illness during delivery hospitalizations in influenza season to describe clinical testing for respiratory viruses and infection prevention practices. Women had nasal swabs tested for influenza and other respiratory viruses. Among 91 enrolled women, 22 (24%) had clinical testing for influenza. Based on clinical and study testing combined, 41/91 (45%) women had samples positive for respiratory viruses. The most common virus was influenza (17/91, 19%); 53% (9/17) of influenza virus infections were identified through study testing alone. Only 16% of women were on droplet precautions. Peripartum respiratory infections may be underrecognized. |
Slide guard effectiveness on steep-sloped roofs
Bobick TG , McKenzie EA Jr , Powers JR Jr . Prof Saf 2021 66 (2) 28-33 Key Takeaways: 1) Roofing contractors should consider using a slide guard as a supplemental means of fall protection when working on roof slopes that are 34 degrees (8 in 12) or less, but a slide guard should never be considered as the sole means to achieve work site fall protection compliance. 2) Using a slide guard on a 45 degree roof slope (12 in 12) would not be an effective fall protection supplement to comply with OSHA's fall protection requirements. 3) Contractors should consider purchasing and using synthetic underlayment materials with higher coefficient-of-friction values. This type of information may be available from the suppliers of underlayment materials that are used on steep-sloped roofs. |
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