Search

Introduction Ebola virus (EBOV), species Zaire ebolavirus, may persist in the semen of male survivors of Ebola virus disease (EVD). We conducted a study of male survivors of the 2014–2016 EVD outbreak in Liberia and evaluated their immune responses to EBOV. We report here findings from the serologic testing of blood for EBOV-specific antibodies, molecular testing for EBOV in blood and semen, and serologic testing of peripheral blood mononuclear cells (PBMCs) in a subset of study participants. Methods We tested for EBOV RNA in blood by quantitative reverse transcription polymerase chain reaction (qRT-PCR), and for anti-EBOV-specific immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies by enzyme-linked immunosorbent assay (ELISA) for 126 study participants. We performed PBMC analysis on a subgroup of 26 IgG-negative participants. Results All 126 participants tested negative for EBOV RNA in blood by qRT-PCR. The blood of 26 participants tested negative for EBOV-specific IgG antibodies by ELISA. PBMCs were collected from 23/26 EBOV IgG-negative participants. Of these, 1/23 participants had PBMCs that produced anti-EBOV-specific IgG antibodies upon stimulation with EBOV-specific glycoprotein (GP) and nucleoprotein (NP) antigens. Conclusions The blood of EVD survivors, collected when they did not have symptoms meeting the case definition for acute or relapsed EVD, is unlikely to pose a risk for EBOV transmission. We identified 1 IgM/IgG negative participant who had PBMCs that produced anti-EBOV-specific antibodies upon stimulation. Immunogenicity following acute EBOV infection may exist along a spectrum, and absence of antibody response should not be exclusionary in determining an individual’s status as a survivor of EVD.

The epidemiology of Rift Valley fever virus (RVFV) and Crimean-Congo hemorrhagic fever virus (CCHFV) in Jordan is unknown. Our investigation showed 3% of 989 tested dairy cattle, sheep, and goats were RVFV seropositive and 14% were CCHFV seropositive. Ongoing surveillance is needed to assess risk to humans and protect public health.

Background: Long-term persistence of Ebola virus (EBOV) in immunologically privileged sites has been implicated in recent outbreaks of Ebola Virus Disease (EVD) in Guinea and the Democratic Republic of Congo. This study was designed to better understand how the acute course of EVD, convalescence, and host immune and genetic factors may play a role in prolonged viral persistence in semen. Methods: In 2017, a cohort of 131 male survivors of the 2014-2016 outbreak of EVD in Liberia were recruited from a national semen testing program into this case-case study. “Early clearers” were defined as those with two consecutive negative EBOV semen tests by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) at least two weeks apart within 1 year after discharge from the Ebola Treatment Unit (ETU). “Late clearers” had detectable EBOV RNA by rRT-PCR more than one year after ETU discharge. Retrospective histories of their EVD clinical course were collected by questionnaire, followed by complete physical exams and blood work including chemistries, blood counts, hemoglobinopathies, human leukocyte antigen typing, and immunologic/inflammatory markers. Findings: Among 131 participants, 30 demonstrated long-term detection of EBOV RNA in semen by rRT-PCR, up to 852 days after ETU discharge. Compared to early clearers, late clearers were more likely to be older (median 42.5 years, p=0.0001) and reported having experienced fewer severe clinical symptoms (median 2, p=0.006). Late clearers were found to more frequently have lens opacifications (OR 3.9, 95%CI 1.1-13.3, p=0.03), after accounting for age. Finally, late clearers also exhibited elevated total serum IgG3 titers (p=0.007) and increased expression of the HLA-C*03:04 allele (OR 0.14, 95% CI 0.02-0.70, p=0.007). Interpretation: Older age and decreased illness severity may play a significant role in determining the clearance of EBOV from the semen of survivors. Immune and host genetic factors including total serum IgG3 and HLA-C*03:04 may mediate that interaction. In addition, our finding that late clearers were more likely to have lens opacifications after accounting for age suggests that individuals with EBOV persistence in semen may also have EBOV persistence in other immunologically protected sites, such as the eye. Identifying risk factors for viral persistence in semen among male survivors is critical to inform screening efforts and prevent recurrent outbreaks of EBOV. Funding Information: This was work was supported by funds from the CDC Foundation and the Centers for Disease Control and Prevention. This project has also been funded in whole or in part with federal funds from the Frederick National Laboratory for Cancer Research, under Contract No. HHSN261200800001E. Declaration of Interests: The authors report no competing interests. Ethics Approval Statement: The study protocol was approved by the CDC and Liberia MoH Institutional Review Boards and Meharry Medical College (for hemoglobinopathy testing).

ImportanceReported cases of SARS-CoV-2 infection likely underestimate the prevalence of infection in affected communities. Large-scale seroprevalence studies provide better estimates of the proportion of the population previously infected.ObjectiveTo estimate prevalence of SARS-CoV-2 antibodies in convenience samples from several geographic sites in the United States.DesignSerologic testing of convenience samples using residual sera obtained for routine clinical testing by two commercial laboratory companies.SettingConnecticut (CT), south Florida (FL), Missouri (MO), New York City metro region (NYC), Utah (UT), and Washington State’s (WA) Puget Sound region.ParticipantsPersons of all ages with serum collected during intervals from March 23 through May 3, 2020.ExposureSARS-CoV-2 virus infection.Main outcomes and measuresWe estimated the presence of antibodies to SARS-CoV-2 spike protein using an ELISA assay. We standardized estimates to the site populations by age and sex. Estimates were adjusted for test performance characteristics (96.0% sensitivity and 99.3% specificity). We estimated the number of infections in each site by extrapolating seroprevalence to site populations. We compared estimated infections to number of reported COVID-19 cases as of last specimen collection date.ResultsWe tested sera from 11,933 persons. Adjusted estimates of the proportion of persons seroreactive to the SARS-CoV-2 spike protein ranged from 1.13% (95% confidence interval [CI] 0.70-1.94) in WA to 6.93% (95% CI 5.02-8.92) in NYC (collected March 23-April 1). For sites with later collection dates, estimates ranged from 1.85% (95% CI 1.00-3.23, collected April 6-10) for FL to 4.94% (95% CI 3.61-6.52) for CT (April 26-May 3). The estimated number of infections ranged from 6 to 24 times the number of reported cases in each site.Conclusions and relevanceOur seroprevalence estimates suggest that for five of six U.S. sites, from late March to early May 2020, >10 times more SARS-CoV-2 infections occurred than the number of reported cases. Seroprevalence and under-ascertainment varied by site and specimen collection period. Most specimens from each site had no evidence of antibody to SARS-CoV-2. Tracking population seroprevalence serially, in a variety of specific geographic sites, will inform models of transmission dynamics and guide future community-wide public health measures.Key findingsQuestionWhat proportion of persons in six U.S. sites had detectable antibodies to SARS-CoV-2, March 23-May 3, 2020?FindingsWe tested 11,933 residual clinical specimens. We estimate that from 1.1% of persons in the Puget Sound to 6.9% in New York City (collected March 23-April 1) had detectable antibodies. Estimates ranged from 1.9% in south Florida to 4.9% in Connecticut with specimens collected during intervals from April 6-May 3. Six to 24 times more infections were estimated per site with seroprevalence than with case report data.MeaningFor most sites, evidence suggests >10 times more SARS-CoV-2 infections occurred than reported cases. Most persons in each site likely had no detectable SARS-CoV-2 antibodies.

Since emergence of SARS-CoV-2 in late 2019, there has been a critical need to understand prevalence, transmission patterns, to calculate the burden of disease and case fatality rates. Molecular diagnostics, the gold standard for identifying viremic cases, are not ideal for determining true case counts and rates of asymptomatic infection. Serological detection of SARS-CoV-2 specific antibodies can contribute to filling these knowledge gaps. In this study, we describe optimization and validation of a SARS-CoV-2-specific-enzyme linked immunosorbent assay (ELISA) using the prefusion-stabilized form of the spike protein [1]. We performed receiver operator characteristic (ROC) analyses to define the specificities and sensitivities of the optimized assay and examined cross reactivity with immune sera from persons confirmed to have had infections with other coronaviruses. These assays will be used to perform contact investigations and to conduct large-scale, cross sectional surveillance to define disease burden in the population.

Seoul orthohantavirus (SEOV) infections, uncommonly reported in the United States, often result in mild illness. We report a case of hemophagocytic lymphohistiocytosis secondary to SEOV infection that was domestically acquired in Washington, DC.

Introduction In October 2017, a blood sample from a resident of Kween District, Eastern Uganda, tested positive for Marburg virus. Within 24 hour of confirmation, a rapid outbreak response was initiated. Here, we present results of epidemiological and laboratory investigations. Methods A district task force was activated consisting of specialised teams to conduct case finding, case management and isolation, contact listing and follow up, sample collection and testing, and community engagement. An ecological investigation was also carried out to identify the potential source of infection. Virus isolation and Next Generation sequencing were performed to identify the strain of Marburg virus. Results Seventy individuals (34 MVD suspected cases and 36 close contacts of confirmed cases) were epidemiologically investigated, with blood samples tested for MVD. Only four cases met the MVD case definition; one was categorized as a probable case while the other three were confirmed cases. A total of 299 contacts were identified; during follow- up, two were confirmed as MVD. Of the four confirmed and probable MVD cases, three died, yielding a case fatality rate of 75%. All four cases belonged to a single family and 50% (2/4) of the MVD cases were female. All confirmed cases had clinical symptoms of fever, vomiting, abdominal pain and bleeding from body orifices. Viral sequences indicated that the Marburg virus strain responsible for this outbreak was closely related to virus strains previously shown to be circulating in Uganda. Conclusion This outbreak of MVD occurred as a family cluster with no additional transmission outside of the four related cases. Rapid case detection, prompt laboratory testing at the Uganda National VHF Reference Laboratory and presence of pre-trained, well-prepared national and district rapid response teams facilitated the containment and control of this outbreak within one month, preventing nationwide and global transmission of the disease., Author summary Marburg virus disease (MVD) is caused by the virus that belongs to the same family as that of Ebola Virus disease. The disease is characterized by severe clinical symptoms such as high fever, diarrhoea and vomiting, and severe bleeding from most body openings. On average, 54% of the people who get infected with Marburg virus die from it [1]. In October 2017, Uganda reported an outbreak of MVD in the eastern district of Kween that borders Kenya in the Mount Elgon plains. This was the first time MVD was being detected in this part of the country since previous MVD outbreaks had been reported in the western part of Uganda. The Ministry of Health together with partners instituted rapid outbreak response for control. Investigations revealed a cluster of one family involving four cases whereby only one survived. The outbreak was traced back into bat caves in Kween district. Rock salt mining in the cave led to a spill-over of the virus into the human population with the subsequent person-to-person transmission. Through contact tracing and isolation of the infected people among other approaches, the outbreak was brought under control as explained in this article.

Importance Reported cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection likely underestimate the prevalence of infection in affected communities. Large-scale seroprevalence studies provide better estimates of the proportion of the population previously infected. Objective To estimate prevalence of SARS-CoV-2 antibodies in convenience samples from several geographic sites in the US. Design, Setting, and Participants This cross-sectional study performed serologic testing on a convenience sample of residual sera obtained from persons of all ages. The serum was collected from March 23 through May 12, 2020, for routine clinical testing by 2 commercial laboratory companies. Sites of collection were San Francisco Bay area, California; Connecticut; south Florida; Louisiana; Minneapolis-St Paul-St Cloud metro area, Minnesota; Missouri; New York City metro area, New York; Philadelphia metro area, Pennsylvania; Utah; and western Washington State. Exposures Infection with SARS-CoV-2. Main Outcomes and Measures The presence of antibodies to SARS-CoV-2 spike protein was estimated using an enzyme-linked immunosorbent assay, and estimates were standardized to the site populations by age and sex. Estimates were adjusted for test performance characteristics (96.0% sensitivity and 99.3% specificity). The number of infections in each site was estimated by extrapolating seroprevalence to site populations; estimated infections were compared with the number of reported coronavirus disease 2019 (COVID-19) cases as of last specimen collection date. Results Serum samples were tested from 16 025 persons, 8853 (55.2%) of whom were women; 1205 (7.5%) were 18 years or younger and 5845 (36.2%) were 65 years or older. Most specimens from each site had no evidence of antibodies to SARS-CoV-2. Adjusted estimates of the proportion of persons seroreactive to the SARS-CoV-2 spike protein antibodies ranged from 1.0% in the San Francisco Bay area (collected April 23-27) to 6.9% of persons in New York City (collected March 23-April 1). The estimated number of infections ranged from 6 to 24 times the number of reported cases; for 7 sites (Connecticut, Florida, Louisiana, Missouri, New York City metro area, Utah, and western Washington State), an estimated greater than 10 times more SARS-CoV-2 infections occurred than the number of reported cases. Conclusions and Relevance During March to early May 2020, most persons in 10 diverse geographic sites in the US had not been infected with SARS-CoV-2 virus. The estimated number of infections, however, was much greater than the number of reported cases in all sites. The findings may reflect the number of persons who had mild or no illness or who did not seek medical care or undergo testing but who still may have contributed to ongoing virus transmission in the population.

The selection of vertebrate hosts by Culex mosquitoes relative to West Nile virus (WNV) transmission in neotropical countries such as Guatemala is not described. This study determined the feeding patterns of Cx. quinquefasciatus and Cx. nigripalpus and estimated the relative contribution of two common and frequently infected wild bird species, Turdus grayi and Quiscalus mexicanus, to WNV transmission. Engorged mosquitoes were collected from rural and urban habitats after the dry and wet seasons in the Department of Izabal in 2007. Host selection by Cx. nigripalpus varied significantly between urban and rural habitats. Both Cx. quinquefasciatus and Cx. nigripalpus fed predominantly on chickens and other domestic animals. Blood meals from wild birds were rare, accounting for 1.1% of blood meals identified from Cx. quinquefasciatus and 6.5% of blood meals from Cx. nigripalpus. Transmission of WNV by these two mosquito species may be dampened by extensive feeding on reservoir-incompetent hosts.

Background In February 2009, a group of Guatemalan school children developed acute gastroenteritis (AGE) after participating in a school excursion. Objectives We conducted a retrospective cohort investigation to characterize the outbreak and guide control measures. Study design A case was defined as an illness with onset of diarrhea or vomiting during February 25–March 5, 2009. Participants were interviewed using a standardized questionnaire, and stool specimens were collected. We inspected the excursion site and tested water samples for total coliforms and Escherichia coli . Results We identified 119 excursion participants, of which 92 (77%) had been ill. Fifty-six (62%) patients sought care for their illness, and three (3%) were hospitalized. Eighteen (90%) of the 20 specimens from ill children tested positive for norovirus. Among these, 16 (89%) were of the genogroup I (GI.7) and two (11%) were genogroup II (GII.12 and GII.17). One (8%) of the 12 food handlers had norovirus (GI.7). Drinking water samples had 146 most probable numbers (MPN)/100 ml of total coliforms and five MPN/100 ml of E. coli . Conclusion We describe the first laboratory-confirmed norovirus outbreak in Guatemala. The high illness attack rate, detection of multiple norovirus strains in sick persons, and presence of fecal contamination of drinking water indicate likely waterborne transmission.

Investigators have used a variety of techniques to sample resting, engorged mosquitoes for the purposes of studying mosquito blood-feeding behavior. However, evidence exists that mosquito blood-feeding patterns may vary according to collection method. Engorged mosquitoes were collected from rural and urban habitats after the 2007 dry (July) and wet (December) seasons in the Department of Izabal, Guatemala, with the use of Centers for Disease Control and Prevention (CDC) light traps, gravid traps, and aspiration from plastic pots and vegetation. We evaluated the utility of plastic pots as sampling tools for engorged Culex mosquitoes and compared Cx. quinquefasciatus blood host identities among collection methods. The array of vertebrate hosts supplying blood to Cx. quinquefasciatus did not differ significantly by method of collection. The density of engorged Cx. quinquefasciatus per trap-night was not significantly different between CDC light traps, gravid traps, and plastic pots; however, there was a significantly higher proportion of total mosquitoes that were engorged collected from pots than from either CDC light traps or gravid traps.

West Nile virus ecology has yet to be rigorously investigated in the Caribbean Basin. We identified a transmission focus in Puerto Barrios, Guatemala, and established systematic monitoring of avian abundance and infection, seroconversions in domestic poultry, and viral infections in mosquitoes. West Nile virus transmission was detected annually between May and October from 2005 to 2008. High temperature and low rainfall enhanced the probability of chicken seroconversions, which occurred in both urban and rural sites. West Nile virus was isolated from Culex quinquefasciatus and to a lesser extent, from Culex mollis/Culex inflictus, but not from the most abundant Culex mosquito, Culex nigripalpus. A calculation that combined avian abundance, seroprevalence, and vertebrate reservoir competence suggested that great-tailed grackle (Quiscalus mexicanus) is the major amplifying host in this ecosystem. West Nile virus transmission reached moderate levels in sentinel chickens during 2007, but less than that observed during outbreaks of human disease attributed to West Nile virus in the United States.

The role wild bird species play in the transmission and ecology of avian influenza virus (AIV) is well established; however, there are significant gaps in our understanding of the worldwide distribution of these viruses, specifically about the prevalence and/or significance of AIV in Central and South America. As part of an assessment of the ecology of AIV in Guatemala, we conducted active surveillance in wild birds on the Pacific and Atlantic coasts. Cloacal and tracheal swab samples taken from resident and migratory wild birds were collected from February 2007 to January 2010.1913 samples were collected and virus was detected by real time RT-PCR (rRT-PCR) in 28 swab samples from ducks (Anas discors). Virus isolation was attempted for these positive samples, and 15 isolates were obtained from the migratory duck species Blue-winged teal. The subtypes identified included H7N9, H11N2, H3N8, H5N3, H8N4, and H5N4. Phylogenetic analysis of the viral sequences revealed that AIV isolates are highly similar to viruses from the North American lineage suggesting that bird migration dictates the ecology of these viruses in the Guatemalan bird population.

West Nile virus (WNV) is an emerging mosquito-borne flavivirus, which has rapidly spread and is currently widely distributed. Therefore, efforts for WNV early detection and ecological surveillance of this disease agent have been increased around the world. Although virus isolation is known to be the standard method for detection and identification of viruses, the use of RT-PCR assays as routine laboratory tests provides a rapid alterative suitable for the detection of viral RNA on field-collected samples. A method for WNV RNA genome detection in field-collected mosquitoes is presented in this chapter. This method has been designed for virus surveillance in tropical regions endemic for other flaviviruses. Reverse Transcriptase-PCR (RT-PCR) assays, both standard and real time, to detect WNV and other flaviviruses are described. A first screening for flavivirus RNA detection is performed using a conventional RT-PCR with two different sets of flavivirus consensus primers. Mosquito samples are then tested for WNV RNA by a real-time (TaqMan) RT-PCR assay. Sample preparation and RNA extraction procedures are also described.

To the Editor: West Nile virus (WNV, Flaviviridae: Flavivirus) is emerging as a public health and veterinary concern. Since its introduction into North America in 1999, it has spread rapidly, reaching the Caribbean Basin in 2001, Mexico in 2002, El Salvador in 2003, and Colombia in 2004 (1). However, reports of equine illness and deaths in Latin America are inconclusive. With the exception of viral isolates from a dead bird, a human, and a mosquito pool in Mexico (2,3), all reports of WNV presence in Latin America have relied on serologic evidence. WNV is a member of the Japanese encephalitis serocomplex, which in the Western Hemisphere includes St. Louis encephalitis virus (SLEV) (4). Serologic investigations for WNV in Latin America must use highly specific assays to differentiate WNV infection from potentially cross-reactive viruses such as SLEV or possibly additional unknown viruses. In particular, SLEV is of concern since it was previously isolated from Guatemalan mosquitoes (5). Alerted by the findings of WNV transmission in the region (1), we collected serum samples from horses from 19 departments of Guatemala from September 2003 to March 2004, to initially estimate the extent of WNV spread and its potential public health risk. Because no animals exhibited signs of neurologic illness at the time of the survey, only healthy horses were sampled. Before 2005, equine WNV vaccines were prohibited and unavailable in Guatemala (Unidad de Normas y Regulaciones, Ministerio de Agricultura Ganaderia y Alimentacion, Guatemala, pers. comm.); as such, cross-reactivity due to prior vaccination is highly unlikely. Samples were initially tested for WNV-reactive antibodies by using an epitope-blocking enzyme-linked immunosorbent assay (blocking ELISA) (6). The ability of the test sera to block the binding of the monoclonal antibodies to WNV antigen was compared to the blocking ability of control horse serum without antibody to WNV. Data were expressed as relative percentages and inhibition values >30% were considered to indicate the presence of viral antibodies. A subset of positive samples was further confirmed by plaque-reduction neutralization test (7). Of 352 samples, 149 (42.3%) tested positive with the 3.1112G WNV-specific monoclonal antibody. Of 70 blocking ELISA–positive samples, the neutralization tests indicated the infecting agent was WNV, SLEV, and undifferentiated flavivirus in 9, 33, and 21 samples, respectively. Titers were expressed as the reciprocal of serum dilutions yielding >90% reduction in the number of plaques in a plaque-reduction neutralization test (PRNT90). PRNT90 titers of horses seropositive for WNV ranged from 80 to 320. PRNT90 titers of horses seropositive for SLEV ranged from 40 to 2,560. For the differential diagnosis of samples with neutralizing antibody titers against both WNV and SLEV in this test, a >4-fold titer difference was used to identify the etiologic agent. The undifferentiated flavivirus-reactive specimens had

Dengue is caused by any of the four serotypes of dengue virus (DENV-1 to 4). Each serotype is genetically distant from the others, and each has been subdivided into different genotypes based on phylogenetic analysis. The study of dengue evolution in endemic regions is important since the diagnosis is often made by nucleic acid amplification tests, which depends upon recognition of the viral genome target, and natural occurring mutations can affect the performance of these assays. Here we report for the first time a detailed study of the phylogenetic relationships of DENV-2 from Central America, and report the first fully sequenced DENV-2 strain from Guatemala. Our analysis of the envelope (E) protein and of the open reading frame of strains from Central American countries, between 1999 and 2009, revealed that at least two lineages of the American/Asian genotype of DENV-2 have recently circulated in that region. In occasions the co-circulation of these lineages may have occurred and that has been suggested to play a role in the observed increased severity of clinical cases. Our time-scale analysis indicated that the most recent common ancestor for Central American DENV-2 of the American/Asian genotype existed about 19 years ago. Finally, we report positive selection in DENV-2 from Central America in codons of the genes encoding for C, E, NS2A, NS3, and NS5 proteins. Some of these identified codons are novel findings, described for the first time for any of the DENV-2 genotypes.