Care of Ebola Survivors and Factors Associated With Clinical Sequelae-Monrovia, Liberia.

BACKGROUND: The Eternal Love Winning Africa (ELWA) Clinic was the first clinic to provide free, comprehensive care to Ebola virus disease (EVD) survivors in Liberia. The objectives of this analysis were to describe the demographics and symptoms of EVD survivors at ELWA from January 2015 through March 2017 and to identify risk factors for development of sequelae. METHODS: Patients' demographic and clinical information was collected by chart review in June 2016 and March 2017. Associations with clinical sequelae were analyzed using the chi-square test, t test, and multivariate logistic regression. RESULTS: From January 2015 to March 2017, 329 EVD survivors were evaluated at ELWA. Most survivors experienced myalgia/arthralgia (73%; n = 239) and headache (53%; n = 173). The length of time from Ebola Treatment Unit (ETU) discharge to first clinic visit ranged from 0 to 30 months. Many visits (30%) occurred 24 or more months after ETU discharge. The proportion of visits for headache, weight loss, joint pain, visual problems, insomnia, fatigue, memory loss, decreased libido, depression, and uveitis decreased over time. More men than women had visits for depression; however, these differences were not significant. Symptom prevalence differed in adults and children; significantly more adults experienced myalgia/arthralgia (77% vs 44%), visual problems (41% vs 12%), post-EVD-related musculoskeletal pain (42% vs 15%), and insomnia (17% vs 2%). CONCLUSIONS: EVD survivors frequented ELWA for EVD-related symptoms many months after ETU discharge, indicating a long-term need for care. Reported symptoms changed over time, which may reflect eventual resolution of some sequelae.

Evolving epidemiology of Nipah virus infection in Bangladesh: evidence from outbreaks during 2010-2011.

Drinking raw date palm sap is the primary route of Nipah virus (NiV) transmission from bats to people in Bangladesh; subsequent person-to-person transmission is common. During December 2010 to March 2011, we investigated NiV epidemiology by interviewing cases using structured questionnaires, in-depth interviews, and group discussions to collect clinical and exposure histories. We conducted a case-control study to identify risk factors for transmission. We identified 43 cases; 23 were laboratory-confirmed and 20 probable. Thirty-eight (88%) cases died. Drinking raw date palm sap and contact with an infected person were major risk factors; one healthcare worker was infected and for another case transmission apparently occurred through contact with a corpse. In absence of these risk factors, apparent routes of transmission included drinking fermented date palm sap. For the first time, a case was detected in eastern Bangladesh. Identification of new epidemiological characteristics emphasizes the importance of continued NiV surveillance and case investigation.

Integrated cluster- and case-based surveillance for detecting stage III zoonotic pathogens: an example of Nipah virus surveillance in Bangladesh.

This paper explores the utility of cluster- and case-based surveillance established in government hospitals in Bangladesh to detect Nipah virus, a stage III zoonotic pathogen. Physicians listed meningo-encephalitis cases in the 10 surveillance hospitals and identified a cluster when ⩾2 cases who lived within 30 min walking distance of one another developed symptoms within 3 weeks of each other. Physicians collected blood samples from the clustered cases. As part of case-based surveillance, blood was collected from all listed meningo-encephalitis cases in three hospitals during the Nipah season (January-March). An investigation team visited clustered cases' communities to collect epidemiological information and blood from the living cases. We tested serum using Nipah-specific IgM ELISA. Up to September 2011, in 5887 listed cases, we identified 62 clusters comprising 176 encephalitis cases. We collected blood from 127 of these cases. In 10 clusters, we identified a total of 62 Nipah cases: 18 laboratory-confirmed and 34 probable. We identified person-to-person transmission of Nipah virus in four clusters. From case-based surveillance, we identified 23 (4%) Nipah cases. Faced with thousands of encephalitis cases, integrated cluster surveillance allows targeted deployment of investigative resources to detect outbreaks by stage III zoonotic pathogens in resource-limited settings.

Genomic analysis of filoviruses associated with four viral hemorrhagic fever outbreaks in Uganda and the Democratic Republic of the Congo in 2012.

In 2012, an unprecedented number of four distinct, partially overlapping filovirus-associated viral hemorrhagic fever outbreaks were detected in equatorial Africa. Analysis of complete virus genome sequences confirmed the reemergence of Sudan virus and Marburg virus in Uganda, and the first emergence of Bundibugyo virus in the Democratic Republic of the Congo.

Nipah virus outbreak with person-to-person transmission in a district of Bangladesh, 2007.

In February 2007 an outbreak of Nipah virus (NiV) encephalitis in Thakurgaon District of northwest Bangladesh affected seven people, three of whom died. All subsequent cases developed illness 7-14 days after close physical contact with the index case while he was ill. Cases were more likely than controls to have been in the same room (100% vs. 9.5%, OR undefined, P<0.001) and to have touched him (83% vs. 0%, OR undefined, P<0.001). Although the source of infection for the index case was not identified, 50% of Pteropus bats sampled from near the outbreak area 1 month after the outbreak had antibodies to NiV confirming the presence of the virus in the area. The outbreak was spread by person-to-person transmission. Risk of NiV infection in family caregivers highlights the need for infection control practices to limit transmission of potentially infectious body secretions.

Risk factors for Nipah virus encephalitis in Bangladesh.

Nipah virus (NiV) is a paramyxovirus that causes severe encephalitis in humans. During January 2004, twelve patients with NiV encephalitis (NiVE) were identified in west-central Bangladesh. A case-control study was conducted to identify factors associated with NiV infection. NiVE patients from the outbreak were enrolled in a matched case-control study. Exact odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by using a matched analysis. Climbing trees (83% of cases vs. 51% of controls, OR 8.2, 95% CI 1.25-infinity) and contact with another NiVE patient (67% of cases vs. 9% of controls, OR 21.4, 95% CI 2.78-966.1) were associated with infection. We did not identify an increased risk for NiV infection among persons who had contact with a potential intermediate host. Although we cannot rule out person-to-person transmission, case-patients were likely infected from contact with fruit bats or their secretions.

Histopathologic and immunohistochemical characterization of Nipah virus infection in the guinea pig.

Mortality rate in humans infected with Nipah virus (NiV) has been reported as high as 92%. Humans infected with NiV show a widespread multisystemic vasculitis with most severe clinical and pathologic manifestations in the brain, lungs, and spleen. The purpose of this study was to study pathologic and immunohistochemical findings in guinea pigs infected with NiV. Of 28 animals inoculated intraperitoneally, only 2 survived the infection, and most died between 4 and 8 days postinoculation (dpi). Viral antigen with minimal pathologic changes was first detected 2 dpi in lymph nodes and spleen. More severe changes were noted in these organs 4-8 dpi, where pathologic damage had a vasocentric distribution and viral antigen was abundant in vascular endothelium, tunica media, adventitia, as well as in macrophages lining sinuses. The urinary bladder, uterus, and ovaries were also affected with necrosis and acute inflammation. In these organs, immunohistochemical positive staining was intense in blood vessels, epithelial cells, and ovarian follicles. Approximately 50% of the animals that died or were euthanized in extremis had evidence of viral antigen and histopathologic changes in brain, especially involving meninges and ependymal cells, with lesser changes in the neural parenchyma. A unifying feature of the damage for all affected tissues was necrosis and inflammation of the vasculature, chiefly in arterioles, capillaries, and venules. Inoculation of guinea pigs intraperitoneally with NiV produces a disease with considerable resemblance to the disease in humans, but with reduced pulmonary involvement and marked infection of urinary bladder and the female reproductive tract.

Spatial and temporal patterns of Zaire ebolavirus antibody prevalence in the possible reservoir bat species.

To characterize the distribution of Zaire ebolavirus (ZEBOV) infection within the 3 bat species (Epomops franqueti, Hypsignathus monstrosus, and Myonycteris torquata) that are possible reservoirs, we collected 1390 bats during 2003-2006 in Gabon and the Republic of the Congo. Detection of ZEBOV immunoglobulin G (IgG) in 40 specimens supports the role of these bat species as the ZEBOV reservoirs. ZEBOV IgG prevalence rates (5%) were homogeneous across epidemic and nonepidemic regions during outbreaks, indicating that infected bats may well be present in nonepidemic regions of central Africa. ZEBOV IgG prevalence decreased, significantly, to 1% after the outbreaks, suggesting that the percentage of IgG-positive bats is associated with virus transmission to other animal species and outbreak appearance. The large number of ZEBOV IgG-positive adult bats and pregnant H. monstrosus females suggests virus transmission within bat populations through fighting and sexual contact. Our study, thus, helps to describe Ebola virus circulation in bats and offers some insight into the appearance of outbreaks.

A serological survey of Ebola virus infection in central African nonhuman primates.

We used an ELISA to determine the prevalence of IgG antibodies specific for the Zaire subtype of Ebola virus in 790 nonhuman primates, belonging to 20 species, studied between 1985 and 2000 in Cameroon, Gabon, and the Republic of Congo. The seroprevalence rate of Ebola antibody in wild-born chimpanzees was 12.9%, indicating that (1) Ebola virus circulates in the forests of a large region of central Africa, including countries such as Cameroon, where no human cases of Ebola infections have been reported; (2) Ebola virus was present in the area before recent outbreaks in humans; (3) chimpanzees are continuously in contact with the virus; and (4) nonlethal Ebola infection can occur in chimpanzees. These results, together with the unexpected detection of Ebola-specific IgG in other species (5 drills, 1 baboon, 1 mandrill, and 1 Cercopithecus), may help to narrow the search for the reservoir of Ebola virus. They also suggest that future Ebola outbreaks may occur anywhere in the central African forest region.

Isolation of Kaeng Khoi virus from dead Chaerephon plicata bats in Cambodia.

A virus isolated from dead Chaerephon plicata bats collected near Kampot, Cambodia, was identified as a member of the family Bunyaviridae by electron microscopy. The only bunyavirus previously isolated from Chaerephon species bats in South-East Asia is Kaeng Khoi (KK) virus (genus Orthobunyavirus), detected in Thailand over 30 years earlier and implicated as a public health problem. Using RT-PCR, nucleotide sequences from the M RNA segment of several virus isolates from the Cambodian C. plicata bats were found to be almost identical and to differ from those of the prototype KK virus by only 2.6-3.2 %, despite the temporal and geographic separation of the viruses. These results identify the Cambodian bat viruses as KK virus, extend the known virus geographic range and document the first KK virus isolation in 30 years. These genetic data, together with earlier serologic data, show that KK viruses represent a distinct group within the genus Orthobunyavirus.

A survey of Nipah virus infection among various risk groups in Singapore.

Following the Nipah virus (NV) outbreak in March 1999 in Singapore, a serological survey was undertaken to screen individuals potentially exposed to NV. Blood samples were tested for NV IgM, IgG and neutralizing antibodies. Twenty-two (1.5%) of 1469 people tested had antibodies suggesting NV infection. Although 12 of the 22 infected people (54.6%) were symptomatic, the remaining 10 (45.4%) were clinically well and had no past history of compatible pulmonary or neurological disease. Clinical and serological findings suggested three people had been infected with NV before the outbreak was recognized. All those who were infected were male abattoir workers. None of the people who had contact with horses, and no healthcare workers exposed to infected patients and their specimens had detectable antibodies. This study provides evidence that NV causes asymptomatic infection. All of the antibody positive individuals had direct contact with pigs and there was no evidence of human to human transmission.

Discriminators between hantavirus-infected and -uninfected persons enrolled in a trial of intravenous ribavirin for presumptive hantavirus pulmonary syndrome.

To provide a potentially therapeutic intervention and to collect clinical and laboratory data during an outbreak of hantavirus pulmonary syndrome (HPS), 140 patients from the United States with suspected HPS were enrolled for investigational intravenous ribavirin treatment. HPS was subsequently laboratory confirmed in 30 persons and not confirmed in 105 persons with adequate specimens. Patients with HPS were significantly more likely than were hantavirus-negative patients to report myalgias from onset of symptoms through hospitalization, nausea at outpatient presentation, and diarrhea and nausea at the time of hospitalization; they were significantly less likely to report respiratory symptoms early in the illness. The groups did not differ with regard to time from the onset of illness to the point at which they sought care; time from onset, hospitalization, or enrollment to death was significantly shorter for patients with HPS. At the time of hospitalization, patients with HPS more commonly had myelocytes, metamyelocytes, or promyelocytes on a peripheral blood smear, and significantly more of them had thrombocytopenia, hemoconcentration, and hypocapnia. Patterns of clinical symptoms, the pace of clinical evolution, and specific clinical laboratory parameters discriminated between these 2 groups.

Multiplex analysis of cytokines in the blood of cynomolgus macaques naturally infected with Ebola virus (Reston serotype).

Ebola virus (EBO) causes the most severe form of viral hemorrhagic fever in humans and nonhuman primates with up to 90% of infections culminating in death. The requirement of maximum containment laboratories for Ebola virus research has limited opportunities to study the pathogenesis of EBO infections. While tissue damage does occur, often it would appear not to be sufficient to explain death, indicating that soluble mediators play an important role in disease progression. In previous studies, fatal human infections with the Zaire subtype of Ebola (EBO-Z) were associated with an increase in the levels of inflammatory cytokines. In this investigation, a new multiplex assay was developed and used to measure circulating levels of cytokines and chemokines in cynomolgus macaques infected with the Reston subtype of EBO (EBO-R). Increased levels of IL-6, TNF-alpha, IFN-gamma, IL-2, IL-4, IL-8, IL-10, and GM-CSF were detected in infected animals, and the increase in circulating cytokines correlated with an increase in circulating viral antigen. Blood samples from animals showing high levels of cytokines were also tested for the chemokines: MCP-1, IL-1beta, MIP-1alpha, MIP-1beta, IP-10, and RANTES. High levels of MCP-1 and MIP-1beta, and RANTES were found in infected primates and, while levels were more variable, IL-1beta was detected only in infected animals.

Molecular characterization of the polymerase gene and genomic termini of Nipah virus.

In 1998, Nipah virus (NV) emerged in peninsular Malaysia, causing fatal encephalitis in humans and a respiratory disease in swine. NV is most closely related to Hendra virus (HV), a paramyxovirus that was identified in Australia in 1994, and it has been proposed that HV and NV represent a new genus within the family Paramyxoviridae. This report describes the analysis of the sequences of the polymerase gene (L) and genomic termini of NV as well as a comparison of the full-length, genomic sequences of HV and NV. The L gene of NV is predicted to be 2244 amino acids in size and contains the six domains found within the L proteins of all nonsegmented, negative-stranded (NNS) RNA viruses. However, the GDNQ motif found in most NNS RNA viruses was replaced by GDNE in both NV and HV. The 3' and 5' termini of the NV genome are nearly identical to the genomic termini of HV and share sequence homology with the genomic termini of other members of the subfamily Paramyxovirinae. At 18,246 nucleotides, the genome of NV is 12 nucleotides longer than the genome of HV and they have the largest genomes within the family Paramyxoviridae. The comparison of the structures of the genomes of HV and NV is now complete and this information will help to establish the taxonomic position of these novel viruses within the family Paramyxoviridae.

Hantavirus infection induces the expression of RANTES and IP-10 without causing increased permeability in human lung microvascular endothelial cells.

Sin Nombre virus (SNV) and Hantaan virus (HTN) infect endothelial cells and are associated with different patterns of increased vascular permeability during human disease. It is thought that such patterns of increased vascular permeability are a consequence of endothelial activation and subsequent dysfunction mediated by differential immune responses to hantavirus infection. In this study, the ability of hantavirus to directly induce activation of human lung microvascular endothelial cells (HMVEC-Ls) was examined. No virus-specific modulation in the constitutive or cytokine-induced expression of cellular adhesion molecules (CD40, CD54, CD61, CD62E, CD62P, CD106, and major histocompatibility complex classes I and II) or in cytokines and chemokines (eotaxin, tumor necrosis factor alpha, interleukin 1beta [IL-1beta], IL-6, IL-8, MCP-1, MIP-1alpha, and MIP-1beta) was detected at either the protein or message level in hantavirus-infected HMVEC-Ls. Furthermore, no virus-specific enhancement of paracellular or transcellular permeability or changes in the organization and distribution of endothelial intercellular junctional proteins was observed. However, infection with either HTN or SNV resulted in detectable levels of the chemokines RANTES and IP-10 (the 10-kDa interferon-inducible protein) in HMVEC-Ls within 72 h and was associated with nuclear translocation of interferon regulatory factor 3 (IRF-3) and IRF-7. Gamma interferon (IFN-gamma)-induced expression of RANTES and IP-10 could also be detected in uninfected HMVEC-Ls and was associated with nuclear translocation of IRF-1 and IRF-3. Treatment of hantavirus-infected HMVEC-Ls with IFN-gamma for 24 h resulted in a synergistic enhancement in the expression of both RANTES and IP-10 and was associated with nuclear translocation of IRF-1, IRF-3, IRF-7, and NF-kappaB p65. These results reveal a possible mechanism by which hantavirus infection and a TH1 immune response can cooperate to synergistically enhance chemokine expression by HMVEC-Ls and trigger immune-mediated increases in vascular permeability.

Low levels of interleukin-8 and interferon-inducible protein-10 in serum are associated with fatal infections in acute Lassa fever.

To investigate the role of inflammatory mediators in the pathogenesis of Lassa fever, the levels of a number of pro- and anti-inflammatory cytokines and chemokines in serum samples collected from hospitalized patients with fatal and nonfatal acute Lassa fever were compared with those from 2 control groups: patients with other febrile illnesses and uninfected individuals. Serum interleukin (IL)-8 and interferon (IFN)-inducible protein (IP)-10 levels were significantly higher in patients with acute nonfatal Lassa fever than in control subjects. In striking contrast, levels of these chemokines were low or undetectable in patients with fatal Lassa fever. IFN-gamma, IL-12, IL-6, and RANTES levels were elevated in all the febrile study groups. Tumor necrosis factor-alpha levels were not elevated in patients with fatal or nonfatal Lassa fever. These data indicate that acute nonfatal Lassa fever is associated with high levels of circulating IL-8 and IP-10 and that low levels or absence of these mediators correlates with a poor outcome.

Monocyte-derived human macrophages and peripheral blood mononuclear cells infected with ebola virus secrete MIP-1alpha and TNF-alpha and inhibit poly-IC-induced IFN-alpha in vitro.

Ebola virus infection of humans is associated with high levels of circulating inflammatory chemokines and cytokines. We demonstrate that direct infection of human PBMC results in the induction of MCP-1, MIP-1alpha, RANTES, and TNF-alpha as early as 24 h p.i. in response to live virus. Monocyte-derived macrophages infected with live Ebola-virus secreted MIP-1alpha and TNF-alpha specifically while RANTES and MCP-1 were secreted by with both live or inactivated virus stimulation and do not require viral replication. Type I interferons (IFN-alpha and -beta), IL-1beta and IL-10, were not induced by Ebola virus. Furthermore, live virus infection of both PBMCs and monocytes-derived macrophages inhibited IFN-alpha induced by double-stranded RNA in vitro. These data provide the first direct evidence of a role for macrophages in the pathogenesis to Ebola virus and suggest that Ebola virus can inhibit cellular antiviral mechanisms mediated by type I interferons.

Passive transfer of antibodies protects immunocompetent and imunodeficient mice against lethal Ebola virus infection without complete inhibition of viral replication.

Ebola hemorrhagic fever is a severe, usually fatal illness caused by Ebola virus, a member of the filovirus family. The use of nonhomologous immune serum in animal studies and blood from survivors in two anecdotal reports of Ebola hemorrhagic fever in humans has shown promise, but the efficacy of these treatments has not been demonstrated definitively. We have evaluated the protective efficacy of polyclonal immune serum in a mouse model of Ebola virus infection. Our results demonstrate that mice infected subcutaneously with live Ebola virus survive infection and generate high levels of anti-Ebola virus immunoglobulin G (IgG). Passive transfer of immune serum from these mice before challenge protected upto 100% of naive mice against lethal Ebola virus infection. Protection correlated with the level of anti-Ebola virus IgG titers, and passive treatment with high-titer antiserum was associated with a delay in the peak of viral replication. Transfer of immune serum to SCID mice resulted in 100% survival after lethal challenge with Ebola virus, indicating that antibodies alone can protect from lethal disease. Thus antibodies suppress or delay viral growth, provide protection against lethal Ebola virus infection, and may not require participation of other immune components for protection.

Lassa fever in Guinea: I. Epidemiology of human disease and clinical observations.

The arenavirus Lassa is found in West Africa, where it sometimes causes a severe illness called Lassa fever. Lassa fever has been seldom investigated outside of a few hyperendemic regions, where the described epidemiology may differ from that in areas of low or moderate incidence of disease. Through a prospective cohort study, we investigated the epidemiology and clinical presentation of Lassa fever in Guinea, where the disease has been infrequently recognized. A surveillance system was established, and suspected cases were enrolled at five Guinean hospitals. Clinical observations were made, and blood was taken for enzyme-linked immunosorbent assay testing and isolation of Lassa virus. Lassa fever was confirmed in 22 (7%) of 311 suspected cases. Another 43 (14%) had Lassa IgG antibodies, indicating past exposure. Both sexes and a wide variety of age and ethnic groups were affected. The disease was more frequently found, and the IgG seroprevalence generally higher, in the southeastern forest region. In some areas, there were significant discrepancies between the incidence of Lassa fever and the prevalence of antibody. Clinical presentations between those with Lassa fever and other febrile illnesses were essentially indistinguishable. Clinical predictors of a poor outcome were noted, but again were not specific for Lassa fever. Case-fatality rates for those with Lassa fever and non-Lassa febrile illnesses were 18% and 15%, respectively. Seasonal fluctuation in the incidence of Lassa fever was noted, but occurred similarly with non-Lassa febrile illnesses. Our results, perhaps typical of the scenario throughout much of West Africa, indicate Lassa virus infection to be widespread in certain areas of Guinea, but difficult to distinguish clinically.

Lassa fever in Guinea: II. Distribution and prevalence of Lassa virus infection in small mammals.

Rodents of the genus Mastomys form the reservoir for Lassa virus (LV), an arenavirus that causes a potentially severe hemorrhagic illness, Lassa fever (LF). Although Mastomys rodents exist throughout sub-Saharan Africa, areas of human LF appear to be quite focal. The distribution of small mammals and LV-infected Mastomys has been assessed in only a few countries. We conducted a survey of small mammals in selected regions of Guinea to assess the degree to which LV poses a public health risk in that country. A total of 1,616 small mammals, including 956 (59%) Mastomys, were captured from 444 households and seven bush sites. Mastomys made up > 90% of the captured animals in the savannah, savannah-forest transition, and forest regions of Guinea, while Mus musculus dominated in coastal and urban sites. Animals were analyzed via enzyme-linked immunosorbent assay (ELISA) for LV-specific antigen (blood and spleen homogenate) and IgG antibody (blood only). Virus isolation from spleen homogenates was also performed on a subset of animals. Lassa antibody and antigen were found in 96 (11%) and 46 (5%), respectively, of 884 tested Mastomys. Antibody and antigen were essentially mutually exclusive and showed profiles consistent with vertical transmission of both LV and antibody. LV was isolated only from Mastomys. ELISA antigen constituted an acceptable surrogate for virus isolation, with a sensitivity and specificity when performed on blood of 78% (95% confidence interval: 68-83%) and 98% (95-99%), respectively. The proportion of LV-infected Mastomys per region ranged from 0 to 9% and was highest in the savannah and forest zones. The proportion of infected animals per village varied considerably, even between villages in close proximity. Infected animals tended to cluster in relatively few houses, suggesting the existence of focal "hot spots" of LV-infected Mastomys that may account for the observed heterogeneous distribution of LF.