Identification of Laboratory Biomarkers for Early Detection and Clinical Management of Post-Acute Syndrome Among Survivors of the 2013-2016 West Africa Ebola Outbreak in Sierra Leone.

Background: The clinical management of persistent medical conditions affecting Ebola survivors, generally described as a post-Ebola syndrome, remains a public health concern. We aimed to analyze Ebola survivors' laboratory biomarkers as compared to their non-infected household relatives to identify biomarkers that could guide the identification of survivors at increased risk of developing severe at odds with the non-severe post-Ebola syndrome. Materials and Methods: Data were extracted from medical records of the Ebola survivors clinic, and we included only Ebola survivor's parameters recorded during the first baseline follow-up visit 2 weeks interval after their second negative PCR result. Moreover, household non-infected family contacts of survivors visiting the clinic during the same period were recruited as community control. Results: The mean age of survivors was 32.65 (IQR: 15.5, 38.25) years, and Ebola IgG immunoglobulin was detected in all, thus confirming their status. The statistical significance (all p < 0.05) observed in monocyte percentage (MONO%), cluster of differentiation 4 percentage (CD4%), alanine aminotransferase (ALT), creatinine (CREA), and creatinine kinase (C-kinase) proved to be clinically significant as compared to the household relatives' group. Interestingly, the linear regression analysis indicated that the duration at ETU was negatively associated with lymphocyte percentage with a 5% lymphocyte decrease per day spent at ETU. Finally, there was a significant (p < 0.05) association between hematological (Hb, PCV, MCV, MCH), biochemical (ALT, CREA, C-kinase, T-cholesterol, triglycerides) parameters and the risk of developing severe complications. Conclusion: We recommend clinicians closely monitor Hb, PCV, MCV, MCH, ALT, CREA, C-kinase, T-cholesterol, triglycerides and lymphocytes as clinically relevant laboratory biomarkers to identify survivors at higher risk of developing severe post-acute syndrome upon discharge from Ebola treatment unit including headache, abdominal pain, chest pain, ocular complication, arthralgia, hearing difficulty and erectile dysfunction which can impact health-related quality of life among Ebola survivors.

HIV prevalence in suspected Ebola cases during the 2014-2016 Ebola epidemic in Sierra Leone.

BACKGROUND: The 2014-2016 Ebola virus epidemic in West Africa was the largest outbreak of Ebola virus disease (EVD) in history. Clarifying the influence of other prevalent diseases such as human immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS) will help improve treatment and supportive care of patients with EVD. CASE PRESENTATION: We examined HIV and hepatitis C virus (HCV) antibody prevalence among suspected EVD cases from the Sierra Leone-China Friendship Biological Safety Laboratory during the epidemic in Sierra Leone. HIV and HCV antibodies were tested in 678 EVD-negative samples by enzyme-linked immunosorbent assay. A high HIV prevalence (17.6%) and low HCV prevalence (0.22%) were observed among the suspected cases. Notably, we found decreased HIV positive rates among the suspected cases over the course of the epidemic. This suggests a potentially beneficial effect of an improved public health system after assistance from the World Health Organization and other international aid organizations. CONCLUSIONS: This EVD epidemic had a considerable impact on the public health system and influenced the prevalence of HIV found among suspected cases in Sierra Leone, but also provided an opportunity to establish a better surveillance network for infectious diseases.

Intra-host Ebola viral adaption during human infection.

The onsite next generation sequencing (NGS) of Ebola virus (EBOV) genomes during the 2013-2016 Ebola epidemic in Western Africa provides an opportunity to trace the origin, transmission, and evolution of this virus. Herein, we have diagnosed a cohort of EBOV patients in Sierra Leone in 2015, during the late phase of the outbreak. The surviving EBOV patients had a recovery process characterized by decreasing viremia, fever, and biochemical parameters. EBOV genomes sequenced through the longitudinal blood samples of these patients showed dynamic intra-host substitutions of the virus during acute infection, including the previously described short stretches of 13 serial T>C mutations. Remarkably, within individual patients, samples collected during the early phase of infection possessed Ts at these nucleotide sites, whereas they were replaced by Cs in samples collected in the later phase, suggesting that these short stretches of T>C mutations could emerge independently. In addition, up to a total of 35 nucleotide sites spanning the EBOV genome were mutated coincidently. Our study showed the dynamic intra-host adaptation of EBOV during patient recovery and gave more insight into the complex EBOV-host interactions.

Serological Investigation of Laboratory-Confirmed and Suspected Ebola Virus Disease Patients During the Late Phase of the Ebola Outbreak in Sierra Leone.

This study aimed to investigate the serological characteristics of Ebola virus (EBOV) infection during the late phase of the Ebola outbreak in Sierra Leone. In total, 877 blood samples from 694 suspected Ebola virus disease (EVD) cases assessed from March to December 2015, were analyzed via real-time reverse transcription polymerase chain reaction (RT-PCR) for viral RNA and enzyme-linked immunosorbent assay (ELISA) and Luminex to detect antibodies against EBOV. Viral load and EBOV-specific IgM/IgG titers displayed a declining trend during March to December 2015. Viral RNA load decreased rapidly at earlier stages after disease onset, while EBOV-specific IgM and IgG still persisted in 58.1% (18/31) and 93.5% (29/31) of the confirmed EVD patients and in 3.8% (25/663) and 17.8% (118/663) of the RNA-negative suspected patients in the later phase, respectively. Dynamic analysis of longitudinally collected samples from eight EVD patients revealed typically reversed trends of declining viral load and increasing IgM and/or IgG titers in response to the EBOV infection. The present results indicate that certain populations of Sierra Leone developed immunity to an EBOV infection in the late phase of the outbreak, providing novel insights into the risk assessment of EBOV infections among human populations.

Transplacental transmission: A rare case of Ebola virus transmission.

During the mid-transmission period of the Ebola virus disease (EVD) outbreak in Sierra Leone, a 19-year-old pregnant woman, who was a petty trader in a health facility in Freetown, noticing no fetal movement for the past 3 days, reported to a health facility. Medical history and laboratory testing showed no abnormalities except that she was positive for sickle cell. She was not known to any surveillance team of having any epidemiological link to EVD case. She was induced with oral medications as well as IV infusion. EVD test showed that the fetus was positive to EVD with a high threshold value of 21, while the woman was negative for EVD with a threshold value of 42. The woman was positive to EVD IgG but negative to EVD IgM by ELISA technique. This is a rare EVD case in the period of medium transmission. We conclude that the woman may have come into contact with a low dose of virus not enough to cause a full blown EVD and that her immune system was able to stop the virus from further replication.

Mapping the clinical outcomes and genetic evolution of Ebola virus in Sierra Leone.

Sierra Leone was the most severely affected country in Western Africa during the 2013-2016 outbreak of Ebola virus disease (EVD). Previous genome surveillance studies have revealed the origin, diversity, and evolutionary dynamics of the Ebola virus (EBOV); however, the information regarding EBOV sequences is insufficient, especially the clinical outcomes, given that the correlation between the clinical outcomes and the genetic evolution of EBOV is still not clear. Here, we collected and curated a comprehensive data set that includes 514 EBOV genome sequences from patients with confirmed EVD (including 60 sequences not previously studied), >87.5% of which have residence information and definitive clinical outcomes. Phylogenetic reconstruction revealed 11 lineages of EBOV in Sierra Leone. The median-joining haplotype network showed that haplotypes that are associated with lethal outcomes tend to contribute more to the spread of the EBOV in Sierra Leone than those with live outcomes. Analyses of the spatial-temporal distribution unraveled the lineage-distinctive distribution patterns. Different viral lineages have different case fatality rates (CFRs) during the same stage of the outbreak, implying that several lineages featuring SNPs may correlate with increased/decreased CFRs. This study provides invaluable data sets of EBOV infection and highlights the potential SNPs for further in-depth investigation.

Quantifying the risk of nosocomial infection within Ebola Holding Units: a retrospective cohort study of negative patients discharged from five Ebola Holding Units in Western Area, Sierra Leone.

OBJECTIVES: A central pillar in the response to the 2014 Ebola virus disease (EVD) epidemic in Sierra Leone was the role of Ebola Holding Units (EHUs). These units isolated patients meeting a suspect case definition, tested them for EVD, initiated appropriate early treatment and discharged negative patients to onward inpatient care or home. Positive patients were referred to Ebola Treatment Centres. We aimed to estimate the risk of nosocomial transmission within these EHUs. METHODS: We followed up a cohort of 543 patients discharged with a negative EVD test from five EHUs in the Western Area, Sierra Leone, and examined all line-listed subsequent EVD tests from any facility in the Western Area to see whether the patient was retested within 30 days, matching by name, age and address. We defined possible readmissions as having the same name and age but uncertain address, and confirmed readmissions where name, age and address matched. RESULTS: We found a positive readmission rate of 3.3% (18 cases), which included 1.5% confirmed readmissions (8 cases) and 1.8% possible readmissions (10 cases). This is lower than rates previously reported. We cannot ascertain whether EVD was acquired within the EHUs or from re-exposure in the community. No demographic or clinical variables were identified as risk factors for positive readmission, likely due to our small sample size. CONCLUSIONS: These findings support the EHU model as a safe method for isolation of suspect EVD patients and their role in limiting the spread of EVD.

Environmental Contamination and Persistence of Ebola Virus RNA in an Ebola Treatment Center.

BACKGROUND: Ebola viruses (EBOVs) are primarily transmitted by contact with infected body fluids. Ebola treatment centers (ETCs) contain areas that are exposed to body fluids through the care of patients suspected or confirmed to have EBOV disease. There are limited data documenting which areas/fomites within ETCs pose a risk for potential transmission. This study conducted environmental surveillance in 2 ETCs in Freetown, Sierra Leone, during the 2014-2016 West African Ebola outbreak. METHODS: ETCs were surveyed over a 3-week period. Sites to be swabbed were identified with input from field personnel. Swab samples were collected and tested for the presence of EBOV RNA. Ebola-positive body fluid-impregnated cotton pads were serially sampled. RESULTS: General areas of both ETCs were negative for EBOV RNA. The immediate vicinity of patients was the area most likely to be positive for EBOV RNA. Personal protective equipment became positive during patient care, but chlorine solution washes rendered them negative. CONCLUSIONS: Personal protective equipment and patient environs do become positive for EBOV RNA, but careful attention to decontamination seems to remove it. EBOV RNA was not detected in general ward spaces. Careful attention to decontamination protocols seems to be important in minimizing the presence of EBOV RNA within ETC wards.

Transmission dynamics of Ebola virus disease and intervention effectiveness in Sierra Leone.

Sierra Leone is the most severely affected country by an unprecedented outbreak of Ebola virus disease (EVD) in West Africa. Although successfully contained, the transmission dynamics of EVD and the impact of interventions in the country remain unclear. We established a database of confirmed and suspected EVD cases from May 2014 to September 2015 in Sierra Leone and mapped the spatiotemporal distribution of cases at the chiefdom level. A Poisson transmission model revealed that the transmissibility at the chiefdom level, estimated as the average number of secondary infections caused by a patient per week, was reduced by 43% [95% confidence interval (CI): 30%, 52%] after October 2014, when the strategic plan of the United Nations Mission for Emergency Ebola Response was initiated, and by 65% (95% CI: 57%, 71%) after the end of December 2014, when 100% case isolation and safe burials were essentially achieved, both compared with before October 2014. Population density, proximity to Ebola treatment centers, cropland coverage, and atmospheric temperature were associated with EVD transmission. The household secondary attack rate (SAR) was estimated to be 0.059 (95% CI: 0.050, 0.070) for the overall outbreak. The household SAR was reduced by 82%, from 0.093 to 0.017, after the nationwide campaign to achieve 100% case isolation and safe burials had been conducted. This study provides a complete overview of the transmission dynamics of the 2014-2015 EVD outbreak in Sierra Leone at both chiefdom and household levels. The interventions implemented in Sierra Leone seem effective in containing the epidemic, particularly in interrupting household transmission.

Comparative Evaluation of the Diagnostic Performance of the Prototype Cepheid GeneXpert Ebola Assay.

The Ebola virus disease (EVD) outbreak in West Africa has highlighted an urgent need for point-of-care (POC) assays for the diagnosis of this devastating disease in resource-limited African countries. The diagnostic performance characteristics of a prototype Cepheid GeneXpert Ebola POC used to detect Ebola virus (EBOV) in stored serum and plasma samples collected from suspected EVD cases in Sierra Leone in 2014 and 2015 was evaluated. The GeneXpert Ebola POC is a self-contained single-cartridge automated system that targets the glycoprotein (GP) and nucleoprotein (NP) genes of EBOV and yields results within 90 min. Results from 281 patient samples were compared to the results of a TaqMan real-time reverse transcription-PCR (RT-PCR) targeting the polymerase gene and performed on two real-time PCR machines. Agreement between the three platforms was 100% at cycle threshold (CT) values of ≤34.99, but discordant results were noted between CT values of 35 and 45.The diagnostic sensitivity of the three platforms was 100% in 91 patient samples that were confirmed to be infectious by virus isolation. All three molecular platforms detected viral EBOV RNA in additional samples that did not contain viable EBOV. The analytical sensitivity of the GeneXpert Ebola POC for the detection of NP was higher, and comparable to that of polymerase gene detection, than that for the detection of GP when using a titrated laboratory stock of EBOV. There was no detectable cross-reactivity with other hemorrhagic fever viruses or arboviruses. The GeneXpert Ebola POC offers an easy to operate and sensitive diagnostic tool that can be used for the rapid screening of suspected EVD cases in treatment or in holding centers during EVD outbreaks.

Rapid outbreak sequencing of Ebola virus in Sierra Leone identifies transmission chains linked to sporadic cases.

To end the largest known outbreak of Ebola virus disease (EVD) in West Africa and to prevent new transmissions, rapid epidemiological tracing of cases and contacts was required. The ability to quickly identify unknown sources and chains of transmission is key to ending the EVD epidemic and of even greater importance in the context of recent reports of Ebola virus (EBOV) persistence in survivors. Phylogenetic analysis of complete EBOV genomes can provide important information on the source of any new infection. A local deep sequencing facility was established at the Mateneh Ebola Treatment Centre in central Sierra Leone. The facility included all wetlab and computational resources to rapidly process EBOV diagnostic samples into full genome sequences. We produced 554 EBOV genomes from EVD cases across Sierra Leone. These genomes provided a detailed description of EBOV evolution and facilitated phylogenetic tracking of new EVD cases. Importantly, we show that linked genomic and epidemiological data can not only support contact tracing but also identify unconventional transmission chains involving body fluids, including semen. Rapid EBOV genome sequencing, when linked to epidemiological information and a comprehensive database of virus sequences across the outbreak, provided a powerful tool for public health epidemic control efforts.

Transmission network of the 2014-2015 Ebola epidemic in Sierra Leone.

Understanding the growth and spatial expansion of (re)emerging infectious disease outbreaks, such as Ebola and avian influenza, is critical for the effective planning of control measures; however, such efforts are often compromised by data insufficiencies and observational errors. Here, we develop a spatial-temporal inference methodology using a modified network model in conjunction with the ensemble adjustment Kalman filter, a Bayesian inference method equipped to handle observational errors. The combined method is capable of revealing the spatial-temporal progression of infectious disease, while requiring only limited, readily compiled data. We use this method to reconstruct the transmission network of the 2014-2015 Ebola epidemic in Sierra Leone and identify source and sink regions. Our inference suggests that, in Sierra Leone, transmission within the network introduced Ebola to neighbouring districts and initiated self-sustaining local epidemics; two of the more populous and connected districts, Kenema and Port Loko, facilitated two independent transmission pathways. Epidemic intensity differed by district, was highly correlated with population size (r = 0.76, p = 0.0015) and a critical window of opportunity for containing local Ebola epidemics at the source (ca one month) existed. This novel methodology can be used to help identify and contain the spatial expansion of future (re)emerging infectious disease outbreaks.

Genotypic anomaly in Ebola virus strains circulating in Magazine Wharf area, Freetown, Sierra Leone, 2015.

The Magazine Wharf area, Freetown, Sierra Leone was a focus of ongoing Ebola virus transmission from late June 2015. Viral genomes linked to this area contain a series of 13 T to C substitutions in a 150 base pair intergenic region downstream of viral protein 40 open reading frame, similar to the Ebolavirus/H.sapiens-wt/SLE/2014/Makona-J0169 strain (J0169) detected in the same town in November 2014. This suggests that recently circulating viruses from Freetown descend from a J0169-like virus.

Molecular Signature of the Ebola Virus Associated with the Fishermen Community Outbreak in Aberdeen, Sierra Leone, in February 2015.

We report the complete genome sequence of Ebola virus from a health worker linked to a cluster of cases occurring in the fishing community of Aberdeen, Sierra Leone (February 2015), which were characterized by unusually severe presentation. The sequence, clustering in the SL subclade 3.2.4, harbors mutations potentially relevant for pathogenesis.

Characteristics and clinical management of patients admitted to cholera wards in a regional referral hospital during the 2012 epidemic in Sierra Leone.

BACKGROUND AND OBJECTIVES: In 2012, Sierra Leone suffered a nationwide cholera epidemic which affected the capital Freetown and also the provinces. This study aims to describe the characteristics and clinical management of patients admitted to cholera isolation wards of the main referral hospital in the Northern Province and compare management with standard guidelines. DESIGN: All available clinical records of patients from the cholera isolation wards were reviewed retrospectively. There was no active case finding. The following data were collected from the clinical records after patients had left the ward: date of admission, demographics, symptoms, dehydration status, diagnoses, tests and treatments given, length of stay, and outcomes. RESULTS: A total of 798 patients were admitted, of whom 443 (55.5%) were female. There were 18 deaths (2.3%). Assessment of dehydration status was recorded in 517 (64.8%) of clinical records. An alternative or additional diagnosis was made for 214 patients (26.8%). Intravenous (IV) fluids were prescribed to 767 patients (96.1%), including 95% of 141 patients who had documentation of being not severely dehydrated. A history of vomiting was documented in 92.1% of all patients. Oral rehydration solution (ORS) was given to 629 (78.8%) patients. Doxycycline was given to 380 (47.6%) patients, erythromycin to 34 (4.3%), and other antibiotics were used on 247 occasions. Zinc was given to 209 (26.2%). DISCUSSION: This retrospective study highlights the need for efforts to improve the quality of triage, adherence to clinical guidance, and record keeping. CONCLUSIONS: Data collection and analysis of clinical practices during an epidemic situation would enable faster identification of those areas requiring intervention and improvement.

Phenotypic and genotypic characteristics of Vibrio cholerae O1 isolated from the Sierra Leone cholera outbreak in 2012.

BACKGROUND: This study describes phenotypic, genotypic and antibiotic susceptibility patterns of the strains isolated from the 2012 Sierra Leone cholera outbreak. Rectal swabs were collected from patients and cultured for Vibrio cholerae O1. METHODS: The isolates were subjected to multiplex PCR, mismatch amplification mutation assay (MAMA) PCR, pulsed field gel electrophoresis (PFGE), and antibiotic sensitivity tests using disk diffusion and minimum inhibitory concentration (MIC) E-test following standard procedures. RESULTS: Out of 17 rectal swabs tested, 15 yielded V. cholerae O1 biotype El Tor, serotype Ogawa. All the strains belonged to 'altered' variants as MAMA PCR result showed the presence of classical cholera toxin B. PFGE result revealed four pulse types. Using antibiotic disk diffusion, all the isolates were resistant to erythromycin, chloramphenicol, furazolidone, and trimethoprim/sulfamethoxazole (SXT) except SL1 which was sensitive to chloramphenicol and SXT. All the isolates were sensitive to nalidixic acid, tetracycline, doxycycline, azithromycin, and ciprofloxacin except SL2 which was resistant to nalidixic acid. However, variable sensitivity patterns were observed for kanamycin. The ranges of MIC were 0.125-0.50 mg/l, 0.003-0.023 mg/l and 0.38-0.75 mg/l for azithromycin, ciprofloxacin and tetracycline, respectively. CONCLUSIONS: This study demonstrates that altered variants of V. cholerae O1 of four clonal types were responsible for the 2012 outbreak of cholera in Sierra Leone.

Establishing an enteric bacteria reference laboratory in Sierra Leone.

In 2012, Sierra Leone experienced its worst cholera outbreak in over 15 years affecting 12 of the country's 13 districts. With limited diagnostic capability, particularly in bacterial culture, the cholera outbreak was initially confirmed by microbiological testing of clinical specimens outside of Sierra Leone. During 2012 - 2013, in direct response to the lack of diagnostic microbiology facilities, and to assist in investigating and monitoring the cholera outbreak, diagnostic and reference services were established in Sierra Leone at the Central Public Health Reference Laboratory focusing specifically on isolating and identifying Vibrio cholerae and other enteric bacterial pathogens. Sierra Leone is now capable of confirming cholera cases by reference laboratory testing.

Cholera epidemic associated with consumption of unsafe drinking water and street-vended water--Eastern Freetown, Sierra Leone, 2012.

During 2012, Sierra Leone experienced a cholera epidemic with 22,815 reported cases and 296 deaths. We conducted a matched case-control study to assess risk factors, enrolling 49 cases and 98 controls. Stool specimens were analyzed by culture, polymerase chain reaction (PCR), and pulsed-field gel electrophoresis (PFGE). Conditional logistic regression found that consuming unsafe water (matched odds ratio [mOR]: 3.4; 95% confidence interval [CI]: 1.1, 11.0), street-vended water (mOR: 9.4; 95% CI: 2.0, 43.7), and crab (mOR: 3.3; 95% CI: 1.03, 10.6) were significant risk factors for cholera infection. Of 30 stool specimens, 13 (43%) showed PCR evidence of toxigenic Vibrio cholerae O1. Six specimens yielded isolates of V. cholerae O1, El Tor; PFGE identified a pattern previously observed in seven countries. We recommended ensuring the quality of improved water sources, promoting household chlorination, and educating street vendors on water handling practices.