Metagenomic surveillance uncovers diverse and novel viral taxa in febrile patients from Nigeria.

Effective infectious disease surveillance in high-risk regions is critical for clinical care and pandemic preemption; however, few clinical diagnostics are available for the wide range of potential human pathogens. Here, we conduct unbiased metagenomic sequencing of 593 samples from febrile Nigerian patients collected in three settings: i) population-level surveillance of individuals presenting with symptoms consistent with Lassa Fever (LF); ii) real-time investigations of outbreaks with suspected infectious etiologies; and iii) undiagnosed clinically challenging cases. We identify 13 distinct viruses, including the second and third documented cases of human blood-associated dicistrovirus, and a highly divergent, unclassified dicistrovirus that we name human blood-associated dicistrovirus 2. We show that pegivirus C is a common co-infection in individuals with LF and is associated with lower Lassa viral loads and favorable outcomes. We help uncover the causes of three outbreaks as yellow fever virus, monkeypox virus, and a noninfectious cause, the latter ultimately determined to be pesticide poisoning. We demonstrate that a local, Nigerian-driven metagenomics response to complex public health scenarios generates accurate, real-time differential diagnoses, yielding insights that inform policy.

Circulation of Lassa virus across the endemic Edo-Ondo axis, Nigeria, with cross-species transmission between multimammate mice.

We phylogenetically compared sequences of the zoonotic Lassa virus (LASV) obtained from Mastomys rodents in seven localities across the highly endemic Edo and Ondo States within Nigeria. Sequencing 1641 nt from the S segment of the virus genome, we resolved clades within lineage II that were either limited to Ebudin and Okhuesan in Edo state (2g-beta) or along Owo-Okeluse-Ifon in Ondo state (2g-gamma). We also found clades within Ekpoma, a relatively large cosmopolitan town in Edo state, that extended into other localities within Edo (2g-alpha) and Ondo (2g-delta). LASV variants from M. natalensis within Ebudin and Ekpoma in Edo State (dated approximately 1961) were more ancient compared to those from Ondo state (approximately 1977), suggesting a broadly east-west virus migration across south-western Nigeria; a pattern not always consistent with LASV sequences derived from humans in the same localities. Additionally, in Ebudin and Ekpoma, LASV sequences between M. natalensis and M. erythroleucus were interspersed on the phylogenetic tree, but those from M. erythroleucus were estimated to emerge more recently (approximately 2005). Overall, our results show that LASV amplification in certain localities (reaching a prevalence as high as 76% in Okeluse), anthropogenically-aided spread of rodent-borne variants amidst the larger towns (involving communal accommodation such as student hostels), and virus-exchange between syntopic M. natalensis and M. erythroleucus rodents (as the latter, a savanna species, encroaches southward into the degraded forest) pose perpetual zoonotic hazard across the Edo-Ondo Lassa fever belt, threatening to accelerate the dissemination of the virus into non endemic areas.

Humoral and cellular immune responses to Lassa fever virus in Lassa fever survivors and their exposed contacts in Southern Nigeria.

Elucidating the adaptive immune characteristics of natural protection to Lassa fever (LF) is vital in designing and selecting optimal vaccine candidates. With rejuvenated interest in LF and a call for accelerated research on the Lassa virus (LASV) vaccine, there is a need to define the correlates of natural protective immune responses to LF. Here, we describe cellular and antibody immune responses present in survivors of LF (N = 370) and their exposed contacts (N = 170) in a LASV endemic region in Nigeria. Interestingly, our data showed comparable T cell and binding antibody responses from both survivors and their contacts, while neutralizing antibody responses were primarily seen in the LF survivors and not their contacts. Neutralizing antibody responses were found to be cross-reactive against all five lineages of LASV with a strong bias to Lineage II, the prevalent strain in southern Nigeria. We demonstrated that both T cell and antibody responses were not detectable in peripheral blood after a decade in LF survivors. Notably LF survivors maintained high levels of detectable binding antibody response for six months while their contacts did not. Lastly, as potential vaccine targets, we identified the regions of the LASV Glycoprotein (GP) and Nucleoprotein (NP) that induced the broadest peptide-specific T cell responses. Taken together this data informs immunological readouts and potential benchmarks for clinical trials evaluating LASV vaccine candidates.

A standardised Phase III clinical trial framework to assess therapeutic interventions for Lassa fever.

BACKGROUND: Only one recommendation currently exists for the treatment of Lassa fever (LF), which is ribavirin administered in conjunction with supportive care. This recommendation is primarily based on evidence generated from a single clinical trial that was conducted more than 30 years ago-the methodology and results of which have recently come under scrutiny. The requirement for novel therapeutics and reassessment of ribavirin is therefore urgent. However, a significant amount of work now needs to be undertaken to ensure that future trials for LF can be conducted consistently and reliably to facilitate the efficient generation of evidence. METHODOLOGY: We convened a consultation group to establish the position of clinicians and researchers on the core components of future trials. A Core Eligibility Criteria (CEC), Core Case Definition (CCD), Core Outcome Set (COS) and Core Data Variables (CDV) were developed through the process of a multi-stakeholder consultation that took place using a modified-Delphi methodology. RESULTS: A consensus position was achieved for each aspect of the framework, which accounts for the inclusion of pregnant women and children in future LF clinical trials. The framework consists of 8 core criteria, as well as additional considerations for trial protocols. CONCLUSIONS: This project represents the first step towards delineating the clinical development pathway for new Lassa fever therapeutics, following a period of 40 years without advancement. Future planned projects will bolster the work initiated here to continue the advancement of LF clinical research through a regionally-centred, collaborative methodology, with the aim of delineating a clear pathway through which LF clinical trials can progress efficiently and ensure sustainable investments are made in research capacity at a regional level.

Lassa fever outcomes and prognostic factors in Nigeria (LASCOPE): a prospective cohort study.

BACKGROUND: Lassa fever is a viral haemorrhagic fever endemic in parts of west Africa. New treatments are needed to decrease mortality, but pretrial reference data on the disease characteristics are scarce. We aimed to document baseline characteristics and outcomes for patients hospitalised with Lassa fever in Nigeria. METHODS: We did a prospective cohort study (LASCOPE) at the Federal Medical Centre in Owo, Nigeria. All patients admitted with confirmed Lassa fever were invited to participate and asked to give informed consent. Patients of all ages, including newborn infants, were eligible for inclusion, as were pregnant women. All participants received standard supportive care and intravenous ribavirin according to Nigeria Centre for Disease Control guidelines and underwent systematic biological monitoring for 30 days. Patients' characteristics, care received, mortality, and associated factors were recorded using standard WHO forms. We used univariable and multivariable logistic regression models to investigate an association between baseline characteristics and mortality at day 30. FINDINGS: Between April 5, 2018, and March 15, 2020, 534 patients with confirmed Lassa fever were admitted to hospital, of whom 510 (96%) gave consent and were included in the analysis. The cohort included 258 (51%) male patients, 252 (49%) female patients, 426 (84%) adults, and 84 (16%) children (younger than 18 years). The median time between first symptoms and hospital admission was 8 days (IQR 7-13). At baseline, 176 (38%) of 466 patients had a Lassa fever RT-PCR cycle threshold (Ct) lower than 30. From admission to end of follow-up, 120 (25%) of 484 reached a National Early Warning Score (second version; NEWS2) of 7 or higher, 67 (14%) of 495 reached a Kidney Disease-Improving Global Outcome (KDIGO) stage of 2 or higher, and 41 (8%) of 510 underwent dialysis. All patients received ribavirin for a median of 10 days (IQR 9-13). 62 (12%) patients died (57 [13%] adults and five [6%] children). The median time to death was 3 days (1-6). The baseline factors independently associated with mortality were the following: age 45 years or older (adjusted odds ratio 16·30, 95% CI 5·31-50·30), NEWS2 of 7 or higher (4·79, 1·75-13·10), KDIGO grade 2 or higher (7·52, 2·66-21·20), plasma alanine aminotransferase 3 or more times the upper limit of normal (4·96, 1·69-14·60), and Lassa fever RT-PCR Ct value lower than 30 (4·65, 1·50-14·50). INTERPRETATION: Our findings comprehensively document clinical and biological characteristics of patients with Lassa fever and their relationship with mortality, providing prospective estimates that could be useful for designing future therapeutic trials. Such trials comparing new Lassa fever treatments to a standard of care should take no more than 15% as the reference mortality rate and consider adopting a combination of mortality and need for dialysis as the primary endpoint. FUNDING: Institut National de la Santé et de la Recherche Médicale, University of Oxford, EU, UK Department for International Development, Wellcome Trust, French Ministry of Foreign Affairs, Agence Nationale de Recherches sur le SIDA et les hépatites virales, French National Research Institute for Sustainable Development.

Immunological screening of Lassa Virus among Health workers and Contacts of patients of Lassa fever in Ondo State.

BACKGROUND: The increasing trends of morbidity and mortality of Lassa fever is becoming more alarming in Nigeria. Information about immune response to the virus is limited. At exposure, the level of immunity plays a vital role in the vulnerability of individuals infected. OBJECTIVE: Investigating the immune status of health workers, infected cases and contacts of infected cases of Lassa fever in Ondo State. STUDY DESIGN: Blood samples were collected from 233 individuals comprising 102 health workers, 22 infected cases and 109 contacts of infected cases from Owo and Ose Local Government Areas and transported in triple level packaging. Plasma samples were analyzed for IgG and IgM markers using ReLASV® Pan-Lassa NP IgG/IgM ELISA Kit (Zalgen Labs, LLC, USA) while RNAs extracted from IgM positive samples were analyzed for LASV RNA according to manufacturers' instructions. RESULT: Among the health workers, 20/102 (19.6%) and 2/102 (2.0%) were IgG and IgM positive respectively. While 16/22 (72.7%) and 14/22 (63.6%) were IgG and IgM positive respectively among the infected cases. Of the contacts of infected cases screened, 64/109 (58.7%) were IgG positive while 4/109 (3.7%) were positive for IgM. There was no detectable LASV RNA in the samples analyzed. CONCLUSION: These findings suggest that majority of the health workers are naïve to the virus and hence may be prone to the viral infection. It could also be suggestive that a good personal protective procedure is been practiced by the health workers, hence the low exposure. However, most of the contacts of infected cases show exposure to the virus.

Association of hypoalbuminemia and reversal of albumin-to-globulin ratio with morbidity outcome among hospitalized Lassa fever infected patients at a dedicated treatment center in Ondo state, south-western Nigeria.

BACKGROUND: As of this present moment, there is paucity of data on report concerning the association between hypoalbuminaemia or reversal of albumin-to-globulin ratio and morbidity outcome in Lassa fever (LF) infection as a crucial determinant prognostic-predictor factor for treatment-survival outcome. AIM: This study was designed to determine the association between hypoalbuminaemia, reversal of albumin-to-globulin ratio and morbidity outcome among confirmed LF infected patients. METHODOLOGY: This was a descriptive retrospective study involving the assessment of records of confirmed LF infected patients that were managed at the center from November 2018 to October 2019. RESULTS: Out of 83 recruited participants with complete records, 66 (79.5%) had hypoalbuminaemia, 74 (89.2%) had reversal of albumin-to-globulin ratio. A higher mean value of total white blood cell (WBC) count was observed among patients with hypoalbuminaemia (p < 0.0001) and reversal of albumin-to-globulin ratio (p < 0.0001) when compared to patients with normal values, respectively. Also, this study showed statistically significant associations between serum albumin level versus total WBC count (p < 0.0001), acute kidney injury (AKI; p = 0.009), bleeding diathesis (p < 0.0001), and occurrence of pregnancy miscarriage (p < 0.0001). CONCLUSION: There is a baseline hypoalbuminaemia and reversal of albumin-to-globulin ratio among confirmed LF infected patients. Based on these findings, the serum level of albumin and albumin-to-globulin ratio at presentation may serve as simple early biomarkers to identify patients at high risk for a complicated clinical course of disease. This study also reveals that those hospitalized LF infected patients with hypoalbuminemia and/or reversal of albumin-to-globulin ratio tend to have leucocytosis and experience prolonged duration of illness.

A critical appraisal of COVID-19 as a nosocomial infection: an African perspective.

The pandemic of Coronavirus disease 19 is not abating since the outbreak began in December 2019. Africa is currently experiencing a surge after an initial low incidence and nosocomial infections could be contributing to this. A dominant factor responsible for this is a weak healthcare system because of many years of neglect due to abysmal budgetary allocation to the sector. The testing capacity for COVID-19 diagnosis in Africa is grossly inadequate coupled with a severe shortage of personal protective equipment and inadequate infectious diseases expert. These factors exposed the frontline health workers and patients to the hazard of nosocomial infection with the attendants´ morbidity and mortality. Deliberate efforts need to be made toward reducing nosocomial COVID-19 infection.

Monitoring of Lassa virus infection in suspected and confirmed cases in Ondo State, Nigeria.

INTRODUCTION: Lassa virus (LASV), the causative agent of Lassa fever (LF), an endemic acute viral haemorrhagic illness in Nigeria, is transmitted by direct contact with the rodent, contaminated food or household items. Person-to-person transmission also occurs and sexual transmission has been reported. Thus, this study investigated the presence of LASV in body fluids of suspected and confirmed cases. METHODS: this was a cross-sectional study between March 2018 and April 2019 involving 112 consenting suspected and post ribavirin confirmed cases attending the Lassa fever treatment center in Ondo State. Whole blood was collected from 57 suspected and 29 confirmed cases. Other samples from confirmed cases were 5 each of High Vaginal Swab (HVS) and seminal fluid; 12 breast milk and 4 urine. All samples were analyzed using reverse transcription-PCR (RT-PCR) targeting the S-gene of LASV. RESULTS: analysis of whole blood by RT-PCR showed that 1/57 (1.8%) suspected and 1/29 (3.4%) confirmed post ribavirin treated cases were positive. While LASV was detected in 2/5 (40%) post ribavirin treated seminal fluids and 1/11 (8.3%) breast milk. However, LASV was not detected in any of the HVS and urine samples. CONCLUSION: the detection of LASV in seminal fluid and breast milk of discharged post ribavirin treated cases suggests its persistence in these fluids of recovering Nigerians. The role of postnatal and sexual transmissions in the perennial outbreak of LF needs to be further evaluated.

Lassa fever clinical course and setting a standard of care for future randomized trials: A protocol for a cohort study of Lassa-infected patients in Nigeria (LASCOPE).

BACKGROUND: Lassa Fever (LF), is a severe viral disease prevalent in Western Africa. It is classified as a priority disease by the World Health Organization (WHO). Ribavirin is the recommended therapy despite weak evidence of its efficacy. Promising therapeutic agents are becoming available for evaluation in human. Before launching therapeutic trials, we need data on the evolution of the disease under the best possible conditions of care. METHODS: We have initiated a prospective study in Nigeria to better understand the clinical course and prognostic factors of LF while implementing high quality standardized care. Inclusion criteria are: suspected or confirmed LF and informed consent. Participants are followed 60 days from admission and receive free of charge standardized supportive care and biological monitoring, as well as intravenous ribavirin for those with confirmed LF. Data are collected using standardized case report forms (CRF). Primary and secondary outcomes are fatality and severe morbidity, with special focus on acute kidney dysfunction and pregnancy complications. Factors associated with outcomes will be investigated. RESULTS: The cohort is planned for 3 years. Inclusions started in April 2018 at the Federal Medical Center Owo in Ondo State. A second site will open in Nigeria in 2020 and discussions are underway to open a site in Benin. 150 to 200 new participants are expected per year. CONCLUSIONS: This cohort will: provide evidence to standardize LF case management; provide key inputs to design future clinical trials of novel therapeutics; and establish clinical research teams capable of conducting such trials in LF-endemic areas. STUDY REGISTRATION: The LASCOPE study was registered on ClinicalTrial.gov (NCT03655561).

Epidemiologic and Clinical Features of Lassa Fever Outbreak in Nigeria, January 1-May 6, 2018.

Lassa fever (LF) is endemic to Nigeria, where the disease causes substantial rates of illness and death. In this article, we report an analysis of the epidemiologic and clinical aspects of the LF outbreak that occurred in Nigeria during January 1-May 6, 2018. A total of 1,893 cases were reported; 423 were laboratory-confirmed cases, among which 106 deaths were recorded (case-fatality rate 25.1%). Among all confirmed cases, 37 occurred in healthcare workers. The secondary attack rate among 5,001 contacts was 0.56%. Most (80.6%) confirmed cases were reported from 3 states (Edo, Ondo, and Ebonyi). Fatal outcomes were significantly associated with being elderly; no administration of ribavirin; and the presence of a cough, hemorrhaging, and unconsciousness. The findings in this study should lead to further LF research and provide guidance to those preparing to respond to future outbreaks.

Increase in Lassa Fever Cases in Nigeria, January-March 2018.

We reviewed data pertaining to the massive wave of Lassa fever cases that occurred in Nigeria in 2018. No new virus strains were detected, but in 2018, the outbreak response was intensified, additional diagnostic support was available, and surveillance sensitivity increased. These factors probably contributed to the high case count.

Arenavirus Diversity and Phylogeography of Mastomys natalensis Rodents, Nigeria.

Mastomys natalensis rodents are natural hosts for Lassa virus (LASV). Detection of LASV in 2 mitochondrial phylogroups of the rodent near the Niger and Benue Rivers in Nigeria underlines the potential for LASV emergence in fresh phylogroups of this rodent. A Mobala-like sequence was also detected in eastern Nigeria.