An unusual two-strain cholera outbreak in Lebanon, 2022-2023: a genomic epidemiology study.

Cholera is a life-threatening gastrointestinal infection caused by a toxigenic bacterium, Vibrio cholerae. After a lull of almost 30 years, a first case of cholera was detected in Lebanon in October 2022. The outbreak lasted three months, with 8007 suspected cases (671 laboratory-confirmed) and 23 deaths. In this study, we use phenotypic methods and microbial genomics to study 34 clinical and environmental Vibrio cholerae isolates collected throughout this outbreak. All isolates are identified as V. cholerae O1, serotype Ogawa strains from wave 3 of the seventh pandemic El Tor (7PET) lineage. Phylogenomic analysis unexpectedly reveals the presence of two different strains of the seventh pandemic El Tor (7PET) lineage. The dominant strain has a narrow antibiotic resistance profile and is phylogenetically related to South Asian V. cholerae isolates and derived African isolates from the AFR15 sublineage. The second strain is geographically restricted and extensively drug-resistant. It belongs to the AFR13 sublineage and clusters with V. cholerae isolates collected in Yemen. In conclusion, the 2022-2023 Lebanese cholera outbreak is caused by the simultaneous introduction of two different 7PET strains. Genomic surveillance with cross-border collaboration is therefore crucial for the identification of new introductions and routes of circulation of cholera, improving our understanding of cholera epidemiology.

New member of Plasmodium (Vinckeia) and Plasmodium cyclopsi discovered in bats in Sierra Leone - nuclear sequence and complete mitochondrial genome analyses.

Malaria remains the most important arthropod-borne infectious disease globally. The causative agent, Plasmodium, is a unicellular eukaryote that develops inside red blood cells. Identifying new Plasmodium parasite species that infect mammalian hosts can shed light on the complex evolution and diversity of malaria parasites. Bats feature a high diversity of microorganisms including seven separate genera of malarial parasites. Three species of Plasmodium have been reported so far, for which scarce reports exist. Here we present data from an investigation of Plasmodium infections in bats in the western Guinean lowland forest in Sierra Leone. We discovered a new Plasmodium parasite in the horseshoe bat Rhinolophus landeri. Plasmodium cyclopsi infections in a member of leaf-nosed bats, Doryrhina cyclops, exhibited a high prevalence of 100%. Phylogenetic analysis of complete mitochondrial genomes and nine nuclear markers recovered a close relationship between P. cyclopsi and the new Plasmodium parasite with the rodent species Plasmodium berghei, a widely used in vivo model to study malaria in humans. The data suggests that the "rodent/bat" Plasmodium (Vinckeia) clade represents a diverse group of malarial parasites that would likely expand with a systematic sampling of small mammals in tropical Africa. Identifying the bat Plasmodium repertoire is central to our understanding of the evolution of Plasmodium parasites in mammals.

Sequence optimized diagnostic assay for Ebola virus detection.

Rapid pathogen identification is a critical first step in patient isolation, treatment, and controlling an outbreak. Real-time PCR is a highly sensitive and specific approach commonly used for infectious disease diagnostics. However, mismatches in the primer or probe sequence and the target organism can cause decreased sensitivity, assay failure, and false negative results. Limited genomic sequences for rare pathogens such as Ebola virus (EBOV) can negatively impact assay performance due to undiscovered genetic diversity. We previously developed and validated several EBOV assays prior to the 2013-2016 EBOV outbreak in West Africa, and sequencing EBOV Makona identified sequence variants that could impact assay performance. Here, we assessed the impact sequence mismatches have on EBOV assay performance, finding one or two primer or probe mismatches resulted in a range of impact from minimal to almost two log sensitivity reduction. Redesigning this assay improved detection of all EBOV variants tested. Comparing the performance of the new assay with the previous assays across a panel of human EBOV samples confirmed increased assay sensitivity as reflected in decreased Cq values with detection of three positive that tested negative with the original assay.

The deployment of mobile diagnostic laboratories for Ebola virus disease diagnostics in Sierra Leone and Guinea.

BACKGROUND: Ebola virus emerged in West Africa in December 2013. The ease of mobility, porous borders, and lack of public health infrastructure led to the largest Ebola virus disease (EVD) outbreak to date. INTERVENTION: The 2013 EVD outbreak signalled the need for laboratory diagnostic capabilities in areas without strong public health systems. As part of the United States' Department of Defense response, MRIGlobal was contracted to design, fabricate, equip, deploy, and operate two mobile diagnostic laboratories (MDLs). The first laboratory analysed blood samples from patients in an adjacent Ebola Treatment Centre (ETC) and buccal swabs from the deceased in the community in Moyamba, Sierra Leone. The second laboratory was deployed to support an ETC in Conakry, Guinea. The Department of Defense provided real-time quantitative reverse transcription polymerase chain reaction assays that were deployed and validated on-site. LESSONS LEARNT: Prompt and accurate molecular diagnostics reduced sample turn-around times from over 24 h to under 4 h. Experienced laboratory staff tested up to 110 samples per day and on-site engineering proved necessary for MDL setup and operation. As the Ebola response slowed, the sustainment of the MDLs' operations was prioritised, including staff training and the transition of the MDLs to local governments. Training programmes for local staff were prepared in Sierra Leone and Guinea. RECOMMENDATIONS: The MRIGlobal MDL team significantly contributed to establishing increased laboratory capacity during the EVD outbreak in West Africa. Using the MDLs for molecular diagnosis is highly recommended until more sustainable solutions can be provided.

Molecular analysis of the 2012 Bundibugyo virus disease outbreak.

Bundibugyo virus (BDBV) is one of four ebolaviruses known to cause disease in humans. Bundibugyo virus disease (BVD) outbreaks occurred in 2007-2008 in Bundibugyo District, Uganda, and in 2012 in Isiro, Province Orientale, Democratic Republic of the Congo. The 2012 BVD outbreak resulted in 38 laboratory-confirmed cases of human infection, 13 of whom died. However, only 4 BDBV specimens from the 2012 outbreak have been sequenced. Here, we provide BDBV sequences from seven additional patients. Analysis of the molecular epidemiology and evolutionary dynamics of the 2012 outbreak with these additional isolates challenges the current hypothesis that the outbreak was the result of a single spillover event. In addition, one patient record indicates that BDBV's initial emergence in Isiro occurred 50 days earlier than previously accepted. Collectively, this work demonstrates how retrospective sequencing can be used to elucidate outbreak origins and provide epidemiological contexts to a medically relevant pathogen.

Sustainable Laboratory Capacity Building After the 2014 Ebola Outbreak in the Republic of Guinea.

Background: The 2014-2016 West Africa Ebola virus disease outbreak heavily impacted the Republics of Guinea, Sierra Leone, and Liberia. The outbreak uncovered the weaknesses of the public health systems, including inadequately trained and insufficient health personnel as well as limited and poorly equipped health infrastructures. These weaknesses represent significant threats to global health security. In the wake of the outbreak, affected countries made urgent requests for international engagement to help strengthening the public health systems. Methods: This work describes the successful multi-year implementation of a laboratory capacity building program in the Republic of Guinea. The program integrated biorisk and quality management systems training, infectious diseases diagnostic training, facility engineering and maintenance training, and mentorship to strengthen Guinea's bio-surveillance capacity. Results: The major outcome of these efforts was an established and local staff-operated public health laboratory that performs disease surveillance and reporting and diagnostic of priority diseases and pathogens of security concerns. Conclusions: This work has improved the Guinea country's capabilities to address country public health issues and preparedness to respond to future infectious disease threats.

HLA-C-restricted viral epitopes are associated with an escape mechanism from KIR2DL2+ NK cells in Lassa virus infection.

BACKGROUND: Lassa virus (LASV) is the etiologic agent of an acute hemorrhagic fever endemic in West Africa. Natural killer (NK) cells control viral infections in part through the interaction between killer cell immunoglobulin-like receptors (KIRs) and their ligands. LASV infection is associated with defective immune responses, including inhibition of NK cell activity in the presence of MHC-class 1+-infected target cells. METHODS: We compared individual KIR and HLA-class 1 genotypes of 68 healthy volunteers to 51 patients infected with LASV in Sierra Leone, including 37 survivors and 14 fatalities. Next, potential HLA-C1, HLA-C2, and HLA-Bw4 binding epitopes were in silico screened among LASV nucleoprotein (NP) and envelope glycoprotein (GP). Selected 10-mer peptides were then tested in peptide-HLA stabilization, KIR binding and polyfunction assays. FINDINGS: LASV-infected patients were similar to healthy controls, except for the inhibitory KIR2DL2 gene. We found a specific increase in the HLA-C1:KIR2DL2 interaction in fatalities (10/11) as compared to survivors (12/19) and controls (19/29). We also identified that strong of NP and GP viral epitopes was only observed with HLA-C molecules, and associated with strong inhibition of degranulation in the presence of KIR2DL+ NK cells. This inhibitory effect significantly increased in the presence of the vGP420 variant, detected in 28.1% of LASV sequences. INTERPRETATION: Our finding suggests that presentation of specific LASV epitopes by HLA-C alleles to the inhibitory KIR2DL2 receptor on NK cells could potentially prevent the killing of infected cells and provides insights into the mechanisms by which LASV can escape NK-cell-mediated immune pressure.

Human Monkeypox in Sierra Leone after 44-Year Absence of Reported Cases.

We note the reemergence of human monkeypox in Sierra Leone following a 44-year absence of reported disease. The persons affected were an 11-month-old boy and, several years later, a 35-year-old man. The reappearance of monkeypox in this country suggests a need for renewed vigilance and awareness of the disease and its manifestations.

Virus-encoded miRNAs in Ebola virus disease.

Ebola virus (EBOV) is a negative-strand RNA virus that replicates in the cytoplasm and causes an often-fatal hemorrhagic fever. EBOV, like other viruses, can reportedly encode its own microRNAs (miRNAs) to subvert host immune defenses. miRNAs are short noncoding RNAs that can regulate gene expression by hybridizing to multiple mRNAs, and viral miRNAs can enhance viral replication and infectivity by regulating host or viral genes. To date, only one EBOV miRNA has been examined in human infection. Here, we assayed mouse, rhesus macaque, cynomolgus macaque, and human samples infected with three EBOV variants for twelve computationally predicted viral miRNAs using RT-qPCR. Ten miRNAs aligned to EBOV variants and were detectable in the four species during disease with several viral miRNAs showing presymptomatic amplification in animal models. miRNA abundances in both the mouse and nonhuman primate models mirrored the human cohort, with miR-1-5p, miR-1-3p, and miR-T3-3p consistently at the highest levels. These striking similarities in the most abundant miRNAs during infection with different EBOV variants and hosts indicate that these miRNAs are potential valuable diagnostic markers and key effectors of EBOV pathogenesis.