Rodent control strategies and Lassa virus: some unexpected effects in Guinea, West Africa.

The Natal multimammate mouse (Mastomys natalensis) is the host of Lassa mammarenavirus, causing Lassa haemorrhagic fever in West Africa. As there is currently no operational vaccine and therapeutic drugs are limited, we explored rodent control as an alternative to prevent Lassa virus spillover in Upper Guinea, where the disease is highly endemic in rural areas. In a seven-year experiment, we distributed rodenticides for 10-30 days once a year and, in the last year, added intensive snap trapping for three months in all the houses of one village. We also captured rodents both before and after the intervention period to assess their effectiveness by examining alterations in trapping success and infection rates (Lassa virus RNA and IgG antibodies). We found that both interventions reduced the rodent population by 74-92% but swiftly rebounded to pre-treatment levels, even already six months after the last snap-trapping control. Furthermore, while we observed that chemical control modestly decreased Lassa virus infection rates annually (a reduction of 5% in seroprevalence per year), the intensive trapping unexpectedly led to a significantly higher infection rate (from a seroprevalence of 28% before to 67% after snap trapping control). After seven years, we conclude that annual chemical control, alone or with intensive trapping, is ineffective and sometimes counterproductive in preventing Lassa virus spillover in rural villages. These unexpected findings may result from density-dependent breeding compensation following culling and the survival of a small percentage of chronically infected rodents that may spread the virus to a new susceptible generation of mice.

Spatio-temporal spread of Lassa virus and a new rodent host in the Mano River Union area, West Africa.

The spread of Lassa virus (LASV) in Guinea, Liberia and Sierra Leone, which together are named the Mano River Union (MRU) area, was examined phylogeographically. To provide a reliable evolutionary scenario, new rodent-derived, whole LASV sequences were included. These were generated by metatranscriptomic next-generation sequencing from rodents sampled between 2003 and 2020 in 21 localities of Guinea and Sierra Leone. An analysis was performed using BEAST to perform continuous phylogeographic inference and EvoLaps v36 to visualize spatio-temporal spread. LASV was identified as expected in its primary host reservoir, the Natal multimammate mouse (Mastomys natalensis), and also in two Guinean multimammate mice (Mastomys erythroleucus) in northern Sierra Leone and two rusty-bellied brush-furred mice (Lophuromys sikapusi) in southern Sierra Leone. This finding is consistent with the latter two species being secondary host reservoirs. The strains in these three species were very closely related in LASV lineage IV. Phylogenetic analysis indicated that the most recent common ancestor of lineage IV existed 316-374 years ago and revealed distinct, well-supported clades from Sierra Leone (Bo, Kabala and Kenema), Guinea (Faranah, Kissidougou-Guekedou and Macenta) and Liberia (Phebe-Ganta). The phylogeographic scenario suggests southern Guinea as the point of origin of LASV in the MRU area, with subsequent spread to towards Mali, Liberia and Sierra Leone at a mean speed of 1.6 to 1.1 km/year.

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.

Prevalence of malaria resistance-associated mutations in Plasmodium falciparum circulating in 2017-2018, Bo, Sierra Leone.

Introduction: In spite of promising medical, sociological, and engineering strategies and interventions to reduce the burden of disease, malaria remains a source of significant morbidity and mortality, especially among children in sub-Saharan Africa. In particular, progress in the development and administration of chemotherapeutic agents is threatened by evolved resistance to most of the antimalarials currently in use, including artemisinins. Methods: This study analyzed the prevalence of mutations associated with antimalarial resistance in Plasmodium falciparum from 95 clinical samples collected from individuals with clinically confirmed malaria at a hospital in Bo, Sierra Leone between May 2017 and December 2018. The combination of polymerase chain reaction amplification and subsequent high throughput DNA sequencing was used to determine the presence of resistance-associated mutations in five P. falciparum genes - pfcrt, pfmdr1, pfdhfr, pfdhps and pfkelch13. The geographic origin of parasites was assigned using mitochondrial sequences. Results: Relevant mutations were detected in the pfcrt (22%), pfmdr1 (>58%), pfdhfr (100%) and pfdhps (>80%) genes while no resistance-associated mutations were found in the pfkelch13 gene. The mitochondrial barcodes were consistent with a West African parasite origin with one exception indicating an isolate imported from East Africa. Discussion: Detection of the pfmdr1 NFSND haplotype in 50% of the samples indicated the increasing prevalence of strains with elevated tolerance to artemeter + lumefantrine (AL) threatening the combination currently used to treat uncomplicated malaria in Sierra Leone. The frequency of mutations linked to resistance to antifolates suggests widespread resistance to the drug combination used for intermittent preventive treatment during pregnancy.

Detection of Lassa Virus-Reactive IgG Antibodies in Wild Rodents: Validation of a Capture Enzyme-Linked Immunological Assay.

The aim of this study was to evaluate the use of a capture enzyme-linked immunosorbent assay (ELISA) for the detection of LASV-reactive IgG antibodies in Mastomys rodents. The assay was used for laboratory-bred Mastomys rodents, as well as for animals caught in the wild in various regions of West Africa. The ELISA reached an accuracy of 97.1% in samples of known exposure, and a comparison to an immunofluorescence assay (IFA) revealed a very strong agreement between the ELISA and IFA results (Cohen's kappa of 0.81). The agreement is valid in Nigeria, and in Guinea and Sierra Leone where the lineages II and IV are circulating, respectively. Altogether, these results indicate that this capture ELISA is suitable for LASV IgG serostatus determination in Mastomys rodents as an alternative to IFA. This assay will be a strong, accurate, and semi-quantitative alternative for rodent seroprevalence studies that does not depend on biosafety level 4 infrastructures, providing great benefits for ecology and epidemiology studies of Lassa fever, a disease listed on the Research and Development Blueprint of the WHO.

Detection of Lassa virus in wild rodent feces: Implications for Lassa fever burden within households in the endemic region of Faranah, Guinea.

Lassa arenavirus (LASV) is the cause of Lassa Fever in humans in West Africa. The multimammate mouse (Mastomys natalensis) is a reservoir host of LASV and the primary source of human infections. Humans are assumed to become infected due to contact with this animal or its excretions. Thus far, the available literature does not describe the sampling of feces as a means to detect LASV in M. natalensis populations. More evidence is needed to know if feces of naturally infected M. natalensis can be LASV-positive and an exposure risk to humans. This study sampled feces deposits in households from three villages in the LASV-endemic region of Faranah, Guinea. PCR analysis found 10 out of 88 samples to be positive for LASV, and sequencing showed clustering to previously identified Yarawelia and Dalafilani strains. We conclude that feces sampling is a viable, non-invasive method for the determination and sequencing of LASV strains.

Comparison of capillary and venous blood for malaria detection using two PCR-based assays in febrile patients in Sierra Leone.

BACKGROUND: Rapid and sensitive diagnostics are critical tools for clinical case management and public health control efforts. Both capillary and venous blood are currently used for malaria detection and while diagnostic technologies may not be equally sensitive with both materials, the published data on this subject are scarce and not conclusive. METHODS: Paired clinical samples of venous and capillary blood from 141 febrile individuals in Bo, Sierra Leone, were obtained between January and May 2019 and tested for the presence of Plasmodium parasites using two multiplexed PCR assays: the FilmArray-based Global Fever Panel (GFP) and the TaqMan-based Malaria Multiplex Sample Ready (MMSR) assay. RESULTS: No significant differences in Plasmodium parasite detection between capillary and venous blood for both assays were observed. The GFP assay was more sensitive than MMSR for all markers that could be compared (Plasmodium spp. and Plasmodium falciparum) in both venous and capillary blood. CONCLUSIONS: No difference was found in malaria detection between venous and capillary blood using two different PCR-based detection assays. This data gives support for use of capillary blood, a material which can be obtained easier by less invasive methods, for PCR-based malaria diagnostics, independent of the platform.

Lassa Virus Circulation in Small Mammal Populations in Bo District, Sierra Leone.

Lassa fever is a viral hemorrhagic fever caused by the Lassa virus LASV, which was first isolated in the rodent Mastomys natalensis in 1974 in Kenema, Sierra Leone. As little is known about the abundance and the presence of LASV in rodents living in the Bo area, we carried out a small mammal longitudinal population survey. A standardized trapping session was performed in various habitats and seasons in six villages over two years (2014-2016) and samples collected were tested for arenavirus IgG and LASV. A Bayesian phylogenetic analysis was performed on sequences identified by PCR. A total of 1490 small mammals were collected, and 16 rodent species were identified, with M. natalensis (355, 24%) found to be the most prevalent species. Forty-one (2.8%) samples were IgG positive, and 31 of these were trapped in homes and 10 in surrounding vegetation. Twenty-nine of 41 seropositive rodents were M. natalensis. We detected four LASV by PCR in two villages, all found in M. natalensis. Phylogenetic analysis showed that the sequences were distributed within the Sierra Leonean clade within lineage IV, distinguishing a Bo sub-clade older than a Kenema sub-clade. Compared to other settings, we found a low abundance of M. natalensis and a low circulation of LASV in rodents in villages around Bo district.

Use of real-time multiplex PCR, malaria rapid diagnostic test and microscopy to investigate the prevalence of Plasmodium species among febrile hospital patients in Sierra Leone.

BACKGROUND: Malaria continues to affect over 200 million individuals every year, especially children in Africa. Rapid and sensitive detection and identification of Plasmodium parasites is crucial for treating patients and monitoring of control efforts. Compared to traditional diagnostic methods such as microscopy and rapid diagnostic tests (RDTs), DNA based methods, such as polymerase chain reaction (PCR) offer significantly higher sensitivity, definitive discrimination of Plasmodium species, and detection of mixed infections. While PCR is not currently optimized for routine diagnostics, its role in epidemiological studies is increasing as the world moves closer toward regional and eventually global malaria elimination. This study demonstrates the field use of a novel, ambient temperature-stabilized, multiplexed PCR assay in a small hospital setting in Sierra Leone. METHODS: Blood samples from 534 febrile individuals reporting to a hospital in Bo, Sierra Leone, were tested using three methods: a commercial RDT, microscopy, and a Multiplex Malaria Sample Ready (MMSR) PCR designed to detect a universal malaria marker and species-specific markers for Plasmodium falciparum and Plasmodium vivax. A separate PCR assay was used to identify species of Plasmodium in samples in which MMSR detected malaria, but was unable to identify the species. RESULTS: MMSR detected the presence of any malaria marker in 50.2% of all tested samples with P. falciparum identified in 48.7% of the samples. Plasmodium vivax was not detected. Testing of MMSR P. falciparum-negative/universal malaria-positive specimens with a panel of species-specific PCRs revealed the presence of Plasmodium malariae (n = 2) and Plasmodium ovale (n = 2). The commercial RDT detected P. falciparum in 24.6% of all samples while microscopy was able to detect malaria in 12.8% of tested specimens. CONCLUSIONS: Wider application of PCR for detection of malaria parasites may help to fill gaps existing as a result of use of microscopy and RDTs. Due to its high sensitivity and specificity, species coverage, room temperature stability and relative low complexity, the MMSR assay may be useful for detection of malaria and epidemiological studies especially in low-resource settings.

Estimating the size of urban populations using Landsat images: a case study of Bo, Sierra Leone, West Africa.

BACKGROUND: This is the third paper in a 3-paper series evaluating alternative models for rapidly estimating neighborhood populations using limited survey data, augmented with aerial imagery. METHODS: Bayesian methods were used to sample the large solution space of candidate regression models for estimating population density. RESULTS: We accurately estimated the population densities and counts of 20 neighborhoods in the city of Bo, Sierra Leone, using statistical measures derived from Landsat multi-band satellite imagery. The best regression model proposed estimated the latter with an absolute median proportional error of 8.0%, while the total population of the 20 neighborhoods was estimated with an error of less than 1.0%. We also compare our results with those obtained using an empirical Bayes approach. CONCLUSIONS: Our approach provides a rapid and effective method for constructing predictive models for population densities and counts utilizing remote sensing imagery. Our results, including cross-validation analysis, suggest that masking non-urban areas in the Landsat section images prior to computing the candidate covariate regressors should further improve model generality.

Seroprevalence of hepatitis B surface antigen (HBsAg) in Bo, Sierra Leone, 2012-2013.

OBJECTIVE: The aim of this study was to determine the prevalence of hepatitis B surface antigen (HBsAg) among febrile individuals tested at Mercy Hospital Research Laboratory (MHRL) in Bo, Sierra Leone. RESULTS: A total of 860 febrile individuals ages 5 years and older were tested by MHRL between July 2012 and June 2013 with a Standard Diagnostics Bioline HBsAg rapid diagnostic test. The overall HBsAg prevalence rate was 13.7%, including a rate of 15.5% among males and 12.6% among females. The HBsAg rate did not differ by child or adult age group (p > 0.5). The prevalence rate in Bo was similar to the 11-15% HBsAg prevalence rates reported in the past decade from other studies across West Africa. Scaling up the infant hepatitis B vaccination program in Sierra Leone will be important for reducing the future burden of disease and premature death attributable to chronic viral hepatitis B disease.

Prevalence of markers of HIV infection among febrile adults and children in Bo, Sierra Leone, 2012-2013.

OBJECTIVE: The goal of this study was to examine the prevalence of HIV among febrile patients seeking care in Mercy Hospital, Bo, Sierra Leone, in 2012-2013. RESULTS: A total of 1207 febrile persons were tested for HIV with Determine™ and SD Bioline rapid diagnostic tests kits that detect the presence of HIV antibodies and HIV p24 antigens. The overall prevalence of HIV among the tested patients was 8.9%, which is considerably higher than the < 2% prevalence of HIV reported previously in the general population. While these results are not sufficient to prove a causal relationship, the obtained data imply that HIV positive individuals may be more likely to suffer from febrile infectious diseases than individuals without HIV infection. Increasing the availability and use of HIV testing services will allow antiretroviral therapy to be accessed in a timely manner and improve health status among people living with HIV.

Surveillance of Vector-Borne Infections (Chikungunya, Dengue, and Malaria) in Bo, Sierra Leone, 2012-2013.

Malaria remains a significant cause of morbidity and mortality in West Africa, but the contribution of other vector-borne infections (VBIs) to the burden of disease has been understudied. We used rapid diagnostic tests (RDTs) for three VBIs to test blood samples from 1,795 febrile residents of Bo City, Sierra Leone, over a 1-year period in 2012-2013. In total, 24% of the tests were positive for malaria, fewer than 5% were positive for markers of dengue virus infection, and 39% were positive for IgM directed against chikungunya virus (CHIKV) or a related alphavirus. In total, more than half (55%) of these febrile individuals tested positive for at least one of the three VBIs, which highlights the very high burden of vector-borne diseases in this population. The prevalence of positives on the Chikungunya IgM and dengue tests did not vary significantly with age (P > 0.36), but higher rates of malaria were observed in children < 15 years of age (P < 0.001). Positive results on the Chikungunya IgM RDTs were moderately correlated with rainfall (r2 = 0.599). Based on the high prevalence of positive results on the Chikungunya IgM RDTs from individuals Bo and its environs, there is a need to examine whether an ecological shift toward a greater burden from CHIKV or related alphaviruses is occurring in other parts of Sierra Leone or the West African region.

Finished Genome Sequence of the Highly Multidrug-Resistant Human Urine Isolate Citrobacter freundii Strain SL151.

Citrobacter freundii is a Gram-negative opportunistic pathogen that is increasingly being recognized as a causative agent of hospital-acquired urinary tract infections and an important reservoir of antimicrobial resistance determinants. In this report, we describe the finished genome sequence of C. freundii strain SL151, a highly multidrug-resistant human urine isolate.

Prevalence of Quinolone Resistance in Enterobacteriaceae from Sierra Leone and the Detection of qnrB Pseudogenes and Modified LexA Binding Sites.

A collection of 74 Enterobacteriaceae isolates found in Bo, Sierra Leone, were tested for quinolone antibiotic susceptibility and resistance mechanisms. The majority of isolates (62%) were resistant to quinolones, and 61% harbored chromosomal gyrA and/or parC mutations. Plasmid-mediated quinolone resistance genes were ubiquitous, with qnrB and aac(6')-Ib-cr being the most prevalent. Mutated LexA binding sites were found in all qnrB1 genes, and truncated qnrB pseudogenes were found in the majority of Citrobacter isolates.

Inpatient healthcare provider bypassing by women and their children in urban Bo, Sierra Leone.

INTRODUCTION: Bypassing refers to a person's decision to seek care at a healthcare facility that is not the nearest one of its type to the person's home. METHODS: This study examined inpatient care facility bypassing in urban Bo, Sierra Leone using data from 1,980 women with children 15 years of age and younger who were interviewed in 2010-2011. The locations of residential structures and hospitals were identified using a geographic information system (GIS), and the road distances from participating households to the nearest and preferred inpatient care facilities were measured. RESULTS: Nine inpatient care facilities serve Bo residents, but more than 70% of the participating women reported that the city's main public hospital (Bo Government Hospital), located in the city center, was their preferred inpatient care provider. Participants resided within a median distance of 0.9 km (Interquartile range (IQR): 0.6, 1.8) from their closest inpatient facility, but they would travel a median distance of 2.4 km (IQR: 1.0, 3.3) to reach their preferred providers. About 87% of the women would bypass their nearest inpatient care facility to access care at a preferred provider. Bypassing rates were similar for various demographic and socioeconomic groups, but higher for women living farther from the city center. CONCLUSION: Although Bo has a diverse healthcare marketplace, access to affordable advanced care options is limited. Most women in Bo would choose to bypass facilities nearer to their homes to seek the low-cost and comprehensive care offered by Bo Government Hospital.

Health-care availability, preference, and distance for women in urban Bo, Sierra Leone.

OBJECTIVES: To examine the diversity of the health-care providers in urban Bo, Sierra Leone, identify the types of health-care facilities preferred by women for fevers, and analyze the road network distances from homes to preferred health-care providers. METHODS: A population-based random sampling method was used to recruit 2419 women from Bo. A geographic information system was used to measure the road distance from each woman's home to her preferred provider. RESULTS: Preferred health-care providers for acute febrile illnesses (commonly referred to as "malaria" in the study communities) were hospitals (62.3 %), clinics (12.6 %), and pharmacies (12.4 %). Participants lived a median distance of 0.6 km from the nearest provider, but on average each woman lived 2.2 km one-way from her preferred provider. Women living farther from the city center had preferred providers significantly farther from home than women living downtown. CONCLUSIONS: The diverse health-care marketplace in Bo allows women to select clinical facilities from across the city. Most women prefer a malaria care provider farther from home than they could comfortably walk when ill.

High prevalence of multidrug resistant Enterobacteriaceae isolated from outpatient urine samples but not the hospital environment in Bo, Sierra Leone.

BACKGROUND: The rising level of antimicrobial resistance among bacterial pathogens is one of the most significant public health problems globally. While the antibiotic resistance of clinically important bacteria is closely tracked in many developed countries, the types and levels of resistance and multidrug resistance (MDR) among pathogens currently circulating in most countries of sub-Saharan Africa are virtually unknown. METHODS: From December 2013 to April 2014, we collected 93 urine specimens from all outpatients showing symptoms of urinary tract infection (UTI) and 189 fomite swabs from a small hospital in Bo, Sierra Leone. Culture on chromogenic agar combined with biochemical and DNA sequence-based assays was used to detect and identify the bacterial isolates. Their antimicrobial susceptibilities were determined using a panel of 11 antibiotics or antibiotic combinations. RESULTS: The 70 Enterobacteriaceae urine isolates were identified as Citrobacter freundii (n = 22), Klebsiella pneumoniae (n = 15), Enterobacter cloacae (n = 15), Escherichia coli (n = 13), Enterobacter sp./Leclercia sp. (n = 4) and Escherichia hermannii (n = 1). Antimicrobial susceptibility testing demonstrated that 85.7 % of these isolates were MDR while 64.3 % produced an extended-spectrum ß-lactamase (ESBL). The most notable observations included widespread resistance to sulphonamides (91.4 %), chloramphenicol (72.9 %), gentamycin (72.9 %), ampicillin with sulbactam (51.4 %) and ciprofloxacin (47.1 %) with C. freundii exhibiting the highest and E. coli the lowest prevalence of multidrug resistance. The environmental cultures resulted in only five Enterobacteriaceae isolates out of 189 collected with lower overall antibiotic resistance. CONCLUSIONS: The surprisingly high proportion of C. freundii found in urine of patients with suspected UTI supports earlier findings of the growing role of this pathogen in UTIs in low-resource countries. The isolates of all analyzed species showed worryingly high levels of resistance to both first- and second-line antibiotics as well as a high frequency of MDR and ESBL phenotypes, which likely resulted from the lack of consistent antibiotic stewardship policies in Sierra Leone. Analysis of hospital environmental isolates however suggested that fomites in this naturally ventilated hospital were not a major reservoir for Enterobacteriaceae or antibiotic resistance determinants.

Child bed net use before, during, and after a bed net distribution campaign in Bo, Sierra Leone.

BACKGROUND: This analysis examined how the proportion of children less than 5-years-old who slept under a bed net the previous night changed during and after a national long-lasting insecticidal net (LLIN) distribution campaign in Sierra Leone in November-December 2010. METHODS: A citywide cross-sectional study in 2010-2011 interviewed the caregivers of more than 3000 under-five children from across urban Bo, Sierra Leone. Chi squared tests were used to assess change in use rates over time, and multivariate regression models were used to examine the factors associated with bed net use. RESULTS: Reported rates of last-night bed net use changed from 38.7 % (504/1304) in the months before the LLIN campaign to 21.8 % (78/357) during the week of the campaign to 75.3 % (1045/1387) in the months after the national campaign. The bed net use rate significantly increased (p < 0.01) from before the campaign to after the universal LLIN distribution campaign in all demographic, socioeconomic, and health behaviour groups, even though reported use during the campaign dropped significantly. CONCLUSION: Future malaria prevention efforts will need to promote consistent use of LLINs and address any remaining disparities in insecticide-treated bed net (ITN) use.

Stratified Sampling of Neighborhood Sections for Population Estimation: A Case Study of Bo City, Sierra Leone.

There is a need for better estimators of population size in places that have undergone rapid growth and where collection of census data is difficult. We explored simulated estimates of urban population based on survey data from Bo, Sierra Leone, using two approaches: (1) stratified sampling from across 20 neighborhoods and (2) stratified single-stage cluster sampling of only four randomly-sampled neighborhoods. The stratification variables evaluated were (a) occupants per individual residence, (b) occupants per neighborhood, and (c) residential structures per neighborhood. For method (1), stratification variable (a) yielded the most accurate re-estimate of the current total population. Stratification variable (c), which can be estimated from aerial photography and zoning type verification, and variable (b), which could be ascertained by surveying a limited number of households, increased the accuracy of method (2). Small household-level surveys with appropriate sampling methods can yield reasonably accurate estimations of urban populations.