Molecular Diagnostics for Lassa Fever at Irrua Specialist Teaching Hospital, Nigeria: Lessons Learnt from Two Years of Laboratory Operation

Background: Lassa fever is a viral hemorrhagic fever endemic in West Africa. However, none of the hospitals in the endemic areas of Nigeria has the capacity to perform Lassa virus diagnostics. Case identification and management solely relies on non-specific clinical criteria. The Irrua Specialist Teaching Hospital (ISTH) in the central senatorial district of Edo State struggled with this challenge for many years. Methodology/Principal Findings A laboratory for molecular diagnosis of Lassa fever, complying with basic standards of diagnostic PCR facilities, was established at ISTH in 2008. During 2009 through 2010, samples of 1,650 suspected cases were processed, of which 198 (12%) tested positive by Lassa virus RT-PCR. No remarkable demographic differences were observed between PCR-positive and negative patients. The case fatality rate for Lassa fever was 31%. Nearly two thirds of confirmed cases attended the emergency departments of ISTH. The time window for therapeutic intervention was extremely short, as 50% of the fatal cases died within 2 days of hospitalization—often before ribavirin treatment could be commenced. Fatal Lassa fever cases were older (p = 0.005), had lower body temperature (p<0.0001), and had higher creatinine (p<0.0001) and blood urea levels (p<0.0001) than survivors. Lassa fever incidence in the hospital followed a seasonal pattern with a peak between November and March. Lassa virus sequences obtained from the patients originating from Edo State formed—within lineage II—a separate clade that could be further subdivided into three clusters. Conclusions/Significance: Lassa fever case management was improved at a tertiary health institution in Nigeria through establishment of a laboratory for routine diagnostics of Lassa virus. Data collected in two years of operation demonstrate that Lassa fever is a serious public health problem in Edo State and reveal new insights into the disease in hospitalized patients.Organismic and Evolutionary Biolog

Molecular diagnostics for lassa fever at Irrua specialist teaching hospital, Nigeria: lessons learnt from two years of laboratory operation.

BACKGROUND: Lassa fever is a viral hemorrhagic fever endemic in West Africa. However, none of the hospitals in the endemic areas of Nigeria has the capacity to perform Lassa virus diagnostics. Case identification and management solely relies on non-specific clinical criteria. The Irrua Specialist Teaching Hospital (ISTH) in the central senatorial district of Edo State struggled with this challenge for many years. METHODOLOGY/PRINCIPAL FINDINGS: A laboratory for molecular diagnosis of Lassa fever, complying with basic standards of diagnostic PCR facilities, was established at ISTH in 2008. During 2009 through 2010, samples of 1,650 suspected cases were processed, of which 198 (12%) tested positive by Lassa virus RT-PCR. No remarkable demographic differences were observed between PCR-positive and negative patients. The case fatality rate for Lassa fever was 31%. Nearly two thirds of confirmed cases attended the emergency departments of ISTH. The time window for therapeutic intervention was extremely short, as 50% of the fatal cases died within 2 days of hospitalization--often before ribavirin treatment could be commenced. Fatal Lassa fever cases were older (p = 0.005), had lower body temperature (p<0.0001), and had higher creatinine (p<0.0001) and blood urea levels (p<0.0001) than survivors. Lassa fever incidence in the hospital followed a seasonal pattern with a peak between November and March. Lassa virus sequences obtained from the patients originating from Edo State formed--within lineage II--a separate clade that could be further subdivided into three clusters. CONCLUSIONS/SIGNIFICANCE: Lassa fever case management was improved at a tertiary health institution in Nigeria through establishment of a laboratory for routine diagnostics of Lassa virus. Data collected in two years of operation demonstrate that Lassa fever is a serious public health problem in Edo State and reveal new insights into the disease in hospitalized patients

Phylogeography of Lassa Virus in Nigeria

ABSTRACT Lassa virus is genetically diverse with several lineages circulating in West Africa. This study aimed at describing the sequence variability of Lassa virus across Nigeria and inferring its spatiotemporal evolution. We sequenced and isolated 77 Lassa virus strains from 16 Nigerian states. The final data set, including previous works, comprised metadata and sequences of 219 unique strains sampled between 1969 and 2018 in 22 states. Most of this data originated from Lassa fever patients diagnosed at Irrua Specialist Teaching Hospital, Edo State, Nigeria. The majority of sequences clustered with the main Nigerian lineages II and III, while a few sequences formed a new cluster related to Lassa virus strains from Hylomyscus pamfi .Within lineages II and III, seven and five sublineages, respectively, were distinguishable. Phylogeographic analysis suggests an origin of lineage II in the southeastern part of the country around Ebonyi State and a main vector of dispersal toward the west across the Niger River, through Anambra, Kogi, Delta, and Edo into Ondo State. The frontline of virus dispersal appears to be in Ondo. Minor vectors are directed northeast toward Taraba and Adamawa and south toward Imo and Rivers. Lineage III might have spread from northern Plateau State into Kaduna, Nasarawa, Federal Capital Territory, and Bauchi. One sublineage moved south and crossed the Benue River into Benue State. This study provides a geographic mapping of lineages and phylogenetic clusters in Nigeria at a higher resolution. In addition, we estimated the direction and time frame of virus dispersal in the country. IMPORTANCE Lassa virus is the causative agent of Lassa fever, a viral hemorrhagic fever with a case fatality rate of approximately 30% in Africa. Previous studies disclosed a geographical pattern in the distribution of Lassa virus strains and a westward movement of the virus across West Africa during evolution. Our study provides a deeper understanding of the geography of genetic lineages and sublineages of the virus in Nigeria. In addition, we modeled how the virus spread in the country. This knowledge allows us to predict into which geographical areas the virus might spread in the future and prioritize areas for Lassa fever surveillance. Our study not only aimed to generate Lassa virus sequences from across Nigeria but also to isolate and conserve the respective viruses for future research. Both isolates and sequences are important for the development and evaluation of medical countermeasures to treat and prevent Lassa fever, such as diagnostics, therapeutics, and vaccines.info:eu-repo/semantics/publishe

Phylogeography of Lassa Virus in Nigeria

Lassa virus is genetically diverse with several lineages circulating in West Africa. This study aimed at describing the sequence variability of Lassa virus across Nigeria and inferring its spatiotemporal evolution. We sequenced and isolated 77 Lassa virus strains from 16 Nigerian states. The final data set, including previous works, comprised metadata and sequences of 219 unique strains sampled between 1969 and 2018 in 22 states. Most of this data originated from Lassa fever patients diagnosed at Irrua Specialist Teaching Hospital, Edo State, Nigeria. The majority of sequences clustered with the main Nigerian lineages II and III, while a few sequences formed a new cluster related to Lassa virus strains from Hylomyscus pamfi Within lineages II and III, seven and five sublineages, respectively, were distinguishable. Phylogeographic analysis suggests an origin of lineage II in the southeastern part of the country around Ebonyi State and a main vector of dispersal toward the west across the Niger River, through Anambra, Kogi, Delta, and Edo into Ondo State. The frontline of virus dispersal appears to be in Ondo. Minor vectors are directed northeast toward Taraba and Adamawa and south toward Imo and Rivers. Lineage III might have spread from northern Plateau State into Kaduna, Nasarawa, Federal Capital Territory, and Bauchi. One sublineage moved south and crossed the Benue River into Benue State. This study provides a geographic mapping of lineages and phylogenetic clusters in Nigeria at a higher resolution. In addition, we estimated the direction and time frame of virus dispersal in the country.IMPORTANCE Lassa virus is the causative agent of Lassa fever, a viral hemorrhagic fever with a case fatality rate of approximately 30% in Africa. Previous studies disclosed a geographical pattern in the distribution of Lassa virus strains and a westward movement of the virus across West Africa during evolution. Our study provides a deeper understanding of the geography of genetic lineages and sublineages of the virus in Nigeria. In addition, we modeled how the virus spread in the country. This knowledge allows us to predict into which geographical areas the virus might spread in the future and prioritize areas for Lassa fever surveillance. Our study not only aimed to generate Lassa virus sequences from across Nigeria but also to isolate and conserve the respective viruses for future research. Both isolates and sequences are important for the development and evaluation of medical countermeasures to treat and prevent Lassa fever, such as diagnostics, therapeutics, and vaccines.status: publishe

Virological and clinical data for Lassa fever patients.

<p>Due to a variable number of missing values, the number (n) of data points that were included in the analysis is indicated with each category.</p><p>Abbreviations:</p>a<p>Patients who were discharged after recovery.</p>b<p>Patients who died during hospitalization.</p>c<p>1+, Lassa virus RT-PCR was only positive with undiluted plasma; 2+, Lassa virus RT-PCR was positive with 1/10-volume plasma, irrespective of whether the undiluted sample was positive or not.</p>#<p>p<0.01 (PCR-positive survived vs. PCR-positive died).</p

Geographic origin of Lassa fever cases.

<p>Top: Map of the southern part of Nigeria showing location of Edo State. Most of the samples came from Edo State and the neighboring Ondo State. FCT, Federal Capital Territory. Bottom: Map of Edo State with borders of the Local Governmental Areas (LGA). LGAs with confirmed Lassa fever cases are shaded from light to dark grey depending on the number of cases. The names of the LGAs with case number in parentheses are given below the map.</p

Molecular testing for Lassa virus at ISTH.

<p>(A) Outline of the diagnostic laboratory with pre- and post-PCR areas (“Clean” and “Dirty”, respectively). (B) Inactivation of plasma samples in a chaotropic buffer in a plexiglas box in the inactivation room. All sample manipulations were done behind a plexiglas shield. The box features a UV light source on top for decontamination. (C) Example of an RT-PCR result. From each patient sample, 140 µl and 14 µl were processed (lanes UD [undiluted] and 1∶10, respectively).</p

Phylogenetic analysis of Lassa virus sequences from Edo State and Ondo State.

<p>Clusters A, B, and C collapsed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001839#pntd-0001839-g005" target="_blank">Figure 5</a> are shown in detail here. The upper part of cluster C has been moved to the right (dashed line) to facilitate representation of all sequences. Published sequences are identified by GenBank accession numbers. The posterior probability of monophyly of the corresponding clade is indicated on the branches if the probability is ≥0.5. If known, State, Local Governmental Area (C, Central; W, West; E, East; NE, North-East; SE, South-East), and city is shown with the strains. Sequences from fatal cases are marked with (F). Sequences highlighted in boldface have been submitted to GenBank (accession nos. JN651366-JN651400). Note, the tree contains some negative branch length at nodes with low posterior probability. This is a correct computational result which arises from calculation of the branch lengths in the consensus tree.</p

Box-plot representation of statistically significant differences between survivors and fatal cases of Lassa fever.

<p>Urea and creatinine blood levels were drawn at admission. Comparison of unpaired groups was performed with the Mann-Whitney test. The number of cases per group is given below the plots in parentheses.</p

Seasonality of Lassa fever.

<p>(A) Number of cases tested, number of positive cases, and percentage of positive cases per month. (B) Average of incidence figures. The curves in A were smoothened by a sliding window covering a 3-month interval and subsequent averaging of the two years. Error bars indicate the range. The rainfall in Benin City is shown as a bar chart in the background in relative units (July = 360 mm). Climate data were taken from <a href="http://www.climatedata.eu/climate.php?loc=nizz0004&lang=en" target="_blank">http://www.climatedata.eu/climate.php?loc=nizz0004&lang=en</a> (accessed 1 June 2012).</p