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

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.

<p>The sequences of the PCR fragments obtained from positive cases were aligned with published sequences. The latter are identified by GenBank accession numbers. For clarity of presentation, only Nigerian strains are shown. The clusters A, B, and C comprising strains from Edo State and Ondo State were collapsed; these strains are shown separately in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001839#pntd-0001839-g006" target="_blank">Figure 6</a>. Posterior probability values are indicated on the branches. The country of origin of Lassa virus strains is indicated by a prefix: IC, Ivory Coast; NIG, Nigeria. If known, State and city is also shown with the strains (FCT, Federal Capital Territory). Sequences highlighted in boldface have been submitted to GenBank (accession nos. JN651366-JN651400). Lassa virus lineages are indicated left. The novel putative lineage/sub-lineage represented by strain Nig05-A08 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001839#pntd.0001839-Ehichioya2" target="_blank">[49]</a> is indicated with a question mark.</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