Lassa hemorrhagic fever in a late term pregnancy from northern sierra leone with a positive maternal outcome: case report

This study aimed to characterize a hospitalized acute LF case from onset of diagnosis to near full recovery using advanced rapid diagnostics and state-of-the-art technologies to dissect immune and metabolic responses in real time at the KGH LFL in Sierra Leone.

Suspected LF patients, close contacts, and healthy volunteers were eligible to participate in these studies as outlined in Tulane University's Institutional Review Board (IRB) protocol for this project, National Institutes of Health/National Institutes of Allergy and Infectious Diseases guidelines governing the use of human subject for research, and Department of Health and Human Services/National Institutes of Health/National Institute of Allergy and Infectious Diseases Challenge and Partnership Grant Numbers AI067188 and AI082119. This project was approved by the Tulane University IRB. The patients in this manuscript have given written informed consent to the publication of their case details. Patient G-1442 consented to have photographs taken at the time of admission and was informed that they may be used for illustrative purposes in scientific publications.

Small blood volumes, typically five milliliters (mL) for serum separation and two mL uncoagulated sample were collected daily from patient G-1442, with consent from the attending physician (Donald S. Grant, M.D.), except on day nineteen. A serum sample obtained from a 20-year old pregnant woman who succumbed to LF at the KGH Maternity Ward on August 29, 2010 was used as positive control. A single sample was collected from this subject before her expiration and assigned the coded designation G-1177. Four additional sera from patients who succumbed to LF at the KGH LFW between September and December 2010 were also partially characterized (G-1209, G-1220, G-1380, and G-1401). One close contact of G-1442 was tested for Ag, IgM, and IgG and assigned the coded designation G-1446. Finally sera from healthy Sierra Leonean volunteers were used as normal controls, and assigned the coded designations LS0xx. Blood was collected in serum vacutainer tubes from patients and control donors and allowed to coagulate for 20 minutes at room temperature. Serum was separated from coagulated blood by centrifugation. The serum fraction was collected for analysis and aliquots were stored in cryovials at -20°C.

Serum levels of LASV nucleoprotein (NP)-specific Ag were initially measured using LASV Antigen Rapid Test cassettes and dipstick LFI currently under pre-clinical development by Corgenix Medical Corp., Broomfield, CO, U.S.A. and the Viral Hemorrhagic Fever Consortium (see acknowledgements). Both Rapid Test strip designs utilize two NP specific murine monoclonal antibodies (Autoimmune Technologies, L.L.C., New Orleans, LA, U.S.A.) in a capture and gold-conjugated detection format. An anti-murine IgG polyclonal antibody is included as a control line. The LASV Antigen Rapid Test cassettes can detect LASV NP in serum and plasma. Twenty five μL of sample were added to the sample well then chased with 100 μL of buffer. Strong titers could be detected as early as 5 minutes but final visual interpretation was conducted between 15-25 minutes of development time. The LASV Antigen Rapid Test dipsticks are similar in construction to the LFI but include a plasma separation sample pad. Whole blood from a finger stick or blood collection tubes (EDTA, citrate) was diluted 1:3 with sample buffer in a test tube followed by addition of LASV Antigen Rapid Test dipsticks. Alternatively, one drop of whole blood was added directly to the sample pad, and once the whole blood absorbed into the plasma separator material, the dipstick was placed in a test tube containing chase buffer to initiate strip development. In this format strong titers could also be detected as early as 5 minutes but final visual interpretation was conducted between 15-25 minutes of development time. Results were recorded photographically and reflectance scans were taken with a QIAGEN ESE-Quant GOLD LFI reader (QIAGEN GmbH, Hilden, Germany). Test line reflectance and Test to Control ratios (T/C Ratio) were calculated for each sample, and compared to a curve generated with recombinant quantified NP spiked into normal human serum.

The positive LF diagnosis was then confirmed with a sensitive antigen-capture ELISA employing either a murine monoclonal or caprine polyclonal capture antibody (Autoimmune Technologies, L.L.C., New Orleans, LA, U.S.A.) followed by a peroxidase-labeled caprine reagent and tetramethylbenzidine (TMB) substrate. Capture antibodies were coated in stripwell plates, blocked, dried, and packaged with desiccating packs (Corgenix Medical Corp.). A standard curve was generated with recombinant LASV NP for quantitation of serum levels of virus-associated nucleoprotein by ELISA. Sera from previously confirmed LF cases were used as positive controls. Sera from healthy Sierra Leonean and normal U.S. sera panels were used as negative controls. For analysis, sera were diluted 1:10 and incubated in wells for 60 minutes at 37°C, washed, followed by incubation with optimized HRP-labeled anti-LASV NP conjugates for an additional 30 minutes. After washing, detection was performed with TMB substrate for 15 minutes at room temperature, stopped with sulfuric acid, and read at A₄₅₀ in a BioTek ELISA plate reader (BioTek, Winooski, VT, U.S.A.). The generation of recombinant full length LASV NP has been described elsewhere [35].

Individual recombinant LASV proteins (Vybion, Inc., Ithaca, NY, U.S.A.) and combinations optimized for detection of virus-specific IgM and IgG levels in serum were coated in stripwell plates, as outlined above. The generation of recombinant mammalian cell-expressed full length LASV GP1 and GP2 have been described elsewhere [36]. Bacterially-expressed LASV Z matrix protein was kindly provided by Dr. Erica O. Saphire, The Scripps Institute, La Jolla, CA, U.S.A. Sera from suspect and convalescent LF cases previously characterized for LASV antigen-specific IgM and IgG responses were used as positive controls in respective ELISA formats. Sera from healthy Sierra Leonean volunteers without significant titers against LASV antigens, and normal U.S. sera panels were used as negative controls. For analysis, sera were diluted 1:100 and incubated in wells for 30 minutes at room temperature, washed, followed by incubation with optimized HRP-labeled anti-human IgG or IgM conjugates for an additional 30 minutes. After washing, detection was performed with TMB substrate for 10 minutes at room temperature, and read as described above.

The kinetics of fourteen serum analyses were analyzed daily using a Piccolo^® blood chemistry analyzer (Abaxis, Inc., Union City, CA, U.S.A.) with Comprehensive Metabolic Reagent Discs, as per manufacturer's recommendations.

Kinetics of eleven serum cytokines were analyzed with an Accrue C6^® benchtop cytometer (Accrue Cytometers Inc., Ann Harbor, MI, U.S.A.) and an eBioscience FlowCytomix Human Th1/Th2 11-plex Kit (Bender MedSystems GmbH, Vienna, Austria). Serum aliquots collected and frozen throughout the timeline were analyzed concurrently at the end of the study.

Ten separate urinalysis tests were performed daily within 20 minutes of urine collection, except for the last two days of this study timeline, using a VWR^® Urine Reagent Strips (VWR, Arlington Heights, IL, U.S.A.).

RNA was extracted from serum using QIAmp Viral RNA Mini kit (QIAGEN, Valencia, CA, U.S.A.). RT-PCR was performed using SuperScript III (Invitrogen, Carlsbad, CA, U.S.A.) and qPCR was performed with PerfeCTa SYBR Green (Quanta Biosciences, Gaithersburg, MD, U.S.A.) using primers 36E2 and 80F2 directed against the LASV GPC gene [37]. A seed stock of Josiah LASV strain (kindly provided by Dr. Lisa E. Hensley, Viral Therapeutics Branch, Virology Division, USAMRIID Diagnostic Systems Division, Fort Detrick, MD, U.S.A.) was used as a standard for calculating RNA copies of LASV present in the serum samples.

The entire LASV S segment was amplified using primer CGCACAGTGGATCCTAGGCAT. Standard Sanger sequencing was then performed using primer G2 targeting the glycoprotein complex (GPC) gene [38]. Alignments from patient G-1442 and 73 partial GPC sequences were created using Muscle [39] followed by manual adjustments. A Neighbor-joining tree was created using LASV Pinneo as an outgroup, and bootstrapped over 1000 replicates.

ELISA data were plotted in MS Excel as mean ± SD, N = 2, with error bars. Analysis between time points was performed with Analysis of Variance (ANOVA). Cytokine levels were calculated by curve fitting analysis of data generated with quantified standards for each analyte.

On January 20^th, 2011 the KGH Maternity Ward alerted the LF team of a suspected case from Tongo, lower Bambara chiefdom in Kenema district, Sierra Leone. A blood sample was collected and sent to the KGH LFL for testing. LASV NP antigen LFI diagnostic confirmed LF within 20 minutes of sample processing (Figure 1). The patient was a 22-year old pregnant woman, estimated gestational age 32 weeks, who had recently travelled to Tongo from Mabineh 1 village, Kunike chiefdom, Tonkolili district, northern Sierra Leone (Additional File 1, Figure 1). She arrived in Tongo on January 10^th experiencing fever and lower abdominal pain. She was taken to Tongo Maternal Health Post on January 15^th for observation where she was referred to KGH on January 19^th as a maternity case after failing to respond to treatment with antibiotics (Ampicillin and Gentamycin). The KGH Maternity Ward staff suspected LF upon arrival and referred the case to the LFW and LFL. This patient was assigned the coded designation G-1442, which will be used henceforth.

Case G-1442 presented with symptoms of fever, sore throat, headache, red eyes, weakness, facial edema, retrosternal pain, generalized abdominal pain, epistaxis and haemoptysis (Additional File 2, Figure 2). On examination, her body temperature was 36.5°C, pulse rate of 96 beats/minute, respiration rate of 26/min, and blood pressure of 90/40 mm Hg (Additional File 3, Figure 3). Respiratory findings included nasal flaring and bibasal crepitations. Abdominal findings included a hard uterus that was tender to palpation with an estimated symphysis fundal height of 30-32 weeks. There was marked epigastric tenderness. Minute bilateral conjunctival hemorrhages were also noted. The differential diagnosis included probable LF, pneumonia, and a possible concealed antepartum hemorrhage (concealed placental abruption).

The case patient had travelled from Mabineh 1 to Waterloo (south of Freetown) three weeks prior to her illness. Upon returning from Waterloo she resided in Massingbi (a neighboring town to Mabineh 1) for one week before returning to Mabineh 1. She remained in Mabineh 1 for five days before departing for Tongo. According to relatives it is estimated that the case patient left Mabineh 1 between January 6^th and 8^th bound for Tongo (Additional File 1, Figure 1C). She had no known exposure to an ex-LF patient or contact with rodents prior to her illness. However, an assessment of her previous dwelling in Mabineh 1 revealed evidence of rodent waste, and rat holes in a structure constructed with mud and with large open spaces in walls. The patient had not been seen by a medical professional throughout her pregnancy as nurses at the Mabineh Health Post could not account for her visiting the center at anytime over the previous eight months. The date of onset of LF was recorded as January 13^th, with fever, headache, and lower abdominal pain, after failure to respond to treatment with anti-malarials. The patient tested positive for malaria parasites while in Tongo (verbal communication). The conclusion from the investigation conducted by the LF outreach team points to infection with LASV in the northern towns of Massingbi and Mabineh 1 where the case patient resided during most of the early stage of the incubation period of the disease.

Contacts of the case patient were identified and none have developed symptoms of LF to date. All contacts were monitored throughout the incubation period (21 days) from date of last reported exposure. Contacts were family members from Mabineh 1, nursing staff at the health clinic in Tongo, and the patient's brother who resides in Tongo township. The brother of G-1442, designated G-1446, with whom she resided while in Tongo, accompanied her to the KGH and was tested for LASV antigen, IgM, and IgG. He tested negative for all three (Figure 2). Testing of G-1446 was prompted by his close contact with G-1442 in Tongo for 9 days, and given the hemorrhagic presentation, with vomiting by the latter at the time of admission to KGH. Additionally, G-1442's mother traveled from Mabineh 1 to Kenema to assist with her daughter's care during hospitalization at the KGH LFW. The mother did not develop a fever and did not feel ill at any time over the course of nearly two weeks of permanence in Kenema; therefore, she was not tested for LASV antigen or immunoglobulin levels. The patient revealed that she travelled from Masingbi to Tongo by motorcycle over the course of 2 days. The motorcycle operator was an unidentified male, and further information on his whereabouts and health status is not known.

The geographical location where G-1442 contracted LF is of particular importance. In recent months several cases have been identified by our field research team in the northern districts of Bombali and Tonkolili (Additional File 1, Figure 1A), which have not been previously considered endemic regions for the illness. Since the Fall of 2010, however, two cases of severe hemorrhagic LF have been identified in these two northern districts, both with fatal outcomes. In addition, several other LF cases from the same districts have been confirmed with subsequent treatment at the KGH LFW and positive outcomes. During the preparation of this manuscript, additional LF cases had been diagnosed at the Magbeneth Hospital in Makeni using LASV Ag Rapid LFI diagnostics provided by Tulane University and Corgenix Medical Corporation.

A blood specimen collected on patient G-1442's day of admission was positive for LASV NP Ag by LFI diagnostic (Figure 1), and by quantitative NP capture ELISA, with a level of 2.265 μg/mL NP (Figure 2). The LFI platform confirmed acute LF within 20 minutes of sample collection. IgM levels to recombinant LASV proteins (NP, GP1, GP2, Z) were determined by ELISA, with low but detectable levels of immunoglobulin to NP and Z (Figure 2B). This data suggests the patient was naive to LASV exposure prior to this incident. Statistically significant levels of low IgM response to GP1 and GP2 were detected on days 11-20 when compared to naïve negative controls and G-1142 sera from days 7 - 10 (p < 0.05) (Figure 2B). Low levels of NP-specific IgG were not detected until at least day 12 post onset of illness (Figure 2C). During the monitoring period G-1442 did not develop significant IgG titers against GP1, GP2, and Z.

LASV NP antigen dropped rapidly over 3 days following treatment with ribavirin, and was below the limit of detection (LOD) of the assay by day 10 in an NP Ag capture ELISA (Figure 2A). RNA was isolated from serum on the day of collection and analyzed by qPCR for amplification of a conserved 300 nt segment of the LASV GPC gene. PCR confirmed and detected viral RNA in serum samples at least 2 days beyond the NP Ag ELISA (Figure 3). Overall, the NP Ag capture ELISA, LFI diagnostic, and qPCR assay showed the same trend with decreasing titers of LASV following the start of ribavirin treatment.

On presentation to KGH LFW, the liver function panel revealed highly elevated levels of aspartate aminotransferase (AST) >2000 U/L, alanine transaminase (ALT) of 643 U/L, alkaline phosphatase (ALP) of 541 U/L, and total bilirubin (TBIL) of 35 micromoles per liter (μM/L) (2.05 mg/dL) (Figure 4). Levels of sodium, potassium, chloride, calcium, carbon dioxide (TCO₂), blood urea nitrogen (BUN), and total protein were within or near normal levels, and albumin was below normal range (Additional File 4, Figure 4). At presentation the hemoglobin level (Hb) was 12.7 g/dL (Figure 4).

Intravenous ribavirin was administered upon NP Ag positive diagnosis by LFI: a loading dose of 30 mg/kg followed by 15 mg/kg every six hours for four days, followed by 7.5 mg/kg every eight hours for six days. Amoxicillin, intravenous quinine in 5% dextrose, acetaminophen, and routine vitamins (multivitamin, ferrous sulphate, folic acid) were commenced upon admission to KGH. Patient G-1442 developed bleeding from the oral mucosa on day eight which resolved on day ten. On day ten she was much improved and was able to stand unaided. Fetal demise was confirmed with the aid of a fetal heart Doppler on day twelve. She had an uncomplicated vaginal delivery of a stillborn fetus on day thirteen, at which time she was started on ampicillin and metronidazole. Artesunate was begun on day fifteen. IV fluid boluses of five percent and 50% dextrose and Ringer's Lactate solution were given as needed.

The sodium and chloride levels gradually decreased during the hospitalization period, following IV fluids management, and G-1442 remained hyponatremic between days 13 and 20 and hypochloremic between days 16 and 20. Creatinine and BUN remained normal during her entire hospital stay resulting in a BUN:Cr ratio between 10 and 20 except for a transient increase to 24 on day 17 (Figure 4). The ALT declined steadily during hospitalization, and was within normal levels on day 16 (42 U/L) and through the rest of the monitoring period, while the AST remained >2000 U/L for three days before declining to 100 U/L on day 20. The ALP remained elevated as well, decreasing to 259 U/L on day 20. The TBIL initially increased to 88 μM/L (5.15 mg/dL), before dramatically declining two days after delivery (day 14) to 32 micromoles per liter (1.87 mg/dL). Bilirubin continued to decrease over the remainder of the observation period, to within normal levels between days 16 and 20. The Hb decreased from an initial normal level of 12.7 g/dL to between 10.4 and 11.1 g/dL throughout the hospitalization period (Figure 4B). These Hb levels did not prompt the doctor to administer a blood transfusion.

Cytokine profiles were performed on serum samples collected daily (Figure 5). G-1442's IFN-γ levels were highly elevated on the day of admission, but decreased to baseline levels by the following day and did not rise above normal levels over the ensuing 12 days of monitoring (Figure 5B). A significant decrease in IL-6 and IL-10 levels was noted on day 8, but levels fluctuated throughout the course of hospitalization. IL-8 levels dropped significantly on days 9 and 10, followed by a spike on day 11, and a steady decrease thereafter. TNF-β was present at elevated levels at the time of admission and decreased to near background levels by the following day, but increased significantly and steadily throughout the hospitalization period. Interleukins -1β, -2, -4, -5, 12p70, and TNF-α were not detected or were present in the serum of G-1442 at very low levels at all time points analyzed. On day 20, the last day of monitoring, all cytokines with the exception of TNF-β were at or near normal levels. Interleukin-8 and TNF-α were elevated in one healthy control serum (LS004) but were within normal levels in the other healthy control serum (LS022). All other cytokines were at baseline levels in both control sera (Figure 5A, B).

Urinalysis revealed ongoing proteinuria (30 mg/dL) that peaked on the day of delivery (300 mg/dL) and subsequently decreased (trace to 30 mg/dL). It is unclear whether proteinuria resulted from LASV infection, pregnancy, or a combination of factors. There was no hypertension to suggest a diagnosis of pre-eclampsia. The presence of large blood was noted on all urinalysis results. Microscopy was not performed to examine for red blood cells or casts. Bilirubin (small to large) was present prior to and including the day of delivery, after which time it was absent, consistent with resolving biliary obstruction as indicated by the decrease in serum TBIL levels and ALP. Leukocyte esterase was absent to trace presence prior to delivery, after which time small to moderate results were noted. Since analysis was performed on catch specimens, the possibility of contamination from vaginal fluid after delivery cannot be excluded. Nitrites were positive on day 18. No symptoms of urinary tract infection were noted (Additional File 5, Table 1).

In order to get a better idea of the geographical location of the isolated strain, a 800nt fragment of the GPC gene from the serum of patient G-1442 was sequenced and compared to other strains circulating in Sierra Leone and West Africa. Phylogenetic analysis showed that the LASV from patient G-1442 clustered with other strains from Sierra Leone, but was significantly different from all of them, forming its own sub-group (Figure 6). The strain was only 89% identical to the prototypical Sierra Leonean LASV Josiah strain at the nucleotide level. In comparison, we have found other currently circulating strains from Sierra Leone to be more than 95% identical to Josiah (Figure 6 and data not shown). This suggest that a new strain of LASV may be responsible for the recent outbreak of LF in the North of Sierra Leone, although more complete sequencing studies are required to firmly establish this.

In order to establish the capabilities and reliability of the Piccolo^®, complete chemistries were performed on blood drawn from two healthy Sierra Leonean volunteers as well as on samples from five patients who succumbed to LF (Additional File 6, Table 2). Patient G-1177's metabolic and biochemical characterization was described in detail elsewhere [40]. The chemistries of the two healthy volunteers were in the normal range as specified by the manufacturer (Abaxis, Inc.) (Additional File 6 Table 2).

LF patients G-1209, G-1220, G-1380, G-1401, and G-1177 had extremely abnormal labs prior to expiration (Additional File 6, Table 2). Along with dysregulated serum electrolytes, all subjects had a highly elevated liver panel, and, with the exception G-1380, all had elevated levels of BUN. Additionally, all subjects had low serum albumin and total protein levels. The cytokine profiles between healthy volunteers and subjects who succumbed to LF were largely unremarkable, with the single exception of IL-10, which was recorded at elevated levels in all cases (Additional File 6, Table 2 and unpublished data).