LASV Ag Rapid Test detected acute LASV infection in G-1442 within 20 minutes of serum collection and processing at the KGH LFL (Figure ). The patient was immediately transferred from the KGH Maternity Ward to the LFW upon diagnosis, permitting isolation and appropriate medical intervention including IV ribavirin administration, currently the only drug used in viremic cases of LF. LFI diagnostic detected LASV NP Ag on the first two days at the KGH LFW (Figure ), whereas antigen capture ELISA diagnostic detected the protein in the serum of G-1442 for three days following admission (Figure ). Quantitative PCR extended detection of LASV RNA sequences for two days beyond the limit of detection of LASV NP Ag ELISA, thus establishing a role for each platform from sensitive and rapid point of care LFI diagnostic to ultrasensitive and time extended qPCR detection of very low levels of arenaviral RNA in the blood.
ELISA data suggest that patient G-1442 was naïve to infection as she presented with very low LASV-specific IgM to all viral proteins analyzed at 7 days after onset of symptoms; she then began showing a consistent increase in NP-specific IgM, and a low level IgM response against the glycoproteins starting on day 11, which continued through all days monitored. Only IgG to NP developed over the analysis timeline (Figure ). The predominant, mature, humoral response in LF is against the viral NP Ag [41-43, unpublished data].
The metabolic panel of G-1442 as well as a previously characterized severe hemorrhagic LF case, G-1180, who also survived [40
], show important differences with patients who succumb to the disease. Despite hepatic and renal dysfunction during the course of LF infection, neither patient developed elevated levels of serum CRE, which are usually associated with a poor outcome [18
]. In G-1442 the BUN:Cr ratio remained within normal levels throughout (10-20:1), with the notable exception of day 17, when it rose above 20 (24.2). These data suggest that in G-1442 renal function was not significantly affected by LF. Conversely, G-1177, a late term pregnant woman diagnosed with LF in August 2010, succumbed to the disease with a CRE level of 818 μmol/L and a BUN:Cr of 5.6 prior to expiring, which is indicative of significant intrarenal damage Additional File 6
, Table 2]. Another significant discrepancy between the two pregnant LF cases was the measured levels of AST. In G-1177 the single sample AST level was zero, whereas G-1442 had a highly elevated level of AST at the time of admission (>2,000 U/L), which rapidly resolved over the course of treatment (Figure ). Levels of AST are commonly highly elevated in LF cases, thus the undetectable level in G-1177 may have been indicative of severe liver failure near the time of expiry and not a representative hepatic metabolic state in late term pregnancies afflicted by LASV infection. Both surviving patients, G-1442 and G-1180, showed rapid resolution of severe hepatic dysregulation, measured by ALP, ALT, and AST, to within normal or near normal levels at the conclusion of ribavirin treatment.
At the time of admission G-1442 presented with elevated serum levels of IFN-γ, IL-6, IL-8, and TNF-β (Figure ). Elevated IFN-γ and IL-6 levels are common in non-lethal LF and other febrile illnesses alike, but are highly variable in fatal cases of LF [18
]. Elevated IL-8 levels have been associated with positive outcomes in acute LF, but are also common in native Sierra Leonean healthy controls [44, unpublished data]. Spontaneous cytokine production in acutely ill and healthy persons living in endemic areas for Human Immunodeficiency Virus, Malaria, Yellow Fever, Dengue, and assorted parasitic infections, has been reported [45
], thus prompting evaluation of such immunomodulatory molecules in the context of specific disease states. Measurable and sustainable levels of TNF-β in G-1442 are a distinguishing feature among the LF cases characterized to date. Detection of TNF-β in G-1442 but not in any of the approximately 100 additional LF patients analyzed in our studies thus far (unpublished data) may represent a rare immunological response to the febrile illness, may be associated with the pregnant status of this patient, may have manifested because of a response to a co-infecting pathogen, or may be a combination of factors. The anti-inflammatory cytokine IL-10 was elevated in G-1442's serum throughout the treatment period. Interleukin-10, a stimulator of B cell maturation and antibody production, is commonly recorded in LF patients when IgM and IgG responses to LASV antigens emerge [18
], irrespective of outcome. Interleukin-1β was not detected in G-1442 throughout the course of recovery from LF. This observation generally contrasts with previous LF studies showing that IL-1β was significantly elevated in non-fatal versus fatal LF and non-LF febrile illness, but not in healthy controls [44
Patient G-1442's test results, in conjunction with those obtained for G-1180 [40
], strengthen the hypothesis, as previously proposed by others, that an imbalance between pro- and pre-inflammatory cytokines plays an important role in the development of Lassa hemorrhagic shock, with poor outcome [18
]. As observed with G-1180, the marked absence of TNF-α, a potent inducer of endothelial damage via apoptosis [46
] and thrombocytopenia [47
], throughout the monitored course of G-1442's illness, suggests a regulated and effective immune response at play. These studies also suggest that lack of specific physiological responses, e.g. elevated TNF-α, serum CRE, and BUN levels, may be relevant, early predictors of outcome in hemorrhagic LF. It is also noteworthy that G-1442 did not present with high core temperature, which remained at or below 36.5°C throughout the acute phase of the illness despite a febrile diagnosis (Additional File 3
, Figure ) and high IFN-γ levels (Figure ). Her body temperature then fluctuated between 36°C and 37.5°C from day 15 onward.
Together, these data strengthen the potential for increased positive outcomes in cases of severe hemorrhagic LF. More importantly, it outlines the possibility of adequate disease management with positive outcome in third trimester pregnancies, particularly for the mother [48
]. Despite severe and prolonged multi-organ dysregulation, pro- and anti-inflammatory cytokine up- and down-regulation, management of a 32 week-pregnancy, a stillbirth delivery, and overall poor health, patient G-1442 was recovering well on day 20 and was discharged on day 25. A quick diagnosis of acute LF followed by prompt treatment with IV ribavirin, IV fluids management, maintenance of electrolyte balance to counter hypovolemia, hemorrhagic shock, malnutrition, and adequate control of secondary infections, even 7 days post onset of symptoms in a severe case of the illness, can meet with a positive outcome.
Additionally, this study highlights the emergence of LF cases in the northern districts of Sierra Leone, where the disease has not been widely reported or identified. Recent collaborative efforts with staff at the Magbeneth Hospital in Makeni includes beta-testing of LASV Ag Rapid Test LFI diagnostic modules, community sensitization, and prompt reporting of antigen positive LF diagnoses to the KGH LFW for patient transport, isolation, and treatment and may be a contributing factor to the elevated number of reported cases in northern Sierra Leonean districts. With promising new diagnostics, we are able to both enhance care of patients in the clinical setting and increase our understanding of the range and impact of this devastating disease. The continuous capacity building at the KGH LFL also permits real time analysis of viral RNA levels by qPCR, cDNA generation, followed by high-throughput next-generation sequencing. Although more extensive studies will be required before confirming the emergence of new LASV strains, particularly in the historically non-endemic northern districts of Sierra Leone, sequencing efforts in this case point to divergence of circulating strains throughout the country, with possible widening in geographical distribution.