article: Convulsions with fever in children are a common neurologic emergency in the tropics, and determining the contribution of endemic viral infections can be challenging. In particular, there is a dearth of data on the prevalence and clinical differentiation of Lassa virus disease (LVD) in febrile children in endemic areas of Nigeria, which has multiple lineages of the virus. The aim of this study was to determine the prevalence and presentation of LVD in febrile children with and without convulsions. This was a prospective study of consecutive febrile children aged ≥1 month– 15 years admitted to the Children’s Emergency Room of Irrua Specialist Teaching Hospital over a period of 1 year. Febrile children with convulsions (Cases) were compared with those without convulsions (Controls). LVD was defined by the presence of a positive Lassa virus RT-PCR test. Rates were compared between groups using χ2 or Fisher’s exact tests and p <0. 05 taken as significant. 373 (40. 9%) of 913 admissions had fever. Of these, 108/373 (29%) presented with convulsions. The overall prevalence of LVD was 13/373 (3. 5%; 95% CI = 1. 9%, 5. 7%) in febrile admissions, 3/108 (2. 8%) in Cases and 10/265 (3. 8%) in Controls [ (Odds Ratio (95% Confidence Interval) (OR (95% CI) ) of LVD in Cases versus Controls = 0. 73 (0. 2,2. 7) ]. Only vomiting (OR (95% CI) = 0. 09 (0. 01,0. 70) ) and bleeding (OR (95% CI) = 39. 56 (8. 52,183. 7) ) were significantly associated with an increased prevalence of LVD. LVD is an important cause of fever, including undifferentiated fever in children in endemic areas, but it is not significantly associated with convulsions associated with fever. Its prevalence, and lack of clinical differentiation on presentation, underscores the importance of a high index of suspicion in diagnosis. Screening of febrile children with undifferentiated fever in endemic areas for LVD could be an important medical and public health control measure.
  The causes of convulsions associated with fever (CAWF), a common paediatric emergency worldwide [1,2], are dependent on age, climate and clinical presentation [3]. Viral infections in temperate countries [4,5], and malaria in tropical countries [3,6–8] are the main aetiologic agents in febrile convulsions but bacterial infections are nonetheless also important causes in both temperate and tropical countries [9–13]. However, while the prevalence, presentation and outcome of malaria and bacterial infections as causes of CAWF in the tropics have been fairly well defined [3,6–8,11], there are only a few reports on the contribution of viral infections [14,15]. Specifically, there is little information on the contribution of Lassa virus disease (LVD). More broadly, reports of the prevalence of LVD in children are few and knowledge of the clinical manifestations limited. In addition, a majority of the studies on the prevalence and clinical features of LVD in children and adults are from the West African countries of Sierra Leone, Liberia and Guinea [16–20], as are the few reports on paediatric LVD [21–23], and little is known about the situation in Nigeria. There are 4 major lineages reported thus far from sequencing of the Lassa virus (LASV): 3 are in circulation in Nigeria and 1 in other West African countries [24,25]. Whether the lineages are associated with different clinical manifestations is largely unknown. Nigeria is one of the LVD-endemic countries in West Africa and reports indicate that it may account for up to 6% of febrile admissions in endemic areas in the country [26]. Its initial clinical manifestations are difficult to differentiate from those of other common febrile illnesses, such as malaria [16,18,27], and a high index of clinical suspicion is required in the diagnosis [27]. Convulsions have been described in LVD as a possible marker of disease severity and a frequent herald of death alongside coma [28,29]. These convulsions could be caused by several mechanisms, including infection of neural tissue, fever and metabolic changes among others as in other viral haemorrhagic fevers [30,31]. Few reports, if any, are available from endemic areas on the contribution of LVD to the common neurologic emergency of CAWF in children, and its true prevalence in childhood CAWF has not been determined. Addressing this should add to our understanding of the manifestations of this emerging/remerging infectious disease with respect to CAWF in the tropics. Perhaps, undiagnosed LVD could contribute to the relatively poor outcome of febrile convulsions in endemic areas in the tropics. Several factors help account for why so little is known about the role of LVD in childhood CAWF: the low awareness of LVD as a possible aetiology in childhood fever and CAWF; the difficulties in differentiating it from other common causes of fever; and the lack of accessible diagnostic facilities. However, better understanding of the role of LVD could provide a useful guide to clinicians in the management of this common neurologic emergency. This is aside its public health importance. This study therefore sought to address these gaps in knowledge. Specifically, we had two aims: to systematically determine the prevalence of LVD in children with CAWF compared to those with fever but no convulsions, and to compare the clinical manifestations of LVD with that of other common febrile illnesses in children. In addition, we sought to determine the current status of malaria as a contributor to CAWF in endemic areas.
  This study was approved by the Research and Ethics Committee of Irrua Specialist Teaching Hospital, via approval number ISTHREC09/14/12/2009 dated 7th January 2010. This was a descriptive, prospective case-control study of consecutive febrile children with convulsions presenting to the Children Emergency Room (CHER), Irrua Specialist Teaching Hospital (ISTH), Irrua, Edo State, NIgreia. Irrua is the headquarters of Esan-Central Local Government Area (LGA), Edo State, one of the local government areas with the highest prevalence of LVD in Nigeria [32]. The Research and Ethics Committee of ISTH approved the study, and written informed consent was obtained from the parents/guardians of the children involved in the study. The institutional review boards of the collaborating institutions from which the co-authors were drawn also approved the study. Enrolment consisted of all consecutive admissions of children aged >1 month to ≤15 years, admitted over a period of one year (December 1st, 2009 to November 30th, 2010) into the CHER with complaints of fever or fever and convulsions. The attending physician had responsibility for the admissions, which were not associated with any particular diagnosis, including LVD, and the authors had no input in the selection of patients for admission. The inclusion criteria for the study included temperature ≥38°C and written informed consent given by the parents or guardians. On admission a detailed history, clinical examination and laboratory tests were undertaken for each patient, after which the data obtained were entered into a standardized form. For the purpose of this study, a ‘Case’ was defined as a child aged ≥1month to ≤15 years, with temperature ≥38°C on admission or a history of fever + temperature ≥38°C within 8 hours of admission, and a history of convulsions during the illness. A ‘Control’ met the same criteria but had no history of convulsions. Both Cases and Controls were given the standard management appropriate for their diagnosis. Patients with LVD were given intravenous ribavirin at 33mg/kg body weight stat, and maintained on 16mg/kg body weight/dose 6 hourly for the first 4 days and thereafter 8mg/kg body weight/dose 8 hourly for another 6 days. In all, therapy with ribavirin was for 10 days [33]. Patients with a diagnosis of sepsis were commenced on empiric antibiotics after appropriate samples had been taken for culture. Thus patients with a diagnosis of meningitis were commenced on intravenous ceftriaxone and those with severe sepsis on ceftriaxone, or cefotaxime, or ceftazidime, and an aminoglycoside (gentamicin or amikacin), pending the receipt of culture results. The history included presenting complaints, drug history, history of recent immunizations, and history of the pattern, frequency and duration of convulsions, if any. Physical examination included a careful examination of the skin and mucous membranes for petechiae, purpura or ecchymosis and examination to exclude a focus of infection such as pharyngotonsilitis, otitis media, pneumonia and meningitis. Presence of signs of meningeal irritation (nuchal rigidity, Kerning’s and Brudzinski’s signs and/or bulging and tense fontanel) was considered as indicative of meningitis. The level of consciousness was assessed using the Blantyre [34] and Glasgow [35] Coma Scales, depending on the child’s age. Blood, cerebrospinal fluid (CSF) and urine samples were collected on admission and processed at the respective service laboratories of the hospital using standard methods. The laboratory tests done included LASV-reverse transcriptase-polymerase chain reaction (RT-PCR) test, haematocrit, white blood cell count (WBC), dipstick urinalysis, microscopic urinalysis [36], blood film for malaria parasites (MP), random blood sugar, blood urea nitrogen, serum creatinine and blood culture. Lumbar puncture and CSF examination were carried out on all children who presented with fever and convulsions [37–39] as well as those with signs of meningeal irritation, except where there were contraindications to lumbar puncture [40,41]. Blood culture was carried out by inoculation of blood drawn aseptically from a peripheral vein into a pair of culture bottles containing glucose broth and sodium thioglycollate, which were then incubated at 37°C. Routine Gram staining and subcultures onto MacConkey and blood agar plates were carried out at 2 and 7 days or whenever a growth became evident. CSF samples were collected in sterile bottles and cultured by inoculation onto blood, chocolate and MacConkey agar plates, and incubated at 37°C for 48 hours under both aerobic and anaerobic conditions. Both blood and CSF cultures were processed manually as our hospital does not, so far, have automated systems. Malaria was defined as the presence of asexual forms of MP in a peripheral blood smear. Bacterial meningitis was defined as the presence of elevated CSF WBC count (>5/mm3) plus CSF/blood glucose ratio <40% or CSF glucose <40 mg/dl and CSF protein >40 mg/dl plus a positive CSF Gram stain or culture [37,42,43]. Possible bacterial meningitis was defined as the presence of elevated CSF WBC count (>5/mm3) plus CSF/blood glucose ratio <40% or CSF glucose <40 mg/dl and CSF protein >40 mg/dl but negative CSF Gram stain or culture [43]. The use of CSF white blood cell count of >5/mm3 as the threshold for the diagnosis of meningitis could be attended with increased sensitivity in very young infants in whom the normal counts are higher. However, the diagnosis of possible bacterial meningitis was not based only on this parameter but on both total and differential white blood cell count and the presence of typical biochemical changes [42,44,45], which are known to have a high sensitivity and specificity for bacterial meningitis [44,45]. We adopted the term ‘possible bacterial meningitis’ in deference to the lack of confirmatory findings by way of positive Gram stain or culture or antigen tests [43]. A child was taken to have unidentified infection in the absence of a focal infection, as indicated by the presence of localising signs, malaria parasitaemia, positive blood culture, bacterial or possible bacterial meningitis or a positive LASV-RT-PCR test, or clinical explanation/s for the febrile illness such as gastroenteritis, HIV/AIDS, or upper respiratory tract infection. Severe anaemia was defined as haematocrit ≤20% [46], leucopaenia as peripheral WBC <3000/mm3, leukocytosis as WBC ≥15000/mm3 [9] and hypoglycaemia as blood glucose ≤2. 2 mmol/L [47]. All the febrile admissions had LASV-RT-PCR test, which was carried out at the Research and Diagnostic Laboratory of the Institute of Lassa Fever Research and Control (ILFRC), ISTH. The ribonucleic acid (RNA) was prepared from 140 μL of serum and conventional PCR tests carried out as described elsewhere [48]. LASV-RT-PCR test was carried out on the serum of both Cases and Controls. RNA was purified with QIA amp viral RNA KIT (Qiagen, Hilden, Germany). RT-PCR was performed with superscript II RT/Platinum taq polymerase 1–step reagents (Invitrogen, Karlsruhe, Germany). This afforded a 95% detection limit of 2,500 copies/ml, which is highly sensitive. A positive LASV-RT-PCR test was taken as confirmatory of the diagnosis of LVD [48]. Children with fever were classified into Cases and Controls. Cases were sub-classified into two groups: children with febrile convulsions (FC) [1] and children with non-febrile convulsions (NFC). The latter included children outside the defined age range of 6 months– 5 years for FC [1] and children with convulsions associated with central nervous system infection as indicated by abnormal findings on CSF examination. Both Cases and Controls were further classified based on clinical presentation into 2 groups: clinically differentiated fever (children with fever with localizing signs of infection and children without localizing signs but with plausible clinical explanation/s for the fever, such as the development of fever within 24–48 hours of vaccination with diphtheria, pertussis and tetanus (DPT) vaccine or a clinical diagnosis of pertussis) and children with fever without localizing signs (undifferentiated fever). The statistical significance of differences in the prevalence of LVD between the groups, and significance of the association of symptoms and signs with LVD, was determined using χ2 test with Yates’ correction or Fisher Exact Test as appropriate. Two-tailed p values <0. 05 were taken as significant.
  Overall, 130 (34. 9%) children had clinically differentiated fever, and 243 (65. 1%) undifferentiated fever. The proportion of children with differentiated fever was lower among the Cases (28. 7%) than among the Controls (37. 4%) but the difference did not attain statistical significance (p = 0. 141). The pattern of illnesses in children with differentiated fever is shown in Table 2. Overall, 52. 3% had focal extra-cranial infections, and 15. 4% possible bacterial meningitis while 32. 3% had neither infection but had other clinical explanations for their febrile illnesses. None had confirmed bacterial meningitis. The focal extra-cranial infections included bronchopneumonia, acute otitis media, acute suppurative otitis media, and abscesses. The diagnosis in children with neither focal extra-cranial infections nor meningitis but with clinical explanation/s for their febrile illnesses included gastroenteritis, HIV/AIDS, upper respiratory tract infection, myocarditis, organophosphorus insecticide poisoning, diabetic ketoacidosis, lymphoma, pertussis, cholestatic jaundice, chronic renal failure, Kawasaki disease, severe malnutrition, neuroblastoma, and pelvic inflammatory disease. The proportion of children with possible bacterial meningitis was significantly higher among the Cases (32. 3% versus 10. 1%, p = 0. 01) (Table 2). The difference between the proportions of Cases and Controls with focal extra-cranial infections and the other illnesses was not statistically significant (Table 2). Children with possible bacterial meningitis had a significantly higher mean total CSF white blood cell count (p = 0. 01), CSF glucose (p <0. 001), CSF/simultaneous blood glucose ratio (p <<0. 001) and CSF protein (p <0. 001) (Table 3). The overall contribution of LVD and malaria parasitaemia in children with undifferentiated fever is shown in Table 4. The overall prevalence of LVD in undifferentiated fever was 5. 4% (95% CI = 3. 0%, 8. 7%) while the prevalence of malaria parasitaemia was 35. 8% (95% CI = 29. 96%, 41. 98%). Four children (1. 6%), including 4/13 (30. 8%) with LVD or 4/87 (4. 6%) with malaria had LVD/malaria comorbidity. All the malaria parasites were Plasmodium falciparum. The youngest child with LVD was 6 months old and the oldest 7 years. There was no significant difference between the overall prevalence of LVD in Cases and Controls (3. 9% versus 6. 0%, p = 0. 729). However, the prevalence of LVD/malaria parasitaemia was significantly higher among the Cases than the Controls with LVD (3. 9% versus 0. 6%, p = 0. 028). Two of the 3 Cases with LVD had generalised tonic-clonic and 1 focal convulsions; the numbers were too small for further analysis. The prevalence of malaria parasitaemia was significantly higher among the Cases (61. 0% versus 21. 7%, p <<0. 001) while the prevalence of children with unidentified infections was significantly lower among the Cases (35. 1% versus 72. 3%, p <<0. 001). Sixty-one Cases (56. 5%) had FC and 47 (43. 5%) NFC, giving a ratio of FC: NFC of 1. 3: 1. The prevalence of undifferentiated fever and pattern of infections in children with FC versus NFC is shown in Table 5. By definition, none of the children with FC had meningitis whereas 10 (21. 3%) of those with NFC had possible bacterial meningitis. Children with FC had a significantly higher prevalence of undifferentiated fever (80. 3% versus 59. 6%, p = 0. 032) but there was no significant difference between the two groups of children in the pattern of infections among children with undifferentiated fever. Cases and Controls were merged in analysis for the purpose of determining the association between age, gender, season and clinical presentation and the prevalence of LVD because the number of Cases (n = 3) as well as the number of Controls (n = 10) with LVD was small. The association between the prevalence of LVD and age, gender, season and clinical presentation in terms of the duration of fever on presentation, clinical status with regard to the presence versus absence of localising signs of infection, initial diagnosis and differential diagnosis and result of urinalysis is shown in Table 6. Overall, 9. 4% febrile admissions presented with a history of fever ≥7 days, 76. 4% were reported as acutely ill looking on presentation, and 77. 5% had no localizing signs of infection, while Lassa fever was the initial diagnosis or differential diagnosis on admission in 2. 9%. The association between the prevalence of LVD and the initial diagnosis/differential diagnosis was highly significant (p <<0. 001) whereas none of the other associations attained statistical significance (Table 6). The association between non-localizing symptoms and signs on presentation and the prevalence of LVD is shown in Table 7. Only the lack of a history of vomiting (p = 0. 01) and presence of bleeding manifestations (p <0. 001) had a statistically significant association with LVD (Table 7). Twelve (3. 2%) children with fever (3,2. 8% Cases and 9,3. 4% Controls; p >0. 5) were discharged against medical advice. None had LVD. Thirteen (3. 6%) of the 361 children whose hospital outcome was known died. Case fatality was 7/105 (6. 7%) among the Cases and 6/256 (2. 3%) among the Controls. The difference in case fatality rate did not attain statistical significance (p = 0. 098). The deaths among the Cases included 1 child with LVD and 6 with malaria parasitaemia while the deaths among the Controls included 2 children with LVD, and 1 child each with malaria parasitaemia, meningitis, diabetic ketoacidosis, and myocarditis. Overall case fatality rate was 3/13 in children with LVD and 10/348 (2. 9%) in children with other febrile illnesses. The difference was statistically significant (p = 0. 017). Case fatality was 1/3 among the Cases versus 2/10 among the Controls with LVD (p >0. 999).
  LVD is an important cause of fever, including undifferentiated fever in children in endemic areas, but it is not significantly associated with convulsions in febrile children. Its prevalence, and lack of clinical differentiation on presentation, underscores the importance of a high index of suspicion in diagnosis. Screening of febrile children with undifferentiated fever in endemic areas for LVD could be an important medical and public health control measure.
summary: There has, perhaps, been undue focus on malaria as a cause of childhood fever and convulsions, often with delayed/missed diagnosis of other serious prevalent infections, and correspondingly very little published data on the contribution of Lassa virus disease (LVD) in endemic areas. There is also very little published data on the contribution of LVD to childhood morbidity and mortality in Nigeria, a large LVD-endemic country that has in circulation 3 of the 4 currently known lineages of the Lassa virus. This study was carried out to address these gaps. The results should also be of relevance in the formulation of policies for the treatment and control of viral haemorrhagic fevers. The prevalence of LVD was 5. 4% among children with clinically undifferentiated fever (n = 243); 3. 9% among those with convulsions associated with fever (n = 77) and 6. 0% among those with fever but no convulsions (n = 166). The results underscore the importance of LVD as a cause of acute undifferentiated fever. The results also underscore the need of diagnostic testing for LVD in children with acute undifferentiated fever in endemic areas in order to facilitate control, including the prevention of nosocomial transmission.
keywords: inflammatory diseases
  medicine and health sciences
  body fluids
  pathology and laboratory medicine
  nervous system
  pathogens
  tropical diseases
  microbiology
  parasitic diseases
  parasitic protozoans
  viruses
  bacterial diseases
  protozoans
  signs and symptoms
  rna viruses
  convulsions
  infectious diseases
  infectious diseases of the nervous system
  malarial parasites
  medical microbiology
  lassa virus
  bacterial meningitis
  microbial pathogens
  diagnostic medicine
  anatomy
  meningitis
  fevers
  physiology
  neurology
  arenaviruses
  viral pathogens
  biology and life sciences
  malaria
  cerebrospinal fluid
  organisms
title: Prevalence of Lassa Virus Disease (LVD) in Nigerian children with fever or fever and convulsions in an endemic area