Abstract:
Globally, the threat of emerging and re-emerging arboviral infections continues to be a
major public health concern. The detection and diagnosis of many of these diseases
remains a challenge, and it is clear that novel approaches to their identification and
control must be continually developed. Clearly there is an overarching need to
accurately detect priority febrile illnesses and their causative agents in Kenya. This
study designed and optimized a Reverse-Transcription Isothermal Loop Mediated Assay
(RT-LAMP) for yellow fever virus (YFV) assay, developed and optimized ELISA
systems using anti-yellow fever virus monoclonal antibody (2D12) as well as used the
developed assays to determine the seroprevalence of YFV in selected health facilities in
Western Kenya during the period 2010-2012. The study design was experimental. RTLAMP was done isothermally at 62 °C using a real-time turbidimeter which allowed
detection within 1 h. RT-PCR and real time RT-PCR using YFV primers was also
performed. Specificity of RT-PCR, real-time RT-PCR and RT-LAMP assays was
determined using RNA from other related flaviviruses (WNV, DENV1-4, JEV) as well
as RVFV and CHIKV where only YFV RNA was detected. In addition, equal sensitivity
was also observed when the RT-LAMP assay and the quantitative real-time RT-PCR
were compared using YFV spiked human serum samples with a detection limit of 0.29
PFU. The RT-LAMP assay further showed equal detection limit sensitivity of 0.29 PFU
while using two local YFV wild isolates and the vaccine strain YFV 17D. A total of 469
serum samples from febrile patients were tested for YFV antibodies using in-house IgM-
xxi
capture ELISA, in-house IgG indirect ELISA and focus reduction neutralization test
(FRNT50). The present study did not yield any IgM ELISA positive cases an indication
of absence of recent YFV infection in the area. Twenty eight samples (6%) tested
positive for YFV IgG. These 28 cases detected during the study were due to either YFV
vaccination or past exposure to various flaviviruses including YFV. There were five
cases confirmed by FRNT50, of these, four cases were either vaccination or natural
infection during YF outbreak (1992-93) or another period and one case was confirmed
as a West Nile virus infection. The YFV NS1 recombinant antigen was successfully
cloned and expressed in a pET 28a system. Sequencing data showed 99% homogeneity
with the consensus YFV 17D strain. Confirmation of the expressed YFV NS1 was
performed using a penta-his tag in a Western blot.
