Abstract:
O'nyong’nyong fever, caused by infection with a mosquito-borne East African
alphavirus, is an acute, non-fatal illness characterized by fever and polyarthralgia.
Alpha viruses such as Chikungunya and O’nyong’nyong are endemic in East Africa
and have caused extensive epidemics and therefore their rapid diagnosis is a priority.
In this study, a novel, cheap and rapid method of gene amplification known as
Reverse Transcriptase Loop Amplification Test was optimized for ONNV detection.
Other methods such as; RT-PCR, Cell culture, plaque assay were also performed for
purposes of comparison with RT-LAMP. The objective of this work was to optimize
and evaluate an RT-LAMP assay for detection of O’nyong’nyong virus. One hundred
samples were prepared. 40 of the samples were spiked with a high concentration of
ONNV antigen (1:1000) while 40 these samples were spiked with low concentration
of ONNV antigen (1:10000) and 20 few of the samples were not spiked with the
ONNV antigen. All the samples were blinded and coded to avoid bias. These samples
were analyzed using plaque assay, RT-PCR, and RT-LAMP. Plaque assay was used
as the gold standard in this study. Out of the 100 samples tested by plaque assay 40
(11-30 pfu/ml) tested positive, 30 (0 pfu/ml) negative and 30 (1-10 pfu/ml)
indeterminate. Using conventional RT-PCR 40 samples were positive, 30 negative
and 30 indeterminate. By RT-LAMP 40 samples were positive, 23 negative and 37
indeterminate. RT-LAMP detected 7 samples as indeterminate that conventional PCR
and plaque assay did not detect. The sensitivity and specificity of RT-PCR and RTLAMP was calculated using plaque assay as the gold standard. The sensitivity of RT-
LAMP was 100% whereas that of RT-PCR was 85%. The specificity of RT-LAMP
was 77% whereas that of RT-PCR was 100%. RT-LAMP is the best screening test for
sensitivity whereas RT-PCR is the best screening test for specificity. Reliability
testing was done by calculating the positive (PPV) and negative (NPV) predictive
values. PPV of RT-LAMP was 85% whereas that of RT-PCR was 100%. NPV of RTLAMP was 100% and that of RT-PCR was 77%. In terms of cost and more detailed
analysis RT-LAMP emerged as the best test amongst the three assays carried out in
this study. The research findings reported in this work will contribute towards the
diagnosis of viral infections especially ONNV and other emerging arboviruses
particularly in outbreaks. Further optimization and evaluation should be done to
establish RT-LAMP as a tool for surveillance of not only O’nyong’nyong virus but
also other emerging viruses.