Search

Background A single-tube one-step real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of chikungunya virus (CHIKV) targeting the conserved 6K-E1 target region was developed. The assay was validated with sera collected from a CHIKV outbreak in Senegal in 2015. Methodology/Principal findings A novel design approach by combining Principal Component Analysis and phylogenetic analysis of 110 available CHIKV sequences and the LAMP oligonucleotide design software LAVA was used. The assay was evaluated with an External Quality Assessment panel from the European Network for Diagnostics of "Imported" Viral Diseases and was shown to be sensitive and specific and did not cross-detect other arboviruses. The limit of detection as determined by probit analysis, was 163 molecules, and 100% reproducibility in the assays was obtained for 103 molecules (7/8 repetitions were positive for 102 molecules). The assay was validated using 35 RNA samples extracted from sera, and results were compared with those obtained by quantitative RT-PCR carried out at the Institut Pasteur Dakar, demonstrating that the RT-LAMP is 100% sensitive and 80% specific, with a positive predictive value of 97% and negative predictive value of 100%. Conclusions/Significance The RT-LAMP appeared to show superior performance with material stored for months compared to qRT-PCR and can be therefore recommended for use in infrastructures with poor settings., Author summary Current chikungunya virus (CHIKV) outbreaks highlight the necessity of sensitive techniques to allow the virus detection even at an early stage (before the onset of clinical symptoms). In addition, CHIKV sometimes is misdiagnosed with other pathogens (i.e., dengue virus or malaria), which implies that specific methods have to be developed. Apart from specificity and sensitivity, these techniques have to be affordable for laboratories with very limited resources, and reactions should be easily performed without the need of experienced researchers and expensive equipment. Finally, because of the increase in number of publicly available sequences, the assay should cover all the possible variations observed in those sequences. We have considered all these premises, and we were able to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) by designing primers using a combination of Principal Component Analysis, phylogenetic analysis and LAVA algorithm. Our assay is specific and does not cross-react with other arboviruses tested, sensitive and has been validated at the Institut Pasteur Dakar, showing very good performance parameters.

Background Zika virus (Flavivirus genus) is the first mosquito-borne virus known to cause high rates of microcephaly and abortion in humans. Typically, Zika virus causes a self-limiting, systemic illness; however, the current outbreak of Zika virus in the Americas has been associated with increased rates of fetal malformations and Guillain-Barré syndrome. Very few Zika virus isolates have been described in the literature, and live viruses are needed to perform studies of pathogenesis and to develop vaccines and treatments. Methodology/Clinical findings We isolated Zika virus, strain FLR, directly from the serum of an individual infected in Barranquilla, Colombia (December, 2015). Here, we describe the patient’s clinical course and characterize strain FLR by its growth characteristics in mosquito and mammalian cells and its partial resistance to UV-inactivation. The full genome sequence of FLR was also analyzed (including the 3’ un-translated region), to determine its probable geographic origin, and to pinpoint structural differences from other Zika virus strains. Conclusions/Significance We anticipate that the study of this low passage, clinical isolate of Zika virus, which is available for worldwide distribution, will help uncover the mechanisms of viral replication and host immune responses contributing to the varied and sometimes severe clinical presentations seen during the current epidemic in the Americas., Author Summary Zika virus (ZIKV) is a mosquito-transmitted virus that causes a febrile illness characterized by a pronounced rash and occasional arthralgia. Serious complications from ZIKV infections were rarely reported until 2015, when Brazilian officials first reported an association between ZIKV infections and a dramatic increase in microcephaly and Guillian-Barré syndrome. Since then, ZIKV has been found in 39 countries in the Americas. Thus, the identification and characterization of ZIKV strains from the current outbreak are of utmost importance. In this study, we successfully isolated a ZIKV strain (ZIKV-FLR) from an individual who was bitten by mosquitoes while traveling in an area of Colombia that had ongoing dengue, chikungunya, and ZIKV transmission. Our analysis of the full genome sequence revealed that this virus strain may have originated in the Caribbean and was not directly imported from Brazil. We also discovered that this virus strain was partially resistant to inactivation by ultraviolet irradiation, when compared to dengue virus. Further work is needed to elucidate the underlying mechanisms behind this increased resistance and potential to cause more severe disease.

Given the current absence of specific drugs or vaccines for Ebola virus disease (EVD), rapid, sensitive, and reliable diagnostic methods are required to stem the transmission chain of the disease. We have developed a rapid detection assay for Zaire ebolavirus based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and coupled with a novel portable isothermal amplification and detection platform. The RT-LAMP assay is based on primer sets that target the untranscribed trailer region or nucleoprotein coding region of the viral RNA. The test could specifically detect viral RNAs of Central and West African Ebola virus strains within 15 minutes with no cross-reactivity to other hemorrhagic fever viruses and arboviruses, which cause febrile disease. The assay was evaluated using a total of 100 clinical specimens (serum, n = 44; oral swab, n = 56) collected from suspected EVD cases in Guinea. The specificity of this diagnostic test was 100% for both primer sets, while the sensitivity was 100% and 97.9% for the trailer and nucleoprotein primer sets, respectively, compared with a reference standard RT-PCR test. These observations suggest that our diagnostic assay is useful for identifying EVD cases, especially in the field or in settings with insufficient infrastructure., Author Summary Rapid and accurate diagnostic tests are required to prevent transmission of Ebola virus disease (EVD). Nucleic acid diagnostic tests are highly sensitive and specific for detecting the virus. The RT-LAMP assay using a portable platform equipped with a battery is a useful technique for detecting the virus in the field or laboratories with limited infrastructure. In the present study, RT-LAMP assay detected Zaire ebolavirus in clinical specimens with diagnostic accuracies corresponding to that of the standard RT-PCR test. In addition, the assay could detect positive samples within 15 minutes. These findings have raised the prospects for developing a rapid molecular diagnostic test for EVD. With further improvements in methods for pretreatment of clinical specimens, the RT-LAMP assay may provide a highly accurate rapid diagnostic test for EVD at point-of-care in endemic rural areas.