Simple technique for in field samples collection in the cases of skin rash illness and subsequent PCR detection of orthopoxviruses and varicella zoster virus.

Catherine Dumont,Jean-Jacques T Muyembe,Mostafa Bentahir,Jean-Luc Gala,Edmond K Magazani,Daniel Garin,Leonid M Irenge,Pierre Roques

doi:10.1371/journal.pone.0096930

Abstract

BackgroundIn case of outbreak of rash illness in remote areas, clinically discriminating monkeypox (MPX) from severe form of chickenpox and from smallpox remains a concern for first responders.ObjectiveThe goal of the study was therefore to use MPX and chickenpox outbreaks in Democratic Republic of Congo (DRC) as a test case for establishing a rapid and specific diagnosis in affected remote areas.MethodsIn 2008 and 2009, successive outbreaks of presumed MPX skin rash were reported in Bena Tshiadi, Yangala and Ndesha healthcare districts of the West Kasai province (DRC). Specimens consisting of liquid vesicle dried on filter papers or crusted scabs from healing patients were sampled by first responders. A field analytical facility was deployed nearby in order to carry out a real-time PCR (qPCR) assay using genus consensus primers, consensus orthopoxvirus (OPV) and smallpox-specific probes spanning over the 14 kD fusion protein encoding gene. A PCR-restriction fragment length polymorphism was used on-site as backup method to confirm the presence of monkeypox virus (MPXV) in samples. To complete the differential diagnosis of skin rash, chickenpox was tested in parallel using a commercial qPCR assay. In a post-deployment step, a MPXV-specific pyrosequencing was carried out on all biotinylated amplicons generated on-site in order to confirm the on-site results.ResultsWhereas MPXV proved to be the agent causing the rash illness outbreak in the Bena Tshiadi, VZV was the causative agent of the disease in Yangala and Ndesha districts. In addition, each on-site result was later confirmed by MPXV-specific pyrosequencing analysis without any discrepancy.ConclusionThis experience of rapid on-site dual use DNA-based differential diagnosis of rash illnesses demonstrates the potential of combining tests specifically identifying bioterrorism agents and agents causing natural outbreaks. This opens the way to rapid on-site DNA-based identification of a broad spectrum of causative agents in remote areas.

Highlights

Orthopoxviruses (OPV) are large double-stranded DNA viruses which replicate exclusively in the cytoplasm of host cells [1,2]
This experience of rapid on-site dual use DNA-based differential diagnosis of rash illnesses demonstrates the potential of combining tests identifying bioterrorism agents and agents causing natural outbreaks
Varicella is a febrile rash illness characterized by the absence of a significant febrile prodrome and by lesions at different stages, with bumps, blisters, and scabbed lesions existing at the same time [14]

Summary

Introduction

Orthopoxviruses (OPV) are large double-stranded DNA viruses which replicate exclusively in the cytoplasm of host cells [1,2]. Symptoms of orthopoxvirus infections range from mild skin lesions to fatal systemic disease, as is the case for the major form of smallpox. This disease is characterized by a generalized rash associated with a high mortality rate in unvaccinated persons [3,4,5,6,7]. The development of rapid sampling method and simple identification methods which can be implemented in the field could substantially improve rapid identification of causative agents, pending definitive confirmation by the LNSP or by any other reference laboratory This potential was illustrated during 2008–2009 outbreaks of skin rash illness in West Kasai province, which were presumably attributed to MPXV. In case of outbreak of rash illness in remote areas, clinically discriminating monkeypox (MPX) from severe form of chickenpox and from smallpox remains a concern for first responders

Methods

Results

Conclusion