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


OBJECTIVE
The 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.


METHODS
In 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.


RESULTS
Whereas 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.


CONCLUSIONS
This 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.
Royal Military Academy, Bruxelles, Belgium; Center for Applied Molecular Technologies, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain, Brussels, Belgium.
Catherine
C
Dumont
Defense Laboratories Department, ACOS Ops&Trg, Belgian Armed Forces, Peutie, Belgium; Center for Applied Molecular Technologies, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain, Brussels, Belgium.
Leonid M
LM
Irenge
Ministère de la Santé Publique, Gombe, République Démocratique du Congo.
Edmond K
EK
Magazani
IRBA, Institut de Recherches Biomédicales des Armées, Service de Santé des Armées, Bretigny-sur-Orge, Cedex, France.
Daniel
D
Garin
Laboratoire National de Santé Publique, Institut National de Recherche Biomedicale, Kinshasa, Democratic Republic of Congo.
Jean-Jacques T
JJ
Muyembe
Defense Laboratories Department, ACOS Ops&Trg, Belgian Armed Forces, Peutie, Belgium; Center for Applied Molecular Technologies, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain, Brussels, Belgium.
Mostafa
M
Bentahir
Defense Laboratories Department, ACOS Ops&Trg, Belgian Armed Forces, Peutie, Belgium; Center for Applied Molecular Technologies, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain, Brussels, Belgium.
Jean-Luc
JL
Gala
D002644:Chickenpox / Q000821:virology; D004279:DNA, Viral / Q000235:genetics; D005076:Exanthema / Q000821:virology*; D014645:Herpesvirus 3, Human / Q000235:genetics* / Q000472:pathogenicity; D006801:Humans; D008993:Monkeypox virus / Q000235:genetics* / Q000472:pathogenicity; D018155:Orthopoxvirus / Q000235:genetics* / Q000472:pathogenicity; D016133:Polymerase Chain Reaction / Q000379:methods; D012901:Variola virus / Q000235:genetics / Q000472:pathogenicity
D004279:DNA, Viral