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

Abstract

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.

Conclusion: 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.

Figure 1. Blotting papers showing six 6 spots with dried liquid exudate from pustules collected in one patient.

One spot (arrow) was removed and processed for DNA-based identification of causative agents of the rash illness.

Figure 2. Multiple alignment of 14 kD DNA targets and design of primers and probes for the qPCR assay.

Primers for qPCR and for the conventional PCR are shown in plain boxes whereas the pan-orthopoxvirus and specific variola virus probes are highlighted in a dashed boxes. The monkepoxvirus specific SNP (C→T) is arrowed. The pyrosequencing probe is indicated by a plain arrow.

Figure 3. MPXV qPCR amplification standard curve.

Figure 4

A) Gel electrophoresis of the conventional PCR amplification products showing the undigested 384-bp OPV amplicon DNA ladder (lane 1); negative controls (lanes 2–3); human specimens from 13 patients of the Bena Tshiadi healthcare district (lanes 4–13 and 15–18), and from 1 patient of the Yangala healthcare district (lane 14). B) BsrGI digestion profile of 384-bp MPXV amplicons: banding patterns with 210- and 174-bp fragments.

Figure 5. Map of West Kasai Province.

Areas affected by rash illness outbreaks are highlighted as follows: ▴: Bena Tshiadi healthcare zone (13 patients in 7 different villages). All cases were confirmed as monkeypox cases. Hourglass Symbol: Yangala healthcare zone (8 patients clustered in Tshikongo village). All were confirmed as varicella cases.♦: Ndesha healthcare zone in the outskirts of Kananga (3 patients clustered in Lubuyi village, several kilometers north of Kananga city). All were confirmed as varicella cases.