Dried-Blood Spots: A Cost-Effective Field Method for the Detection of Chikungunya Virus Circulation in Remote Areas

<div><p>Background</p><p>In 2005, there were outbreaks of febrile polyarthritis due to Chikungunya virus (CHIKV) in the Comoros Islands. CHIKV then spread to other islands in the Indian Ocean: La Réunion, Mauritius, Seychelles and Madagascar. These outbreaks revealed the lack of surveillance and preparedness of Madagascar and other countries. Thus, it was decided in 2007 to establish a syndrome-based surveillance network to monitor dengue-like illness.</p><p>Objective</p><p>This study aims to evaluate the use of capillary blood samples blotted on filter papers for molecular diagnosis of CHIKV infection. Venous blood samples can be difficult to obtain and the shipment of serum in appropriate temperature conditions is too costly for most developing countries.</p><p>Methodology and principal findings</p><p>Venous blood and dried-blood blotted on filter paper (DBFP) were collected during the last CHIKV outbreak in Madagascar (2010) and as part of our routine surveillance of dengue-like illness. All samples were tested by real-time RT-PCR and results with serum and DBFP samples were compared for each patient. The sensitivity and specificity of tests performed with DBFP, relative to those with venous samples (defined as 100%) were 93.1% (95% CI:[84.7–97.7]) and 94.4% (95% CI:[88.3–97.7]), respectively. The Kappa coefficient 0.87 (95% CI:[0.80–0.94]) was excellent.</p><p>Conclusion</p><p>This study shows that DBFP specimens can be used as a cost-effective alternative sampling method for the surveillance and monitoring of CHIKV circulation and emergence in developing countries, and probably also for other arboviruses. The loss of sensitivity is insignificant and involved a very small number of patients, all with low viral loads. Whether viruses can be isolated from dried blood spots remains to be determined.</p></div

Sensitivity and specificity of test performed on DBFP for the diagnosis of Chikungunya virus infection^a.

a<p>Sensitivity, 93.1% (68/73, (95% CI:[84.7–97.7]); specificity, 94.4% (102/108, (95% CI:[88.3–97.7]); Kappa coefficient, 0.87 (95% CI:[0.80–0.94]).</p

Ct Differences in RNA amplification from 15 µl aliquots of whole blood and DBFP.

<p>Ct Differences in RNA amplification from 15 µl aliquots of whole blood and DBFP.</p

Primers and probes used in amplification of Chikungunya RNA as previously described by Laurent et al. 2007 [28] (modifications of primers are indicated in bold).

<p>Primers and probes used in amplification of Chikungunya RNA as previously described by Laurent et al. 2007 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002339#pntd.0002339-Laurent1" target="_blank">[28]</a> (modifications of primers are indicated in bold).</p

Genetic relatedness of geographically distinct WNV isolates determined by using the nucleotide sequence data from a 228 bp region of the E gene.

<p>The tree was constructed with the PAUP by using the neighbor-joining distance program of Mega 5 software. Node values were determined for 1,000 replicates. Isolates are labeled as follows: strain identification, country, date of isolation, genbank accession number.</p

Seroprevalence analysis of West Nile virus in domestic birds.

<p>(A) in the Mitsinjo district (B) in the Antsalova district. Numbers in parentheses corresponded to the number of animals in each category and in the y axis represented the percentage of animals presenting WNV antibodies. P-values were generated from the regression analysis.</p

Additional file 2 of Enterovirus detection in different regions of Madagascar reveals a higher abundance of enteroviruses of species C in areas where several outbreaks of vaccine-derived polioviruses occurred

Additional file 2: Figure S1. Detection of NPEVs on RD and HEp-2c cells

Additional file 3 of Enterovirus detection in different regions of Madagascar reveals a higher abundance of enteroviruses of species C in areas where several outbreaks of vaccine-derived polioviruses occurred

Additional file 3: Figure S2. Phylogenetic trees of Madagascan EV-Cs based on the 5′UTR, the VP1- and the 3D-encoding sequences. The isolates are colour-coded according to their respective type; triangles indicate isolates from this study, circles isolates from previous works

Additional file 1 of Enterovirus detection in different regions of Madagascar reveals a higher abundance of enteroviruses of species C in areas where several outbreaks of vaccine-derived polioviruses occurred

Additional file 1: Table S1. Accession numbers of the sequences used to draw the tree displayed in Fig. 7

Incidence of ILI during each regional peak.

<p>Incidence of ILI during each regional peak.</p