Short Report: Dengue-3 Outbreak in Paraguay: Investigations Using Capillary Blood Samples on Filter Paper Severine Matheus,* Jean-Baptiste Meynard, Anne Lavergne, Romain Girod, David Moua, Bhety Labeau, Philippe Dussart, Vincent Lacoste,† and Xavier Deparis† Centre National de Référence des Arbovirus, Institut Pasteur de la Guyane, Cayenne, French Guiana; Unité d’épidemiologie, Institut Pasteur de la Guyane, Cayenne, French Guiana; Unité d’entomologie Institut Pasteur de la Guyane, Cayenne, French Guiana; Laboratoire des Interactions Virus Hôtes, Institut Pasteur de la Guyane, Cayenne, French Guiana; Institut de Médecine Tropicale du Service de Santé des Armées, Le Pharo, Marseille, France Abstract. During a dengue-3 outbreak in Paraguay at the beginning of 2007, capillary blood samples absorbed onto filter papers were collected from 44 suspected cases. These samples were subjected to three molecular and serologic tests, and 31 of the 44 samples gave a positive result by at least one of the techniques used. Molecular analyses detected the dengue-3 serotype in 22 patients and additionally the dengue-2 serotype in two patients. Therefore two different serotypes were co-circulating during this outbreak. Overall, this study validates the use of dried-blood samples for field screening investigations. Indeed, all types of laboratory studies of dengue were possible with samples consisting of a few drops of dried blood from finger pricks. Dengue fever (DF) is a common viral disease that is a major public health problem in most tropical countries. There are cur- rently about 100 million dengue virus infections annually, with more than 500,000 cases of the severe forms of the disease, dengue hemorrhagic fever (DHF) or dengue shock syndrome. 1 Several factors have contributed to the spread of this arbovi- rus in South America since the 1970s. In particular, the sus- pension of Aedes aegypti vector eradication programs, and the increase of human population movements within and be- tween endemic regions have led to the resurgence of epidemic DF and the emergence of DHF in South America. 2–4 Four distinct serotypes of dengue virus (DENV-1 to DENV-4) cur- rently co-circulate in this sub-continent and dengue virus out- breaks are frequent. 5 The persistence of dengue outbreaks during recent years has become a public health problem in Paraguay, a country adjacent to Argentina, Brazil, and Bo- livia. In early 2007, the Ministry of Public Health and Social Welfare of Paraguay declared a nationwide epidemiologic alert after the confirmation of 390 cases of dengue infection during the first week of January. This outbreak has been the worst in the country’s history in terms of the number of cases and percentage mortality. By March 2007, 19,000 suspected dengue cases, including 46 DHF and 10 deaths, had been reported. The DENV-3 serotype was identified as the sero- type responsible. 6,7 To help in managing this outbreak and to evaluate the epidemiologic situation, the Institut Pasteur de la Guyane set up a scientific expedition in March 2007. We re- cently proposed that capillary blood samples absorbed on fil- ter papers could be an alternative to venous blood samples for the diagnosis of dengue virus infection. 8 This expedition was an opportunity to validate the usefulness of collecting capil- lary blood samples onto filter papers for field screening stud- ies, in particular in an area where the storage and transport conditions were incompatible with the molecular diagnosis of dengue infection from venous blood samples. Blood samples dried onto filter paper have the advantage of simplicity of collection (from the finger), and allow the identification of the viral serotype by molecular methods and the detection of dengue virus–specific immunoglobulin (IgM) antibodies and antigens. 8 Capillary blood samples were collected, in four hospitals in Asuncion, between March 3 and 13, 2007, from 44 patients sus- pected of dengue virus infection and who agreed to participate in the study. 9 For each patient, three drops of about 20 L of capillary blood were obtained from a finger and absorbed onto filter papers (Schleicher & Schuell, Germany) for sub- sequent reverse transcriptase-polymerase chain reaction (RT-PCR), and antigen (NS1) and IgM detection. All the samples were stored at room temperature until arrival at the Centre National de Référence des Arbovirus, Institut Pasteur de la Guyane, on March 17, 2007, when they were transferred to -80°C until studied. All capillary blood samples were first analyzed by RT-PCR: filter papers were cut into strips, placed into sterile tubes, and total RNA was extracted as previously described. 8 Dengue virus genome detection and typing was then done according to Lanciotti and others. 10 Although NS1 antigen detection is still not considered by the World Health Organization (WHO) to be a reference test for confirming dengue infection, it is widely used for diagnosis. We therefore decided to use it as a confirmatory test for the diagnosis of dengue virus infection. The Platelia dengue NS1 antigen cap- ture ELISA kit (Bio-Rad Laboratories, Marnes la Coquette, France) was used to detect the presence of the NS1 antigen secreted by dengue virus during infection in capillary blood samples from all acute cases and from those convalescent cases for which sufficient biologic material was available. 11 For each patient, one drop of capillary blood absorbed onto filter paper was cut and placed in a sterile tube containing 150 L of the dilution buffer provided in the kit. In addition to the controls provided in the kit, we also included two NS1- positive and -negative sera as internal controls: 10 L of these sera were absorbed onto filter paper and thereafter processed in the same way as the capillary blood samples. After 30 minutes of incubation at room temperature, 100 L of the capillary blood samples or controls were incubated with 100 L of diluted conjugate for 90 minutes. The subsequent steps were performed according to the manufacturer’s recommen- dations. 12,13 Serum samples were tested for IgM antibodies to * Address correspondence to Severine Matheus, Centre National de Référence des Arbovirus, Institut Pasteur de la Guyane, 23 Avenue Pasteur, BP 6010, 97306 Cayenne Cedex, French Guiana. E-mail: smatheus@pasteur-cayenne.fr † Senior authorship: Vincent Lacoste and Xavier Deparis share se- nior authorship. Am. J. Trop. Med. Hyg., 79(5), 2008, pp. 685–687 Copyright © 2008 by The American Society of Tropical Medicine and Hygiene 685