Dengue Diagnosis Tested in Urine and Saliva Specimens

Dengue is the most important arthropod-borne disease affecting humans, representing a huge public health burden in affected countries. Symptoms are often nonspecific, hence the need for an early, sensitive, and specific diagnosis of dengue for appropriate management.

Direct diagnosis of Dengue virus (DENV) infection is based on virus isolation, detection of the viral genome by reverse transcription polymerase chain reaction (RT-PCR) or detection of nonstructural protein-1 (NS1) antigen. Indirect diagnosis using serological methods to detect anti-DENV immunoglobulin M (IgM) and IgG is

Scientists at the Institut Pasteur du Cambodge (Phnom Penh, Cambodia) collected and analyzed a total, 913 plasma, 1,555 urine and 1,564 saliva samples obtained from 267 patients with confirmed dengue infection and complete records. The clinical specimens obtained from 20 patients hospitalized for a non-dengue febrile illness were also used to assess the specificity of the different diagnostic methods in urine and saliva samples. These controls showed no biological evidence of on-going DENV infection (DENV qRT-PCR negative, NS1 test negative and no anti-DENV antibodies in paired plasma.

A capture enzyme-linked immunosorbent assay (ELISA) was used to detect NS1 in plasma, non-concentrated urine, and undiluted saliva. In plasma and urine, the NS1 protein was detected by the two-step ELISA method. In-house capture ELISAs were used to detect anti-DENV IgM (MAC-ELISA) and IgA (AAC-ELISA) in plasma, non-concentrated urine and undiluted saliva specimens. The viral ribonucleic acid (RNA) was extracted from plasma, saliva mixed with viral transport medium (VTM) and concentrated urine, using the QIAmp Viral RNA Mini kit (Qiagen; Hilden, Germany). Urine samples were concentrated using the 100K Microsep ultrafiltration device (Pall Corporation; Port Washington, NY, USA) to convert an initial volume of 5 mL urine to a final volume of 280 µL of concentrated urine.

The RT-PCR and NS1 tests demonstrated an overall sensitivity of 85.4%/63.4%, 41.6%/14.5%, and 39%/28.3%, in plasma, urine and saliva specimens, respectively. When urine and saliva samples were collected at the same time-points and tested concurrently, the diagnostic sensitivity of RNA and NS1 detection assays was 69.1% and 34.4%, respectively. IgG/IgA detection assays had an overall sensitivity of 54.4%/37.4%, 38.5%/26.8%, and 52.9%/28.6% in plasma, urine, and saliva specimens, respectively. IgM were detected in 38.1% and 36% of the plasma and saliva samples but never in urine.

The authors concludes that although the performance of the different diagnostic methods were not as good in saliva and urine as in plasma specimens, the results obtained by qRT-PCR and by anti-DENV antibody ELISA could well justify the use of these two body fluids to detect dengue infection in situations when the collection of blood specimens is not possible. The study was published on September 25, 2015, in the journal Public Library of Science Neglected Tropical Diseases.

Related Links:
Institut Pasteur du Cambodge 
Qiagen 
Pall Corporation