This provided an opportunity to perform a follow-up on these patients of which 20 were found 3 to 6 months after attending the clinic either by direct observation or indirectly through discussion with relatives. revealed none had persistent VL symptoms. The Cohen kappa index comparing blood and serum was 0.88 indicating excellent concordance. Conclusion Although the concordance was excellent, it is possible to miss rK39 positive individuals when using blood and the titer of anti-rK39 antibodies is low. We recommend that when an individual from an endemic area has obvious clinical symptoms of VL and the whole blood rK39 RDT is negative, that the test should be redone 2C3 weeks later if RG14620 the symptoms persist. Author Summary Visceral leishmaniasis (VL), is a neglected tropical disease that is highly endemic in the Indian subcontinent and in East Africa and is the second most fatal parasitic disease after malaria. There currently exists several effective treatments for VL RG14620 and it is therefore essential that the diagnosis be as accessible, sensitive and specific as possible. The current diagnostic test, known as the rK39 rapid diagnostic test (RDT) involves detection of antibodies against the K39 protein antigen from (in the Indian subcontinent . The current method of VL diagnosis involved evaluating clinical symptom that include fever for more than 2 week, the presence of splenomegaly, and a positive serological rK39 immunochromatographic rapid diagnostic test (RDT) , . The rK39 RDT is used to detect the presence of antibodies against the antigen K39 that contains a repetitive 39 amino acid sequence from the kinesin protein. Clinical RG14620 features of VL however can be mistaken for other febrile illnesses such as malaria and enteric fever. Therefore, accurate serological diagnosis with the rK39 RDT is essential. Although a number of rK39 RDTs are commercially available and have recently been shown to be effective on the Indian continent, these tests have been developed for use with serum . These includes the Kalazar Detect test which, is the most widely used test in India. However, in order to be used at RG14620 the point of care, the rK39 RDTs are routinely performed on blood instead of serum in the endemic regions of India, Nepal and Bangladesh . It was therefore necessary in this study to establish whether the rK39 RDT is as sensitive when using blood as serum. This is a critical issue because performing the rK39 RDT on blood allows the test to be point of care at the level of primary health care centers close to the endemic villages, whereas performing the test on serum would require the test be performed at a district hospital which is generally much further from the endemic communities. Methods Patients The study and informed consent forms were approved by the Rajendra Memorial Research Institute of Medical Sciences (RMRIMS) ethics review board. Parents provided written consent on behalf of participants under the age of 18. None of patients enrolled previously had VL or PKDL. Clinical suspicion for VL was defined as fever for more than 14 days and signs of splenomegaly. All RG14620 suspected patients attended the out-patient department between August 2011 and April 2012. rK39 rapid test The rK39 immunochromatographic RDT, Kalazar Detect (InBios International, USA) was performed at RMRIMS according to manufacturers instructions. At room temperature, 20 ul of serum prepared from venous blood or one drop of fingerstick blood was added to the dipstick. A single drop of blood was used in this study because this is what is routinely performed in the field. Three drops of the chase buffer solution was added to a test tube followed by addition of the dipstick into the test tube containing the chase buffer. The results were read after 10 minutes. The test was considered positive when both the control line and the test line appeared red in color. The level of agreement between the tests performed on serum versus blood was calculated using Cohen’s kappa index. ELISA against recombinant K39 protein Recombinant K39 protein was kindly provided by Dr. Steve Reed from the Infectious Diseases Research Institute, Seattle USA. Ninety six well microtiter plates were coated with 100 ul of 5 ug/ml rK39 in carbonate/bicarbonate buffer overnight. Wells were then washed extensively in 0.05% PBS-T and then blocked in 5% non fat dry milk +0.1% PBS-T for 1 h at 37C followed by washing again in 0.05% PBS-T. Human sera samples (100 ul) at various dilution from 150 to 16400 was added for TET2 2 hours at room temperature and then wells washed 3 times in 0.05% PBS-T. 100 uL of HRP-linked anti-human.