The leishmanin skin test (LST) measures delayed-type hypersensitivity to Leishmania and is frequently used for clin- ical diagnosis, and as an epidemiologic tool on the prevalence of exposure to Leishmania parasites to characterize popu-

The aim of the present preliminary study was to investigate the potential of measurement of IFN-γ secretion by T cells into blood plasma using QuantiFERON assay with leishmanial antigens to determine the presence of Leishmania infection. Blood samples from cured visceral ( N = 18), and cutaneous ( N = 20) leishmaniasis cases, and 20 healthy controls were tested. The IFN-γ responses to Leishmania major H2B and Leishmania infantum H2B antigens were detected from the majority of treated old visceral leishmaniasis cases, but not from controls. Future studies using larger groups will be required to establish the true potential of the assay for epidemiological screening of leishmaniasis. * Address correspondence to Nevin Turgay, Ege University Medical School, Department of Parasitology, Bornova, Izmir 35100. E-mail: nevin-turgay@ege.edu.tr 823 GAMMA INTERFERON TEST FOR LEISHMANIA to mitogen (PHA) was strong in all samples from all participants. The mean IFN productions to mitogen (PHA) were strong as 60.58, 66.13 and 26.40 IU/mL in samples from old CL, VL cases and controls, respectively. The leishmanin skin test has been widely used as an epidemiologic tool, but using not good manufacturing practice ( GMP) products in most parts of the endemic areas as an antigen, influence of subjectivity on reaction size measurement, need to return in 3 days to read the result, and the unwillingness of individuals to accept the test limited the use of LST. The QuantiFERON-TB test was developed to overcome some of the limitations of the tuberculin skin test (TST) for tuberculosis. 5– 8 Although TST and LST both measure delayedtype hypersensitivity on the forearm, QFN actually measures in vitro antigen-specific IFN-γ production. The IFN-γ is one of the key cytokines involved in the cell-mediated immune response against both pathogens. Thus, a QFN test for leishmaniasis could provide a rapid and convenient alternative test to measure specific T-cellmediated immune responses and IFN-γ production of people living in endemic areas to Leishmania . Whole blood incubated for 16–24 hrs with parasite antigens should cause IFN-γ secretion from previously primed memory T cells in people previously exposed to the leishmaniasis, but not from naive T cells of healthy volunteers. This study shows that use of L. major or L. infantum H2B as antigens stimulated the highest IFN-γ production from subjects with a previous history of VL, whereas people who had CL caused by L. tropica in the past produced less IFN-γ in a response to these antigens. The reason for the cells from old CL cases not responding to lysate might be caused by either movement of these cases out of the endemic region and not being exposed to parasite, losing memory T cells or isolated lesion sites, such as mostly on the face and low parasite burden. It is possible that these antigens may induce T-cell proliferation, which was not preferred to be included in this study because of being a less friendly procedure, but they may not cause IFN-γ production. The combination of all four antigens yielded maximal responses and a number of positives. The H2B molecules were superior to lysate for use as an epidemiological screening marker in leishmaniasis endemic/sporadic areas. The H2B antigens did not produce IFN-γ responses from healthy volunteers. Even though studies have shown lifelong T cell responses to leishmanial antigens measured with in vitro tests, others have pointed to the possibility of loss of positivity with time. 2, 9 Two of the three VL cases with negative H2B IFN-γ responses had disease in 1992 and 1997, respectively, and hence their response may have waned with time. This new test can be used for epidemiological studies as an alternative to the LST presently unavailable as a GMP product in most endemic regions, including Europe. Because of increased IL-10 secretion during the acute phase VL might inhibit IFN-γ production; we are not expecting this test to be useful in diagnosing this disease, though additional studies are needed. For people with no history of symptomatic VL, high IFN-γ production results using QFN may be presumed to indicate prior asymptomatic leishmaniasis infection or exposed to Leishmania parasite without developing infection. In such asymptomatic individuals with leishmaniasis, the infection might even reactivate and cause active disease if such people become immunosuppressed later in life. Even though commercial in vitro tests are unlikely to be affordable in most markets, if IFN-γ production in QFN assay could give the information about cumulative leishmanial exposure experienced by the community, who remained negative, it might indicate the susceptible segment of the population, which could be suitable candidates for the future vaccine trials or prophylactic procedures for those in endemic areas. This work is a preliminary study aimed to examine the potential usefulness of the technique for Leishmania screening and more work needs to be carried out to explain the dynamic kinetics related to different clinical forms of leishmaniasis. Received October 9, 2009. Accepted for publication May 3, 2010. Acknowledgments: We are indebted to Steve Reed (Infectious Disease Research Institute, Seattle, WA) and Emanuela Handman (Walter and Eliza Hall Institute for Medical Research, Melbourne, Australia) for provision of leishmanial antigens for initial screening studies. Cellestis provided QuantiFERON kits, antigens, and related materials, and financial support. We also thank Charles L. Jaffe and M. Ziya Alkan for reading the manuscript. Authors’ addresses: Nevin Turgay, Seray Ozensoy Toz, and Yusuf Ozbel, Department of Parasitology, Ege University Medical School, Izmir, Turkey, E-mails: nevin.turgay@ege.edu.tr , seray.ozensoy.toz@ ege.edu.tr , and yusuf.ozbel@ege.edu.tr . I. Cuneyt Balcioglu, Department of Parasitology, Celal Bayar University Medical School, Manisa, Turkey, E-mail: drcbal@yahoo.com . Stephen L. Jones, Cellestis Limited, Carnegie, Australia, E-mail: sjones@cellestis.com .