Little is known about CD8 T cells in human visceral leishmaniasis (VL) and it is unclear if these cells have a protective, pathological and/or suppressive function. In experimental VL CD8 T cells have been shown to contribute to parasite control and play an important role in vaccine-generated immunity. To better understand the role of CD8 T cells in human VL, we examined molecules associated with anergy and cytotoxic T lymphocytes (CTL) in peripheral blood mononuclear cells (PBMC) and splenic aspirates (SA), and in CD8 cells derived from these tissues. Gene and surface marker expression suggest that splenic CD8 cell predominantly display an anergic phenotype, whereas CD8-PBMC have features of both anergic cells and CTLs. CD8 cells contribute to the baseline IFNγ levels in whole blood (WB) and SA cultures, but not to the Leishmania induced IFNγ release that is revealed using WB cultures. Blockade of CTLA-4 or PD1 had no effect on IFNγ production or parasite survival in SA cultures. Following cure, CD8 T cells contribute to the Leishmania induced IFNγ production observed in Leishmania stimulated cell cultures. We suggest CD8 T cells are driven to anergy/exhaustion in human VL, which affect their ability to contribute to protective immune responses.

IntroductionAsymptomatic persons infected with the parasites causing visceral leishmaniasis (VL) usually outnumber clinically apparent cases by a ratio of 4–10 to 1. We describe patterns of markers of Leishmania donovani infection and clinical VL in relation to age in Bihar, India.MethodsWe selected eleven villages highly endemic for Leishmania donovani. During a 1-year interval we conducted two house to house surveys during which we collected blood samples on filter paper from all consenting individuals aged 2 years and above. Samples were tested for anti-leishmania serology by Direct Agglutination Test (DAT) and rK39 ELISA. Data collected during the surveys included information on episodes of clinical VL among study participants.ResultsWe enrolled 13,163 persons; 6.2% were reactive to DAT and 5.9% to rK39. Agreement between the tests was weak (kappa = 0.30). Among those who were negative on both tests at baseline, 3.6% had converted to sero-positive on either of the two tests one year later. Proportions of sero-positives and sero-converters increased steadily with age. Clinical VL occurred mainly among children and young adults (median age 19 years).DiscussionAlthough infection with L. donovani is assumed to be permanent, serological markers revert to negative. Most VL cases occur at younger ages, yet we observed a steady increase with age in the frequency of sero-positivity and sero-conversion. Our findings can be explained by a boosting effect upon repeated exposure to the parasite or by intermittent release of parasites in infected subjects from safe target cells. A certain proportion of sero-negative subjects could have been infected but below the threshold of antibody abundance for our serologic testing.

Summary Background Visceral leishmaniasis, also known on the Indian subcontinent as kala-azar, is a fatal form of leishmaniasis caused by the protozoan parasite Leishmania donovani and transmitted by the bites of the vector sandfly Phlebotomus argentipes . To achieve and sustain elimination of visceral leishmaniasis, the transmission potential of individuals exposed to L donovani from across the infection spectrum needs to be elucidated. The aim of this study was to evaluate the relative infectiousness to the sandfly vector of patients with visceral leishmaniasis or post-kala-azar dermal leishmaniasis, before and after treatment, and individuals with asymptomatic infection. Methods In this prospective xenodiagnosis study done in Muzaffarpur district of Bihar, India, we included patients with clinically confirmed active visceral leishmaniasis or post-kala-azar dermal leishmaniasis who presented to the Kala-Azar Medical Research Center. These participants received treatment for L donovani infection. We also included asymptomatic individuals identified through a serosurvey of 17 254 people living in 26 high-transmission clusters. Eligible participants were aged 12–64 years, were HIV negative, and had clinically or serologically confirmed L donovani infection. During xenodiagnosis, the forearms or lower legs of participants were exposed to 30–35 female P argentipes sandflies for 30 min. Blood-engorged flies were held in an environmental cabinet at 28°C and 85% humidity for 60–72 h, after which flies were dissected and evaluated for L donovani infection by microscopy and quantitative PCR (qPCR). The primary endpoint was the proportion of participants with visceral leishmaniasis or post-kala-azar dermal leishmaniasis, before and after treatment, as well as asymptomatic individuals, who were infectious to sandflies, with a participant considered infectious if promastigotes were observed in one or more individual flies by microscopy, or if one or more of the pools of flies tested positive by qPCR. Findings Between July 12, 2016, and March 19, 2019, we recruited 287 individuals, including 77 with active visceral leishmaniasis, 26 with post-kala-azar dermal leishmaniasis, and 184 with asymptomatic infection. Of the patients with active visceral leishmaniasis, 42 (55%) were deemed infectious to sandflies by microscopy and 60 (78%) by qPCR before treatment. No patient with visceral leishmaniasis was found to be infectious by microscopy at 30 days after treatment, although six (8%) were still positive by qPCR. Before treatment, 11 (42%) of 26 patients with post-kala-azar dermal leishmaniasis were deemed infectious to sandflies by microscopy and 23 (88%) by qPCR. Of 23 patients who were available for xenodiagnosis after treatment, one remained infectious ...