Nitric oxide (NO) has been demonstrated to be a principal effector molecule responsible for mediating intracellular killing of Leishmania parasites, the causative organism of leishmaniasis. As measurement of intracellular NO remains a challenge to biologists, we have developed a flow cytometric approach to perform real time biological detection of NO within Leishmania parasites and parasitized macrophages using a membrane permeable derivative of diaminofluorescein [4,5-diaminofluorescein diacetate (DAF-2DA)]. Initially, assay optimization was performed in Leishmania donovani promastigotes, assay specificity being confirmed using both a NO donor [S-nitroso-N-acetyl-penicillamine (SNAP)] and a NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, C-PTIO]. Using 40 lM DAF-2DA, basal levels of intracellular NO were measured which varied in different Leishmania species; addition of conventional anti-leishmanial drugs, antimony and miltefosine translated into a dramatic increase in DAF-2T fluorescence. Furthermore, the assay also measured levels of NO in macrophages, but needed a 20 fold lower concentration of DAF-2DA, being 2 lM. Following parasitization, levels of NO decreased which was normalized following treatment with anti-leishmanial drugs. Similarly monocytes of patients with visceral leishmaniasis at disease presentation showed decreased levels of NO which too reverted on completion of treatment. Taken together, this study opens new perspectives of research regarding monocyte function and provides a real time approach for monitoring the effect of anti-leishmanial compounds. ' International Society for Advancement of Cytometry Key termsanti-leishmanial; DAF-2DA; flow cytometry; leishmaniasis; nitric oxide LEISHMANIA are intracellular protozoan parasites that infect host mononuclear phagocytes, the resultant complex of diseases being leishmaniasis affecting 12 million people world wide in 88 countries (1). This disease is characterized by development of dermal lesions that may be cutaneous leishmaniasis (CL), diffuse cutaneous leishmaniasis (DCL), or mucocutaneous leishmaniasis (MCL) as also there can be systemic involvement as in the case of visceral leishmaniasis (VL), which is potentially fatal if left untreated (2).This obligate intracellular parasite resides and multiplies within macrophages, and therefore it should be able to evade the cytotoxic mechanisms innately operative within the macrophages for its survival (3). One of the predominant cytotoxic mechanisms includes production of reactive nitrogen intermediates, which the Leishmania parasite effectively decreases to ensure its survival (4,5).

Abstract. This report presents three cases where the rK39 strip test failed to diagnose two cases of post-kala-azar dermal leishmaniasis and one case of visceral leishmaniasis. However, a strong clinical suspicion prompted further evaluation by polymerase chain reaction (PCR), which established the etiology. The present case series highlights the usefulness of PCR in the diagnosis of leishmaniasis.

Background: Bacterial pathogens using the type III secretion system (T3SS) require class II chaperones (LcrH) for infection. Results: The modular TPR fold of LcrH produces a weakly folded structure held together by its weak dimeric interface. Conclusion: Under physiological conditions, LcrH populates partially folded structures, thus suggesting a mechanism for cargo binding. Significance: This work is important for understanding one essential part of the T3SS.

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