Genome Analysis of Endotrypanum and Porcisia spp., Closest Phylogenetic Relatives of Leishmania, Highlights the Role of Amastins in Shaping Pathogenicity

While numerous genomes of Leishmania spp. have been sequenced and analyzed, an understanding of the evolutionary history of these organisms remains limited due to the unavailability of the sequence data for their closest known relatives, Endotrypanum and Porcisia spp., infecting sloths and porcupines. We have sequenced and analyzed genomes of three members of this clade in order to fill this gap. Their comparative analyses revealed only minute differences from Leishmaniamajor genome in terms of metabolic capacities. We also documented that the number of genes under positive selection on the Endotrypanum/Porcisia branch is rather small, with the flagellum-related group of genes being over-represented. Most significantly, the analysis of gene family evolution revealed a substantially reduced repertoire of surface proteins, such as amastins and biopterin transporters BT1 in the Endotrypanum/Porcisia species when compared to amastigote-dwelling Leishmania. This reduction was especially pronounced for δ-amastins, a subfamily of cell surface proteins crucial in the propagation of Leishmania amastigotes inside vertebrate macrophages and, apparently, dispensable for Endotrypanum/Porcisia, which do not infect such cells.

Design, Synthesis and In Vitro Investigation of Novel Basic Celastrol Carboxamides as Bio-Inspired Leishmanicidal Agents Endowed with Inhibitory Activity against Leishmania Hsp90

The natural triterpene celastrol (CE) is here used as lead compound for the design and synthesis of a panel of eleven CE carboxamides that were tested in vitro for their growth inhibitory activity against Leishmania infantum and L.tropica parasites. Among them, in vitro screening identified four basic CE carboxamides endowed with nanomolar leishmanicidal activity, against both the promastigotes and the intramacrophage Leishmania amastigotes forms. These compounds also showed low toxicity toward two human (HMEC-1 and THP-1) and one murine (BMDM) cell lines. Interestingly, the most selective CE analogue (compound 3) was also endowed with the ability to inhibit the ATPase activity of the Leishmania protein chaperone Hsp90 as demonstrated by the in vitro assay conducted on a purified, full-length recombinant protein. Preliminary investigations by comparing it with the naturally occurring Hsp90 active site inhibitor Geldanamycin (GA) in two different in vitro experiments were performed. These promising results set the basis for a future biochemical investigation of the mode of interaction of celastrol and CE-inspired compounds with Leishmania Hsp90.

Visceral Dissemination of Mucocutaneous Leishmaniasis in a Kidney Transplant Recipient

Intracellular protozoan of the genus Leishmania, endemic in the Mediterranean basin, are the cause of cutaneous (CL), mucocutaneous (MCL), and visceral leishmaniasis (VL). A 75-year-old woman was admitted nine years after a second kidney transplant (KT), due to persistent pancytopenia and fever. She presented edema and erythema of the nose in the last two years and an exophytic nodular lesion located on the left arm, with areas of peripheral necrosis and central ulceration in the last 18 months. A bone marrow biopsy revealed features compatible with Leishmania amastigotes, and polymerase chain reaction test (PCR) for Leishmania infantum was positive. Moreover, biopsy and PCR for L. infantum of the cutaneous lesion on the patient’s left arm and nose and PCR from peripheral blood were positive. Thus, a diagnosis of CL, MCL, and VL was made, and liposomal amphotericin B was initiated, but the patient had an unfavorable outcome and died. This is the first report of a KT recipient presenting with the entire spectrum of leishmaniasis. In Portugal, this infection is rare—so a high degree of clinical suspicion is required for its diagnosis, especially in endemic regions, as visceral leishmaniasis is a potentially life-threatening infection.

The Hematological and Biochemical Manifestations of Cutane-ous Leishmaniasis in a Shih Tzu-Terrier Dog with Severe Infection: A Case Report

Cutaneous involvement in canine leishmaniasis, caused by Leishmania infantum, is the most frequent clinical manifestation of the zoonotic infectious disease. A 4-month-old female Shih Tzu-terrier dog with significant weight loss and depression and chronic erosive skin lesions around eyes and the area above the nose was presented to the Semnan University Veterinary Hospital teaching, Semnan, Iran. The main clinicopathological findings included marked leukocytosis, neutrophilia, left shift, monocytosis, mild hypoproteinemia, and hypoalbuminemia. The diagnosis of leishmaniasis was performed based on the presence of a large number of Leishmania amastigotes in skin Fine Needle Aspiration (FNA). The dog was euthanized and sent to the autopsy department for further investigation.

Microscopic Image Segmentation to Quantification of Leishmania Infection in Macrophages

The determination of infection rate parameter from in vitro macrophages infected by Leishmania amastigotes is fundamental in the study of vaccine candidates and new drugs for the treatment of leishmaniasis. The conventional method that consists in the amastigotes count inside macrophages, normally is done by a trained microscope technician, which is liable to misinterpretation and sampling. The objective of this work is to develop a method for the segmentation of images to enable the automatic calculation of the infection rate by amastigotes. Segmentation is based on mathematical morphology in the context of a computer vision system. The results obtained by computer vision system presents a 95% accuracy in comparison to the conventional method. Therefore, the proposed method can contribute to the speed and accuracy of analysis of infection rate, minimizing errors from the traditional methods, especially in situations where exhaustive repetitions of the procedure are required from the technician.

Comparative Study of Promastigote- and Amastigote-Initiated Infection of Leishmania infantum (Kinetoplastida: Trypanosomatidae) in Phlebotomus perniciosus (Diptera: Psychodidae) Conducted in Different Biosafety Level Laboratories

Sand flies (Diptera: Psychodidae) are natural vectors of Leishmania. For the initiation of sand fly experimental infections either Leishmania amastigotes or promastigotes can be used. In order to obtain comparable results, it is necessary to adjust and standardize procedures. During this study, we conducted promastigote- and amastigote-initiated infections of Leishmania infantum Nicolle, 1908 parasites in Phlebotomus (Larroussius) perniciosus Newstead, 1911 in two laboratories with different levels of biosafety protection. Protocol originally designed for a biosafety level 2 facility was modified for biosafety level 3 facility and infection parameters were compared. Particularly, specially designed plastic containers were used for blood feeding; feeders were placed outside the sand fly cage, on the top of the mesh; feeding was performed inside the climatic chamber; separation of engorged females was done in Petri dishes kept on ice; engorged females were kept in the cardboard containers until dissection. All experiments, conducted in both laboratories, resulted in fully developed late stage infections with high number of parasites and colonization of the stomodeal valve. We demonstrated that protocol originally designed for biosafety level 2 facilities can be successfully modified for other biosafety facilities, depending on the special requirements of the individual institution/laboratory.

Circulating and bone marrow myeloid cells containingLeishmaniaamastigotes in a case of advanced canine leishmaniosis

A 5-y-old male Poodle mix was presented with intermittent vomiting, anorexia, and weight loss. Physical examination revealed emaciation, lethargy, dehydration, hypothermia, respiratory distress, and splenomegaly. Based on clinicopathologic, serologic, and parasitologic findings, diagnoses of severe leishmaniosis and dirofilariasis were made. Extracellular, intraneutrophilic, and intramonocytic Leishmania amastigotes were observed on blood smear and buffy coat smear examination. In blood smears, 0.2% of neutrophils were observed to be infected; in buffy coat smears, 0.5% of neutrophils and 0.1% of monocytes were found to be infected. Leishmania amastigotes were also found engulfed by eosinophils and neutrophil precursors in bone marrow aspiration cytology. The detection of Leishmania amastigotes in blood smears is rare, and the clinical significance is uncertain. In circulating blood, Leishmania amastigotes are primarily found phagocytized by neutrophils. Although debatable, there is growing evidence that neutrophils are used as carriers enabling the “silent entry” of the protozoa into macrophages (“Trojan horse” theory). To date, cytologic screening of blood smears for the diagnosis of canine leishmaniosis is not a routine practice. Clinical pathologists and practitioners should be aware that Leishmania amastigotes may be present in neutrophils and less frequently monocytes during blood smear evaluation; neutrophil precursors and eosinophils may also be parasitized in bone marrow specimens.