Infectivity of five different types of macrophages by Leishmania infantum

Leishmania are intracellular parasites that multiply as the amastigote form in the macrophages of their vertebrate hosts. Since vaccines against leishmaniases are still under development, the control of these diseases relies on prompt diagnosis and chemotherapy in infected humans as well as in dogs, which are the main reservoir of Leishmania infantum, in Mediterranean countries. To establish the macrophage type to be used as an in vitro model for antileishmanial chemotherapeutic studies, we analysed the susceptibility of human peripheral blood derived macrophages, macrophages derived from mouse bone marrow, mouse peritoneal macrophages and macrophages differentiated from cell lines U-937 and DH82 to infection by two L. infantum strains, one obtained from a human leishmanial infection and other from a canine infection. Both strains displayed comparable behaviour in their capacity of infecting the different macrophage types. Human peripheral blood macrophages and DH82 cells were less infectable by both strains. U-937, mouse peritoneal macrophages and mouse bone marrow derived macrophages are the most active cells to phagocytose the parasites. However, U-937 cell line appears to be the most useful as Leishmania infection model providing an unlimited source of homogeneous host cells with reproducibility of the results, is less time consuming, less expensive and tolerate high doses of first line drugs for human and canine visceral leishmaniasis treatment.     

Short report: Treatment failure in Hansen's disease

Areas of low endemicity of Hansen's disease, such as Texas, California, and Hawaii, exist due to immigration and rare autochthonous infections. Managing this disease in these areas of low endemicity is difficult, especially in observing for relapse. The accurate diagnosis of relapse is imperative so that appropriate therapy can be promptly reinstituted and unnecessary treatment can be avoided. To assess treatment failures in an area of low endemicity, we retrospectively evaluated 113 patients with Hansen's disease treated in southern Texas. Of 57 patients who completed therapy, 11 were later restarted on medications for this disease for presumed relapse. However, nine of the 11 were found not to have true relapses of Hansen's disease. The accurate diagnosis of relapse of this disease is essential not only in the individual patient but also for prospective treatment trials to establish best practices.     

Localized mucosal leishmaniasis due to Leishmania (Leishmania) infantum: clinical and microbiologic findings in 31 patients

The clinical and microbiologic characteristics of 31 patients with mucosal leishmaniasis due to Leishmania (Leishmania) infantum are described. Twenty-eight (90%) patients were male. Mean age at presentation was 48 +/- 14 years. Thirteen (42%) patients had no underlying disease, while 18 (58%) patients had several other medical conditions. Fifteen (48%) patients were immunocompromised, 7 patients were infected with human immunodeficiency virus (HIV), and 3 were graft recipients. The primary location of lesions was the larynx in 11 (35%) patients, oral mucosa in 10 (32%) patients, and the nose in 5 (16%) patients. Mucosal lesions were painless in all patients but 2 and consisted of whitish, red, or violaceous nodular swelling or tumorlike masses. Ulceration was reported in 6 patients. Pathologically, the lesions showed a chronic inflammatory infiltrate. Granuloma may be seen.The localization of the lesions determined the symptomatology of the disease. Symptoms included hoarseness, difficulty swallowing, and nasal obstruction. The disease presentation was usually protracted, with a mean time from the onset of symptoms to diagnosis of 13 months (range, 3 wk-4.5 yr), and the clinical diagnosis was usually mistaken for neoplasia of the upper aerodigestive tract. No laboratory abnormalities were found in these patients due to the localized disease, apart from those attributed to underlying diseases.Parasites were easily identified in smears or sections by Giemsa stain or hematoxylin-eosin stain. Leishmania was grown in culture in 12 (60%) patients; culture was negative in 8 (40%) patients. Leishmania (Leishmania) infantum was identified in only 9 instances. The following zymodemes were reported: MON-1 (2 patients), MON-24 (2 patients), MON-27 (1 patient), and MON-34 (1 patient). Serologic test results were known in 25 patients. Serology was usually positive at low titer; 6 (24%) patients had negative serologic test results.Twenty patients were treated with antimonial compounds for between 3 and 36 days. Three patients were given drugs other than antimonial drugs. Five patients were treated only locally, by surgery (3 patients) or topical medical therapy. One patient received no therapy, and treatment was not reported in 2 cases. Patients were cured in 25 (89%) cases, and sequelae were uncommon (14%). Relapse was detected in 2 individuals and 1 patient developed visceral leishmaniasis after treatment. Two HIV-coinfected patients died of causes unrelated to leishmaniasis.The results of the present report stress the clinical importance of searching for the presence of Leishmania in patients with suspected neoplasia of the upper respiratory tract if they have visited or resided in zones endemic for Leishmania (Leishmania) infantum. The treatment of choice for these patients is not established yet, but most patients respond to antimonial compounds given for 28 days or less.     

Short report: fluorescent Leishmania: application to anti-leishmanial drug testing

Classic techniques for detecting the susceptibility of Leishmania to different drugs are time-consuming, laborious, and require the use of macrophages. The use of flow cytometry for monitoring Leishmania susceptibility to drugs is beginning to be implemented. Using green fluorescent protein (GFP), we have improved and simplified the screening procedure. We introduced a GFP marker into field strains of Leishmania causing kala-azar (visceral leishmaniasis) and explored the suitability of transgenic L. donovani promastigotes that constitutively express GFP in their cytoplasm as target cells for in vitro screening of anti-leishmanial drugs.     

Experimental canine leishmaniasis: evolution of infection following re-challenge with Leishmania infantum

The aim of the study was to assess the clinical, parasitological and immunological effect of a second inoculation of amastigotes in dogs previously inoculated with Leishmania infantum. Three dogs primarily inoculated with amastigotes (Group I) and four with cultured virulent stationary phase promastigotes (Group II) were afterwards re-inoculated with 2x10(9) amastigotes per kg. Three other groups of dogs were used as controls: Group III was infected only once with amastigotes, Group IV only once with promastigotes and Group V was non infected. The animals were followed up by clinical and parasitological examinations, hematological and serum protein analysis, anti-leishmanial antibody levels and proliferative assays of specific peripheral blood mononuclear cells over a period up to 50 months. Parasites were isolated from lymph node of three animals during primary amastigote infection and in five animals (Group I and II) after re-challenge. Group I dogs presented a strong increase of the humoral immune response while Group II animals displayed no significant or significantly low antileishmanial antibodies titres, after re-challenge. The detection, only after challenge, of positive specific lymphoproliferation in two animals of Group II that had the longest primary infection interval (more than 26 months), indicates the requirement of a long time interval to obtain specific lymphocyte sensitization. A previous exposure to virulent cultured L. infantum promastigotes seems to confer some degree of resistance against an amastigote infection.     

Leishmania infantum chagasi in northeastern Brazil: asymptomatic infection at the urban perimeter

Visceral leishmaniasis (VL) is endemic in large cities in Brazil, including Natal. We determined the prevalence of asymptomatic human infection with Leishmania infantum chagasi and associated environmental risks around Natal. Infection was detected by Leishmania skin test (LST) and anti-leishmanial antibodies in humans and anti-leishmanial antibodies in dogs. Amongst 345 humans, 24.6% were seropositive, and 38.6% were LST-positive. Prevalence of positive serology was similar in both sexes and across all ages. However, positive LST responses increased with age, suggesting that LST is long-lasting and cumulative. Multinomial logistic analysis showed that LST response varied with location (P = 0.007) and that males were more frequently LST-positive (P = 0.027). Indicators of lower socioeconomic status associated significantly with human infection. Furthermore, there was geographic coincidence of seropositive humans and dogs (r = 0.7926, P = 0.011). These data suggest that dog and human L. i. chagasi infection are intimately interrelated in environmental conditions associated with low income.     

Leukemoid reaction due to mixed malaria infection; report of a case

A case of mixed malarial infection with a leukemoid blood picture is reported.The literature is reviewed.

     

Leishmaniasis in Portugal: enzyme polymorphism of Leishmania infantum based on the identification of 213 strains

This study reports isoenzyme polymorphism of Leishmania strains isolated in different regions of Portugal between 1982 and 2005. A total of 213 strains were obtained from cases of visceral and cutaneous leishmaniasis isolated from immunocompetent patients (adults and children) and immunocompromised adults, as well as from dogs and sandflies. Four zymodemes were identified: MON-1, MON-24, MON-29 and MON-80. Zymodeme MON-1 was identified in 96.7% of the strains, predominating in both immunocompetent and immunocompromised human patients, and it was the only zymodeme isolated from dogs. Isoenzyme diversity in HIV-infected patients was higher than in the immunocompetent group, in which all the strains from visceral leishmaniasis were MON-1. The domestic dog was confirmed as the reservoir host of zoonotic leishmaniasis in Portugal and Phlebotomus perniciosus and Phlebotomus ariasi as vectors. The overall low enzyme polymorphism observed in the Portuguese foci contrasts with the neighbouring foci in Spain.     

Ileal intussusception due to a parasite egg: A case report

Ileal intussusception is the invagination of the small intestine within itself and accounts for 1% of cases of acute obstruction. However, physicians do not initially consider intussusception as a possible diagnosis of obstruction due to its rarity in adults. Herein, we report the case of a 22-year-old male who was admitted to the Emergency Department with continuous abdominal pain. Ultrasonography and computed tomography revealed an ileal intussusception. The patient underwent surgical removal of the segment of the small bowel. Unexpectedly, pathology revealed that the invagination occurred due to a parasite egg, with features suggestive of Schistosoma species. Schistosomiasis, although considered a parasitic disease in tropical countries, is not absent from Europe and though it is highly improbable, it may be responsible for cases of intussusception in adults.     

First report on natural Leishmania infection of Phlebotomus sergenti due Leishmania tropica by high resolution melting curve method in Southeastern Iran

Objective:To identify the Leishmania species in infected sand flies by Real-time PCR coupled with HRM analysis.Methods:Real-time PCR coupled with HRM analysis targeting the first internal transcribed spacer(ITS1)of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish Lelthmania species in sand flies specimens.Results:Three out of 115females of Phlebotomus sergenti(P.sergenti)(2.6%)were positive to Leishmania tropica(L.tropica).Conclusions:This is the first report on P.sergenti as the main and proven vector of anthroponitic cutaneous leishmaniasis in Dehbakri County using Real-time PCR coupled with HUM analysis.This method is rapid,sensitive and specific for diagnosing of parasites in infected Sand flies and ideal for large scale genotyping projects.     

Experimental infection of Phlebotomus perniciosus and determination of the natural infection rates of Leishmania infantum in dogs

Direct xenodiagnosis has been used to determine the infection rates in sand flies of 12 dogs parasitized by Leishmania infantum and classified in function of clinical signs. The dogs were divided into three clinical groups and no statistically significant differences in the infection rates of female sand flies were observed among them. This result indicates the epidemiological importance of asymptomatic and oligosymptomatic dogs. Indirect xenodiagnosis uses sand flies fed on the blood of dogs ex vivo. This technique was not found to be useful as an alternative to the direct method for the detection of dogs parasitized by L. infantum. The clinical worsening of the disease was not reflected in the increasing number of parasites detected in the animals' blood by the indirect technique, thereby rejecting its utility as an indicator of the prognosis of the disease.     

Canine Leishmania infantum enzymatic polymorphism: a review including 1023 strains of the Mediterranean area, with special reference to Algeria

This bibliographic review reports the isoenzyme polymorphism of 1023 Leishmania infantum strains isolated from dogs that have been characterized by multilocus enzyme electrophoresis in the Leishmania Reference Centre of Montpellier, or in other laboratories, to which this typification technique has already been transferred. Between 1981 and 2010, a total of 12 zymodemes were identified around the Mediterranean basin: MON-1, MON-24, MON-34, MON-72, MON-77, MON-80, MON-98, MON-105, MON-108, MON-199, MON-199 var NP1130 and MON-281, of which 6 were present in Algeria. The zymodeme MON-1 was predominant (86.5% of the strains). The dog was confirmed as the main reservoir of L. infantum MON-1, while the reservoir of the other zymodemes has not yet been identified. The enzymatic polymorphism is relatively high in Algeria and in Spain in contrast to other Mediterranean countries. The reasons for this polymorphism are discussed.     

Leishmania donovani: immunization against infection as a function of parasite growth phase

C57BL/6 mice were immunized against Leishmania donovani infection with a subcutaneous vaccination protocol. Groups received 3 injections at 4-day intervals combining glucan and killed promastigotes harvested from either logarithmic or stationary phase cultures. Controls were immunized with glucan alone, stationary or log phase promastigotes alone, or were untreated. All groups were challenged intravenously with stationary phase promastigotes at day 45 post-immunization. Results revealed that animals immunized with the glucan-killed parasite vaccine, utilizing promastigotes derived from either log (GPL) or stationary phase cultures (GPS), demonstrated significant resistance against infection as compared to controls or untreated mice. Additionally, the reduction in hepatic amastigote proliferation in mice immunized with GPS was significantly greater than in mice immunized with GPL.     

甘肃文县婴儿利什曼原虫无症状感染犬的检测

目的评价PCR法、ELISA法和试条法检测我国婴儿利什曼原虫无症状感染犬的潜能。方法用两组PCR引物RV1-RV2和K13A-K13B检测动物源型黑热病疫区健康犬静脉血和骨髓中利什曼原虫特异DNA，以利什曼原虫可溶性抗原为包被抗原的ELISA法和rk39-dipstick试条法分别检测利什曼原虫特异抗体，并比较各种检测方法的敏感性差异。结果PCR法检测抗凝静脉血和骨髓的阳性率分别为50．63％（40／79）和69．62％（55／79），两种样本总检出率为77．21％（61179）；ELISA法检测的阳性率为22．22％（16／72），而rk39-dipstick试条检测的阳性率为33．33％（19／57）。结论我国动物源性黑热病疫区利什曼原虫无症状感染犬的比例相当高，以骨髓为样本的PCR检测法为较精确的犬无症状感染检测方法。     

Canine neutrophils cooperate with macrophages in the early stages of Leishmania infantum in vitro infection

Leishmania infantum is the aetiological agent of human visceral leishmaniasis and canine leishmaniasis, both systemic and potentially fatal diseases. Polymorphonuclear neutrophils (PMN) are the first cells to phagocyte this parasite at the inoculation site, but macrophages (MØ) are the definitive host cells, ensuring parasite replication. The interaction between dog MØ, PMN and L infantum promastigotes was in vitro investigated. It was observed that promastigotes establish contact with blood monocyte‐derived MØ mainly by the tip of the flagellum. These cells, that efficiently bind and internalize parasites, underwent major morphological changes, produced nitric oxide (NO) and released histone H1 in order to inactivate the parasite. Transfer of intracellular parasites from PMN to MØ was confirmed by flow cytometry, using L infantum expressing a green fluorescent protein. The interaction of MØ with L infantum‐infected PMN lead to NO production and release of extracellular traps, which may contribute to parasite containment and inactivation. This study highlights for the first time the diversity of cellular and molecular events triggered by the interaction between canine PMN and MØ, which can promote a reduction of parasite burden in the early phase of L infantum infection.     

Pathogen inactivation technology applied to a blood component collected from an asymptomatic carrier of Leishmania infantum: a case report

Asymptomatic Leishmania infections have been the main cause of transfusion transmission in endemic areas. Polymerase chain reaction has been used to detect L. infantum DNA in the peripheral blood of asymptomatic Leishmania carriers. In our region, the prevalence of asymptomatic L. infantum infection in donors is markedly high (5·9% of donors studied). We investigated the ability of pathogen inactivation technology, using amotosalen and UVA illumination, to eliminate L. infantum in a blood component collected from an asymptomatic L. infantum infected donor. This is the first report of the INTERCEPT system being used to eliminate a parasite from a component collected from a donor.     

6例利什曼原虫感染所致噬血细胞综合征的临床特点

目的分析6例表现为噬血细胞综合征的内脏利什曼病患者临床特点,为提高临床医师的诊治水平提供依据。方法收集我院于2017年1月—2018年11月收治确诊的6例表现为噬血细胞综合征的内脏利什曼病患者的临床资料,并进行分析。结果 6例患者均有明确的流行病学史,临床表现主要为发热、三系下降及肝脾肿大;免疫球蛋白、甘油三酯、血清铁蛋白、可溶性CD25表达水平的各项平均值均明显升高,血清白蛋白及NK细胞活性均明显下降;细胞因子IL-1β、IL-6、IL-10、IL-18、IP-10、TNF-α以及IFN-γ含量明显升高,但IL-12p70未见明显变化;利什曼原虫抗体均为阳性,骨髓细胞学涂片或培养均可查见利什曼原虫。结论利什曼原虫感染所致的噬血细胞综合征表现复杂,采集流行病学史,明确血液细胞学、骨髓细胞学、免疫学及病原学检查对于明确诊断具有重要意义。     

Experimental transmission of Leishmania infantum by the bite of Phlebotomus perniciosus from Switzerland

The possible role of the sandfly Phlebotomus perniciosus in the transmission of Leishmania infantum in southern Switzerland was investigated. It was found that the Syrian golden hamster (Mesocricetus auratus) could be infected by the bite of experimentally infected P. perniciosus females from canton Ticino, in southern Switzerland. This showed that this sandfly species could indeed be a vector for L. infantum in this area. However, as the population density of P. perniciosus is relatively low in canton Ticino, it is extremely unlikely that leishmaniasis could become endemic there.