Epidemiology of visceral leishmaniasis in Atbara River area,eastern Sudan: the outbreak of Barbar El Fugara village (1996-1997)

An outbreak of visceral leishmaniasis (VL) started in 1995 in the Atbara River area in eastern Sudan. This article reports on this outbreak and on the clinical and immunological studies that were carried out in a village, with the highest incidence of VL cases, from 1996 to 1997. A significant increase in VL incidence was recorded in a dozen villages in this area; one village, Barbar El Fugara accounted for half of the total number of cases recorded at the regional hospital. A total of 152 VL and 61 post kala-azar dermal lesion (PKDL) cases were diagnosed and treated in Barbar. Household (n = 671) and school (n = 276) surveys were performed using the leishmanin skin test (LST) and the direct agglutination test (DAT). LST positivity was 23.1 and 15.7%, whereas DAT positivity was 8.9 and 26.4% in both surveys, respectively. No gender differences were observed in either test. Unlike DAT, LST positivity was predominant in the higher age groups that also exhibited lower prevalence of VL. Few individuals were positive by both tests (1.3%, 5.2%) while the majority (68.8%, 64.8%) had no evidence of acquired immune response, suggesting either a role of innate immunity in preventing parasite establishment or, unexpectedly, lack of exposure to Leishmania. Subclinical parasitism was also demonstrated, as evidence of both acquired humoral and cellular immune responses was observed in individuals with no past history of the disease. The wide spectrum of L. donovani/human interactions may be explained by differential exposure to environmental risk factors, parasite strain polymorphisms or host genetic makeup.     

Susceptibility to visceral leishmaniasis in the domestic dog is associated with MHC class II polymorphism

Zoonotic visceral leishmaniasis (VL) is a disease of dogs, humans and other animals caused by the intracellular macrophage parasite Leishmania infantum. We examined the relationship between DLA class II alleles ( DRB1, DQA1, DQB1) and the course of infection in a cohort of Brazilian mongrel dogs exposed to natural L. infantum infection. DLA alleles were typed by sequence-based typing. DLA-DRB1 genotype was significantly associated with levels of anti- Leishmania IgG and parasite status assessed by PCR. Dogs with DLA-DRB1*01502 had higher levels of specific IgG and an increased risk of being parasite positive compared with dogs without this allele, controlling for other alleles and significant variables. No significant associations were seen for DLA-DQA1 or DLA-DQB1 alleles. These results suggest that the DLA-DRB1 locus plays a role in determining susceptibility to canine VL. As the domestic dog is the main reservoir for human infection, the identification of genetic factors influencing canine resistance or susceptibility to VL may provide insights into the immunology and potential control through vaccination of VL.     

Upregulation of surface proteins in Leishmania donovani isolated from patients of post kala-azar dermal leishmaniasis

Five to fifteen percent of visceral leishmaniasis (VL) patients in India develop post kala-azar dermal leishmaniasis (PKDL), usually 1-2 years after apparent clinical cure. There is evidence pointing to a role played by the host immune responses in the disease pathogenesis, however, the contribution of changes in parasite gene expression has not been explored. Highly sensitive gene expression microarray technology was employed to identify genes that are differentially expressed in Leishmania parasites isolated from PKDL patients in comparison with those from VL. Hybridization on Leishmania donovani genomic microarray comprised of unique clones allowed us to identify 46/2268 (2%) clones that showed statistically significant (P<0.05) changes in expression (1.5-3.5-fold) in parasites of PKDL origin compared to those of VL origin. Sequence analysis of six genomic clones, consistently showing approximately 2-fold higher expression in PKDL parasites, revealed significant homology with gp63, gp46, putative amastin, a putative reductase and a possible calpain-like protein. The gene products showing upregulated expression in PKDL isolates may be candidates playing a role in the altered clinical manifestation in PKDL. Such differentially expressed genes hold the key to understanding the parasite genetic factors that contribute to the persistence after clinical cure of VL.     

Structure-guided approach to identify a novel class of anti-leishmaniasis diaryl sulfide compounds targeting the trypanothione metabolism

Leishmania protozoans are the causative agent of leishmaniasis, a neglected tropical disease consisting of three major clinical forms: visceral leishmaniasis (VL), cutaneous leishmaniasis, and mucocutaneous leishmaniasis. VL is caused by Leishmania donovani in East Africa and the Indian subcontinent and by Leishmania infantum in Europe, North Africa, and Latin America, and causes an estimated 60,000 deaths per year. Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against leishmaniasis. This enzyme is fundamental for parasite survival in the human host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize the hydrogen peroxide produced by host macrophages during infection. Recently, we solved the X-ray structure of TR in complex with the diaryl sulfide compound RDS 777 (6-(sec-butoxy)-2-((3-chlorophenyl)thio)pyrimidin-4-amine), which impairs the parasite defense against the reactive oxygen species by inhibiting TR with high efficiency. The compound binds to the catalytic site and engages in hydrogen bonds the residues more involved in the catalysis, namely Glu466′, Cys57 and Cys52, thereby inhibiting the trypanothione binding. On the basis of the RDS 777–TR complex, we synthesized structurally related diaryl sulfide analogs as TR inhibitors able to compete for trypanothione binding to the enzyme and to kill the promastigote in the micromolar range. One of the most active among these compounds (RDS 562) was able to reduce the trypanothione concentration in cell of about 33% via TR inhibition. RDS 562 inhibits selectively Leishmania TR, while it does not inhibit the human homolog glutathione reductase.     

Heterogeneity among zymodemes of Leishmania infantum from HIV-positive patients with visceral leishmaniasis in south Italy

Abstract Eleven zymodemes of Leishmania infantum were identified among 38 parasite stocks isolated from Italian HIV-positive patients with visceral leishmaniasis (VL). Only one zymodeme is a common agent of Mediterranean VL in HIV-negative individuals, five zymodemes usually cause simple, self-resolving cutaneous leishmaniasis (CL), and five belong to unique genotypes which have not been previously reported from either VL or CL cases in immunocompetent individuals. This last group of parasites showed reassortaient patterns within electromorphs frequently observed in dermotropic L. infantum zymodemes. The highest zymodeme heterogeneity was found in south Italy (Sicily), with six zymodemes identified among 12 HIV-positive patients surveyed.     

A major susceptibility locus on chromosome 22q12 plays a critical role in the control of kala-azar

Kala-azar (KA) is a life-threatening protozoal disease caused by Leishmania parasites (L. donovani, L. chagasi, and L. infantum). The disease, which is also called "visceral leishmaniasis," is prevalent in Africa, South America, Asia, and the Mediterranean basin. Epidemics occur periodically, killing a large number of infected individuals. Factors determining whether a patient remains asymptomatic or develops KA are still largely unknown. In a previous study that was performed during an outbreak of KA in a village on the Ethiopian-Sudanese border, we showed that KA was more frequent in certain families and ethnic groups, thereby suggesting that host genetic factors play an important role in the development of the disease. Here, we report the results of a genomewide linkage study performed on 63 Sudanese families selected from the most affected ethnic group and including 169 children with KA. Significant linkage (LOD score 3.50 [P=3x10-5] in all patients; LOD score 3.90 [P=10-5] in patients who were affected early in the outbreak) was obtained with markers on chromosome 22q12. These results are the first evidence of a major genetic effect on the development of human KA. They may lead to identification of genes critical in the pathogenesis of this disease and to new therapeutic interventions against this parasite, which is developing resistance to available drugs.     

Persistent dermal lesions in a patient with previous history of visceral leishmaniasis

Post-kala-azar dermal leishmaniasis (PKDL) is a complication of visceral leishmaniasis (VL) that most frequently occurs after an episode of VL caused by Leishmania donovani. In this case report, we present a 21-year-old male patient with persistent skin lesions and recurrent visceral leishmaniasis (VL) due to Leishmania infantum. The patient did not respond to multiple lines of anti-leishmanial treatment (including Liposomal amphotericin B and miltefosine) and later died from cerebral lesions presumed to be secondary to persistent VL.     

Comparative genomics: from genotype to disease phenotype in the leishmaniases

Recent progress in sequencing the genomes of several Leishmania species, causative agents of cutaneous, mucocutaneous and visceral leishmaniasis, is revealing unusual features of potential relevance to parasite virulence and pathogenesis in the host. While the genomes of Leishmania major, Leishmania braziliensis and Leishmania infantum are highly similar in content and organisation, species-specific genes and mechanisms distinguish one from another. In particular, the presence of retrotransposons and the components of a putative RNA interference machinery in L. braziliensis suggest the potential for both greater diversity and more tractable experimentation in this Leishmania Viannia species.     

Leishmania chagasi/infantum: further investigations on Leishmania tropisms in atypical cutaneous and visceral leishmaniasis foci in Central America

In Central America, apparently genetically identical Leishmania chagasi/infantum parasites cause cutaneous (CL) and visceral leishmaniasis (VL), the latter being more frequent in young children. The present study investigated if there were pathology-related differences in virulence between Honduran CL and VL strains using Mediterranean L. infantum strains as a reference. Macrophage infectivity and serum sensitivity, properties thought to be associated with virulence, were similar between CL and VL strains from both regions. Attention focused on the genome organisation of genes for two candidate virulence factors: Leishmania mitogen activated protein kinase (LMPK) and cysteine proteinase b (Cpb). Interestingly, the Mediterranean strains exhibited restriction enzyme polymorphisms associated with tropism for both LMPK and Cpb genes whereas no differences were observed for the Honduran strains. We also report relative genetic homogeneity of the Honduran strains as compared to the Mediterranean strains and discuss it in terms of the probable origin for the Central American L. chagasi/infantum.     

A novel mechanism for an old drug: amphotericin B in the treatment of visceral leishmaniasis

Visceral leishmaniasis (VL) is caused by various species of the genus Leishmania. Internalization of Leishmania into host cells is facilitated by a large number of receptors, and therefore no panacea is available for the treatment of leishmaniasis. We previously demonstrated the requirement of host membrane cholesterol in the entry of Leishmania into macrophages by cholesterol depletion using methyl-β-cyclodextrin (MβCD). We recently showed that leishmanial infection is inhibited upon sequestration of host membrane cholesterol using amphotericin B (AmB), considered as the best existing drug against VL. The reason for the antileishmanial activity of AmB is generally believed to be its ability to bind ergosterol in parasite membranes. Our recent results offer the opportunity to reexamine the mechanism behind the effectiveness of current AmB-based therapeutic strategies to treat leishmaniasis. We propose here a novel mechanism in which the effectiveness of AmB treatment could be partly based on its ability to sequester cholesterol in the host membrane, thereby abrogating macrophage-parasite interaction.     

Survey of dogs from Vietnam for antibodies to visceralizing Leishmania spp

Cases of visceral leishmaniasis, one of the most neglected tropical diseases, are increasing globally. Dogs are considered an important reservoir host for visceral leishmaniasis in people. The first cases of human visceral leishmaniasis in Vietnam have recently been reported. Blood samples were collected from 41 dogs in rural Vietnam. Sera were examined for antibodies to visceralizing Leishmania spp. by canine immunochromatographic strip assays based on recombinant K39 antigen. Antibodies to Leishmania spp. were not detected in any of the dogs tested. Results from this study suggest that rural dogs are not likely to be involved in the emergence of human visceral leishmaniasis in Vietnam.     

Heme oxygenase-1 promotes the persistence of Leishmania chagasi infection

Visceral leishmaniasis (VL) remains a major public health problem worldwide. This disease is highly associated with chronic inflammation and a lack of the cellular immune responses against Leishmania. It is important to identify major factors driving the successful establishment of the Leishmania infection to develop better tools for the disease control. Heme oxygenase-1 (HO-1) is a key enzyme triggered by cellular stress, and its role in VL has not been investigated. In this study, we evaluated the role of HO-1 in the infection by Leishmania infantum chagasi, the causative agent of VL cases in Brazil. We found that L. chagasi infection or lipophosphoglycan isolated from promastigotes triggered HO-1 production by murine macrophages. Interestingly, cobalt protoporphyrin IX, an HO-1 inductor, increased the parasite burden in both mouse and human-derived macrophages. Upon L. chagasi infection, macrophages from Hmox1 knockout mice presented significantly lower parasite loads when compared with those from wild-type mice. Furthermore, upregulation of HO-1 by cobalt protoporphyrin IX diminished the production of TNF-α and reactive oxygen species by infected murine macrophages and increased Cu/Zn superoxide dismutase expression in human monocytes. Finally, patients with VL presented higher systemic concentrations of HO-1 than healthy individuals, and this increase of HO-1 was reduced after antileishmanial treatment, suggesting that HO-1 is associated with disease susceptibility. Our data argue that HO-1 has a critical role in the L. chagasi infection and is strongly associated with the inflammatory imbalance during VL. Manipulation of HO-1 pathways during VL could serve as an adjunctive therapeutic approach.     

Imaging host cell-Leishmania interaction dynamics implicates parasite motility, lysosome recruitment, and host cell wounding in the infection process

Leishmania donovani causes human visceral leishmaniasis. The parasite infectious cycle comprises extracellular flagellated promastigotes that proliferate inside the insect vector, and intracellular nonmotile amastigotes that multiply within infected host cells. Using primary macrophages infected with virulent metacyclic promastigotes and high spatiotemporal resolution microscopy, we dissect the dynamics of the early infection process. We find that motile promastigotes enter macrophages in a polarized manner through their flagellar tip and are engulfed into host lysosomal compartments. Persistent intracellular flagellar activity leads to reorientation of the parasite flagellum toward the host cell periphery and results in oscillatory parasite movement. The latter is associated with local lysosomal exocytosis and host cell plasma membrane wounding. These findings implicate lysosome recruitment followed by lysosome exocytosis, consistent with parasite-driven host cell injury, as key cellular events in Leishmania host cell infection. This work highlights the role of promastigote polarity and motility during parasite entry.     

Impact of ASHA Training on Active Case Detection of Visceral Leishmaniasis in Bihar,India

Background

One of the major challenges for management of visceral leishmaniasis (VL) is early diagnosis of cases to improve treatment outcome and reduce transmission. We have therefore investigated active case detection of VL with the help of accredited social health activists (ASHA). ASHAs are women who live in the community and receive performance-based incentives for overseeing maternal and other health-related issues in their village.

Methods and Principal Finding

Through conducting interviews with 400 randomly selected ASHAs from four primary health care centers (PHCs), it was observed that their level of knowledge about visceral leishmaniasis (VL) regarding transmission, diagnosis, and treatment was limited. The baseline data indicated that less than 10% of VL cases seeking treatment at the PHCs were referred by ASHAs. To increase the knowledge and the referral rate of VL cases by ASHAs, training sessions were carried out during the monthly ASHA meetings at their respective PHCs. Following a single training session, the referral rate increased from less than 10% to over 27% and the overall knowledge about VL substantially improved. It was not possible, however, to demonstrate that ASHA training reduced the time that individuals had fever before treatment at the PHC.

Conclusions

Training ASHAs to identify VL cases in villages for early diagnosis and treatment at the local PHC is feasible and should be undertaken routinely to improve knowledge about VL.     

Proteomic analysis of the secretome of <Emphasis Type="Italic">Leishmania donovani</Emphasis>

Background  

Leishmania and other intracellular pathogens have evolved strategies that support invasion and persistence within host target cells. In some cases the underlying mechanisms involve the export of virulence factors into the host cell cytosol. Previous work from our laboratory identified one such candidate leishmania effector, namely elongation factor-1α, to be present in conditioned medium of infectious leishmania as well as within macrophage cytosol after infection. To investigate secretion of potential effectors more broadly, we used quantitative mass spectrometry to analyze the protein content of conditioned medium collected from cultures of stationary-phase promastigotes of Leishmania donovani, an agent of visceral leishmaniasis.     

Interpretation of laboratory data during cryptic leishmaniasis in dog

Leishmaniasis is a zoonosis caused by an intracellular parasite belonging to the genus Leishmania. In Europe, Africa, South America and China, visceral leishmaniasis is caused by L. infantum. The vectors of leishmaniasis are phlebotomine sandflies belonging to the genera Phlebotomus. According to the World Health Organization there are 2 million new cases each year and 1/10 of the world's population is at risk of infection. Leishmaniasis is considered a zoonosis and human are generally accidental hosts. The animal reservoir includes rodents, dog and other mammals. Several studies have indicate that half of the dogs with antileishmanial antibodies have no signs of disease although, animal with subclinical infections are potentially infectious to sand flies. The factors determining susceptibility or resistence to visceral leishmaniasis remain unclear, but the genetics of the host may play a major role. Clinical signs are: intermittent fever, hepatosplenomegaly, skin lesions and ulcers, alopecia, onychogryphosis, anemia, thrombocytopenia and hypergammaglobulinemia. In mice, the outcome of infection depends on the polarized activation of one of two subsets of CD4+ T cells, Th1 or Th2, the subdivision into Th1 and Th2 cells is based on the pattern of cytokines that they produce. Th1 cells produce gamma interferon (IFN-gamma) and interleukin -2 (IL-2), whereas Th2 cells produce IL-4, IL-5, and IL-10. An important difference between susceptible and resistant mice is that the resistant mice are able to switch to a Th1 profile and control the disease. An important factor in the "decision" to form a Th1 or Th2 phenotype is the early cytokine environment, and IL-12 is one of the cytokines that contributes significantly to the establishment of the Th1 phenotype. Canine leishmaniosis is endemic in the Mediterranean basin and, in most cases is caused by the parasite Leishmania infantum. The main clinical findings are skin lesions, local or generalized lymphoadenopathy, loss of body weight, glomerulopathy, ocular lesions, epistaxis and lameness. Non pruritic skin lesions are the usual manifestation and several forms have been described, such as exfoliative dermatitis and alopecia, and ulcerative, nodular and pustular dermatitis. Seroepidemiological studies of canine leishmaniasis have revealed a large number of asymptomatic seropositive animals. Moreover in areas where leishmaniasis is highly endemic, high proportion of apparently healthy animals show low levels of anti-Leishmania antibodies. Others have regressive forms of the desease, and their antibody levels will decrease in the following months or years; still others maintain low levels of antibodies without developing the desease for many years. However, the total number of infected animals is unknown. Canine leishmaniasis is a major zoonosic parasitic disease, enzootic in the Mediterranean area, caused by the intracellular protozoan Leishmania infantum. The dog is the main reservoir host of the parasite. However, most infected dogs do not present any clinical signs, and there is evidence that Leishmania infection prevalence rates in areas of endemicity are higher than those ascertained by serological studies. Visceral leishmaniasis is becoming a real problem of public health because it is an opportunistic infection in immunocompromised patients and in human immunodeficiency virus-positive subjects. The detection of the extent of the infection, particularly among asymptomatic dogs, is of great importance for the control of leishmaniasis. PCR has been applied successfully in recent years to detect Leishmania spp. even in the cases with any of the clinical manifestation of leishmaniasis. Very recently, real-time PCR for Leishmania has been applied to evaluate the parasitic load of dog tissues both at the time of the diagnosis and during follow-up of the therapy and to measure cytokine mRNA levels in different clinical samples of infected and uninfected dogs.     

Emergence of Zoonotic Canine Leishmaniasis in the United States: Isolation and Immunohistochemical Detection of Leishmania infantum from Foxhounds from Virginia.

ABSTRACT. Previously considered an exotic disease, canine leishmaniasis caused by Leishmania infantum has recently been detected within the foxhound population in the United States and parts of Canada. Leishmania infantum is the etiologic agent of visceral leishmaniasis in many areas of the world and dogs are considered a major reservoir host for human Leishmania infections. Human visceral leishmaniasis has recently emerged as an opportunistic infection among individuals co-infected with HIV/AIDS and in persons taking immunosuppressive drugs. We report the isolation of L. infantum from 3 naturally infected foxhounds from Virginia by culture of popliteal lymph node and bone marrow, and the development of an immunohistochemical test to detect the parasite in tissues.     

Visceral leishmaniasis in eastern Sudan: parasite identification in humans and dogs; host-parasite relationships

In 1996, an epidemic outbreak of visceral leishmaniasis (VL) started in Barbar el Fugara, a village in Gedarif State (eastern Sudan). From 1997 to 2000, regular epidemiological studies were carried out in the human population, as well as in mammals and sand flies. In symptomatic patients, 46/69 lymph node, 6/20 post kala-azar dermal leishmaniasis (PKDL) and 1/4 cutaneous cultures in NNN medium were positive. In 69 dogs, 23/79 lymph node cultures were positive. In other mammals (47 rodents, five donkeys, one mongoose and one monkey) spleen and/or blood cultures were negative. Characterization of isolated strains (by starch gel electrophoresis and isoelectrofocusing) identified three zymodemes of Leishmania donovani, two of L. infantum and two of L. archibaldi complexes from patient samples and three zymodemes of L. donovani, three of L. infantum and two of L. archibaldi complexes from dog samples. Five of them were present in both man and dog. For the first time, a strain from a PKDL case was identified as L. infantum, and a child had the same L. infantum zymodeme in VL and in subsequent PKDL. Blood samples from dogs were studied by immunofluorescent antibody test (IFAT). The seroprevalence in dogs was 72.5%, 74.3% and 42.9% in 1998, 1999 and 2000, respectively. By using CDC miniature light traps 12 745 sand flies were collected and then identified. Phlebotomus papatasi (7%) and P. orientalis (5%) were sympatric, mainly inside homes (85% and 75%, respectively). These results, the relative stability of seroprevalence in dogs and the intradomiciliar presence of P. orientalis, known as a vector of VL in Sudan, suggest several hypotheses: (i) man is responsible for the disease in dogs, (ii) the dog is the reservoir of VL, (iii) the dog is an intermediate host between a possible sylvatic cycle and the anthroponotic cycle. More extensive studies are needed to assess the transmission cycle of VL in this area of Sudan.     

Genes and environment in susceptibility to visceral leishmaniasis

Kala azar (KA) is a lethal disease caused by Leishmania parasites (Leishmania donovani s.l.) that multiply in large numbers in deep organs such as spleen and liver. The host immunological response to these organisms is complex and experimental studies in animals have detected a large number of genetic loci involved in the control of infection and disease. We report here on a study in a human population of Sudan carried out during an outbreak of KA. The following conclusions are presented: (1) environmental factors that could have affected the distribution of the insect vector, influenced progression of KA in the initial phase of the epidemics - but they became less important later at the peak of transmission, probably after infected phlebotomies had spread to all parts of the village -; (2) Leishmania population during the epidemics was heterogeneous, suggesting a possible parasite evolution during the outbreak; (3) the incidence of KA varied markedly among age groups, families and ethnic groups. Susceptibility to KA was shown to depend on a locus on chromosomes 22q12 and on NRAMP1 on chromosome 2q35; the data also suggested a third locus in the region 2q23-q24. Overall, this study indicates complex interactions between host genes and environment in the spreading of KA in that population. It is also suspected that the large parasite diversity observed in the outbreak has contributed to disease spreading across host genetic barriers.