Mucosal relapse of visceral leishmaniasis in a child treated with anti-TNFα.

Visceral leishmaniasis is an enzootic parasitosis present across the Mediterranean Basin. Some consider it an opportunistic parasite. We report the case of a girl treated with anti-tumour necrosis factor alpha (anti-TNFα) for juvenile idiopathic arthritis who had previously presented with visceral leishmaniasis. Two and a half years later, she presented a tumour-like mass in the nasal mucous membrane caused by Leishmania parasites. Leishmania infantum is classically responsible for visceral leishmaniasis, but pure mucocutaneous leishmaniasis has also been described. To our knowledge, this is the first observation of a recurrence of visceral leishmaniasis in the mucocutaneous form. The occurrence of atypical forms and presentations in those on anti-TNF therapy should be considered.

Canine visceral leishmaniasis caused by Leishmania infantum in Senegal: risk of emergence in humans?

In the context of global warming and the risk of spreading arthropod-borne diseases, the emergence and reemergence of leishmaniasis should not be neglected. In Senegal, over the past few years, cases of canine leishmaniasis have been observed. We aim to improve the understanding of the transmission cycle of this zoonosis, to determine the responsible species and to evaluate the risk for human health. An epidemiological and serological study on canine and human populations in the community of Mont Rolland (Thiès area) was conducted. The data showed a high seroprevalence of canine leishmaniasis (>40%) and more than 30% seropositive people. The dogs' seroprevalence was confirmed by PCR data (concordance > 0.85, Kappa > 0.7). The statistical analysis showed strong statistical associations between the health status of dogs and seropositivity, the number of positive PCRs, clinical signs and the number of Leishmania isolates. For the first time, the discriminative PCRs performed on canine Leishmania strains clearly evidenced that the pathogenic agent is Leishmania infantum. The results obtained show that transmission of this species is well established in this area. That the high incidence of seropositivity in humans may be a consequence of infection with this species is discussed.

Frequency of drug resistance gene amplification in clinical leishmania strains.

Experimental studies about Leishmania resistance to metal and antifolates have pointed out that gene amplification is one of the main mechanisms of drug detoxification. Amplified genes code for adenosine triphosphate-dependent transporters (multidrug resistance and P-glycoproteins P), enzymes involved in trypanothione pathway, particularly gamma glutamyl cysteine synthase, and others involved in folates metabolism, such as dihydrofolate reductase and pterine reductase. The aim of this study was to detect and quantify the amplification of these genes in clinical strains of visceral leishmaniasis agents: Leishmania infantum, L. donovani, and L. archibaldi. Relative quantification experiments by means of real-time polymerase chain reaction showed that multidrug resistance gene amplification is the more frequent event. For P-glycoproteins P and dihydrofolate reductase genes, level of amplification was comparable to the level observed after in vitro selection of resistant clones. Gene amplification is therefore a common phenomenon in wild strains concurring to Leishmania genomic plasticity. This finding, which corroborates results of experimental studies, supports a better understanding of metal resistance selection and spreading in endemic areas.

A battery of 12 microsatellite markers for genetic analysis of the Leishmania (Viannia) guyanensis complex.

We used 12 microsatellite markers developed for Leishmania braziliensis to genotype 28 strains of the main species of the Leishmania guyanensis complex (i.e. L. guyanensis and L. panamensis) collected in Ecuador and Peru. The important heterozygote deficits observed in these populations are similar with the previous data obtained in L. braziliensis and raise again the debate on the reproductive mode of these protozoan parasites. The data showed genetic polymorphism and geographical differentiation giving information on population structure of the L. guyanensis complex. Regarding the two species, this study enhances again the debate on the taxonomic status of the different isolates belonging to L. guyanensis s.l. since the results showed substantial heterogeneity within this species complex. In conclusion, this study increases the number of available microsatellite loci for L. guyanensis species complex and raises fundamental biological questions. It confirms that microsatellite markers constitute good tools for population genetic studies on parasites of this complex.

Localized Cutaneous Leishmaniasis due to Leishmania infantum in a Patient Treated with Infliximab.

We report the first case of cutaneous leishmaniasis in a patient treated with infliximab. The species was Leishmania infantum, agent of both cutaneous leishmaniasis and visceral leishmaniasis. Cutaneous leishmaniasis occurred after the 9th infusion of infliximab in a patient who was suffering from ankylosing spondylitis.

[Pseudotumoral-like recurrence of visceral leishmaniasis in a seven-year-old girl]. / Récidive sous forme pseudotumorale d'une leishmaniose viscérale.

Visceral leishmaniasis is endemic around Mediterranean and is considered by certain authors as an opportunist disease. We report on the case of a 7-year-old girl treated by anti-TNF for an idiopathic juvenile arthritis which has presented a visceral leishmaniasis. Four years later, she presented a pseudotumoral-like recurrence located in a nasal mucous membrane. Leishmania infantum is classically responsible for visceral leishmaniasis but pure mucocutaneous leishmaniasis has been described. It is, for our knowledge, the first observation of a recurrence of visceral leishmaniasis in a mucocutaneous location. Atypical leishmaniasis in the endemic zones can appear in immunodepressed patients and must be evoked when in doubt.

Visceral leishmaniasis infection in a rheumatoid arthritis patient treated with infliximab.

Anti-TNFalpha strategies can result in significant clinical benefits in rheumatoid arthritis (RA), but with an increased rate of opportunistic infections. Visceral leishmaniasis (VL) is a severe disease that can develop in immunocompromised hosts, principally in HIV patients. VL in RA patients treated with TNFalpha antagonists is an extremely rare event, and only one case has been described. Here we report a case of VL, occurring after 9 infusions of infliximab in association with azathioprine, in a patient who developed blood cytopenia, fluctuant fever, and splenomegaly.

Enzymatic polymorphism during Leishmania/HIV co-infection: a study of 381 Leishmania strains received between 1986 and 2000 at the international cryobank in Montpellier, France.

Between 1986 and 2000, 381 Leishmania strains isolated from 288 HIV-positive patients were studied at the international cryobank in Montpellier, France. Most (95.1%) of the strains came from cases of visceral leishmaniasis but 4.9% were from HIV-positives with cutaneous leishmaniasis. The majority of the strains came from patients infected in the Mediterranean region, with a few originating in sub-Saharan Africa and South America. Isoenzymatic characterization revealed 28 zymodemes in four different species: L. infantum (which was predominant), L. donovani, L. major and L. guyanensis. The strains belonging to the L. infantum complex included 20 zymodemes, some of which have so far only been found in cases of Leishmania/HIV co-infection.

Visceral leishmaniasis. Persistence of parasites in lymph nodes after clinical cure.

Visceral leishmaniasis (VL) is generally associated with severe immunodeficiency (AIDS; renal, liver, and heart transplantations; haemopoietic malignancies). More rarely it can be related to an immunotolerence status such as pregnancy. Various observations report the development of leishmaniasis several months or even years after exposure to the parasite. Relapses occur rarely in patients not known to be immunocompromised, but are common after incomplete treatment. They are frequent in patients with Leishmania/HIV co-infection. Asymptomatic phases and relapses suggest that parasite can exist in the tissues for a long time before and/or after clinical onset of the disease. The mechanisms of onset of clinical leishmaniasis following exposure and infestation are highly relevant to understanding the pathology of the disease. The survival of Leishmania parasite between infection and disease or after cure is a very important issue for clinicians and epidemiologists. We describe two cases of VL occurring in a patient with lymphoma and in a pregnant woman. In both cases, parasites remained present in the lymph nodes after clinical cure.

The sandfly fauna in the visceral-leishmaniasis focus of Gedaref, in the Atbara-River area of eastern Sudan.

Visceral leishmaniasis (VL) is an acute public-health problem in Sudan. Between 1997 and 2000, four, brief entomological surveys were carried out in Barbar El Fugarra, a village in the state of Gedaref, in the Atbara-River area of eastern Sudan. Between 1996 and 1999, 658 cases of VL occurred among the village's population of about 4000. CDC miniature light-traps set inside and outside human dwellings were used to collect a total of 12,745 sandflies, including five species of the genus Phlebotomus and 19 of Sergentomyia. Phlebotomus papatasi and P. orientalis made up 7% and 5% of the collected sandflies, respectively. Seasonal variation was observed in the numbers of P. orientalis, P. papatasi, S. schwetzi and S. magna caught. Almost all (88%) of the sandflies collected were caught inside houses or granaries and there appeared to be particularly large indoor populations of P. orientalis, P. papatasi, S. schwetzi, S. magna and S. clydei. Phlebotomus orientalis could be responsible for the indoor transmission of the parasites causing the local VL, between humans and between humans and local dogs (which have been found infected by some of the Leishmania zymodemes found in humans). The co-occurrence in this focus of P. papatasi and Arvicanthis niloticus, which are known vectors and reservoir hosts, respectively, of L. major, indicates the possibility that outbreaks of human cutaneous leishmaniasis might occur in the area.

Value of two PCR methods for the diagnosis of canine visceral leishmaniasis and the detection of asymptomatic carriers.

The value of 2 PCR methods, targeting genomic and kinetoplast minicircle DNA respectively, was investigated for both diagnosis and prevalence studies of canine visceral leishmaniasis (CVL). The first method (R) was 5000-fold less sensitive than the second (method KRV). Both were tested for diagnosis of CVL in 44 sick dogs with confirmed disease using different biological samples. Method R was highly efficient when using invasive samples, but the use of method KRV proved necessary for a 100% sensitive diagnosis using peripheral blood. This method was applied to peripheral blood and skin samples in 263 dogs during a mass survey in the Cévennes focus. PCR was compared to serology and all results were analysed according to clinical status. The 'CVL-infection' prevalence was found to be 79.8% by PCR compared with 29.6% by serology: 89.4% of symptomatic and 65.2% of asymptomatic dogs harboured parasites in peripheral blood. This study confirms the high prevalence of asymptomatic carriers of Leishmania. In total, for the diagnosis of CVL in sick dogs, method R is recommended in view of its 100% positive predictive value (compared with 30% for method KRV). A strategy best adapted for prevalence surveys might combine serology and highly sensitive PCR on peripheral blood.

Sudan: the possible original focus of visceral leishmaniasis.

Fifty-two Leishmania strains, obtained from human patients and dogs in a visceral leishmaniasis focus in Sudan, were characterized by isoenzyme electrophoresis (15 enzymes). The phylogenetic analysis showed that the 7 Leishmania zymodemes obtained hold ancestral positions on the phylogenetic tree, supporting the hypothesis of an East African origin of visceral leishmaniasis.

Trypanosomatidae from wild mammals in the neotropical rainforest of French Guiana.

The initial filling of the reservoir behind the Petit Saut hydro-electric dam, on the Sinnamary River in French Guiana, threatened the terrestrial and arboreal animals living in the neotropical rainforest being flooded. During a rescue programme between 24 October and 12 November in 1994, many of these animals were checked for infection with trypanosomatids. Overall, 45 blood samples and 54 skin biopsies were collected from 53 mammals (of 13 species representing five orders) and blood samples were also taken from each of nine reptiles (six species from four families). When the skin biopsies and the buffy-coats from the blood samples were cultured in NNN medium, 10 of the cultures, each initiated with mammalian blood, were found to be positive for trypanosomatids. Multilocus enzyme electrophoresis (MLEE) on cellulose acetate plates, with 20 enzyme systems, was then used to investigate each of the positive cultures. The results were analysed by clustering from a genetic distance matrix, using the unweighted pair group method with arithmetic averages (UPGMA), and applying a bootstrap procedure to Wagner parsimony trees. A stock obtained from Didelphis marsupialis was identified as a zymodeme of Trypanosoma cruzi (Miles' zymodeme 1) known to cause Chagas disease in French Guiana. Five stocks (one each from Bradypus tridactylus, Tamandua tetradactyla and Alouatta seniculus and two from Saguinus midas) were of a single zymodeme close to Trypanosoma rangeli reference stock RGB. This is the first confirmation of the presence of Tr. rangeli in French Guiana, and the first time that it has been identified, by iso-enzyme analysis, in the neotropical primates A. seniculus and S. midas. Two other stocks, isolated from Choloepus didactylus, were related to Endotrypanum schaudinni reference stock LEM 2790. Although the remaining stocks, one from C. didactylus and the other from A. seniculus, clustered together on UPGMA and in a Wagner tree, they did not appear to be related to any of the reference stocks included in the UPGMA dendrogram.

[Visceral leishmaniasis in Cameroon. Seroepidemiologic survey in the Kousseri region, north Cameroon]. / Leishmaniose viscérale au Cameroun. Enquête séro-épidémiologique dans la région de Kousseri, Nord-Cameroun.

A sero-epidemiological survey of school children was carried out in Kousseri, a focus for visceral leishmaniasis. Sero-immunological assays for the detection of anti-Leishmania antibodies were based on the indirect immunofluorescence assay test and counter-immunoelectrophoresis. 9 out of 223 school children tested positive for visceral leishmaniasis (seroprevalence rate of 4%). These 9 cases had no history of the disease. The data obtained confirm the endemicity of visceral leishmaniasis in this focus and call for extensive studies in order to determine the prevalence of the disease in the entire population as well as the main components of the transmission cycle.

Lutzomyia whitmani (Diptera: Psychodidae) as vector of Leishmania (V.) braziliensis in Paraná state, southern Brazil.

The phlebotomine sandflies in the northern areas of the state of Paraná, Brazil, particularly those in the '16a' health region, were investigated over a 3-year period. Using CDC light traps (with and without hamster bait) and Shannon traps (with lights and horse or human bait), 16 species were collected from seven municipal districts which were known foci for cutaneous leishmaniasis: Arapongas; Apucarana; Cambira; Marumbi; Faxinal; Florestópolis; and Sabáudia. Although the frequency at which each species was collected varied with the collection site, Lutzomyia whitmani predominated (62.0% of all the sandflies collected), followed by Lu. fischeri (13.3%), Lu. pessoai (10.8%), Lu. migonei (8.2%) and Lu. intermedia (2.8%). Lutzomyia monticola, Lu. shanonni, Lu. firmatoi, Lu. lanei, Lu. alphabetica, Lu. misionensis, Lu. correalimai, Lu. cortellezzii, Lu. longipenis, Brumptomyia brumpti and B. nitzulescui together represented the remaining 3.0% of the collected sandflies. Three of the 1961 female sandflies collected and dissected in the municipal district of Cambira, where a recent case of cutaneous leishmaniasis had been registered, were found to have flagellates in their guts. All three were Lu. whitmani. The parasites from each of these infections were successfully isolated in NNN and 'Tobie and Evans' media and/or by inoculation into a hind foot of a golden hamster. The results of isoenzyme electrophoresis indicated that all three isolates were of Leishmania (Viannia) braziliensis.

Optimized PCR using patient blood samples for diagnosis and follow-up of visceral Leishmaniasis, with special reference to AIDS patients.

We developed a highly sensitive PCR method that enables the diagnosis and posttherapeutic follow-up of visceral leishmaniasis with patient blood. The PCR assay was thoroughly optimized by successive procedural refinements to increase its sensitivity and specificity. It was compared to in vitro cultivation as well as to direct examination of bone marrow and to serology. Two hundred thirty-seven patients presenting with clinical signs compatible with visceral leishmaniasis were included in the study. Thirty-six were diagnosed as having Mediterranean visceral leishmaniasis (MVL). Twenty-three of them, including 19 AIDS patients, were monitored during and after treatment over a period from 2 weeks to 3 years. Our PCR assay proved more sensitive than in vitro cultivation, direct examination, and serology for all patients. It is simple and can be adapted to routine hospital diagnostic procedures. For the primary diagnosis of MVL, the sensitivity of PCR versus that of cultivation was 97 versus 55% with peripheral blood and 100 versus 81% with bone marrow samples. Regarding posttherapeutic follow-up, overall, 48% of positive samples were detected by PCR only. Seven patients presented with a clinical relapse during the study; six relapses were detected at first by PCR only, sometimes a few weeks before the reappearance of signs or symptoms. We conclude that an optimized and well-mastered PCR assay with a peripheral blood sample is sufficient to provide a secure diagnosis for all immunocompromised patients and most immunocompetent patients. We also suggest systematic posttherapeutic monitoring by PCR with peripheral blood for immunocompromised patients.

[Canine leishmaniasis from Leishmania infantum: value and production of the latex test. Ecoepidemiologic applications]. / Leishmaniose canine à Leishmania infantum: intérêt et réalisation du test au latex. Applications en éco-épidémiologie.

The authors relate the realization, evaluation and eco-epidemiological applications of a "field-test": the agglutination of latex particles coated with a soluble antigen of Leishmania infantum in the presence of homologous antibodies. Evaluated on 1,035 canine sera, the sensitivity of the latex agglutination test (LAT) was 93.4% compared to the indirect fluorescent antibody test (IFAT). 90 node cultures were carried out on dogs with positive or negative LAT and/or positive or negative IFAT. The frequency of positive node cultures (70%) as versus positive LAT came between the results obtained for an IFAT > 1/40 (64%) and IFAT > 1/80 (73%). 32/33 (97%) dogs had positive node culture, LAT and IFIAT(> 1/80). 6 dogs had negative LAT but positive node culture: 5 of these had also an IFAT < 1/160. This test was used in the field on several eco-epidemiological surveys in leishmanian enzootic areas. Node cultures were made on the dogs with positive TL. 39 strains were isolated: 18 in Algeria, 15 in Morocco, 2 in Syria and 4 in Yemen. 13/39 strains were obtained from dogs with IFI < 1/160: 2 at 1/20, 8 at 1/40 and 3 at 1/80. In Algeria this test was also used for the diagnosis of human visceral leishmaniasis in a child. This quick, simple, sensitive and specific test could be usefully carried out on "field" surveys for the diagnosis of visceral-leishmaniasis in animals and human beings.