Molecular identification of Leishmania tropica and L. infantum isolated from cutaneous human leishmaniasis samples in central Morocco.

BACKGROUND & OBJECTIVES: Cutaneous leishmaniasis (CL) in Marrakesh-Safi region located in the central-south part of Morocco is a public health problem. This study assessed the efficiency of a microscopic examination method in establishing the diagnosis of CL and PCR for the characterization and identification of the circulating Leishmania strains in different CL foci of the study area. METHODS: A total of 297 smears obtained from cutaneous lesions of suspected patients with CL were stained with May-Grünwald Giemsa (MGG) for microscopic examination. For each positive smear, genomic DNA was extracted and PCR-analysed, targeting the small subunit ribosomal ribonucleic acid (ssu rRNA) gene to detect Leishmania DNA. Then, the internal transcribed spacer 1 (ITS1) was amplified and sequenced in order to identify the Leishmania species. The sensitivity and specificity of the conventional microscopy with ssu rRNA gene were compared by Leishmania nested PCR (LnPCR) and ITS1 gene by ITS-PCR. RESULTS: A total of 257 smears were positive in the microscopic examination, i.e. the detection rate of amastigotes by optical microscopy was 86.53% (257/297). The LnPCR was found to have a specificity and a sensitivity of 100%, each. Interestingly, the sequencing results showed that 99.61% (256/257) of the isolates had Leishmania tropica and 0.39% (1/257) had L. infantum infection. INTERPRETATION & CONCLUSION: Though, classical microscopic examination is useful and economical, it is not sensitive enough, especially in endemic regions where several Leishmania species coexist. In such situations, PCR constitutes a complementary method for the identification of the causal species. The results indicate that both the L. tropica (dominant) and L. infantum are the causative agents of CL in the Marrakesh-Safi region. The rate of CL infection is high in Imintanout, and Chichaoua provinces. Hence, early diagnosis and prompt treatment of CL patients is necessary to prevent its extension to neighboring localities.

Asymptomatic carriers of Leishmania infantum in patients infected with human immunodeficiency virus (HIV) in Morocco.

In Morocco, visceral leishmaniasis (VL) is a parasitic disease caused by the flagellated protozoan parasite Leishmania infantum. L. infantum is transmitted by the bite of female phlebotomine sandflies, and its main reservoir hosts are domestic dogs. Asymptomatic infection with L. infantum is more frequent than clinically apparent disease. In HIV-infected patients, the risk of clinical VL is increased due to immunosuppression that may reactivate latent infections. However, coinfected subjects do not necessarily develop VL and may remain as asymptomatic carriers depending on their immune status. The present study investigates the asymptomatic carriers of L. infantum in HIV-infected patients in central Morocco, where human cases of visceral leishmaniasis by L. infantum have been reported. A total of 200 HIV-infected patients attending the Infectious Diseases Unit of the Ibn Zohar Hospital of Marrakech participated in the study. Parasitological and serological blood analyses included a direct microscopic examination (DME), culture in Novy-McNeal-Nicolle (NNN) medium, and serology by indirect immunofluorescence (IFI). We found prevalence rates of 5% (10/200) by IFI, 3% (6/200) by DME, and 2.5% (5/200) by culture. The parasite was identified as L. infantum by PCR from positive cultures.

Mucosal leishmaniasis mimicking squamous cell carcinoma in a liver transplant recipient.

Organ transplant recipients living in endemic regions are at increased risk of Leishmania infections. Visceral leishmaniasis is the most common kind of presentation in the Mediterranean basin. Rarely, Leishmania infantum may cause localized mucosal disease. We present the first case, to our knowledge, of a liver transplant recipient with localized mucosal leishmaniasis. Twenty-two years after transplantation, a painless, very slow growing ulcer appeared on the inner side of the patient's upper lip. A biopsy performed in the community hospital showed non-specific chronic inflammation without neoplastic signs. Because of a high suspicion of malignancy, the patient was transferred to the referral hospital to consider complete excision. The excisional biopsy revealed a granulomatous inflammatory reaction together with intracellular Leishmania amastigotes within macrophages. Leishmaniasis was confirmed by the nested polymerase chain reaction assay. The clinical and laboratory findings did not suggest visceral involvement. The patient received meglumine antimoniate for 21 days without relevant adverse effects.

Molecular typing of Leishmania infantum isolates from a leishmaniasis outbreak in Madrid, Spain, 2009 to 2012.

Leishmaniasis is endemic in south-west Europe. Recent data point to the spread and (re-)emergence of this disease in previously endemic and non-endemic European countries. A recent example is the urban community outbreak of cutaneous and visceral leishmaniasis in the south-west of Madrid autonomous community, Spain, which began on 1 July 2009. A total of 446 cases associated to this outbreak were reported up to 31 December 2012. We show molecular typing data for 73 Leishmania infantum isolates obtained from January 2008 to July 2012 from different areas of Madrid, including those affected by the outbreak. Seven different genotypes were identified by combining data from two targets: the ribosomal internal transcribed spacers (ITS)-1 and -2 and the haspb (k26) gene. The results contribute to a better understanding of the parasite population circulating in the region, and indicate that most of the outbreak-associated isolates (22/31) were infected by parasites with the same combined genotype. Additional data from 82 L. infantum isolates typed as either MON-1 or MON-24 by isoenzyme analysis indicate that far from concluding that the outbreak was caused by a 'new' emerging genotype, further molecular typing-based surveillance studies are required to better understand the epidemiology of leishmaniasis in the region.

Implications of zoonotic and vector-borne parasites to free-roaming cats in central Spain.

Cats are definitive hosts and reservoirs for several parasites, some of which are responsible for serious zoonotic diseases. We conducted a case-control study of data from a trap-neuter-return (TNR) programme (years 2014-2017) designed to examine the prevalence of zoonotic parasites in free-roaming cats living in urban areas of central Spain. In the animal population tested (n = 263), we detected a 29.2% prevalence of endoparasites, including high rates of cestodes (12.9%) and Toxocara cati (11.7%). While faecal samples showed no Toxoplasma gondii oocysts, the seroprevalence of T. gondii infection was 24.2%. Antibodies to Leishmania infantum were detected in 4.8% of the animals, though all skin and blood samples analyzed were PCR negative for this parasite. Ectoparasites (ticks and fleas) were found in 4.6% of the cat population, and 10.6% of the cats were detected with Otodectes cynotis. Finally, 6.3% and 7.9% cats tested positive for feline leukaemia virus and feline immunodeficiency virus, respectively. Our study provides useful information for animal-welfare and public-health, as the parasites detected can affect native wild animals through predation, competition and disease transmission. Our detection of zoonotic parasites such as L. infantum, T. gondii, T. cati, Giardia duodenalis and several ectoparasites prompts an urgent need for health control measures in stray cats.

Acidic pH stress induces protein tyrosine phosphorylation in Leishmania pifanoi.

In order to determine whether in vitro Leishmania exposure to conditions comparable to those encountered inside the host cell would induce specific signals, we have studied tyrosine phosphorylation patterns in Leishmania pifanoi. Incubation of L. pifanoi at acidic pH resulted in the phosphorylation of several proteins including three of 27, 43 and 51 kDa, as well as the dephosphorylation of a 175 and a 39 kDa proteins in promastigotes recently transformed. In contrast, heat shock at 37 degrees C did not change the tyrosine phosphorylation pattern. Phosphorylation only occurs at pH 5.0 or lower and reached completion after 1 h. Changes returned to the initial conditions in 2 h after pH medium neutralization, indicating a reversible mechanism of phosphorylation.

Molecular epidemiology of Leishmania infantum on the island of Majorca: a comparison of phenotypic and genotypic tools.

In the Mediterranean basin, Leishmania infantum is the causative agent of both visceral and cutaneous leishmaniasis, and is an important opportunistic parasite in patients infected with human immunodeficiency virus (HIV). The commonest method used to study the variability of Leishmania spp. is isoenzyme analysis. In addition to this, we employed 3 assays based on the polymerase chain reaction (PCR): random amplified polymorphic deoxyribonucleic acid (RAPD), intergenic region typing (IRT), based on the amplification of ribosomal ribonucleic acid internal transcribed spacers and restriction fragment length polymorphism (PCR-RFLP). We used 54 L. infantum stocks isolated from HIV co-infected patients, 38 isolated from dogs, 3 isolated from immunocompetent patients and 3 isolated from 1826 sand files in the island of Majorca (Spain), a closed ecological niche. Zymodemes MON-1 (70%), MON-24 (11%) and MON-34 (18%) were found among the human isolates, and MON-1 (95%) and MON-108 (5%) among those from dogs. RAPD and IRT could not discriminate among the strains as they all gave the same pattern, even when different zymodemes were examined. In contrast, PCR-RFLP was able to distinguish the strains and, furthermore, a dendrogram (unweighted pair group method with arithmetic average [UPGMA]) was constructed from the genetic distances derived from RFLP data. The Leishmania isolates from HIV-infected subjects formed a single cluster, supporting the existence of an artificial anthroponotic cycle previously proposed by our group, in which syringes have been substituted for sand flies, and in which certain clones have been spread among intravenous drug users. This contrasts with the clusters representing a zoonotic cycle, involving dogs, sand flies and both immunocompetent and immunocompromised humans.

Molecular tracking of infections by Leishmania infantum.

Leishmania infantum is a major opportunistic parasite in patients with acquired immune deficiency syndrome and is very variable in these subjects. Isoenzyme characterization is not able to explain this variability, since half of the stocks isolated from patients co-infected with human immunodeficiency virus and Leishmania belong to zymodeme MON-1. Amplification of L. infantum minicircles by the polymerase chain reaction (PCR) and digestion of the amplified product to reveal restriction fragment length polymorphisms (RFLP) has proved very useful in distinguishing between relapses and reinfections in co-infected, treated patients. We have confirmed the existence of a leishmaniasis outbreak among intravenous drug users in north-east Spain, previously detected by isoenzymatic analysis. We have documented persistence of the same strain of Leishmania in 2 treated co-infected patients throughout several years, regardless of the theoretical rapid evolution ascribed to kinetoplast deoxyribonucleic acid minicircle sequences. We suggest using this PCR-RFLP technique to detect reinfections in treated co-infected subjects.

A nested polymerase chain reaction (Ln-PCR) for diagnosing and monitoring Leishmania infantum infection in patients co-infected with human immunodeficiency virus.

We investigated a Leishmania-specific nested polymerase chain reaction (Ln-PCR) for the diagnosis and treatment monitoring of L. infantum infections in patients co-infected with human immunodeficiency virus (HIV). Peripheral blood and bone marrow samples from 89 HIV patients in Spain suspected of having leishmaniasis were examined by different diagnostic techniques (Ln-PCR, microscopy, NNN culture and indirect fluorescent antibody test). The sensitivity of Ln-PCR compared with microscopy and culture of bone marrow was 95.45% using blood and 100% when using bone marrow. 38 of these patients with confirmed leishmaniasis were entered in a chemotherapy trial (reported elsewhere), and samples from them were collected before treatment, one month after treatment ended and during follow-up (1-20 months), and examined similarly. Ln-PCR was shown to be a good method for testing efficacy of treatment and for predicting relapses after treatment (relapses were predicted on average 5 months earlier than when using classical diagnostic techniques). We suggest that Ln-PCR (especially using peripheral blood) should be the technique of choice for diagnosis, monitoring the success of treatment, and predicting relapses in patients with HIV and suspected or confirmed L. infantum infection.

Genetic diversity of human zoonotic leishmaniasis in Iberian Peninsula.

Leishmaniasis caused by Leishmania (Leishmania) infantum is a zoonotic disease endemic in South Europe, from Portugal to the Middle East. The aim of the present study was to investigate the genetic diversity of L. infantum parasites in Iberian Peninsula. Twenty-four L. infantum strains isolated from immunocompetent patients with leishmaniasis from several localities of Portugal and Spain were studied. The use of kinetoplast DNA-PCR-restriction fragment length polymorphism as a molecular marker revealed intra-specific variation. No association was found between genotype and clinical form of the disease or patients age group. Two main clusters were identified with this marker: (i) zymodeme MON-1 strains and (ii) non-MON-1 strains. However, no association was found between strains variability and geographical distribution suggesting that parasite populations of different regions in the Iberian Peninsula are homogenous.

[Pilomatrixoma: unusual clinical presentation]. / Pilomatrixoma: una presentación clínica inusual.

Pilomatrixoma is a rare, benign neoplasm that is derived from hair matrix cells. It is more frequent during childhood, especially between the ages of 5 and 15 years. Pilomatrixoma is usually a hard, well circumscribed, solitary lesion, and can be located on any part of the body, except palms and soles, with a predilection for maxillofacial region. Multiple pilomatrixomas are rare and they have been associated with various systemic syndromes, of which myotonic dystrophy has been the most described. The diagnosis of pilomatrixoma is fundamentally clinical. Complementary studies such as ultrasound can be useful to confirm the diagnosis. Excisional surgery is the standard curative treatment. We present a 17-year-old female patient with an extremely uncommon clinical presentation of pilomatrixoma: multiple lesions, anetodermic, and rapid growth.

Genomic organization and expression of the HSP70 locus in New and Old World Leishmania species.

Heat shock is believed to be a developmental inductor of differentiation in Leishmania. Furthermore, heat shock genes are extensively studied as gene models to decipher mechanisms of gene regulation in kinetoplastids. Here, we describe the organization and expression of the HSP70 loci in representative Leishmania species (L. infantum, L. major, L. tropica, L. mexicana, L. amazonensis and L. braziliensis). With the exception of L. braziliensis, the organization of the HSP70 loci was found to be well conserved among the other Leishmania species. Two types of genes, HSP70-I and HSP70-II, were found to be present in these Leishmania species except for L. braziliensis that lacks HSP70-II gene. Polymorphisms in the HSP70 locus allow the differentiation of the Old and New World species within the subgenus Leishmania. A notable discrepancy between our data and those of the L. major genome database in relation to the gene copy number composing the L. major HSP70 locus was revealed. The temperature-dependent accumulation of the HSP70-I mRNAs is also conserved among the different Leishmania species with the exception of L. braziliensis. In spite of these differences, analysis of the HSP70 synthesis indicated that the HSP70 mRNAs are also preferentially translated during heat shock in L. braziliensis.

Immunization with H1, HASPB1 and MML Leishmania proteins in a vaccine trial against experimental canine leishmaniasis.

The protective capabilities of three Leishmania recombinant proteins - histone 1 (H1) and hydrophilic acylated surface protein B1 (HASPB1) immunized singly, or together as a protein cocktail vaccine with Montanide, and the polyprotein MML immunized with MPL-SE adjuvant - were assessed in beagle dogs. Clinical examination of the dogs was carried out periodically under blinded conditions and the condition of the dogs defined as asymptomatic or symptomatic. At the end of the trial, we were able to confirm that following infection with L. infantum promastigotes, five out of eight dogs immunized with H1 Montanide, and four out of eight dogs immunized with either the combination of HASPB1 with Montanide or the combination of H1+HASPB1 with Montanidetrade mark, remained free of clinical signs, compared with two out of seven dogs immunized with the polyprotein MML and adjuvant MPL-SE, and two out of eight dogs in the control group. The results demonstrate that HASPB1 and H1 antigens in combination with Montanide were able to induce partial protection against canine leishmaniasis, even under extreme experimental challenge conditions.

Pilomatrixoma: una presentación clínica inusual / Pilomatrixoma: unusual clinical presentation

El pilomatrixoma, una neoplasia benigna poco frecuente, se origina de la matriz del folículo piloso. Es más frecuente durante la infancia, especialmente entre los 5 y 15 años de edad. La forma múltiple de pilomatrixoma es rara y su presencia se asocia a síndromes sistémicos, entre los cuales destaca la distrofa miotónica. El diagnóstico de pilomatrixoma es fundamentalmente clínico. Estudios complementarios como la ecografía pueden ser útiles para confrmar su diagnóstico. La resección quirúrgica de la lesión corresponde el tratamiento curativo, con baja tasa de recurrencia. Se presenta una paciente de sexo femenino, de 17 años de edad, por su pilomatrixoma de características clínicas inusuales en edad pediátrica: variante anetodérmica, forma múltiple y crecimiento rápido.

Pilomatrixoma is a rare, benign neoplasm that is derived from hair matrix cells. It is more frequent during childhood, especially between the ages of 5 and 15 years. Pilomatrixoma is usually a hard, well circumscribed, solitary lesion, and can be located on any part of the body, except palms and soles, with a predilection for maxilofacial region. Multiple pilomatrixomas are rare and they have been associated with various systemic syndromes, of which myotonic dystrophy has been the most described. The diagnosis of pilomatrixoma is fundamentally clinical. Complementary studies such as ultrasound can be useful to confrm the diagnosis. Excisional surgery is the standard curative treatment. We present a 17-year-old female patient with an extremely uncommon clinical presentation of pilomatrixoma: multiple lesions, anetodermic, and rapid growth.

Lower trypanosomatids in HIV/AIDS patients.

Although the family Trypanosomatidae includes parasites of plants, insects and vertebrates, only two genera in the family, Leishmania and Trypanosoma, are usually found in humans. Since 1995, however, other monoxenous trypanosomatids have been isolated from several HIV-positive individuals, in whom the parasites cause either visceral or cutaneous lesions. These odd cases are reviewed here. It appears that immunocompromised patients may be vulnerable to infection with trypanosomatids (and other parasites) that either fail to survive or never cause detectable morbidity in the immunocompetent.

Iso-enzymatic variability of Leishmania infantum in Spain.

In many areas of the Mediterranean basin, leishmaniasis can now be found in HIV-positive individuals. Such cases of Leishmania/HIV co-infection are relatively common in southern Europe, Spain being the country that has reported the greatest number. Since 1984, 359 Spanish isolates of Leishmania infantum have been characterized at the Instituto de Salud Carlos III in Madrid. Most (94.6%) of the isolates came from HIV-positive patients. The results of iso-enzymatic analysis indicated a high level of variability among the isolates, the visceralization in HIV-positive individuals of variants considered to be dermotropic in the immunocompetent, and the appearance of new zymodemes among the HIV-positive human population.

Leishmania-infected macrophages sequester endogenously synthesized parasite antigens from presentation to CD4+ T cells.

CD4+ T cell lines raised against the protective leishmanial antigens GP46 and P8 were used to study the presentation of endogenously synthesized Leishmania antigens by infected cells. Using two different sources of macrophages, the I4.07 macrophage cell line (H-2k) which constitutively expresses major histocompatibility complex (MHC) class II molecules, and elicited peritoneal exudate cells, we found that cells infected with Leishmania amastigotes presented little, if any endogenously synthesized parasite antigens to CD4+ T cells. In contrast, promastigote-infected macrophages did present endogenous parasite molecules to CD4+ T cells, although only for a limited time, with maximal presentation occurring within 24 h of infection and decreasing to minimal antigen presentation at 72 h post-infection. These observations suggest that once within the macrophage, Leishmania amastigote antigens are sequestered from the MHC class II pathway of antigen presentation. This allows live parasites to persist in infected hosts by evading the activation of CD4+ T cells, a major and critical anti-leishmanial component of the host immune system. Studies with drugs that modify fusion patterns of phagosomes suggest that the mechanism of this antigen sequestration includes targeted fusion of the parasitophorous vacuole with certain endocytic compartments.

N-terminal fatty acid substitution increases the leishmanicidal activity of CA(1-7)M(2-9), a cecropin-melittin hybrid peptide.

In order to improve the leishmanicidal activity of the synthetic cecropin A-melittin hybrid peptide CA(1-7)M(2-9) (KWKLFKKIGAVLKVL-NH(2)), a systematic study of its acylation with saturated linear fatty acids was carried out. Acylation of the N(epsilon)-7 lysine residue led to a drastic decrease in leishmanicidal activity, whereas acylation at lysine 1, in either the alpha or the epsilon NH(2) group, increased up to 3 times the activity of the peptide against promastigotes and increased up to 15 times the activity of the peptide against amastigotes. Leishmanicidal activity increased with the length of the fatty acid chain, reaching a maximum for the lauroyl analogue (12 carbons). According to the fast kinetics, dissipation of membrane potential, and parasite membrane permeability to the nucleic acid binding probe SYTOX green, the lethal mechanism was directly related to plasma membrane permeabilization.

Septicemia neonatal: incidencia, letalidad y características bacteriológicas: comparación de dos períodos / Neonatal septicemia: incidence, mortality and microbiological findings along 2 different periods

Objetivos: describir y comparar la incidencia y letalidad de la septicemia neonatal, en dos períodos en un hospital universitario. Pacientes y método: se compararon recién nacidos que tuvieron septicemia entre los meses de julio de 1987 a junio de 1989 con los del período de enero de 1995 a diciembre de 1996. Resultados: la incidencia de septicemia neonatal aumentó (2,9 a 8,7 por mil) y la letalidad (44 por ciento a 2,2 por ciento) disminuyó en el segundo período. Las poblaciones estudiadas y los métodos de diagnóstico fueron similares. En las septicemias connatales el germen preponderante fue el Streptococcus grupo B sin diferencias entre períodos (50 por ciento ante 52 por ciento). Entre las septicemias nosocomiales, aumentaron las producidas por estafilococo (desde 25 por ciento a 91 por ciento), siendo Staphylococcus epidermidis devino el principal agente durante el segundo período (57 por ciento del total), disminuyendo la sensibilidad a cloxacilina (100 por ciento a 33 por ciento). Conclusiones: se demuestra un aumento de la incidencia y mejoría del pronóstico de las septicemias neonatales con un cambio en sus características bacteriológicas entre los períodos estudiados, similar a lo observado en otros países