Changes in the microbiological diagnosis and epidemiology of cutaneous leishmaniasis in real-time PCR era: A six-year experience in a referral center in Barcelona.

BACKGROUND: Leishmaniasis is a neglected disease caused by different species of the protozoa Leishmania spp. Cutaneous lesions are the most common clinical manifestation. This disease is prevalent in tropical and subtropical areas, including the Mediterranean basin. In Spain, Leishmania (L.) infantum is the only endemic species, but imported cases are often diagnosed. Different classical parasitological methods can be performed for cutaneous leishmaniasis (CL) diagnosis; but currently molecular techniques serve as a relevant tool for the detection and characterization of Leishmania parasites. We aimed to evaluate clinical and epidemiological characteristics of CL diagnosed patients by real-time PCR in a tertiary hospital over a six-year period. METHODOLOGY/PRINCIPAL FINDINGS: Clinical, epidemiological and microbiological data were retrospectively collected and analyzed. In our study, CL was confirmed in 59 (31.4%) out of 188 patients by real-time PCR, showing an increase over recent years: 11 cases of CL between 2014 and 2016 and 48 between 2017 and 2019. Real-time PCR was performed on skin swabs and/or biopsies samples, with a positivity of 38.5% and 26.5%, respectively. Results were 100% concordant when biopsy and skin swab were performed simultaneously. L. (L.) infantum was the most frequent species detected (50%), followed by L. (L.) major (45%) and Viannia subgenus (5%), which were detected only in imported cases. L. (L.) major was almost entirely detected in travelers/migrants from Morocco. Multiple and atypical skin lesions were more common in imported cases than in autochthonous cases (44.4% vs. 21.8%). CONCLUSIONS/SIGNIFICANCE: An increase in both autochthonous and imported CL cases has been observed in past years in our hospital. Molecular techniques assist in improving CL diagnosis and characterization of the Leishmania species, mainly in imported cases.

Cutaneous leishmaniasis: A pathological study of 360 cases with special emphasis on the contribution of immunohistochemistry and polymerase chain reaction to diagnosis.

BACKGROUND: Traditional methods for the diagnosis of leishmaniasis yield poor sensitivity, which limits its effectiveness in lesions with a low parasite burden. METHODS: Retrospective pathologic study of 360 cases of cutaneous leishmaniasis and analysis of the different diagnostic methods used. RESULTS: In 93% of the lesions, histopathology showed a dense and diffuse inflammatory infiltrate, consisting of lymphocytes, histiocytes and plasma cells, which occupied the superficial and mid dermis and variably extended to deep dermis and superficial subcutis (standard pattern). The remaining cases exhibited atypical features, such as perivascular, interstitial or perifollicular inflammatory patterns, folliculitis or panniculitis. Granulomas were identified in 84% of biopsies, most of them as small, poorly formed, non-necrotizing histiocytic aggregates. Amastigotes were visualized by routine histopathologic exam in 36% of biopsies. Immunohistochemistry stained 17 of 26 lesions (65%) negative by conventional stains. PCR provided the correct diagnosis in 218 cases (58% of the series) negative for Leishmania by other techniques. CONCLUSIONS: Biopsies negative for Leishmania by traditional diagnostic methods that show the histopathologic standard pattern, those with atypical features from patients with clinical suspicion of cutaneous leishmaniasis in endemic areas, should be studied by immunohistochemistry and/or PCR for Leishmania in order to reach the definitive diagnosis.

Molecular typing reveals the co-existence of two transmission cycles of American cutaneous leishmaniasis in the Andean Region of Venezuela with Lutzomyia migonei as the vector

BACKGROUND The transmission routes for American cutaneous leishmaniasis (ACL) are in flux, so studies examining its transmission in humans, mammalian hosts, and sand fly vectors are urgently needed. OBJECTIVES The aim of this work was understand the epidemiological cycles of Leishmania spp., which causes ACL in the Andean Region of Venezuela, by identifying the Leishmania and the sand fly species involved in human and dog infections. METHODS Thirty-one biopsies from patients in Mérida and Táchira states with suspected ACL were studied by both parasitological tests (cultures and hamster inoculation) and a molecular test [Internal transcribed spacer 1 (ITS1) nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)]. We also conducted a survey to detect Leishmania infection in dogs (Immunifluorescence antibody test and ITS1 nested PCR-RFLP) and sand flies (ITS1 nested PCR-RFLP) from El Carrizal, a highly endemic focus of ACL in Venezuela. FINDINGS Three different Leishmania species were identified in the clinical samples from humans (Leishmania braziliensis, L. guyanensis, and L. mexicana) and dogs (L. guyanensis and L. mexicana). The predominant sand fly species found were those from the Verrucarum group (infected with L. mexicana) and Lutzomyia migonei (infected with L. guyanensis and L. mexicana). MAIN CONCLUSIONS We show that Lu. migonei may be the putative vector in two ACL epidemiological cycles, involving L. guyanensis and L. mexicana. We also report for the first time the presence of L. guyanensis in domestic animals.

LEISHMANIA INFANTUM INFECTION IN BENNETT'S WALLABIES (MACROPUS RUFOGRISEUS RUFOGRISEUS) IN A SPANISH WILDLIFE PARK.

Although dogs are the main reservoir for human Leishmania infantum infection, the disease has also been reported in other domestic and wild mammals. In 2011, a fatal case of naturally acquired leishmaniosis was described for the first time in a Bennett's wallaby (Macropus rufogriseus rufogriseus) kept in a wildlife park in Madrid (Spain). This study was designed to assess the infection status of twelve Bennett's wallabies in the same park one year after this incident. Phlebotomus perniciosus, the main vector of L. infantum in Spain, was screened for using sticky and Centers for Disease Control miniature light traps. L. infantum infection was confirmed by molecular diagnosis in four animals, but only one wallaby returned a positive serology result. The presence of the sand fly vector was also confirmed in this habitat. These results suggest that the first case of L. infantum in a wallaby in this park was not an isolated incident and stress the need for further work to determine the role of this parasite in the morbidity and mortality of these macropods. Madrid was recently the scene of an outbreak of human cutaneous and visceral leishmaniosis. Epidemiological studies have so far revealed the widespread presence of L. infantum infection in animals other than the dog. Our ongoing work suggests a risk of L. infantum infection not only among captive animals in Madrid, but also among threatened species or even species that are already extinct in the wild.

Hemophagocytic lymphohistiocytosis in children with visceral leishmaniasis.

Acquired hemophagocytic lymphohistiocitosis (HLH) syndrome can be a complication of visceral leishmaniasis (VL). A multicenter prospective study was conducted to determine the frequency of HLH syndrome in children with VL. Twenty-four children with VL were identified, and 10 (41%) developed HLH syndrome. VL should be ruled out in all children with HLH criteria living in or coming from endemic areas.

Comparison of new diagnostic tools for management of pediatric Mediterranean visceral leishmaniasis.

New techniques are available for diagnosing leishmaniasis, but their efficacy in the identification of pediatric visceral leishmaniasis (VL) has not been compared with that of traditional methods. Blood, bone marrow, and urine samples were taken from 25 children with VL during their first clinical episode, 22 days after the start of treatment with liposomal amphotericin B (3 mg/kg/day on 6 days over a 10-day period), and when a relapse was suspected during follow-up. The results obtained suggest that antibody detection techniques, the antigen detection in urine (KAtex kit), and Leishmania nested PCR (LnPCR) analysis of the blood could be used for diagnosis of the first clinical episode. After treatment, clinical improvement was associated with negativization of Novy-MacNeal-Nicolle culture and microscopy of bone marrow aspirate, KAtex test, and LnPCR blood analysis results. Interestingly, LnPCR analysis of the bone marrow aspirate showed that sterile cure was not achieved in eight patients, two of which suffered a relapse within 10 to 20 weeks. All of the new noninvasive techniques tested showed high diagnostic sensitivity. However, LnPCR analysis of the bone marrow was the most sensitive; this test was able to detect the persistence of parasites and predict potential relapses.

Comparison of molecular markers for strain typing of Leishmania infantum.

The epidemiology of Leishmania infantum, the etiological agent of visceral leishmaniasis, is changing rapidly; hence powerful typing tools are required in order to monitor the parasite populations spreading and to adapt adequate control measures. We compared here the resolving power of four molecular methods at the zymodeme level: PCR-RFLP analysis of kDNA minicircles (kDNAPCR-RFLP) and antigen genes (cysteine proteinase b and major surface protease, cpb- and gp63PCR-RFLP), multilocus microsatellite typing (MLMT) and random amplification of polymorphic DNA (RAPD) were applied to samples of 25 L. infantum MON-1 strains obtained from different hosts (HIV+ patients, HIV- patients and dogs) coming from three Spanish foci: Madrid, Mallorca and Ibiza. While RAPD was not sufficiently resolving, the other three methods allowed genotyping within the zymodeme. KDNAPCR-RFLP and MLMT were the most discriminatory and appeared the most adequate for strain fingerprinting. In an eco-geographical context, cpbPCR-RFLP, MLMT and kDNAPCR-RFLP were all informative: they showed here a similar picture, with the existence of cluster(s) of isolates from the islands and other one(s) of mixed composition (Madrid and the islands). None of the markers revealed an association with the host type or the clinical form. In general, there was a significant correlation between each pair of distances calculated from the cpb, microsatellite and kDNA data, respectively, but visual inspection of the trees revealed a better congruence between cpb and microsatellite trees. The methods used here are complementary and each adapted to answer specific epidemiological questions. Their choice should be the result of a compromise between the required resolving power, the genetic features of the respective markers and the technical aspects.