Genetic diversity in Leishmania infantum and Leishmania tropica isolates from human and canine hosts in northern Morocco.

This study investigated nine provinces in northern Morocco and collected 275 skin scraping, 22 bone marrow aspirates, and 89 fine needle aspirations from suspected cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) patients and potentially infected dogs. Molecular analysis using ITS1 RFLP PCR and RT-PCR revealed a higher prevalence of L. infantum (66.18 %; χ2 = 28.804; df = 1; P-value = 8.01e-08) than L. tropica in skin scraping, with L. infantum being the sole causative agent for both VL and canine leishmaniasis. L. infantum was predominantly found in most provinces, while L. tropica was relatively more dominant in Taza Province. Discriminant Analysis of Principal Components (DAPC) revealed distinct clustering between L. tropica and the other three species. However, no small subset of SNPs could clearly differentiate between Infantum_CL, Infantum_VL, and CanL, as they likely share a significant genetic background. The high rate of L. infantum could be attributed to the abundance of sand fly species transmitting VL. In Taza Province, Phlebotomus sergenti, responsible for anthroponotic CL, is the most abundant species. DNA sequencing demonstrated sequence heterogeneity in L. infantum (variants 1-9) and L. tropica (variants 1-7). Phylogenetic analysis showed a distinct separation between L. tropica and L. infantum strains, with an overlap among L. infantum strains isolated from cutaneous, visceral, and canine cases, and dogs serving as the central population for L. infantum.

Inflammatory Nasal Swelling due to Leishmania tropica.

Since its discovery in the 19th century, cutaneous leishmaniasis has been a major public health problem, especially with the appearance of more and more unusual cases of cutaneous lesions due to this parasite. Indeed, the present study joins the previous studies and describes a typical case of a nasal lesion due to Leishmania infection. This is a 20-year-old young man, with no particular pathological history, from an epidemic focus who presented with inflammatory nasal swelling similar to a mucocutaneous form. However, the X-ray data showed that no lysis of the bones proper to the nose was detected and no damage to the underlying mucosa was observed. Nevertheless, the parasitological diagnosis confirmed the presence of amastigotes, and the results of the molecular study showed that the electrophoretic profile was comparable to that of L. tropica. After diagnosis and confirmation, treatment with meglumine antimonate at the rate of two ampoules/injection (one ampoule = 5 ml) of antimony salt for one month was administered intramuscularly with favorable outcome. Atypical forms of cutaneous leishmaniasis constitute a rare and unusual entity often leading to diagnostic delay. For this, the clinical examination must take into account both exceptional presentations of Leishmania infection, in particular in subjects living or having stayed in an endemic area, in order to ensure appropriate and early treatment.

Risk Factors Associated with Leishmaniasis in the Most Affected Provinces by Leishmania infantum in Morocco.

BACKGROUND: Human leishmaniasis, both visceral and cutaneous, has been reported in Morocco for centuries and constitutes a serious public health problem. However, the evolution of this pathology depends on several factors such as ecological, socioeconomic, and climatic conditions. The risk study of the affected foci is of great value for the control and surveillance of this endemic disease, especially in the provinces where Leishmania infantum predominates. METHODS: This study concerned nine provinces located in the extreme and central north of Morocco (Taounate, Taza, Chefchaouen, Al Hoceima, Larache, Tétouane, Tanger-Assilah, M'diq-Fnideq, and Fahs-Anjra Provinces). In this work, leishmaniasis cases (VL and CL) were subjected to an epidemiological study which was performed using a linear regression model to identify the impact as well as the interaction between all predictor variables on the distribution of leishmaniasis in this region. RESULTS: During the period 1997-2018, a total of 6 128 cases of VL and CL were recorded in the study area. Our results showed that among demographic factors studied, urbanization showed significance for both cutaneous and visceral forms (P < 0.05). Regarding the environmental factors, the humidity and the altitude were significant for both CL and VL (P < 0.05), while the temperature and the normalized difference vegetation index (NDVI) showed a significance only for VL. Moreover, trends in season of occurrence revealed that wet season (October to April) had a higher incidence of leishmaniasis compared to the dry season (May to September) specifically for CL. As for socioeconomic factors, poverty was the only factor that influences the spread of VL. Finally, the distance from endemic foci showed significance for both VL and LC (P < 0.05). CONCLUSION: Our study revealed that the risk factor associated with cutaneous and visceral leishmaniasis in northern Morocco could help in the establishment of a prediction program.

Leishmaniasis in Northern Morocco: Predominance of Leishmania infantum Compared to Leishmania tropica.

In Morocco, Leishmania infantum species is the main causative agents of visceral leishmaniasis (VL). However, cutaneous leishmaniasis (CL) due to L. infantum has been reported sporadically. Moreover, the recent geographical expansion of L. infantum in the Mediterranean subregion leads us to suggest whether the nonsporadic cases of CL due to this species are present. In this context, this review is written to establish a retrospective study of cutaneous and visceral leishmaniasis in northern Morocco between 1997 and 2018 and also to conduct a molecular study to identify the circulating species responsible for the recent cases of leishmaniases in this region. Data concerning leishmaniases cases were collected from the Epidemiology and Disease Control Directorate from 1997 to 2018. Human samples obtained from peripheral laboratories were examined using PCR-ITS1 method. The ITS1 products were subjected to digestion with the restriction endonuclease Mn1-I. Between 1997 and 2018, a total of 1,255 cases of cutaneous and visceral leishmaniasis were recorded in Tangier-Tetouan-Al Hoceima Region, i.e., 1.56% of the reported cases in Morocco (1,255/80,299). Concerning the geographical study covering the period 2007-2018, 79.5% (105/132) of the sectors were affected by leishmaniases. The molecular results showed that Humans were found to be infected with the L. infantum species with a high infection rate compared to L. tropica infection. Moreover, molecular characterization using ITS1 PCR-RFLP showed that the density of L. infantum was significantly higher (n = 68/81; 84%) than that of L. tropica (n = 13/81; 16%) (P-value 9.894e-10). While regarding visceral leishmaniasis, L. infantum was the only species responsible of this form. These findings of this study showed the emergence of L. infantum in Morocco and suggest that this species might be more prevalent than previously thought. Furthermore, the molecular determination of L. infantum will be helpful for control strategies by taking into consideration the reservoir of this species.

Accuracy of a Rapid Diagnostic Test Based on Antigen Detection for the Diagnosis of Cutaneous Leishmaniasis in Patients with Suggestive Skin Lesions in Morocco.

In rural areas in Morocco, diagnosing cutaneous leishmaniasis (CL) can be challenging. We evaluated the accuracy of a rapid diagnostic test (RDT) based on antigen detection, CL Detect Rapid Test™ (Inbios International Inc., Seattle, WA), in this setting. We consecutively recruited patients with new skin ulcers in nine primary health centers. We took a dental broach sample for the RDT and two other tissue samples by scraping the border and center of the lesion with a scalpel and smearing it on a slide. We duplicated each smear by pressing a clean slide against it and processed the slides by microscopy, polymerase chain reaction (PCR) internal transcribed spacer 1, and kDNA minicircle PCR. In a subgroup with positive PCR, the Leishmania species was identified using PCR-restriction fragment length polymorphism and PCR-sequencing of hsp70 genes. A participant with positive microscopy and/or PCR was considered a confirmed CL case. We computed sensitivity (Se) and specificity (Sp) of the RDT compared with this reference standard (ClinicalTrials.gov registration: NCT02979002). Between December 2016 and July 2017, we included 219 patients, 50% of them were under 18 years old. Rapid diagnostic test Se was 68% [95% confidence interval (CI): 61-74], Sp 94% [95% CI: 91-97], positive predictive value 95% [95% CI: 92-98], and negative predictive value 64% [95% CI: 58-70]. Despite its low Se, this novel RDT is a useful addition to clinical management of CL in Morocco, especially in isolated localities. Rapid diagnostic test-positive lesions can be treated as CL; but when RDT negative, microscopy should be done in a second step. The Se of the RDT can probably be optimized by improving the sampling procedure.

Comparative evaluation of immunochromatographic dipstick test (ICT) rk39, soluble antigen ELISA and IFAT for the sero-diagnosis of visceral leishmaniasis in Morocco.

A rapid, sensitive and specific tool for detection of Leishmania infantum infection in Humans would be highly desirable, because it would allow control interventions in endemic areas of visceral leishmaniasis. This study was carried out at the Reference National Laboratory of Leishmaniasis (RNLL) in National Institute of Hygiene (NIH) Morocco, in order to evaluate the diagnostic potential of immunochromatographic dipstick test (ICT) rk39 in Moroccan suspected VL patients. A total of 49 admitted patients with strong clinical suspicion of VL and 40 healthy controls were investigated for the performance of the ICT rk39. Bone marrow smears were examined for microscopic detection of Leishmania amastigotes obtained from the admitted patients. Only PCR and smear positive cases were considered as gold standard as well as confirmed cases of VL. Out of 49 suspected patients, twenty four (48.9%) were found PCR and smear-positive and twenty three (46.9%) were positive for ICT rk39. Voluntary healthy controls, which included twenty persons from the endemic zone and twenty from non-endemic zone of VL, were found all negative for the strip test. The sensitivity in sera was 75% by ELISA and 87.5% by IFAT, compared with 95.8% for ICT rk39. Specificity was 95.8%, with both tests ELISA and IFAT, and 100% by ICT rk39 respectively. Present study findings again reinforce that the ICT rk39 is a simple, reliable and easy-to-perform non-invasive diagnostic tool for visceral leishmaniasis in the endemic area of Morocco.

Risk factors and prediction analysis of cutaneous leishmaniasis due to Leishmania tropica in Southwestern Morocco.

Cutaneous leishmaniasis is currently a serious public health problem in northern Africa, especially in Morocco. The causative parasite is transmitted to a human host through the bite of infected female sandflies of the genus Phlebotomus. The objective of the present study is to characterize the causative organisms and to predict the risk of cutaneous leishmaniasis (CL) cases in six provinces in southwestern Morocco, based on the spatial distribution of cases in relation to environmental factors and other risk factors such as socio-economic status and demographics. A molecular study was carried out using ITS1 PCR-RFLP method of the ribosomal DNA of Leishmania. An epidemiological study on CL cases was reported between 2000 and 2016 in this current investigation in six provinces in southwestern Morocco. Statistical analysis was performed using a linear regression model to identify the impact as well as the interaction between all predictor variables on the distribution of CL in the studied provinces. The forecast Holt-Winters (HW) method was used to describe the trend and seasonality of CL cases. The ITS1-PCR- RFLP analysis revealed the presence of Leishmania tropica in all studied provinces. The spatial distribution of CL cases documented in all studied provinces during the sixteen years showed a heterogeneous pattern and fluctuation trend with an average prevalence of 9.92 per 100,000 inhabitants. In addition, the forecast HW model predicts continued variability of trend and seasonality of CL cases in the upcoming years. This study confirmed the importance of socioeconomic factors, in particular poverty and the vulnerability rate, on distribution and emergence of CL. This study revealed a relationship between increasing risk of CL occurrence due to Leishmania tropica, as well as the distribution and emergence thereof, and socioeconomic factors in the investigated area.

[Assessment of a supervision grid being used in the laboratories of cutaneous leishmaniasis in Morocco]. / Evaluation d'une grille de supervision des laboratoires des leishmanioses cutanées au Maroc.

INTRODUCTION: The aim of our study was to assess a standardized supervisory grid as a new supervision tool being used in the laboratories of leishmaniasis. METHODS: We conducted a pilot trial to evaluate the ongoing performances of seven provincial laboratories, in four provinces in Morocco, over a period of two years, between 2006 and 2014. This study detailed the situation in provincial laboratories before and after the implementation of the supervisory grid. A total of twenty-one grids were analyzed. RESULTS: In 2006, the results clearly showed a poor performance of laboratories: need for training (41.6%), staff performing skin biopsy (25%), shortage of materials and reagents (65%), non-compliant document and local management (85%). Several corrective actions were conducted by the National Reference Laboratory (LNRL) of Leishmaniasis during the study period. In 2014, the LNRL recorded a net improvement of the performances of the laboratories. The need for training, the quality of the biopsy, the supply of tools and reagents were met and an effective coordination activity was established between the LNRL and the provincial laboratories. CONCLUSION: This trial shows the effectiveness of the grid as a high quality supervisory tool and as a cornerstone of making progress on fight programs against leishmaniases.

Chemical composition of Mentha pulegium and Rosmarinus officinalis essential oils and their antileishmanial, antibacterial and antioxidant activities.

The aim of the study was the determination of the chemical composition of Mentha pulegium L. and Rosmarinus officinalis L. essential oils and the evaluation of their antileishmanial, antibacterial and antioxidant activities. Essential oils (EOs) were isolated using steam distillation and the chemical composition was determined using GC-MS analysis. The antibacterial activity was tested against ten pathogenic strains using the diffusion method, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) by microtitration assay. The antioxidant activity was estimated by DPPH free radical scavenging ability and ferric-reducing power. The antileishmanial activity was tested against Leishmania major, Leishmania tropica and Leishmania infantum using MTT (3-(4.5-dimethylthiazol-2yl)-2.5-diphenyltetrazolium bromide) assay. The yield of essential oils (v/w %) M. puleguim and R. officinalis based on dry weight were 5.4 and 2.7% respectively. GC/MS analysis of R. officinalis essential oil (ROEO) revealed the presence of 29 components, mainly represented by oxygenated monoterpenes (63.743%) and hydrocarbons monoterpenes (21.231%). Mentha pulegium essential oil (MPEO) revealed 21 components, mainly represented by oxygenated monoterpenes (83.865%). The major components of ROEO were α-pinene (14.076), 1,8-Cineole (23.673) and camphor (18.743), while menthone (21.164) and pulegone (40.98) were the main major components of MPEO. M. pulegium and R. officinalis EOs showed a significant antioxidant activity compared with ascorbic acid and Trolox to the IC50 values of 58.27 ± 2.72 and 85.74 ± 7.57 µg/mL respectively revealed by reducing power assay. As for the antibacterial effect, the highest zone diameters were shown by the MPEO against Bacillus subtilis (30 ± 1.43 mm) and Proteus mirabilis (28 ± 1.32 mm). These values are significantly important compared with those of the commercialized antibiotic (Erythromycin and Chlorophenicol). The lowest MIC and MBC values were obtained with MPEO against S. aureus MBLA (MIC = MBC = 0.25% (v/v)). While, ROEO has exhibited a bactericidal effect against Listeria monocytogenes (MIC = MBC = 0.5% (v/v)), Bacillus subtilis (MIC = MBC = 1% (v/v)) and Escherichia coli (MIC = MBC = 1% (v/v)). For the antileishmanial effect, ROEO is the most active against L. major (IC50 = 1.2 ± 0.36 µg/mL. While, the MPEO has the most leishmanicidal effect against L. major (IC50 = 1.3 ± 0.45 µg/mL). These findings show that the EOs of M. pulegium and R. officinalis are good sources of bioactive molecules that could have potential applications in the food and pharmaceutical industries.

Circulating species of Leishmania at microclimate area of Boulemane Province, Morocco: impact of environmental and human factors.

BACKGROUND: Cutaneous leishmaniasis (CL) is widely distributed in Morocco where its geographical range and incidence are related to environmental factors. This study aimed to examine the impact of several factors on the distribution of CL in Boulemane Province, which is characterized by several microclimates, and to identify the Leishmania species circulating in these areas. METHODS: Ordinary least squares regression (OLSR) analysis was performed to study the impact of poverty, vulnerability, population density, urbanization and bioclimatic factors on the distribution of CL in this province. Molecular characterization of parasites was performed using a previously described PCR-RFLP method targeting the ITS1 of ribosomal DNA of Leishmania. RESULTS: A total of 1009 cases were declared in Boulemane Province between the years 2000 and 2015 with incidences fluctuating over the years (P = 0.007). Analyzing geographical maps of the study region identified four unique microclimate areas; sub-humid, semi-arid, arid and Saharan. The geographical distribution and molecular identification of species shows that the Saharan microclimate, characterized by the presence of Leishmania major was the most affected (47.78%) followed by semi-arid area where Leishmania tropica was identified in three districts. Among several environmental factors included in the study, poverty had the greatest influence on the spatial extension of the disease in this province. CONCLUSIONS: The incidence of CL in Boulemane Province varies between microclimate areas, and environmental factors partly explain this variation. However, the existence of CL in the most affected districts is mainly related to poverty, population movement and human behavior. To our knowledge, this the first study utilizing molecular techniques to confirm L. tropica and L. major as the causative agents of CL in Boulemane Province. Our findings indicate that the spatial and temporal distribution of CL in Boulemane Province is strongly related to poverty and population movement.

Epidemiological characteristics of visceral leishmaniasis in Morocco (1990-2014): an update.

Leishmaniases are parasitic diseases frequent in the Mediterranean Basin. Visceral leishmaniasis (VL) is a notifiable parasitic disease that increased in incidence in Morocco over the past few years and has recently emerged in several new foci, causing a public health problem in Morocco. The aim of this study is to describe the spatio-temporal distribution of VL in Morocco between 1990 and 2014 period in order to highlight important features and trends of VL and its epidemiology and to assess whether the activity of the unit reflects the situation of the disease at the national level and whether it could constitute an indicator of public health relevance. Two thousand four hundred and twenty one cases were reported in Morocco between 1990 and 2014 with an average annual reported incidence rate of 0.4 cases per 100.000 inhabitants. Before 1996 the average annual incidence of VL was 50 cases on average. After this date the number of cases increased and then remained stable with around 100-150 cases per year. Children whose age varies between 1 and 4 years old are the most affected with 1327 (74%) of total cases; nevertheless the adult starts to be affected by the disease. In 2000, 65% of positive cases of VL are concentrated at both northern regions: Taza-Al Hoceima- Taounate with 45% of cases, Tanger- Tetouan mainly represented by Chefchaoun with 20% of cases. The Fez-Boulemane region located in the center recorded 12% of cases. Throughout the years the map VL distribution has been progressively changed and spatial spread of the disease to the center is noted in 2007. 2014 has been marked by an even greater extension of the disease to the center and south of Morocco. Nationally in 2014, 34 of 75 provinces and prefectures are affected compared to 2000, when 22 out of 82 provinces and prefectures were affected. Leishmania infantum was identified the causative agent based on species- specific PCR-Lei70 assay. VL remains a sporadically endemic parasitic disease in Morocco with a progressive extension of its range of distribution. Such a situation would relate to the geographical succession of Phlebotomine sand fly vectors, the difficulty of actions against the canine population reservoirs of L. infantum and unfavorable socio-economic factors.

New epidemiological aspects of visceral and cutaneous leishmaniasis in Taza, Morocco.

BACKGROUND: Leishmaniasis is considered among the main endemic diseases in Morocco. However, further knowledge about epidemiological aspects of this disease is needed in several provinces to plan control and preventive strategies to tackle the disease. The present study aims to determine the epidemiological aspect of cutaneous and visceral leishmaniasis in Taza Province from 2007-2014 and to identify the circulating species in this province. RESULTS: The temporal study from 2007 to 2014 showed that the number of cutaneous leishmaniasis cases increased since 2010. During the period of study, most leishmaniasis cases were detected in both urban and rural areas with 34% of cases detected in two urban localities, Bab Zitouna and Bab tété with 297 and 106 cases, respectively. The molecular study of cutaneous leishmaniasis showed the presence of non-sporadic Leishmania infantum and Leishmania tropica in this province. Regarding visceral leishmaniasis, Leishmania infantum is the species that has been identified. CONCLUSIONS: The epidemio-molecular study of leishmaniasis in Taza Province showed the coexistence of two species of Leishmania in the same foci. They also indicated that CL due to Leishmania infantum is more prevalent than reported in the literature. These results will be helpful for the implementation of control strategies by targeting dogs that constitute a reservoir of Leishmania infantum.

Schistosoma haematobium detection in snails by DraI PCR and Sh110/Sm-Sl PCR: further evidence of the interruption of schistosomiasis transmission in Morocco.

BACKGROUND: This is the first study in Morocco to estimate snail infection rates at the last historic transmission sites of schistosomiasis, known to be free from new infection among humans since 2004. Screening of large numbers of snails for infection is one way to confirm that Schistosoma haematobium transmission has stopped and does not resurge. METHODS: A total of 2703 Bulinus truncatus snails were collected from 24 snail habitats in five provinces of Morocco: Errachidia, El Kelaa des Sraghna, Tata, Beni Mellal, and Chtouka Ait Baha. All visible snails were collected with a scoop net or by hand. We used waders and gloves as simple precautions. Snails were morphologically identified according to Moroccan Health Ministry guide of schistosomiasis (1982).All snails were analyzed in pools by molecular tool, using primers from the newly identified repeated DNA sequence, termed DraI, in the S. haematobium group. To distinguish S. bovis and S. haematobium, the snails were analyzed by Sh110/Sm-Sl PCR that was specific of S. haematobium. RESULTS: The results showed that snails from Errachidia, Chtouka Ait Baha, sector of Agoujgal in Tata and sector of Mbarkiya in El kelaa des Sraghna were negative for DraI PCR; but, snails from remaining snail habitats of El Kelaa des Sraghna, Tata and Beni Mellal were positive. This led to suggest the presence of circulating schistosome species (S. haematobium, S. bovis or others) within these positive snail habitats. Subsequently, confirmation with S. haematobium species specific molecular assay, Sh110/Sm-Sl PCR, showed that none of the collected snails were infected by S. haematobium in all historic endemic areas. CONCLUSION: The absence of S. haematobium infection in snails supports the argument of S. haematobium transmission interruption in Morocco.

National serologic survey of Haematobium schistosomiasis in Morocco: evidence for elimination.

The Moroccan Health Ministry launched a Process of Eliminating Schistosomiasis in 1994. During 2005-2009, the epidemiologic status showed a clear interruption of disease transmission at the national level; only a few residual cases were recorded. Our present study is the first systematic serologic survey to evaluate the transmission status in remaining disease-endemic foci. A study population of 2,382 children born after the date of the last autochthonous cases were selected from provinces with histories of high schistosomiasis transmission (Tata, Chtouka Ait Baha, Errachidia, El Kelaa Des Sraghna, and Beni Mellal). To identify the presence of disease, specific antibodies directed against Schistosoma haematobium adult worm microsomal antigens were detected by using an enzyme-linked immunoelectrotransfer blot assay. The results showed an absence of antibodies in all serum samples. Consequently, our findings confirm either a low transmission status or an interruption of schistosomiasis transmission within the last disease endemic foci.

New clinico-epidemiologic profile of cutaneous leishmaniasis, Morocco.

During the past 20 years, cutaneous leishmaniasis has emerged as a major public health threat in Morocco. We describe distribution of Leishmania major and L. tropica in Morocco and a new focus of cutaneous leishmaniasis due to L. infantum. We recommend using molecular techniques to diagnose suspected leishmaniasis cases.