Geographical distribution and molecular survey of freshwater snail intermediate hosts of Schistosoma haematobium by DraI/Sh73 PCR in eliminated foci of Morocco.

Morocco had reached the level of schistosomiasis elimination 16 years ago. However the spread of freshwater snails in several breeding sites, and imported schistosome infection, still exist. Therefore, snail survey is a crucial component to sustain elimination progress. This study aimed to evaluate and to incorporate DraI/Sh73 PCR, for detecting early prepatent Schistosoma haematobium infection in snail host, into epidemiologic surveillance for schistosomiasis, particularly in reportedly eliminated foci where S.bovis overlaps with S. haematobium. The geographical distribution and the density of Bulinus truncatus and Planorbarius metidjensis were monitored for six years (2014-2019) and snail sampling were conducted in Fkih Ben Saleh province. All snails were analyzed in pools by DraI/Sh73 PCR. Results showed absence of Planorbarius metidjensi and none of the collected Bulinus truncatus snails were infected by S. haematobium. DraI/Sh73 PCR using pooled snail extracts is specific, feasible and suitable in routine malacological survey in the post elimination phase of schistosomiasis in Morocco.

High performance of a novel point-of-care blood test for Toxoplasma infection in women from diverse regions of Morocco.

Point-of-care (POC) testing for Toxoplasma infection has the potential to revolutionize diagnosis and management of toxoplasmosis, especially in high-risk populations in areas with significant environmental contamination and poor health infrastructure precluding appropriate follow-up and preventing access to medical care. Toxoplasmosis is a significant public health challenge in Morocco, with a relatively heavy burden of infection and, to this point, minimal investment nationally to address this infection. Herein, we analyse the performance of a novel, low-cost rapid test using fingerstick-derived whole blood from 632 women (82 of whom were pregnant) from slums, educational centres, and from nomad groups across different geographical regions (i.e. oceanic, mountainous) of Morocco. The POC test was highly sensitive and specific from all settings. In the first group of 283 women, sera were tested by Platelia ELISA IgG and IgM along with fingerstick whole blood test. Then a matrix study with 349 women was performed in which fingerstick - POC test results and serum obtained by venipuncture contemporaneously were compared. These results show high POC test performance (Sensitivity: 96.4% [IC95 90.6-98.9%]; Specificity: 99.6% [IC95 97.3-99.9%]) and high prevalence of Toxoplasma infection among women living in rural and mountainous areas, and in urban areas with lower educational levels. The high performance of POC test confirms that it can reduce the need for venipuncture and clinical infrastructure in a low-resource setting. It can be used to efficiently perform seroprevalence determinations in large group settings across a range of demographics, and potentially expands healthcare access, thereby preventing human suffering.

Retrospective study on imported schistosomiasis in Morocco between 2005 and 2017.

Since 2004, no indigenous cases of schistosomiasis have been found in Morocco; only imported cases have been detected. The aim of the present study was to describe and analyse the epidemiological profile of imported schistosomiasis between 2005 and 2017, and, by this, attract attention to the probability of a reintroduction of this disease. During this period, 27 cases were recorded in Morocco, with a male predominance (13:1). All cases reported were found among African immigrants from Mauritania (37%), Mali (18%) and Senegal (15%). Schistosoma heamatobium was the most dominant specie. Most cases were reported in Rabat and Agadir, where there are many snail habitats. To prevent a re-emergence of the disease, the main challenge would be to consolidate and maintain a sustainable surveillance and control system of the importation of bilharzia. The frequency of asymptomatic schistosomiasis justifies a systematic health check-up for all travellers, migrants and immigrants.

Cystic Echinococcosis in Three Locations in the Middle Atlas, Morocco: Estimation of the Infection Rate in the Dog Reservoir.

A longitudinal study was carried out in Middle atlas, Morocco (locality of Had Oued Ifrane) in a population of 255 dogs from three localities, including two categories of dogs (owned and stray dogs). The dogs were investigated three times over a period ranging from 4 to 8 months between December and August. At each investigation, dogs were treated with arecoline, inducing defecation and allowing feces collection. Dogs were further treated with praziquantel to clear them from Echinococcus granulosus. Microscopic examination of feces was performed to assess the infection status of dogs at each investigation, and positive samples underwent copro-PCR to determine the circulating strain of E. granulosus. A high prevalence of infestation ranging from 23.5% to 38.8% and from 51.3% to 68.5% was, respectively, found in owned and in stray dogs. The PCR results revealed the presence of G1 strain in all positive samples. A logistic regression model was used to determine the incidence of infestation and showed that stray dogs underwent a significantly higher risk of infection (odds ratio = 14; 95% confidence interval: 6-30; p < 0.001) compared with owned dogs. Only anthelmintic treatment intervals of 2 months efficiently prevented egg shedding in owned and stray dogs. The seasonal effect was also significant, with the highest risk of reinfestation in winter and the lowest risk in summer. This study confirms that stray dogs undergo an increased risk of infestation by E. granulosus and indicate that infective pressure is influenced by season.

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.

[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.

Selecting accurate post-elimination monitoring tools to prevent reemergence of urogenital schistosomiasis in Morocco: a pilot study.

BACKGROUND: After alleged stop of transmission of schistosomiasis and further down the line in post elimination settings, sensitive tools are required to monitor infection status to prevent potential re-emergence. In Rahala, where transmission cycle of Schistosoma haematobium is interrupted since 2004 but where 30% of snails are still infected by S. bovis, potential human S. bovis infection can't be excluded. As methods based on egg-counts do not provide the required sensitivity, antibody or antigen assays are envisaged as the most appropriate tools for this type of monitoring. METHODS: In this pilot study, the performances of three assays were compared: two commercially available antibody tests (ELISA and haemagglutination format) indicating exposure, and an antigen test (lateral flow strip format) demonstrating active infection. All 37 recruited study participants resided in Rahala (Akka, province Tata, Morocco). Participants had been diagnosed and cured from schistosomiasis in the period between 1983 and 2003. In 2015 these asymptomatic participants provided fresh clinical samples (blood and urine) for analysis with the aforementioned diagnostics tests. RESULTS: No eggs were identified in the urine of the 37 participants. The haemagglutination test indicated 6 antibody positives whereas the ELISA indicated 28 antibody positives, one indecisive and one false positive. ELISA and haemagglutination results matched for 18 individuals, amongst which 5 out of 6 haemagglutination positives. With the antigen test (performed on paired serum and urine samples), serum from two participants (cured 21 and 32 years ago) indicated the presence of low levels of the highly specific Schistosoma circulating anodic antigen (CAA), demonstrating low worm level infections (less than 5 pg/ml corresponding to probably single worm pair). One tested also CAA positive with urine. ELISA indicated the presence of human anti-Schistosoma antibodies in these two CAA positive cases, haemagglutination results were negative. CONCLUSIONS: To prevent reemergence of schistosomiasis in Morocco current monitoring programs require specific protocols that include testing of antibody positives for active infection by the UCP-LF CAA test, the appropriate diagnostic tool to identify Schistosoma low grade infections in travelers, immigrants and assumed cured cases. The test is genus specific will also identify infections related to S. bovis.

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.