Establishing African genomics and bioinformatics programs through annual regional workshops.

The African BioGenome Project (AfricaBP) Open Institute for Genomics and Bioinformatics aims to overcome barriers to capacity building through its distributed African regional workshops and prioritizes the exchange of grassroots knowledge and innovation in biodiversity genomics and bioinformatics. In 2023, we implemented 28 workshops on biodiversity genomics and bioinformatics, covering 11 African countries across the 5 African geographical regions. These regional workshops trained 408 African scientists in hands-on molecular biology, genomics and bioinformatics techniques as well as the ethical, legal and social issues associated with acquiring genetic resources. Here, we discuss the implementation of transformative strategies, such as expanding the regional workshop model of AfricaBP to involve multiple countries, institutions and partners, including the proposed creation of an African digital database with sequence information relating to both biodiversity and agriculture. This will ultimately help create a critical mass of skilled genomics and bioinformatics scientists across Africa.

Chromosome Abnormalities Related to Reproductive and Sexual Development Disorders: A 5-Year Retrospective Study.

Chromosomal abnormalities are the main genetic risk factor associated with reproductive and sexual development disorders (DSD). The goal of this study is to retrospectively evaluate the frequency of chromosomal aberrations in Moroccan subjects with problems of procreation or sexual ambiguity. A total of 1005 individuals, including 170 infertile couples, underwent cytogenetic analysis in the Cytogenetic Laboratory of the Pasteur Institute of Morocco. Heparinized blood samples were processed according to the standard karyotype method. A total (81.5%) of the patients studied had a normal karyotype, while the remaining (18.5%) patients had an abnormal karyotype. Female patients had more chromosomal abnormalities (52%) than male patients (48%). These chromosomal aberrations included 154 cases (83%) of sex chromosomal abnormalities, the most common being Turner's syndrome and Klinefelter's syndrome, and 31 cases (17%) had autosomal aberrations, especially chromosome 9 reversal (inv(9)(p12;q13)). The present data shows that among 170 couples, 10.6% had chromosomal abnormalities mainly involved in the occurrence of recurrent miscarriages. Genotype-phenotype correlations could not be made, and therefore, studies using more resolutive molecular biology techniques would be desirable.

Epidemiological features of a recent zoonotic cutaneous leishmaniasis outbreak in Zagora province, southern Morocco.

BACKGROUND: Leishmania major is an endemic vector-borne disease in Morocco that causes zoonotic cutaneous leishmaniasis (ZCL), especially in arid pre-Saharan regions where its unique vector and reservoir are Phlebotomus papatasi and Meriones shawi, respectively, and may cause epidemics. In late 2017, the Zagora province, an endemic focus for ZCL in southern Morocco, had CL outbreak. The main objective of our investigation was to analyze the epidemiological features of this latest ZCL outbreak. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed epidemiological features of this latest ZCL outbreak. The Regional Delegation of Health, Zagora, recorded 4,402 CL patients between October 2017 and end of March 2018. Our findings showed that 24 municipalities were affected and majority (55.1%) of infected cases belonged to the Tinzouline rural municipality. Majority of patients were females (57.2%). While all age group patients were affected, those aged <10 years were the most affected (42.1%). During this outbreak over 5 days in December 2017, we conducted a survey in Tinzouline and recruited and sampled 114 CL patients to confirm CL diagnosis by parasitological (direct examination and culture) and molecular (ITS1-PCR) methods and identify the etiological agent of infection using ITS1-PCR-RFLP and sequencing. We completed a detailed questionnaire including clinical and epidemiological data for each patient and found 72.8% of patients presenting multiple lesions (≥2), with an average number of lesions of 5.16 ± 0.5. Lesions were more prevalent in the upper limbs, with the most common type being the ulcerocrusted lesion (60.5%). We detected no associations between lesion type and patients' sex or age. CONCLUSIONS/SIGNIFICANCE: Among 114 clinically diagnosed CL patients, we confirmed 90.35% and identified L. major as the species responsible for this outbreak. Self-medication using various products caused superinfection and inflammation of lesions and complicated the diagnosis and treatment. Thus, ZCL remains a major public health problem in the Zagora province, and commitment of all stakeholders is urgently required to implement a sustainable regional control program.

Genetic diversity of Leishmania tropica in Morocco: does the dominance of one haplotype signify its fitness in both predominantly anthropophilic Phlebotomus sergenti and human beings?

In Morocco, cutaneous leishmaniasis (CL) caused by Leishmania tropica is endemic to locations where the predominantly anthropophilic blood-feeding Phlebotomus sergenti and humans co-perpetuate. The objective of this study was to explore whether the range of epidemiological features of CLcould be linked to the range of L. tropica genetic heterogeneity and to further explore the relationships between the genetic diversity of L. tropica in both P. sergenti and humans. L. tropica DNAwas extracted from dermal scarping smears of 125 CLpatients. Genetic polymorphisms were analyzed by sequencing the internal transcribed spacer (ITS) 1 and 5.8S rDNAgene. Nucleotide diversity (π), haplotype diversity (Hd) and Tajima's D test for neutrality, as well as overall and pairwise FSTvalues, were calculated using Arlequin ver 3.5 software. Out of the 125 amplified DNAsequences, 93 were completely sequenced and 13 L. tropica haplotypes were identified, which confirmed the significant genetic heterogeneity of L. tropica in Morocco. The most common haplotype included 74 out of 93 sequences; this haplotype is not only widely represented but was also detected in P. sergenti, which is known to be the most abundant species in the studied foci. Considering the negative value calculated using Tajima's D index, we briefly discussed the hypothesis that the L. tropica common haplotype propagation could be a sign of its fitness in P. sergenti and human hosts. Furthermore, analysis of molecular variance (AMOVA) shows significant correlations between intraspecific variants of L. tropica and patients' geographic origins. The long-term goals of the present pilot study are to further explore the relationships between the genetic diversity of L. tropica in human and P. sergenti populations.

Leishmania Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification.

Protozoan parasites of the genus Leishmania adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern Leishmania genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new Leishmania clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to in vitro culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various Leishmania isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast in vitro growth. Together our data draw a complex picture of Leishmania genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain.IMPORTANCE Protozoan parasites of the genus Leishmania cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of Leishmania biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the L. donovani, L. major, and L. tropica complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery.

First Molecular Identification and Phylogeny of Moroccan Anopheles sergentii (Diptera: Culicidae) Based on Second Internal Transcribed Spencer (ITS2) and Cytochrome c Oxidase I (COI) Sequences.

Anopheles sergentii known as the "oasis vector" or the "desert malaria vector" is considered the main vector of malaria in the southern parts of Morocco. Its presence in Morocco is confirmed for the first time through sequencing of mitochondrial DNA (mDNA) cytochrome c oxidase subunit I (COI) barcodes and nuclear ribosomal DNA (rDNA) second internal transcribed spacer (ITS2) sequences and direct comparison with specimens of A. sergentii of other countries. The DNA barcodes (n = 39) obtained from A. sergentii collected in 2015 and 2016 showed more diversity with 10 haplotypes, compared with 3 haplotypes obtained from ITS2 sequences (n = 59). Moreover, the comparison using the ITS2 sequences showed closer evolutionary relationship between the Moroccan and Egyptian strains than the Iranian strain. Nevertheless, genetic differences due to geographical segregation were also observed. This study provides the first report on the sequence of rDNA-ITS2 and mtDNA COI, which could be used to better understand the biodiversity of A. sergentii.

Genetic polymorphism in Leishmania infantum isolates from human and animals determined by nagt PCR-RFLP.

BACKGROUND: Leishmania infantum is the causative agent of human visceral leishmaniasis (VL) and sporadic human cutaneous leishmaniasis (CL) in the Mediterranean region. The genetic variation of the Leishmania parasites may result in different phenotypes that can be associated with the geographical distribution and diversity of the clinical manifestations. The main objective of this study was to explore the genetic polymorphism in L. infantum isolates from human and animal hosts in different regions of Morocco. METHODS: The intraspecific genetic variability of 40 Moroccan L. infantum MON-1 strains isolated from patients with VL (n = 31) and CL (n = 2) and from dogs (n = 7) was evaluated by PCR-RFLP of nagt, a single-copy gene encoding N-acetylglucosamine-1-phosphate transferase. For a more complete analysis of L. infantum polymorphism, we included the restriction patterns of nagt from 17 strains available in the literature and patterns determined by in-silico digestion of three sequences from the GenBank database. RESULTS: Moroccan L. infantum strains presented a certain level of genetic diversity and six distinct nagt-RFLP genotypes were identified. Three of the six genotypes were exclusively identified in the Moroccan population of L. infantum: variant M1 (15%), variant M2 (7.5%), and variant M3 (2.5%). The most common genotype (65%), variant 2 (2.5%), and variant 4 (7.5%), were previously described in several countries with endemic leishmaniasis. Phylogenetic analysis segregated our L. infantum population into two distinct clusters, whereas variant M2 was clearly distinguished from both cluster I and cluster II. This distribution highlights the degree of genetic variability among the Moroccan L. infantum population. CONCLUSION: The nagt PCR-RFLP method presented here showed an important genetic heterogeneity among Moroccan L. infantum strains isolated from human and canine reservoirs with 6 genotypes identified. Three of the six Moroccan nagt genotypes, have not been previously described and support the particular genetic diversity of the Moroccan L. infantum population reported in other studies.

Molecular identification of Leishmania infection in the most relevant sand fly species and in patient skin samples from a cutaneous leishmaniasis focus, in Morocco.

BACKGROUND: Cutaneous leishmaniasis (CL) is an infectious disease caused by various species of Leishmania and transmitted by several species of sand flies. CL is among the most neglected tropical diseases, and it has represented a major health threat over the past 20 years in Morocco. The main objectives of this study were to identify relevant sand fly species and detect Leishmania infection in the most prevalent species and patient skin samples in Taza, a focus of CL in North-eastern Morocco. METHODOLOGY AND FINDING: A total of 3672 sand flies were collected by CDC miniature light traps. Morphological identification permitted the identification of 13 species, namely 10 Phlebotomus species and 3 Sergentomyia species. P. longicuspis was the most abundant species, comprising 64.08% of the total collected sand flies, followed by P. sergenti (20.1%) and P. perniciosus (8.45%). Using nested-kDNA PCR, seven pools of P. sergenti were positive to Leishmania tropica DNA, whereas 23 pools of P. longicuspis and 4 pools of P. perniciosus tested positive for Leishmania infantum DNA. The rates of P. longicuspis and P. perniciosus Leishmania infection were 2.51% (23/915) and 7.27% (4/55), respectively, whereas the infection prevalence of P. sergenti was 3.24%. We also extracted DNA from lesion smears of 12 patients suspected of CL, among them nine patients were positive with enzymatic digestion of ITS1 by HaeIII revealing two profiles. The most abundant profile, present in eight patients, was identical to L. infantum, whereas L. tropica was found in one patient. The results of RFLP were confirmed by sequencing of the ITS1 DNA region. CONCLUSION: This is the first molecular detection of L. tropica and L. infantum in P. sergenti and P. longicuspis, respectively, in this CL focus. Infection of P. perniciosus by L. infantum was identified for the first time in Morocco. This study also underlined the predominance of L. infantum and its vector in this region, in which L. tropica has been considered the causative agent of CL for more than 20 years.

New epidemiological pattern of cutaneous leishmaniasis in two pre-Saharan arid provinces, southern Morocco.

Three Leishmania species are responsible of cutaneous leishmaniasis (CL) in Morocco. Zoonotic CL due to Leishmania major and Leishmania infantum, the first is known as established in the eastern arid regions, whereas the latter evolves sporadically, especially in the North. While Leishmania tropica, classically considered anthroponotic, is endemic in the semi-arid regions and is largely distributed throughout the country. The aim of this study was to identify the Leishmania species causing CL in two Provinces in arid pre-Saharan region known as zoonotic CL foci, and to contribute an update to the national data concerning the distribution of Leishmania species in both regions. The recruitment of patients was done in six localities in Ouarzazate and Zagoura provinces in 2015 and 2016. Out of 81 samples collected, 66 were positive (81%) by ITS1-PCR amplification of Leishmania DNA extracted from stained smears. The highest rate of Leishmania infection was registered in children aged 9 years or less (71,2%). The ITS1-PCR- RFLP analysis revealed the predominance of L. major infecting 52 patients (79%), followed by L. tropica in 12 patients (18%) and L. infantum in 2 patients who had no history of travel outside the studied area (3%). The sequencing of the ITS1 of both L. infantum, showed 100% similarities with L. infantum strains isolated from dogs and visceral leishmaniasis patients from the south and north of Morocco. The coexistence of the 3 Leishmania species in the same focus, and the difficult distinction of infections associated to the different Leishmania species based only on clinical lesions' aspects complicate the diagnosis and then the national control strategy, as well as the therapeutic management. The epidemiological pattern of CL in the studied areas appears to have changed during the last decades, from a predominant zoonotic CL caused by L. major to a polymorphic disease that can be due to any of the 3 Leishmania species. The expansion of L. infantum and L. tropica in southern parts of Morocco, calls for in depth epidemiological investigations for a better understanding of the CL situations in Southern parts of the country and for an assessment of the climate impact and environment changes on the leishmaniasis transmission system.

Intraspecific genetic variability in a population of Moroccan Leishmania infantum revealed by PCR-RFLP of kDNA minicircles.

In Morocco, Leishmania infantum is the main etiologic agent of human and canine visceral leishmaniasis (VL). This species has been proven to be an opportunistic agent in HIV+ patients and is also responsible of sporadic cutaneous leishmaniasis (CL).This work aims to evaluate the genetic variability of Moroccan L. infantum strains based on PCR-RFLP analysis of the kinetoplastid DNA (kDNA) minicircles. A total of 75 DNA samples extracted from positive Giemsa-stained smears (n=32) and from L. infantum cultures (n=43) was studied. The samples have been taken from VL patients infected (n=7) or not (n=56) by HIV, patients with CL (n=2) and finally from infected dogs (n=10). An hypervariable region of kDNA was amplified using the primers MC1 and MC2; the PCR products were digested separately by a panel of nine restriction enzymes. The presence or absence of restriction fragments was scored in a binary matrix and the SplitsTree4 software was used for the construction of a Neighbor-Net network. Moroccan L. infantum population showed an important level of variability with the identification of 6 genotypes. For each genotype a PCR product was sequenced, confirming the presence of all the expected restriction sites. The predominant profile was the genotype B. A new genotype, named Q was detected for the first time, whereas the four other genotypes (G, K, N and O) were reported sporadically in the Mediterranean basin. The Neighbor-Net network segregates our L. infantum population into 3 clusters: Cluster I includes genotype B, cluster II grouping the genotypes O, Q and G and finally the cluster III contains the genotype N. The kDNA-PCR-RFLP assay is suitable for use directly on biological samples; it reveals an important degree of genetic variability among L. infantum strains even those belonging to the same zymodeme what is of great epidemiological interest.