Clonality of Mycobacterium ulcerans by Using VNTR-MIRU Typing in Ivory Coast (Côte d'Ivoire), West Africa

International audienceBuruli ulcer (BU) is neglected skin disease caused by Mycobacterium ulcerans. The lack of early diagnosis and treatment causes severe disability. In Central and in West Africa, BU is endemic and its control is difficult because the most cases occur in rural regions. The molecular particularity of M. ulcerans was the acquisition of the virulence plasmid pMUM001. Genetic analyses have demonstrated the high diversity with variable number tandem repeats (VNTR) and Mycobacterial Interspersed Repetitive Units (MIRU) in M. ulcerans and in mycolactone producing Mycobacteria (MPMs). Objective: The objective of this study was to investigate the molecular diversity by using MIRU-VNTR method in clinical samples of BU patients in Côte d'Ivoire. Study Design: 21 clinical samples were collected from BU patients in different sites and were first analyzed in molecular diagnosis of BU using two targets insertion sequence IS2404 and keto reductase-B-domain (KR). In a second step, we have analyzed the strains by PCR typing for four specific and sensitive markers MIRU1, VNTR6, ST-1 and VNTR19. Results and Conclusion: 100% of clinical samples were positive in molecular tests for IS2404 and 95% for KR and confirm M. ulcerans in the samples. By PCR typing, we have found 61.9 % positive for MIRU1 and 52%, 85.7%, and 61.9% for VNTR6, ST-1 and VNTR19 respectively. One of sample was negative for all genotyping markers. Two different genetic profiles were identified by MIRU1 and ST-1 loci by gel-analyzed of the amplified products. The VNTR profile C (3,1,1) corresponding of 3 copies MIRU1, 1 copy VNTR6 and 1 copy ST-1 was detected in 28.5% of samples and confirms the West African genotype in Côte d'Ivoire. Different genetic strains of M. ulcerans were co-circulated in the same endemic region in the country. This study has described first the circulating of different genetic strains of M. ulcerans in Côte d'Ivoire

Community-based geographical distribution of Mycobacterium ulcerans VNTR-genotypes from the environment and humans in the Nyong valley, Cameroon

Background: Genotyping is a powerful tool for investigating outbreaks of infectious diseases and it can provide useful information such as identifying the source and route of transmission, and circulating strains involved in the outbreak. Genotyping techniques based on variable number of tandem repeats (VNTR) are instrumental in detecting heterogeneity in Mycobacterium ulcerans (MU) and also for discriminating MU from other mycobacteria species. Here, we describe and map the distribution of MU genotypes in Buruli ulcer (BU) endemic communities of the Nyong valley in Cameroon. We also tested the hypothesis of whether the suspected animal reservoirs of BU that share the human microhabitat are shedding contaminated fecal matters and saliva into their surrounding environments. Methods: Environmental samples from suspected MU-risk factors and lesion swabs from human patients were sampled in BU-endemic communities and tested for the presence of MU by qPCR targeting three independent sequences (IS 2404 , IS 2606 , KR-B). Positive samples to MU were further genotyped by VNTR with confirmation by sequencing of four loci (MIRU1, Locus 6, ST1, Locus 19). Results: MU was detected in environmental samples including water bodies (23%), biofilms (14%), detritus (10%), and in human patients (73%). MU genotypes D, W, and C were found both in environmental and human samples. The micro geo-distribution of MU genotypes from communities showed that genotype D is found both in environmental and human samples, while genotypes W and C are specific to environmental samples and human lesions, respectively. No obvious focal grouping of MU genotypes was observed at the community scale. An additional survey in the human microhabitat suggests that domestic and wild animals do not shed MU in their saliva and feces in sampled communities. Conclusions: VNTR typing uncovered different MU genotypes circulating in the endemic communities of the Akonolinga district. A MU environmental genotype was found in patients, yet the mechanism of contamination remains to be investigated; and recovering MU in culture from the environment remains key priority to enable a better understanding of the mode of transmission of BU. We also conclude that excretions from suspected animals are unlikely to be major sources of MU in the Nyong Valley in Cameroon.</p

Distribution of MU DNA targets in animal lesions according to animal species and BU locality in Benin.

<p>Distribution of MU DNA targets in animal lesions according to animal species and BU locality in Benin.</p

BU-like infected lesions in roaming domesticated animals.

<p>The lesions are shown as captured before any treatment on infected animals. <b>A) MU infected lesion on the abdominal part of a 3 years old female goat</b>. This BU-like lesion (1.8 cm diameter) appears reddish with undermined borders, a well circumscribed ulceration and a necrotic base. <b>B) MU infected lesion on the nape area of the neck of a 20 months aged female dog</b>. The type 1 lesion (1.4 cm diameter) characteristic of BU appears reddish in the center and whitish at the borders. The borders of this well-circumscribed lesion remain undermined.</p

Phylogenetic reconstruction of animal and human MU isolates and comparison with reference MU strains using MIRU1 orthologs.

<p>The evolutionary history was inferred using the UPGMA method in MEGA 6. The optimal tree with the sum of branch length = 1.459786 in shown. Bootstrapping values (1000 replicates) are shown in percentage next to the branches. MIRU1 reference orthologs (white triangle) were retrieved from GenBank with accession numbers given in the tree. Sequences of animal (black circle) and human (white diamond) MU isolates are clustered at the top of the tree.</p

Location of the sampling sites in Western and Central Africa.

<p><b>A)</b> BU endemic locality of Sedje-Denou in Benin; <b>B)</b> BU endemic locality of Akonolinga in Cameroon.</p