Mise en place de la culture in vitro de Mycobacterium ulcerans à partir d’échantillons cliniques versus recherche de BAAR et détection du génome bactérien à Abidjan, Côte d’Ivoire

Abstract

Mycobacterium ulcerans infections are a public health problem in Céte d'Ivoire. The etiological diagnosis of this disease made by culture remains a big concern due to the slowness and difficulties encountered. This detection by culture of M. ulcerans represents a big interest as it allows obtaining the circulating strains for research. The purpose of this study was to determine on a routine basis in a poorly equipped laboratory, in vitro culture of M. ulcerans from exudates of skin ulcerations and from biopsy of patients with suspected Buruli ulcer. A particular attention was paid to the conditioning of the sample forwarded to the laboratory and inoculation in Lowenstein-Jensen medium supplemented with glycerol. The results of the three methods for the analysis showed 26.7, 57.4 and 17.8% positive rate respectively in the microscopy examination by nested PCR and by culture. In all the analysis, the positive rate from biopsy is higher than that obtained from exudates. The overall contamination rate by invasion of the three tubes of culture by fungi is 15.8 with 14.3 and 19.4% respectively,from exudates and biopsies. All positive samples in Ziehl-Neelsen staining and in culture were also positive by nested PCR. The nested PCR confirmed the positive strains found in culture, which were responsible for skin ulcerations. After culture, only one strain was nPCR negative. This strain was identified as Mycobacterium Gordonae. Our culture conditions showed that M. ulcerans was not the only strain identified and that other strains were present in the culture. We can conclude that the culture of M. ulcerans, in spite of the growth difficulties of the bacterium can be performed in laboratory in developing countries despite the lack of reagent and consumables. The implementation of this culture is the only way to determine sensitivity tests in vitro and in vivo in order to treat patients with Buruli ulcer.

First isolation of Mycobacterium ulcerans from Swabs and Fine-Needle-Aspiration Specimens in Togo.

“…The rate observed in this study is similar to the WHO recommendation. However in other studies some authors found a positivity rate high [18,19] or low to compare to our rate [9,20]. The different of positivity rate observed could be explained by the number of samples cultured but especially by the decontamination method.…”

“…There is no difference between the positivity rate of the direct examination (44.28%) and culture (36%) (P = 0.37) but the positivity rate of the two techniques were lower than the qPCR. These rates are online with some studies and also the recommendations of WHO [9,20,21,26]. This isexplainedbythe capacity of IS2404-qPCR to reliably detect low genome copies of the bacteria in samples containing live or dead bacteria [27].…”

“…The isolation of M. ulcerans from clinical specimens is a slow and difficult process due to many factors, including bacteria growing extremely slowly [6-8 weeks] and growing on media that are often contaminated by other fast-growing bacteria. This makes the culture technique difficult to rapid confirmation of BU cases in the laboratory [7][8][9][10][11]. Despite this, the culture of M. ulcerans is of great epidemiological interest because it makes it possible to characterize the circulating strains, an essential step in the determination of the resistance of M. ulcerans to antibiotics.…”