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

Background Buruli ulcer is a skin disease caused by a mycobacterium called Mycobacterium ulcerans . It is prevalent in more than 33 countries on several continents but West Africa is the most affected. The isolation in culture of the bacteria is difficult because of its slow growth and the facilities required. In Togo, studies have been done on the risk factors for Mycobacterium ulcerans infection and the detection of cases by the Ziehl-Neelsen and PCR technique on clinical and environmental samples, but to date no data of isolates from clinical samples are available. The purpose of this study was to perform an in vitro culture of M. ulcerans from swab and fine needle aspiration samples through the confirmation stages of direct examination and IS2404 -PCR. Method A total of 70 clinical samples from Togo and 10 clinical isolates from Benin are analyzed by the three techniques indicated in the diagnosis, in particular the direct examination of acid-fast bacilli (AFB) using the Ziehl-Neelsen staining, the PCR targeting the IS2404 sequence, and the culture after transport of the samples in a transport medium made of Middlebrook 7H9 medium supplemented with a mixture of PANTA and OADC and decontamination by the modified Petroff method. Results The application of the three techniques of diagnosis for clinical samples yielded 44.28% of positivity rates on direct examination of AFB, 35.71% on culture and 77.14% on qPCR IS2404 with a significantly higher rate for qPCR (0.0001). All samples positive for Ziehl-Neelsen staining and culture were also positive for qPCR. Conclusion : Our results show that the culture, despite it difficulty and the slow growth of the bacteria, can be carried out with recommended tools of the mycobacteria culture and a good method of decontamination of the samples can improve the positivity rates. Its realization will allow the assessment of the in vitro sensitivity to the antibiotics used in the treatment and the discovery of new strains of Mycobacterium ulcerans .

[1].The outbreaks are geographically almost always circumscribed around an aquatic ecosystem (river, artificial or natural lake, marsh area, irrigation system) [2,3]. This disease is the third most common mycobacterial infection in human after tuberculosis and leprosy [4] The notification of Buruli ulcer cases is based on confirmation in the laboratory by the WHO recommended tests for the diagnosis of the disease, including direct examination of smears for acidfast bacilli (AFB); in vitro culture and gene amplification (PCR) targeting the genome sequence IS2404. According to WHO, 70% of BU cases should be confirmed by the PCR-IS2404 gene amplification technique [5,6]. 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. There are applications of the culture of M.ulcerans in several studies [12][13][14][15].
In Togo, studies have been done on the risk factors for Mycobacterium ulcerans infection and the detection of cases by the Ziehl-Neelsen and PCR technique on clinical and environmental samples [2,16,17], and to date no data of isolates from clinical samples are available. The objective of this study is to perform an in vitro culture method to isolate circulating clinical strains of M. ulcerans in Togo from fine needle aspiration (FNA) and swabs samples.

Study sites
The study was conducted from January 2018 to September 2019 at the National Reference Center for the Treatment of Buruli Ulcer located at the regional hospital of Tsévie (CNRT-UB) where patients were samples were collected. The laboratory of this center was used for direct examination of the smears. The culture was performed at the reference laboratory for mycobacteria at the Sylvanus Olympio teaching hospital. The DNA amplification using PCR technique was conducted at the national reference laboratory for Buruli ulcer at the National Institute of Hygiene (INH).

Sampling
Control strains of M. ulcerans Ten strains of M. ulcerans received from the Reference laboratory of mycobacteria of Benin (RLM) were used as control for identification of our isolates and as quality of culture of clinical specimens.
Clinical samples 70 samples were collected from suspected patients of Buruli ulcer who visited the national center for treatment of BU according to WHO criteria. These samples are the FNAs collected from non-ulcerated lesions (nodules, plaques or edema) and swabs from ulcers [5,6].For each type of lesion, three samples were collected. The first was used for culture and put in a screw-cap tube containing a transport medium consisted of 2 mL of Middlebrook 7H9 broth medium (Becton Dickinson) supplemented with a mixture of PANTA (Polymyxin B, Amphotericin B, Nalidixic acid, Trimethoprim and Azlocillin) and OADC (Oleic acid, Albumin, Dextrose and Catalase) (Becton Dickinson). The second sample was used for DNA amplification by qPCR is collected in a tube containing cell lysis solution (CLS, Qiagen Germany) and the third for a smear for Ziehl-Neelsen staining. All samples were taken after the consent of the patients was obtained [5,6].

Laboratory analysis
Ziehl-Neelsen staining Direct smears for microscopy were prepared from swab/FNA samples and control strains then subjected to Ziehl-Neelsen staining for detection of acid fast bacilli. Slides were analyzed by microscopy according to the WHO recommended grading system [5].

Control strains
Benin strains used as growth control previously stored at -80°C were thawed and then subcultured in Middlebrook 7H9 liquid medium (Becton Dickinson) supplemented with a mixture of PANTA and OADC.
After four weeks of incubation at 31°C, theses cultures were subcultured onto Lowenstein-Jensen medium (Becton Dickinson Dyfco TM ) supplemented with glycerol prepared according to the   [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. Indeed, the transport medium used in our study is the Middlebrook 7H9 supplemented with PANTA and OADC which conserve the mycobacteria in the sample for a long time [6,21]. However, the method of decontamination with sodium hydroxide 4% and Sodium Chloride 0.85% (modified Petroff) used has a great impact on the viability not only for the non-acid-fast contaminants bacteria but it also kills 60-70 % of the mycobacteria present in the sample due to NaOH toxicity [22,23]. Compared to our decontamination method described above, other studies have employed the oxalic acid or the method with 2% cetylpyridinium chloride/4% sodium chloride which provided more positive culture and less contamination rate [19,24,25].
The culture has been compared to the Ziehl Nelssen and IS2404-qPCR. 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].
In our study, the incubation time required to obtain positive cultures of M. ulcerans from clinical specimens was 8 weeks compare to subcultures of control strains (3weeks). In both cases, the colonies observed were yellowish, rough corresponding to the African strains of M. ulcerans more yellowish than the Australian strains [5,6].

Conclusion
The