[Detection of the IS2404 insertion sequence and ketoreductase produced by Mycobacterium ulcerans in the aquatic Heteroptera in the health districts of Dabou and Tiassalé in Côte d'Ivoire]. / Mise en évidence de la séquence d'insertion IS2404 et de la kétoréductase (Kr) produite par Mycobacterium ulcerans chez les Hétéroptères aquatiques dans les districts sanitaires de Dabou et Tiassalé en Côte d'Ivoire.

Buruli ulcer (BU) disease, caused by Mycobacterium ulcerans, is a major public health problem in Côte d'Ivoire. Until now, the mode of BU transmission was unknown, but recent studies implicate aquatic Heteroptera in the chain of transmission. This study was launched in Côte d'Ivoire to search for specific genetic markers for M. ulcerans in these bugs, including the insertion sequence IS2404 and ketoreductase (Kr), both involved in the synthesis of mycolactone, a toxin produced by these mycobacteria. Samples of aquatic Heteroptera were collected monthly with deep nets from ponds near villages in the health districts of Dabou and Tiassalé. After identification and enumeration of the bugs, batches of the same taxon underwent real-time PCR to search for the IS2404 target and Kr. Saliva of 69 specimens of Diplonychus sp randomly selected in the samples was also analyzed by PCR. In all, 283 single-taxon batches were created. Thus, PCR identified 26 batches belonging to the families of Belostomatidae, Naucoridae, Corixidae, Ranatridae, and Nepidae as positive for both targets. The IS2404 insertion sequence and Kr were present in 6 of the 69 samples analyzed in the saliva of Diplonychus sp. These aquatic Heteroptera suspected of infection by M. ulcerans might release it into the environment because of their ability to fly. They might thus be the source of human contamination.

[Micronecta sp (Corixidae) and Diplonychus sp (Belostomatidae), two aquatic Hemiptera hosts and/or potential vectors of Mycobacterium ulcerans (pathogenic agent of Buruli ulcer) in Cote d'Ivoire]. / Micronecta sp (Corixidae) et Diplonychus sp (Belostomatidae), deux hémiptères aquatiques hôtes et/ou vecteurs potentiels de Mycobacterium ulcerans agent pathogène de l'ulcère de Buruli en Côte d'Lvoire.

Buruli ulcer is currently a major public health problem in Côte d'Ivoire. It is a neglected tropical disease closely associated with aquatic environments. Aquatic insects of the Hemiptera order have been implicated in human transmission of Mycobacterium ulcerans, the pathogenic agent of Buruli ulcer. The purpose of this preliminary study using the polymerase chain reaction (PCR) method was to evaluate aquatic insects in Sokrogbo, a village in the Tiassalé sanitary district where Buruli ulcer is endemic. Findings identified two water bugs hosting Mycobacterium ulcerans, i.e., one of the Micronecta genus in the Corixidae family and another of the Diplonychus genus in the Belostomatidae family. The PCR technique used revealed the molecular signatures of M. ulcerans in tissue from these two insects. Based on these findings, these two water bugs can be considered as potential hosts and/or vectors of M. ulcerans in the study zone. Unlike Diplonychus sp., this is the first report to describe Micronecta sp as a host of M. ulcerans. Further investigation will be needed to assess the role of these two water bugs in human transmission of M. ulcerans in Côte d'Ivoire.

[Implementation of in vitro culture of Mycobacterium ulcerans from clinical samples versus detection of acid-fast bachilli and bacterial genome in Abidjan, Côte d'Ivoire]. / Mise en place de la culture in vitro de Mycobacterium ulcerans à partir d'éhantillons cliniques versus recherche de BAARet détection du génome bactérien à Abidjan, Côte d'Ivoire

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.

Detection of viral RNA by RT-PCR from serum for molecular diagnosis of measles.

Measles continues to be a cause of morbidity and mortality in Côte d'Ivoire although the death rates are weak (2.4%). The monitoring and elimination programme of this disease require a laboratory confirmation testing by diverse methods of diagnosis needing diverse biological products. Serum is usually used for IgM detection. This study has therefore assessed the importance of the measles virus RNA detection from sera of measles suspected cases for confirmation of the case and determination of the genotype. A total of 45 sera tested were split into two groups according to the interval between the rash appearance and the day of blood collection: Group 1 (day 1 to day 3); group (2 day 4 to day 7). Four sera from Group 1 of the 45 (8.9 %) were positive by RT-PCR technique while 10 (22.2%) sera were positive for IgM anti- measles virus by ELISA test. RT-PCR and ELISA showed the same performance in group 1 with a positivity rate of 13.79 %. The B3 genotype was found. This result showed that the viral RNA can be detected in the serum but only from those sera collected the first 3 days after the rash appearance and could be used as palliative in case it is impossible to obtain other biological products.

[Qnr-type quinolone resistance in extended-spectrum beta-lactamase producing enterobacteria in Abidjan, Ivory Coast]. / Résistance aux quinolones de type qnr chez les entérobactéries productrices de bêta-lactamases à spectre élargi à Abidjan en Côte d'Ivoire.

The aim of the study was to show the emergence of the qnr genes in extended spectrum beta-lactamases producing enterobacteria in Abidjan between 2005 and 2006. The whole of 151 strains of extended spectrum beta-lactamases producing enterobacteria were studied: 64 Escherichia coli, 66 Klebsiella pneumoniae, seven Klebsiella oxytoca and 14 Enterobacter spp. isolated from various biological products and from in- and out-patients. The techniques of disks diffusion, double-disk synergy, E-test were respectively used for the antimicrobial susceptibility test, the detection of extended spectrum beta-lactamases and the minimal inhibiting concentration. The bla genes(SHV, TEM, CTXM groups 1, 2, 8, 9), and AmpC were determined by PCR and characterized by sequencing. A global prevalence of 27,2 % (41/151) and rates of 9,9, 14,6, 2,7 % for the qnr genes A, B, A and S were observed. The distribution was 42,9 % for Enterobacter spp, 31,2 % for Escherichia coli, 20,5 % for Klebsiella; 30 strains expressed at least two bla genes; four strains were associated with AmpC. The strains were resistant to the cotrimoxazole (97,6 %), to the céfépime (73,2 %), to the céfoxitine (56,1 %), to the imipénème (0 %) and 43,9 % to all the aminosides. This high qnr gene prevalence associated with several types of bla genes in epidemic matter, the high level of resistance to antibiotics make fear a high risk of the transmission of multi-resistants bacteria and challenge the authorities for a resistance monitoring policy.

[Detection by RT-PCR of the 1st cases of Astrovirus in human stools in Abidjan, Côte d'Ivoire]. / Détection par RT-pCR des premiers cas d'Astrovirus dans les selles humaines à Abidjan, Côte d'Ivoire.

Viral gastroenteritis are a problem of public health because of the high rate of morbidity and mortality, particularly in children. Among the etiologic agents, human Astroviruses are the third agents most often incriminated after Rotaviruses and Caliciviruses. Symptoms of gastroenteritis caused by Astroviruses are generally moderated compared with those observed with Rotaviruses and rarely involve hospitalization. In sub-Saharan Africa, particularly in Côte d'Ivoire, the majority of viral gastroenteritis is attributed to Rotavirus with rates varying from 20 to 26%. No study on the circulation of human Astroviruses has been carried out in Côte d'Ivoire. Our objective was to detect human Astroviruses in the diarrhoeal stools in Abidjan. Seventy-two samples of human diarrhoeal stools were collected in ambulatory patients. This population was made up of 44 patients from 0 to 15 and 28 patients over 15 years old. The concentration of the viral particles of the samples was followed by the extraction of the RNA by the modified method of Boom. The extracted RNA were amplified by RT-PCR by using specific primers targeting a portion of the 3' end of the open reading frame ORF la of the genome of human Astroviruses. The amplified fragment was 192 pb. The genome of human Astroviruses was detected in 3 stools out of the 72 samples. That is a frequency of 4%. Among these 3 stools, 2 came from 4 month and 3 year-old children and the 3rd stool came from a 33 year-old patient. For the first time this survey has pointed out the circulation of human Astroviruses in the Côte d'Ivoire population. This survey also showed that human Astroviruses could be found in children as well as in adults.

[Direct sputa examination limits in tuberculosis active detection in 200 immigrant candidates in United States of America]. / Limites de l'examen direct des expectorations dans le dépistage actif de la tuberculose chez 200 candidats à l'émigration aux Etats-Unis.

The Côte-d'Ivoire Pasteur Institute unit of Tuberculous and Nontuberculous Mycobacteria carried out 600 smears stained by Ziehl-Neelsen in 200 immigrant candidates for the U.S.A. The sputa of 44 of them were put in culture on Lowenstein-Jensen medium. Eight (4%) candidates had active pulmonary tuberculosis among whom 5 had smear negative sputum. The pulmonary tuberculosis active detection performed on target population with accessible and sensitive tool can contribute to strengthen the fight against tuberculosis in endemic areas.

[Detection of transfusion transmitted virus (TTV) DNA in sera among three different population groups in Abidjan, Côte d'Ivoire in 2001]. / Détection de l'ADN du virus transmis par la transfusion (VTT) dans le sérum de trois populations différentes à Abidjan, Côte d'Ivoire, en 2001.

The aim of this study was to determine the transfusion transmitted Virus (TTV) prevalence in three groups of population from Abidjan, Côte d'Ivoire. The A group contained 39 multitransfused patients, the B group contained 10 blood donors supposed to be healthy persons which have never been transfused and the group C contained 43 patients with chronic liver pathology. In this last group, 33 patients had HBV positive serology and the 10 others, HCV positive serology. We used PCR to investigate TTV in patients serum. Detection rates were comprised between 67% and 82%. This is the first study to provide information about the high portage of TTV in ivorian population.

[Contribution of gene amplification in Mycobacterium ulcerans detection in exudates and cutaneous biopsies in Côte d'Ivoire]. / Apport de l'amplification génique dans la détection de Mycobacterium ulcerans dans les exsudats et les biopsies cutanées en Côte d'Ivoire.

Mycobacterium ulcerans skin ulceration is a major issue of public health in Côte d'Ivoire. The diagnosis of M. ulcerans infection is hampered by the slow growth of the bacterium in culture, implying a delay of several weeks before a specific diagnosis can be obtained. In Côte d'Ivoire the diagnosis of Buruli ulcer is almost based on clinical features. During the last decade, many studies have demonstrated the extremely high capacity of PCR for rapidly and specifically detecting bacteria and genes of interest. That ability has revealed PCR as a powerful tool in clinical microbiology studies. In this study we evaluated the M. ulcerans detection in specimens of exudates and biopsies collected from patients clinically suspected of Buruli ulcer and treated in "Raoul Follereau" centre of Manikro in the North-central region of Côte d'Ivoire. The microscopic research of BAAR in 185 swabs loaded with skin lesions collected from these patients showed a positive rate of 14.6%. The PCR detection in 48 h or 72 h of the M. ulcerans IS2404 and IS2606 in the swabs and in the 26 biopsies, from these patients, showed positive rates of 15.7% and 84.6% respectively and in the same samples. These results obtained with PCR detection of M. ulcerans insertions sequences suggest that this technique performed with exudates and biopsy can be used to confirm a routine specific diagnosis of M. ulcerans and early screening of Buruli ulcer in Côte d'Ivoire.

Characterization of Brucella suis clpB and clpAB mutants and participation of the genes in stress responses.

Pathogens often encounter stressful conditions inside their hosts. In the attempt to characterize the stress response in Brucella suis, a gene highly homologous to Escherichia coli clpB was isolated from Brucella suis, and the deduced amino acid sequence showed features typical of the ClpB ATPase family of stress response proteins. Under high-temperature stress conditions, ClpB of B. suis was induced, and an isogenic B. suis clpB mutant showed increased sensitivity to high temperature, but also to ethanol stress and acid pH. The effects were reversible by complementation. Simultaneous inactivation of clpA and clpB resulted in a mutant that was sensitive to oxidative stress. In B. suis expressing gfp, ClpA but not ClpB participated in degradation of the green fluorescent protein at 42 degrees C. We concluded that ClpB was responsible for tolerance to several stresses and that the lethality caused by harsh environmental conditions may have similar molecular origins.

Geographical distribution of HIV-1 group O viruses in Africa.

OBJECTIVE: To determine to what extent HIV-1 group O strains are present in different African countries. MATERIALS AND METHODS: A total of 14,682 samples of sera from a range of patients from 12 different African countries were tested. All the sera were tested with an enzyme-linked immunosorbent assay (ELISA) using a combination of V3 peptides from ANT-70 and MVP-5180. Samples reactive in ELISA were retested in a line immunoassay (LIA-O). Samples reactive in ELISA were also retested with an in-house Western blot to determine the presence of antibodies to gp120 of HIV-1 ANT-70. Polymerase chain reaction was performed on HIV-1 group O and group O indeterminate sera. RESULTS: Of all the sera samples tested, only 19 sera had antibodies to group O V3 peptides exclusively and 46 were indeterminate for group O infection in LIA-O. The highest prevalence of HIV-1 group O infection among HIV-positive sera was observed in Cameroon (2.1%) and neighbouring countries, 1.1% in Nigeria and 0.9% in Gabon. The lowest rates were seen in west Africa: 0.07% in Senegal, 0.14% in Togo, 0.16% in Chad and 0.3% in Niger. Group O sera were observed in almost all the population categories tested. The ANT-70 V3 peptide in LIA-O was reactive with all of the sera considered to be HIV-1 group O antibody positive by LIA, versus 78.9% for the MVP-5180 peptide. Thirteen out of 19 group O samples of sera were tested in PCR. Eight samples were identified as group O by specific group O pol and/or V3 primers; in the remaining five samples no HIV RNA could be detected. Of the indeterminate sera samples, two were identified as group O. CONCLUSION: In eight of the 12 countries tested, antibodies to group O viruses were identified. Numbers of HIV-1 group O viruses are low. Their presence is not restricted to Cameroon and neighbouring countries but can also be found in west and south-east Africa.

PIP: An enzyme-linked immunosorbent assay (ELISA), using a combination of V3 peptides and ANT-70 and MVP-5180, was used to test 14,682 sera samples from people living in Burkina Faso, Burundi, Cameroon, Chad, Congo, Gabon, Mali, Niger, Nigeria, Senegal, Togo, and Zambia to examine the geographic spread of HIV-1 group O viruses in Africa. An in-house Western blot and a line immunoassay (LIA-O) were used to detect the presence of antibodies to gp120 of HIV-1 ANT-70 of samples reactive in ELISA and then a polymerase chain reaction (PCR) on HIV-1 group O and group O indeterminant sera. HIV-1 group O antibodies were present in 8 countries (Cameroon, Chad, Gabon, Niger, Nigeria, Senegal, Togo, and Zambia). Among these 8 countries, the prevalence of HIV-1 group O sera ranged from 2.1% in Cameroon to 0.07% in Senegal. Cameroon and its neighboring countries had a higher prevalence than the West African countries (0.9-2.1% vs. 0.07-0.3%) and Zambia. HIV-1 group O virus was more or less evenly distributed among the population groups tested. The ANT-70 V3 peptide in LIA-O had a higher reactivity rate with HIV-1 group O sera than MVP-5180 V3 peptide in LIA-O (100% vs. 78.9%). 8 of the 13 samples tested in PCR were identified as group O by specific group O pol and/or V3 primers. Among the remaining 5 indeterminant sera samples, 2 were identified as group O. Prospective studies are needed to monitor the true prevalence of HIV-1 group O viruses in Cameroon, its neighboring countries, and West Africa. They are also needed to determine the risk factors associated with group O infection. Monitoring these viruses will allow adaptation of HIV testing strategies for blood screening and serodiagnosis if required.