Isolation and partial characterization of bacteria (Pseudoalteromonas sp.) with potential antibacterial activity from a marine costal environment from New Caledonia.

UNLABELLED: Marine bacteria are a rich source of bioactive metabolites. However, the microbial diversity of marine ecosystem still needs to be explored. The aim of this study was to isolate and characterize bacteria with antimicrobial activities from various marine coastal environment of New Caledonia. We obtained 493 marine isolates from various environments and samples of which 63 (12.8%) presented an antibacterial activity against a panel of reference pathogenic strains (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis). Ten out of the most promising strains were cultured, fractionated and screened for antibacterial activity. Four of them (NC282, NC412, NC272 and NC120) showed at least an activity against reference and multidrug-resistant pathogenic strains and were found to belong to the genus Pseudoalteromonas, according to the 16S phylogenetic analysis. The NC282 strain does not belong to any described Pseudoalteromonas species and might be of interest for further chemical and biological characterization. These findings suggest that the identified strains may contribute to the discovery for new sources of antimicrobial substances to develop new therapies to treat infections caused by multidrug-resistant bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: With the constant increasing of bacterial resistance against known antibiotics in worldwide public health, it is now necessary to find new sources of antimicrobials. Marine bacteria from New Caledonia were isolated, tested for antibacterial activity and characterized to find new active molecules against multidrug-resistant bacteria. This study illustrates the diversity of the marine ecosystem with potent new bacteria species. Also the potential of marine bacteria as a rich source of bioactive molecule, for example antibiotics, is highlighted.

Partial characterization of an exopolysaccharide secreted by a marine bacterium, Vibrio neocaledonicus sp. nov., from New Caledonia.

AIMS: Exopolysaccharides (EPS) are industrially valuable molecules with numerous useful properties. This study describes the techniques used for the identification of a novel Vibrio bacterium and preliminary characterization of its EPS. METHODS AND RESULTS: Bioprospection in marine intertidal areas of New Caledonia followed by screening for EPS producing brought to selection of the isolate NC470. Phylogenetic analysis (biochemical tests, gene sequencing and DNA-DNA relatedness) permitted to identify NC470 as a new member of the Vibrio genus. The EPS was produced in batch fermentation, purified using the ultrafiltration process and analysed by colorimetry, Fourier Transform Infrared spectroscopy, gas chromatography, Nuclear Magnetic Resonance and HPLC-size exclusion chromatography. This EPS exhibits a high N-acetyl-hexosamines and uronic acid content with a low amount of neutral sugar. The molecular mass was 672 × 10(3)  Da. These data are relevant for possible technological exploitation. CONCLUSIONS: We propose the name Vibrio neocaledonicus sp. nov for this isolate NC470, producing an EPS with an unusual sugar composition. Comparison with other known polymers permitted to select applications for this polymer. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to evaluate the marine biodiversity of New Caledonia. It also highlights the biotechnological potential of New Caledonia marine bacteria.

Outbreak of leptospirosis in New Caledonia: diagnosis issues and burden of disease.

A leptospirosis epidemic affected New Caledonia during the first semester of 2008. A total of 135 cases were diagnosed with a relatively low fatality rate of 3.7%. Heavy rainfalls, related to La Niña, favoured this epidemic. The PCR, routinely used, confirmed 54% of the cases, and the microagglutination test 56%. Epidemiological and economical data on this epidemic are presented and discussed.

Field evaluation of rapid diagnostic tests for meningococcal meningitis in Niger.

OBJECTIVE: To evaluate dipstick rapid diagnostic tests (RDTs) for meningococcal meningitis in basic health facilities. METHODS: Health facility staff received a one-day training. During the meningitis season, they performed RDTs on cerebrospinal fluid (CSF) specimens from suspected cases of meningitis. A frozen aliquot of CSF was later tested using polymerase chain reaction (PCR) to establish the reference diagnosis. RDTs used in health facilities were archived to allow checking the concordance between reported diagnosis and observed results. Reported diagnosis was also compared to PCR diagnosis. A second RDT was performed on each CSF specimen at the reference laboratory. RESULTS: Using RDTs, health facilities reported 382 negative results (73.9%), 114 NmA (22.1%), 12 NmW135 (2.3%) and nine uninterpretable results (1.7%), the latter corresponding to the misuse of a reagent by three agents. The agreement between reported diagnosis and archived dipsticks was excellent (kappa = 0.98). The agreement between PCR diagnosis and reported RDTs results was strong (kappa = 0.82). In health facilities, the sensitivity of RDTs for N. meningitidis A was Se = 0.91. The kappa coefficient measuring the agreement between RDTs operated in the reference laboratory and RDTs operated in health facilities was kappa = 0.78. CONCLUSION: We confirmed that dipstick RDTs to identify N. meningitidis serogroups A, C, W135 and Y can be reliably operated by non-specialized staff in basic health facilities. RDTs proved very useful to recommend vaccination in NmA epidemics, and also to avoid vaccination in epidemics due to serogroups not included in vaccines (NmX).

[ Bancroftian lymphatic filariasis: toward its elimination from the Pacific?]. / Filariose lymphatique de Bancroft: vers l'elimination dans le Pacifique?

The region of the Pacific is historically affected by lymphatic filariasis (LF). Following the World Health Assembly resolution in 1997, the Global Program to Eliminate Lymphatic Filariasis (GPELF) was launched. In the Pacific, the World Health Organization (WHO) has implemented from 1999, the Pacific Program to Eliminate Lymphatic Filariasis (PacELF) bringing together the 22 countries and territories, in a common effort to eliminate the disease. The strategy is based on Mass Drug Administration (MDA); in annual single dose during 5 years of a Diethycarbamazine/Albendazole association distributed to all the population at risk. Among the 22 countries and territories of the Pacific, 16 are endemic and 6 are non endemic. The classification is based according to the filarian antigen prevalence upper or lower than 1%. MDA are decided when the rate of the filarian antigen prevalence is > 1%. The objective of PacELF is to reduce this rate down to < 1%, threshold under which the transmission is supposed to be stopped. From 1999 to 2007, 14 of the 16 endemic countries organized MDA. Eleven of them completed the cycle of 5 treatments and even beyond. But, these MDA reached only 19% of the at risk population, because of logistic difficulties in Papua New Guinea, the most populated country in the Pacific. The investigations carried out in sentinel sites showed a public health impact, by the fall of the number of microfilaria carriers, often down to a rate < 1%. However the rate of circulating antigen prevalence remains often above the required threshold of 1%. Prevalence surveys carried out in 2007, in different endemic countries, revealed the necessity to intensify efforts and to refine strategy for elimination of FL from the Pacific. A lot of progress were obtained, but few problems were identified. Reflexions are imperative and in progress about: the MDA coverage rates while at the same time a certain lassitude appears in the populations and among health staff, the methods to evaluate the effectiveness of MDA, the reliability of the diagnostic tools to decide of the stop of MDA and to certify the absence of the transmission, the relevance of univocal biological criteria for the whole Pacific area, the need for an active surveillance during several years after stopping MDA, particularly in the countries affected by the very efficient vector Aedes polynesiensis. Seven years after its launching, despite undeniable success, the PacELF program did not achieve its very ambitious goal of stopping the transmission. Three years before its term, strong efforts have to be done and additional strategies be implemented. However; it is reasonable to expect the prolongation of the program in order to achieve the final objective. Beyond, in some countries, it will be still necessary to ensure a sustained global drug pressure and an active surveillance to prevent the re-emergence of the disease.

HIV seroprevalence surveys in Nigerien pregnant women: a comparison between 2002 and 2006.

In the framework of HIV serosurveillance, serosurveys in pregnant women are a good alternative to population-based surveys, which are more difficult to implement. In 2002 and 2006, surveys were conducted in Niger to assess the HIV seroprevalence in pregnant women and to evaluate the trend of the HIV epidemic. The overall seroprevalence was 0.96% (95% CI 0.5-1.7%) and 1.3% (95% CI 0.9-1.8%) in 2002 and 2006, respectively, showing no significant change. In the 2006 overall sample, women living in urban areas were significantly more infected than those in rural environments, with prevalences of 1.9% and 0.7%, respectively (P=0.006). Women with higher school attainment were more often HIV-positive than other women (4.6% vs. 1.7%; P<0.001). The 2006 prevalence, which is among the lowest of the sub-Saharan region, was not significantly different from the national seroprevalence measured in adults in 2002 (0.87%, 95% CI 0.5-1.3%). Close monitoring of the epidemic must be continued.

High sensitivity and specificity of the Pastorex latex agglutination test for Neisseria meningitidis serogroup A during a clinical trial in Niger.

There is a great need for a rapid diagnostic test to guide vaccine choice during outbreaks of meningococcal meningitis in resource-poor countries. During a randomised clinical trial conducted during an epidemic of Neisseria meningitidis serogroup A in Niger in 2003, the sensitivity and specificity of the Pastorex latex agglutination test for this serogroup under optimal field conditions were assessed, using culture and/or PCR as the gold standard. Results from 484 samples showed a sensitivity of 88% (95% CI 85-91%) and a specificity of 93% (95% CI 90-95%). Pastorex could be a good alternative to current methods, as it can be performed in a local laboratory with rapid results and is highly specific. Sensitivity can be improved with prior microscopy where feasible. A study specifically to evaluate the Pastorex test under epidemic conditions, using laboratories with limited resources, is recommended.

Evaluation of the Pastorex meningitis kit for the rapid identification of Neisseria meningitidis serogroups A and W135.

The recent emergence of Neisseria meningitidis W135 as a cause of epidemic bacterial meningitis and the availability of a trivalent ACW135 vaccine have created a need for accurate and timely meningococcal serogroup determination for organization of epidemic vaccine response. The sensitivity and specificity of the Pastorex meningitis kit (Bio-Rad) to identify serogroups A and W135 in the African meningitis belt was assessed using PCR testing as the gold standard. The sensitivity and specificity for serogroups A and W135 were 87 and 85%, respectively, while the specificities were 93 and 97%. The positive and negative likelihood ratios for A were 12 and 0.14 and for W135 were 33 and 0.16. The positive and negative predictive values, computed to simulate an epidemic of meningococcal meningitis with an estimated 70% prevalence of N. meningitidis among suspected cases, were 97% and 75% for A and 99% and 73% for W135. In remote locations of the African meningitis belt, latex agglutination is the only currently available test that can rapidly determine meningococcal serogroup. This study showed that latex agglutination performs well and could be used during the epidemic season to determine appropriate vaccine response.

[Prevalence of tuberculous meningitis in Niamey's Hospital, Niger]. / Prevalence de la méningite tuberculeuse a l'hôpital de Niamey, Niger.

Tuberculosis is hyperendemic in Niger. In Niamey between June 2002 and May 2004, 996 cerebro-spinal fluids (CSF) collected from meningitis suspected patients have been analysed by PCR for the detection of Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae: the aetiologic diagnosis was obtained for 208 cases (20.9%). The Mycobacterium tuberculosis PCR assay performed on the negative samples was positive for 4 CSF: 0.4% prevalence among suspected cases of meningitis or 1.9% among confirmed bacterial meningitis.

[From genomics to surveillance, prevention and control: new challenges for the African meningitis belt]. / Ceinture africaine de la méningite: de la génomique aux stratégies de surveillance, de lutte et de prévention.

An international conference was held in Niamey, Niger, in November 2005. It aimed at reviewing the current situation in the meningitis belt. This region stretches from Senegal to Ethiopia and is characterized by high levels of seasonal endemicity with large epidemics of meningococcal meningitis occurring cyclically, generally caused by N. meningiditis serogroup A. WHO currently recommends a reactive strategy based on rapid detection of epidemics, intervention with antibiotics to treat cases and mass vaccination with a meningococcal polysaccharide vaccine to halt the outbreak. Epidemiological patterns of the disease in Africa have been changing with the occurrence of outbreaks outside the meningitis belt and with the emergence of serogroup W135, which first caused an epidemic among Hajj pilgrims in 2000 and then a large-scale meningitis outbreak in Burkina Faso in 2002. Consequently enhanced laboratory surveillance and confirmation of the strain responsible for the outbreak are required. New rapid dipstick tests have been developed through a collaboration between Institut Pasteur and CERMES. They are designed for bedside diagnosis and detect meningococcal antigens present in CSF using immunochromatography. The treatment of meningococcal meningitis during epidemics is based on short-course, long-acting oily chloramphenicol. An alternative is the use of ceftriaxone, which is equally effective and can be used in pregnant women and infants. A low-cost, monovalent serogroup A meningococcal conjugate vaccine for large-scale use in Africa is under development. In spite of the emergence of W135 strains in the meningitis belt, N. meningiditis A continues to be the principal strain isolated during the epidemic seasons and elimination of outbreaks of N. meningiditis serogroup A can still be considered as the primary objective of a preventive vaccination strategy.

[Assessment of five rapid tests and two algorithms for the HIV infection diagnosis in Niger]. / Evaluation de cinq tests rapides et de deux algorithmes pour le diagnostic de l'infection par le VIH au Niger.

Niger is a Sahelian country of 1,267,000 km2 and 11 M inhabitants (84% living in rural area). The national seroprevalence of the HIV infection is low (2.08% in urban area and 0. 64% in rural area). Because of limited resources, laboratories and qualified personnel are scarce, so the classical strategy using ELISA methods to diagnose HIV is available only in the capital Niamey. In order to define a strategy suitable to the Nigerian field conditions, we have determined the diagnostic value of five commercialised rapid assays (Génie II, InoCheck, Determine, Capillus and Doublecheck). A panel of plasma, whole blood and dried blood spot from 42 positive and 160 negative control individuals was tested and the sensitivity and specificity of each assay were calculated. The two best algorithms using two tests were selected: a algorithm discriminating HIV1 & 2 combining Determine and Genie II showing an excellent sensitivity and specificity (100%) but with many practical constraints; and a non discriminative algorithm combining Determine and Double-check less specific but workable on capillary blood and fully compatible with the field conditions. Its negative predictive value was 100% and its positive predictive value was > 97% for urban population and > 91% for rural population. Dried blood spots on filter paper proved to be very practical and efficient to use by the reference laboratory for the quality control of the peripheral diagnostic centres.

Current epidemiology of human plague in Madagascar.

From 1996 to 1998, 5,965 patients with suspected plague were identified in 38 districts of Madagascar (40% of the total population are exposed). Using standard bacteriology, 917 of them were confirmed or presumptive (C + P) cases. However, more than 2,000 plague cases could be estimated using F1 antigen assay. Two out of the 711 Yersinia pestis isolates tested were resistant to chloramphenicol and to ampicillin (both isolates found in the harbour of Mahajanga). Urban plague (Mahajanga harbour and Antananarivo city) accounted for 37.4% of the C + P cases. Bubonic plague represented 97.2% of the cases, and the lethality rate was still high (20%). In comparing the exposed population, plague was more prevalent in males (M:F sex ratio 1.3:1) and patients under 20 years (2.7% babies under two years). Buboes were mainly localised in the inguinal/femoral regions (55.8%). The epidemiological risk factors are discussed.

Wuchereria bancrofti filariasis in French Polynesia: age-specific patterns of microfilaremia, circulating antigen, and specific IgG and IgG4 responses according to transmission level.

The age-specific patterns of microfilaremia, Og4C3 antigenemia, anti-Brugia malayi IgG and IgG4 were assessed in 3 villages of low, medium and high transmission level for Wuchereria bancrofti filariasis. The prevalence rates for each of the 4 markers were clearly age dependent and their patterns strongly associated with the transmission level. The antigenemia prevalence rate was consistently higher than the microfilaremia prevalence rate, in all age groups. The prevalences of anti-B. malayi IgG and IgG4 responses were very similar and much higher than those of microfilaremia or antigenemia. Antibody responses reached the plateau at an earlier age and at a higher prevalence with increased intensity of transmission. For all the markers, the prevalence rates were significantly higher in males than in females.

Follow-up of tuberculosis patients undergoing standard anti-tuberculosis chemotherapy by using a polymerase chain reaction.

We have analysed the clearance of Mycobacterium tuberculosis in sputum specimens from pulmonary tuberculosis patients undergoing 6-month chemotherapy, using the polymerase chain reaction (PCR) and standard microbiological methods. In a group of 19 patients, 11 (58%) were smear- or culture-positive and 13 (74%) were PCR-positive before treatment. Of the 16 patients followed from 2 months after the start of treatment and thereafter, all became smear-negative and culture-negative, whereas, with PCR, 4 (27%), 2 (13%) and 1 (7%) of these patients remained positive after 2, 3 and 6 months, respectively. These results suggest the possible usefulness of PCR in monitoring the efficacy of treatment when bacteriological tests are negative, so as to identify patients with a high risk of relapse.

Transmission of tuberculosis in the prison of Antananarivo (Madagascar).

The prevalence of tuberculosis in the Antananarivo prison is 16 times higher than that in the general population of Madagascar. We compared the clustering of Mycobacterium tuberculosis strains within and outside the prison and studied the transmission of strains in the prison. M. tuberculosis strains isolated in 1994 to 1995 from 146 prisoners and from 260 nonprisoner patients from Antananarivo were typed using the genetic markers IS6110 and direct repeat. We compared the strains isolated from prisoners and nonprisoners and found that the clustering rate was higher within (58.9%) than outside the prison (40%) suggesting that the transmission rate was higher in prison. Of the 146 incarcerated patients, 82 were grouped into 22 clusters. We checked for possible tuberculosis transmission between prisoners with identical strains by epidemiological investigation of the various prison clusters. We found that 9.5% of the incarcerated patients could have been sources of infection and that only 15.1% could have been infected in the prison. One hundred and twenty-seven prison patients were new cases. Epidemiological data suggested that 37% of them resulted from a reactivation of an old infection, due to poor living conditions or recent transmission from an index case outside the prison.

The blackfly Simulium buissoni and infection by hepatitis B virus on a holoendemic island of the Marquesas archipelago in French Polynesia.

The hematophagous blackfly Simulium buissoni causes skin lesions on an island in the Marquesas archipelago that is holoendemic for hepatitis B virus (HBV). To test the hypothesis of the possible role of this fly in the transmission of hepatitis B, 506 children (age range 2-11 years) were examined for the presence of skin lesions, and attempts were made to detect HBV DNA in and on blackflies using two polymerase chain reaction methods. The mean number of skin lesions showed a positive correlation with the age of these children (r = 0.12, P < 0.05). Furthermore, it was significantly higher in the rural zone than in the urban zone (mean +/- SD 41.02 +/- 31.71 versus 17.73 +/- 13.43; P < 0.05), and showed a correlation with a higher infection rate (73.9% versus 41.3%). Of the 45 pools of 10 insects tested, HBV DNA was not detectable on the inside of the insect, but was detectable on the flies (1-10 particles/insect in three positive pools). Infection by HBV conveyed by the flies is theoretically possible, but their indirect role via the numerous skin lesions caused on children is likely to explain such a high level of transmission.

Ivermectin plus diethylcarbamazine: an additive effect on early microfilarial clearance.

The effects of ivermectin, diethylcarbamazine (DEC), and the combination of both drugs on levels of microfilaremia (mf) were studied in 30 male Polynesian Wuchereria bancrofti carriers. Microfilarial densities were measured 30 min (H1/2), 1 hr (H1), and 2, 4, 8, 24, and 96 hr (H2, H4, H8, H24, and H96) after supervised single doses of ivermectin plus DEC (400 micrograms/kg plus 1 mg/kg, respectively, 400 micrograms/kg plus 3 mg/kg, respectively, and 400 micrograms/kg plus 6 mg/kg, respectively), DEC (6 mg/kg) alone, and ivermectin (400 micrograms/kg and 100 micrograms/kg, respectively) alone given to six groups of five patients each. The results showed that 1) DEC alone or combined with ivermectin induced a rapid clearance of mf after drug intake; at H1/2, the number of circulating microfilariae was reduced to 16%, 8%, 28%, and 31%, respectively, of pretreatment values in the groups receiving ivermectin plus DEC (400 micrograms/kg plus 1 mg/kg, 400 micrograms/kg plus 3 mg/kg, and 400 micrograms/kg plus 6 mg/kg) and DEC (6 mg/kg) alone; 2) ivermectin alone induced a rapid increase of mf densities during the first 2 hr, followed by a sharp decrease from H4 to H96; and 3) between H8 and H96, mf clearance was almost complete with the combination of ivermectin and DEC. A comparison among groups did not show any synergistic interaction between ivermectin and DEC on the clearance of microfilaria, with the effect of each drug being additive to each another.

A polymerase chain reaction assay for detection of the parasite Wuchereria bancrofti in human blood samples.

To identify Wuchereria bancrofti DNA sequences that could be used as the basis for a simple and rapid parasite detection assay, a genomic library of W. bancrofti was constructed and screened for highly repeated DNA. The repeat found with the highest copy number was 195 basepairs (bps) long, 77% AT, and 300 copies per haploid genome. This sequence was designated the Ssp I repeat because it has a unique recognition site for that restriction endonuclease in all or most of the repeat copies. The Ssp I repeat DNA family is dispersed, genus-specific, and exists in all of the different geographic isolates of W. bancrofti tested. Based on DNA sequence analysis of this repeat, we have developed an assay to detect very small quantities of W. bancrofti DNA using the polymerase chain reaction (PCR). With this PCR assay, the Ssp I repeat was detected in as little as 1 pg of w. bancrofti genomic DNA (about 1% of the DNA in one microfilaria) added to 100 microliters of human blood. The PCR assay also amplified Ssp I repeat DNA from geographic isolates of W. bancrofti from around the world but not from other species of filariae or from human or mosquito DNA. Microfilaria-positive human blood samples collected in Mauke, Cook Islands were shown to be Ssp I PCR-positive, while microfilaria-negative samples were PCR-negative. The specificity and sensitivity of the Ssp I PCR assay indicates that this approach has significant potential for improved screening of large human populations for active W. bancrofti infection.

Predictive values of the ICT Tuberculosis test for the routine diagnosis of tuberculosis in Madagascar.

The rapid commercialised ICT Tuberculosis test has been tested in Madagascar for the diagnosis of smear-positive pulmonary (SM+) and extra-pulmonary tuberculosis (EPT), using microscopy, culture and histopathology as reference tests. Specificity was 83.3% and sensitivity 68.2% for SM+ patients and 65.2% for EPT patients. With a prevalence of 22.2% for SM+ patients and 52.4% for confirmed EPT among consulting patients in the National Reference Laboratory, the ICT Tuberculosis assay was not sufficiently predictive for application in the tuberculosis control programme.

45/47 kilodalton (APA) antigen capture and antibody detection assays for the diagnosis of tuberculosis.

SETTING: APA complex (45/47 kDa) is an antigen specifically excreted by Mycobacterium tuberculosis and could therefore be a good candidate for diagnosis. OBJECTIVES: To develop three APA immunocapture ELISA assays using monoclonal antibodies (Mabs) and one IgG anti-APA ELISA test, and to determine their usefulness for the diagnosis of tuberculosis in Madagascar. DESIGN: For the Ag assays, 23 negative sputum and serum samples and 64 pairs of sputum and serum from active smear-positive patients (PTM+) were tested. For antibody assay, 116 negative controls, 143 PTM+ and 54 extra-pulmonary tuberculosis patients were tested. RESULTS: The sensitivities of the APA antigen detection assays were low (less than 40%) for a specificity of 95.6%, using either monoclonal antibodies or clinical specimens. The anti-APA serology was more sensitive (76.9% for PTM+ patients) but less specific (73.2%). Due to their poor predictive values, these tests cannot be recommended for the routine diagnosis of tuberculosis in Madagascar.