Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
Diagnosis of toxoplasmic encephalitis (TE) in patients with AIDS is not straightforward because clinicians rely initially on an empiric diagnosis based on clinical and radiographic improvement to specific anti-Toxoplasma gondii therapy. There is therefore a need for biological tools to improve the diagnosis of TE, especially in tropical areas where this diagnosis is likely to be underestimated. The use of PCR testing in blood samples for the diagnosis of TE has been limited by its poor sensitivity in the studies conducted in Europe. In tropical South America, the results of PCR sensitivity in blood samples were controversial. Considering that T. gondii strains from tropical South America have substantial genetic and pathogenic differences with those from USA and Europe, it is therefore important to re-evaluate the performance of the PCR assay in blood samples for the diagnosis of TE in AIDS patients from this region. Our results showed that the only interest of PCR would be in the most severe forms of TE with altered consciousness because PCR is more likely to be positive. We also provided important genotyping data on T. gondii strains isolated in human cases of toxoplasmosis in the Caribbean and in South America.
ISSN: 1935-2727

hal-01503761
https://hal-unilim.archives-ouvertes.fr/hal-01503761
https://hal-unilim.archives-ouvertes.fr/hal-01503761/document
https://hal-unilim.archives-ouvertes.fr/hal-01503761/file/journal.pntd.0004790.pdf
DOI : 10.1371/journal.pntd.0004790

Éditeur(s) : HAL CCSD American Society for Microbiology
Résumé : International audience
This study aimed to evaluate the repeated sequence REP-529 compared to the B1 gene for the molecular diagnosis of toxoplasmosis by quantitative PCR (qPCR) in routine diagnosis.Over a ten-year period (2003-2013), all patients prospectively diagnosed with a positive REP-529 qPCR result for toxoplasmosis were included. All DNA samples (76 samples from 56 patients) were simultaneously tested using the two qPCR methods (REP-529 and B1).The mean Ct obtained with the B1 qPCR was significantly higher (+4.71 cycles) than that obtained with REP-529 qPCR (p<0.0001). Thirty-one out of 69 extracts (45.6%) positive with REP-529 qPCR were not amplified with the B1 qPCR (relative sensitivity 54.4%, compared to REP-529), yielding false negative results on 15/28 placentas, 5 cord blood, 2 amniotic fluids, 4 cerebrospinal fluids, 1 aqueous humor, 2 lymph node punctures and 1 abortion product. This defect in sensitivity would have left 20/56 patients undiagnosed, distributed as follows: 12/40 congenital toxoplasmosis, 4/5 cerebral toxoplasmosis, 2/8 patients with retinochoroiditis, and both patients with chronic lymphadenopathy. This poor performance of B1 qPCR could be related to low parasite loads, since the mean Toxoplasma quantification in extracts with B1 false negative results was 0.4 parasite/reaction.These results clearly show the superiority of the REP-529 sequence in the diagnosis of toxoplasmosis by PCR suggest that this target should be adopted as part of standardization of the PCR assay.
ISSN: 0095-1137

hal-01118293
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01118293
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01118293/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01118293/file/A%2010-Year%20Retrospective%20Comparison%20of%20Two%20Target%20Sequences-belaz2015.pdf
PUBMEDCENTRAL : PMC4365238

Éditeur(s) : HAL CCSD American Society for Microbiology
Résumé : International audience
Toxoplasmosis is a life-threatening infection in immunocompromised patients (ICPs). The definite diagnosis relies on parasite DNA detection, but the incidence and burden of disease are ill-known in HIV-negative patients. A three-year retrospective study was conducted in 15 reference laboratories from the network of the French National Reference Center for Toxoplasmosis, to record the frequency of Toxoplasma DNA detection in ICPs, to review the molecular methods used for diagnosis and the prevention measures implemented in transplant patients. During the study period, out of 31,640 PCR performed in ICPs, 610 were positive (323 patients). Blood (n=337), cerebrospinal fluid (n=101) and aqueous humor (n=100) samples were more frequently positive. Chemoprophylaxis schemes in transplant patients differed between centers. PCR follow-up of allogeneic hematopoietic stem-cell transplant (allo-HSCT) patients was implemented in 8/15 centers. Data from 180 patients (13 centers) were further analyzed regarding clinical setting and outcome. Only 68/180 (38%) patients were HIV+; the remaining 62% consisted in 72 HSCT, 14 solid-organ transplants, and 26 miscellaneous immunodeficiencies. Cerebral toxoplasmosis and disseminated toxoplasmosis were most frequently observed in HIV and transplant patients, respectively. Of 72 allo-HSCT patients with a positive PCR, 23 were asymptomatic; all were diagnosed in centers performing systematic blood PCR follow-up, and received specific treatment. Overall survival of allo-HSCT patients at two months was better in centers with PCR follow-up than in other centers (p<0.01). This study provides updated data on the frequency of toxoplasmosis in HIV-negative ICPs and suggests that regular PCR follow-up of allo-HSCT patients could guide pre-emptive treatment and improve outcome.
ISSN: 0095-1137

hal-01134189
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01134189
DOI : 10.1128/JCM.03282-14
PUBMED : 25762774

Éditeur(s) : HAL CCSD
Résumé : International audience
This article describes a previously unreported mutation at position 210 (C210T) of the mitochondrial large subunit ribosomal RNA (mtLSUrRNA) gene of Pneumocystis jirovecii, which led to a false-negative result of a real-time polymerase chain reaction (PCR) assay. Since the aforementioned real-time PCR assay is widely used in France, a French multicenter study was conducted to estimate the mutation frequency and its potential impact on the routine diagnosis of Pneumocystis pneumonia (PCP). Through analysis of data obtained from eight centers, the mutation frequency was estimated at 0.28%. This low frequency should not call into question the routine use of this PCR assay. Nonetheless, the occurrence of the false-negative PCR result provides arguments for maintaining microscopic techniques combined to PCR assays to achieve PCP diagnosis.
Medical Mycology

hal-01477200
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01477200
DOI : 10.1093/mmy/myw051
PUBMED : 27489302

Éditeur(s) : HAL CCSD American Society for Microbiology
Résumé : International audience
Molecular diagnosis of congenital toxoplasmosis or disseminated toxoplasmosis is based mainly on PCR. The repeated DNA element rep529 has become the main DNA target used in most PCR methods, whether laboratory developed or commercial. In this multicenter study, we evaluated the Toxoplasma ELITe MGB (Elitech) commercial kit by comparison with three reference quantitative PCR assays (RAs) used routinely in three proficient laboratories of the French National Reference Center for Toxoplasmosis network, using Toxoplasma calibrated suspensions diluted to obtain a range of concentrations from 0.1 to 10,000 parasites/ml. These suspensions were extracted with either the DNA extraction kit (EXTRAblood; Elitech) recommended by the manufacturer or the QIAamp DNA minikit (Qiagen). The Toxoplasma ELITe MGB assay was also evaluated on a panel of 128 clinical samples, including 56 amniotic fluid samples, 55 placenta samples, and various other samples, of which 95 originated from patients with proven toxoplasmosis. The ELITe MGB assay amplified low-concentration replicates (<10 parasites/ml) of calibrated suspensions less frequently than the RAs of 2/3 laboratories. Additionally, the combination of EXTRAblood and Toxoplasma ELITe MGB yielded poorer sensitivity than the combination of QIAamp DNA minikit and ELITe MGB for low parasite concentrations (P < 0.001 for 1 parasite/ml). On clinical samples, the sensitivity and the specificity of the commercial assay were 89% and 100%, respectively. The sensitivity ranged from 79% (placenta samples) to 100% (amniotic fluid samples). Overall, this study shows that the Toxoplasma ELITe MGB assay is suitable for the diagnosis of toxoplasmosis from non-cell-rich or non-hemoglobin-rich samples and that the EXTRAblood kit is not optimal.
ISSN: 0095-1137

hal-01526432
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01526432
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01526432/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01526432/file/Evaluation%20of%20the%20Toxoplasma.pdf
DOI : 10.1128/JCM.02379-16

Éditeur(s) : HAL CCSD Elsevier Masson
Résumé : International audience
The diagnosis of diabetic foot infections is difficult due to limitations of conventional culture-based techniques. The objective of this study was to evaluate the contribution of denaturing gradient gel electrophoresis (DGGE) in the microbiological diagnosis of diabetic foot ulcers in comparison to conventional techniques, and also to evaluate the need to perform a biopsy sample for this diagnosis. Twenty diabetic patients (types 1 and 2) with foot ulcers (grades 1-4) were included. After debridement of their wounds, samples were taken in duplicate by surface swabbing and deep-tissue biopsy. The samples were analyzed by conventional culture and by a new molecular biology tool, DGGE technology. Polymerase chain reaction (PCR)-DGGE led to the identification of more bacteria than did conventional cultures (mean: 2.35 vs 0.80, respectively). In 11 cases, the technology detected pathogenic species not isolated by classical cultures. PCR-DGGE also identified significantly more pathogenic species at deep levels compared with species detected at superficial levels (87% vs 58%, respectively; P = 0.03). In 9/20 cases, pathogenic bacteria were detected only in deep samples, revealing the need to perform tissue biopsy sampling. DGGE, achievable in 48h, could be a useful technique for the bacteriological diagnosis of diabetic foot infections. It may help to identify pathogenic bacteria in deeply infected ulcers, thereby contributing to a more appropriate use of antibiotics.
ISSN: 1262-3636

hal-01122240
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01122240
DOI : 10.1016/j.diabet.2014.03.002
PUBMED : 24751989

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Whilst it used to affect mostly intravenous drug users and patients who underwent valvular surgery with suboptimal infection control procedures, fungal endocarditis is now mostly observed in patients with severe immunodeficiency (onco-haematology), in association with chronic central venous access and broad-spectrum antibiotic use. The incidence of fungal endocarditis has probably decreased in most developed countries with access to harm-reduction policies (i.e. needle exchange programmes) and with improved infection control procedures during cardiac surgery. Use of specific blood culture bottles for diagnosis of fungal endocarditis has decreased due to optimisation of media and automated culture systems. Meanwhile, the advent of rapid techniques, including fungal antigen detection (galactomannan, mannan/anti-mannan antibodies and β-1,3-d-glucans) and PCR (e.g. universal fungal PCR targeting 18S rRNA genes), shall improve sensitivity and reduce diagnostics delays, although limited data are available on their use for the diagnosis of fungal endocarditis. New antifungal agents available since the early 2000s may represent dramatic improvement for fungal endocarditis: (i) a new class, the echinocandins, has the potential to improve the management of Candida endocarditis owing to its fungicidal effect on yeasts as well as tolerability of increased dosages; and (ii) improved survival in patients with invasive aspergillosis with voriconazole compared with amphotericin B, and this may apply to Aspergillus sp. endocarditis as well, although its prognosis remains dismal. These achievements may allow selected patients to be cured with prolonged medical treatment alone when surgery is considered too risky.
ISSN: 0924-8579

hal-01118441
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01118441
DOI : 10.1016/j.ijantimicag.2014.07.003
PUBMED : 25178919

Éditeur(s) : HAL CCSD Lippincott, Williams & Wilkins
Résumé : International audience
Purpose of review Toxoplasmosis in immunocompromised patients is associated with a high mortality rate. Molecular techniques are important tools to diagnose acute disease in immunocompromised patients, but there are various methods with variable efficiency. Some of them have been validated for the diagnosis of congenital toxoplasmosis, but the impact of their use has not been evaluated in immunocompromised patients. Recent findings Toxoplasmosis is of increasing importance in non-HIV immunocompromised patients. In addition, the picture of disease shows greater severity in South America, both in immunocompetent study participants and in congenitally infected infants. These epidemiological differences could influence the sensitivity of diagnostic methods. This review analyzes recent data on molecular diagnosis and compares them with older ones, in light of progress gained in molecular techniques and of recent epidemiological findings. Most recent studies were conducted in South America and used PCR targeting the B1 gene. PCR on blood could allow diagnosing a significant proportion of patients with ocular toxoplasmosis in Brazil. Summary Quantitative PCR methods with specific probes should be used to improve sensitivity and warrant specificity. Performance of quantitative PCR targeting the repeated 529 bp sequence for the diagnosis of toxoplasmosis in immunocompromised patients needs evaluation in field studies in South America and in western countries.
ISSN: 0951-7375

hal-01390970
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01390970
DOI : 10.1097/QCO.0000000000000275
PUBMED : 27191201

Éditeur(s) : HAL CCSD European Centre for Disease Prevention and Control
Résumé : International audience
Leishmaniasis is endemic in southern Europe, and in other European countries cases are diagnosed in travellers who have visited affected areas both within the continent and beyond. Prompt and accurate diagnosis poses a challenge in clinical practice in Europe. Different methods exist for identification of the infecting Leishmania species. Sixteen clinical laboratories in 10 European countries, plus Israel and Turkey, conducted a study to assess their genotyping performance. DNA from 21 promastigote cultures of 13 species was analysed blindly by the routinely used typing method. Five different molecular targets were used, which were analysed with PCR-based methods. Different levels of identification were achieved, and either the Leishmania subgenus, species complex, or actual species were reported. The overall error rate of strains placed in the wrong complex or species was 8.5%. Various reasons for incorrect typing were identified. The study shows there is considerable room for improvement and standardisation of Leishmania typing. The use of well validated standard operating procedures is recommended, covering testing, interpretation, and reporting guidelines. Application of the internal transcribed spacer 1 of the rDNA array should be restricted to Old World samples, while the heat-shock protein 70 gene and the mini-exon can be applied globally.
ISSN: 1560-7917

hal-01460743
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01460743
DOI : 10.2807/1560-7917.ES.2016.21.49.30418
PUBMED : 27983510

Éditeur(s) : Inter-Research
Résumé : Experimental infections of Penaeus (Litopenaeus) stylirostris were performed with a Vibrio penaeicida strain (AM101) isolated in New Caledonia from Syndrome 93 diseased shrimp. Cumulative mortalities resulting from intramuscular injection or immersion of shrimp in bacterial suspensions demonstrated high virulence for this bacterial strain and suggested that V. penaeicida could be the etiological agent of Syndrome 93. The median lethal dose (LD50) for AM101 was 1.3 x 104 CFU (colony forming units) ml-1 by immersion and less than 5 CFU shrimp-1 by intramuscular challenge, with mortality outbreaks at 48 and 22 h after challenge, respectively. A polymerase chain reaction (PCR) detection assay using a primer set designed from the 16S ribosomal RNA gene of V. penaeicida was developed. It gave an expected amplicon of approximately 310 bp in ethidium bromide-stained agarose gels. The specificity of these primers was assessed with different Vibrio species. Furthermore, DNA extracted by the ChelexTM method could be used to detect fewer than 20 cultured Vibrio cells in seawater or shrimp hemolymph by this assay. It appears to be a reliable screening method for detecting V. penaeicida in shrimp and from the aquatic environment.
Diseases of aquatic organisms (0177-5103) (Inter-Research), 2000-03 , Vol. 49 , N. 2 , P. 109-115

Droits : Inter-Research 2000
http://archimer.ifremer.fr/doc/2000/publication-4436.pdf
DOI:10.3354/dao040109
http://archimer.ifremer.fr/doc/00000/4436/

Éditeur(s) : HAL CCSD Publisher : Paris : Baillière [1980-. Latest Publisher : Paris : Elsevier
Résumé : National audience
Pneumocystis jiroveci (anciennement P. carinii) est un champignon opportuniste responsable de pneumopathies chez les patients immunodéprimés. Les pneumocystoses des patients non infectés par le VIH se distinguent des pneumocystoses du sida par des difficultés diagnostiques accrues, et un pronostic plus sombre. Il est donc indispensable de cibler les immunodépressions les plus à risque, qui bénéficieront d’un traitement par triméthoprime-sulfaméthoxazole à faible dose au long cours, très efficace en prophylaxie des pneumocystoses : ainsi, les vascularites des moyens et petits vaisseaux, les hémopathies lymphoïdes B (leucémie lymphoïde chronique ou aiguë, lymphome malin non hodgkinien) et les cancers solides sous corticothérapie prolongée doivent faire envisager cette prophylaxie. À l’inverse, la généralisation de la prophylaxie à l’ensemble des patients porteurs de pathologies inflammatoires sous corticothérapie n’est pas justifiée. La prise en charge des pneumocystoses des patients non infectés par le VIH suit les principes établis pour les pneumocystoses du sida. Le diagnostic de certitude repose sur la mise en évidence de kystes de P. jiroveci sur des prélèvements respiratoires, tandis que la PCR ne permet pas formellement, en 2015, de différencier colonisation et infection. Le traitement repose sur le triméthoprime-sulfaméthoxazole à fortes doses. Par contre, l’intérêt de la corticothérapie adjuvante pour les patients hypoxémiques, parfaitement documenté au cours du sida, est discuté pour les autres immunodépressions, d’autant que les patients sont en général déjà sous corticoïdes au moment du diagnostic de pneumocystose. Celle-ci est cependant préconisée par plusieurs experts et sociétés savantes, compte tenu du bénéfice démontré sur la morbi-mortalité au cours du sida. Abstract Pneumocystis jiroveci (formerly P. carinii) is an opportunistic fungus responsible for pneumonia in immunocompromised patients. Pneumocystosis in non-HIV-infected patients differs from AIDS-associated pneumocystosis in mostly two aspects: diagnosis is more difficult, and prognosis is worse. Hence, efforts should be made to target immunocompromised patients at higher risk of pneumocystosis, so that they are prescribed long-term, low-dose, trimethoprime-sulfamethoxazole, highly effective for pneumocystosis prophylaxis. Patients at highest risk include those with medium and small vessels vasculitis, lymphoproliferative B disorders (chronic or acute lymphocytic leukaemia, non-Hodgkin lymphoma), and solid cancer on long-term corticosteroids. Conversely, widespread use of prophylaxis in all patients carrier of inflammatory diseases on long-term corticosteroids is not warranted. The management of pneumocystosis in non-AIDS immunocompromised patients follows the rules established for AIDS patients. The diagnosis relies on the detection of P. jiroveci cyst on respiratory samples, while PCR does not reliably discriminate infection from colonization, in 2015. High-doses trimethoprim-sulfamethoxazole is, by far, the treatment of choice. The benefit of adjuvant corticosteroid therapy for hypoxic patients, well documented in AIDS patients, has a much lower level of evidence in non-HIV-infected patients, most of them being already on corticosteroid by the time of pneumocystosis diagnosis anyway. However, based on its striking impact on morbi-mortality in AIDS patients, adjuvant corticosteroid is recommended in hypoxic, non-HIV-infected patients with pneumocystosis by many experts and scientific societies
ISSN: 0248-8663

hal-01237080
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01237080
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01237080/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01237080/file/Pneumocystose%20chez%20les%20patients%20immunod%C3%A9prim%C3%A9s%20non%20infect%C3%A9s%20par%20le%20VIH.pdf
DOI : 10.1016/j.revmed.2015.10.002

Éditeur(s) : HAL CCSD Oxford University Press
Résumé : International audience
Chronic rhinosinusitis (CRS) rank second at chronic inflammatory diseases in industrialized countries and are an important public health concern. Diagnosis relies on a set of arguments including clinical signs, imaging, histopathologic and mycological analyses of sinus specimens, collected during nasal endoscopy. The sensitivity of fungal cultures is reported to be poor, even when direct examination is positive, thus the epidemiology of fungal chronic sinusitis is ill-known. This study evaluated the sensitivity of molecular diagnosis in 70 consecutive samples (61 patients with CRS) analysed at the University Hospital of Rennes during a 3-year period. DNA detection was performed using a conventional PCR method targeting the ITS1/ITS2 sequence and the resulting amplification products were sequenced. Fungal CRS was proven in 42 patients (69%), of which only 20 (48%) had a positive culture. 37/42 (88%) patients were diagnosed with a fungus ball, 3 with allergic fungal CRS and 2 with undetermined fungal CRS. PCR was positive in all 42 cases and direct sequencing allowed to identify fungi in all cases but one, and detected multiple infection in 3. Aspergillus fumigatus was present in 69% of patients; Cladosporium cladosporoides in 9.5%, Scedosporium sp., A. nidulans and A. flavus in 7% each. In 2/19 patients with negative direct examination, sequencing analysis revealed the presence of Capnobotryella sp. and C. cladosporoides, in clinical settings compatible with fungal sinusitis. In conclusion, ITS1/ITS2 PCR had a twice better sensitivity than culture, and combined sequencing provides accurate epidemiological data on fungal CRS.
ISSN: 1369-3786

hal-01383632
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01383632
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01383632/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01383632/file/Contribution%20of%20molecular%20tools%20for%20the%20diagnosis.pdf
DOI : 10.1093/mmy/myw041

Éditeur(s) : HAL CCSD Lippincott, Williams & Wilkins
Résumé : International audience
BACKGROUND:Fever in infants younger than 3 months is generally a cause for concern because of the risk for a serious bacterial infection. The aim of this study was to describe clinical and biological features of Chikungunya infection in infants < 3 months of age hospitalized in Cayenne Hospital during the 2014-2015 outbreak.METHODS:We performed a preliminary retrospective study followed by a prospective study from March 2014 to February 2015. All infants younger than 3 months presenting with fever and hospitalized in Cayenne Hospital were included. The main diagnostic criteria were fever and positive Chikungunya PCR.RESULTS:One hundred and twenty infants were hospitalized with fever. The mean age was 46 days (SD± 22 days). The mean hospitalization duration was 7.4 days (SD± 6.1 days). Chikungunya infection was diagnosed in 26 children. The most important clinical findings were high (80.8% [77.5-84]) and prolonged fever (76.9%, [73.4-80.4]), irritability (96.2% [94.5-97.7]) and skin rash. (69.2%, [65.4-73]). Half of the infants presented edema of the extremities (hands and feet principally). However, in 15 %, Chikungunya infection was associated with a serious bacterial infection. Infants who presented with irritability, high fever and elevated PCT were at high risk for Chikungunya: OR 39, [9.2-243] (p < 0.001), with a specificity of 96.7 % and a negative predictive value of 89.4%. The area of the ROC curve was 0.96 CONCLUSIONS:: Our results confirm that Chikunguyna infection is a cause of high fever in infants younger than 3 months. Our data should be confirmed by larger studies.
ISSN: 0891-3668

inserm-01476019
http://www.hal.inserm.fr/inserm-01476019
http://www.hal.inserm.fr/inserm-01476019/document
http://www.hal.inserm.fr/inserm-01476019/file/2017%2C%20Elenga%20-%20Chikungunya%20Infection%20in%20hospitalized%20febrile%20infants.pdf
DOI : 10.1097/INF.0000000000001541

Éditeur(s) : HAL CCSD Wiley-Blackwell
Résumé : International audience
AIMS: Virus detection has often been difficult due to a low concentration in water. In this study, we developed a new procedure based on concentration of virus particles on an innovative support: poly-L-lysine dendrigrafts (DGL), coupled with directed nucleic acid extraction and real-time PCR quantification. METHODS AND RESULTS: This method was evaluated using the bacteriophage MS2 as a model virus. This virus exhibited the size and structural properties of human pathogenic enteric viruses and has often been used to assess new supports of concentration. Moreover, this bacteriophage is also a faecal contamination indicator. In this study, many water filtration conditions were tested (volume of water, concentration, etc.), and more than 80% of bacteriophage were recovered after filtration on polymer, in most conditions. We demonstrated that the method was linear (slope = 0*99 ± 0*04 and Y intercept when x = -0*02 ± 0*28), valid (as manipulators, tested concentrations, volumes of sample and batch of polymer did not have any influence on concentration) and sensitive (allowing to concentrate up to 16,600-fold 1 l of sample and to detect and quantify down to 750 GC l(-1) and 7500 GC l(-1), respectively). CONCLUSIONS: To conclude, this support exhibits high interest to retain viruses and to allow to detect low concentration of virus in water. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives valuable advance in the methods of concentration and diagnosis of virus in water.
Journal of Applied Microbiology

hal-00828987
https://hal.archives-ouvertes.fr/hal-00828987
DOI : 10.1111/jam.12209
PUBMED : 23551794

Éditeur(s) : Springer
Résumé : Two recurring syndromes threaten the viability of the shrimp industry in New Caledonia, which represents the second largest export business. The "Syndrome 93" is a cold season disease due to Vibrio penaeicida affecting all shrimp farms, while the "Summer Syndrome" is a geographically restricted vibriosis caused by a virulent lineage of Vibrio nigripulchritudo. Microbiological procedures for diagnosis of these diseases are time-consuming and do not have the ability to discriminate the range of virulence potentials of V. nigripulchritudo. In this study, we developed a multiplex PCR method to simultaneously detect these two bacterial species and allow for pathotype discrimination. The detection limits of this assay, that includes an internal amplification control to eliminate any false-negative results, were determined at 10 pg purified DNA and 200 cfu/ml. After confirming the effectiveness of our method using experimentally infected animals, its accuracy was compared to standard biochemical methods during a field survey using 94 samples collected over 3 years from shrimp farms encountering mortality events. The multiplex PCR showed very high specificity for the detection of V. penaeicida and V. nigripulchritudo (inclusivity and exclusivity 100%) and allowed us to detect the spreading of highly pathogenic isolates of V. nigripulchritudo to a farm adjoining the "Summer Syndrome area." This assay represents a simple, rapid, and cost-effective diagnostic tool for implementing timely risk management decisions but also understanding the seasonal and geographical distribution of these pathogens.
Microbial Ecology (0095-3628) (Springer), 2012-01 , Vol. 63 , N. 1 , P. 127-138

Droits : Springer Science+Business Media, LLC 2011
http://archimer.ifremer.fr/doc/00063/17398/15169.pdf
DOI:10.1007/s00248-011-9951-3
http://archimer.ifremer.fr/doc/00063/17398/

Éditeur(s) : HAL CCSD Elsevier Masson
Résumé : International audience
PURPOSE: Two hundred and fifty-three patients were included in a study on the etiology of encephalitis, carried out in France in 2007. Tuberculosis was the second most frequently identified cause, after HSV and with the same number of cases as VZV. The authors report the specific features of patients presenting with tuberculosis encephalitis (TE). METHODS: TE patients were defined as patients presenting with encephalitis, with positive culture or PCR for Mycobacterium tuberculosis, or the association of clinical, biological, imaging, and epidemiological evidence (possible cases). Clinical, microbiological, and brain imaging data was analyzed and compared to that of other included patients. RESULTS: Twenty cases of TE were identified. The M/F sex-ratio was 1.5, the mean age 53 years. Four (20%) patients had a history of tuberculosis before the encephalitis. The median delay between the onset of general and neurological symptoms was significantly longer for tuberculosis cases than for others (10 days vs. 2; P<10(-10)). The median CSF protein level was significantly higher for tuberculosis cases (2.1 g/L vs. 0.8 g/L, P=0.002). CT scan and MRI were normal on admission for eight patients out of 17. Fourteen isolated strains of M. tuberculosis were susceptible to first-line anti-tuberculosis drugs and one was rifampicin-resistant. Six (33%) patients died during hospitalization and two were lost to follow-up. Ten out of 12 (78.6%) had persisting neurological symptoms on discharge. DISCUSSION: Despite non-multiresistant MT strains, the case fatality rate among TE patients was high in our series. Early brain imaging is poor contributive for the diagnosis of TE.
ISSN: 0399-077X

hal-00874835
https://hal.archives-ouvertes.fr/hal-00874835
DOI : 10.1016/j.medmal.2013.05.003
PUBMED : 23816146

Éditeur(s) : Wiley-blackwell Publishing, Inc
Résumé : Aims: A description of bacterial pathogens in shrimp ponds is necessary to understand their pathogenesis. Vibrio nigripulchritudo was shown to contain saprophytic and pathogenic strains among New Caledonian isolates. We established a method to map the development of V. nigripulchritudo in pond sediments at three different genetic levels: the species level, then at the pathogenic cluster level and finally at the plasmid level, present only in all highly pathogenic isolates. Methods and Results: PCR methods were applied to shrimp pond sediments both before and after a mortality outbreak. Using crude samples, the species V. nigripulchritudo is not detected at first (0/42 samples at day 56 post stocking) but appears frequently in the sediments after the mortality event (30/42 at day 107). The distribution of strains from the pathogenic cluster of V. nigripulchritudo also follows this pattern. In contrast, the pSFn1 virulence-associated plasmid was detected in one sample at day 56 and none at day 107. An enrichment method was developed to lower the detection limits of our assays. After enrichment, the species V. nigripulchritudo was detected in all samples at both dates. The number of samples positive for pSFn1 was 42/42 samples at day 56 and 29/42 at day 107. Conclusions: These results show that the sediments contain V. nigripulchritudo, notably pathogenic strains. Surprisingly, the virulence-associated plasmid pSFn1 found in all V. nigripulchritudo isolated from moribund shrimp appears less frequently in sediments, possibly being useless or even detrimental to its recipient bacteria in this environment. Significance and Impact of the Study: This study confirms the presence of pathogenic V. nigripulchritudo strains in shrimp pond sediment before a mortality outbreak complying with a previous hypothesis that sediments could be the infecting reservoir. After the outbreak, both total V. nigripulchritudo and pathogenic strains populations have largely increased, possibly contributing to the recurrent mortality observed in this shrimp vibriosis.
Journal Of Applied Microbiology (1364-5072) (Wiley-blackwell Publishing, Inc), 2010-06 , Vol. 108 , N. 6 , P. 2003-2011

Droits : 2010 The Society for Applied Microbiology - Wiley-Blackwell
http://archimer.ifremer.fr/doc/00004/11484/8177.pdf
DOI:10.1111/j.1365-2672.2009.04601.x
http://archimer.ifremer.fr/doc/00004/11484/

Éditeur(s) : HAL CCSD
Résumé : International audience
Early antifungal therapeutic strategies are proposed during invasive fungal infection (IFI), but antifungal stewardship programs should institute a systematic reevaluation of prescriptions, particularly in the context of empirical treatment. Here, we aimed to evaluate the performances and particularly the negative predictive value (NPV) of diagnostic strategies, including a whole blood panfungal quantitative PCR assay (PF-qPCR) in a high risk population for IFI. The first step was to standardize and optimize a new PF- rtPCR targeting ITS2 region. Then, this method was evaluated in a multicenter prospective study including 313 patients with suspected IFI for whom an early antifungal treatment was prescribed. All patients enrolled at day 0 of their treatment benefited from serum Aspergillus galactomannan (GM) antigen detection twice a week, weekly PF-qPCR assay, and when indicated and feasible, CT-scan and mycological sampling. In total, 125 of 313 patients were diagnosed with IFI: 68 invasive aspergillosis (eight proven, 48 probable and 12 possible), one fusariosis, 47 candidemia, three disseminated candidiasis and six cryptococcosis. Globally, the sensitivity of the PF-qPCR assay was only 40%, but the specificity, PPV and NPV were 96%, 88% and 69%, respectively. In the population of patients at high risk for invasive aspergillosis who also benefited from Aspergillus GM detection, the sensitivity and the NPV of the combined detection reached to 78% and 84%, respectively. Even higher NPV were obtained when combining negative PF-qPCR and CT scan (95%) as well as negative GM and CT scan (93%), thus allowing to rationalize and re-evaluate the prescription of empirical treatment in such highly selected population.
The Journal of Infection

hal-01146845
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01146845
DOI : 10.1016/j.jinf.2015.04.005
PUBMED : 25896093

Éditeur(s) : HAL CCSD
Résumé : International audience
Background Mycobacterium ulcerans infection is the third most common mycobacterial disease in the world after tuberculosis and leprosy. To date, transmission pathways from its environmental reservoir to humans are still unknown. In South America, French Guiana has the highest reported number of M ulcerans infections across the continent. This empirical study aimed to characterise the epidemiology of M ulcerans infection in French Guiana between 1969 and 2013.MethodsData were collected prospectively mainly by two dermatologists at Cayenne Hospital’s dermatologydepartment between Jan 1, 1969, and Dec 31, 2013, for age, date of diagnosis, sex, residence, location of the lesion,type of lesion, associated symptoms, and diagnostic method (smear, culture, PCR, or histology) for all confirmed andsuspected cases of M ulcerans. We obtained population data from censuses. We calculated mean M ulcerans infectionincidences, presented as the number of cases per 100 000 person-years.Findings 245 patients with M ulcerans infections were reported at Cayenne Hospital’s dermatology department duringthe study period. M ulcerans infection incidence decreased over time, from 6·07 infections per 100 000 person-years(95% CI 4·46–7·67) in 1969–83 to 4·77 infections per 100 000 person-years (3·75–5·79) in 1984–98 and to3·49 infections per 100 000 person-years (2·83–4·16) in 1999–2013. The proportion of children with infections alsodeclined with time, from 42 (76%) of 55 patients in 1969–83 to 26 (31%) of 84 in 1984–98 and to 22 (21%) of 106 in1999–2013. Most cases occurred in coastal areas surrounded by marshy savannah (incidence of 21·08 per100 000 person-years in Sinnamary and 21·18 per 100 000 person-years in Mana). Lesions mainly affected limbs (lowerlimbs 161 [66%] patients; upper limbs 60 [24%] patients). We diagnosed no bone infections.Interpretation The decrease of M ulcerans infection incidence and the proportion of children with infections over a45 year period in this ultra-peripheral French territory might have been mostly driven by improving living conditions,prophylactic recommendations, and access to health care.
The Lancet Planetary Health

inserm-01545603
http://www.hal.inserm.fr/inserm-01545603
http://www.hal.inserm.fr/inserm-01545603/document
http://www.hal.inserm.fr/inserm-01545603/file/2017%2C%20Douine%20-%20Mycobacterium%20ulcerans%20infection%20%28Buruli%20ulcer%29.pdf
http://www.hal.inserm.fr/inserm-01545603/file/Comments.pdf

Éditeur(s) : HAL CCSD Wiley-Blackwell
Résumé : International audience
Virus detection has often been difficult due to a low concentration in water. In this study, we developed a new procedure based on concentration of virus particles on an innovative support: poly-L-lysine dendrigrafts (DGL), coupled with directed nucleic acid extraction and real-time PCR quantification. Methods and Results: This method was evaluated using the bacteriophage MS2 as a model virus. This virus exhibited the size and structural properties of human pathogenic enteric viruses and has often been used to assess new supports of concentration. Moreover, this bacteriophage is also a faecal contamination indicator. In this study, many water filtration conditions were tested (volume of water, concentration, etc.), and more than 80% of bacteriophage were recovered after filtration on polymer, in most conditions. We demonstrated that the method was linear (slope = 0 99 0 04 and Y intercept when x = 0 02 0 28), valid (as manipulators, tested concentrations, volumes of sample and batch of polymer did not have any influence on concentration) and sensitive (allowing to concentrate up to 16 600-fold 1 l of sample and to detect and quantify down to 750 GC l 1 and 7500 GC l 1, respectively). Conclusions: To conclude, this support exhibits high interest to retain viruses and to allow to detect low concentration of virus in water. Significance and Impact of the Study: This study gives valuable advance in the methods of concentration and diagnosis of virus in water.
Journal of Applied Microbiology

hal-00932328
https://hal.archives-ouvertes.fr/hal-00932328
DOI : 10.1111/jam.12209