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Resume Au cours de la mise au point d'une methode immunoenzymatique de depistage des Escherichia coli porteurs d'adhesines CFA/I ou CFA/II dans des selles diarrheiques, la methode de fixation des anticorps sur le polystyrene par covalence a ete comparee a l'adsorption passive. La fixation covalente des anticorps par covalence permet de conserver les plaques 9 semaines au lieu de 6 par simple adsorption. La sensibilite pour les deux techniques est de 10 5 bacteries/ml pour l'antigene CFA/I et 10 6 pour le CFA/II.

Resume Une technique immunoenzymatique ELISA en micromethode de depistage direct dans les selles des Escherichia coli porteurs des adhesines CFA/I ou CFA/II (Colonization Factor Antigens) est decrite. La methode est comparee a la seroagglutination des subcultures des E. coli. La sensibilite de l'epreuve est de 105 E. coli/ml pour le CFA/I et de 106 pour le CFA/II. La seroagglutination est positive tout au long de la maladie et l'ELISA est positive pendant une periode plus courte.

Summary Objective To determine the causative organisms and characteristics of patients presenting with meningitis in Bangui in order to provide guidance to physicians for case management. Methods Adults with proven or suspected meningitis were enrolled in this prospective study. Laboratory tests Full blood count, blood chemistry, and HIV tests were performed. Cerebrospinal fluid (CSF) was submitted for routine microbiology, chemistry (glucose, protein), and hematology testing. When classical microbiology analyses were negative, a broad-range bacterial polymerase chain reaction (BRBPCR) was used. Results and conclusions Of the 276 patients enrolled, 215 (77.9%) were HIV positive. In HIV-positive patients cryptococcal meningitis (CM) was the most common cause of meningitis (39.1%) followed by pyogenic meningitis (PM) (30.7%), mononuclear meningitis (MM) (28.8%), and tuberculous meningitis (TM) (1.4%). In HIV-negative patients, PM was the most common cause (60.7%) followed by MM (37.7%) and CM (1.6%, one case). In-hospital mortality was higher in HIV-positive patients (73/128 = 57%) compared to those HIV negative (3/18 = 16.7%) ( p = 0.001). Streptococcus pneumoniae ( n = 26) was the most common bacterial diagnosis, mainly in HIV-positive patients ( n = 22, 10.2%). Meningococcal meningitis (14 Neisseria meningitidis of group A and one W135) was diagnosed in nine (4.2%) HIV-positive and six (9.8%) HIV-negative patients. Gram-negative rods were isolated from five HIV-positive and two HIV-negative patients, respectively. The bacteria and fungi involved in meningitis did not display high levels of in vitro resistance. Conventional microbiology techniques failed to detect the causative agent in 55 (53.4%) PM cases. Broad-range bacterial PCR detected DNA from S. pneumoniae in three samples, N. meningitidis in two, Escherichia coli in one, Listeria monocytogenes in two and Staphylococcus aureus in one sample. In the CSF of five (three HIV negative and two HIV positive), PCR products were not identified with the oligonucleotide probes specific for the usual species of bacteria found in CSF, or genera commonly considered potential contaminants of clinical samples. Among the MM cases, 77 (90.5%) probable viral meningitis (54 HIV positive and 23 HIV negative) and eight TM (HIV positive) were suspected.

Objectives: A survey was conducted in Dakar, Senegal, to identify major types and prevalences of bacteria, parasites, fungi, and Rotaviruses associated with diarrhea in relation to human immunodeficieny virus (HIV) serostatus with the goal to provide guidance to physicians for case management. Methods: Etiologic agents were identified in a case-control study: cases were HIV-infected patients with diarrhea (HIV+ D+) and HIV seronegative patients with diarrhea (HIV− D+); controls were HIV-infected patients without diarrhea (HIV+ D−) and seronegative controls without diarrhea (HID− D−). Ordinary enteric pathogens were identified by conventional methods. Different Escherichia coli pathotypes were characterized by polymerase chain reaction (PCR), identification of HEp-2 cell adherence pattern, Sereny test, GIvl1-ELISA, and the suckling mouse assay. Opportunistic parasites, such as Cryptosporidium and Microsporidium , were identified by the Kinyoun method and trichromic stain of Weber, respectively. Rotaviruses were identified with a commercial latex agglutination kit. Antimicrobial susceptibility testing was carried out by the disk diffusion method. Results: Among the 594 patients examined, 158 were HIV+ D+, 121 were HIV− D+, 160 were HIV+ D−, and 155 were HIV− D−. The main etiologies of diarrhea were different according to HIV serostatus of patients. In immunocompetent adults the main causes of diarrhea were Shigella sp (12.4%), Entamoeba histolytica (10.7%), Salmonella enterica (6.6%), and Giardia (4.9%). In the immunocompromised host the more frequent pathogens were enteroaggregative E. coli (19.6%), Microsporidium (9.4%), Cryptosporidium sp (8.2%), Rotavirus (8.2%), Shigella sp (7.6%), Candida albicans (7.6%), E. histolytica (5.1%), S. enterica (4.4%), and Isospora belli (4.4%). Also, Blastocystis hominis has to be considered as an opportunistic parasite, because it was identified only in HIV-infected patients, with higher prevalence in adults with diarrhea (2.5% in HIV+ D+ patients; 0.6% in HIV+ D− patients). High level of asymptomatic carriage of Ascaris lumbricoides and Trichuris trichiura and some cases of multiple infections were observed. Fungi, Cryptosporidium sp and Microsporidium sp, were often identified in patients with low CD4 counts (range, 79–250 cells/mL). Independently from HIV-serostatus, CD4 count was lower in diarrheic persons, suggesting that diarrhea is a debilitating illness and that effective management of diarrhea can prevent immunosuppression. Isolated enteropathogenic strains displayed high resistance to most antibiotics used in Senegal for treating diarrhea (ampicillin, tetracycline, cotrimoxazole); they were susceptible to amikacin, gentamicin, and norfloxacin. Conclusion: These epidemiologic data suggest that guidelines for the management of diarrhea during HIV infection in Dakar should be updated.

We investigated the possibility of using a sheep erythrocyte-antibody conjugate as reagent in a sandwich erythroimmunoassay (SERIA) procedure to detect and titrate Escherichia coli heat-labile enterotoxin (LT) with the naked eye. In this assay, which is based on the immunological similarity between Vibrio cholerae toxin (CT) and LT, rabbit anti-CT IgG was used as immunosorbent, and sheep erythrocytes, sensitized with the rabbit anti-CT antibodies, were used as indicator. The sensitivity of the test was demonstrated by a comparative study using an enzyme-linked immunosorbent assay (ELISA). The results obtained by SERIA with 130 samples correlated well with those of a Vero cell assay and a GM1-ELISA. The test is easy, relatively cheap and as sensitive as other standard techniques; it is particularly suited for field laboratories, especially in tropical countries, and a large number of strains may be examined daily.

Covalently prepared chimera antibodies were tested in a ganglioside GM1 erythro-immunoassay (CERIA) for E. coli heat-labile enterotoxin (LT) detection. The antibody specific for LT was conjugated with a polyclonal antibody specific for sheep erythrocytes. The assay is based on the specific binding of LT to polystyrene-adsorbed GM1 and subsequent erythro-adsorption via chimera antibody by which the bound toxin is visualized. Enterotoxin titers determined with this CERIA method were similar to those obtained with the Vero cell assay and with ELISA. 5 ng of cholera toxin/ml may be detected with the assay. The CERIA, as described, may be used either qualitatively or quantitatively and is well suited for routine laboratory diagnosis of LT in a culture supernatant of E. coli.

A GM1 ganglioside erythroimmunoassay for the detection of heat-labile Escherichia coli enterotoxin (LT) was developed for use in poorly equipped laboratories in developing countries. This assay is based on the immunological similarity between Vibrio cholerae toxin and LT and uses cholera toxin antiserum and sheep anti-rabbit immunoglobulin covalently coupled to sheep erythrocytes as conjugate. This assay has the following advantages over other currently available techniques: the reagents it uses are stable, in particular, tanned and sensitized sheep erythrocytes; GM1 ganglioside is commercially available; erythro-adsorption can be read with the naked eye; the test can be completed in 1 day; and as little as 4 ng of V. cholerae toxin or LT per ml can be detected accurately. The GM1 ganglioside erythroimmunoassay showed good quantitative and qualitative correlation with the Vero cell assay and the conventional GM1 enzyme-linked immunosorbent assay. The GM1 ganglioside erythroimmunoassay was somewhat less sensitive than the GM1 enzyme-linked immunosorbent assay but more sensitive than the Vero cell assay. Results obtained for 12 LT-positive and 138 LT-negative E. coli strains correlated with results obtained with GM1 enzyme-linked immunosorbent and Vero cell assays.

A study of the etiologies of diarrhea in adults in relation to their human immunodeficiency virus (HIV) serostatus and number of CD4+ cells was carried out in the Central African Republic. In cases and controls, multi-parasitism was observed. Salmonella spp. were identified mainly during acute diarrhea, with 50% of the S. enteritidis isolated during the study being responsible for septicemia and/or urinary tract infection in immunodeficient patients. Enteroaggregative Escherichia coli (EAggEC) were the most frequently identified agent in HIV+ patients with persistent diarrhea; 42.8% of the patients with EAggEC as sole pathogens had bloody diarrhea, and these strains were negative for the presence of a virulence plasmid. Coccidia were found in those with acute and persistent diarrhea. Blood was observed in 53.3% of infections involving coccidia as the sole pathogen. Microsporidium spp. and Blastocystis hominis were found only in HIV+ patients with persistent diarrhea. Shigella spp., Campylobacter spp., and Entamoeba histolytica were found in HIV+ and HIV- dysenteric patients; bacteria resembling spirochetes that could not be cultivated were identified only in HIV+ cases with dysentery. Shiga-like toxin-producing E. coli O157:H- was isolated from two cases with hemolytic-uremic syndrome. Fungi were identified as the sole pathogen in 6.4% of the HIV+ patients with persistent diarrhea. Most of enteropathogenic bacteria identified were resistant to ampicillin and trimethoprim-sulfamethoxazole, remained susceptible to ampicillin plus clavulanic acid, and were susceptible to amikacin, gentamicin, and ciprofloxacin.

Nowadays, Rapid Diagnostic Tests (RDTs) are essential tools for an urgent response in infectious diseases. Many tests are available to search for different pathogens (HIV, group A streptococcus, plasmodium ...) in various biological samples (urine, cerebrospinal fluid or CSF, blood ...). The main advantages of this mode of diagnosis are speed, simplicity of implementation, including by non-specialists or outside a laboratory structure, and reasonable cost. In this “CIFRE” (industrial) thesis, we present three RDTs based on lateral flow immunochromatography (LFIA) that we contributed to develop and evaluate.The first TDR targets Neisseria meningitidis, a bacterium responsible for severe outbreaks of meningitis in resource-limited countries, in CSF samples. This RDT is the only LFIA-type commercial test that can detect 5 of the 6 major serogroups involved in the disease (A/C/W/X/Y). A study published under the authority of the meningococci reference centre at the Institut Pasteur of Paris showed the excellent performances of this test on nearly 560 CSF samples collected from 6 countries including 5 in Africa.The second TDR targets Streptococcus pneumoniae in urine and CSF; it is also intended to the diagnosis of bacterial meningitis. This test, coupled with the previous one, is the object of a multicentric study presently conducted in West Africa under cover of WHO.The third TDR is an avatar of the previous one but was dedicated to respiratory secretions. Called PneumoResp, it introduces the concept of semi-quantitative RDT. It proposes to perform the test on undiluted secretions and, in the case of positive result, on 1:100-diluted secretions. We present an algorithm (which is the object of a patent pending appraisal) that aims to differentiate S. pneumoniae carriage from invasive infection by this germ in children. Compared to conventional techniques (semi-quantitative culture and qPCR assays), the test performed on 196 respiratory specimens showed an excellent sensitivity and a very good negative predictive value, allowing to exclude or suspect an active S. pneumoniae infection as soon as the first day., Les tests de diagnostic rapide (TDR) sont aujourd’hui des outils indispensables pour une réponse urgente en pathologie infectieuse. De nombreux tests sont disponibles pour rechercher différents agents pathogènes (VIH, streptocoque du groupe A, plasmodium …) dans des prélèvements biologiques variés (urine, liquide cérébrospinal ou LCS, sang …). L’avantage de ce mode de diagnostic est leur rapidité, leur simplicité de mise en œuvre, y compris par des non-spécialistes ou à l’extérieur d’une structure de laboratoire, et leur coût raisonnable. Dans ce travail de thèse CIFRE, nous présentons trois TDR que nous avons contribué à développer et à évaluer, basés sur l’immunochromatographie à flux latéral (LFIA). Le premier TDR cible Neisseria meningitidis dans le LCS, bactérie responsable de redoutables épidémies de méningites dans les pays à ressources limitées. Ce TDR est le seul test commercial de type LFIA qui permette de détecter 5 des 6 principaux sérogroupes impliqués dans la maladie (A/C/W/X/Y). Une étude publiée sous l’égide du CNR des méningocoques à l’Institut Pasteur de Paris montre les excellentes performances de ce test sur près de 560 échantillons de LCS provenant de 6 pays. Le deuxième TDR cible Streptococcus pneumoniae dans l’urine et le LCS, également dans le cadre du diagnostic des méningites bactériennes. Ce test, couplé au précédent, fait l’objet d’une étude multicentrique en Afrique de l’ouest sous couvert de l’OMS. Le troisième TDR est un avatar du précédent dédié aux sécrétions respiratoires. Dénommé PneumoResp, il introduit le concept de TDR semi-quantitatif en proposant d’effectuer le test sur sécrétions non diluées et, en cas de résultat positif, sur sécrétions diluées au 1 :100ème. Nous proposons un algorithme (qui fait l’objet d’un brevet en cours d’expertise) qui vise à différencier le portage de l’infection invasive à S. pneumoniae chez l’enfant. Par rapport aux techniques conventionnelles (culture semi-quantitative et qPCR), nous montrons sur quelque 200 échantillons respiratoires une excellente sensibilité et une très bonne valeur prédictive négative de ce test pour exclure ou suspecter une infection active à S. pneumoniae chez l’enfant dès le premier jour.

Nowadays, Rapid Diagnostic Tests (RDTs) are essential tools for an urgent response in infectious diseases. Many tests are available to search for different pathogens (HIV, group A streptococcus, plasmodium ...) in various biological samples (urine, cerebrospinal fluid or CSF, blood ...). The main advantages of this mode of diagnosis are speed, simplicity of implementation, including by non-specialists or outside a laboratory structure, and reasonable cost. In this “CIFRE” (industrial) thesis, we present three RDTs based on lateral flow immunochromatography (LFIA) that we contributed to develop and evaluate.The first TDR targets Neisseria meningitidis, a bacterium responsible for severe outbreaks of meningitis in resource-limited countries, in CSF samples. This RDT is the only LFIA-type commercial test that can detect 5 of the 6 major serogroups involved in the disease (A/C/W/X/Y). A study published under the authority of the meningococci reference centre at the Institut Pasteur of Paris showed the excellent performances of this test on nearly 560 CSF samples collected from 6 countries including 5 in Africa.The second TDR targets Streptococcus pneumoniae in urine and CSF; it is also intended to the diagnosis of bacterial meningitis. This test, coupled with the previous one, is the object of a multicentric study presently conducted in West Africa under cover of WHO.The third TDR is an avatar of the previous one but was dedicated to respiratory secretions. Called PneumoResp, it introduces the concept of semi-quantitative RDT. It proposes to perform the test on undiluted secretions and, in the case of positive result, on 1:100-diluted secretions. We present an algorithm (which is the object of a patent pending appraisal) that aims to differentiate S. pneumoniae carriage from invasive infection by this germ in children. Compared to conventional techniques (semi-quantitative culture and qPCR assays), the test performed on 196 respiratory specimens showed an excellent sensitivity and a very good negative predictive value, allowing to exclude or suspect an active S. pneumoniae infection as soon as the first day.; Les tests de diagnostic rapide (TDR) sont aujourd’hui des outils indispensables pour une réponse urgente en pathologie infectieuse. De nombreux tests sont disponibles pour rechercher différents agents pathogènes (VIH, streptocoque du groupe A, plasmodium …) dans des prélèvements biologiques variés (urine, liquide cérébrospinal ou LCS, sang …). L’avantage de ce mode de diagnostic est leur rapidité, leur simplicité de mise en œuvre, y compris par des non-spécialistes ou à l’extérieur d’une structure de laboratoire, et leur coût raisonnable. Dans ce travail de thèse CIFRE, nous présentons trois TDR que nous avons contribué à développer et à évaluer, basés sur l’immunochromatographie à flux latéral (LFIA). Le premier TDR cible Neisseria meningitidis dans le LCS, bactérie responsable de redoutables épidémies de méningites dans les pays à ressources limitées. Ce TDR est le seul test commercial de type LFIA qui permette de détecter 5 des 6 principaux sérogroupes impliqués dans la maladie (A/C/W/X/Y). Une étude publiée sous l’égide du CNR des méningocoques à l’Institut Pasteur de Paris montre les excellentes performances de ce test sur près de 560 échantillons de LCS provenant de 6 pays. Le deuxième TDR cible Streptococcus pneumoniae dans l’urine et le LCS, également dans le cadre du diagnostic des méningites bactériennes. Ce test, couplé au précédent, fait l’objet d’une étude multicentrique en Afrique de l’ouest sous couvert de l’OMS. Le troisième TDR est un avatar du précédent dédié aux sécrétions respiratoires. Dénommé PneumoResp, il introduit le concept de TDR semi-quantitatif en proposant d’effectuer le test sur sécrétions non diluées et, en cas de résultat positif, sur sécrétions diluées au 1 :100ème. Nous proposons un algorithme (qui fait l’objet d’un brevet en cours d’expertise) qui vise à différencier le portage de l’infection invasive à S. pneumoniae chez l’enfant. Par rapport aux techniques conventionnelles (culture semi-quantitative et qPCR), nous montrons sur quelque 200 échantillons respiratoires une excellente sensibilité et une très bonne valeur prédictive négative de ce test pour exclure ou suspecter une infection active à S. pneumoniae chez l’enfant dès le premier jour.