Yves Germani

Strategy for the detection of Helicobacter species by amplification of 16S rRNA genes and identification of H. felis in a human gastric biopsy

The aim of the present work was to develop polymerase chain reactions (PCRs) based on the conserved nucleotide sequence of the 16S rRNA gene for detection of bacteria of the Helicobacter genus in human antral biopsy samples. The assay for Helicobacter spp was developed by amplifying a 399-bp 16S rRNA gene sequence specific to the genus Helicobacter. The identity of the amplicon was confirmed by hybridization with an internal probe and by restriction by endonuclease VspI showing two expected fragments of 295 and 104 base pairs. A total of 65 dyspeptic patients from France and New Caledonia were screened for Helicobacter spp infection through the use of the following diagnostic assays on biopsy specimens collected through endoscopy: direct detection of bacteria in histological sections by Giemsa and Warthin Starry staining, urease test and bacterial isolation, PCR for Helicobacter pylori ureC/glmM gene, and PCR targeted to 16S rRNA genes. The 16S rRNA gene PCR assay was able to detect down to 680 bacterial cells, as assessed by agarose gel electrophoresis, and down to 4 bacterial cells by hybridization of amplicon with the internal probe. The 16S rRNA PCR test was 100% specific and sensitive; results obtained with this test were in agreement with the visualization of bacteria by histology. Urease test and culture were 86.4% and 22.7% sensitive, and 96.5 and 100% specific, respectively. The H. pylori ureC/glmM gene-based PCR was 100% specific and only 95.4% sensitive, since one biopsy from a Melanesian patient contained a Helicobacter strain other than H. pylori. For this Melanesian patient, a branch-specific PCR targeting the epsilon branch of Proteobacteria was used to amplify a 967-bp amplicon. This amplicon was sequenced and matched with the H. felis sequence. This was confirmed using an H. felis-specific urease PCR test.

Ordinary and Opportunistic Enteropathogens Associated with Diarrhea in Senegalese Adults in Relation to Human Immunodeficiency Virus Serostatus

Abstract 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.

Chronic Diarrhea, Hemorrhagic Colitis, and Hemolytic-Uremic Syndrome Associated with HEp-2 Adherent Escherichia coli in Adults Infected with Human Immunodeficiency Virus in Bangui, Central African Republic

ABSTRACT In human immunodeficiency virus (HIV)-infected adults from the Central African Republic, the occurrence of chronic diarrhea due to HEp-2 adherent Escherichia coli (EAEC) harboring virulence markers (eaeA, BFP, EAF, astA determinant of EAST/1, positive FAS test, enteropathogenic E. coli O serogroup) was shown to be associated with AIDS. We also show that EAEC that produce verotoxin (Stx2) but do not harbor the genetic markers for classical enterohemorrhagic E. coli are involved in hemorrhagic colitis and hemolytic-uremic syndrome in patients with HIV.

Dipstick for Rapid Diagnosis of Shigella flexneri 2a in Stool

Background Shigellosis or bacillary dysentery, an acute bloody diarrhoea, is a major public health burden in developing countries. In the absence of prompt and appropriate treatment, the infection is often fatal, particularly in young malnourished children. Here, we describe a new diagnostic test for rapid detection, in stool, at the bedside of patients, of Shigella flexneri 2a, the most predominant agent of the endemic form of the disease. Methodology/Principal Findings The test is based on the detection of S.flexneri 2a lipopolysaccharide (LPS) using serotype 2a-specific monoclonal antibodies coupled to gold particles and displayed on one-step immunochromatographic dipstick. A concentration as low as 20 ng/ml of LPS is detected in distilled water and in reconstituted stools in under 15 minutes. The threshold of detection corresponds to a concentration of 5×107 CFU/ml of S. flexneri 2a, which provides an unequivocal positive reaction in three minutes in distilled water and reconstituted stools. The specificity is 100% when tested with a battery of Shigella and unrelated strains, in culture. When tested in Vietnam, on clinical samples, the specificity and sensitivity were 99.2 and 91.5%, respectively. A decrease of the sensitivity during the evaluation on stool samples was observed after five weeks at room temperature and was due to moistening of the dipsticks caused by the humidity of the air during the fifth week of the evaluation. This drawback is now overcome by improving the packaging and providing dipsticks individually wrapped in waterproof bags. Conclusion This simple dipstick-bases test represents a powerful tool for case management and epidemiological surveys.

Broad spectrum of coreceptor usage and rapid disease progression in HIV-1-infected individuals from Central African Republic.

To study the progression of HIV-1 infection and coreceptor usages in Central African Republic, clinical data, plasma viral load, and coreceptor usage of sequential HIV-1 isolates were analyzed in a seroincident prospective cohort (PRIMOCA). Twenty-three HIV-1 infected individuals from the Central African Armed Forces were followed from 1995 to 2000. Viruses were isolated from 17 patients at various time points after seroconversion and their coreceptor usage was examined using GHOST cells expressing CD4 and one of the HIV-1 chemokine coreceptors CCR5, CXCR4, BOB/GPR15, and Bonzo/STRL33/CXCR6. Eleven patients died from AIDS. Eight of them died between 2 and 5 years after seroconversion, after a brief symptomatic stage. Patients who rapidly progressed to AIDS and death displayed the highest viral loads after seroconversion. All isolates obtained soon after seroconversion used CCR5, albeit, in some cases, CXCR4, BOB, or Bonzo were also used. Most isolates remained R5 (59 out of 61 isolates), although viruses using CXCR4 appeared in some cases of progression to AIDS. In several cases, a broad tropism was observed during the course of infection, with a frequent usage of BOB and Bonzo in addition to CCR5. Rapid progression to disease and short survival time among Central African HIV-1 patients appear more frequent than those reported in industrialized countries. Viral coreceptor used was mainly CCR5, but, interestingly, a large part of isolates also used BOB and Bonzo. However, there was no strict correlation between the clinical outcome and extended viral tropism.

Role of Deoxyribose Catabolism in Colonization of the Murine Intestine by Pathogenic Escherichia coli Strains

ABSTRACT We previously suggested that the ability to metabolize deoxyribose, a phenotype encoded by the deoK operon, is associated with the pathogenic potential of Escherichia coli strains. Carbohydrate metabolism is thought to provide the nutritional support required for E. coli to colonize the intestine. We therefore investigated the role of deoxyribose catabolism in the colonization of the gut, which acts as a reservoir, by pathogenic E. coli strains. Molecular and biochemical characterization of 1,221 E. coli clones from various collections showed this biochemical trait to be common in the E. coli species (33.6%). However, multivariate analysis evidenced a higher prevalence of sugar-metabolizing E. coli clones in the stools of patients from countries in which intestinal diseases are endemic. Diarrhea processes frequently involve the destruction of intestinal epithelia, so it is plausible that such clones may be positively selected for in intestines containing abundant DNA, and consequently deoxyribose. Statistical analysis also indicated that symptomatic clinical disorders and the presence of virulence factors specific to extraintestinal pathogenic E. coli were significantly associated with an increased risk of biological samples and clones testing positive for deoxyribose. Using the streptomycin-treated-mouse model of intestinal colonization, we demonstrated the involvement of the deoK operon in gut colonization by two pathogenic isolates (one enteroaggregative and one uropathogenic strain). These results, indicating that deoxyribose availability promotes pathogenic E. coli growth during host colonization, suggest that the acquisition of this trait may be an evolutionary step enabling these pathogens to colonize and persist in the mammalian intestine.

Dipstick Test for Rapid Diagnosis of Shigella dysenteriae 1 in Bacterial Cultures and Its Potential Use on Stool Samples

Background We describe a test for rapid detection of S. dysenteriae 1 in bacterial cultures and in stools, at the bedside of patients. Methodology/Principal Findings The test is based on the detection of S. dysenteriae 1 lipopolysaccharide (LPS) using serotype 1-specific monoclonal antibodies coupled to gold particles and displayed on a one-step immunochromatographic dipstick. A concentration as low as 15 ng/ml of LPS was detected in distilled water and in reconstituted stools in 10 minutes. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 1.6×106 CFU/ml and 4.9×106 CFU/ml of S. dysenteriae 1, respectively. Optimal conditions to read the test have been determined to limit the risk of ambiguous results due to appearance of a faint yellow test band in some negative samples. The specificity was 100% when tested with a battery of Shigella and unrelated strains in culture. When tested on 328 clinical samples in India, Vietnam, Senegal and France by laboratory technicians and in Democratic Republic of Congo by a field technician, the specificity (312/316) was 98.7% (95% CI:96.6–99.6%) and the sensitivity (11/12) was 91.7% (95% CI:59.8–99.6%). Stool cultures and the immunochromatographic test showed concordant results in 98.4 % of cases (323/328) in comparative studies. Positive and negative predictive values were 73.3% (95% CI:44.8–91.1%) and 99.7% (95% CI:98–100%). Conclusion The initial findings presented here for a simple dipstick-based test to diagnose S. dysenteriae 1 demonstrates its promising potential to become a powerful tool for case management and epidemiological surveys.

Rapid Diagnosis of Diarrhea Caused by Shigella sonnei Using Dipsticks; Comparison of Rectal Swabs, Direct Stool and Stool Culture

Background We evaluated a dipstick test for rapid detection of Shigella sonnei on bacterial colonies, directly on stools and from rectal swabs because in actual field situations, most pathologic specimens for diagnosis correspond to stool samples or rectal swabs. Methodology/Principal Findings The test is based on the detection of S. sonnei lipopolysaccharide (LPS) O-side chains using phase I-specific monoclonal antibodies coupled to gold particles, and displayed on a one-step immunochromatographic dipstick. A concentration as low as 5 ng/ml of LPS was detected in distilled water and in reconstituted stools in 6 minutes. This is the optimal time for lecture to avoid errors of interpretation. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 4 x 106 CFU/ml of S. sonnei. The specificity was 100% when tested with a battery of Shigella and different unrelated strains. When tested on 342 rectal swabs in Chile, specificity (281/295) was 95.3% (95% CI: 92.9% - 97.7%) and sensitivity (47/47) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 95.5 % of cases (328/342) in comparative studies. Positive and negative predictive values were 77% (95% CI: 65% - 86.5%) and 100% respectively. When tested on 219 stools in Chile, Vietnam, India and France, specificity (190/198) was 96% (95% CI 92%–98%) and sensitivity (21/21) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 96.3 % of cases (211/219) in comparative studies. Positive and negative predictive values were 72.4% (95% CI 56.1%–88.6%) and 100 %, respectively. Conclusion This one-step dipstick test performed well for diagnosis of S. sonnei both on stools and on rectal swabs. These data confirm a preliminary study done in Chile.

Molecular characterization of enterotoxigenic Escherichia coli (ETEC) isolated in New Caledonia (value of potential protective antigens in oral vaccine candidates)

The role of enterotoxigenic Escherichia coli (ETEC) in childhood diarrhoea in New Caledonia was demonstrated in previous epidemiological works. This study was undertaken in order to characterize these strains and to determine whether bacterial components of current vaccine candidates (toxin, colonization factor antigens, O:H antigens) would be useful in our region. A total of 24 ETEC strains were studied: 5 strains produced heat-labile enterotoxin, 17 strains produced heat-stable enterotoxin (9 STp and 8 STh), and 2 strains produced both toxins (1 LT/STp/STh and 1 LT/STh). E. coli strains were screened for the presence of genes encoding for enterotoxins (DNA dot blot and Southern hybridization assays); results obtained with probes were closely correlated and were in agreement with biological assays. No two ETEC strains possessed similar plasmid profiles, and DNA sequences encoding for enterotoxins were located on plasmids ranging from 58 to 75 MDa. The O:H (O1:H-,O2:H7, O6:H16, O25:H-, O27:H7, O28ab:H9, O52:H10, O64:H5, O70:H-, O78:H12, O88:H25, O99:H6, O101:H-, O126:H12, O166:H30) serotypes are presented (all the strains were typable, but some ETEC serotypes were unusual). By using antisera against colonization factor antigens (CFA) I and II, results showed that 9 of the 24 ETEC strains expressed CFA (2 CFA/II and 7 CFA/I). These strains possessed high bacterial surface hydrophobicity. Fifteen ETEC did not possess CFA; among these, 11 did not exhibit high hydrophobicity or show haemagglutination activity. Four of the 15 CFA-negative strains exhibited high hydrophobicity (two O64:H45, one O70:H- and one O88:H25) but no haemagglutination in the presence or absence of mannose.(ABSTRACT TRUNCATED AT 250 WORDS)

Enterohemorrhagic Escherichia coli O157, Kinshasa.

To the Editor: During the rainy season, from April to September 2003, 463 children ≤15 years of age (median 10 months) with severe diarrhea were admitted to the Pediatric Hospital of Kalembelembe in Kinshasa, the capital of the Democratic Republic of Congo. The population of the outbreak area was approximately one million. Several children with bloody diarrhea without fever were treated. They came from six districts of Kinshasa (Bumbu, Selembao, Makala, Kimbanseke, Masina, and Ndjili). Abdominal cramps, nausea, vomiting, and dehydration were uncommon. The duration of illness ranged from 5 days to 2 weeks. Available antiparasitic drugs, trimethoprim-sulfamethoxazole, and ampicillin showed no effect against the illness. Fifty-six infants died between June and July. Symptoms of hemolytic-uremic syndrome developed in most of them. Stool samples from 32 patients were screened for parasites, enteropathogenic bacteria, rotavirus, and adenovirus. Three samples were positive for rotavirus. In contrast, all stool cultures were positive for Escherichia coli which always grew as pure cultures on purple bromocresol agar, a nonselective medium containing lactose. The E. coli isolates appeared sorbitol negative when tested on MacConkey sorbitol; they were agglutinated by O157 and H7 antisera (Difco Laboratories, Detroit, MI) and lacked expression of β-glucuronidase. All E. coli isolates were sent to the Pasteur Institute in Bangui, Central African Republic, for further characterization. Polymerase chain reaction allowed detection of Shiga-like toxin slt-1 and slt-2 genes (1,2) in isolates from all patients. The Vero cell assay phenotypically confirmed cytotoxicity of these isolates, with most of them being seroneutralized by rabbit antisera against Shiga toxin (3). Thus, all E. coli isolates responded to the definition of enterohemorrhagic E. coli. Before 2003, sporadic infections or outbreaks caused by enterohemorrhagic E. coli were not reported as a cause of bloody diarrhea in the Democratic Republic of Congo. A case-control study could not be performed because of political unrest in Kinshasa. Although reported outbreaks of E. coli O157 in sub-Saharan Africa have been few to date, available information indicates that the pathogen has wide geographic distribution. E. coli O157–related diarrhea outbreaks that occurred before 2003 have been reported in South Africa, Swaziland (4), and Malawi (5) in 1992; Central African Republic (6) and Kenya (7) in 1996; Cameroon in 1998 (8); and Nigeria (9) and Ivory Coast (10) in 2000. In the Central African Republic and in Zemio, a small village located on the Democratic Republic of Congo border, outbreaks of bloody diarrhea in 1996 were attributed to E. coli O157 from molecular test results (6). Since 2001, an increasing number of cases of acute bloody diarrhea have been reported in Kinshasa between June and August. During this 2003 outbreak, an investigation could not be conducted; possible routes of transmission would include person-to-person contact related to lack of hygiene, and contaminated food and water. In 1996 in the Central African Republic and in 1998 in Cameroon, the major contributing factors of the E. coli O157 outbreak were consumption of smoked zebu meat and contaminated drinking water. Studies of E. coli O157 carriage rates among livestock, food, and environment in this central African area might be useful in assessing the potential for future outbreaks. Hemolytic-uremic syndrome occurs in approximately 8% of children and an unknown proportion of adults infected with E. coli O157 and can be fatal without hemodialysis. The high death rate of infants during this outbreak was linked to the lack of treatment (mainly hemodialysis) at the beginning of the epidemic. Obviously, more work is needed to better define the incidence and epidemiology of E. coli–associated diarrhea in the Democratic Republic of Congo so that optimal recommendations for preventing and managing illness can be developed.