Jalel Boukadida

Distribution of uropathogenic virulence genes in Escherichia coli isolated from patients with urinary tract infection

BACKGROUND Escherichia coli is the predominant pathogen causing urinary tract infection (UTI), the most common bacterial infectious disease encountered in clinical practice, accounting for significant morbidity and high medical costs. The severity of UTI produced by E. coli is due to the expression of a wide spectrum of virulence factors. In this study we evaluated the role of E. coli virulence determinants in the pathogenesis of UTI. METHODS A total of 90 uropathogenic E. coli strains were screened by PCR for the prevalence of seven virulence genes encoding type 1 fimbriae (fimH), pili associated with pyelonephritis (pap), S and F1C fimbriae (sfa and foc), afimbrial adhesins (afa), cytotoxic necrotizing factor (cnf), hemolysin (hly), and aerobactin (aer). RESULTS The prevalence of genes coding for fimbrial adhesive systems was 68% for fimH, 41% for pap, and 34% for sfa/foc. The operons coding for afa afimbrial adhesins were identified in 20% of strains. The hly and cnf genes coding for toxins were amplified in 19% and 3% of strains, respectively. A prevalence of 52% was found for the aer gene. The various combinations of detected genes were designated as virulence patterns. The strains isolated from hospitalized patients displayed a great diversity of gene associations compared to those isolated from ambulatory patients. CONCLUSIONS Our study showed that investigation of the bacterial pathogenicity associated with UTI may contribute to a better medical intervention.

Interferon Gamma +874T/A Polymorphism Is Associated with Susceptibility to Active Pulmonary Tuberculosis Development in Tunisian Patients

Interferon gamma (IFN-γ) is a key cytokine involved mainly in the defense against intracellular pathogens such as Mycobacterium tuberculosis. Given its key role in the control of tuberculosis (TB), in the present article we have investigated a possible association between IFN-γ gene single-nucleotide polymorphism linked to high and low producer phenotypes (IFN-γ [+874T(high) → A(low)]) (rs2430561) and risk development of active TB in Tunisian patients. Genomic DNA samples were obtained from 223 patients with active TB (168 pulmonary and 55 extrapulmonary cases) and 150 healthy blood donors. Genotypes were analyzed using polymerase chain reaction-restriction fragment length polymorphism method. The +874 AA genotype (low IFN-γ producer) was significantly associated with increased risk of developing of active pulmonary TB (odds ratio [OR] = 2.18; 95% confidence intervals [CI], 1.33-3.57; P corrected for the number of genotypes [Pc] = 0.003). By contrast, the AT genotype was found to be significantly associated with resistance to pulmonary TB (OR = 0.46; 95% CI, 0.28-0.74; Pc = 0.0018) and extrapulmonary TB development (OR = 0.46; 95% CI, 0.23-0.91; Pc = 0.045). Collectively, our data showed that the IFN-γ +874T/A polymorphism is a determinant in the resistance or susceptibility to the development of active TB in the studied population.

Molecular analysis of HBV genotypes and subgenotypes in the Central-East region of Tunisia

BackgroundIn Tunisia, country of intermediate endemicity for Hepatitis B virus (HBV) infection, most molecular studies on the virus have been carried out in the North of the country and little is known about other regions. The aim of this study was to determine HBV genotype and subgenotypes in Central-East Tunisia. A total of 217 HBs antigen positive patients were enrolled and determination of genotype was investigated in 130 patients with detectable HBV DNA. HBV genotyping methods were: PCR-RFLP on the pre-S region, a PCR using type-specific primers in the S region (TSP-PCR) and partial sequencing in the pre-S region.ResultsThree genotypes (D, B and A) were detected by the PCR-RFLP method and two (D and A) with the TSP-PCR method, the concordance between the two methods was 93%. Sequencing and phylogenetic analysis of 32 strains, retrieved the same genotype (D and A) for samples with concordant results and genotype D for samples with discordant results. The sequences of discordant genotypes had a restriction site in the pre-S gene which led to erroneous result by the PCR-RFLP method. Thus, prevalence of genotype D and A was 96% and 4%, respectively. Phylogenetic analysis showed the predominance of two subgenotypes D1 (55%) and D7 (41%). Only one strain clustered with D3 subgenotype (3%).ConclusionsPredominance of subgenotype D7 appears to occur in northern regions of Africa with transition to subgenotype D1 in the East of the continent. HBV genetic variability may lead to wrong results in rapid genotyping methods and sequence analysis is needed to clarify atypical results.

Evaluation of an immunochromatographic assay for rapid identificationof Mycobacterium tuberculosis complex in clinical isolates

Identification of Mycobacterium tuberculosis complex (MTC) remains slow. Over the years, several new technologies have been proposed to accelerate and simplify the detection of MTC. In this context, we evaluated an immunochromatographic assay (ICA) (BIO-LINE SD Ag MPT64 TB) for rapid identification of MTC, based on detection of a specific MPT64 antigen of MTC. We have tested it on i) mycobacterial cultures: 210 MTC strains and 28 nontuberculous mycobacteria; ii) M. bovis bacille Calmette-Guérin strain SSI (Statens Serum Institut, Denmark); and iii) 22 microorganisms other than mycobacteria, isolated from cultures. We concluded that this kit has an excellent specificity (100%) and sensitivity (99%) from isolated cultures. The ICA (BIO-LINE SD Ag MPT64 TB) allows excellent MTC identification from clinical isolates. It is a rapid, simple, and inexpensive test, and has a definite contribution in the rapid laboratory diagnosis of tuberculosis.

Contribution of the P2X7 1513A/C loss-of-function polymorphism to extrapulmonary tuberculosis susceptibility in Tunisian populations

The P2X7 receptor has been found to be linked to an increased risk for tuberculosis in some populations. In this study, we investigate whether the P2X7 receptor plays a role in increasing susceptibility to tuberculosis in Tunisia. We examined two 1513A/C and -762T/C polymorphisms at the P2X7 receptor in 168 patients with pulmonary TB (pTB), 55 patients with extrapulmonary TB (epTB) and 150 blood donors from Tunisia. Genotyping of 1513A/C and -762T/C polymorphisms was performed in purified genomic DNA using PCR-restriction fragment length polymorphism and allele-specific PCR, respectively. The 1513C, CC and AC loss-of-function allele and genotypes were overrepresented in the epTB group compared with the control group (45% vs. 17%, P=10(-8) ; 24% vs. 4%, P=3 × 10(-7) ; 42% vs. 27%, P=10(-3) , respectively). Additionally, they were associated with 3.83-, 11.86- and 3.15-fold risks of developing this clinical tuberculosis form, respectively. No associations between the -762T/C polymorphism and tuberculosis disease, as well as disease anatomic location were observed. Collectively, our results suggest that the P2X7 1513A/C loss-of-function polymorphism may contribute to susceptibility to epTB in Tunisian populations.

Polymorphisms in the RANTES Gene Increase Susceptibility to Active Tuberculosis in Tunisia

RANTES plays a pivotal role in attracting and activating various leukocyte populations that control Mycobacterium tuberculosis infection. The present study investigated the relationship between the RANTES polymorphisms (-28C/G; rs2280788, and -403G/A; rs2107538) and susceptibility to active tuberculosis (TB) in Tunisian populations. A total of 168 patients with pulmonary TB (pTB), 55 with extrapulmonary TB (epTB), and 150 control subjects were studied. Genotype analyses were carried out using polymerase chain reaction-restriction fragment length polymorphism method. We found that the -28 GG genotype was significantly associated with susceptibility to pTB (odds ratio [OR]=11.19; 95% confidence intervals [CI], 5.14-25; P corrected for the number of genotypes [Pc]=10(-8)) and epTB (OR=11.67; 95% CI, 4.74-29.33; Pc=10(-8)). However, the -28 CC genotype was found to be significantly associated with resistance to pTB (OR=0.08; 95% CI, 0.04-0.16; Pc=10(-8)) and epTB development (OR=0.11; 95% CI, 0.05-0.27; Pc=10(-8)). -403A allele was associated with increased risk development of epTB (OR=2.21; 95% CI, 1.18-4.14; p=0.007). G-G and A-C haplotypes and the AG/GC diplotype were associated with increase susceptibility to pTB (OR=7.88, 95% CI, 5.38-11.55; Pc=3.10(-8); OR=2.32, 95% CI, 1.32-4.11; Pc=3.10(-3); OR=13.26, 95% CI, 6.06-29.89; Pc=3.10(-8); respectively) and epTB (OR=6.64, 95% CI, 4-11.05; Pc=3.10(-8); OR=2.6, 95% CI, 1.26-5.35; Pc=12.10(-3); OR=11.26, 95% CI, 4.44-29.28; Pc=3.10(-8); respectively). Collectively, our findings suggested an association of the RANTES -28C/G and -403G/A functional polymorphisms with susceptibility to Mycobacterium tuberculosis infection in Tunisian populations.

Hépatite virale B chez les femmes enceintes tunisiennes : facteurs de risque et intérêt de l’étude de la réplication virale en cas d’antigène HBe négatif

OBJECTIVE To evaluate the seroprevalence and the risk factors of hepatitis B virus (HBV) infection in 2303 Tunisian pregnant women and to estimate the risk of perinatal transmission in women positive for hepatitis B surface antigen (HBsAg) but negative for hepatitis B e-antigen (HBeAg). MATERIAL AND METHODS Positive samples were tested for HBeAg and anti-HBe antibody using enzyme immunoassays. Serum HBV-DNA was determined by real time PCR assay. RESULTS Overall, 4% of women were HBsAg positive and for the majority of them (96.8%) this status was unknown. Only 1.4% of studied population were vaccinated previously against hepatitis B. Study of risk factors revealed association between the HBsAg status and presence of intrafamilial hepatitis cases (p<0.05). Only four women were positive for HBeAg. Among patients with HBeAg negative status, only 11% were negative for HBV DNA. For the others, DNA level ranged from 34 to 10(8)copies/ml; it was greater than 10(4)copies/ml in 26.5% of them. CONCLUSION Hepatitis B virus (HBV) prevalence in pregnant women is of intermediate endemicity in Tunisia. Universal vaccination before pregnancy and antenatal screening is recommended. Pregnant women who are found to be HBsAg positive and HBeAg negative should be tested systematically for DNA level to evaluate the risk of perinatal infection and to prevent it by sero-prophylactic for babies or by treatment during the third trimester of pregnancy.

Identification of clinically relevant Corynebacterium strains by Api Coryne, MALDI-TOF-mass spectrometry and molecular approaches

We evaluated the Bruker Biotyper matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) for the identification of 97 Corynebacterium clinical in comparison to identification strains by Api Coryne and MALDI-TOF-MS using 16S rRNA gene and hypervariable region of rpoB genes sequencing as a reference method. C. striatum was the predominant species isolated followed by C. amycolatum. There was an agreement between Api Coryne strips and MALDI-TOF-MS identification in 88.65% of cases. MALDI-TOF-MS was unable to differentiate C. aurimucosum from C. minutissimum and C. minutissimum from C. singulare but reliably identify 92 of 97 (94.84%) strains. Two strains remained incompletely identified to the species level by MALDI-TOF-MS and molecular approaches. They belonged to Cellulomonas and Pseudoclavibacter genus. In conclusion, MALDI-TOF-MS is a rapid and reliable method for the identification of Corynebacterium species. However, some limits have been noted and have to be resolved by the application of molecular methods.

Rapid detection of Mycobacterium tuberculosis in sputum by Patho-TB kit in comparison with direct microscopy and culture.

The usefulness of a new rapid diagnostic test (Patho-TB) using antibodies specific to mycobacterial antigens was evaluated for the rapid discrimination between pulmonary tuberculosis (TB) and non-TB pulmonary diseases on sputa. One hundred sputa collected from 79 active TB patients and from 21 patients with non-TB pulmonary diseases (asthma and chronic obstructive pulmonary disease) were enrolled into the study and tested for the presence of Mycobacterium tuberculosis by Ziehl-Neelsen smear, Patho-TB kit, and Löwenstein-Jensen culture. The sensitivity, specificity, positive predictive value, and negative predictive value of the Patho-TB test were 95%, 100%, 100%, and 84%, respectively. Patho-TB test is simple, quick, and easy to perform. Its sensitivity, specificity, and positive predictive value are satisfactory. Therefore, it could be used as a screening test in poorly equipped laboratories of TB endemic areas.

Association of an HLA-G 14-bp Insertion/Deletion polymorphism with high HBV replication in chronic hepatitis.

Identification of an HLA‐G 14‐bp Insertion/Deletion (Ins/Del) polymorphism at the 3′ untranslated region of HLA‐G revealed its importance in HLA‐G mRNA stability and HLA‐G protein level variation. We evaluated the association between the HLA‐G 14‐bp Ins/Del polymorphism in patients with chronic Hepatitis B virus (HBV) infection in a case–control study. Genomic DNA was extracted from 263 patients with chronic HBV hepatitis and 246 control subjects and was examined for the HLA‐G 14‐bp Ins/Del polymorphism by PCR. The polymorphic variants were genotyped in chronic HBV seropositive cases stratified according to HBV DNA levels, fibrosis stages and in a control population. There was no statistical significant association between the 14‐bp Ins/Del polymorphism and increased susceptibility to HBV infection neither for alleles (P = 0.09) nor for genotypes (P = 0.18). The stratification of HBV patients based on HBV DNA levels revealed an association between the 14‐bp Ins/Del polymorphism and an enhanced HBV activity with high HBV DNA levels. In particular, the Ins allele was significantly associated with high HBV DNA levels (P = 0.0024, OR = 1.71, 95% CI 1.2–2.4). The genotype Ins/Ins was associated with a 2.5‐fold (95% CI, 1.29–4.88) increased risk of susceptibility to high HBV replication compared with the Del/Del and Ins/Del genotypes. This susceptibility is linked to the presence of two Ins alleles. No association was observed between the 14‐bp Ins/Del polymorphism and fibrosis stage of HBV infection. We observed an association between the 14‐bp Ins/Del polymorphism and high HBV replication characterized by high HBV DNA levels in chronic HBV patients. These results suggest a potential prognostic value for disease outcome evaluation.