Naila Hannachi

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.

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.

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.

Comparison of LED and conventional fluorescence microscopy for detection of acid-fast bacilli in an area with high tuberculosis incidence

The objective of the study is to compare the performance of conventional fluorescence microscopy (CFM) and light-emitting diode (LED) fluorescence microscopy (FM) for detection of acid-fast bacilli (AFB) in clinical samples. We included AFB smears, stained using the auramine O method and blindly examined with both CFM and LED-FM. Culture results were used as reference for evaluating the reliability of the FM. We included 180 culture positive specimens and an equal number of culture negative specimens. Sensitivities for the CFM and LED-FM were 79.4% and 82.2%, respectively. Both microscopes had a high specificity (97.2%). The negative-positive (>1 cross) inter-reader agreement of LED-FM and CFM was excellent. Therefore, detection of scanty AFB was higher with LED-FM. Both microscopes were equivalent with respect to time required to read smears. Although it was not faster than CFM, the higher detection of scanty AFB smears combined with ease of use supports the consideration of LED microscopy by all tuberculosis diagnostic laboratories, as a replacement for conventional fluorescence microscopes.

Prévalence de l’hépatite virale C chez le personnel de santé au Centre tunisien

Les professionnels de santé sont confrontés au risque de ontamination par les virus de l’hépatite virale B et C, et ce par ’intermédiaire du sang et des liquides biologiques. Dans la littéature, les résultats de la prévalence de l’hépatite virale C (HVC) hez le personnel de santé sont discordants. En Tunisie, aucune tude de prévalence de l’HVC n’a été publiée. L’objectif de ce ravail était de déterminer la prévalence des anticorps antivirus e l’hépatite C (VHC) chez les personnels de santé de l’hôpital arhat-Hached de Sousse.

Hepatitis D Virus Infection Among Hepatitis B Surface Antigen Carriers and in “Isolated anti-HBc” Antibodies Profile in Central Tunisia

Background: Hepatitis D Virus (HDV) causes accelerated liver diseases in patients with Hepatitis B Virus (HBV) infection. There is lack of data about its prevalence, related risk factors and interaction with HBV carriers in our country. Objectives: The aim of this study was to estimate the prevalence of hepatitis delta and associated risk factors among Hepatitis B surface antigen (HBsAg) and “isolated anti-HBc” profile carriers in central Tunisia. Patients and Methods: In this cross-sectional study, 540 patients with positive HBsAg and 109 “isolated anti-HBc” profile receiving care in a teaching hospital were tested for the presence of HDV serum-markers using commercially available enzyme immunoassay kit. HBV-DNA was detected by nested PCR in “isolated anti-HBc” profile group. Results: Prevalence of HDV was 8.1% in HBsAg carriers group, but it was significantly higher in active than inactive hepatitis (30.2% and 4.5%, respectively, OR = 9, 95% CI: [4.48-18.58]). There was no significant association between studied risk factors and HDV infection. In the “isolated anti-HBc” profile group, prevalence of HDV was 4.6% and HBV-DNA had negative result in all patients with positive results for HDV. Conclusions: Although HDV had low prevalence in our area, it is vital to plan preventive strategies for HDV spread as well as HBV prevention. It is particularly important to suspect HDV infection in active HBV carriers to manage a particularly severe dual infection. HDV infection should be suspected even in negative HBsAg patients having “isolated anti-HBc” profile.

HLA-E polymorphism and soluble HLA-E plasma levels in chronic hepatitis B patients

Chronic hepatitis B virus (HBV) infection occurs in association to a deregulation of immune system. Human leukocyte antigen E (HLA‐E) is an immune‐tolerant nonclassical HLA class I molecule that could be involved in HBV progression. To measure soluble (s) HLA‐E in patients with chronic HBV hepatitis (CHB). We tested the potential association of HLA‐E*01:01/01:03 A > G gene polymorphism to CHB. Our cohort consisted of 93 Tunisian CHB patients (stratified in CHB with high HBV DNA levels and CHB with low HBV DNA levels) and 245 healthy donors. Plasma sHLA‐E was determined using enzyme‐linked immunosorbent assay (ELISA). Genotyping was performed using polymerase chain reaction sequence‐specific primer. No association between HLA‐E*01:01/01:03 A > G polymorphism and HBV DNA levels in CHB patients was found. G/G genotype is less frequent in CHB patients without significance. sHLA‐E is significantly enhanced in CHB patients compared with healthy controls (P = 0.0017). Stratification according to HBV DNA levels showed that CHB patients with low HBV DNA levels have higher sHLA‐E levels compared with CHB patients with high HBV DNA levels. CHB patients with G/G genotype have enhanced sHLA‐E levels compared with other genotypes (P = 0.037). This significant difference is maintained only for CHB women concerning G/G genotypes (P = 0.042). Finally, we reported enhanced sHLA‐E in CHB patients with advanced stages of fibrosis (P = 0.032). We demonstrate, for the first time, the association of sHLA‐E to CHB. Owing to the positive correlation of HLA‐E*01:01/01:03 A > G polymorphism and the association of sHLA‐E to advanced fibrosis stages, HLA‐E could be a powerful predictor for CHB progression. Further investigations will be required to substantiate HLA‐E role as a putative clinical biomarker of CHB.

Naturally Occurring Surface Antigen Variants of Hepatitis B Virus in Tunisian Patients

In Tunisia, the prevalence of naturally occurring surface (S) gene variants of hepatitis B virus (HBV) has not been determined. In the present study, the prevalence of these variants was examined in terms of the clinical and viral state in a series of 99 Tunisian patients with HBV infection. The S genes were amplified and directly sequenced. Genotype D was predominant (98%), 40.4% isolates belonged to subgenotypes D7 and 1 to subgenotype D2. The most common subtype was ayw2 (95.9%). In total, 60.6% of the studied strains harbored S mutations. Several novel mutation patterns were detected. Interestingly, the presence of S mutations was significantly correlated with the D7 subgenotype, low HBV DNA and advancing age (≥35 years), and tended to be higher in liver cirrhosis than in chronic infection. The global prevalence of the major hydrophilic region variants was 12.1%, with substitution S143L/T as the most frequent (4%). Only 33.9% of S substitutions produced amino acid changes in the polymerase gene. In conclusion, a high prevalence of naturally occurring HBsAg variants was observed among Tunisian HBV carriers. Natural viral variability in a geographical region and duration of infection are among the major factors associated with the occurrence of S mutations.