Hanen Sellami

Chlamydia trachomatis infection increases the expression of inflammatory tumorigenic cytokines and chemokines as well as components of the Toll-like receptor and NF-κB pathways in human prostate epithelial cells

Inflammation has been reported to play a major role in prostate carcinogenesis. Several bacterial infections can lead to prostate inflammation; however, until now, the precise molecular and cellular mechanisms linking inflammation to carcinogenesis have remained unclear. We therefore investigated the initiation of inflammation induced by Chlamydia trachomatis (C. trachomatis) infection in human prostate epithelial cells using an in vitro culture system in which human androgen-independent PC-3 prostate cancer epithelial cells were infected with C. trachomatis serovar L2. The expression levels of VEGF, ICAM-1, IL-6, IL-8, IL-1β, TNFα, CCL5, CCL2 and iNOS inflammation-related genes, as well as genes involved in the Toll-like receptor (TLR) pathway (TLR2, TLR4, CD14 and MyD88), were evaluated at the mRNA level in infected PC-3 cells 24 h after infection with C. trachomatis serovar L2. The expression levels of components of the NF-κB pathway (p65 and IκBα) were evaluated at the mRNA level in infected PC-3 cells at different time points (1, 6, 12 and 24 h) after infection. The expression levels of inflammation-related genes, components of the Toll-like receptor pathway and genes involved in NF-κB activation were analyzed in infected and uninfected cells using semi-quantitative RT-PCR. We detected a significant increase (p < 0.001) in inflammation-related cytokines in infected PC-3 cells. During infection, PC-3 cells elicited a proinflammatory response, as shown by NF-κB activation, TLR2 and TLR4 upregulation and the increased expression of inflammation-related genes. Furthermore, we observed significant upregulation of the adhesion molecules ICAM-1 and VEGF, which are two biomarkers correlated with tumor progression and immune system evasion. The present study suggests that human prostate cancer epithelial cells are susceptible to C. trachomatis infection and upregulate proinflammatory markers during infection.

Chlamydia trachomatis genovar distribution in clinical urogenital specimens from Tunisian patients: high prevalence of C. trachomatis genovar E and mixed infections

BackgroundThis epidemiological study was carried out in Sfax (south of Tunisia) and focused on genital Chlamydia trachomatis (C. trachomatis) genovar distribution.MethodsOne hundred and thirty seven genital samples from 4067 patients (4.2%) attending the Habib Bourguiba University hospital of Sfax over 12 years (from 2000 to 2011) were found to be C. trachomatis PCR positive by the Cobas Amplicor system. These samples were genotyped by an in house reverse hybridization method.ResultsOne hundred and eight (78.8%) samples contained only one genovar and 29 (21.2%) samples contained two or three genovars. Genovar E was the most prevalent (70.8%) single genovar and it was detected in 90.6% of all the cases. Genovars J, C and L1-L3 were not detected in our samples whereas ocular genovars A and B were in 5 cases. All the five cases were mixed infections. Men had more mixed infections than women (p=0.02) and were more frequently infected by genovars F and K (p<0.05). No associations between current infection, infertility and the genovar distribution were observed. Patients coinfected with Neisseria gonorrhoeae were also significantly more frequently infected with mixed genovars (p=0.04).ConclusionsIn conclusion, we have reported a high prevalence of genovar E and of mixed infections in our study population. Such data could have implications for the control and vaccine development of C. trachomatis in Tunisia.

Assessment of Chromatin Maturity in Human Spermatozoa: Useful Aniline Blue Assay for Routine Diagnosis of Male Infertility

During spermatogenesis, sperm chromatin undergoes structural changes and results in a high condensation. This nuclear compaction would be useful as a predictor of sperm fertilization capacity and pregnancy outcome. We purpose to evaluate firstly the relationship among chromatin maturity assessed by aniline blue staining (AB) and the semen parameters in infertile men. Secondly, we analyzed whether the sperm gradient density centrifugation is effective to select mature spermatozoa. Fifty-one ejaculates were investigated by semen analysis and stained for chromatin condensation with AB to distinguish between unstained mature sperm and stained immature sperm. AB was applied also on 12 ejaculates which proceeded by density gradient centrifugation to compare the rates of immature sperm before and after selection. Neat semen were divided into two groups: G1 (n = 31): immature sperm <20% and G2 (n = 20): immature sperm ≥20%. No significant differences were detected in sperm concentration, motility, and normal morphology between G1 and G2. However, the rates of some morphology abnormalities were higher in G2: head abnormalities (P = 0.01) and microcephalic sperm (P = 0.02). We founded significant correlation between sperm immaturity and acrosome abnormalities (r = 0.292; P = 0.03). Sperm selection has significantly reduced the rates of immature sperm. A better understanding of chromatin structure and its impact on the sperm potential is needed to explore male infertility.

Molecular diagnosis of Pneumocystis jiroveci pneumonia in immunocompromised patients.

Pneumocystis jiroveci is the major cause of pneumonia in immunocompromised patients. To evaluate the performance of single and nested‐polymerase chain reaction (PCR) methods compared with immunofluorescent assay (IFA) and cytological staining for diagnosis of P. jiroveci infection, the bronchoalveolar lavage (BAL) and sputum samples from 60 immunocompromised patients were studied. Between January 2005 and March 2008, 75 respiratory specimens (41 BAL and 34 sputum samples) were examined for P. jiroveci identification. We used the clinical classification as our diagnostic standard and we considered true positive the definite or probable Pneumocystis pneumonia. Fourteen patients (23.3%) developed Pneumocystis pneumonia. Eleven patients had a positive IFA but only nine were positive by cytological staining. Sixteen patients had a positive detection of P. jiroveci by PCR and nested‐PCR. Thirteen of these patients were considered as having a definite Pneumocystis pneumonia and one patient with a probable Pneumocystis pneumonia. Five other patients had a positive detection only by nested‐PCR. These patients were classified as no Pneumocystis pneumonia. PCR detection of P. jiroveci is a very sensitive test and will offer a powerful technique in clinical laboratories for the routine diagnosis of Pneumocystis pneumonia. Using the nested‐PCR, additional clinical cases can be diagnosed, but there is then an obvious risk of detecting subclinical colonisation by P. jiroveci.

Molecular detection of Chlamydia trachomatis and other sexually transmitted bacteria in semen of male partners of infertile couples in Tunisia: the effect on semen parameters and spermatozoa apoptosis markers.

This study was undertaken to determine the prevalence of Chlamydia trachomatis, Mycoplasmas, and Ureaplasmas in semen samples of the male partners of infertile couples and to investigate whether Chlamydia trachomatis could initiate apoptosis in human spermatozoa. A total of 85 males partners of infertile couples undergoing routine semen analysis according to World Health Organization guidelines were included. Specimens were examined for the presence of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum and Ureaplasma parvum by Real time PCR (qPCR). Semen specimens were analysed for the appearance of apoptotic markers (sperm DNA fragmentation, activated caspase 3 levels, mitochondrial membrane potential (ΔΨm)) using flow cytometry. C. trachomatis, N. gonorrhoeae, U. urealyticum, M genitalium were detected in semen samples of 13 (15.2%), 5 (5.8%), 5 (5.8%) and 3 (3.5%) male partners of infertile couples, respectively. M. hominis and U. parvum were detected in semen sample of only one patient (1.1%). The semen of infertile men positive for C. trachomatis showed lower mean of semen count and lower rapid progressive motility (category [a]) of spermatozoa compared to uninfected men with statistically significances (p = 0.02 and p = 0.04, respectively). Flow cytometry analyses demonstrated a significant increase of the mean rate of semen with low ΔΨm and caspase 3 activation of infertile men positive for C. trachomatis compared to uninfected men (p = 0.006 and p = 0.001, respectively). DNA fragmentation was also increased in sperm of infertile men positive for C. trachomatis compared to uninfected men but without statistical significances (p = 0.62). Chlamydial infection was associated to loss of ΔΨm and caspase 3activation. Thus, C. trachomatis infection could be incriminated in apoptosis induction of spermatozoa. These effects may explain the negative direct impact of C. trachomatis infection on sperm fertilizing ability.

Comparison of Two Quantitative Real Time PCR Assays for Rickettsia Detection in Patients from Tunisia

Background and objectives Quantitative real time PCR (qPCR) offers rapid diagnosis of rickettsial infections. Thus, successful treatment could be initiated to avoid unfavorable outcome. Our aim was to compare two qPCR assays for Rickettsia detection and to evaluate their contribution in early diagnosis of rickettsial infection in Tunisian patients. Patients and methods Included patients were hospitalized in different hospitals in Tunisia from 2007 to 2012. Serology was performed by microimmunofluorescence assay using R. conorii and R. typhi antigens. Two duplex qPCRs, previously reported, were performed on collected skin biopsies and whole blood samples. The first duplex amplified all Rickettsia species (PanRick) and Rickettsia typhi DNA (Rtt). The second duplex detected spotted fever group Rickettsiae (RC00338) and typhus group Rickettsiae DNA (Rp278). Results Diagnosis of rickettsiosis was confirmed in 82 cases (57.7%). Among 44 skin biopsies obtained from patients with confirmed diagnosis, the first duplex was positive in 24 samples (54.5%), with three patients positive by Rtt qPCR. Using the second duplex, positivity was noted in 21 samples (47.7%), with two patients positive by Rp278 qPCR. Among79 whole blood samples obtained from patients with confirmed diagnosis, panRick qPCR was positive in 5 cases (6.3%) among which two were positive by Rtt qPCR. Using the second set of qPCRs, positivity was noted in four cases (5%) with one sample positive by Rp278 qPCR. Positivity rates of the two duplex qPCRs were significantly higher among patients presenting with negative first serum than those with already detectable antibodies. Conclusions Using qPCR offers a rapid diagnosis. The PanRick qPCR showed a higher sensitivity. Our study showed that this qPCR could offer a prompt diagnosis at the early stage of the disease. However, its implementation in routine needs cost/effectiveness evaluation.

A Proposed Mouse Model to Study Male Infertility Provoked by Genital Serovar E, Chlamydia trachomatis

Chlamydia trachomatis is a common sexually transmitted pathogen. The impact of chlamydial infection on male infertility is controversial. The aim of this study was to assess the role of C trachomatis human genital serovar E on sperm function, induction of apoptosis in spermatozoa, and reproductive performance, using the Swiss male mice model. Fertile mice were inoculated in the meatus urethra with 10(6) C trachomatis inclusion-forming units at day 0. The studied parameters were evaluated 7, 15, 21, and 30 days postinoculation (pi) in infected and sham-infected controls. Semen parameters of the infected mice groups were significantly lower than those of the control groups at the different days pi. DNA fragmentation study indicated that the mean percentages of apoptotic spermatozoa in the infected mice groups were significantly higher than those in the control groups 7 and 15 days pi, whereas the mean percentages of necrotic spermatozoa in the infected mice groups were significantly higher than those in the control group on the 30th day pi. A decrease in reproductive performance was observed at different days pi in infected male mice groups when compared to the control groups. Furthermore, a statistically significant decrease in the mean number of infant mice was observed at 21 and 30 days pi. In conclusion, our data showed that inoculation of fertile male Swiss mice in the meatus urethra with C trachomatis could lead to alteration of semen parameters, induction of apoptosis in spermatozoa, and decrease of the reproductive performance of male mice.

Development and application of an in-house reverse hybridization method for Chlamydia trachomatis genotyping

To develop and evaluate an in‐house reverse hybridization technique for Chlamydia trachomatis genotype identification.

Isolation and Identification of Campylobacter spp. from Poultry and Poultry By-Products in Tunisia by Conventional Culture Method and Multiplex Real-Time PCR

Thermophilic Campylobacter spp. are one of the primary causes of bacterial human diarrhea. The consumption of poultry meats, by-products, or both is suspected to be a major cause of human campylobacteriosis. The aims of this study were to determine the prevalence of thermophilic Campylobacter spp. in fresh poultry meat and poultry by-products by conventional culture methods and to confirm Campylobacter jejuni and Campylobacter coli isolates by using the multiplex PCR assay. Two hundred fifty fresh poultry samples were collected from a variety of supermarkets and slaughterhouses located in Sfax, Tunisia, including chicken (n =149) and turkey (n =101). The samples were analyzed using conventional microbiological examinations according to the 2006 International Organization for Standardization method (ISO 10272-1) for Campylobacter spp. Concurrently, a real-time PCR was used for identification of C. jejuni and C. coli . Of the 250 samples of poultry meat and poultry by-products, 25.6% (n = 64) were contaminated with Campylobacter spp. The highest prevalence of Campylobacter spp. was found in chicken meat (26.8%) followed by turkey meat (23.7%). Among the different products, poultry breasts showed the highest contamination (36.6%) followed by poultry by-products (30%), poultry wings (28%) and poultry legs (26%) showed the lowest contamination, and no contamination was found on neck skin. Of the 64 thermophilic Campylobacter isolates, C. jejuni (59.7%) was the most frequently isolated species and 10.9% of the isolates were identified as C. coli . All of the 64 Campylobacter isolates identified by the conventional culture methods were further confirmed by PCR. The seasonal peak of Campylobacter spp. contamination was in the warm seasons (spring and summer). The study concluded that high proportions of poultry meat and poultry by-products marketed in Tunisia are contaminated by Campylobacter spp. Furthermore, to ensure food safety, poultry meats must be properly cooked before consuming.

Molecular Detection of the Three Major Pathogenic Vibrio Species from Seafood Products and Sediments in Tunisia Using Real-Time PCR

Vibrio spp. have emerged as a serious threat to human health worldwide. V. parahaemolyticus , V. cholerae , and V. vulnificus pose a considerable public health risk in Tunisia because they cause sporadic and epidemic foodborne infections associated with the consumption of raw or undercooked contaminated seafood. More recently, toxR-positive V. alginolyticus was also reported to be a potential source of contaminated seafood. A total of 247 samples, including 113 fishes ( Labrus viridis , Penaeus kerathurus , Diplodus annularis , Diplodus sparaillon , Scorparna porcus , Sarpa salpa , Dentex dentex , Scorparna scrofa , Sardinella aurita , Trachurus trachurus , Synodus saurus , Pagellus erythrinus , and Metapenaeus monoceros ), 83 clams ( Ruditapes decussatus species), 30 seawater samples, and 21 sediment samples were analyzed using traditional culture methods (ISO/TS 21872-1; International Organization for Standardization 2007) and a conventional PCR method for Vibrio spp. IDENTIFICATION A rapid, sensitive, and highly reproducible real-time PCR assay was developed to detect the three major Vibrio spp. pathogenic for humans in Tunisian seafood products and sediments. A conventional culture method found 102 (41.3%) of 247 analyzed samples positive for Vibrio spp.; a conventional PCR method found 126 (51%) of the 247 samples positive. Real-time PCR assay found 126 (51.1%) samples positive; V. alginolyticus toxR was the most common, found in 99 (78.57%) of samples, followed by V. parahaemolyticus in 26 (20.63%) and V. cholerae in 1 (0.7%). All culture-positive samples were PCR positive. However, 24 samples that were positive by conventional PCR and real-time PCR were culture negative. Our findings indicate that retail seafood is commonly contaminated with Vibrio spp. and presents a potential risk to human health in Tunisia. These data also indicate that real-time PCR can provide sensitive species-specific detection of Vibrio spp. in seafood without prior isolation and characterization of the bacteria by traditional microbiological methods.