Human miscarriage and infection in Tunisia: Role of Mycoplasma hominis and high Waddlia seroprevalence.

INTRODUCTION: Miscarriage is one of the most common adverse pregnancy outcomes. The aim of this study was to investigate the relationship between miscarriage in humans and infections caused by zoonotic bacteria and genital pathogens. METHODOLOGY: Cervicovaginal swabs and placenta samples from 132 women with miscarriage (patient group: PG), and cervicovaginal swabs from 54 women with normal pregnancy (control group:CG), were subjected to bacteriological culture and real time PCRs detecting Coxiella burnetii, Brucella spp, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, Chlamydia trachomatis, Waddlia chondrophila and Parachlamydia acanthamoebeae DNA. Serology of C. burnetii, C. trachomatis and W. chondrophila was also performed. RESULTS: Placenta samples were positive for E. coli, S. agalactiae, U. urealyticum, M. hominis and C. trachomatis in 4.7%, 3.1%, 3.1%, 0.7% and 0.7% of cases, respectively. For cervicovaginal swabs, M. hominis was more frequently detected among PG than CG with a significant statistical difference (p = 0.02). C. trachomatis was detected in 3.3% and 5.5% among PG and CG, respectively. U. urealyticum DNA was detected with high percentages in the two groups. Samples from both groups showed negatives results for C. burnetii, Waddlia, and Brucella qPCRs. A high rate of W. chondrophila seroprevalence (42%) was noted with significant difference among women with early miscarriage. CONCLUSIONS: C. trachomatis, S. agalactiae and M. hominis may play a role in miscarriage. However, the full characterization of the vaginal flora using other technologies such as NGS-based metagenomics is needed to clarify their role in miscarriage. Finally, further investigations should be performed to explain high W. chondrophila seroprevalence.

Detection of Shigella spp. nucleic acids in the synovial tissue of Tunisian rheumatoid arthritis patients and other forms of arthritis by quantitative real-time polymerase chain reaction.

Enterobacterial components in the joints of patients are believed to contribute to a perpetuating inflammation leading to a reactive arthritis (ReA), a condition in which microbial agents cannot be recovered from the joint. At present, it is unclear whether nucleic acids from Shigella spp. are playing a pathogenic role in causing not only ReA but also other forms of arthritis. Quantitative real-time polymerase chain reaction assay (qPCR) is the method of choice for the identification of bacteria within the synovium. The aim of our study was to detect the presence of Shigella spp. nucleic acids in the synovial tissue (ST) of Tunisian arthritis patients. We investigated 57 ST samples from rheumatoid arthritis (RA) n = 38, undifferentiated oligoarthritis (UOA) n = 12, and spondyloarthritis (SpA) n = 7 patients; 5 ST samples from healthy individuals were used as controls. Shigella spp. DNA and mRNA transcripts encoding the virulence gene A (VirA) were examined using an optimized qPCR with newly designed primers and probes. Using qPCR, Shigella spp. DNA was found in 37/57 (65%) ST samples (24/38, i.e., 63.2% of RA, 8/12, i.e., 67% of UOA, and 5/7, i.e., 71.4% of SpA patients). Paired DNA and mRNA were extracted from 39 ST samples, whose VirA cDNA was found in 29/39 (74.4%) patients. qPCR did not yield any nucleic acids in the five healthy control ST samples. The qPCR assay was sensitive and showed a good intra- and inter-run reproducibility. These preliminary findings generated by an optimized, highly sensitive PCR assay underline a potential role of past gastrointestinal infections. In Tunisian patients, a bacterial etiology involving Shigella spp. in the manifestation of arthritic disorders including RA might be more common than expected.

Sero-epidemiological survey of Crimean-Congo hemorrhagic fever virus in Tunisia.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease associated with a high case fatality rate and transmitted mainly by Hyalomma marginatum. The geographical distribution of H. marginatum covers most of the Western Mediterranean basin. We aimed to investigate whether CCHF virus (CCHFv) is circulating in Tunisia. Samples from unexplained acute febrile patients (n = 181) and a high risk group of humans, mainly slaughter workers (n = 38), were collected in the summer of 2014 and analyzed for exposure to CCHFv using serological tests and real-time RT-PCR. Ticks were collected from Northern and Southern Tunisia during May-June 2014 and examined for the presence of CCHFv by real-time RT-PCR. Of the 181 febrile patients, 5 showed only high titers of IgM suggesting a recent exposure to CCHFv. Among 38 slaughter workers, 2 had IgG anti-CCHFv responses yielding a seroprevalence of 5.2%. No CCHFv was detected in ticks and sera. Our results provide evidence of human exposure to CCHFv in Tunisia.

Molecular diagnosis of Rickettsia infection in patients from Tunisia.

Diagnosis of rickettsioses had largely benefited from the development of molecular techniques. Unfortunately, in Tunisia, despite the large number of rickettsial cases registered every year, the Rickettsia species remain unidentified. In this study, we aimed to detect the Rickettsia species in clinical samples using molecular tests. A study was established to analyze skin biopsies, cutaneous swabs, and cerebrospinal fluid samples taken from clinically suspected patients to have rickettsial infection. Two molecular techniques were used to detect Rickettsia DNA: quantitative real time PCR (qPCR) and reverse line blot test (RLB). An analysis of the RLB hybridization assay results revealed the presence of Rickettsia DNA in skin biopsies (40.6%) and swabs (46.7%). Rickettsia conorii was the most prevalent identified species among tested samples. Other species of interest include Rickettsia typhi and Rickettsia massiliae. Using qPCR positivity rates in skin biopsies was 63.7% against 80% in swabs. R. conorii was the most frequently detected species, followed by R. typhi. The agreement between the two techniques was 68.6% (kappa=0.33). Molecular tests, especially using specific probes qPCR, allow for a rapid, better and confident diagnosis in clinical practice. They improve the survey of Mediterranean spotted fever which is considered to be the most important rickettsial infection in humans in Tunisia.

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.

Coxiella burnetii-positive PCR in febrile patients in rural and urban Africa.

OBJECTIVES: Q fever has been reported throughout the African continent. The objective of this study was to detect the presence of Coxiella burnetii in febrile patients from Africa. METHODS: Blood samples from febrile and non-febrile patients from six African countries and from France were investigated retrospectively for Q fever infection by molecular assays targeting the IS1111 and IS30A spacers. RESULTS: We tested 1888 febrile patients from Senegal, Mali, Tunisia, Algeria, Gabon, and Morocco and found one male adult patient (0.3%) infected with C. burnetii in Algeria and six positive patients (0.5%) in Senegal. For one patient from Senegal we determined that the infection was caused by C. burnetii genotype 35. In Senegal, more patients were infected with C. burnetii in Keur Momar Sarr (p=0.002) than in the other locations. Blood samples taken from 500 (51% males) non-febrile people from Senegal and France were all negative. CONCLUSIONS: The installation of point-of-care laboratories in rural Africa can be a very effective tool for studying the epidemiology of many infectious diseases.

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.

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-1ß, 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.

Detection of Rickettsia in Rhipicephalus sanguineus ticks and Ctenocephalides felis fleas from southeastern Tunisia by reverse line blot assay.

Ticks (n = 663) and fleas (n = 470) collected from domestic animals from southeastern Tunisia were screened for Rickettsia infection using reverse line blot assay. Evidence of spotted fever group Rickettsia was obtained. We detected Rickettsia felis in fleas, Rickettsia massiliae Bar 29 and the Rickettsia conorii Israeli spotted fever strain in ticks, and Rickettsia conorii subsp. conorii and Rickettsia spp. in both arthropods. The sensitivity of the adopted technique allowed the identification of a new association between fleas and R. conorii subsp. conorii species. The presence of these vector-borne Rickettsia infections should be considered when diagnosing this disease in humans in Tunisia.

Common epidemiology of Rickettsia felis infection and malaria, Africa.

This study aimed to compare the epidemiology of Rickettsia felis infection and malaria in France, North Africa, and sub-Saharan Africa and to identify a common vector. Blood specimens from 3,122 febrile patients and from 500 nonfebrile persons were analyzed for R. felis and Plasmodium spp. We observed a significant linear trend (p<0.0001) of increasing risk for R. felis infection. The risks were lowest in France, Tunisia, and Algeria (1%), and highest in rural Senegal (15%). Co-infections with R. felis and Plasmodium spp. and occurrences of R. felis relapses or reinfections were identified. This study demonstrates a correlation between malaria and R. felis infection regarding geographic distribution, seasonality, asymptomatic infections, and a potential vector. R. felis infection should be suspected in these geographical areas where malaria is endemic. Doxycycline chemoprophylaxis against malaria in travelers to sub-Saharan Africa also protects against rickettsioses; thus, empirical treatment strategies for febrile illness for travelers and residents in sub-Saharan Africa may require reevaluation.

Multispacer typing of Rickettsia isolates from humans and ticks in Tunisia revealing new genotypes.

BACKGROUND: Rickettsioses are important remerging vector born infections. In Tunisia, many species have been described in humans and vectors. Genotyping is important for tracking pathogen movement between hosts and vectors. In this study, we characterized Rickettsia species detected in patients and vectors using multispacer typing (MST), proposed by Founier et al. and based on three intergenic spacers (dksA-xerC, rmpE- tRNA(fMet), mppA-pruC) sequencing. METHODS: Our study included 25 patients hospitalized during 2009. Ticks and fleas were collected in the vicinity of confirmed cases. Serology was performed on serum samples by microimmunofluorescence using Rickettsia conorii and Rickettsia typhi antigens. To detect and identify Rickettsia species, PCR targeting ompA, ompB and gltA genes followed by sequencing was performed on 18 obtained skin biopsies and on all collected vectors. Rickettsia positive samples were further characterized using primers targeting three intergenic spacers (dksA-xerC, rmpE- tRNA(fMet) and mppA-purC). RESULTS: A rickettsial infection was confirmed in 15 cases (60%). Serology was positive in 13 cases (52%). PCR detected Rickettsia DNA in four biopsies (16%) allowing the identification of R. conorii subsp israelensis in three cases and R. conorii subsp conorii in one case. Among 380 collected ticks, nine presented positive PCR (2.4%) allowing the identification of six R. conorii subsp israelensis, two R. massiliae and one R. conorii subsp conorii. Among 322 collected fleas, only one was positive for R. felis. R. conorii subsp israelensis strains detected in humans and vectors clustered together and showed a new MST genotype. Similarly, R. conorii subsp conorii strains detected in a skin biopsy and a tick were genetically related and presented a new MST genotype. CONCLUSIONS: New Rickettsia spotted fever strain genotypes were found in Tunisia. Isolates detected in humans and vectors were genetically homogenous despite location differences in their original isolation suggesting epidemiologic circulation of these strains.

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

BACKGROUND: This epidemiological study was carried out in Sfax (south of Tunisia) and focused on genital Chlamydia trachomatis (C. trachomatis) genovar distribution. METHODS: One 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. RESULTS: One 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). CONCLUSIONS: In 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.

[The role of neutrophils in the formation of peritoneal adhesions]. / Rôle des polynucléaires neutrophiles dans la formation des adhérences post opératoires.

BACKGROUND: The use of antibiotics during peritonitis appears to decrease the formation of postoperative intra peritoneal adhesions and reduce their severity. The effect of this antibiotic is still controversial. AIM: To study the relationship between the decrease postoperative adhesions induced by rifamycin, and the number of neutrophils and the number of intraperitoneal bacteria. METHODS: This is an experimental prospective, randomized singleblind study performed on adult male rats. The product used for the peritoneal lavage was rifamycin s. The animals were randomized into three groups: Group S: intra peritoneal lavage with saline to 9%, R25 Group: intra peritoneal lavage with rifamycin at a dose of 25 mg / kg group and 12.5 R: intra peritoneal lavage with rifamycin at a dose of 12.5 mg / kg. Adhesions score was evaluated according to Zulkhé by the same operator. RESULTS: The adhesion score was significantly lower between groups S and R12.5 (p = 0.000) and group S and group R25 (P = 0.01). However, the difference was not significant between the two groups R 25 and R12.5 compared to S group (p = 0.655). The number of bacteria between the time of caecal resection (before peritoneal lavage) and the time of death or sacrifice was significantly decreased significantly in the groups R25, comparing the group S (p = 0.003). However, there is no significant difference between groups S and R12, 5 (p = 0.106). The number of neutrophils between the time of cecal resection (before peritoneal lavage) and the time of death or sacrifice decreased significantly in the groups R25 and R12, 5 in comparison to the group S. Between the group R25 and the S group, the difference is significant (p = 0.037) as well between the group R12, 5 and S (p = 0.026). However, there is no significant difference between the two groups R 25 and R12, 5 (p = 0.712). CONCLUSION: The action of rifamycin sodium on neutrophils seems to be independent of its antibacterial action. These findings deserve to be explored at the end to clarify the mechanism of neutropenia by intra peritoneal washing with rifamycin and the relationship between neutropenia and post-operative adhesions.

Diversity of ß-lactamases in Pseudomonas aeruginosa isolates producing metallo-ß-lactamase in two Tunisian hospitals.

This study was conducted to identify the ß-lactamase content of 30 metallo-ß-lactamase-producing Pseudomonas aeruginosa isolated in 2007 from two Tunisian hospitals and to investigate their genetic relatedness. All these isolates produced VIM-2. bla(PER-1), bla(PSE-1), bla(OXA-2), and bla(OXA-10) were identified in 17, 5, 21, and 1 isolates, respectively. These enzymes were often associated in the same isolate: 26 isolates had at least two ß-lactamases. The predominant serotype was O12. Pulsed-field gel electrophoresis revealed genetic diversity among the metallo-ß-lactamase-producing P. aeruginosa isolates. This is the first report on the existence of bla(PER-1), bla(PSE-1), bla(OXA-2), and bla(OXA-10) in Tunisia.

Sexually transmitted infections among female sex workers in Tunisia: high prevalence of Chlamydia trachomatis.

OBJECTIVES: The aim of this study was to determine the prevalence of Chlamydia trachomatis infection and other sexually transmitted infections (STI) in female sex workers (FSW) in Tunisia. METHODS: 188 prostitutes from three Tunisian towns were enrolled at their weekly medical visit. Demographic and sexual behaviour data were collected. C trachomatis, Neisseria gonorrhoeae, herpes simplex virus 2 (HSV-2) and human papillomavirus (HPV) were detected by PCR. Blood samples were tested for the presence of HIV, hepatitis B core, hepatitis C virus (HCV), HSV-2, C trachomatis and syphilis antibodies and Hbs antigen. RESULTS: The mean age of the FSW was 34 years. They had worked in the sex industry for 6.6 years on average. Nearly all FSW (98.9%) had at least one marker of STI. A current infection was found in 86.7% of cases. Only one STI was noted in 37.2% and two or more in 49.5% of FSW. C trachomatis, N gonorrhoeae, HPV and HSV-2 PCR were positive in 72.9%, 11.2%, 44.1% and 1.1% of cases, respectively. Syphilis, HCV antibodies and Hbs antigen were detected in poor percentages, 2.7%, 1.1% and 0.5% of cases, respectively. No case of HIV infection was noted. No epidemiological or clinical factors were associated with STI. Only disturbed bacterial vaginal flora was found to be associated with C trachomatis infection. CONCLUSION: In this study, a high rate of C trachomatis infection was observed. The detection of this microorganism should be introduced in systematic surveillance of Tunisian FSW.

Diagnostic value of enzyme-linked immunosorbent assays using hypothetical proteins CT226 and CT795 as antigens in Chlamydia trachomatis serodiagnosis.

The CT226 and the CT795 proteins were produced as purified recombinant proteins and were used as antigens in enzyme-linked immunosorbent assay (ELISA) tests for the detection of Chlamydia trachomatis IgG antibodies. The performances of the developed ELISA tests were compared with our in-house microimmunofluorescence test and the species-specific pELISA test using a panel of 342 sera. Our results indicate that the performance of the CT795 ELISA test was higher than that of the CT226 ELISA test according to the microimmunofluorescence and to the pELISA. To assess whether a combination of tests could improve the serodiagnosis of C. trachomatis infections, we associated results obtained with these tests to that using the previously developed CT694 ELISA test. Combining ELISA test results did not improve significantly the performances of these ELISA tests. The CT795 ELISA test, showing the highest performance, may be used for the serodiagnosis of C. trachomatis infections.

Rifamycin lavage in the treatment of experimental intra-abdominal infection.

HYPOTHESIS: Peritoneal lavage with rifamycin reduces the number of intraperitoneal bacteria and adhesions and improves the outcome of intra-abdominal infection (IAI). MATERIAL AND METHODS: Experimental IAI was induced in Wistar rats. After 24 h, the animals underwent relaparotomy. A peritoneal fluid sample was obtained and lavage of the abdominal cavity was performed. Animals were randomly assigned to the three following groups: lavage with 0.9% sodium chloride solution (S group); lavage with rifamycin at the dose of 25 mg/kg (R25 group); and lavage with rifamycin at the dose of 12.5 mg/kg (R12.5 group). Mortality was recorded every 8 h for 7 d. All animals that died had a necropsy. Surviving rats were later sacrificed and also underwent a necropsy. At necropsy, intraperitoneal adhesions were noted and a peritoneal fluid sample was obtained for bacterial analysis. RESULTS: Peritoneal lavage with rifamycin improved survival from 50% in the S group to 91.7 and 100% in the R25 group and R12.5 group, respectively. Adhesion formation was significantly reduced in the R25 group and R12.5 group compared with the S group (P < or = 0.01 and P < 0.01, respectively). There was a greater reduction in bacterial counts in peritoneal fluid in the R25 group compared with the S group (P = 0.003) but there was no significant difference in the reduction of bacterial count between R25 group and R12.5 group. CONCLUSION: These results suggest that peritoneal lavage with rifamycin improves the outcome of IAI.