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

AIM: To develop and evaluate an in-house reverse hybridization technique for Chlamydia trachomatis genotype identification. METHODS AND RESULTS: The evaluation of the developed and optimized reverse hybridization method on reference strains showed the specific detection of all genotypes. This technique showed its ability to type one inclusion-forming unit of C. trachomatis genotype E and equivalent sensitivity to the Cobas TaqMan assay. It was also able to detect mixed infections in vitro. Application of the reverse hybridization method on 38 isolated C. trachomatis strains and their respective swabs allowed the detection of six urogenital genotypes D, E, F, G, H and K and one trachoma genotype B. Genotype E was the most prevalent, detected in 73% of the swab samples. Mixed infections were detected in 26% of swab cases. CONCLUSION: The reverse hybridization technique is simple and does not require specialized instruments. It is powerful in the diagnosis of mixed infections and is suitable for use in epidemiological studies. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique allowed rapid C. trachomatis genotype identification.

Diagnostic value of an enzyme-linked immunosorbent assay using the recombinant CT694 species-specific protein of Chlamydia trachomatis.

AIM: To study the performance of the CT694 protein in relation to the microimmunofluorescence (MIF) and the pELISA tests for the serodiagnosis of Chlamydia trachomatis infections. METHODS AND RESULTS: The CT694 protein was produced as recombinant protein and was used as antigen in ELISA test for the detection of C. trachomatis IgG antibodies. The performance of the developed ELISA test was compared to the MIF test at two cut-off values of 16 and 64, and to the specific pELISA test using a panel of 342 sera. These sera were from children MIF C. trachomatis and Chlamydophila pneumoniae negative, patients MIF C. pneumoniae positive, patients MIF C. trachomatis positive, patients suspected to have chlamydial infections diagnosed by the Cobas Amplicor test, healthy blood donors and prostitutes. Our results indicate that the developed ELISA test has performed better compared with the MIF and the pELISA tests. The highest performance was obtained when comparing the developed ELISA test in relation to the pELISA, yielding an overall sensitivity and specificity of 85% and 87% respectively. CONCLUSIONS: The CT694 ELISA showed the best performance when compared to the species-specific pELISA test and may be used for the serodiagnosis of C. trachomatis infections. SIGNIFICANCE AND IMPACT OF THE STUDY: The CT694 ELISA test responds to the criteria of both sensitivity and specificity according to the MIF and pELISA tests and may be used for serodiagnosis of C. trachomatis infections.

Prosthetic valve endocarditis: management strategies and prognosis: A ten-year analysis in a tertiary care centre in Tunisia.

BACKGROUND: Prosthetic valve endocarditis (PVE) is a rare and serious complication after heart valve replacement; its optimal management strategy, though, still needs to be defined. OBJECTIVE: To study the clinical, microbiological and echocardiographic characteristics of PVE and to analyse the influence of the adopted therapeutic strategy (medical or surgical) on short- and midterm outcome in a tertiary care centre in a developing country (Tunisia). METHODS: All cases of PVE treated in our institution between 1997 and 2006 were retrospectively analysed according to the modified DUKE criteria. RESULTS: A total of 48 PVE episodes were diagnosed (30 men and 18 women), mean age was 37.93 years. Twenty-eight patients (58.33%) were exclusively medically treated, whereas 20 (41.66%) were treated by a combined surgical and medical strategy. Indications for surgery were haemodynamic deterioration in eight patients (40%), annular abscess in six (30%) and persisting sepsis in six (30%). In comparison with those from the medical group, operated patients had a longer delay to diagnosis (p=0.025), were more frequently in heart failure (p=0.04) and experienced more early complications (p=0.011); they also more frequently had prosthetic dehiscence (p=0.015), annular abscesses (p=0.039) and vegetations >10 mm (p=0.008). Conversely, no differences were found between the groups in terms of age, sex, or nature of involved organisms. In-hospital mortality for the medical group was 14.28% and for the surgical group 35% (p=0.09). CONCLUSION: PVE is a very serious condition carrying high mortality rates regardless of the adopted strategy. Our study demonstrates that, in selected patients, medical treatment could be a successful and acceptable approach. (Neth Heart J 2009;17: 56-60.).

Assessment of Chlamydia trachomatis, Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis, and Mycoplasma genitalium in semen and first void urine specimens of asymptomatic male partners of infertile couples.

The purpose of this study was threefold: to compare semen and first void urine (FVU) specimens from asymptomatic infertile men for the detection of Chlamydia trachomatis, genital ureaplasma, and genital mycoplasma infections using in-house inhibitor-controlled polymerase chain reaction (PCR)-microtiter plate hybridization assay; to determine the prevalence of those organisms in infertile men in Tunisia; and to study the relationship between these bacteria and male infertility. Paired urine and semen specimens from 104 patients were examined by in-house PCR for the presence of DNA of Chlamydia trachomatis, genital ureaplasmas (Ureaplasma urealyticum and Ureaplasma parvum) and genital mycoplasmas (Mycoplasma hominis and Mycoplasma genitalium). Semen analysis was assessed according to the guidelines of the World Health Organization. Nominal scale variables, the Mann-Whitney test, and the Kruskal-Wallis nonparametric analysis of variance test were used for statistical analysis. There was a very high concordance (>95%) and a very good agreement (kappa > 0.9) between the detection of Chlamydia trachomatis, genital ureaplasmas, and Mycoplasma hominis in semen and corresponding FVU specimens. Our findings also show a high concordance (81.1%) and a good agreement (kappa = 0.79) between the detection of Mycoplasma genitalium in both specimens. C trachomatis, genital mycoplasmas, and genital ureaplasmas were found to be widespread among infertile male patients in Tunisia, as shown by their respective prevalences of 43.3%, 18.3%, and 14.4%. The mean values of seminal volume, sperm concentration, sperm viability, sperm motility, sperm morphology, and leukocyte count were not significantly related either to the detection of C trachomatis DNA or to that of genital ureaplasma or mycoplasma DNA in semen specimens. Using our in-house PCR, both semen and FVU were found to be sensitive diagnostic specimens for the detection of C trachomatis, ureaplasmas, and mycoplasmas. The FVU, a less invasive and self-collected specimen, can serve as a marker for the presence of these organisms in the genital tract and can be used as a reliable way of detecting asymptomatic carriers of infection.

Screening for bacterial pathogens in semen samples from infertile men with and without leukocytospermia.

We aimed to study the correlation between leukocyte counts in semen and bacterial pathogens in seminal samples of infertile men, and to establish the minimum leukocyte count associated with significant bacteriospermia. A total of 116 patients who underwent evaluation of fertility were investigated using routine semen analysis according to the guidelines of the WHO and bacterial pathogens analysis by culture and in-house PCR assay. The overall prevalence of bacteriospermia in semen samples was 56.9% independent of the presence of leukocytes. The most common bacterial species detected were Chlamydia trachomatis (41.4%), Ureaplasma urealyticum (15.5%) and Mycoplasma hominis (10.3%). The receiver operating characteristic curve analysis demonstrated that the sensitivity/specificity for detecting bacteria at a cut off level of >or=1 x 10(6) leukocytes per ml (which is the WHO defined level for leukocytospermia) was 20.3%/81.5%. The highest sensitivity/specificity ratio was found in semen samples with a cut-off level of >or=0.275 x 10(6) leukocytes per ml, which is best shown with the odds ratio of 2.47. A significant correlation was found between bacteriospermia and leukocytospermia at the cut-off level of >or=0.275 x 10(6) leukocytes per ml of semen samples (P = 0.032). We proposed that this is a possible new cut-off level to predict the presence of bacteria in semen of infertile men.

[Resistance to third generation cephalosporins in Sfax hospitals, Tunisia (1999-2005)]. / Etude de la résistance des entérobactéries aux céphalosporines de troisième génération dans les hôpitaux de Sfax, Tunisie (1999-2005).

OBJECTIVES: The authors wanted to assess the prevalence and to monitor the trends of resistance to broad-spectrum cephalosporins among various species of enterobacteria in the region of Sfax (Tunisia). METHODS: A retrospective study was carried out in the microbiology laboratory at the Habib-Bourguiba Teaching Hospital in Sfax. Data concerning a seven-year period (1999-2005) were analyzed with the Whonet 5.4 software. All clinical isolates of enterobacteria were identified with the API 20 E system. Antimicrobial susceptibilities were determined by disk diffusion on Mueller Hinton agar according to CA-SFM recommendations. RESULTS: During the study period, 24,702 non-duplicate clinical strains of enterobacteria were identified. Fifteen percent (3,826) clinical isolates showed acquired resistance to third generation cephalosporins (3rdGC). The overall frequency of resistance increased from 10% in 1999 to 18% in 2005. This increase was statistically significant. High prevalence rates of 3rdGC resistance have been observed in intensive care units (48%), hematology and oncology wards (27%) and pediatric wards (25%). Klebsiella pneumoniae, Indole positive Proteus and Enterobacter showed high prevalence rates of broad-spectrum cephalosporin resistance. CONCLUSION: This study revealed a high rate of 3rdGC resistance enterobacteria in our region, particularly in intensive care units. The frequency of acquired resistance to broad-spectrum cephalosporins seemed to be increasing. Implementation of infection control measures and identification of the mechanism responsible for third generation cephalosporins resistance are necessary to limit the spreading of these resistant enterobacteria in hospitals and community settings.

Culture-independent genome sequencing of Coxiella burnetii from a native heart valve of a Tunisian patient with severe infective endocarditis.

We report draft genome of a Coxiella burnetii strain sequenced from the native valve of a patient presenting with severe endocarditis in Tunisia. The genome could be sequenced without a cellular or axenic culture step. The MST5 strain was demonstrated to be closely related to the published reference genome of C. burnetii CbuK_Q154.

[Virus and Atypical Pathogens Detected in Community-Acquired Lower Respiratory Tract Infection in Infants and Children of Sfax Region, Tunisia]. / Virus et bactéries atypiques détectés dans les infections respiratoires basses communautaires de l'enfant dans la région de Sfax en Tunisie.

Little is known about viral and atypical bacteria pathogen spectra of community-acquired lower respiratory tract infection in children in Tunisia. Thus, a prospective study was carried out between January 2009 and March 2010 in Sfax. Nasopharyngeal aspirates collected from 368 patients (78 with pneumonia and 290 with acute bronchiolitis) were analyzed by indirect immunofluorescence assay and PCR to detect influenza viruses, parainfluenza viruses, respiratory syncytial virus (RSV), human metapneumovirus, human rhinovirus, human enterovirus, adenovirus, coronavirus, Mycoplasma pneumonia (Mpn) and Chlamydia pneumonia (Cpn). One or more etiology was documented in 319 cases (86.7%). The most detected viruses were RSV (42.7%), rhinovirus (32.9%) and adenovirus (28.5%). Co-detection of two or three pathogens was found in 40% of positive samples. This study highlights the importance of respiratory viruses in lower respiratory tract infection in children of Sfax region as well as the high rate of co-detection of multiple viruses, resulting in challenges in clinical interpretation.

Le profil étiologique microbien des infections respiratoires basses (IRB) communautaires de l'enfant a été peu étudié en Tunisie. Une étude prospective a été menée à Sfax entre janvier 2009 et mars 2010 sur 368 enfants hospitalisés pour pneumonie (n = 78) ou bronchiolite aiguë (n = 290). Les aspirations nasopharyngées ont été analysées par immunofluorescence et par PCR à la recherche des virus influenza, virus para-influenza, virus respiratoire syncytial (VRS), métapneumovirus, rhinovirus, entérovirus, adénovirus, coronavirus, Mycoplasma pneumoniae (Mpn) et Chlamydia pneumoniae (Cpn). Une étiologie ou plus a été retrouvée dans 319 cas (86,7 %) : principalement le VRS (42,7 %), des rhinovirus (32,9 %) et des adénovirus (28,5 %). Dans 40 % des prélèvements positifs, deux ou trois agents pathogènes ont été codétectés. Cette étude a permis de montrer la prévalence élevée des virus dans les IRB de l'enfant dans la région de Sfax et leur détection fréquente en co-infection posant la question sur leur rôle pathogène réel.

[Bacteriological profile of infectious endocarditis in the area of Sfax (Tunisia)]. / Profil bactériologique des endocardites infectieuses dans la region de Sfax (Tunisie).

We performed a study to analyze epidemiological characteristics and bacteriological profile of infectious endocarditis (I.E) in the area of Sfax (Tunisia). We analyzed, retrospectively, all cases of I.E, according to Duke Criteria, hospitalized in the CHU Hédi Chaker of Sfax between January 1997 and December 2000. Bacteriological investigation included blood culture, cardiac valve culture and serology. Seventy-two cases of I.E were diagnosed. The average of age was 32.3 years. 47.3% of the patients did not have cardiac disease, 25% had a valvular prosthesis, 20.8% a native valvulopathy and 6.9% a congenital cardiopathy. Antecedent of acute rheumatic fever was noted in 66% of I.E on native valvulopathy and in 55.5% of I.E on prosthesis. The mitral valve was involved in 39%, the aortic in 27.5% and the two in 26% of the cases. The origin of bacteremia was found or supposed in 55.5% of the cases and was commonly dental (33 % of EI). The bacteriological diagnosis was positive in 51 cases (70.8%). Staphylococci were isolated in 17 cases (23.6%), Streptococci in 17 cases (23.6%) and dominated by oral streptococci (12 cases). Chlamydial serology was positive in 8 cases (11.1%). Diagnosis of infectious endocarditis due to Chlamydia pneumoniae was confirmed in a case by genomic amplification (PCR) and in situ hybridization on the valve. Endocarditis in Tunisia remains frequent. It reaches with predilection the young person in particular with rheumatic heart diseases. The bacteriological profile remains dominated by Streptococci and the Staphylococci.

Serologic evidence of exposure to Rift Valley fever virus detected in Tunisia.

Rift Valley fever virus (RVFv) is capable of causing dramatic outbreaks amongst economically important animal species and is capable of causing severe symptoms and mortality in humans. RVFv is known to circulate widely throughout East Africa; serologic evidence of exposure has also been found in some northern African countries, including Mauritania. This study aimed to ascertain whether RVFv is circulating in regions beyond its known geographic range. Samples from febrile patients (n = 181) and nonfebrile healthy agricultural and slaughterhouse workers (n = 38) were collected during the summer of 2014 and surveyed for exposure to RVFv by both serologic tests and PCR. Of the 219 samples tested, 7.8% of nonfebrile participants showed immunoglobulin G reactivity to RVFv nucleoprotein and 8.3% of febrile patients showed immunoglobulin M reactivity, with the latter samples indicating recent exposure to the virus. Our results suggest an active circulation of RVFv and evidence of human exposure in the population of Tunisia.

Macrophagic activation syndrome related to an infection by Rickettsia conorii in a child.

Since the Arab Spring, a resurgence of zoonotic diseases such as rickettsiosis, endemic in the Mediterranean basin, has been observed. It preferentially infects microvascular endothelial cells of mammalian hosts inducing vasculitis with endothelial injury. Rickettsioses are considered benign infectious diseases. Severe systemic manifestations have been reported and are often explained by a delay in diagnosis. We present a case of hemophagocytic syndrome occurring in a 4-year-old Libyan girl as a complication of Mediterranean spotted fever. Rickettsial infection was confirmed by serology and the patient was treated with clarithromycin, with a favorable outcome.

Murine typhus in Tunisia: a neglected cause of fever as a single symptom.

OBJECTIVES: We had for aim to describe the epidemiologic and clinic characteristics of murine typhus in a series of 43 serologically confirmed cases, in our region. PATIENTS AND METHODS: Serologic screening for IgG and IgM against Rickettsia typhi was performed in 1024 patients during three years (2006-2008). The characteristics of patients with a positive serology were examined retrospectively. One hundred and seventy sera obtained from blood donors were tested to detect IgG against R. typhi to determine the seroprevalence of the infection. RESULTS: There was evidence of recent R. typhi infection in 43 patients (4.2%) during the study period, and 3.7% of blood donors had IgG against R. typhi. The mean age of patients was 43.1 years and the sex-ratio was 1.04. Among the patients, 58.1% were from rural areas. No patient reported any exposure to rats or rat-fleas. There were more cases during the summer and fall. The most frequent complaint was fever as a single symptom (67.5%). A cutaneous rash was reported in 44.1% and headache in 60.5% of patients. Among the patients, 44.1% presented with thrombopenia and 47.2% with elevated liver enzymes. CONCLUSION: Murine typhus seems to be frequent in Tunisia. This infection could be a threat for travellers. Serology should be performed systematically in patients with fever as a single symptom since its clinical presentation is non-specific.

Usefulness of enzyme linked immunosorbent assays species specific in the detection of Chlamydia trachomatis and Chlamydophila pneumoniae IgG antibodies in patients with genital infections or respiratory tract infections.

Chlamydia trachomatis (Ct) and Chlamydophila pneumoniae (Cpn) are obligate intracellular bacteria causing genital tract infections (GTI) and respiratory tract infections (RTI), respectively. Antigenic cross-reactivity between the two species may complicate serologic diagnosis. In this study, we compared the performance of two ELISA tests in relation to microimmunofluorescence (MIF) for the detection of Ct and Cpn IgG antibodies. We also explored the degree of cross-reactivity by ELISA and MIF. Among 278 positive sera for Cpn and/or Ct IgG antibodies in the MIF, 153 were from patients with GTI and 125 were from patients with RTI. These sera were tested by our in house MIF test and by two commercial ELISA: SeroCP and SeroCT for the detection of anti-Cpn IgG antibodies and anti-Ct IgG antibodies, respectively. In sera from patients with RTI, correlation between MIF and SeroCP was 92%. The specificity of this test was 38.5%. In fact, among the 140 sera from patients with GTI and that cross-reacted in MIF, only six were confirmed by the two ELISA tests as having IgG antibodies to Ct. The correlation between MIF and SeroCT was 80%. The specificity of this test was 100%. Indeed, among the 65 sera from patients with RTI with cross-reactions in MIF, 30 sera showed a negative SeroCT test. SeroCT was highly specific and could diminish considerably the extent of cross-reactions. Whilst, SeroCP test was not specific enough to distinguish between the presence of IgG antibodies and Cpn or Ct.