Mohamed Gorcii

Diagnosis of Congenital Toxoplasmosis: Comparison of Targets for Detection of Toxoplasma gondii by PCR

ABSTRACT Three PCR targets (18S ribosomal DNA, B1, and AF146527) and mouse inoculation were compared for 83 samples in the context of congenital toxoplasmosis. These four techniques are not statistically different in terms of sensitivity and specificity. However, further analysis highlighted problems sometimes encountered with PCR diagnosis of congenital toxoplasmosis.

First detection of Leishmania killicki (Kinetoplastida, Trypanosomatidae) in Ctenodactylus gundi (Rodentia, Ctenodactylidae), a possible reservoir of human cutaneous leishmaniasis in Tunisia

BackgroundLeishmania killicki was originally described in 1980 in southeast Tunisia. It was also recently reported in Lybia and Algeria. Nevertheless, neither vector nor reservoirs of this parasite are known. The identification of the vector and the animal reservoir host of L. killicki is critical for the establishment of an efficient control strategy.Findingsblood, popliteal lymph node, spleen, bone marrow, liver and skin were collected from 50 rodents in 2009 in south western Tunisia. Samples were smeared onto glass slides, cultured on NNN medium and tested by polymerase chain reaction for Leishmania detection. Parasites were detected by PCR from 10 Psammomys obesus and from two Ctenodactylus gundi. Parasite identification was performed simultaneously by internal transcribed spacer 1 PCR-RFLP and by PCR sequencing. Both Leishmania major and Leishmania killicki were identified from infected Psammomys and Ctenodactylus gundi respectively.ConclusionThis is the first report of Leishmania killicki identified from Ctenodactylus gundi in Tunisia. This result supports the assumption that C. gundi is a potential reservoir for Leishmania killicki.

Isolation and molecular characterization of recombinant Echinococcus granulosus P29 protein (recP29) and its assessment for the post-surgical serological follow-up of human cystic echinococcosis in young patients.

We synthesized recombinant Echinococcus granulosus protoscolex recP29 antigen to be preliminarily assessed by ELISA and immunoblotting. RecP29-serology was carried out on 54 young patients with cystic echinococcosis (CE). Patients were classified into either cured (CCE) (n=40) or non-cured (NCCE) (n=14) CE patients. RecP29 ELISA showed a gradual decrease of antibody concentrations in all CCE cases that were initially (before treatment) seropositive to this antigen (25 out of 40) or that seroconverted following treatment. A complete seronegativity was reached within 3 years post-surgery in all of these cases. Conventional HCF ELISA yielded seronegativity in only 10% of initially recP29-seropositive CCE patients (P=0.086). Likewise, recP29 immunoblotting yielded seronegativity in 93% of 29 out of 40 initially recP29-immunoblot-positive CCE patients after 3 years follow-up, compared with 72% in the HCF immunoblotting (P=0.060). Eleven out of 14 NCCE patients were initially positive by recP29 ELISA, and 10 out of these maintained a marked anti-recP29 antibody reactivity until the endpoint of the follow-up period. All 14 NCCE cases were initially seropositive by recP29 immunoblotting, and 13 cases remained seropositive until the end of the study. Thus, recombinant P29 protein appears prognostically useful for monitoring those post-surgical CE cases with an initial seropositivity to this marker.

Assessment of Echinococcus granulosus Somatic Protoscolex Antigens for Serological Follow-Up of Young Patients Surgically Treated for Cystic Echinococcosis

ABSTRACT Echinococcus granulosus protoscolex soluble somatic antigens (PSSAs) were assessed for their prognostic value in the serological follow-up of young patients treated for cystic echinococcosis (CE), compared to conventional hydatid fluid (HF) antigen. Based on different clinical courses and outcome of infection, as well as imaging findings, patients were retrospectively classified into two different groups including either cured CE (CCE; i.e., absence of active cysts or presence of inactive cysts, respectively) and noncured CE (NCCE) patients still presenting active cysts at the end of an up to 5-year follow-up period. An immunoglobulin G (IgG)-PSSA enzyme-linked immunosorbent assay (ELISA) showed a gradual decrease in antibody levels in CCE cases, reaching seronegativity in 20% of the cases at least within 5 years postsurgery. In comparison, the conventional IgG-HF ELISA showed a significantly lower progressive decrease in antibody levels, serology becoming negative in only 15% of CCE patients at the endpoint of the follow-up period. Serological analysis of PSSA by immunoblotting yielded an interesting immunoreactive double band of 27 and 28 kDa that, in 15 (75%) of 20 CCE cases, exhibited a rapid decrease and subsequent disappearance of respective antibody reactivities within 3 years postsurgery. Conversely, anti-27- and -28-kDa antibody reactivity strongly persisted until the endpoint of the follow-up period in all of the five NCCE patients. Further analysis of the 27- and 28-kDa doublet by using affinity-purified antibodies showed that the double band was not detectable in HF. Furthermore, a predominantly IgG4 subclass-restricted humoral immune response against the 27- and 28-kDa antigens was demonstrated in seroreactive CE patients. Overall, an anti-27- and -28-kDa response appeared to correlate with cyst activity. In conclusion, PSSA represents a useful candidate to carry out a serologic follow-up of CE subsequent to treatment and deserves further respective evaluation for other age groups of CE patients.

Transmission of Visceral Leishmaniasis in a Previously Non-Endemic Region of Tunisia: Detection of Leishmania Dna in Phlebotomus perniciosus

ABSTRACT: Visceral leishmaniasis (VL) has been endemic in northern Tunisia and has occurred sporadically in the center of Tunisia. Recently, there have been several cases from areas known to be free of VL. We report in this work all human and canine cases of VL recorded between 2003 and 2011 and an entomological study of phlebotomine fauna in a previously non-endemic region. Sixty-three cases of VL were diagnosed and identified as L. infantum using several different methods. Eight species of 179 sand flies were caught and identified by both morphological and molecular methods. Two genera were present, Phlebotomus and Sergentomya, with an abundance of the subgenus Phlebotomus (Larrousius) spp., a classic vector of VL in Tunisia. Moreover, Leishmania DNA was detected in seven unfed Phlebotomus pernicousus and L. infantum was identified in three of them. This result confirms the establishment of a transmission cycle of VL in the studied region by the coexistence of infected vectors with infected hosts.

Twenty-four new human cases of cutaneous leishmaniasis due to Leishmania killicki in Metlaoui, southwestern Tunisia. Probable role of Phlebotomus sergenti in the transmission

Metlaoui district in the South-west of Tunisia is a classical focus of cutaneous leishmaniasis (CL) due to Leishmania major. Since 2005, a single case of CL due to L. killicki has been reported. We report twenty four human cases due to this parasite, affecting men and women from 2 to 70 years old. Leishmania killicki have been typed using molecular techniques: polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and gene sequencing. Four strains from patients have been successfully cultured on NNN medium and isoenzymatically typed as L. killicki MON-8. Our results strongly suggests that Metlaoui is a new L. killicki focus with a stable transmission cycle. Sand flies fauna in the same focus was also studied. 1400 Phlebotomine sand flies (785 males/615 females) have been caught during an entomological survey. Leishmania major DNA has been found in one P. papatasi female, the most abundant species, whereas L. killicki DNA has been found in one Phlebotomus sergenti female emphasizing the probable role of this species as vector of this zoonotic parasite.

Development of a polymerase chain reaction-restriction fragment length polymorphism assay for Leishmania major/Leishmania killicki/Leishmania infantum discrimination from clinical samples, application in a Tunisian focus.

Topoisomerase II gene of Leishmania genus was used to develop a molecular tool for detection and species differentiation of Leishmania from clinical samples. Identification was achieved by a polymerase chain reaction followed by digestion with 2 restriction endonucleases BstU1 and Taq1. Despite the relatively low sensitivity, it is able to differentiate between 3 complexes responsible for cutaneous leishmaniasis.

Post-surgical follow-up (by ELISA and immunoblotting) of cured versus non-cured cystic echinococcosis in young patients

The study was designed to determine comparatively the prognostic value of immunoblotting and ELISA in the serological follow-up of young cystic echinococcosis (CE) patients exhibiting either a cured or a progredient (non-cured) course of disease after treatment. A total of 54 patients (mean age 9 years, range from 3 to 15 years) with surgically, radiologically and/or histologically proven CE were studied for a period up to 60 months after surgery. Additionally, some of the patients underwent chemotherapy. Based on the clinical course and outcome, as well as on imaging findings, patients were clustered into 2 groups of either cured (CCE), or non-cured (NCCE) CE patients. ELISA showed a high rate of seropositivity 4 to 5 years post-surgery for both CCE (57.1%) and NCCE (100%) patients, the difference found between the two groups was statistically not significant. Immunoblotting based upon recognition of AgB subcomponents (8 and 16 kDa bands) showed a decrease of respective antibody reactivities after 4 years post-surgery. Only sera from 14.3% of CCE patients recognized the subcomponents of AgB after 4 years, while none (0%) of these sera was still reactive at 5 years post-surgery. At variance, immunoblotting remained positive for AgB subcomponents in 100% of the NCCE cases as tested between 4 and 5 years after surgical treatment. Immunoblotting therefore proved to be a useful approach for monitoring post-surgical follow-ups of human CCE and NCCE in young patients when based upon the recognition of AgB subcomponents.

Prevalence of Toxoplasmosis in Sheep, Goats and Cattle in Southern Tunisia

The prevalence of Toxoplasma gondii infection in ruminants (sheep, goats, and cattle) in Southern Tunisia is largely unknown. Antibodies and DNA to T. gondii were determined in serum and apex of the heart samples of 261 animals (204 sheep, 32 goats and 25 cattle) using the modified agglutination technique test (MAT) and the PCR respectively. Antibodies to T. gondii (MAT, 1:20) were found in 40.2% (95% CI: 33.4%, 47.2%), 34.5% (95% CI: 19.1%, 53.2%) and 12% (95% CI: 03.15%, 32.13%) in sheep, goats and cattle respectively. Seroprevalence significantly (p 3 years) and females were significantly (p<0.05) more infected than younger and male animals respectively. The seroprevalence was highest in sheep and specifically in the Barbarine breed. The presence of Toxoplasma DNA was detected in 11 of 96 samples (11.5%). The prevalence observed in the present study indicates a widespread exposure to T. gondii in South Tunisia. Results indicate, for the first time in Tunisia, that infected sheep and goats represent a potential source of T. gondii infection for humans in particular in the South of the country (Gafsa area).