Development and evaluation of molecular tools for detecting and differentiating intestinal amoebae in healthy individuals.

Amoebae are single-celled parasites frequently colonizing human gut. However, few molecular tools are available for accurate identification. Here, we evaluated a panel of polymerase chain reactions (PCRs) targeting Entamoeba histolytica, Entamoeba dispar, Entamoeba coli, Entamoeba hartmanni, Entamoeba polecki, Endolimax nana and Iodamoeba bütschlii. Thirty-six faecal samples (18 containing at least one amoeba species by microscopy and 18 microscopy negative for amoebae) were tested. Real-time PCRs were used for detection and differentiation of E. histolytica and E. dispar. Conventional PCR with Sanger sequencing were applied for detection and differentiation of E. coli, E. hartmanni, E. polecki, E. nana and I. bütschlii. All microscopy results were confirmed by DNA-based methods. However, more samples were positive for single and mixed amoebic species by DNA-based assays than by microscopy (22 vs 18 and 7 vs 1, respectively). DNA sequencing allowed identification of E. coli subtypes (ST1 and ST2), showed low intra-specific variation within E. hartmanni, identified two phylogenetically distinct groups within E. nana, and identified Iodamoeba at the ribosomal lineage level. Taking into account the high intra-genetic diversity within some of the species at the small subunit (SSU) rRNA gene level, amplification of SSU rRNA genes with subsequent sequencing represents a useful method for detecting, differentiating and subtyping intestinal amoebae.

Late relapse of imported Plasmodium ovale malaria: a case report.

We report the first case of an imported Plasmodium ovale relapse in a Tunisian man who developed malaria three years after leaving sub- Saharan Africa. A 29-year-old Tunisian man consulted in September 2011 because of a fever, myalgia, and headache that had begun eight days earlier and persisted despite treatment with oral antibiotics. On questioning, the patient stated that he had resided three years ago for six months in Ivory Coast, where he acquired malaria. He was treated with artemether-lumefantrine. The patient said he had no recent travel to any other malaria-endemic area and had not received a blood transfusion. A first microscopy of peripheral blood smears was negative for malaria parasites. The diagnosis was established 17 days after onset of symptoms. A repeat microscopic examination of blood smears confirmed the presence of Plasmodium ovale with a parasitemia lower than 0.1%. The patient was treated with artemether lumefantrine, followed by primaquine. This case emphasizes the possibility of relapse of some plasmodial species. It highlights the importance of repeating microscopic examination of blood when the diagnosis of malaria is suspected.

Diagnosis of mediterranean visceral leishmaniasis by detection of leishmania antibodies and leishmania DNA in oral fluid samples collected using an Oracol device.

Current methods for diagnosis of visceral leishmaniasis (VL) require invasive sampling procedures such as visceral aspiration and/or blood drawing. The use of diagnostic tests using oral fluid, which is easier to collect, would be more simple and practical for VL diagnosis, especially under field conditions. Oral fluids from 37 VL patients and 40 healthy controls were collected using Oracol devices. Blood samples and oral fluid specimens from both groups were analyzed by recombinant protein K39 (rK39) enzyme-linked immunosorbent assay and quantitative real-time PCR. Detection of antibodies in the oral fluid had a sensitivity of 100% and a specificity of 97.5%. Antibody levels measured in serum and oral fluid showed a significant positive correlation (ρ = 0.655 and P = 0.01). Detection of Leishmania DNA in oral fluid had a sensitivity of 94.6% and a specificity of 90%. The median parasite load estimated in blood was 133 parasites/ml (interquartile range [IR], 10 to 1,048), whereas that in oral fluid specimens was 3 parasites/ml (IR, 0.41 to 92). However, there was no significant linear relationship between parasite loads assessed in the two biological samples (ρ = 0.31 and P = 0.06). VL diagnosis based on specific antibody detection and Leishmania DNA identification using oral fluid samples was equivalent in accuracy to that using blood and therefore is promising for clinical use.

Unexpected diagnosis of basal cell carcinoma in a patient presenting with a secondary location of Leishmania parasites in the skin.

We report here a case of simultaneous cutaneous and visceral manifestations due to Leishmania L. infantum diagnosed in an immunocompetent adult. We describe a 74-year-old woman from Tunis, Tunisia, who presented a biologically confirmed visceral leishmaniasis infection concomitant with arm ulceration which appeared 2 years before. Leishmania DNA was detected by ITS PCR in both buffy coat and dermal scrapping of the arm lesion. Sequencing revealed that the 2 isolated strains corresponded to L. infantum and were 100% identical. The symptoms of visceral leishmaniasis responded to amphotericin B with rapid healing. However, the skin lesion did not improve although Leishmania PCR on dermal sample became negative. This location is probably secondarily to lymphatic or blood dissemination during the systemic visceral leishmaniasis infection. It would be favored by the inflammatory environment induced by the basal cell carcinoma subsequently diagnosed.

Prevalence and Subtype Identification of Blastocystis sp. in Healthy Individuals in the Tunis Area, Tunisia.

Blastocystis sp. is currently the most common eukaryotic parasite found in humans. Despite its potential public health impact, epidemiological data regarding its prevalence and molecular subtype (ST) distribution in Maghreb are rarely reported. Therefore, the aim of this study was to determine the prevalence of the parasite in a cohort of healthy food handler Tunisian individuals and to acquire the first molecular data regarding the distribution of Blastocystis sp. STs in this country. Therefore, 524 fecal samples were collected, and 68 of them (13%) were identified as positive for the parasite by direct-light microscopy of smears. Seventeen samples of 100 negative by microscopy were also shown to be positive by real-time quantitative polymerase chain reaction. Among all the positive samples, 61 Blastocystis isolates were subtyped using partial small subunit ribosomal RNA gene analysis. ST3 was the most abundant (51%) followed by ST1 (30%), ST2 (16%), and ST4 and ST7 (both 1.6%).

Natural infection of North African gundi (Ctenodactylus gundi) by Leishmania tropica in the focus of cutaneous leishmaniasis, Southeast Tunisia.

North African gundis (Ctenodactylus gundi) were trapped in the Leishmania (L.) tropica focus of cutaneous leishmaniasis, situated in southeast Tunisia and evaluated for Leishmania infection by real-time kinetoplast DNA polymerase chain reaction (PCR). Species identification was performed by internal transcribed spacer one (ITS1)-PCR-restriction fragment length polymorphism (RFLP) and high-resolution melting (HRM) analysis of the 7SL RNA gene. Real-time PCR on blood was positive in 6 of 13 (46.2%) tested gundis. Leishmania tropica was identified in five infected gundis and Leishmania major in one specimen. Alignments of the ITS-1 DNA sequences and 7S-HRM curves analysis indicated that similar genotypes were present in humans, a sandfly, and gundis from the same region suggesting a potential role of this rodent as reservoir host of L. tropica in southeast Tunisia.

Evaluation of an enzyme-linked immunosorbent assay based on crude Leishmania histone proteins for serodiagnosis of human infantile visceral leishmaniasis.

Human visceral leishmaniasis (VL) is routinely diagnosed by detecting IgG that specifically binds to Leishmania antigens. The enzyme-linked immunosorbent assay (ELISA) remains a widely used method. However, the biggest challenge remains the choice of antigen with the highest specificity and sensitivity. This study is aimed at assessing the diagnostic performances of crude Leishmania histone (CLH) protein-based ELISAs in Mediterranean VL patients. The CLH proteins were biochemically purified from promastigote nuclear extracts. Their reactivities were analyzed by Western blotting (WB) using rabbit polyclonal antibodies against Leishmania recombinant histones and sera from VL patients, respectively. Then, the diagnostic potential of CLH proteins was validated by the CLH-based ELISA using 42 infantile VL patients' sera and 70 control subjects. The CLH-based ELISA performance was compared to that of the soluble Leishmania antigen (SLA)- and the recombinant K39 (rK39)-based ELISAs. Analysis of the WB profile with the use of polyclonal antibodies confirmed the histone origin of low molecular mass proteins (12 to 16 kDa). All VL samples tested presented antibodies reacting against different antigen fractions; however, recognition patterns were different depending on the reactivity of each serum. CLH-based ELISA showed an excellent ability to discriminate between VL cases and healthy controls (97.6% sensitivity and 100% specificity). It had a diagnostic performance similar to that of rK39-based ELISA (97.6% sensitivity and 97.1% specificity, P = 0.5) and a better serodiagnosis accuracy than the SLA-based ELISA (85.7% sensitivity and 90% specificity, P < 0.05). Therefore, crude Leishmania histone extract could be a valuable antigen for clinical use.

Advantages and limits of real-time PCR assay and PCR-restriction fragment length polymorphism for the identification of cutaneous Leishmania species in Tunisia.

Cutaneous leishmaniasis (CL), a public health problem in Tunisia, is associated to three species: Leishmania (L.) infantum, L. major and L. killicki. Accurate and sensitive procedures for the diagnostic of Leishmania infection and for species identification are required to enable adequate treatment and appropriate control measures. Several PCR-methods are applied for the diagnosis and the identification of Leishmania parasites such as PCR-restriction fragment length polymorphism (PCR-RFLP), DNA sequencing, hybridization probes and real-time PCR (RT-PCR). In this study, PCR-RFLP and RT-PCR were performed on skin scrapings from 27 patients with confirmed CL by microscopic examination, in order to compare their usefulness and efficiency for identification of Leishmania species in routine diagnostic laboratories. Identification of Leishmania species was successfully achieved in 96.3% and 81.5% respectively. Agreement between using internal transcribed spacer 1 (ITS1)-PCR-RFLP and kDNA-RT-PCR assays was 70% (19/27). Characterization problems using RT-PCR were mainly due to the difficulties in analyzing the melting temperatures. ITS1-PCR-RFLP and kDNA-RT-PCR presented an interesting alternative to conventional methods for the identification of Leishmania parasites from clinical samples. Both PCR assays can be used in a routine diagnostic, however, further prospective studies including largest sampling, are required to determine their performances in a routine use.

Epidemiologic and clinical features of cutaneous leishmaniasis in southeastern Tunisia.

Species-specific diagnosis was performed in 66 patients with cutaneous leishmaniasis (CL) living in Tataouine focus in southeastern Tunisia. Leishmania DNA was extracted directly from dermal scrapings (n = 66) and from parasites obtained in culture (n = 12). Species were identified by using polymerase chain reaction-restriction fragment length polymorphism analysis for internal transcribed spacer region 1 and isoenzyme analysis. Leishmania tropica and L. major were identified in 31 (47%) and 35 (53%) cases respectively. Leishmania tropica CL cases were geographically scattered, and L. major CL cases were clustered. Lesions caused by L. tropica were mostly single (83.8%) and face-localized (55.8%), and lesions caused by L. major were multiple (57.1%; P < 0.001) and situated on limbs (83.7%; P < 0.001). For both species, most lesion onsets were reported during June-January. However, lesions that emerged during February-May were mainly caused by L. tropica (83.3%; P < 0.01). Moreover, the delay before seeking medical advice was higher for L. tropica infections than for L. major infections (P < 0.05).