Mediterranean visceral leishmaniasis : update on biological diagnosis.

Visceral leishmaniasis (VL) is a severe life threatening parasitosis requiring early management of cases. It is an emerging disease in the Mediterranean region with a spread of endemic areas and an increase in case incidence. The patient profile has also evolved with more affected adults, presenting generally non-specific symptoms. Hence the interest of a systematic biological confirmation. The microscopic detection of Leishmania amastigotes in bone marrow aspirates (BMA) smears is the gold standard diagnostic technique. However, it requires invasive sampling. Serological tests searching for specific antibodies remain highly contributory, but their interpretation must always take into account the epidemiological context and the patient's clinical and biological features. Currently, the Western-Blot represents the most specific serological technique for diagnostic confirmation. VL diagnosis has greatly improved by the introduction of both rapid diagnostic tests (RDTs) and molecular biological techniques. RDTs using recombinant rk39 antigen are easy to perform and deliver results in less than 30 minutes. Real-time PCR (Polymerase Chain Reaction) is currently retained as the best technique for VL diagnosis. It is efficient on simple blood samples, allowing to avoid invasive BMA needed for microscopy. In addition, real time PCR estimates parasite load which is helpful for the post-treatment follow-up. In any case, the choice of techniques to be used should be strategic and adapted to the local epidemiology as well as to the means available.

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.

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.

Polymorphism study of Cryptosporidium hominis gp60 subtypes circulating in Tunisia.

Cryptosporidium spp. are a major cause of gastrointestinal diseases in humans worldwide. While a single subtype of Cryptosporidium hominis has been shown to be responsible for several large outbreaks related to water contamination in developed countries, little is known about the epidemiology of C. hominis in developing countries. This study reports the first genetic characterization of C. hominis at the subtype level in several human populations in Tunisia using the gp60 gene. Eighteen isolates were identified as C. hominis by a restriction fragment length polymorphism (RFLP) analysis. The prevalence of this species in different human populations ranges from 1.53% to 13.04% with a high prevalence being reported in immunocompromised children (13.04%) followed by patients with malignent myeloma (5.5%) and HIV-infected patients (4.59%). The gp60 analysis on C. hominis isolates, performed in 14 cases, showed the presence of a single subtype family: "Ia". Different subtypes were identified within this family (A11G1R1, A12R3, A23G1R1, A26G1R1, A27G1R1, A28G1R1). The IaA26G1R1 subtype was the most dominant subtype described in this area (50%). Despite the high genetic diversity of Cryptosporidium spp, a low heterogeneity at the subtype level was observed within C. hominis circulating in Tunisia. This distribution is an indicator for intensive and stable anthroponotic cryptosporidiosis in this region. Besides, the presence of a unique genotype in 5 HIV-infected patients attending the same hospital ward suggests the possible occurrence of hospital-acquired infection and underlines the need to implement preventive measures to avoid nosocomial transmission.

Malaria outside a living in endemic areas: diagnosis and measures to take.

In spite of its elimination from Tunisia, malaria remains a public health concern due to the severity of the disease and risk of its reintroduction. The vulnerability of our country is related to the persistent anophelism and to the existence of a potential reservoir represented by imported cases. In the absence of a stay in an endemic area, the suspicion of malaria remains a rare event. However, in front of the possibility of other modes of contamination, this parasitosis must be evoked in any fever without obvious etiology. Especially as delayed diagnosis in non immune subjects may cause a severe illness and death. We propose to present the principal clinical and biological aspects of malaria and to specify the action to be taken when the infection is contracted outside a stay in endemic areas, in order to determine the origin of contamination.

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.

Airport malaria: report of four cases in Tunisia.

Four cases of airport malaria were notified for the first time in Tunisia during the summer of 2013. All patients were neighbours living within 2 km of Tunis International Airport. They had no history of travel to malarious countries, of blood transfusion or of intravenous drug use. Although malaria transmission had ceased in Tunisia since 1980, autochthonous infection by local Anopheles mosquitoes was initially considered. However, this diagnostic hypothesis was ruled out due to negative entomological survey and the absence of additional cases.All cases were caused by Plasmodium falciparum. Clinical presentation was severe (important thrombocytopaenia and parasitaemia), because of relatively important delay in diagnosis (average of seven days). This indicates the need to consider malaria while examining airport employees or people living near international airports presenting with fever of unknown origin. It also stresses the need for effective spraying of aircrafts coming from malarious areas.

Evolution of the prevalence of intestinal parasitosis in the region of tunis from 1996 at 2012.

BACKGROUND: The prevalence of intestinal parasitosis is very different according to countries. Therefore, it is always interesting to update the data in Tunisia to better direct control measures. AIM: The objectives of this survey were to estimate the prevalence of intestinal parasitosis in the region of Tunis, to study their evolution and to establish various combinations of intestinal protozoa. METHODS: This is a retrospective study carried out over a period of 17 years from 1996 at 2012 and which involved 20033 individuals. Each subject had one or more stool examination which included a direct microscopic examination and a concentration by modified Ritchie technique. RESULTS: The prevalence of intestinal parasitosis was 12.55%. Entamoeba histolytica/dispar and Giardia intestinalis accounted respectively a prevalence of 0.51% and 1.48%. Hymenolepis nana was the most predominant helminth with a prevalence rate of 0.53%, followed by Enterobius vermicularis (0.21%). Two cases of Hookworms and seven cases of Strongyloides stercoralis were diagnosed. Polyparasitism concerned 16.59% of infected individuals. Significant combinations occured mainly for amoeba in particular Entamoeba histolytica/dispar and Entamoeba coli (r=0.232). CONCLUSION: Our study confirms the decrease of the prevalence of giardiasis and amebiasis, whereas helminthiases with direct transmission remain frequent.

Contribution of Polymerase Chain Reaction for detection of malaria in Tunisia.

BACKGROUND: In Tunisia, detection of Plasmodium in asymptomatic individuals from endemic countries is a critical measure in national program of malaria eradication. The screening is based on microscopic examination of thick and thin blood smears. However, the performance of this diagnosis is closely related to the experience of biologist and the parasitaemia. AIM: The objective of this study was to evaluate the contribution of the PCR in the screening of malaria. METHODS: This prospective study involved 260 students from malaria endemic areas who were screened for malaria between september 2011 and june 2013. Each subject had a blood sample which was examined for malaria by microscopy and nested multiplex PCR. RESULTS: PCR detected the presence of Plasmodium in 13 blood samples (5%). While microscopy was positive only in nine cases (3.5%). The discordances involved five negative samples at microscopy and which were positive in PCR and a negative sample in PCR which was positive at microscopy. A mixed infection with Plasmodium falciparum and Plasmodium malariae was identified by PCR. For this case, microscopy diagnosed only Plasmodium falciparum specie. CONCLUSION: PCR is more efficient than microscopy in detecting low parasitaemia ; particularly observed in asymptomatic subjects. This technique allows to reduce asymptomatic carriage of Plasmodium and reduce the risk of a resumption of transmission of malaria in our country.

An atypical strain associated with congenital toxoplasmosis in Tunisia.

We report the identification and typing of a congenital toxoplasmosis case in a diabetic pregnant young woman living in Tunis. The Toxoplasma DNA extracted from amniotic fluid was detected by Real Time PCR and subjected to a multilocus genetic characterisation of the strain at markers: 3'SAG2, 5'SAG2, New SAG2, SAG3, GRA6, BTUB, APICO, PK1, KT850 and UPRT1. An atypical genotype of T.gondii with unusual genetic composition was revealed. It is the first time that an atypical strain has been associated with congenital toxoplasmosis in Africa. Atypical strains are associated with severe clinical manifestations so systematic genotyping should be investigated with the amniocentesis.

Tunisian Toxoplasma gondii strains genotyping by the use of AK69 marker.

BACKGROUND: Clinical manifestation due to infection by Toxoplasma gondii is closely linked to the infecting strain of the parasite. Several genetic markers are available to determinate its genotype but few of them are able to discriminate between the three predominant lineages, namely types I, II and III. The number of markers decreases when atypical, recombinant/mixed genotypes need to be identified. FINDINGS: In our study, the contribution of sequence polymorphisms in the AK69 gene as typing markers for T. gondii was investigated for the first time in an epidemiological study. The coding region of the marker was amplified, sequenced and aligned for different Toxoplasma strains. The identified nucleotide polymorphism at 12 positions was able to highly discriminate between the different congenital toxoplasmosis Tunisian strains. Moreover the high detection sensitivity level of the marker enabled unambiguous identification of mixed/recombinant genotypes directly. CONCLUSION: It can be, thus, very useful for direct typing in areas where such genotypes are frequently encountered, mainly in the African continent.

Direct genotypic characterization of Toxoplasma gondii strains associated with congenital toxoplasmosis in Tunisia (North Africa).

Here, we determined the Toxoplasma gondii genotype in amniotic fluid, placenta, and cerebrospinal fluid samples from 14 congenital toxoplasmosis cases in Tunisia, North Africa. Direct genotypic characterization of T. gondii strains was performed by polymerase chain reaction (PCR) amplification of six genetic markers (3'SAG2, 5' SAG2, SAG3, BTUB, GRA6, and APICO) and thereafter, was analyzed by restriction fragment-length polymorphism (RFLP). Samples were sequenced to resolve strain type whenever there were unclear enzyme digestion results. Multilocus analysis revealed that only one specimen harbored the type I allele in all studied loci, whereas the 13 others gave mixed genotype results with different alleles at different markers. Seven specimens produced RFLP profile of the recombinant strains I/III, and three produced a profile of I/II recombinant strains. The last three specimens produced complex digestion patterns. In these cases, sequence analysis revealed double peaks at known polymorphic sites, indicating the presence of multiple alleles.

[Relapse of Plasmodium malariae malaria 20 years after living in an endemic area]. / Paludisme a Plasmodium malariae, une rechute 20 ans après un séjour en zone impaludée.

INTRODUCTION: Malaria has been eradicated in Tunisia since 1979. Although it continues to be evoked in the case of fever after travel to an endemic zone, its diagnosis is however difficult during relapses, notably when they are delayed. OBSERVATION: A 50 year-old man having lived in Mauritania from 1978 to 1982 was hospitalized for interstitial pneumopathy and urarthritis. In spite of treatment with broad spectrum antibiotics, the fever accompanied by abundant sweating persisted. A thick blood drop and blood smear was requested and led to the diagnosis of Plasmodium malariae malaria. DISCUSSION: This observation recalls the possibility of parasitic upsurge of some plasmodial species. It should prompt physicians to be careful and evoke malaria in the case of fever in subjects having stayed, even several years before, in an endemic zone. This would permit early diagnosis and treatment.

[Value of reagent strips in screening urinary infection in children]. / Intérêt des bandelettes réactives dans le dépistage de l'infection urinaire chez l'enfant.

The aim of this study is to specify the role of rapid tests in the screening of childhood urinary tract infection. During the period between july to december 1998, 572 urinary samples were collected from pediatric out-patient in Hôpital d'Enfants de Tunis and aged from 1 month to 15 years. Only 75 samples (12.5%) were culture positive. The predictive value of leucocytes or nitrites test was 97.2%. These results allowed the use of rapid test in the screening of urinary tract infection in children. However, if clinical symptoms are present, the culture of urine must be associated to the rapid test.