Assessment of microscopic and molecular tools for the diagnosis and follow-up of cryptosporidiosis in patients at risk.

Cryptosporidiosis is an important though underreported public health concern. Molecular tools might be helpful in improving its diagnosis. In this study, ZR Fecal DNA MiniPrep™ Kit (ZR) and NucliSens® easyMAG® (EM) were compared using four Cryptosporidium-seeded feces and 29 Cryptosporidium-positive stools. Thereafter, ZR was selected for prospective evaluation of Cryptosporidium detection by 18S rDNA and LAXER quantitative PCR (qPCR) in 69 stools from 56 patients after Cryptosporidium detection by glycerin, modified Ziehl-Neelsen (ZN) and auramine-phenol (AP) stainings. The combination of any of the two extraction methods with 18S qPCR yielded adequate detection of Cryptosporidium in seeded stools, but the ZR kit showed the best performance. All 29 Cryptosporidium-positive samples were positive with 18S qPCR, after both ZR and EM extraction. However, false-negative results were found with LAXER qPCR or nested PCR. Cryptosporidiosis was diagnosed in 7/56 patients. All the microscopic methods enabled the initial diagnosis, but Cryptosporidium was detected in 12, 13, and 14 samples from these seven patients after glycerin, ZN, and AP staining respectively. Among these samples, 14 and 12 were positive with 18S and LAXER qPCR respectively. In two patients, Cryptosporidium DNA loads were found to be correlated with clinical evolution. Although little known, glycerin is a sensitive method for the initial detection of Cryptosporidium. When combined with 18S qPCR, ZR extraction, which had not been evaluated so far for Cryptosporidium, was an accurate tool for detecting Cryptosporidium and estimating the oocyst shedding in the course of infection.

Multicentric evaluation of a new real-time PCR assay for quantification of Cryptosporidium spp. and identification of Cryptosporidium parvum and Cryptosporidium hominis.

Cryptosporidium is a protozoan parasite responsible for gastroenteritis, especially in immunocompromised patients. Laboratory diagnosis of cryptosporidiosis relies on microscopy, antigen detection, and nucleic acid detection and analysis. Among the numerous molecular targets available, the 18S rRNA gene displays the best sensitivity and sequence variations between species and can be used for molecular typing assays. This paper presents a new real-time PCR assay for the detection and quantification of all Cryptosporidium species associated with the identification of Cryptosporidium hominis and Cryptosporidium parvum. The sensitivity and specificity of this new PCR assay were assessed on a multicentric basis, using well-characterized Cryptosporidium-positive and -negative human stool samples, and the efficiencies of nine extraction methods were comparatively assessed using Cryptosporidium-seeded stool samples and phosphate-buffered saline samples. A comparison of extraction yields showed that the most efficient extraction method was the Boom technique in association with mechanical grinding, and column extraction showed higher binding capacity than extraction methods based on magnetic silica. Our PCR assay was able to quantify at least 300 oocysts per gram of stool. Satisfactory reproducibility between laboratories was observed. The two main species causing human disease, Cryptosporidium hominis and Cryptosporidium parvum, were identified using a duplex real-time PCR assay with specific TaqMan minor-groove-binding ligand (MGB) probes for the same amplicon. To conclude, this one-step quantitative PCR is well suited to the routine diagnosis of cryptosporidiosis since practical conditions, including DNA extraction, quantification using well-defined standards, and identification of the two main species infecting humans, have been positively assessed.

Molecular method for the diagnosis of imported pediatric malaria.

Malaria is a polymorphous disease; it can be life threatening especially for children. We report a case of imported malaria in a boy, illustrating the epidemiological and clinical aspects of severe pediatric malaria. In this case real-time PCR was used to quantify Plasmodium falciparum DNA levels, to monitor the evolution under treatment, and to determine genetic mutations involved in chloroquine resistance. The major epidemiological features of imported malaria, and the difficulty to diagnose childhood severe malaria are described. The contribution of molecular methods for the diagnosis of imported malaria is discussed.

[Relevance of the toxoplasma IgG avidity test in the serological surveillance of pregnant women]. / Intérêt de l'indice d'avidité des IgG pour le diagnostic d'exclusion d'une toxoplasmose récente : comparaison de la trousse Toxo IgG Avidité SFRI à la trousse Toxo IgG Avidity Vidas.

In addition to the serological systematic screening tests, kits to measure the avidity of toxoplasma IgG antibodies are currently available. Since high-avidity IgG toxoplasma antibodies have been shown to exclude recent infection, IgG avidity determination is especially useful in ruling out acute infection having occurred in the 3-4 prior months of pregnancy. We therefore compared the efficacy of two toxoplasma IgG avidity ELISA kits: SFRI (SFRI Laboratoire) and VIDAS Toxo-IgG avidity kit (bioMérieux). The agreement of the results from the 2 commercial assays were analysed using 55 serum samples, in terms of global mother-child Toxoplasma results and outcome, specially with light of the results of Toxoplasma antenatal, postnatal assays and of clinical follow up of children.

[Massive haemoptysis associated with pulmonary Strongyloides stercoralis hyperinfestation]. / Hémoptysie massive associée à une hyperinfestation pulmonaire à Strongyloides stercoralis.

INTRODUCTION: Pulmonary infestation with Strongyloides stercoralis is an exceptionally rare cause of haemoptysis, the diagnosis being difficult and often delayed. CASE REPORT: We report the case of a retired coal miner suffering from pneumoconiosis who presented with acute respiratory insufficiency and massive haemoptysis, with a fatal outcome, associated with pulmonary stongyloidosis. The only identified source of infestation with Strongyloides stercoralis was his period in the coal mine and the only risk factors for the hyperinfestation were a short course of systemic corticosteroid therapy and the presence of a peritoneal-auricular valve. CONCLUSION: This observation illustrates the importance of a systematic search for anguillosis in ex coal miners prior to any immunosuppressant treatment in order to avoid the serious and frequently fatal form of hyperinfestation with Strongyloides stercoralis.

[Clinical studies using the combination atovaquone-proguanil as malaria prophylaxis in non-immune adult and child travelers]. / Etudes cliniques de l'association atovaquone-proguanil en prophylaxie du paludisme chez les voyageurs adultes et enfants non-immuns.

Prophylaxis for short-term travel in malaria-endemic areas can be difficult for two reasons. The first is that currently available antimalarial drugs are becoming less effective because of the ability of the parasite to adapt to drug pressure. The second involves poor compliance with chemoprophylactic regimens due to the highly restrictive conditions of administration and adverse drug side-effects, especially in "healthy" subjects. The combination of atovaquone/proguanil (Malarone) could provide an answer to both these problems since it is not only effective on multiresistant strains of Plasmodium falciparum but also simplifies the conditions of administration and shows good tolerance in adults and children.

Case report: recovery of Calliphora vicina first-instar larvae from a human traumatic wound associated with a progressive necrotizing bacterial infection.

Human myiasis caused by Calliphora vicina is rare in Europe. Here we report a case of C. vicina infection occurring in the traumatic leg wound of a healthy 21-year-old man. Firstly, a progressive necrotizing infection developed in the wound despite administration of antibiotics. Aeromonas hydrophila was isolated from the wound samples. Secondly, during debridement, C. vicina first-instar larvae were isolated from the wound. To our knowledge, this is the first European case of C. vicina wound myiasis associated with severe A. hydrophila infection.

[Cyclospora cayetanensis: review of an emerging intestinal pathogen]. / Cyclospora cayetanensis, un agent pathogène intestinal émergent.

Cyclospora cayetanensis is an emerging pathogen. It is a new human coccidian agent of intestinal disease. Twenty years ago, the first known human cases of cyclosporiasis were reported in the medical literature. Cyclosporiasis occurs in persons of all ages and either in immunocompetent or immunocompromised hosts. The most characteristic feature of this infection is a syndrome of acute or chronic diarrhea. This parasite has a world-wide distribution. In previous reports, Cyclospora cayetanensis was associated with prolonged diarrhea in travellers, returning from developing countries. However, Cyclospora infection has recently been reported in non travellers in the United States and Canada. Cyclospora can be transmitted by ingestion of water or food contaminated with oocysts. The life cycle of Cyclospora cayetanensis is not fully known. Diagnosis of cyclosporiasis is made by direct examination of stool samples. To date, oral trimethoprim-sulfamethoxazole is the only effective treatment for Cyclospora infection.

[Diagnostic value of the demonstration of specific antigens of Fasciola hepatica by western blot technique]. / Intérêt diagnostique de la mise en évidence d'antigènes spécifiques de Fasciola hepatica par la technique du western blot.

A western blot assay was performed for the detection of Fasciola hepatica specific antigens for the diagnostic of fasciolasis; 72 sera were tested, 28 coming from patients with the parasitic disease and 44 from persons either healthy or presenting other diseases; 11 different antigenic bands were detected using sera from patients with fasciolasis. The 57 and 29 kDa specific antigens are considered like major, their specificity is about 100% and their respective sensibility, 79 and 93%. Band of 9-12 kDa is also appeared specific but is revealed only in 47% of the cases; 27 out of the 28 sera from patients with fasciolasis were able to recognize at least one of the 57, 29 or 9-12 kDa specific antigens. The present results suggest that western blot could be useful for the diagnosis of this parasitic disease as far as the criteria of positivity is based on the recognition of at least one of the major specific antigens.

Specific diagnostic antigens of Echinococcus granulosus detected by western blot.

A western blot assay was performed for the detection of Echinococcus granulosus specific antigens useful for the diagnostic of hydatic disease. 191 sera were tested, 105 coming from patients with different localizations of hydatic cysts and 86 from persons either healthy or presenting other diseases. 48 different antigenic bands were detected using sera from patients with hydatidosis. A 35 kDa antigen co-migrating with a band labeled by a McAb specific of antigen 5 was recognized in western blot by only 68% of the sera able to precipitate antigen 5 in immunoelectrophoresis. A 8 kDa antigen corresponding to the specific E. granulosus antigen already described has been recognized by 80% of the sera coming from patients with hydatidosis and not by the 86 control sera. Bands of 21, 30, and 92 kDa appeared also specific and were recognized by at least 50% of tested sera. These antigens appeared unrelated one to each other. 103 out of the 105 sera from patients with hydatidosis were able to recognize at least one of the 8, 21, 30, 35 or 92 kDa specific antigens. The present results suggest that western blot could be useful for the diagnosis of hydatidosis as far as the criteria of positivity is based on the recognition of at least one of the major specific antigens.

[Strongyloides stercoralis myelitis]. / Myélite à anguillule.

A 66 year-old man presented with a myelitis associated with eosinophilia in blood and cerebrospinal fluid. Specific serological procedures in blood and stool examinations led to the diagnosis of myelitis due to Strongyloides stercoralis. Physiopathology of medullary lesions is discussed. To our knowledge, this is the first report of myelitis in the course of Strongyloides infection.

[Blastocystis hominis: in search of a disease, a misunderstood organism]. / Blastocystis hominis: à la recherche d'une maladie, un organisme incompris.

Blastocystis hominis is a micro-organism which remains somewhat mysterious. Having defined its present position in the classification of Protozoa, the authors describe its ultrastructure and morphology. Its epidemiology and pathogenicity are discussed in the light of experimental studies and human clinical data, especially in AIDS patients. Metronidazole seems to be the most active drug against this organism, but extreme caution must be exerted when the possible pathogenic property of B. hominis is discussed.

[Autochthonous strongyloidiasis in the north of France]. / Anguillulose autochtone dans le nord de la France.

The authors discuss four cases of indigenous strongyloidiasis, which were detected in northern France during the past twenty years. In our hemisphere, the limits of this helminthiasis range between the 50th and the 53rd parallels of latitude. In two cases, indoor contamination must be suspected; in the third case, transmission has been facilitated by insalubrity and crowding; the fourth case was related to the activities of a dustman in camping sites. Nose bleedings were noticed in two cases and the haemorrhagic manifestations in strongyloidiasis are mentioned.

Imaging of systemic Candida albicans infections with a radioiodinated monoclonal antibody: experimental study in the guinea pig.

Guinea pigs intravenously infected with Candida albicans were scanned to evaluate the use of radioiodinated monoclonal antibodies (MAb) to fungal antigens for detecting tissue infection sites. A total of 18 infected and 8 uninfected animals were used. MAb and F(ab')2 fragments directed against cell wall glycoproteins of C. albicans were labeled with 131I. Another MAb directed against a Schistosoma mansoni glycoprotein was labeled with 125I and used as a nonspecific control. Radiolabeled MAbs were injected at a dose of 12.5 micrograms (500 kBq) per animal. Images were acquired 24 h later. Animals were then killed and the dissected organs were separately gamma-counted. The number of C. albicans colony forming units (cfu) per gram was determined in each organ. A clear relationship was found between the anatomic distributions of C. albicans and 131I. The biodistribution of 131I radioactivity associated with anti-Candida MAb was greater in infected animals than in healthy animals and increased with the number of cfu per g in each organ. The distribution was highly specific in animals with Candida endophthalmitis, a pathognomic feature of organ involvement during hematogenous dissemination. In contrast, the distribution of 125I radioactivity associated with the nonspecific MAb was similar in healthy and infected animals. In infected animals, it was totally independent of the intensity of fungal infection.