Intestinal parasites infections in hospitalized AIDS patients in Kinshasa, Democratic Republic of Congo.

To determine the prevalence and the species spectrum of intestinal parasites (IP) involved in hospitalized AIDS patients, a prospective observational and cross-sectional study was carried out in the four main hospitals in Kinshasa, Democratic Republic of the Congo. From November 2006 through September 2007, a single stool sample was collected from 175 hospitalized AIDS patients older than 15 years. Parasites were detected by light microscopy, including Ziehl-Neelsen, Fungi-Fluor, modified trichrome stains, and by immunofluorescence antibody tests and PCR for species diagnosis of microsporidia. At baseline, 19 patients (10.8%) were under antiretroviral therapy and 156 (89.2%) were eligible for ART. The main diagnosis for justifying hospitalization was intestinal infection associated with diarrhea in 87 out of 175 (49.7%). 47 out of 175 (26.9%) were found to harbor an IP, and 27 out of 175 (15.4%) were infected with at least one opportunistic IP (OIP). Prevalence rate for OIP were 9.7%, 5.1%, 1.7% and 0.6% for Cryptosporidium sp., Enterocytozoon bieneusi, Isospora belli and Encephalitozoon intestinalis respectively. Considering patients with diarrhea only, prevalence rate were 12.6%, 4.6%, 3.4% and 1.1% respectively. The other IP observed were Entamoeba histolytica/Entamoeba dispar in nine cases (5.1%), Ascoris lumbricoides in seven cases (4.0%), Giardia intestinalis in three cases (1.7%), hookworm in two cases (1.1%) and Trichiuris trichiura, Enterobius vermicularis, Schistosoma mansoni in one patient each (0.6%). No significant relationship was established between any individual IP and diarrhea. These results underline the importance of OIP in symptomatic AIDS patients regardless of diarrhea at the time of the hospitalisation, and showed that routine microscopic examination using stains designed for Cryptosporidium spp. or the microsporidia should be considered due to the absence of clinical markers.

[Prevalence of opportunistic digestive parasitic infections in Kinshasa, Democratic Republic of Congo. Results of a preliminary study in 50 AIDS patients]. / Prévalence des infections opportunistes digestives parasitaires a Kinshasa (République Démocratique du Congo), résultats d'une enquête préliminaire chez 50 patients au stade sida.

In the Democratic Republic of Congo (DRC), as in many African countries, AIDS and its procession of opportunistic infections are a major cause of morbidity and mortality. In Kinshasa, the estimated prevalence rate of HIV-infected persons is between 4 and 5%, corresponding to more than 200,000 people. Due to the lack of trained laboratory personnel and appropriate diagnostic equipment, no local investigation has been carried out to determine the prevalence of the opportunistic digestive parasitic infection in HIV-infected persons. As a step to obtaining this information that is needed for implementation of an adequate care policy, a preliminary investigation was carried out in Paris, France on 50 stool samples from 50 AIDS-patients hospitalized in 3 reference hospitals in Kinshasa. Eleven patients (22%) had digestive symptoms with a diarrhea syndrome. Further study using specialized techniques demonstrated 2 cases of digestive infection related to opportunistic parasites (4%). The first involved a Cryptosporidium sp. The second represented the first case of Enterocytozoon bieneusi infection reported in the literature from the DRC.

Prevalence des infections opportunistes digestives parasitaires a Kinshasa (Republique Democratique du Congo). Resultats d'une enquete preliminaire chez 50 patients au stade SIDA

En Republique Democratique du Congo (RDC); comme dans de nombreux pays africains; le sida et son cortege d'infections opportunistes sont une cause majeure de morbidite et de mortalite. A Kinshasa; on estime entre 4 et 5le taux de prevalence de sujets infectes par le VIH; soit plus de 200 000 personnes (chiffres du Programme National de Lutte contre le Sida; PNLS 2005). A ce jour; faute de personnels formes et de moyens diagnostiques adaptes; aucune enquete n'a encore ete menee sur la prevalence des parasites opportunistes digestifs dans la population des patients infectes par leVIH; prealable indispensable a la mise en place d'une politique de soin adaptee. Une enquete preliminaire a ete realisee a Paris sur 50 echantillons de selles de 50 patients malades du sida; hospitalises dans 3 hopitaux de references de Kinshasa. Onze patients (22) avaient une symptomatologie digestive avec un syndrome diarrheique. La realisation des examens specialises a mis en evidence 2 cas d'infection digestive par des parasitoses opportunistes (4); une a Cryptosporidium sp. et une a Enterocytozoon bieneusi; premier cas decrit dans la litterature en RDC

Diagnosis of malaria using thick bloodsmears: definition and evaluation of a faster protocol with improved readability.

The value of some inexpensive modifications to the standard method of preparing thick bloodsmears, involving rapid drying, an isotonic fixative and a haemolysing solution containing saponin, was evaluated. The drying, haemolysing, fixing and staining steps, together called the fast-thick-smear method (FTS), can be completed in < 10 min. The FTS and a more classical thick-smear method (CTS) were both used on each of 1185 samples of venous blood samples from 1034 cases of suspected malaria (all international travellers returning to France). The results indicated that there was no statistically significant differences between the two methods in terms of their sensitivity, specificity or predictive values for parasite detection. However, estimates of the intensities of the Plasmodium falciparum infections observed, based on counts of trophozoites against 200 leucocytes, were markedly higher (37.8% higher overall) with the FTS than with the CTS (P < 0.0001). Moreover, the concordance between results obtained by inexperienced and experienced microscopists was excellent when the FTS was used, with a kappa value of 0.96 (95% confidence interval = 0.93-0.98).

Evaluation of an immunofluorescent-antibody test using monoclonal antibodies directed against Enterocytozoon bieneusi and Encephalitozoon intestinalis for diagnosis of intestinal microsporidiosis in Bamako (Mali).

A 2-month study was carried out in Mali to evaluate an immunofluorescent-antibody test (IFAT) using monoclonal probes specific for Enterocytozoon bieneusi or Encephalitozoon intestinalis. Sixty-one human immunodeficiency virus (HIV)-seropositive adult patients and 71 immunocompetent children were enrolled. Microsporidia were detected in stools from 8 of 61 patients (13.1%) seropositive for HIV. A single species, E. bieneusi, was identified. All the children were negative for microsporidia. The sensitivity and specificity of IFAT were 100% compared with those of PCR, which was used as the "gold standard." Moreover, species identification by IFAT was more rapid and less expensive than that by PCR. These results show the suitability of IFAT for detection of microsporidia in developing countries.

One-step purification of Enterocytozoon bieneusi spores from human stools by immunoaffinity expanded-bed adsorption.

An original, reliable, and reproducible method for the purification of Enterocytozoon bieneusi spores from human stools is described. We recently reported the production of a species-specific monoclonal antibody (MAb) 6E52D9 immunoglobulin G2a (IgG2a) raised against the exospore of E. bieneusi spore walls. The MAb was used as a ligand to develop an immunoaffinity matrix. The mouse IgG2a MAb was bound directly to a Streamline rProtein A adsorbent, used for expanded-bed adsorption of immunoglobulins, for optimal spatial orientation of the antibody and maximum binding efficiency of the antigen. The complex was then cross-linked covalently using dimethyl pimelimidate dihydrochloride. After incubation of the immunoaffinity matrix with filtered stool samples containing numerous E. bieneusi spores and before elution with 6 M guanidine HCl, the expansion of the adsorbent bed eliminated all the fecal contaminants. The presence of spores in the elution fractions was determined by an indirect immunofluorescence antibody test (IFAT). E. bieneusi spores were found in the elution fraction in all four experiments and were still highly antigenic as indicated by IFAT. Smears examined by light microscopy contained very clean spores with no fecal debris or background bacterial and fungal contaminants. However, spore recovery rates were relatively low: an average of 10(7) spores were purified per run. This technique for isolating E. bieneusi spores directly from human stool samples with a high degree of purity opens up new approaches for studying this parasite.

Production of monoclonal antibodies directed against the microsporidium Enterocytozoon bieneusi.

Several hybridomas producing antibodies detected by indirect immunofluorescence antibody test (IFAT) were established by fusion of mouse myeloma SP2/O with spleen cells from BALB/c mice immunized against whole spores (protocol 1) or chitinase-treated spores (protocol 2) of Enterocytozoon bieneusi and were cloned twice by limiting dilutions. Two monoclonal antibodies (MAbs), 3B82H2 from protocol 1, isotyped as immunoglobulin M (IgM), and 6E52D9 from protocol 2, isotyped as IgG, were expanded in both ascites and culture. IFAT with the MAbs showed that both MAbs reacted exclusively with the walls of the spores of E. bieneusi, strongly staining the surface of mature spores, and produced titers of greater than 4,096. Immunogold electron microscopy confirmed the specific reactivities of both antibodies. No cross-reaction, either with the spores of the other intestinal microsporidium species Encephalitozoon intestinalis or with yeast cells, bacteria, or any other intestinal parasites, was observed. The MAbs were used to identify E. bieneusi spores in fecal specimens from patients suspected of having intestinal microsporidiosis. The IFAT was validated against standard staining methods (Chromotrope 2R and Uvitex 2B) and PCR. We report here the first description and characterization of two MAbs specific for the spore wall of E. bieneusi. These MAbs have great potential for the demonstration and species determination of E. bieneusi, and their application in immunofluorescence identification of E. bieneusi in stool samples could offer a new diagnostic tool for clinical laboratories.

Specific PCR assay for direct detection of intestinal microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis in fecal specimens from human immunodeficiency virus-infected patients.

A routine assay based on the PCR was developed for the detection of Enterocytozoon bieneusi and Encephalitozoon intestinalis in fecal samples. Two oligonucleotide primer pairs from a conserved region in the small-subunit rRNA genes of E. bieneusi (primer pair V1 and EB450) and E. intestinalis (primer pair V1 and SI500) were used to amplify microsporidian DNA. We achieved specific amplification of a 382-bp DNA fragment in E. intestinalis and a 353-bp DNA fragment in E. bieneusi. Boiling of the samples appeared to be most effective for DNA extraction. Fecal samples containing fewer than 10 microsporidia gave a positive result in the PCR assay. Fecal specimens from 30 human immunodeficiency virus-infected patients with microsporidiosis and fecal specimens from 42 patients suspected of having microsporidiosis were investigated by the PCR assay. The PCR assay was validated against standard staining methods (the Uvitex 2B and Chromotrope 2R staining methods) and immunofluorescence assay specific for E. intestinalis. This comparative study has shown that PCR improved species determination and can thus be considered a fast and reliable method for the detection and identification of each intestinal species.

Treatment of Strongyloides stercoralis infection with ivermectin compared with albendazole: results of an open study of 60 cases.

Ivermectin is highly effective against animal intestinal nematodes and is used in the treatment of onchocerciasis in humans. A study was undertaken to compare the efficacy of the drug with that of albendazole in the treatment of uncomplicated strongyloidiasis. Sixty patients with confirmed Strongyloides stercoralis infection were enrolled in an open randomized study and given either albendazole, 400 mg/d for 3 d or ivermectin, 150-200 micrograms/kg in a single dose. Efficacy and tolerance were evaluated on days 7, 30 and 90. Each visit included a parasitological examination of 3 stool specimens, using saline and Kato smears and formalin-ether and Baermann concentrations. Fifty-three patients were eligible for evaluation. Parasitological cure was obtained in 24 of the 29 patients treated with ivermectin (83%) and in 9 of the 24 patients who were given albendazole (38%); ivermectin was significantly more effective than albendazole (P < 0.01). Clinical and biological adverse reactions were negligible in both treatment groups. The 20 patients who failed therapy were given a second treatment course with 150-200 micrograms/kg of ivermectin in a single dose or on 2 consecutive days. Sixteen patients were cured and the other 4 had only incomplete follow-up. Ivermectin therefore constitutes an acceptable therapeutic alternative for uncomplicated strongyloidiasis.