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.

Nosocomial Pneumocystis jirovecii infections.

Airborne transmission of Pneumocystis sp. from host to host has been demonstrated in rodent models and several observations suggest that interindividual transmission occurs in humans. Moreover, it is accepted that the Pneumocystis organisms infecting each mammalian species are host specific and that the hypothesis of an animal reservoir for Pneumocystis jirovecii (P. jirovecii), the human-specific Pneumocystis species, can be excluded. An exosaprophytic form of the fungus cannot be strictly ruled out. However, these data point toward the potential for the specific host to serve as its own reservoir and for Pneumocystis infection in humans as an anthroponosis with humans as a reservoir for P. jirovecii. This review highlights the main data on host-to-host transmission of Pneumocystis in rodent models and in humans by the airborne route and provides a rationale for considering the occurrence of nosocomial infections and measures for their prevention

A matched case-control study of toxoplasmosis after allogeneic haematopoietic stem cell transplantation: still a devastating complication.

Toxoplasmosis (TXP) is a life-threatening complication of allogeneic haematopoietic stem cell transplantation (AHSCT). Little is known about the risk factors and there is no consensus on prophylactic measures. To investigate the risk factors, we conducted a single-centre, retrospective matched case-control study among adults who underwent AHSCT from January 2006 to March 2015 in our hospital. TXP cases were identified from the prospectively maintained hospital's database. The 1:2 control population consisted of the two patients who received an AHSCT immediately before and after each case with similar donor relationship (related, unrelated) but who did not develop TXP. Risk factors were identified by conditional logistic regression. Clinical features and outcome of TXP were examined. Twenty-three (3.9%) cases of TXP (20 diseases, three infections) were identified among 588 AHSCT recipients. Twenty (87%) cases had a positive pre-transplant Toxoplasma gondii serology. In comparison with 46 matched control patients, risk factors were the absence of effective anti-Toxoplasma prophylaxis (odds ratio (OR) 11.95; 95% CI 3.04-46.88; p <0.001), high-grade (III-IV) acute graft-versus-host-disease (OR 3.1; 95% CI 1.04-9.23; p 0.042) and receipt of the tumour necrosis factor-α blocker etanercept (OR 12.02; 95% CI 1.33-108.6; p 0.027). Mortality attributable to TXP was 43.5% (n = 10). Non-relapse mortality rates during the study period of cases and controls were 69.6% (n = 16) and 17.4% (n = 8), respectively. Lung involvement was the dominant clinical feature (n = 14). Two cases were associated with graft failure, one preceded by haemophagocytic syndrome. Given TXP-related morbidity and attributable mortality, anti-Toxoplasma prophylaxis is essential for optimized management of seropositive AHSCT recipients.

Prevalence of intestinal protozoans in French patients infected with HIV.

To assess the prevalence of intestinal protozoans in French HIV-infected patients, stool samples, duodenojejunal biopsies, and/or colorectal biopsies from 81 patients were studied for parasites, viruses, and bacteria. Pathogens were found in 70.6% of AIDS patients with diarrhea or malabsorption. The respective prevalence of protozoa in AIDS patients with diarrhea was Cryptosporidium sp.: 37.3%, Blastocystis hominis: 13.7%, Giardia intestinalis: 5.8%, Isospora belli: 2%, Enterocytozoon bieneusi: 2%. Microsporidia were noted in one patient with severe malabsorption but no diarrhea. Other pathogens included cytomegalovirus in 27.4% and Mycobacterium avium in 5.8%. Patients with identified pathogens were more immunosuppressed and more severely malnourished than those with unexplained diarrhea. Multiple pathogens were found in 13 of 81 patients (16%). Twenty-six of 66 identified pathogens (40%) were diagnosed only on biopsy specimens. Chronic diarrhea in HIV patients could be explained in the vast majority by appropriate gastrointestinal investigations. Cryptosporidia played a major role, while microsporidia appeared to be less common.

Intestinal microsporidiosis occurring in two renal transplant recipients treated with mycophenolate mofetil.

BACKGROUND: Intestinal microsporidiosis is a major cause of chronic diarrhea and malabsorption in patients with human immunodeficiency virus. Its occurrence in transplant recipients has exceptionally been reported to date. METHODS: We report what we believe are the first two cases of intestinal microsporidiosis in renal transplant recipients. The patients were treated with mycophenolate mofetil. RESULTS: The clinical presentation was chronic diarrhea with massive weight loss. Stool analysis revealed microsporidian spores, identified as Enterocytozoon bieneusi spores by polymerase chain reaction. The onset of this opportunistic infection in these two patients is believed to be secondary to an increase in immunosuppression after azathioprine replacement by mycophenolate mofetil. The withdrawal of mycophenolate mofetil led to clinical recovery. CONCLUSION: The incidence of microsporidiosis will probably increase in transplant recipients treated with powerful immunosuppressants. Therefore, we recommend a systematic search for microsporidian spores in stool specimens in cases of unexplained diarrhea in these patients.

Detection of Pneumocystis carinii DNA by PCR amplification in various rat organs in experimental pneumocystosis.

Pneumocystosis is usually a disease of the lungs, but the number of cases of extrapulmonary pneumocystosis has greatly increased during the AIDS epidemic. Much remains unknown about the frequency and mechanisms of dissemination. In the present study, a systematic search for Pneumocystis carinii by PCR with primers specific for mitochondrial rRNA was performed in the lung, liver, spleen and kidney of 12 immunosuppressed rats and two immunocompetent rats. The amplified products were analysed by Southern hybridisation with a digoxigenin-11-dUTP labeled probe. P. carinii DNA was found in lungs in all 14 rats and in at least one organ other than lung in 11 immunosuppressed rats and the two control rats. We suggest that extrapulmonary dissemination may not be an exceptional phenomenon in the course of pneumocystosis, but rather part of the natural evolution of the disease.

Real time quantitative PCR and RT--PCR for analysis of Pneumocystis carinii hominis.

Pneumocystis carinii hominis is a common cause of pneumonia in immunocompromised patients and particularly in those infected by HIV. Giemsa- and Gomori--Grocott-stained smears are widely used for detection and quantification of this opportunistic fungus obtained from biological samples or from in vitro culture. But these methods are fastidious and time-consuming. Thus, instead of performing a count of organisms, we focused our attention on the level of specific DNA by a quantitative PCR technique. This procedure has the advantage of greater precision and more objectivity. To verify the presence of organisms, quantitative RT--PCR based on DHFR and a cell cycle mRNA have been developed. In this current study, we present a detailed description of these methods and their applications for analysis of P. carinii hominis.

[Tracheal stenosis of inflammatory aspect]. / Sténose trachéale d'aspect inflammatoire.

We report a case of tracheal leishmaniasis with stenosis in a 52 year-old female originative from the Gard region (South of France). The unusual localization acknowledges for the difficulties met in setting the diagnosis which was established through cytological examination of bronchial brushing. Though rare, leishmaniasis infection must be suspected in all cases of mucosal lesions occurring in patients living in endemic areas.

Paromomycin: an effective treatment for cryptosporidial diarrhea in patients with AIDS.

Cryptosporidiosis is a common cause of diarrhea in patients with AIDS but remains one of the most challenging AIDS-related conditions to treat. We therefore carried out an open, uncontrolled, prospective study to evaluate the efficacy of oral paromomycin for the treatment of chronic diarrhea due to Cryptosporidium in 24 successive patients infected with human immunodeficiency virus. Twenty-two of the 24 patients responded clinically, with complete remission occurring in 18 cases and marked reduction of the diarrhea occurring in four. Clearance of cryptosporidia from stools and/or intestinal biopsy specimens occurred within 2-4 weeks. Furthermore, all 18 patients who achieved complete remission gained weight. Ten patients relapsed either during administration of maintenance therapy or after discontinuation of treatment, but diarrhea resolved again after increasing or restarting paromomycin therapy in all but two. Paromomycin appeared to be an active and well-tolerated treatment for cryptosporidiosis in patients with AIDS. Efficacy is dose-related, and continuous maintenance therapy is required to prevent relapses. These data suggest that paromomycin might be given as first-line therapy in AIDS-related cryptosporidiosis, but this should be confirmed by the findings of a controlled, randomized trial.