Selecting a multiplex PCR panel for accurate molecular diagnosis of intestinal protists: a comparative study of Allplex® (Seegene®), G-DiaParaTrio (Diagenode®), and RIDA®GENE (R-Biopharm®) assays and microscopic examination.

Commercial multiplex PCR assay panels were developed to overcome the limitations of microscopic examination for parasitological diagnosis on stool samples. However, given the increased supply of this diagnostic approach, these assays must be evaluated to position them in a diagnostic algorithm. Analytical performances of the multiplex PCR assay G-DiaParaTrio, Allplex® GI parasite and RIDA®GENE parasitic stool panel for detecting Blastocystis sp., Entamoeba histolytica, Giardia duodenalis, Cryptosporidium spp., Dientamoeba fragilis, and Cyclospora cayetanensis, were assessed through a retrospective comparative study on 184 stool samples initially sent for parasitological investigation. The composite reference method for parasitological diagnosis was microscopic observation and Entamoeba histolytica-specific adhesion detection when necessary. Multiplex PCR assays were performed on extracted DNA from each stool, following the manufacturer's recommendations. Discrepant results with the composite reference method were investigated with species-specific PCR to approach a final parasitological diagnosis. Overall sensitivity/specificity for the multiplex PCR assays was 93.2%/100% for G-DiaParaTrio, 96.5%/98.3% for Allplex® GI parasite and 89.6%/98.3% for RIDA®GENE, whereas the composite reference method presented an overall sensitivity/specificity of 59.6%/99.8%. These results confirmed the added diagnostic value of the multiplex PCR approach for gastrointestinal protists. Nevertheless, the PCR procedure and the analytical performance for each protist of interest, variable depending on the multiplex PCR assay, must be considered when implementing a PCR-based diagnostic approach.

TITLE: Sélection d'un panel PCR multiplex pour un diagnostic moléculaire précis des protistes intestinaux : étude comparative des tests Allplex® (Seegene®), G-DiaParaTrio (Diagenode®) et RIDA®GENE (R-Biopharm®) et de l'examen microscopique. ABSTRACT: Des panels commerciaux de tests PCR multiplex ont été développés pour dépasser les limites de l'examen microscopique pour l'examen parasitologique des selles. Cependant, compte tenu de l'offre croissante de cette approche diagnostique, ces tests doivent être évalués pour les positionner dans un algorithme de diagnostic. Les performances analytiques des tests PCR multiplex G-DiaParaTrio, Allplex® GI parasite et RIDA®GENE parasitic stool panel pour la détection de Blastocystis sp., Entamoeba histolytica, Giardia duodenalis, Cryptosporidium spp., Dientamoeba fragilis et Cyclospora cayetanensis, ont été évaluées à travers une étude comparative rétrospective sur 184 échantillons de selles envoyés initialement pour un examen parasitologique. La méthode composite de référence pour le diagnostic parasitologique était l'observation microscopique et la détection d'adhérence spécifique d'Entamoeba histolytica lorsque cela était nécessaire. Des tests PCR multiplex ont été effectués sur l'ADN extrait de chaque selle conformément aux recommandations du fabricant. Les résultats discordants avec la méthode de référence composite ont été étudiés par PCR spécifique d'espèce pour approcher un diagnostic parasitologique final. La sensibilité/spécificité globale des tests PCR multiplex est respectivement de 93,2 %/100 % pour G-DiaParaTrio, 96,5 %/98,3 % pour Allplex® GI et 89,6 %/98,3 % pour RIDA® GENE alors que la méthode de référence composite présente une sensibilité/spécificité globale de 59,6 %/99,8 %. Ces résultats ont confirmé la valeur diagnostique ajoutée de l'approche PCR multiplex pour les protistes gastro-intestinaux. Néanmoins, la procédure de PCR et les performances analytiques pour chaque protiste d'intérêt, variables selon les tests PCR multiplex, doivent être prises en compte lors de la mise en œuvre d'une approche de diagnostic basée sur la PCR.

Characteristics and outcome according to underlying disease in non-AIDS patients with acute respiratory failure due to Pneumocystis pneumonia.

In the non-AIDS group, several underlying conditions and immune defects could lead to different PCP presentations. This study compared PCP presentation and outcome according to the underlying disease. A secondary analysis of a previously published prospective observational study including 544 PCP patients was done. Only non-AIDS patients were included. Underlying disease was defined as chronic lymphocytic leukemia (CLL), organ transplantation, solid cancer, allogeneic hematopoietic stem cell transplant (AHSCT), other hematological diseases, and immunosuppressive treatment. Clinical characteristics and outcomes were compared between groups. Multiple correspondent analyses compared clinical characteristics at diagnosis. Day 30 mortality was analyzed. Three hundred and twenty-one patients were included in the study. The underlying diseases were hematological malignancy (n = 75), AHSCT (n = 14), CLL (n = 19), solid organ transplant (n = 94), solid tumor (n = 39), and immunosuppressive treatment (n = 57). Compared with other underlying diseases, PCP related to CLL was closer to PCP related to AIDS presentation (long duration of symptoms before diagnosis, high level of dyspnea, and low oxygen saturation at diagnosis). Day 30 mortality was associated with underlying disease, oxygen flow, and shock at ICU admission. PCP presentations may vary according to the underlying reason for immunosuppression. Response to treatment and adjuvant steroid therapy should be analyzed regarding this result.

Assessment of the first commercial multiplex PCR kit (ParaGENIE Crypto-Micro Real-Time PCR) for the detection of Cryptosporidium spp., Enterocytozoon bieneusi, and Encephalitozoon intestinalis from fecal samples.

Cryptosporidium spp. Enterocytozoon bieneusi and Encephalitozoon intestinalis are opportunistic pathogens responsible for gastrointestinal diseases. We evaluated the ParaGENIE Crypto-Micro Real-Time PCR kit (Ademtech, France), the first CE-IVD compliant PCR assay available for these pathogens. This study was conducted blindly against a reference panel of 115 stool specimens including positive samples for Cryptosporidium spp. (n = 48) and E. bieneusi (n = 38) as well as negative or positive samples for other parasites to test for cross-reactivity. An additional set of samples corresponding to 8 rare Cryptosporidium species was also included. Discrepancies were evaluated with external in-house PCR tests. The ParaGENIE Crypto-Micro PCR assay displayed a sensitivity/specificity of 91.7%/100% and 97.3%/98.7% for Cryptosporidium spp. and E. bieneusi, respectively, and was able to detect all 12 Cryptosporidium species of the reference panel, including rare species. This new CE-IVD assay will facilitate the diagnosis of intestinal cryptosporidiosis and microsporidiosis, a major concern in immunocompromised patients and travelers.

Rapid diagnostic tests relying on antigen detection from stool as an efficient point of care testing strategy for giardiasis and cryptosporidiosis? Evaluation of a new immunochromatographic duplex assay.

Microscopy is the gold standard for the diagnosis of gastrointestinal parasites but is time-consuming and dependent on operator skills. Rapid diagnostic tests represent alternative methods but most evaluations have been conducted on a limited number of samples preventing their implementation in the clinical setting. We evaluated a new CE-IVD marked immunochromatographic assay (Crypto/Giardia K-SeT®, Coris Bioconcept) for the detection of G. intestinalis and Cryptosporidium spp. in 2 phases (retrospective and prospective) on a set of 482 stool samples including rare Cryptosporidium species. Besides G. intestinalis, this test could represent a rapid and reliable alternative to the modified Ziehl-Neelsen staining for the diagnosis of cryptosporidiosis (sensitivity/specificity were 89.2%/99.3% and 86.7%/100% for G. intestinalis and Cryptosporidium resp.), reducing diagnostic delays. Such strategy would also be time-saving by avoiding wet mount microscopy and concentrations steps, being particularly appropriate for laboratories having little expertise in microscopy or not able to implement molecular diagnostic methods.

Pneumocystis jirovecii pneumonia in patients with or without AIDS, France.

Pneumocystis jirovecii pneumonia (PCP) in patients without AIDS is increasingly common. We conducted a prospective cohort study of consecutive patients with proven PCP; of 544 patients, 223 (41%) had AIDS (AIDS patients) and 321 (59%) had other immunosuppressive disorders (non-AIDS patients). Fewer AIDS than non-AIDS patients required intensive care or ventilation, and the rate of hospital deaths--17.4% overall--was significantly lower for AIDS versus non-AIDS patients (4% vs. 27%; p<0.0001). Multivariable analysis showed the odds of hospital death increased with older age, receipt of allogeneic bone marrow transplant, immediate use of oxygen, need for mechanical ventilation, and longer time to treatment; HIV-positive status or receipt of a solid organ transplant decreased odds for death. PCP is more often fatal in non-AIDS patients, but time to diagnosis affects survival and is longer for non-AIDS patients. Clinicians must maintain a high index of suspicion for PCP in immunocompromised patients who do not have AIDS.

Multilocus sequence typing of Pneumocystis jirovecii from clinical samples: how many and which loci should be used?

Pneumocystis jirovecii pneumonia (PCP) is an opportunistic infection with airborne transmission and remains a major cause of respiratory illness among immunocompromised individuals. In recent years, several outbreaks of PCP, occurring mostly in kidney transplant recipients, have been reported. Currently, multilocus sequence typing (MLST) performed on clinical samples is considered to be the gold standard for epidemiological investigations of nosocomial clusters of PCP. However, until now, no MLST consensus scheme has emerged. The aim of this study was to evaluate the discriminatory power of eight distinct loci previously used for the molecular typing of P. jirovecii (internal transcribed spacer 1 [ITS1], cytochrome b [CYB], mitochondrial rRNA gene [mt26S], large subunit of the rRNA gene [26S], superoxide dismutase [SOD], ß-tubulin [ß-TUB], dihydropteroate synthase [DHPS], and dihydrofolate reductase [DHFR]) using a cohort of 33 epidemiologically unrelated patients having respiratory samples that were positive for P. jirovecii and who were admitted to our hospital between 2006 and 2011. Our results highlight that the choice of loci for MLST is crucial, as the discriminatory power of the method was highly variable from locus to locus. In all, the eight-locus-based scheme we used displayed a high discriminatory power (Hunter [H] index, 0.996). Based on our findings, a simple and alternative MLST scheme relying on three loci only (mt26S, CYB, and SOD) provides enough discriminatory power (H-index, 0.987) to be used for preliminary investigations of nosocomial clusters of PCP.

Phaeohyphomycosis due to Alternaria infectoria: a single-center experience with utility of PCR for diagnosis and species identification.

The term phaeohyphomycosis refers to a rare group of fungal infections characterized by the presence of dark-walled hyphae or yeast-like cells in affected tissues. Herein, we report on the clinical and epidemiological characteristics of six cases of phaeohyphomycosis due to Alternaria spp. that occurred in our hospital over a 30-month period (from January 2008 to June 2010). Interestingly, whereas histopathological examinations were positive and fungal cultures yielded molds in all cases, mycological identification using conventional phenotypic methods was never possible despite prolonged incubation of the isolates. Identification of Alternaria infectoria species complex was obtained for each isolate by amplification and sequencing of the internal transcribed spacer of the ribosomal DNA (ITS rDNA). All patients had favourable outcomes following the introduction of azole-based antifungal therapy. This case series describes the clinical course of these six patients and highlights the utility of molecular identification to help in the identification of the etiologic agent when classical mycological methods have failed.

Trichomonads in pleural effusion: case report, literature review and utility of PCR for species identification.

Trichomonas tenax is a flagellated protozoan commonly found in the human oral cavity but of unusual occurrence in pulmonary infections. We describe a case of a 67-year-old patient with glioblastoma who presented with severe pleurisy in the post-operative period while she was receiving high-dose corticotherapy. Several motile flagellated protozoa were identified in the pleural fluid. Trichomonas tenax was identified by molecular methods. Pulmonary infections with Trichomonads might be underestimated because of diagnostic difficulties. The utility of molecular biology for species identification is underlined and the pathogenicity of Trichomonad parasites in human lungs is discussed in light of previously reported cases.