No abstract

Alterations in the composition of commensal bacterial populations, a phenomenon known as dysbiosis, are linked to multiple gastrointestinal disorders, such as inflammatory bowel disease and irritable bowel syndrome, or to infections by diverse enteric pathogens. Blastocystis is one of the most common single-celled eukaryotes detected in human faecal samples. However, the clinical significance of this widespread colonization remains unclear, and its pathogenic potential is controversial. To address the issue of Blastocystis pathogenicity, we investigated the impact of colonization by this protist on the composition of the human gut microbiota. For that purpose, we conducted a cross-sectional study including 48 Blastocystis-colonized patients and 48 Blastocystis-free subjects and performed an Ion Torrent 16S rDNA gene sequencing to decipher the Blastocystis-associated gut microbiota. Here, we report a higher bacterial diversity in faecal microbiota of Blastocystis colonized patients, a higher abundance of Clostridia as well as a lower abundance of Enterobacteriaceae. Our results contribute to suggesting that Blastocystis colonization is usually associated with a healthy gut microbiota, rather than with gut dysbiosis generally observed in metabolic or infectious inflammatory diseases of the lower gastrointestinal tract.

Human and animal fungal pathogens are a growing threat worldwide leading to emerging infections and creating new risks for established ones. There is a growing need for a rapid and accurate identification of pathogens to enable early diagnosis and targeted antifungal therapy. Morphological and biochemical identification methods are time-consuming and require trained experts. Alternatively, molecular methods, such as DNA barcoding, a powerful and easy tool for rapid monophasic identification, offer a practical approach for species identification and less demanding in terms of taxonomical expertise. However, its wide-spread use is still limited by a lack of quality-controlled reference databases and the evolving recognition and definition of new fungal species/complexes. An international consortium of medical mycology laboratories was formed aiming to establish a quality controlled ITS database under the umbrella of the ISHAM working group on "DNA barcoding of human and animal pathogenic fungi." A new database, containing 2800 ITS sequences representing 421 fungal species, providing the medical community with a freely accessible tool at http://www.isham.org/ and http://its.mycologylab.org/ to rapidly and reliably identify most agents of mycoses, was established. The generated sequences included in the new database were used to evaluate the variation and overall utility of the ITS region for the identification of pathogenic fungi at intra-and interspecies level. The average intraspecies variation ranged from 0 to 2.25%. This highlighted selected pathogenic fungal species, such as the dermatophytes and emerging yeast, for which additional molecular methods/genetic markers are required for their reliable identification from clinical and veterinary specimens.

To obtain a rapid genotyping method of Candida albicans, three polymorphic microsatellite markers were investigated by multiplex PCR. The three loci, called CDC3, EF3, and HIS3, were chosen because they are on different chromosomes so as to improve the chances of finding polymorphisms. One set of primers was designed for each locus, and one primer of each set was dye-labeled to read PCR signals by using an automatic sequencer. Amplifications were performed directly from the colonies harvested on the agar plate without a sophisticated DNA extraction step. At total of 27 reference strains and 73 clinical independent isolates were tested. The numbers of allelic associations were 10, 22, and 25 for the loci CDC3, EF3, and HIS3, respectively. The combined discriminatory power of the three microsatellites markers was 0.97. The markers were stable after 25 subcultures, and the amplifications were specific for C. albicans. An initial study of 17 clinical isolate pairs, including blood culture and peripheral sites, showed a similar genotype for 15 of them, confirming that candidemia usually originates from the colonizing isolate. Therefore, microsatellite marker analysis with multiplex PCR and automated procedures has a high throughput and should be suitable for large epidemiologic studies of C. albicans.Among the yeasts that have emerged as major fungal pathogens in recent years (3), the commensal Candida albicans is the most prevalent and acts as an opportunistic agent in immunocompromised patients. The ability to discriminate strains of the organism has been developed for a better understanding of the epidemiology of this yeast. Thus, the route of acquisition (21), nosocomial transmission (16), or the emergence of antifungalresistant strains (6, 25) can be identified by using DNA-based methods already used for typing C. albicans. Strain-typing techniques such as restriction length polymorphic DNA (RFLP) with hybridization with a C. albicans-specific probe and the random amplified polymorphic DNA (RAPD) have been recently reviewed (23). The RFLP technique is very informative but is time-consuming since Southern blots are needed. The PCR-based RAPD technique is rapid but poorly reproducible, especially between laboratories. Other authors have developed allele-specific oligonucleotide probes in Southern hybridizations with PCR-amplified DNA regions (6). Another PCRbased method is the analysis of microsatellites, defined as tandemly repetitive stretches of two to five nucleotides. Since most microsatellites show a substantial level of polymorphism between individuals, microsatellites are extensively used for physical mapping in humans (28). Moreover, since microsatellites test the presence of different alleles at a given locus, distinguishing heterozygotes in diploid organisms such as C. albicans is possible in contrast to the RFLP and RAPD methods. Several studies have already reported the application of this technique for the genotyping of C. albicans (4,7,10,13,22).One microsatellite marker in the EF3 promoter sequence...

Overall survival after cryptococcosis has dramatically improved at the cART era. Immune restoration and low serum cryptococcal antigen titres are associated with lower cryptococcosis relapse rates.

To improve the diagnosis of invasive aspergillosis (IA), we developed a LightCycler PCR assay targeted to Aspergillus fumigatus and A. flavus mitochondrial DNA. To avoid contamination, fully automated nucleic acid extraction with the MagNA Pure LC apparatus was used. The linearity of the results was achieved over a 6-log range of input A. fumigatus DNA, from 0.3 ng to 3 fg/10 l of water. We retrospectively compared the LightCycler PCR and an enzyme-linked immunosorbent assay for the detection of galactomannan (GM) in serum from 14 patients with IA. The GM assay was more frequently positive (57 of 109; 52%) than the PCR assay (49 of 109; 45%). The LightCycler PCR assay, combined with automated DNA extraction, could be used in association with the GM assay to improve the reliability of IA diagnosis.The incidence of invasive aspergillosis (IA) is difficult to assess, as at least 30% of the cases that occur remain undiagnosed and untreated at death (7). Two approaches have been investigated to improve the sensitivity of aspergillosis detection by noninvasive techniques; these are antigen detection and DNA detection (13). Among the antigen tests, detection of galactomannan (GM) by an enzyme-linked immunosorbent assay (ELISA) has been shown to have good sensitivity, but a false-positivity rate of 10 to 15% has been reported (5, 16). Nevertheless, this method is now included in the IA diagnosis criteria (1).PCR technology is not as acceptable a diagnostic tool as the GM assay because very divergent results have been reported (3, 9). These discrepancies are most likely due to technical reasons because the PCR techniques used are not standardized. Real-time PCR assays dramatically decrease the risk of false-positive results. Indeed, contamination with previously amplified products is reduced because the reaction tubes need not be opened following amplification, thus avoiding potential contamination of the environment with amplicons (6, 15). We therefore developed a PCR assay based on the LightCycler technology (19). We also employed a fully automated nucleic acid extraction technique with the MagNA Pure LC apparatus (12). This apparatus can purify DNA from a broad variety of samples by magnetic bead technology, thus eliminating the need for vacuum pumps, centrifugation, or other manual steps that may result in cross contamination. We subsequently compared the performance of the GM assay and the real-time PCR test with samples from selected patients.LightCycler PCR test development. The LightCycler PCR test was targeted at the Aspergillus fumigatus mitochondrial gene (GenBank accession number L37095) (4). The amplicon comprises a 91-bp fragment. The two hybridization probes (TibMolBiol, Berlin, Germany) used are the 24-mer 5Ј-CTGT TAGTGCGGGAGTTCAAAXTCT-3Ј, where X represents the internal labeling of the T with LC-Red 640, and the 28-mer 5Ј-CTGAGCTAATTTCTTTCAACCCAAGGGA-3Ј, which is labeled at the 3Ј end with fluorescein isothiocyanate. Because of the thermodynamic constraints on the choice of the primers and probes, the LC...

BackgroundBlastocystis sp. is the most common intestinal parasite of humans. Despite its potential public health impact, epidemiological data regarding the prevalence and molecular subtype distribution of Blastocystis sp. in Europe are rarely reported. Therefore, the first multi-center epidemiological survey performed in Europe was conducted in France to diagnose and subtype Blastocystis sp. and to identify risk factors for infection.MethodsStool samples from 788 patients were collected either in summer or winter in 11 hospitals throughout France together with patient data. All stool samples were tested for the presence of Blastocystis sp. by quantitative PCR targeting the SSU rDNA gene. Positive samples were sequenced to determine the distribution of the subtypes in our cohort. Statistical analyses were performed to identify potential risk factors for infection.ResultsUsing quantitative PCR, the overall prevalence of Blastocystis sp. was shown to reach 18.1 %. The prevalence was significantly higher in summer (23.2 %) than in winter (13.7 %). Travellers or subjects infected with other enteric parasites were significantly more infected by Blastocystis sp. than non-travellers or subjects free of other enteric parasites, respectively. Different age-related epidemiological patterns were also highlighted from our data. The prevalence of Blastocystis sp. was not significantly higher in patients with digestive symptoms or diagnosed with chronic bowel diseases. Among symptomatic patients, Blastocystis sp. infection was significantly associated with abdominal pain. Gender, socioeconomic status, and immune status were not identified as potential risk factors associated with infection. Among a total of 141 subtyped isolates, subtype 3 was predominant (43.3 %), followed by subtype 1 and subtype 4 (20 %), subtype 2 (12.8 %), subtype 6 and subtype 7 (2.1 %). No association between ST and clinical symptoms was statistically evidenced.ConclusionsA high prevalence of Blastocystis sp. infection was found in our French patient population. Seasonal impact on the prevalence of Blastocystis sp. was highlighted and recent travels and age were identified as main risk factors for infection. Most cases were caused by subtypes 1 to 4, with a predominance of subtype 3. Large variations in both prevalence and ST distribution between hospitals were also observed, suggesting distinct reservoirs and transmission sources of the parasite.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-016-1776-8) contains supplementary material, which is available to authorized users.

Background Data on incidence of ventilator-associated pneumonia (VAP) and invasive pulmonary aspergillosis in patients with severe SARS-CoV-2 infection are limited. Methods We conducted a monocenter retrospective study comparing the incidence of VAP and invasive aspergillosis between patients with COVID-19-related acute respiratory distress syndrome (C-ARDS) and those with non-SARS-CoV-2 viral ARDS (NC-ARDS). Results We assessed 90 C-ARDS and 82 NC-ARDS patients, who were mechanically ventilated for more than 48 h. At ICU admission, there were significantly fewer bacterial coinfections documented in C-ARDS than in NC-ARDS: 14 (16%) vs 38 (48%), p < 0.01. Conversely, significantly more patients developed at least one VAP episode in C-ARDS as compared with NC-ARDS: 58 (64%) vs. 36 (44%), p = 0.007. The probability of VAP was significantly higher in C-ARDS after adjusting on death and ventilator weaning [sub-hazard ratio = 1.72 (1.14–2.52), p < 0.01]. The incidence of multi-drug-resistant bacteria (MDR)-related VAP was significantly higher in C-ARDS than in NC-ARDS: 21 (23%) vs. 9 (11%), p = 0.03. Carbapenem was more used in C-ARDS than in NC-ARDS: 48 (53%), vs 21 (26%), p < 0.01. According to AspICU algorithm, there were fewer cases of putative aspergillosis in C-ARDS than in NC-ARDS [2 (2%) vs. 12 (15%), p = 0.003], but there was no difference in Aspergillus colonization. Conclusions In our experience, we evidenced a higher incidence of VAP and MDR-VAP in C-ARDS than in NC-ARDS and a lower risk for invasive aspergillosis in the former group.