Towards a molecular diagnosis of invasive aspergillosis and disseminated candidosis

A lot of in-house polymerase chain reaction assays have been reported for diagnosis of invasive aspergillosis and disseminated candidosis. Encouraging results have been published to anticipate the diagnosis over the conventional microbiological methods. However, the absence of standardized methods has led to diverging results. As a consequence, these tests are not recognized as consensual diagnostic criteria, in contrast with some antigenemia detection kits. The major breakthrough for improving the results of these methods is the emergence of real-time technologies. This markedly improves the reliability of the PCR results by dramatically decreasing the risk of false positive results due to PCR products carryover. Moreover, using the quantitative results provided by this technique, this allows to rapidly compare the efficiency of primers, probes, and DNA extraction methods. Therefore, the hope is to identify the more specific and sensitive parameters to implement comparative studies. Automated DNA extraction should also be useful to achieve this goal.Whatever sophisticated technology is used, we still have to define the meaning of detecting nucleic acids in a given clinical sample. This seems simple in normally sterile anatomical sites but less obvious for example in respiratory specimens for invasive aspergillosis or in blood for candidosis in heavily colonized patients. Additional studies of the kinetics of fungal DNA are needed. The development of real-time technology should improve our knowledge in order to give the clinicians informative clues for making a decision.     

Development of two real-time quantitative TaqMan PCR assays to detect circulating Aspergillus fumigatus DNA in serum

Several PCR assays have been developed for detecting Aspergillus fumigatus DNA in blood of patients with invasive aspergillosis. However, the best blood fraction to be assayed has not been defined and the multicopy genes used as the DNA targets for amplification not characterized. Firstly, we developed a real-time PCR assays based on the TaqMan technology targeted to a single copy gene. To compare serum, white cell pellet, and plasma for effectiveness as blood assay fractions, we spiked whole blood with A. fumigatus DNA and processed these fractions similarly. The difference between white cell pellet and serum was not significant. In contrast, the yield from plasma was 10 times lower than from serum. Then, we compared serum processed immediately or after 24 h at room temperature and observed a lower yield after 24 h. Secondly, a real-time PCR assay targeted to a mitochondrial gene was also developed. The copy number was estimated between 9 and 10 mitochondrial genes per single copy gene. Therefore, we recommend serum, stored and frozen as soon as possible, to be used for detecting circulating A. fumigatus DNA for diagnosis. Moreover, the mitochondrial multicopy gene was characterized in order to compare results from different patients.     

New Short Tandem Repeat-Based Molecular Typing Method for Pneumocystis jirovecii Reveals Intrahospital Transmission between Patients from Different Wards

Pneumocystis pneumonia is a severe opportunistic infection in immunocompromised patients caused by the unusual fungus Pneumocystis jirovecii. Transmission is airborne, with both immunocompromised and immunocompetent individuals acting as a reservoir for the fungus. Numerous reports of outbreaks in renal transplant units demonstrate the need for valid genotyping methods to detect transmission of a given genotype. Here, we developed a short tandem repeat (STR)-based molecular typing method for P. jirovecii. We analyzed the P. jirovecii genome and selected six genomic STR markers located on different contigs of the genome. We then tested these markers in 106 P. jirovecii PCR-positive respiratory samples collected between October 2010 and November 2013 from 91 patients with various underlying medical conditions. Unique (one allele per marker) and multiple (more than one allele per marker) genotypes were observed in 34 (32%) and 72 (68%) samples, respectively. A genotype could be assigned to 55 samples (54 patients) and 61 different genotypes were identified in total with a discriminatory power of 0.992. Analysis of the allelic distribution of the six markers and minimum spanning tree analysis of the 61 genotypes identified a specific genotype (Gt21) in our hospital, which may have been transmitted between 10 patients including six renal transplant recipients. Our STR-based molecular typing method is a quick, cheap and reliable approach to genotype Pneumocystis jirovecii in hospital settings and is sensitive enough to detect minor genotypes, thus enabling the study of the transmission and pathophysiology of Pneumocystis pneumonia.     

Correlation Between Pneumocystis jirovecii Mitochondrial Genotypes and High and Low Fungal Loads Assessed by Single Nucleotide Primer Extension Assay and Quantitative Real‐Time PCR

We designed a single nucleotide primer extension (SNaPshot) assay for Pneumocystis jirovecii genotyping, targeting mt85 SNP of the mitochondrial large subunit ribosomal RNA locus, to improve minority allele detection. We then analyzed 133 consecutive bronchoalveolar lavage (BAL) fluids tested positive for P. jirovecii DNA by quantitative real‐time PCR, obtained from two hospitals in different locations (Hospital 1 [n = 95] and Hospital 2 [n = 38]). We detected three different alleles, either singly (mt85C: 39.1%; mt85T: 24.1%; mt85A: 9.8%) or together (27%), and an association between P. jirovecii mt85 genotype and the patient's place of hospitalization (p = 0.011). The lowest fungal loads (median = 0.82 × 10³ copies/μl; range: 15–11 × 10³) were associated with mt85A and the highest (median = 1.4 × 10⁶ copies/μl; range: 17 × 10³–1.3 × 10⁷) with mt85CTA (p = 0.010). The ratios of the various alleles differed between the 36 mixed‐genotype samples. In tests of serial BALs (median: 20 d; range 4–525) from six patients with mixed genotypes, allele ratio changes were observed five times and genotype replacement once. Therefore, allele ratio changes seem more frequent than genotype replacement when using a SNaPshot assay more sensitive for detecting minority alleles than Sanger sequencing. Moreover, because microscopy detects only high fungal loads, the selection of microscopy‐positive samples may miss genotypes associated with low loads.     

Towards a molecular diagnosis of invasive aspergillosis and disseminated candidosis

A lot of in-house polymerase chain reaction assays have been reported for diagnosis of invasive aspergillosis and disseminated candidosis. Encouraging results have been published to anticipate the diagnosis over the conventional microbiological methods. However, the absence of standardized methods has led to diverging results. As a consequence, these tests are not recognized as consensual diagnostic criteria, in contrast with some antigenemia detection kits. The major breakthrough for improving the results of these methods is the emergence of real-time technologies. This markedly improves the reliability of the PCR results by dramatically decreasing the risk of false positive results due to PCR products carryover. Moreover, using the quantitative results provided by this technique, this allows to rapidly compare the efficiency of primers, probes, and DNA extraction methods. Therefore, the hope is to identify the more specific and sensitive parameters to implement comparative studies. Automated DNA extraction should also be useful to achieve this goal. Whatever sophisticated technology is used, we still have to define the meaning of detecting nucleic acids in a given clinical sample. This seems simple in normally sterile anatomical sites but less obvious for example in respiratory specimens for invasive aspergillosis or in blood for candidosis in heavily colonized patients. Additional studies of the kinetics of fungal DNA are needed. The development of real-time technology should improve our knowledge in order to give the clinicians informative clues for making a decision.     

Temporal similarity between Candida albicans genotypes in a Tunisian neonatal intensive care unit suggests several nosocomial cross-contamination episodes

The nosocomial transmission of Candida albicans in neonatal intensive care units (NICUs) is an increasing concern and understanding the route of this transmission is critical for adequate infection control measures. The aim of our study was to assess the likeliness of nosocomial acquisition of C. albicans in the NICU of Farhat Hached hospital in Sousse (Tunisia). We genotyped 82 isolates from 40 neonates and 7 isolates from 5 health care workers (HCWs) with onychomycosis, by using CDC3 microsatellite length polymorphism (MLP) and the high-resolution melting (HRM) analysis. Combined MLP and HRM CD3 analysis led to the delineation of 12 genotypes. Five temporal clustering caused by five genotypes occurred during the study period. Three of these genotypes were isolated in both neonates and HCWs. The first clustering included 28 isolates obtained between January 2003 and May 2004 from 16 neonates and 2 HCWs. The second clustering included three isolates collected in 2004 from three neonates and two HCWs. The third clustering included 11 isolates obtained from 6 neonates and 1 HCW in 2006. The two remaining clustering could not be associated with any HCW's contamination. These results argue for the nosocomial transmission of C. albicans in our NICU. The combined MLP and HRM analysis is a rapid first approach for tracking cross-contamination.     

Rapid technique for cryopreservation of Echinococcus multilocularis metacestodes

Cysts of E. multilocularis were minced to prepare a crude homogenate and after addition of glycerol at a final concentration of 10%, cryopreservation was performed at a rate of 1 degree C min-1 in a controlled-rate freezer. The aliquots were subsequently stored in liquid nitrogen. All 22 isolates tested were successfully cryopreserved and their viability maintained.     

Aspergillus fumigatus causes in vitro electrophysiological and morphological modifications in human nasal epithelial cells

The role of the airway epithelium in the development of invasive aspergillosis in immunocompromised hosts has rarely been studied although patients at risk for this infection frequently have epithelial damage. We developed an in vitro model of primary culture of human nasal epithelial cells (HNEC) in air-liquid interface, which allows epithelial cell differentiation and mimics in vivo airway epithelium. We subsequently tested 7-day and 24-hour Aspergillus fumigatus filtrates on the apical side of HNEC to know whether A. fumigatus, the main species responsible for invasive aspergillosis, produces specific damage to the epithelial cells. The results were compared with those obtained with non-pathogenic filamentous fungi. Seven-day culture filtrates of A. fumigatus and Penicillium chrysogenum induced electrophysiological modifications whatever the fungus tested. In contrast, only 24-hour A. fumigatus filtrates induced a specific decrease in transepithelial resistance, hyperpolarization of the epithelium, and cytoplasmic vacuolization of HNEC compared with both A. niger and Penicillium chrysogenum. The inhibition of the A. fumigatus effects with amiloride suggests that the 24-hour fungal filtrate acts through sodium channels of HNEC. These early modifications of the epithelial cells could facilitate colonization of the airways by A. fumigatus. To know whether the molecules involved are specific to A. fumigatus or simply produced more rapidly than by other filamentous fungi warrants further investigation. In this perspective, the primary culture of HNEC represents a suitable model to study the interactions between airway epithelial cells and A. fumigatus.     

Bacterial, viral and parasitic enteric pathogens associated with acute diarrhea in hospitalized children from northern Jordan

To determine the etiology of acute diarrhea in Jordanian children under 5 years of age, we examined stool samples from 265 children admitted to the pediatric ward at Princess Rahma Hospital for Children, Irbid, Jordan, for parasites, rotavirus and enteric bacteria. Using both traditional and molecular diagnostic techniques, we detected enteropathogens in 66.4% of patients with diarrhea. A single enteric pathogen was detected in 50.9% of the children, and multiple pathogens were detected in 15.5%. The prevalence of enteropathogens identified was as follows: rotavirus (32.5%), enteropathogenic Escherichia coli (12.8%), enteroaggregative E. coli (10.2), enterotoxigenic E. coli (5.7%), Shigella spp. (4.9%), Entamoeba histolytica (4.9%), Salmonella spp. (4.5%), Campylobacter jejuni/coli (1.5%), Cryptosporidium spp. (1.5%), enteroinvasive E. coli (1.5%), eae-, Ehly-positive E. coli (0.8%), Giardia lamblia (0. 8%) and Yersinia enterocolitica (0.4%). No Vibrio cholerae, Shiga toxin-producing E. coli, microsporidia, adenovirus or small round virus were detected. Findings from this study demonstrate that rotavirus and several types of diarrheagenic E. coli, which are not screened for during routine examinations of stool samples in public health laboratories, were the most frequently detected enteropathogens in these children. Our findings highlight the value of using a combination of traditional and molecular techniques in the diagnosis of diarrheal disease in this population.