Comparative evaluation of Fungitest, Neosensitabs and broth microdilution method for yeasts susceptibility testing

The need of a simple and reliable method for routine yeasts susceptibility testing led us to evaluate two commercially available methods. We investigated the in vitro susceptibility of 67 clinical isolates (26 C. albicans, 32 C. glabrata, 4 C. krusei, 2 C. tropicalis, 2 C. kefyr and 1 S. cerevisiae) to 6 drugs flucytosine (FC), amphotericin B (A), fluconazole (FZ), itraconazole (IT), kétoconazole (K), miconazole (M), comparing two methods Fungitest (Sanofi Pasteur) and Neosensitabs (Rosco). A broth microdilution adaptation from the NCCLS-M27A procedure was used as reference method. Fungitests consist of individually packed 16 wells microplates containing 6 drugs at two critical concentrations in buffered medium. Reading was performed after 24 and 48h incubation. Neosensitabs is an agar diffusion method on Shadomy agar using antifungals tablets. Reading was performed after 24h. For all strains Neosensitabs was in concordance with NCCLS M27-A for FC (94%), A (98%), FZ (55%) I (53%) with p<0.05. Fungitest correlated with NCCLS method for all antifungals after 24 and 48h incubation time (p<0.05) with respectively 95/95% for FC, 100/100% for A, 80/76% for FZ, 81/55% for I and 93/75% for K, and 88/81 for M. Candida glabrata gave the poorest result with Neosensitabs with 28% concordance for FZand 39% for I; therefore the method can't be recommended for this species. Fungitest concordance observed was 55% for I after 48h. Our results suggest that Fungitest is appropriate for routine yeast susceptibility testing. However itraconazole testing has to be improved with this method

Development and optimization of quantitative PCR for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid

Background: The diagnosis of invasive pulmonary aspergillosis (IPA) remains challenging. Culture and histopathological examination of bronchoalveolar lavage (BAL) fluid are useful but have suboptimal sensitivity and in the case of culture may require several days for fungal growth to be evident. Detection of Aspergillus DNA in BAL fluid by quantitative PCR (qPCR) offers the potential for earlier diagnosis and higher sensitivity. It is important to adopt quality control measures in PCR assays to address false positives and negatives which can hinder accurate evaluation of diagnostic performance. Methods: BAL fluid from 94 episodes of pneumonia in 81 patients was analyzed. Thirteen episodes were categorized as proven or probable IPA using Mycoses Study Group criteria. The pellet and the supernatant fractions of the BAL were separately assayed. A successful extraction was confirmed with a human 18S rRNA gene qPCR. Inhibition in each qPCR was measured using an exogenous DNA based internal amplification control (IAC). The presence of DNA from pathogens in the Aspergillus genus was detected using qPCR targeting fungal 18S rRNA gene. Results: Human 18S rRNA gene qPCR confirmed successful DNA extraction of all samples. IAC detected some degree of initial inhibition in 11 samples. When culture was used to diagnose IPA, the sensitivity and specificity were 84.5% and 100% respectively. Receiver-operating characteristic analysis of qPCR showed that a cutoff of 13 fg of Aspergillus genomic DNA generated a sensitivity, specificity, positive and negative predictive value of 77%, 88%, 50%, 96% respectively. BAL pellet and supernatant analyzed together resulted in sensitivity and specificity similar to BAL pellet alone. Some patients did not meet standard criteria for IPA, but had consistently high levels of Aspergillus DNA in BAL fluid by qPCR. Conclusion: The Aspergillus qPCR assay detected Aspergillus DNA in 76.9% of subjects with proven or probable IPA when the concentrated BAL fluid pellet fraction was used for diagnosis. There was no benefit from analyzing the BAL supernatant fraction. Use of both extraction and amplification controls provided optimal quality control for interpreting qPCR results and therefore may increase our understanding of the true potential of qPCR for the diagnosis of IPA.Supported by NIH grant R01 AI054703 from the National Institute of Allergy and Infectious Diseases

Evaluation of PCR on Bronchoalveolar Lavage Fluid for Diagnosis of Invasive Aspergillosis: A Bivariate Metaanalysis and Systematic Review

BACKGROUND: Nucleic acid detection by polymerase chain reaction (PCR) is emerging as a sensitive and rapid diagnostic tool. PCR assays on serum have the potential to be a practical diagnostic tool. However, PCR on bronchoalveolar lavage fluid (BALF) has not been well established. We performed a systematic review of published studies to evaluate the diagnostic accuracy of PCR assays on BALF for invasive aspergillosis (IA). METHODS: Relevant published studies were shortlisted to evaluate the quality of their methodologies. A bivariate regression approach was used to calculate pooled values of the method sensitivity, specificity, and positive and negative likelihood ratios. Hierarchical summary receiver operating characteristic curves were used to summarize overall performance. We calculated the post-test probability to evaluate clinical usefulness. Potential heterogeneity among studies was explored by subgroup analyses. RESULTS: Seventeen studies comprising 1191 at-risk patients were selected. The summary estimates of the BALF-PCR assay for proven and probable IA were as follows: sensitivity, 0.91 (95% confidence interval (CI), 0.79-0.96); specificity, 0.92 (95% CI, 0.87-0.96); positive likelihood ratio, 11.90 (95% CI, 6.80-20.80); and negative likelihood ratio, 0.10 (95% CI, 0.04-0.24). Subgroup analyses showed that the performance of the PCR assay was influenced by PCR assay methodology, primer design and the methods of cell wall disruption and DNA extraction. CONCLUSIONS: PCR assay on BALF is highly accurate for diagnosing IA in immunocompromised patients and is likely to be a useful diagnostic tool. However, further efforts towards devising a standard protocol are needed to enable formal validation of BALF-PCR

Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph?+?ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1

Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear. This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph + ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10−3 and 36/67 (53%) and 53/67 (79%) at 10−4BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph + ALL

Expression of myeloperoxidase in acute myeloid leukemia blasts mirrors the distinct DNA methylation pattern involving the downregulation of DNA methyltransferase DNMT3B

Myeloperoxidase (MPO) has been associated with both a myeloid lineage commitment and favorable prognosis in patients with acute myeloid leukemia (AML). DNA methyltransferase inhibitors (decitabine and zeburaline) induced MPO gene promoter demethylation and MPO gene transcription in AML cells with low MPO activity. Therefore, MPO gene transcription was directly and indirectly regulated by DNA methylation. A DNA methylation microarray subsequently revealed a distinct methylation pattern in 33 genes, including DNA methyltransferase 3 beta (DNMT3B), in CD34-positive cells obtained from AML patients with a high percentage of MPO-positive blasts. Based on the inverse relationship between the methylation status of DNMT3B and MPO, we found an inverse relationship between DNMT3B and MPO transcription levels in CD34-positive AML cells (P=0.0283). In addition, a distinct methylation pattern was observed in five genes related to myeloid differentiation or therapeutic sensitivity in CD34-positive cells from AML patients with a high percentage of MPO-positive blasts. Taken together, the results of the present study indicate that MPO may serve as an informative marker for identifying a distinct and crucial DNA methylation profile in CD34-positive AML cells

Falciparum malaria molecular drug resistance in the Democratic Republic of Congo: a systematic review

peer reviewedBackground: Malaria cases were estimated to 207 million in 2013. One of the problems of malaria control is the emergence and spread of Plasmodium falciparum strains that become resistant to almost all drugs available. Monitoring drug resistance is essential for early detection and subsequent prevention of the spread of drug resistance by timely changes of treatment policy. This review was performed to gather all data available on P. falciparum molecular resistance in DR Congo, as baseline for future assessments. Methods: The search for this review was undertaken using the electronic databases PubMed and Google Scholar using the terms “malaria”, “Congo”, “resistance”, “molecular”, “antimalarial”, “efficacy”. Articles were classified based on year of collecting, year of publication, sample size and characteristics, molecular markers analysed and polymorphisms detected. Results: Thirteen articles were included and five genes have been analysed in these studies: pfcrt, pfdhps, pfdhfr, pfmdr1 and K13-propeller. The majority of studies included were not representative of the whole country. Conclusion: This systematic review demonstrates the lack of molecular resistance studies in DRC. Only 13 studies were identified in almost 15 years. The MOH must implement a national surveillance system for monitoring malaria drug resistance and this surveillance should be conducted frequently and country-representative

Microbial analysis of in situ biofilm formation in drinking water distribution systems: implications for monitoring and control of drinking water quality.

Biofilm formation in drinking water distribution systems (DWDS) is influenced by the source water, the supply infrastructure and the operation of the system. A holistic approach was used to advance knowledge on the development of mixed species biofilms in situ, by using biofilm sampling devices installed in chlorinated networks. Key physico-chemical parameters and conventional microbial indicators for drinking water quality were analysed. Biofilm coverage on pipes was evaluated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The microbial community structure, bacteria and fungi, of water and biofilms was assessed using pyrosequencing. Conventional wisdom leads to an expectation for less microbial diversity in groundwater supplied systems. However, the analysis of bulk water showed higher microbial diversity in groundwater site samples compared with the surface water site. Conversely, higher diversity and richness were detected in biofilms from the surface water site. The average biofilm coverage was similar among sites. Disinfection residual and other key variables were similar between the two sites, other than nitrates, alkalinity and the hydraulic conditions which were extremely low at the groundwater site. Thus, the unexpected result of an exceptionally low diversity with few dominant genera (Pseudomonas and Basidiobolus) in groundwater biofilm samples, despite the more diverse community in the bulk water, is attributed to the low-flow hydraulic conditions. This finding evidences that the local environmental conditions are shaping biofilm formation, composition and amount, and hence managing these is critical for the best operation of DWDS to safeguard water quality