Marios Arvanitis

Molecular and Nonmolecular Diagnostic Methods for Invasive Fungal Infections

SUMMARY Invasive fungal infections constitute a serious threat to an ever-growing population of immunocompromised individuals and other individuals at risk. Traditional diagnostic methods, such as histopathology and culture, which are still considered the gold standards, have low sensitivity, which underscores the need for the development of new means of detecting fungal infectious agents. Indeed, novel serologic and molecular techniques have been developed and are currently under clinical evaluation. Tests like the galactomannan antigen test for aspergillosis and the β-glucan test for invasive Candida spp. and molds, as well as other antigen and antibody tests, for Cryptococcus spp., Pneumocystis spp., and dimorphic fungi, have already been established as important diagnostic approaches and are implemented in routine clinical practice. On the other hand, PCR and other molecular approaches, such as matrix-assisted laser desorption ionization (MALDI) and fluorescence in situ hybridization (FISH), have proved promising in clinical trials but still need to undergo standardization before their clinical use can become widespread. The purpose of this review is to highlight the different diagnostic approaches that are currently utilized or under development for invasive fungal infections and to identify their performance characteristics and the challenges associated with their use.

PCR in Diagnosis of Invasive Aspergillosis: a Meta-Analysis of Diagnostic Performance

ABSTRACT Invasive aspergillosis is a difficult-to-diagnose infection with a high mortality rate that affects high-risk groups such as patients with neutropenia and hematologic malignancies. We performed a bivariate meta-analysis of diagnostic data for an Aspergillus sp. PCR assay with blood specimens from high-risk hematology patients. We included all studies involving human subjects that assessed the performance of any PCR assay for invasive aspergillosis in whole blood or serum and that used the European Organization for the treatment of Cancer/Mycoses Study Group criteria as a reference standard. Three investigators independently searched the literature for eligible studies and extracted the data. Out of a total of 37 studies, 25 met strict quality criteria and were included in our evidence synthesis. Twenty-five studies with 2,595 patients were analyzed. The pooled diagnostic performance of whole-blood and serum PCR assays was moderate, with a sensitivity and specificity of 84% (95% confidence interval [CI], 75 to 91%) and 76% (95% CI, 65 to 84%), respectively, suggesting that a positive or negative result is unable, on its own, to confirm or exclude a suspected infection. The performance of a PCR assay of serum was not significantly different from that of whole blood. Notably, at least two positive PCR test results were found to have a specificity of 95% and a sensitivity of 64% for invasive infection, achieving a high positive likelihood ratio of 12.8. Importantly, the European Aspergillus PCR Initiative (EAPCRI) recommendations improved the performance of the PCR even further when at least two positive specimens were used to define PCR positivity. In conclusion, two positive PCR results should be considered highly indicative of an active Aspergillus sp. infection. Use of the EAPCRI recommendations by clinical laboratories can further enhance PCR performance.

Invertebrate models of fungal infection

The morbidity, mortality and economic burden associated with fungal infections, together with the emergence of fungal strains resistant to current antimicrobial agents, necessitate broadening our understanding of fungal pathogenesis and discovering new agents to treat these infections. Using invertebrate hosts, especially the nematode Caenorhabditis elegans and the model insects Drosophila melanogaster and Galleria mellonella, could help achieve these goals. The evolutionary conservation of several aspects of the innate immune response between invertebrates and mammals makes the use of these simple hosts an effective and fast screening method for identifying fungal virulence factors and testing potential antifungal compounds. The purpose of this review is to compare several model hosts that have been used in experimental mycology to-date and to describe their different characteristics and contribution to the study of fungal virulence and the detection of compounds with antifungal properties. This article is part of a Special Issue entitled: Animal Models of Disease.

Galactomannan and Polymerase Chain Reaction–Based Screening for Invasive Aspergillosis Among High-Risk Hematology Patients: A Diagnostic Meta-analysis

BACKGROUND Screening of high-risk patients for invasive aspergillosis (IA) has the potential to decrease the use of empiric antifungal agents. However, the performance of different screening methods has not been studied. METHODS We performed a meta-analysis of published studies to assess the diagnostic performance of galactomannan (GM) and polymerase chain reaction (PCR) as weekly screening tests in high-risk populations. The sensitivity and specificity of 6 approaches combining GM and PCR were estimated using the bivariate model. RESULTS Thirteen studies with 1670 patients met our inclusion criteria. Single positive test results had modest sensitivity and specificity for screening (respectively, 92% and 90% for GM; 84% and 76% for PCR). The screening approach with the highest sensitivity was the one that used at least 1 GM- or PCR-positive result to define a positive episode, achieving a sensitivity of 99%, significantly higher than any single test (P = .0018 compared with GM and P < .0001 compared with PCR). Meanwhile, when both GM and PCR were positive for the same patient, the specificity increased to 98%, which was not significantly different compared to the specificity of at least 2 positive GM (95%, P = .56 for the comparison) or PCR results (93%, P = .07 for the comparison). CONCLUSIONS When screening high-risk patients for IA with GM and PCR tests, the absence of any positive test can obviate the need for antifungal agents with a negative predictive value of 100%, whereas the presence of at least 2 positive results is highly suggestive of an active infection with a positive predictive value of 88%.

A Multi-Host Approach for the Systematic Analysis of Virulence Factors in Cryptococcus neoformans

A multi-host approach was followed to screen a library of 1201 signature-tagged deletion strains of Cryptococcus neoformans mutants to identify previously unknown virulence factors. The primary screen was performed using a Caenorhabditis elegans-C. neoformans infection assay. The hits among these strains were reconfirmed as less virulent than the wild type in the insect Galleria mellonella-C. neoformans infection assay. After this 2-stage screen, and to prioritize hits, we performed serial evaluations of the selected strains, using the C. elegans model. All hit strains identified through these studies were validated in a murine model of systemic cryptococcosis. Twelve strains were identified through a stepwise screening assay. Among them, 4 (CSN1201, SRE1, RDI1, and YLR243W) were previously discovered, providing proof of principle for this approach, while the role of the remaining 8 genes (CKS101, CNC5600, YOL003C, CND1850, MLH3, HAP502, MSL5, and CNA2580) were not previously described in cryptococcal virulence. The multi-host approach is an efficient method of studying the pathogenesis of C. neoformans. We used diverse model hosts, C. elegans, G. mellonella, and mice, with physiological differences and identified 12 genes associated with mammalian infection. Our approach may be suitable for large pathogenesis screens.

Nocardiosis of the Central Nervous System: Experience From a General Hospital and Review of 84 Cases From the Literature

AbstractCentral nervous system (CNS) nocardiosis is a rare disease entity caused by the filamentous bacteria Nocardia species. We present a case series of 5 patients from our hospital and a review of the cases of CNS nocardiosis reported in the literature from January 2000 to December 2011. Our results indicate that CNS nocardiosis can occur in both immunocompromised and immunocompetent individuals and can be the result of prior pulmonary infection or can exist on its own. The most common predisposing factors are corticosteroid use (54% of patients) and organ transplantation (25%). Presentation of the disease is widely variable, and available diagnostic tests are far from perfect, often leading to delayed detection and initiation of treatment. The optimal therapeutic approach is still undetermined and depends on speciation, but lower mortality and relapse rates have been reported with a combination of targeted antimicrobial treatment including trimethoprim/sulfomethoxazole (TMP-SMX) for more than 6 months and neurosurgical intervention.

Antimicrobial Lock Solutions as a Method to Prevent Central Line–Associated Bloodstream Infections: A Meta-analysis of Randomized Controlled Trials

BACKGROUND Antimicrobial lock solutions may be an effective strategy to prevent catheter-associated infections. However, there remains concern about their efficacy and safety. METHODS To investigate the efficacy of antimicrobial lock therapy to prevent central line-associated bloodstream infections (CLABSIs), we performed a systematic search of PubMed, Embase, Cochrane Central Register of Controlled Trials, and ClinicalTrials.gov, from the earliest date up to 31 December 2013. Studies were eligible if they were randomized controlled trials comparing antimicrobial lock solutions to heparin and if they provided an appropriate definition of infection. RESULTS The 23 included studies reported data on 2896 patients, who were predominantly adult patients undergoing hemodialysis (16/23 studies), but also adult and pediatric oncology patients, critically ill neonates, and patients receiving total parenteral nutrition. The use of antimicrobial lock solutions led to a 69% reduction in CLABSI rate (relative risk [RR], 0.31; 95% confidence interval [CI], .24-.40) and a 32% reduction in the rate of exit site infections (RR, 0.68; 95% CI, .49-.95) compared with heparin, without significantly affecting catheter failure due to noninfectious complications (RR, 0.83; 95% CI, .65-1.06). All-cause mortality was not different between the groups (RR, 0.84; 95% CI .64-1.12). Neither the type of antimicrobial solution nor the population studied, affected the relative reduction in CLABSIs, which also remained significant among studies reporting baseline infection rates of <1.15 per 1000 catheter-days, and studies providing data for catheter-related bloodstream infections. Publication and selective reporting bias are a concern in our study and should be acknowledged. CONCLUSIONS Antimicrobial lock solutions are effective in reducing risk of CLABSI, and this effect appears to be additive to traditional prevention measures.

Activity of Daptomycin or Linezolid in Combination with Rifampin or Gentamicin against Biofilm-Forming Enterococcus faecalis or E. faecium in an In Vitro Pharmacodynamic Model Using Simulated Endocardial Vegetations and an In Vivo Survival Assay Using Galleria mellonella Larvae

ABSTRACT Enterococci are the third most frequent cause of infective endocarditis. A high-inoculum stationary-phase in vitro pharmacodynamic model with simulated endocardial vegetations was used to simulate the human pharmacokinetics of daptomycin at 6 or 10 mg/kg of body weight/day or linezolid at 600 mg every 12 h (q12h), alone or in combination with gentamicin at 1.3 mg/kg q12h or rifampin at 300 mg q8h or 900 mg q24h. Biofilm-forming, vancomycin-susceptible Enterococcus faecalis and vancomycin-resistant Enterococcus faecium (vancomycin-resistant enterococcus [VRE]) strains were tested. At 24, 48, and 72 h, all daptomycin-containing regimens demonstrated significantly more activity (decline in CFU/g) than any linezolid-containing regimen against biofilm-forming E. faecalis. The addition of gentamicin to daptomycin (at 6 or 10 mg/kg) in the first 24 h significantly improved bactericidal activity. In contrast, the addition of rifampin delayed the bactericidal activity of daptomycin against E. faecalis, and the addition of rifampin antagonized the activities of all regimens against VRE at 24 h. Also, against VRE, the addition of gentamicin to linezolid at 72 h improved activity and was bactericidal. Rifampin significantly antagonized the activity of linezolid against VRE at 72 h. In in vivo Galleria mellonella survival assays, linezolid and daptomycin improved survival. Daptomycin at 10 mg/kg improved survival significantly over that with linezolid against E. faecalis. The addition of gentamicin improved the efficacy of daptomycin against E. faecalis and those of linezolid and daptomycin against VRE. We conclude that in enterococcal infection models, daptomycin has more activity than linezolid alone. Against biofilm-forming E. faecalis, the addition of gentamicin in the first 24 h causes the most rapid decline in CFU/g. Of interest, the addition of rifampin decreased the activity of daptomycin against both E. faecalis and VRE.

Diagnosis of invasive aspergillosis: recent developments and ongoing challenges.

Invasive aspergillosis is an infection with high morbidity and mortality that affects mostly immunocompromised individuals. Early identification and targeted treatment of the infection is essential to improve survival of affected patients. The purpose of our review is to highlight the most recent developments in diagnosis and screening for invasive aspergillosis (IA) along with the challenges associated with the development and validation of novel diagnostic approaches.

C. elegans for anti-infective discovery.

Widespread use of antimicrobial agents has led to the emergence of multidrug resistant pathogens, which are becoming increasingly common in clinical practice. This underscores the need to discover novel anti-infective compounds. The soil-living nematode Caenorhabditis elegans has proved to be an excellent model host in the search for such compounds. Indeed, this alternative host is successfully being used to identify traditional microbicidal agents, including antihelminthic compounds, as well as novel agents that attenuate microbial virulence or enhance the hosts immune response. Thanks to numerous advantageous features, including its amenability to high-throughput automated screens that allow for the detection of bioactive products among thousands of tested substances, the C. elegans model is now in the spotlight of scientific attention.