Image: http://www.bag-healthcare.com/en/diagnostics Top Papers in Diagnostics/Laboratory Session title: Top papers in mycology Michaela Lackner Division of Hygiene and Medical Microbiology Medical University of Innsbruck Austria Disclosures: Lecturing for Forest Pharma, Astellas Pharma, Basilea Pharma. ResearchESCMID grants from Astellas Pharma, MSD, Forest. eLibrary © by author METHODS

Inclusion criteria: Publication Date: March 2016 until April 2017  E-pub before print  Early online  Full publications

Original articles

Diagnostics & n = 69* Diagnostics & fungal infections n = 98* Diagnostics & n = 51* Diagnostics & n = 42* Diagnostics & mycology n = 33* ESCMID eLibrary*(pubmed 12.04.2017) © by author DISCLAIMER

Thanks to all researcher contributing to the field of mycological diagnostics.

Apologies to the many researcher which deserved to be included, but unfortunately were not.

The selection is my personal view, I tried my best to show a wide range of methods and fungal pathogens.

There is no ranking of the selected papers, they are sorted by topic rather than impact. ESCMID eLibrary © by author Content

Identification (n = 2)

Antigen-based tests (n = 3)

PCR-based tests (n = 5) Ʃ = 10 ESCMID eLibrary © by author IDENTIFICATION Title Toward a novel multilocus phylogenetic of the Authors G.S. de Hoog, K. Dukik, M. Monod, A. Packeu, D. Stubbe, M. Hendrickx, C. Kupsch, J.B. Stielow, J. Freeke, M. Göker, A. Rezaei-Matehkolaei, H. Mirhendi, Y. Gräser Journal Mycopathologia doi 10.1007/s11046-016-0073-9

Why did I choose this paper? The long-awaited and long-debated taxonomic revision of the dermatophytes.

What is the study about? Multilocus phylogeny to revise the taxonomy of dermatophytes. Better separation of geophilic, zoophilic, and anthropophilic dermatophytes.

Studied genes: rDNA ITSESCMIDand partial LSU, the ribosomal 60S protein, fragments eLibraryof b-tubulin, and translation elongation factor 3 . © by author Novelty? Anthropophils Thirteen new combinations are proposed!

Two new genera were introduced: Guarromyces (only one ) and Paraphyton

Newly proposed taxonomy: (16 species), Epidermophyton (1; E. floccosum), Nannizzia (9; N. gypseum), Microsporum (3), Lophophyton (1), Arthroderma (21), Ctenomyces (1).

All antropophils are associated to the genera TrichophytonESCMIDand Epidermophyton. eLibrary © by author Title Identification and typing of the emerging pathogen Candida auris by matrix- assisted Laser desorption ionisation time of flight mass spectrometry Authors V. Girard, S. Mailler, M. Chetry, C. Vidal, G. Durand, A. van Belkum, A.L. Colombo, F. Hagen, J.F. Meis, A. Chowdhary Journal Mycosis doi 10.1111/myc.12519

Why did I choose this paper? Candida auris is an emerging pathogen closely resembling C. haemulonii. The authors answer the if C. auris can be accurately identified and genotyped using MS-VITEK MALDI TOF and AFLP.

What is the study about? The authors answer the question if C. auris can be accurately identified and genotyped using MS-VITEK MALDI TOF and ALFP. https://www.cdc.gov/fungal/diseases/candidiasis/candida ESCMID-auris.html eLibrary © by author Novelty?

VITEK MS MALDI combined with AFLP allows rapid and accurate identification and subsequent typing of this emerging pathogen.

Note: Bruker MALDI has been used successfully for the identification of a variety of species including C. auris.1 1Prakash A, Sharma C, Singh A et al. Evidence of genotypic diversity among Candida auris isolates by multilocus sequence typing, matrixassisted laser desorptionESCMID ionization time-of-flight mass spectrometry and amplified fragment eLibrary length polymorphism. Clin Microbiol Infect 2016; 22: 277.e1 – 9. © by author ANTIGEN-BASED TESTS Title An evaluation of the performance of the Dynamiker® (1-3)-β-D-Glucan Assay to assist in the diagnosis of invasive , invasive and Authors P.L. White, J.S. Price, R.P. Posso, R.A. Barnes Journal Medical Mycology doi 10.1093/mmy/myx004

Why did I choose this paper? First independent evaluation of the novel (1-3)-beta D glucan Assay called Dynamiker ® Fungus for IC, PCP and IA for several high-risk patient cohorts. What is the study about? The study was designed to evaluate the performance of the D-BDG assay when testing serum samples from high risk patients (n =121). Compared was the performance of Platelia TM Aspergillus AG (GM), versus Associated of Cape Code (1-3)-beta-D-Glucan assay (ACC-BDG) versus Dynamiker® Fungus (1-3)-beta-D-Glucan assay (D-BDG). performance: ESCMID sensitivity, specificity, positive and negative likelihood eLibrary ratios, and diagnostic odds ratio © by author Novelty? Technical benefit: eight well strips, more cost efficient and flexible than the Associated of Cape Code (1-3)-beta-D-Glucan assay for laboratories with low sample numbers. D-BDG showed fair and good agreement with the Fungitell®, and GM assays (agreement 83.9%), respectively. The highest sensitivity (93.3%) was for the detection of IC, whereas the sensitivity for PCP was only 50%, although cases were limited. The positivity threshold could be lowered to 69 pg/ml. Further, large scale, prospective evaluation is required to confirm ESCMIDthe clinical validity eLibraryand determine clinical utility. © by author Title A prospective study of fungal biomarkers to improve management of invasive diseases in a mixed specialty critical care unit Authors A. Fe Talento, K. Dunne, E.A. Joyce, M. Palmer, E. Johnson, P.L. White, J. Springer, J. Löffler, T. Ryan, D. Collins, T.R. Rogers Journal Journal of Critical Care doi 10.1016/j.jcrc.2017.03.025 Why did I choose this paper? The diagnosis of invasive fungal diseases (IFD) in critical care patients (CrCP) is difficult. This study investigated the performance of a set of biomarkers for diagnosis of IFD in this patient cohort.

What is the study about? A prospective observational study in patients receiving critical care for ≥ 7 days was performed to (1) document incidence of IFD, (2) enumerate host factors, and (3) determine the performance of biomarkers. Serum samples were tested for the presence of: (1-3)-β-D-glucan (BDG), galactomannan (GM), and Aspergillus fumigatus DNA. GM was tested in BALs. The patients were classified using a diagnostic algorithm for invasive pulmonary aspergillosis1.

1Blot SI, et al. A clinical algorithm to diagnose invasive pulmonary aspergillosis in critically ESCMIDill patients. AmeLibrary J Respir Crit Care Med 2012;186(1):56–64. © by author Novelty? Outcome: 36 % of patients suffering from proven or probable IFD died within 30 days, while only 16 % died with possible IFD/colonisation. CrCU stay was longer in patients with proven and probable IFD. Few patients had ≥ 2 positive PCR and/or GM results. Cut-off value of 80 pg/mL was found optimal.

High negative predictive value of BDG (82 % in this studies up to 95 % in other studies) for the detection of IC, BDG could contribute to stewardship in critical care. Interpretation of fungal biomarkers is difficult in these critical care patients who do not have the host risk factors for IFD as outlined in existing published guidelines.  new diagnostic criteria needed which also consider BDG and host factors unique to critical care patientsESCMID. eLibrary © by author Title Application of mass spectrometry technology to early diagnosis of invasive fungal infections Authors A. Mery, B. Sendid, N. Francois, M. Cornu, J. Poissy, Y. Guerardel, D. Poulain Journal Journal of Clinical Microbiology doi 10.1128/JCM.01655-16

Why did I choose this paper? Novel pan-fungal antigen covering also mucormycetes. Mass spectrometry detection of serum disaccharide (MS-DS).

What is the study about? This retrospective study included 48 patients (23 IC patients, 15 IA patients, and 10 MM patients) and 49 appropriate controls (102 serum samples). MS-DS, mannan (Mnn), galactomannan (GM), and (1,3)-D-glucan (BDG) were detected by matrix- assisted laser desorption ionization–time of flight (MALDI-TOF) MS, Platelia, and Fungitell assays, respectively ESCMID. eLibrary © by author Novelty?

The MS-DS index test was positive up to 25 days earlier (median 3 days) than cultures.

MS-DS, BDG and Mnn do not circulate at the same time!

Mortality in IC patients was significantly associated with a high serum MS-DS index and high levels ofESCMID BDG. eLibrary © by author IC patient

Note: GM was one of IA patient the inclusion criteria.

MS-DS index positively complements Mnn and BDG for the diagnosis of IC. The MS -ESCMIDDS index earlier positive than other antigens alsoeLibrary in IA patients. © by author

9/10 patients and 4/5 sera had a high MS- DS index!!

MS-DS fills the gap as a new serological marker for MM!!!

pan-fungal IFD marker!

cost-efficient

easy to implement ESCMID eLibrary © by author PCR-BASED TESTS

Title New panfungal real-time PCR assay for diagnosis of invasive fungal infections Authors C. Valeri, L. de la Cruz-Villar, O. Zaragoza, M.J. Buitrago Journal Journal of Clincial Microbiology doi 10.1128/JCM.01580-16

Why did I choose this paper? New panfungal real-time PCR assay combining intercalating dye, sequence-specific probes, HRM and sequencing. First step to a pan-fungal PCR that does not necessarily require sequencing.

What is the study about? The panfungal real-time PCR assay was standardized by using 11 fungi and validated in 60 clinical samples from patients with proven and probable IFI. A melting curve database was constructed.

Principle: degenerated ITS primer pair + 5 probes (Aspergillus-P, Rhizopus-P, Mucor-P, ‘Histoplasma, Blastomyces Paracoccidioides ESCMID-P’, -P) eLibrary © by author 50 out of 60 samples were positive!

Remark: ESCMID eLibraryCandida probe missing! © by author By the probe-based and HRM-based assay 67.8% of identifications could be made without sequencing, resulting in an ID report within one working day.

Detection limit 0.1 fg -100 fg of DNA.

The overall sensitivity of the technique was 83.3%.

This technique is fast, sensitive, and specific and promises to be useful for improving early diagnosis of IFIs.

Further experiments are warranted in order to enlarge the melting curve database and expand the range of fungal species that can be detected by this technique. ESCMID eLibrary © by author Title Similar efficacy of broad-range ITS PCR and conventional fungal culture for diagnosing fungal infections in non-immunocompromised patients Authors S.K. Rampini, A. Zbinden, R.F. Speck, G.V. Bloemberg Journal BMC Microbiology doi 10.1186/s12866-016-0752-1

Why did I choose this paper? Surgical site infections due to fungal pathogens are increasing. This study evaluates the efficacy of ITS PCR versus fungal culture in non-immunocompromised patients. Standardized evaluation criteria are missing for these less selected patient groups. Gold standard is culture and histopathology. Circulating biomarkers are not useful for localized infections.

What is the study about? The aim of this study was to evaluate the analytical and clinical sensitivity and specificity of ITS PCR in patients without overt immunosuppression in comparison to culture, and to explore its value for diagnosis of fungal infections in daily practice. Studied wereESCMID252 clinical samples. eLibrary © by author Novelty? Results from 251 specimens showed a high concordance of 89.6 % for ITS PCR and fungal culture!! Assessing the clinical probability of a fungal infection based on detailed chart reviews, PCR had a clinical sensitivity of 88.9 %, a specificity of 86.3 %, a PPV of 64.0 % and a NPV of 96.6 %. The overall performance of fungal broad-range PCR was similar to that of culture, as sensitivity of culture in patients under antifungal treatment is surprisingly high.

Compared to culture, PCR has the advantage of a rapid time-to-result, proper identification of rare pathogens, prompt initiation of a species-targeted antifungal treatment, and prospects for automation ESCMID. eLibrary © by author Title Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus? Authors C.O. Morton, P.L. White, R.A. Barnes, L. Klingspor, M. Cuenca-Estrella, K. Lagrou,S. Bretagne, W. Melchers, C. Mengoli, A.M. Caliendo, M. Cogliati, Y. Debets-Ossenkopp, R. Gorton, F. Hagen, C. Halliday, P. Hamal, K. Harvey-Wood, K. Jaton, G. Johnson, S. Kidd, M. Lengerova, C. Lass-Florl, C. Linton, L. Millon, C.O. Morrissey, M. Paholcsek, A. Fe Talento, M. Ruhnke, B. Willinger, J.P. Donnelly, J. Loeffler on behalf of the EAPCRI Journal Medical Mycology doi 10.1093/mmy/myw093

Why did I choose this paper? Clearly shows that Aspergillus PCR assays perform differently for the various Aspergillus species, with best performance for A. fumigatus. What is the study about? A special focus was on the detection of other aspergilli than A. fumigatus. And to evaluate the assays for cross reactivity with other fungal pathogens. Multicenter ESCMIDstudy (n = 28) generating 33 data sets. eLibrary © by author Novelty? The positivity rate for A. fumigatus was significantly greater at both concentrations. false The lowest true positivity rate at positivity rate the 100 fg/µL concentration was was 14.5% seen for the detection of A. versicolor.

Detection rates for non-fumigatus Aspergillus species is concerning. High number of false negatives, not useful for excluding IA! ESCMID eLibrary © by author Title Direct molecular diagnosis of aspergillosis and CYP51A profiling from respiratory samples of French patients Authors Y. Zhao, C. Garnaud, M.-P. Brenier-Pinchart, A Thiébaut-Berntrand, C. Saint-Raymond, B. Camara, R. Hamidfar, O. Cognet, D. Maubon, M. Cornet, D. S. Perlin, Grenoble Aspergillus Committee Journal Frontiers in Microbiology doi 10.3389/fmicb.2016.01164

Why did I choose this paper? A possibility of estimating azole resistance for culture negative cases? Authors rise the question if we use the right respiratory samples for PCR diagnostics! Bronchial aspirates versus BALs?

What is the study about? 97 respiratory samples (BAL, BA, tracheal aspirates, sputa, pleural fluids, lung biopsy) 33 patients (CF, SOT, HAEM, PULM): invasive aspergillosis (n=12), chronic pulmonary aspergillosis (n=3), allergic broncho pulmonary aspergillosis (n=7), or colonization (n=11) and 28 controls. Each specimen was evaluated by (1) culture, (2) pan-Aspergillus qPCR, and (3) CYP51A PCR, and (4) sequencing ESCMID. eLibrary © by author Novelty?

PCR positivity was significantly higher than culture positivity in the IA group.

Aspergillus detection rate was generally higher in all patient groups by pan-Aspergillus real-time PCR than by culture. A. fumigatus CYP51A PCR works directly on respiratory samples (38 out of 44), alternative for culture negatives and faster as culture-based susceptibility testing.

DNA concentration in BA is much higher than in BALs (9 out of 11 samples) in average approx. 50 times moreESCMID concentrated (Ct difference 4.7). eLibrary © by author Title PCR-based detection of Aspergillus fumigatus Cyp51A mutations on bronchoalveolar lavage: a multicentre validation of the AsperGenius assay in 201 patients with haematological disease suspected for invasive aspergillosis

Authors G. M. Chong, M. T. van der Beek, P. A. von dem Borne, J. Boelens, E. Steel, G. A. Kampinga, L. F. R. Span,K. Lagrou, J. A. Maertens, G. J. H. Dingemans, G. R. Gaajetaan, D. W. E. van Tegelen, J. J. Cornelissen, A. G. Vonk, B. J. A. Rijnders Journal Journal of Antimicrobial Chemotherapy doi 10.1093/jac/dkw323

Why did I choose this paper? Evaluation of a commercial multiplex PCR kit AsperGenius® in BALs of patients with haematological disease. No sequencing is required! Aspergillus ID and detection of resistance-associated mutations (TR34/L98/T289A/Y121F). What is the study about? Retrospective study (5 hospitals); 201 patients and BALS samples (88 positive and 113 negatives) (1) determining optimal cut off CT value and diagnostic performance of the species probe (2) treatment failure and 6 week mortality were determined for patients receiving monotherapy and resistance ESCMIDPCR was successful. eLibrary © by author Novelty?

CT-off value of 38 was taken. Sensitivity: 84.09% Specificity: 79.65% PPV: 76.29% NPV: 86.54%  good diagnostic performace!

Azole treatment failure was observed in 12/45 patients with WT A. fumigatus compared with 6/8 patients with A. fumigatus with RAM. Six week mortality was higher (17.8% without versus 50.0%) in patients ESCMIDwith detected RAM. eLibrary © by author Image: http://www.bag-healthcare.com/en/diagnostics

for your attention!

ESCMIDThanks to Ferry Hagen foreLibrarydiscussion! © by author