Next-generation sequencing for hypothesis-free genomic detection of invasive tropical infections in poly-microbially contaminated, formalin-fixed, paraffin-embedded tissue samples – a proof-of-principle assessment

Table 5 Comparison of the NGS Results in Terms of Percentage of Species-specific Reads of Investigated Fungal Species Per Total Number of Reads in the Sample with the Respective Largest Standard Deviation above the Mean of All Samples in Multiples of the Standard Deviation (SD), Depicted for the Samples from Patients with Invasive Mycoses. “Positive” = matching of the molecular results with histology. “Negative” = inconsistency of molecular results with histology. “Partially positive” = detection of both matching and nonmatching molecular results compared with histology. “Contaminated” = Detection of environmental fungi only. “Match” = NGS result matching the histological findings. “Mismatch” = NGS results not matching the histological findings, “No match” = No evidence for relevant fungal species above the respective standard deviation (SD), “Not performed” in “Specific PCR” = No specific PCR performed. Highest standard deviations (SD) are shown

Sample	Histological diagnosis	Specific PCR [13]	Panfungal PCRs [13]	NGS > 1st SD	NGS > 2nd SD	NGS > 3rd SD	NGS > 4th SD
1	Chromoblastomycosis	Not performed	Contaminated	Match^d	No match	No match	No match
2	Mucormycosis	Negative	Contaminated	No match	No match	No match	No match
3	Histoplasmosis	Negative	Contaminated	Mismatch^e	Mismatch^f	No match	No match
4	Mucormycosis	Positive^a	Contaminated	No match	No match	No match	Match^g
5	Histoplasmosis or cryptococcosis	Negative	Contaminated	No match	No match	No match	Match^h
6	Chromoblastomycosis	Not performed	Contaminated	Mismatchⁱ	Match^j	No match	No match
7	Rhinosporidiosis	Not performed	Contaminated	No match	No match	No match	No match
8	Mycetoma	Not performed	Contaminated	Mismatch^k	Mismatch^l	No match	Match^m
9	Rhinosporidiosis	Not performed	Contaminated	Mismatchⁿ	No match	No match	No match
10	Mycetoma	Not performed	Contaminated	Mismatch^o	No match	No match	No match
11	Histoplasmosis	Negative	Contaminated	No match	No match	No match	No match
12	Histoplasmosis	Negative	Contaminated	Mismatch^p	Mismatch^q	Mismatch^r	No match
13	Chromoblastomycosis	Not performed	Contaminated	No match	No match	No match	No match
14	Histoplasmosis	Positive	Partially positive^b	No match	No match	No match	Match^s
15	Coccidioidomycosis	Not performed	Contaminated	Mismatch^t	No match	No match	No match
16	Coccidioidomycosis	Not performed	Contaminated	No match	No match	No match	No match
17	Mycetoma	Not performed	Partially positive^c	No match	No match	No match	Match^u

^aLichtheimia / Absidia corymbifera, ^bHistoplasma capsulatum in 2 out of 5 panfungal PCRs, ^cMadurella mycetomatis in 1 out of 5 panfungal PCRs, ^d0.004% Cladophialophora yegresii, ^e0.0004% Capronia epimyces, ^f0.0002% Cladophialophora carrionii, ^g0.00004% Rhizopus oryzae, ^h0.00001% Cryptococcus carnescens, ⁱ0.00009% Fusarium graminearum, ^j0.0002% Cladophialophora psammophila, ^k0.0004% Bipolaris oryzae, ^l0.0005% Paracoccidioides sp. ‘lutzii’, ^m0.02% Fusarium pseudograminearum, ⁿ0.004% Cladophialophora yegresii, ^o0.004% Cladophialophora yegresii, ^p0.0003% Bipolaris sorokiniana, 0.004% Cladophialophora yegresii, 0.0003% Coccidioides immitis, 0.0004% Paracoccidioides sp. ‘lutzii’, ^q0.0006% Capronia epimyces, 0.0006% Chaetomium globosum, 0.0004% Chaetomium thermophilum var. Thermophilum, 0.0002% Cladophialophora psammophila, 0.0002% Cryptococcus neoformans, 0.0002% Exophiala dermatitidis, 0.0006% Paracoccidioides brasiliensis, ^r0.0005% Coccidioides posadasii, 0.0005% Cyphellophora europaea, 0.0002% Fusarium graminearum, ^s0.02% Histoplasma capsulatum, ^t0.004% Cladophialophora yegresii, ^u0.001% Madurella mycetomatis. Matches with the histological findings are depicted in underlined print