Invasive mycoses are rare but associated with severe diseases with high mortality [
However, culture will fail if potential microbial causes of the pathological findings are not considered at the time of sample acquisition, so formalin-fixed, paraffin-embedded bioptic material is collected for subsequent histological assessment. After formalin-based sample inactivation, histological assessment is usually the method of choice in the diagnostic algorithm. Nevertheless, a recent 10-year single-center study impressively demonstrated an analytical correctness of no more than 79% of histological findings for assessed invasive mycoses [
If culture is not possible for logistic, technical, or other reasons and if there is no alternative sample material available other than the formalin-fixed, paraffin-embedded material, PCR-based diagnostic approaches may be considered. Various PCR protocols for the specific detection of certain invasively growing fungi with human pathogenic potential, for example, Mucorales, in tissue have been introduced [
If there is no explicit suspicion regarding the potential fungal pathogen, broad-range PCRs with consecutive sequence-based analysis have to be performed, usually amplifying longer DNA fragments of several hundred base pairs. Numerous protocols have been described for the sequence-based identification of fungi in formalin-fixed, paraffin-embedded tissue, usually targeting the 18S rRNA gene, the 28S rRNA gene, or ITS (internal transcribed spacer) fragments [
In this study, we evaluated five published PCR/sequencing protocols for the identification of fungal pathogens in formalin-fixed, paraffin-embedded bioptic tissue samples [
In a 30-year period between 1984 and 2013, a specimen collection comprising a total of 17 paraffin-embedded, buffered formalin-fixed bioptic samples demonstrating histologically confirmed invasive mycosis due to tropical or other rare fungal pathogens was established at the Department of Infectious Disease Pathology of the Bernhard Nocht Institute for Tropical Medicine in Hamburg, Germany. Histological diagnoses comprised chromoblastomycosis (
Histological diagnosis, material, and age of the 17 analyzed samples from patients with invasive mycosis.
Sample I.D. | Histological diagnosis | Sample material | Sample age (years) |
---|---|---|---|
Case 1 | Chromoblastomycosis | Skin biopsy | 30 |
Case 2 | Mucormycosis | Material from the ethmoid | 17 |
Case 3 | Histoplasmosis | Lymph node biopsy | 15 |
Case 4 | Mucormycosis | Skin biopsy | 14 |
Case 5 | Histoplasmosis or cryptococcosis | Lymph node biopsy | 13 |
Case 6 | Chromoblastomycosis | Skin biopsy | 12 |
Case 7 | Rhinosporidiosis | Biopsy of a nasal polyp | 12 |
Case 8 | Mycetoma/maduromycosis | Vulva exudate | 11 |
Case 9 | Rhinosporidiosis | Skin biopsy | 11 |
Case 10 | Mycetoma/maduromycosis | Lung tissue | 11 |
Case 11 | Histoplasmosis | Bone, muscular tissue, and connective tissue from the spine extension of third thoracic vertebra | 11 |
Case 12 | Histoplasmosis | Tissue from the tricuspid valve | 11 |
Case 13 | Chromoblastomycosis | Skin biopsy | 8 |
Case 14 | Histoplasmosis | Material from the bottom lip | 8 |
Case 15 | Coccidioidomycosis | Lung tissue | 6 |
Case 16 | Coccidioidomycosis | Lung tissue | 5 |
Case 17 | Mycetoma/maduromycosis | Biopsy from a wound of the foot | 1 |
To exclude microtome-associated contamination, three randomly chosen, recently obtained bioptic samples from patients with diseases other than invasive mycosis were included in the analyses as negative controls. As invasive mycosis is a rare diagnosis [
The small quantities of residual material from the tissue blocks in the specimen collection allowed for only one mode of DNA preparation, which has been reported to be optimal for the recovery of fungal DNA from formalin-fixed, paraffin-embedded tissues [
In addition to thick sections for PCR, thinner 4–6
The DNA amount in the samples was quantified using a Pico 100 Picodrop Microliter Spectrophotometer (Picodrop Ltd., Hinxton, UK) according to the manufacturer’s instructions. DNA quality was assessed in all samples by a Taqman PCR targeting a 155-base-pair fragment of the human 18S rRNA gene as previously described [
Results of inhibition control PCR (phocid herpesvirus PCR), DNA extraction control PCR (18S rRNA gene PCR), and specific PCR/sequencing results for
Sample I.D. | Histological diagnosis | Ct-value of phocid herpesvirus PCR | Ct-value of human 18S rRNA gene PCR | DNA concentration as measured by Picodrop |
|
Mucorales-specific seminested PCR (and consecutive sequencing) |
---|---|---|---|---|---|---|
Case 1 | Chromoblastomycosis | 22 | 23 | 12.3 ng/ |
N.a. | N.a. |
Case 2 | Mucormycosis | 18 | 15 | 14.6 ng/ |
Negative | Negative |
Case 3 | Histoplasmosis | 18 | 14 | 246.3 ng/ |
Negative | Negative |
Case 4 | Mucormycosis | 20 | — | 26.9 ng/ |
Negative | Positive (100% |
Case 5 | Histoplasmosis or Cryptococcosis | 20 | 19 | 157.6 ng/ |
Negative | Negative |
Case 6 | Chromoblastomycosis | 26 | 30 | 414.5 ng/ |
N.a. | N.a. |
Case 7 | Rhinosporidiosis | 26 | 23 | 62.7 ng/ |
N.a. | N.a. |
Case 8 | Mycetoma/maduromycosis | 22 | 33 | 618.9 ng/ |
N.a. | N.a. |
Case 9 | Rhinosporidiosis | 18 | 22 | 21.9 ng/ |
N.a. | N.a. |
Case 10 | Mycetoma/maduromycosis | 25 | 15 | 16.8 ng/ |
N.a. | N.a. |
Case 11 | Histoplasmosis | 21 | 30 | 104.6 ng/ |
Negative | Negative |
Case 12 | Histoplasmosis | 24 | 15 | 44.3 ng/ |
Negative | Positive (no interpretable sequence results due to poor sequence quality) |
Case 13 | Chromoblastomycosis | 23 | 14 | 33.8 ng/ |
N.a. | N.a. |
Case 14 | Histoplasmosis | 20 | 17 | 175.5 ng/ |
Positive (99% |
Negative |
Case 15 | Coccidioidomycosis | 24 | 21 | 16.8 ng/ |
N.a. | N.a. |
Case 16 | Coccidioidomycosis | 24 | 14 | 107.5 ng/ |
N.a. | N.a. |
Case 17 | Mycetoma/maduromycosis | 18 | 13 | 26.3 ng/ |
N.a. | N.a. |
Control 1 | Negative control | 22 | 34 | 609.7 ng/ |
N.a. | N.a. |
Control 2 | Negative control | 23 | 25 | 655.3 ng/ |
N.a. | N.a. |
Control 3 | Negative control | 19 | 31 | 676.6 ng/ |
N.a. | N.a. |
Plasmids containing phocid herpesvirus 1 (PhHV-1) sequences were added to each sample as internal controls to exclude sample inhibition. Dilution was chosen to achieve cycle threshold- (Ct-) values of about 18. Primers and probes were used as previously described [
Samples with histological suspicion of histoplasmosis or Mucorales infection were subjected to specific PCRs with consecutive sequencing for confirmatory testing. Nested PCR for histoplasmosis targeting the gene encoding the unique fungal 100-kDa-like protein [
A total of five panfungal PCRs that have been evaluated for the diagnostic identification of invasive mycoses, including in some cases from formalin-fixed, paraffin-embedded tissue [
DNA of clinical
The sequences obtained were compared with deposited sequence information using the BLAST algorithm (
Thin 4–6
Photographs of representative fungi-containing samples analyzed in this study. (a) Case 1. Chromoblastomycosis of the skin. Fungal elements can be recognized in HE-stained sections as brownish-yellow pigmented bodies (arrows). A separation of the thick-walled fungus is also visible (multiform body). (b) Case 9. Skin lesion in rhinosporidiosis. Numerous spherical structures varying in diameter in an age-dependent manner. Immature forms (trophocytes) contain nucleus (arrow) and cytoplasm. Mature forms (sporangia) contain numerous endospores (asterisk). The rupture of this form is also visible. HE staining. (c) Case 10. Mycetoma. Lobulated grain with light colored center (brown) in lung. Grocott stain. (d) Case 11.
Ethical clearance for the anonymous retrospective molecular assessment of residual materials from formalin-fixed, paraffin-embedded tissues for evaluation purposes was obtained from the Ethics Committee of the Medical Association of Hamburg, Germany (document number WF-028/13).
A total of 17 formalin-fixed, paraffin-embedded tissue specimens from patients with histologically confirmed invasive mycoses, deposited for 1 to 30 years (mean 11.5 years), were included in the analysis. Microscopic assessment allowed the detection of fungal elements on microscopic slides of all mycosis samples. The fungal density varied substantially from slide to slide owing to inhomogeneous distribution of the pathogens in tissue. Furthermore, in the case of filamentous fungi, elements of a multiply-cut filament were indistinguishable from single cuts of multiple filaments. For these reasons, semiquantification of fungi per unit area (160
The mean DNA-amount in the fungal samples was 123.6 ng/
Positive results of the DNA-quality assessment 18S rRNA gene PCR were obtained for all but one sample. However, Ct-values were distributed over a wide range from 14 to 33. Ct-values did not seem to be influenced by sample age alone, as there was also a 17-year-old sample with a Ct-value as low as 15 (Table
Inhibition control PCR was positive for all analyzed samples. However, Ct-values ranged from 18 to 26, corresponding to low to moderate sample inhibition up to about 2.5 decadic logarithmic steps. Again, there was no obvious association between sample age and sample inhibition (Tables
Five samples with histological suspicion of histoplasmosis and two samples with histological suspicion of Mucorales infections were subjected to specific PCR with consecutive sequencing. Only one out of five suspected histoplasmosis cases and one out of two suspected Mucorales infections could be confirmed by this approach (Table
The panfungal PCRs readily reacted with DNA from contaminating spores of environmental fungi, even in the negative control materials, as demonstrated by the sequencing results (Table
Comparison of histological and sequencing results. (Details on PCR results and sequencing quality can be found in supplementary materials 2–5.)
Sample I.D. | Histological diagnosis | PCR 1 | PCR 2 | PCR 3 | PCR 4 | PCR 5 |
---|---|---|---|---|---|---|
Case 1 | Chromoblastomycosis | Multiple species, no discrimination possible | Noninterpretable, poor/overlapping sequences |
|
Noninterpretable, poor/overlapping sequences | Noninterpretable, poor/overlapping sequences |
|
||||||
Case 2 | Mucormycosis |
|
|
Noninterpretable, poor/overlapping sequences | Noninterpretable, poor/overlapping sequences |
|
|
||||||
Case 3 | Histoplasmosis |
|
Noninterpretable, poor/overlapping sequences | Various |
Various |
— |
|
||||||
Case 4 | Mucormycosis |
|
Various |
— | Multiple species, no discrimination possible/ |
— |
|
||||||
Case 5 | Histoplasmosis or cryptococcosis |
|
Noninterpretable, poor/overlapping sequences | Various |
Noninterpretable, poor/overlapping sequences | Noninterpretable, poor/overlapping sequences |
|
||||||
Case 6 | Chromoblastomycosis |
|
Noninterpretable, poor/overlapping sequences | — | Noninterpretable, poor/overlapping sequences | — |
|
||||||
Case 7 | Rhinosporidiosis | Noninterpretable, poor/overlapping sequences | Noninterpretable, poor/overlapping sequences | Noninterpretable, poor/overlapping sequences |
|
— |
|
||||||
Case 8 | Mycetoma/maduromycosis |
|
— | — | Noninterpretable, poor/overlapping sequences | — |
|
||||||
Case 9 | Rhinosporidiosis | Multiple species, no discrimination possible | Noninterpretable, poor/overlapping sequences | Various |
|
Noninterpretable, poor/overlapping sequences |
|
||||||
Case 10 | Mycetoma/maduromycosis | Multiple fungal species including |
Noninterpretable, poor/overlapping sequences | Noninterpretable, poor/overlapping sequences | Various |
Noninterpretable, poor/overlapping sequences |
|
||||||
Case 11 | Histoplasmosis | Noninterpretable, poor/overlapping sequences |
|
Noninterpretable, poor/overlapping sequences |
|
— |
|
||||||
Case 12 | Histoplasmosis |
|
|
|
|
|
|
||||||
Case 13 | Chromoblastomycosis | Multiple fungal species including |
|
|
|
|
|
||||||
Case 14 | Histoplasmosis |
|
Noninterpretable, poor/overlapping sequences |
|
|
— |
|
||||||
Case 15 | Coccidioidomycosis | Multiple species, no discrimination possible |
|
Noninterpretable, poor/overlapping sequences |
|
|
|
||||||
Case 16 | Coccidioidomycosis |
|
|
Noninterpretable, poor/overlapping sequences | Noninterpretable, poor/overlapping sequences | Noninterpretable, poor/overlapping sequences |
|
||||||
Case 17 | Mycetoma/maduromycosis |
|
Noninterpretable, poor/overlapping sequences |
|
|
Noninterpretable, poor/overlapping sequences |
|
||||||
Control 1 | Negative control |
|
Noninterpretable, poor/overlapping sequences | Various |
|
— |
|
||||||
Control 2 | Negative control | Noninterpretable, poor/overlapping sequences | Noninterpretable, poor/overlapping sequences | Various |
Noninterpretable, poor/overlapping sequences | — |
|
||||||
Control 3 | Negative control | Noninterpretable, poor/overlapping sequences | Noninterpretable, poor/overlapping sequences |
|
Noninterpretable, poor/overlapping sequences | — |
Sequences of the microscopically observed pathogens, however, were not detected with the exception of three instances. Panfungal PCRs 1 and 4 identified
All sample materials with correct species identifications were younger than 10 years of age. Further, no significant PCR inhibition was detectable for the respective samples; the Ct-values were ≤20. Of note is that the missed coccidioidomycosis and chromoblastomycosis cases that were younger than 10 years all showed Ct-values ≥23 in the inhibition control PCR (Table
With the exceptions of panfungal PCRs 1 and 4, the other panfungal PCRs led to a varying number of failed reactions in the 17 microscopically positive samples assessed, that is, one failure for PCR 2, three failures for PCR 3, and as many as seven failures for PCR 5. In contrast, PCRs 1–4 showed positive results with negative control samples, while only PCR 5 was not associated with such false-positive reactions (Table
In addition to the requirement for the presence of a critical number of pathogens in the analyzed materials, the histological identification of pathological fungi in tissue sections of patients with invasive mycosis demands specific skills and a high degree of experience, as suggested by the considerable number of mismatches in comparison with cultural diagnostic approaches [
Molecular tests such as panfungal PCR with subsequent sequence analysis might help pathologists confirm the differential diagnosis, but nonspecies-specific diagnostic approaches are easily disturbed by environmental contamination, for example, located within the paraffin, unless protective measures are assured. Species-specific simplex or multiplex PCRs [
Here we assessed the performance of five panfungal PCRs with subsequent sequence analysis that were designed for the diagnosis of invasive mycosis partly from rather difficult material such as formalin-fixed, paraffin-embedded tissues and evaluated in Western industrialized settings [
Some of the assessed panfungal PCR and sequencing protocols showed promising evaluation results in previous studies [
According to our analysis, the few correct sequencing results were observed in comparatively fresh samples that were younger than 10 years of age and that did not show considerable PCR inhibition. Even moderate inhibition prevented the amplification and subsequent sequencing of DNA fragments of the microscopically observed invasive fungal pathogens. The reliability of PCR and sequencing seems to be affected by sample quality and sample storage time. While 20 years of storage time marked the cut-off for diagnostic reliability of target-specific PCR as observed in a previous study [
As in recent reports on fungal specimens [
Panfungal PCR with subsequent sequencing clearly performed less well than classical microscopy of stained sections for the identification of invasive rare and/or tropical fungal infections, at least when histology was performed by experienced pathologists. Matching sequence results were observed only in individual instances, underlining the importance of preserving histological skills in diagnostic routine. As suggested by the results of specific PCR analyses with the samples of histologically suspected histoplasmosis and mucormycosis as well as by previous studies [
We conclude that sequence results obtained after panfungal PCR can only be considered as confirmatory information in case of matching with preliminary histological results owing to the high risk of contamination of paraffin blocks with environmental fungal spores. Other histological techniques such as immunohistochemistry [
If mycological culture is possible, it is the reference method for the diagnosis of mycosis. In many mycoses, histology only allows a presumptive diagnosis that needs confirmation by mycological culture [
The authors declare that there is no conflict of interests regarding the publication of this paper.
Hagen Frickmann and Ulrike Loderstaedt equally contributed to this work.
The authors are grateful to Steffen Lohr and Annett Michel for their excellent technical assistance. The PCR/sequencing analyses were funded by Grant 12K2-S-451315 “optimizing of microbiological diagnostic preanalytics for tropical conditions” of the German Ministry of Defense (MoD). The study was supported by the Körber Foundation to Paul Racz.