Invasive fungal infections appear to have increased over the past few years, mostly in immunocompromised patient 1234. Moreover, the diagnosis of invasive filamentous fungal disease (IFFD) is often difficult in immunodeficient patients. Tissue specimens are difficult to obtain in debilitated patients, but are essential in detecting invasive fungi 5. In the absence of identification of fungal elements in tissue, the diagnosis of a "probable" invasive fungal disease (IFD) can be retained when a host factor, clinical features and mycological evidence are present 5. Additionally, the diagnosis of a "possible" IFD is made when appropriate host factors and sufficient clinical evidence are present 5. Since a high level of mortality occurs in immunocomprised patients with IFFD 678, an accurate and rapid diagnosis is required in order to give the appropriate anti-fungal treatment. In addition, depending of the isolated fungal species, some of these treatments can be inefficient 910. The combination of two or more IFFD occurring simultaneously or sequentially in immunocompromised patients makes diagnosis of these patients very challenging. Combined invasive mucormycosis and aspergillosis has been rarely described, its occurrence has probably been underestimated 111213141516. Predisposing factors and clinical features in association with these mycoses are almost the same 1718. Although mycological analyzes allow definitive identification of the different species, combined infections are diagnosed histologically.

We report a case of an associated aspergillosis and mucormycosis infection responsible for fatal necrotic and extensive pneumonia in a patient with a renal transplant. Mycological analysis identified Aspergillus fumigatus in a culture of bronchoalveolar lavage (BAL) and Rhizopus microsporus in a culture of a lung tissue specimen. Identification of a combined aspergillosis and mucormycois infection was made by PCR performed on formaldehyde pulmonary fixed tissues.

A 66-year-old man, who had received a renal transplant 5 months before, and was on tritherapy [prednisolone (1 mg/kg/day); cyclosporine A (7 mg/kg/day); azathioprine (1 mg/kg/day)], was admitted to the hospital with hemoptysia. A computed tomographic scan of the chest showed a not well-circumscribed and excavated mass mass involving the pulmonary inferior left lobe, bilateral pleural effusions, and a small parenchyma nodule in the pulmonary inferior right lobe (Fig. 1A et 1B). An analysis of the BAL was quickly performed due to the presence of Aspergillus fumigatus in sputum: cytopathological examination showed filamentous hyphae corresponding to aspergillosis (Fig. 1C). These filaments were subsequently identified by culture as A. fumigatus. In addition, pyocyanic bacteria were isolated from the BAL. Treatment associating voriconazole, ceftazidime and ciprofloxacine was started. However, the absence of a clinically improved status and repeated massive hemoptysia, led to surgical resection of the pulmonary left inferior lobe one week later. Gross macroscopy demonstrated a diffuse hemorrhagic infarct of the lung (Fig. 1D). A specimen of lung tissue was sent to the mycology laboratory. The patients died of cardio-pulmonary failure two days after surgery.

Although invasive mycoses have long been recognized for a long time as being caused by significant pathogens, particularly in immunocompromised patients, the frequency of infection with opportunistic fungi is increasing with time and the spectrum of the infectious agent of invasive mycoses is changing 1234. In this regard, advances in therapeutic technologies and in particular the development of novel immunosuppressive therapies, have prolonged the period of risk for many individuals 34. A large number of invasive mycoses have been identified because of the increase in the number of patients at risk 242526. Most of the invasive mycoses are caused by filamentous fungi, rarely by other fungi 6272829. In this regard, a number of IFFD has been reported in renal transplant patients, particularly aspergillosis and mucormycosis 30. Other IFFD have been more rarely described in immunocompromised hosts, such as scedosporiosis 31.

A confirmed diagnosis of IFFD is made on the demonstration of hyphae in the diseased tissue. Pathological identification of the hyphae is based on different well-described criteria: diameter, presence of septa, and branching angle (right or acute branching), pigmentation 3233. It is important to consider the characteristics of the infiltrate at the inflammatory region that is in contact with hyphae: presence of angioinvasion, of giant cells and of eosinophilic necrosis 34. Mycological analysis made on non fixed-tissue specimens, associating microscopic examination and culture allows identification of the species and the study of the sensitivity to anti-fungal drugs. However, the different results of these mycological analyses can be negative in the following circumstances: 1) When there is an error in sampling, the specimen sent to the mycology laboratory does not contain the different fungi, 2) When the specimen containing the hyphae is totally necrotic. In addition, when two filamentous mycoses are present in the same organ, the mycological results are even more inconstant, since only one fungus might be present on the specimen sent to the mycology laboratory. In these different situations, it is essential to compare the culture results and the morphological analysis of the tissue sections. However, histological analyses have some limits, even after histochemical staining.

The distinction between aspergillosis and scedosporiosis, between aspergillosis and fusariosis and certain mucormycosis, such as those caused by Cunninghamella from tissue sections may be difficult or impossible 313536. Other potential pitfalls are more easily eliminated 3738. Ancillary methods using specific antibodies raised against filamentous fungi can be helpful for diagnosis. However, the different antibodies are rarely commercially available 333639. Finally, in situ hybridization has been developed for fungal detection in fixed-tissue sections, but the probes used for this method are rarely available in the pathology laboratory 40

In recent years, PCR methods performed with deparaffinized tissue sections have been used to try to improve the detection and identification of pathogens in fixed specimens 3941. This methodology needs to use strict positive and negative controls and exact comparison with histological features and mycological analysis is required. However, false-negative and false-positive results due to artefacts in amplification, or sampling, and possible exogenous contamination by microorganisms, still are very important pitfalls in molecular diagnostic pathology 4243. In the present case, there was no evidence of contamination by external sources and strict laboratory precautions were applied to avoid carry over and false positive results.

The present case report underlines the usefulness of the PCR method performed with embedded paraffin fixed specimens, which allows simultaneous detection on the same tissue of two types of IFFD. Moreover, the pathogenic status of these pathogens can be confirmed by comparing histopathological and molecular biology results obtained from the same embedded paraffin tissue. In the present case, the absence of mycological diagnosis of aspergillosis from non-fixed biopsy specimen was probably due to an error in sampling. In this regard, histological analysis showed only a few areas with the two associated mycoses. Filaments of aspergillosis, but not of mucorales, were noted by cytopathological analysis of the BAL and identified as A. fumigatus in culture. This can be due to an error in sampling, but also because at that time mucorales was undetectable, since it grew subsequently in the patient treated with voriconazole. However, the clinical distinction between aspergillosis and mucormycosis is crucial since there is an increased incidence of mucormycosis in patients treated with voriconazole for suspected aspergillosis 44. In this regard, a couple of cases of mucormycosis occurring in patients with voriconazole-treated aspergillosis have been reported 944. The diagnosis of IFFD based on hyphae isolated from the BAL is uncertain. In this case, different associated clinical features and predisposing host factors must be present before giving specific treatment, which can be toxic, ineffective, and favor the development of another mycotic disease 4546. Finally, for most clinicians, the identification of aspergillosis or mucormycosis in the BAL is not sufficient to confirm the diagnosis of IFFD 47. The utility of performing transbronchial or transparietal biopsies in debilitated patients should be discussed 4849.

Our observations outline the difficulty of diagnosing associated invasive mycoses in immunocompromised patients. For diagnosis, it is crucial to compare the histological and mycological analyses. However, as demonstrated here, molecular biology performed on FFPE tissue sections, after morphological control on a mirrored stained tissue section, can be the only method for the identification of different pathogens in case of discrepancies between pathological and mycological approaches. In this regard, we strongly believe that molecular biology of FFPE tissues should be developed and available to surgical pathology laboratories for infectious diseases pathology, since, in particular in immunocompromised patients, it is crucial in cases of invasive mycotic diseases to provide a rapid and optimal treatment

The authors declare that they have no competing interests.

VH was involved in the histopathological evaluation and interpreted the histopathology. AD and CB performed the literature search, prepared the materials and supplied the relevant clinical details. BP provided the radiological details. MGT and DGH interpreted the mycology data. MB was involved in molecular biology experiments. GC interpreted the molecular biology data from paraffin sections. NV was involved in surgery. PH outlined the general concept and was involved in drafting and revising the manuscript. All authors have read and approved of the present manuscript.