A definitive IA diagnosis is usually based on the isolation and identification of an Aspergillus species and/or microscopic evidence of fungi in histopathological specimens [5]. However, the low sensitivity of fungus culture from clinical specimens (10% to 30% [6]) and the need for (invasive) biopsies in critically ill patients limit the occasions when definitive diagnoses are possible. Recently, the criteria of the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group (EORTC) have been widely used to diagnose IA, although these criteria are not recommended for use in daily clinical practice [7]. Caution is needed when diagnosing probable IA on the basis of positive galactomannan (GM) test results; the assay lacks both sensitivity and specificity. Numerous instances of false-positive and false-negative GM test results have been reported in patients with suspected IA [8-10]. The following case is a typical example. A 60-year female received an ABO-incompatible kidney transplant. Acute rejection developed soon after transplantation, resulting in graft failure. Four months after transplantation, the patient presented with fever, and a new lung nodule was detected on chest X-ray. Chest computed tomography (CT) revealed a mass-like consolidation (Fig. 1A). The bronchoalveolar lavage (BAL) fluid was positive on the GM assay, although the serum was negative. No other pathogen was isolated and the final mycobacterial culture of BAL fluid (performed 8 weeks later) was negative. She was clinically diagnosed with probable IPA (using the EORTC criteria) and received voriconazole therapy. However, the chest mass-like consolidation increased despite 16 weeks of therapy, and many supraclavicular and mediastinal lymph nodes became enlarged (Fig. 1B). The supraclavicular lymph node and a lung were both biopsied. The specimens exhibited chronic granulomatous inflammation with caseous necrosis. Voriconazole therapy was discontinued and empirical anti-tuberculosis (TB) therapy commenced. Six weeks later, a member of the Mycobacterium tuberculosis complex was cultured from sputum and aspirated lung tissue, and the masslike consolidation in the right lung became reduced in size after 8 weeks of anti-tuberculous therapy (Fig. 1C and 1D). Thus, in this case, TB had been misdiagnosed as IPA because TB may occasionally present with atypical clinical manifestations in immunocompromised patients. Therefore, TB mimicking IPA should be considered in TB-endemic areas (such as South Korea). We recently found that about one-thirds of transplant recipients with TB underwent initial antifungal therapy and 20% to 30% showed positive GM test results [11]. Thus, misdiagnosis should be considered in voriconazole-unresponsive patients in whom IA is indicated based on non-culture-based assays, such as the GM test. It is likely that some patients diagnosed with probable IPA based on positive GM test data have in fact been misdiagnosed and treated with voriconazole in the past, with adverse outcomes. It is worth emphasizing that isolation of an Aspergillus species from even a non-sterile specimen yields important information in terms of IA status. The EORTC criteria state that a positive non-sterile culture result, combined with a positive GM assay outcome, afford the same level of evidence as a single positive mycological factor. Notably, we found that all five probable IPA cases with positive non-sterile culture results were also positive in the aspergillosis immunohistochemistry (IHC) assay only. Of 12 probable IPA cases for whom the GM assays were positive, only five (42%) were positive on the aspergillosis IHC test [6]. We thus infer that non-sterile culture results are more accurate than non-culture-based tests, such as the GM assay. Hence, we propose that the EORTC criteria should be stratified further into probable IPA based on GM test results and highly probable IPA based on non-sterile culture results.

Histomorphological diagnoses of invasive mold infections are usually definitive. However, some limitations should be borne in mind. The following is an extreme example. A 62-year female received a kidney transplant. On postoperative day 8, she developed severe abdominal pain and a sigmoid perforation with panperitonitis, and underwent total colectomy. The pathology of the resected colon (Fig. 2A) was morphologically compatible with mucormycosis (Fig. 2B). She therefore received conventional, amphotericin-B antifungal therapy. Despite 10 days of such therapy, she deteriorated clinically and a second-look operation was performed. Total gastrectomy, splenectomy, graftectomy, and ileal repair were required because the uncontrolled infection had extended to the liver, spleen, stomach, and small bowel. The fungal hyphae seen in the stomach, liver, and spleen branched at an acute angle and were septated, suggestive of aspergillosis rather than mucormycosis (Fig. 2C). The initial pathology was reviewed and the final pathological report was revised from a morphological diagnosis compatible with mucormycosis to a diagnosis compatible with aspergillosis. The patient’s clinical condition deteriorated and she died 6 days after the second-look operation. The aspergillosis IHC test was negative but the mucormycosis IHC test was positive (Fig. 2D). Thus, the morphology of an infecting fungus may be confusing, indicating that morphological diagnosis has certain limitations.

We recently found that the extent of overall agreement in terms of histomorphological diagnoses made by two experienced pathologists was 87% [6]. Hence, disagreements were evident in 13% of cases. In addition, we earlier showed that 25% of pulmonary aspergillosis cases diagnosed based on both fungal morphology and other mycological evidence of aspergillosis were in fact incorrectly diagnosed (pulmonary mucormycosis had been interpreted as pulmonary aspergillosis) [6]. Therefore, the morphology of the infecting fungus alone is of limited utility in the diagnosis of aspergillosis.

Coinfection should be considered in cases of voriconazole-refractory IPA. An earlier study found that respiratory cultures of 13 of 17 patients (76%) with definite or probable pulmonary mucormycosis were also positive for another pathogen, and cultures of six of 13 histologically-proven cases (46%) of pulmonary mucormycosis yielded other molds, including three Aspergillus species and two Fusarium species [12]. We also earlier showed that positive GM test results were not uncommon in patients with proven or probable mucormycosis [6].

We do not know whether other mold infections revealed by a positive GM test result, or culture of another mold, in patients with pulmonary mucormycosis are attributable simply to opportunistic colonization of damaged bronchi or true tissue-invasive coinfection. In addition, we do not know if frequent coinfection with another fungus in mucormycosis patients is exclusive to such patients, and thus absent in patients with aspergillosis; few studies concerning this have appeared. However, it is necessary to be cautious when interpreting non-sterile culture results and non-culture-based assays in patients with voriconazole-refractory IA; coinfection with another mold is a possible differential diagnosis. Combined antifungal therapy (voriconazole plus anidulafungin [13] or liposomal amphotericin plus caspofungin [14,15]), sequential antifungal therapy (voriconazole followed by liposomal amphotericin [16]), and prescription of broader-spectrum azole agents such as isavuconazole [17] and posaconazole [18] may all be valuable in such uncertain situations.