European Journal of Cardiovascular Medicine

Invasive fungal infections (IFIs) represent a growing global health concern and are associated with significant morbidity and mortality, particularly among immunocompromised individuals, critically ill patients, and those with underlying comorbidities such as diabetes mellitus, malignancy, organ transplantation, and prolonged corticosteroid or immunosuppressive therapy [1–3]. Over the past two decades, the incidence of IFIs has increased substantially due to advances in medical care that prolong survival but compromise host immunity, including chemotherapy, hematopoietic stem cell transplantation, and intensive care interventions [4,5].

Despite improvements in antifungal therapy, mortality rates from IFIs remain unacceptably high, often ranging from 30% to 70%, largely attributable to delays in diagnosis and initiation of appropriate treatment [6,7]. Early and accurate diagnosis is therefore crucial, as prompt antifungal therapy significantly improves patient outcomes. However, diagnosis of IFIs remains challenging due to nonspecific clinical manifestations, overlapping radiological features with bacterial or non-infectious conditions, and limitations of available diagnostic modalities [8].

Histopathological examination of tissue specimens has long been considered the cornerstone for the diagnosis of invasive fungal infections. Demonstration of fungal elements invading tissue and vasculature establishes a diagnosis of “proven” IFI according to international consensus definitions [9]. Routine hematoxylin and eosin staining, supplemented by special stains such as Gomori methenamine silver (GMS) and periodic acid–Schiff (PAS), allows visualization of fungal morphology and assessment of tissue invasion, host response, and angioinvasion [10]. Importantly, histopathology remains valuable even in patients who have already received antifungal therapy, as nonviable fungal elements may still be detected within tissues [11].

However, histopathology has inherent limitations. While it provides rapid evidence of fungal invasion, it often lacks the ability to reliably identify fungi to the species or even genus level, particularly in morphologically overlapping groups such as Aspergillus, Fusarium, and Scedosporium species [12]. Furthermore, interpretation may be influenced by sampling error, tissue necrosis, or suboptimal staining, and it does not provide information on antifungal susceptibility patterns.

Microbiological methods, particularly fungal culture, remain essential for etiological diagnosis of IFIs. Culture allows definitive species identification and enables antifungal susceptibility testing, which is increasingly important in the context of emerging antifungal resistance [13]. Nevertheless, culture is limited by low sensitivity, prolonged incubation times, and frequent false-negative results due to prior antifungal exposure, inadequate specimen volume, or fastidious growth requirements of certain fungi such as Mucorales [14,15]. As a result, culture negativity does not exclude invasive fungal disease, especially when histopathological evidence of invasion is present.

In recent years, molecular diagnostic techniques, including polymerase chain reaction (PCR)-based assays and sequencing methods, have emerged as valuable adjuncts in the diagnosis of IFIs. These techniques have demonstrated improved sensitivity and faster turnaround times compared to conventional culture and can be applied to fresh tissue, blood samples, and even formalin-fixed

Study Design This study was designed as a systematic review and meta-analysis to compare the diagnostic yield of histopathology versus microbiological methods in the diagnosis of invasive fungal infections (IFIs). The review methodology was developed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [20]. Search Strategy A comprehensive literature search was conducted across multiple electronic databases, including PubMed/Medline, Embase, Scopus, and PubMed Central (PMC). The search covered studies published from database inception until January 2025. The search strategy employed a combination of Medical Subject Headings (MeSH) terms and free-text keywords, including: “invasive fungal infection”, “histopathology”, “histology”, “fungal culture”, “microbiology”, “tissue biopsy”, “PCR”, “molecular diagnosis”, “Gomori methenamine silver”, and “PAS stain”. Boolean operators (“AND”, “OR”) were used to refine the search. Reference lists of relevant original articles and review papers were manually screened to identify additional eligible studies. Eligibility Criteria Inclusion Criteria Studies were included if they met the following criteria: 1. Original research articles (prospective or retrospective). 2. Studies involving human subjects with suspected or proven invasive fungal infections. 3. Studies reporting paired comparison of histopathology and microbiological methods (culture and/or molecular diagnostics) performed on the same tissue or sterile site specimens. 4. Studies providing data on diagnostic yield, concordance, or diagnostic accuracy of the methods. Exclusion Criteria Studies were excluded if they: 1. Were case reports or small case series (≤5 cases). 2. Included only non-invasive samples (e.g., sputum, BAL, or blood without tissue correlation). 3. Lacked comparative or paired diagnostic data. 4. Were reviews, editorials, conference abstracts, or animal studies. 5. Did not provide sufficient methodological or outcome data. Study Selection Two independent reviewers screened titles and abstracts for relevance. Full-text articles were subsequently assessed for eligibility. Discrepancies between reviewers were resolved through discussion and consensus. The study selection process was documented using a PRISMA flow diagram. Data Extraction Data were independently extracted by two reviewers using a standardized data extraction form. The following variables were recorded: • Author(s) and year of publication • Country and study setting • Study design and sample size • Patient population and underlying risk factors • Type of specimen (lung, sinus, brain, skin, bone, etc.) • Histopathological methods and stains used (H&E, GMS, PAS) • Microbiological methods (direct microscopy, culture, PCR, sequencing) • Diagnostic yield of each modality • Concordance or discordance between histopathology and microbiology

Study Selection

The initial database search identified 1,246 records. After removal of duplicates (n = 318), 928 titles and abstracts were screened. Of these, 850 articles were excluded due to irrelevance to the study objective. Seventy-eight full-text articles were assessed for eligibility, of which 36 were excluded for reasons including lack of paired diagnostic data, case reports or small case series, and absence of tissue-based diagnosis.

Finally, 42 studies fulfilled the inclusion criteria and were included in the qualitative synthesis and quantitative tabulation.

Characteristics of Included Studies

The 42 included studies were published between 2000 and 2025 and comprised both retrospective and prospective observational designs. The majority were single-center studies, with a smaller number of multicenter analyses. Study populations primarily included immunocompromised patients, individuals with diabetes mellitus, malignancy, transplant recipients, and critically ill patients.

Specimens evaluated included lung tissue, paranasal sinus tissue, brain tissue, skin and subcutaneous tissue, bone, and other deep organ biopsies. Histopathological evaluation most commonly employed hematoxylin and eosin (H&E) staining supplemented by special stains such as Gomori methenamine silver (GMS) and periodic acid–Schiff (PAS). Microbiological methods included direct microscopy, fungal culture, and, in more recent studies, molecular assays such as polymerase chain reaction (PCR) and sequencing.

Diagnostic Yield of Histopathology

Histopathology demonstrated a consistently high diagnostic yield across studies, particularly in demonstrating fungal invasion of tissue, which is essential for establishing a diagnosis of proven invasive fungal infection. In several studies, histopathology identified fungal elements even in cases where microbiological culture was negative. This was especially evident in patients who had received prior antifungal therapy and in infections caused by Mucorales and other fastidious fungi.

Histopathological examination also provided valuable information regarding host tissue response, angioinvasion, necrosis, and extent of disease, which were not obtainable through microbiological methods alone.

Diagnostic Yield of Microbiological Methods

Fungal culture demonstrated variable sensitivity across studies, with reported positivity rates generally lower than histopathology. Despite this limitation, culture remained indispensable for species identification and antifungal susceptibility testing. Several studies reported culture negativity despite clear histopathological evidence of fungal invasion.

The incorporation of molecular diagnostic methods significantly improved microbiological yield. PCR-based assays demonstrated higher sensitivity than culture alone and showed improved concordance with histopathological findings, particularly in culture-negative specimens.

Concordance Between Histopathology and Microbiology

Concordance between histopathology and microbiology varied widely across studies. Some studies reported high agreement when molecular diagnostics were included, while others demonstrated low concordance between histopathology and culture alone. Discordant results were attributed to sampling variability, nonviable organisms, prior antifungal exposure, and limitations inherent to each diagnostic modality.

Summary of Diagnostic Performance

Overall, histopathology showed superior performance in confirming invasive disease, while microbiological methods, particularly when augmented by molecular techniques, enhanced etiological diagnosis. The findings underscore the complementary nature of these diagnostic approaches rather than superiority of one over the other.

Table 1. PRISMA Flow Summary of Study Selection

Table 2. General Characteristics of Included Studies (n = 42)

Table 3. Comparative Diagnostic Yield of Histopathology and Microbiology

Table 4. Common Causes of Discordance Between Diagnostic Modalities

Histopathology demonstrated superior ability to establish invasive disease, while microbiological methods, particularly molecular assays, improved etiological identification. A combined diagnostic approach provided the highest diagnostic yield.

Figure 1. Comparative diagnostic yield of histopathology, fungal culture, and molecular methods in invasive fungal infections. The figure illustrates the relative diagnostic yield of histopathology, fungal culture, and molecular methods based on pooled trends observed across included studies. Values represent schematic percentages derived from narrative synthesis and are intended to demonstrate comparative performance rather than exact pooled sensitivity.

Figure 2. PRISMA flow diagram of study selection. PRISMA flow diagram illustrating the process of literature search, screening, eligibility assessment, and inclusion of studies in the systematic review and meta-analysis.

The present systematic review and meta-analysis evaluated the diagnostic yield of histopathology compared with microbiological methods in invasive fungal infections and demonstrated that histopathology remains a cornerstone in establishing proven invasive disease, while microbiological and molecular techniques play a critical complementary role in etiological confirmation and therapeutic guidance. These findings are consistent with and extend observations reported in earlier studies and reviews addressing the diagnostic challenges of IFIs [1–4].

Several previous investigations have emphasized that histopathological demonstration of fungal invasion is central to the diagnosis of invasive fungal infections, particularly in the context of international consensus definitions, where tissue invasion or recovery of fungi from sterile sites is mandatory for a “proven” diagnosis [5,6]. Our findings corroborate these reports, as histopathology consistently demonstrated higher detection rates of invasive disease compared with culture alone, especially in patients who had received prior antifungal therapy or in infections caused by fastidious organisms such as Mucorales [7–9]. Similar observations have been reported in multiple organ-specific studies, including sino-orbital, pulmonary, and cerebral fungal infections, where histopathology identified fungal elements despite negative cultures [10–12].

In contrast, fungal culture, while indispensable for species identification and antifungal susceptibility testing, showed variable and often lower sensitivity across studies included in this review. This variability has been widely reported in earlier literature and attributed to multiple factors, including low fungal burden, nonviable organisms, delayed specimen processing, and the inhibitory effects of antifungal agents administered before tissue sampling [13–15]. Our findings align with prior studies demonstrating that culture negativity does not exclude invasive fungal disease when histopathological evidence of invasion is present [16,17]. This limitation of culture has significant clinical implications, as reliance on microbiology alone may delay diagnosis and treatment initiation.

The increasing incorporation of molecular diagnostic methods into IFI workflows represents a significant advancement in this field. Several studies included in this review demonstrated that polymerase chain reaction–based assays and sequencing techniques substantially improved detection rates, particularly in culture-negative but histopathology-positive cases [18–20]. These findings are in agreement with earlier systematic reviews and multicenter studies reporting higher sensitivity of molecular methods compared with culture and improved concordance with histopathological findings [21–23]. Molecular assays have also been shown to be valuable when applied to formalin-fixed paraffin-embedded tissues, further enhancing their utility in routine diagnostic practice [24].

The variability in concordance between histopathology and microbiology observed across studies mirrors the heterogeneity reported in the literature. Some studies reported high agreement when molecular methods were included, whereas others demonstrated substantial discordance between histopathology and culture alone [25–27]. Such discordance has been attributed to sampling error, uneven distribution of fungal elements within tissues, morphological overlap between different fungal species on histology, and technical limitations inherent to both modalities [28–30]. These findings reinforce the notion that histopathology and microbiology should not be viewed as competing diagnostic approaches but rather as complementary tools that provide different yet essential information.

Indian studies, have highlighted the continued importance of histopathology in resource-limited settings, where advanced molecular diagnostics may not be universally available [31]. These studies emphasize that histopathology often serves as the primary modality for early diagnosis and therapeutic decision-making, while microbiological confirmation, when available, enhances diagnostic confidence and enables targeted antifungal therapy. Our review supports this perspective and underscores the relevance of integrated diagnostic strategies across diverse healthcare settings.

The strengths of this systematic review include a comprehensive literature search across multiple databases, strict inclusion of studies with paired histopathology and microbiology data, and detailed narrative synthesis of diagnostic performance across varied clinical contexts. By focusing on tissue-based diagnosis, this review addresses a clinically critical aspect of IFI diagnosis that directly influences classification, treatment decisions, and outcome assessment.

However, several limitations must be acknowledged. Significant heterogeneity in study design, patient populations, specimen types, diagnostic techniques, and reference standards limited the feasibility of uniform quantitative pooling of diagnostic accuracy metrics. Many studies did not report complete contingency data, precluding robust pooled sensitivity and specificity estimates. Additionally, variations in laboratory protocols, staining techniques, culture methods, and molecular assays may have influenced reported diagnostic yields. Publication bias toward studies demonstrating positive findings cannot be excluded, and temporal changes in diagnostic practices over the long study period may further contribute to heterogeneity.

Despite these limitations, the overall evidence consistently supports the conclusion that histopathology is indispensable for confirming invasive fungal disease, while microbiological and molecular methods enhance etiological diagnosis and antifungal stewardship. Future prospective studies with standardized diagnostic algorithms, uniform reporting of accuracy metrics, and integration of molecular diagnostics are required to refine diagnostic pathways and improve outcomes in patients with invasive fungal infections.

This systematic review and meta-analysis demonstrates that histopathology and microbiological methods serve complementary and indispensable roles in the diagnosis of invasive fungal infections. Histopathological examination remains the most reliable modality for establishing invasive disease through direct demonstration of fungal elements and tissue invasion, thereby fulfilling criteria for proven invasive fungal infection. In contrast, microbiological techniques, including fungal culture and molecular diagnostics, provide essential etiological identification and antifungal susceptibility information that are critical for targeted therapy and optimal patient management. The findings highlight the inherent limitations of relying on a single diagnostic approach, as culture negativity does not exclude invasive fungal infection and histopathology alone may not reliably identify the causative organism. Integration of molecular diagnostic methods has significantly enhanced diagnostic yield, particularly in culture-negative cases, and has improved concordance with histopathological findings. An integrated, multimodal diagnostic strategy combining histopathology, microbiological culture, and molecular techniques offers the highest diagnostic yield and should be considered the standard of care in suspected invasive fungal infections. Future research should focus on standardized diagnostic algorithms, prospective multicenter studies, and broader implementation of validated molecular assays to further improve diagnostic accuracy and patient outcomes.

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