Fungal infections have emerged as a major global health concern, particularly in recent decades, owing to the increasing number of immunocompromised individuals. Advances in medical care, such as organ transplantation, cytotoxic chemotherapy, prolonged corticosteroid therapy, and use of immunomodulatory agents, have significantly improved patient survival but have simultaneously increased susceptibility to opportunistic fungal infections (1,2). Invasive fungal infections (IFIs) caused by Aspergillus, Candida, Mucorales, and Cryptococcus species account for substantial morbidity and mortality worldwide, with mortality rates often exceeding 50% in critically ill patients (3,4). Early and accurate diagnosis is therefore crucial, as delays in initiation of antifungal therapy are strongly associated with poor outcomes (5).

Diagnosis of fungal infections is challenging because clinical features are often nonspecific and may mimic bacterial or viral diseases. Radiological findings, although useful, are rarely diagnostic. Conventional microbiological culture remains the reference method for confirming fungal infection and identifying the species involved, but it is limited by low sensitivity, long turnaround times, and frequent false negatives (6). Culture may fail to detect fungi when the sample contains non-viable organisms, when antifungal therapy has already been started, or when fastidious fungi are involved (7,8). These limitations often necessitate reliance on other laboratory methods such as histopathology or molecular testing to achieve a definitive diagnosis (9).

Histopathological examination continues to play a vital role in the rapid diagnosis of fungal infections. It allows direct visualization of fungal elements within tissues and provides crucial information on tissue reaction, necrosis, and vascular invasion, helping to distinguish colonization from true invasive disease (10). The Hematoxylin and Eosin (H&E) stain, although routinely used in all biopsy specimens, has limited utility in fungal diagnosis because fungal walls may appear faint or indistinct, especially in necrotic or hemorrhagic areas (11). Special stains such as Periodic Acid–Schiff (PAS) and Gomori Methenamine Silver (GMS) significantly enhance detection. PAS highlights the polysaccharide-rich fungal cell wall in bright magenta, improving visibility of yeast and hyphal elements even in scant infections, while GMS provides excellent contrast by staining fungal elements black against a pale background, making it particularly useful in detecting filamentous fungi such as Aspergillus and Mucorales (12,13). Despite these advantages, histopathology cannot reliably determine fungal species, and morphologic similarities between different genera or the presence of artifacts may lead to diagnostic errors (14).

Microbiological culture, though specific, is often insensitive and time-consuming. It remains indispensable for confirming the viability of fungi and for performing antifungal susceptibility testing, but its diagnostic yield is suboptimal in many clinical settings (15,16). The advent of molecular methods such as polymerase chain reaction (PCR) has substantially improved the diagnostic landscape of mycoses. PCR enables detection of fungal DNA directly from tissue, blood, or body fluids, providing high sensitivity and specificity and allowing species-level identification even in culture-negative cases (17). However, PCR-based assays face practical challenges such as lack of standardization, risk of contamination, and high cost, which limit their routine use in many laboratories (18,19).

Given the complementary advantages and limitations of histopathological and microbiological techniques, several studies have attempted to compare their diagnostic performance in fungal infections. Reported sensitivities and specificities vary considerably across studies, depending on the type of infection, sample quality, and diagnostic criteria used (20,21). Histopathology provides rapid preliminary results and identifies tissue invasion, while PCR and culture offer confirmatory and species-level identification. The combined application of these modalities may therefore represent the most effective diagnostic approach (22).

In view of these considerations, this systematic review aims to comprehensively evaluate and compare the diagnostic accuracy of histopathological staining techniques-H&E, PAS, and GMS-with microbiological methods including culture and PCR in the diagnosis of fungal infections. By synthesizing evidence from studies conducted over the past two decades, this review seeks to determine which diagnostic approach, or combination thereof, offers the best balance of sensitivity, specificity, and practicality in clinical practice. The findings are expected to guide the development of integrated diagnostic algorithms for early, accurate detection of fungal infections, ultimately improving patient outcomes (23,24).

METHODS

This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to ensure methodological transparency and reproducibility (25). A comprehensive search strategy was designed to identify studies that compared the diagnostic accuracy of histopathological techniques-Hematoxylin and Eosin (H&E), Periodic Acid–Schiff (PAS), and Gomori Methenamine Silver (GMS) staining-with microbiological methods such as culture and polymerase chain reaction (PCR) for the diagnosis of fungal infections in human clinical specimens.

Search Strategy and Data Sources

An extensive electronic search was carried out in PubMed, Scopus, Embase, and Google Scholar databases for articles published between January 2000 and August 2025. The following search terms and their combinations were used: “fungal infection,” “histopathology,” “PAS,” “GMS,” “H&E,” “culture,” “PCR,” “diagnostic accuracy,” “comparison,” and “sensitivity and specificity.” Boolean operators “AND” and “OR” were applied to combine terms appropriately (26). To ensure comprehensiveness, the reference lists of all selected studies and relevant review articles were manually screened to identify additional eligible studies not retrieved through database searches.

Eligibility Criteria

Studies were included if they met the following criteria: (i) involved human subjects with suspected or confirmed fungal infection; (ii) compared at least one histopathological staining method (H&E, PAS, or GMS) with a microbiological reference standard (culture and/or PCR); (iii) reported data on diagnostic accuracy measures such as sensitivity, specificity, positive predictive value (PPV), or negative predictive value (NPV); and (iv) were published in English. Exclusion criteria comprised experimental animal studies, in vitro analyses without clinical correlation, review articles, conference abstracts, and case reports with fewer than ten cases (27,28). Studies lacking adequate statistical data to calculate diagnostic accuracy parameters were also excluded.

Study Selection and Data Extraction

All retrieved citations were imported into EndNote software, and duplicates were removed. Two independent reviewers screened the titles and abstracts of the articles to determine eligibility. Full-text evaluation was then performed for studies that met inclusion criteria. Disagreements were resolved through discussion or consultation with a third reviewer to minimize selection bias (29).

Data were extracted using a standardized proforma, which included information on study design, country of origin, year of publication, type and number of specimens, fungal species involved, diagnostic methods evaluated, and reported measures of diagnostic performance. Where data were incomplete, corresponding authors were contacted by email for clarification.

Quality Assessment

The methodological quality and risk of bias of the included studies were assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool (30). This tool evaluates four key domains-patient selection, index test, reference standard, and flow/timing-assigning each as having low, high, or unclear risk of bias. Applicability concerns were also assessed across the same domains. Any discrepancies in scoring were resolved through consensus between reviewers.

Data Synthesis and Statistical Analysis

Diagnostic performance metrics (sensitivity, specificity, PPV, NPV) for each histopathological method were extracted or calculated using standard formulas. When not explicitly stated, true positive (TP), false negative (FN), false positive (FP), and true negative (TN) values were derived from reported data. Pooled estimates of sensitivity and specificity, along with 95% confidence intervals (CIs), were computed using a random-effects model (DerSimonian–Laird method) to account for inter-study variability (31). Heterogeneity among studies was quantified using the I² statistic, where values of 25%, 50%, and 75% represented low, moderate, and high heterogeneity, respectively (32).

Subgroup analyses were performed to compare diagnostic accuracy between different histopathological stains (H&E, PAS, GMS) and reference standards (culture versus PCR). Publication bias was assessed visually using Deeks’ funnel plot asymmetry test and quantitatively through Egger’s regression analysis, with a p-value <0.05 indicating significant bias (33,34).

Outcome Measures

The primary outcome of interest was the pooled diagnostic accuracy (sensitivity and specificity) of histopathological stains relative to microbiological reference methods. Secondary outcomes included evaluation of combined testing approaches, such as histopathology plus PCR, and assessment of the diagnostic yield in specific fungal infections like aspergillosis, mucormycosis, and candidiasis (35).

Ethical Considerations

As this study involved a review of previously published data, institutional ethics approval and informed patient consent were not required. However, all included studies were verified to have obtained appropriate ethical clearance from their respective institutions, as indicated in their publications (36).

RESULTS

The initial search identified 1,278 studies, of which 932 remained after removing duplicates. Screening by title and abstract excluded irrelevant or non-comparative studies, leaving 114 articles for full-text assessment. After applying inclusion and exclusion criteria, 26 studies were included in the final analysis (37).

Figure 1. PRISMA Flow Diagram

The PRISMA-based study selection process is summarized in Table 1.

Table 1. PRISMA Summary of Study Selection Process

The included studies, conducted between 2000 and 2025, collectively analyzed 3,428 clinical samples. These included biopsy tissues (62%), bronchoalveolar lavage fluids (18%), cerebrospinal fluids (8%), and other sterile body fluids (12%). The predominant fungal pathogens were Aspergillus spp. (42%), Candida spp. (31%), Mucorales (15%), Cryptococcus spp. (8%), and other rare fungi including Fusarium and Histoplasma (4%) (38).

Among the included studies, 18 compared histopathological staining with fungal culture as the reference standard, while 14 compared histopathology with PCR; six studies evaluated all four diagnostic modalities. Table 2 summarizes the diagnostic performance of H&E, PAS, and GMS stains across included studies.

Table 2. Diagnostic Accuracy of Histopathological Stains Compared with Culture/PCR

When culture was considered the reference standard, H&E exhibited the lowest pooled sensitivity (68%) and specificity (82%), mainly due to difficulties in identifying faint or fragmented fungal elements in necrotic tissue (39). PAS showed moderate to high accuracy, particularly for Candida and Histoplasma, with sensitivity and specificity of 82% and 88%, respectively (40). GMS staining outperformed both, achieving pooled sensitivity and specificity of 89% and 91%, respectively (41).

The diagnostic performance of culture and molecular methods is summarized in Table 3.

Table 3. Diagnostic Accuracy of Culture and PCR in Fungal Detection

Culture remained highly specific (97%) but poorly sensitive (58%), reflecting frequent false negatives in cases of antifungal pretreatment or necrotic samples (42). In contrast, PCR demonstrated excellent diagnostic performance with pooled sensitivity of 93% and specificity of 95% (43). PCR targeting fungal-specific gene regions (ITS, 18S rRNA, β-tubulin) showed the highest yield, particularly for Aspergillus and Mucorales infections (44).

Subgroup analysis of diagnostic performance across major fungal genera is presented in Table 4.

Table 4. Subgroup Analysis of Diagnostic Accuracy by Fungal Type

The analysis revealed that the diagnostic yield of histopathological stains varies depending on fungal morphology and tissue characteristics. PAS staining was particularly effective for yeast-like fungi, while GMS provided superior detection of filamentous fungi such as Aspergillus and Mucorales. H&E, though widely available, lacked adequate contrast and failed to detect fungal elements in up to one-third of confirmed cases (45,46).

Heterogeneity assessment using the I² statistic demonstrated moderate heterogeneity (I² = 52% for sensitivity, 47% for specificity), indicating acceptable variability among studies (47). Funnel plot symmetry and Egger’s regression test (p = 0.21) showed no significant publication bias, confirming the robustness of the pooled estimates (48).

When histopathology was combined with PCR as an integrated diagnostic strategy, pooled sensitivity improved to 96% and specificity to 94% (49). This combined approach reduced diagnostic delay and provided species-level identification in culture-negative invasive fungal infections, particularly in immunocompromised patients. A summary comparison of standalone and combined diagnostic approaches is shown in Table 5.

Table 5. Comparison of Standalone versus Combined Diagnostic Approaches

Overall, the findings from this systematic review demonstrate that while histopathological stains remain indispensable for rapid and cost-effective detection of fungi, their diagnostic accuracy is enhanced significantly when combined with molecular techniques. PCR provides confirmation and species-level identification, while PAS and GMS staining offer morphological evidence of invasion. Together, they form the most efficient and clinically relevant diagnostic strategy for timely management of fungal infections (50).

DISCUSSION

The findings of this systematic review highlight the complementary value of histopathological and microbiological methods in the diagnosis of fungal infections. Among the 26 studies analyzed, histopathological staining with Periodic Acid–Schiff (PAS) and Gomori Methenamine Silver (GMS) proved to be highly sensitive and specific, whereas routine Hematoxylin and Eosin (H&E) staining demonstrated lower diagnostic yield. These results reaffirm the continued importance of histopathology as a rapid and widely available tool for the detection of fungal elements in clinical specimens (51).

The pooled sensitivity and specificity of GMS (89% and 91%, respectively) observed in this review are consistent with previously published data, where GMS was reported to have sensitivity ranging from 85–95% in invasive fungal infections (52). The ability of GMS to clearly delineate fungal hyphae against a contrasting background enables accurate visualization even in necrotic or hemorrhagic tissue. PAS, though slightly less sensitive, remains a valuable adjunct, particularly for identifying yeast forms such as Candida and Histoplasma species due to its ability to stain fungal cell wall polysaccharides in bright magenta hues (53). In contrast, H&E—while indispensable for assessing overall tissue architecture—often fails to highlight hyphae and spores distinctly, leading to under-detection in up to one-third of cases, as also reported by Guarner and Brandt (54).

Despite their diagnostic utility, histopathological stains have inherent limitations. Morphological overlap between different fungal genera (e.g., Aspergillus versus Fusarium) and the inability to provide species-level identification restrict their role in guiding specific antifungal therapy (55). Moreover, artifacts such as necrotic debris or cotton fibers may mimic fungal elements, resulting in false-positive interpretations. Nevertheless, histopathology provides the unique advantage of confirming tissue invasion, which is critical for differentiating colonization from infection—a distinction that microbiological tests alone cannot establish (56).

In contrast, microbiological culture remains the traditional gold standard for confirming fungal infections, primarily because it provides species identification and antifungal susceptibility data. However, the pooled sensitivity of culture in this review was only 58%, despite its excellent specificity (97%). Several studies attribute this poor sensitivity to factors such as antifungal pretreatment, inadequate specimen size, or non-viable organisms (57). In invasive infections such as mucormycosis, the yield of culture can be as low as 30–50%, even when histopathology clearly demonstrates fungal elements (58). The slow growth rate of certain fungi further limits the timeliness of culture-based diagnosis, often delaying targeted therapy.

Molecular techniques, particularly polymerase chain reaction (PCR), have revolutionized fungal diagnostics. The pooled sensitivity (93%) and specificity (95%) of PCR obtained in this review corroborate previous meta-analyses showing PCR’s superior diagnostic accuracy, especially for invasive aspergillosis and mucormycosis (59,60). PCR enables the detection of fungal DNA directly from tissue or body fluids and remains effective even when culture results are negative due to non-viable fungi. Furthermore, PCR assays targeting conserved gene regions such as 18S rRNA, ITS, and β-tubulin allow species-level identification and have proven valuable for differentiating morphologically similar organisms (61). However, despite its high diagnostic performance, PCR’s routine clinical use remains limited by cost, technical complexity, and lack of standardization across laboratories (62).

A key finding of this review is that a combined diagnostic approach using histopathology (PAS or GMS) alongside PCR achieves the highest diagnostic yield, with pooled sensitivity and specificity of 96% and 94%, respectively. This finding is in agreement with studies by Springer et al. and Jenks et al., who demonstrated that integrating molecular and histological methods significantly increases diagnostic accuracy, particularly in culture-negative invasive fungal infections (63,64). Histopathology provides immediate evidence of infection and tissue invasion, while PCR confirms the pathogen at the species level, ensuring both rapid diagnosis and targeted treatment. Such combined strategies are particularly valuable in cases of disseminated fungal infections or deep-seated lesions where culture yield is low (65).

Subgroup analyses further revealed diagnostic nuances among fungal species. For Aspergillus infections, GMS and PCR showed excellent concordance, consistent with the characteristic narrow, septate, dichotomously branching hyphae visualized on GMS (66). In Candida infections, PAS provided better sensitivity due to its ability to stain budding yeast and pseudohyphal forms. For Mucorales, GMS proved indispensable, as the broad, aseptate hyphae often lack distinct cell wall staining on H&E or PAS (67). In Cryptococcus infections, the mucopolysaccharide capsule occasionally hindered PAS and GMS staining, but PCR compensated by providing rapid, species-specific detection (68). These findings suggest that the optimal diagnostic strategy may vary depending on the suspected fungal pathogen.

The presence of moderate heterogeneity (I² = 52% for sensitivity and 47% for specificity) among included studies reflects differences in specimen types, reference standards, and assay protocols. However, the lack of significant publication bias indicates that results are methodologically reliable and generalizable across settings (69). Importantly, most included studies emphasized that diagnostic turnaround time is a critical determinant of patient survival. Histopathology typically provides results within 24 hours, while PCR can deliver species confirmation within a similar timeframe—offering a marked advantage over culture, which requires several days (70).

The clinical implications of these findings are significant. In immunocompromised or critically ill patients, where delay in antifungal therapy may prove fatal, early tissue-based diagnosis through histopathology can guide empiric antifungal initiation. Simultaneous use of PCR can refine treatment by identifying the exact pathogen, allowing targeted antifungal selection and dose optimization (71). The combined diagnostic workflow thus ensures both speed and accuracy, addressing the limitations inherent to individual methods.

Despite these advantages, standardization of molecular assays and uniform interpretation criteria for histopathological diagnosis remain areas for improvement. Future research should focus on developing consensus protocols for multiplex PCR panels, exploring next-generation sequencing for direct fungal identification, and enhancing digital pathology-based image analysis for automated detection of fungal elements (72,73). Additionally, cost-effective diagnostic algorithms tailored to resource-limited settings are needed to ensure broader clinical applicability (74).

In summary, this systematic review demonstrates that while traditional histopathological methods continue to serve as the backbone of fungal diagnosis, their accuracy is significantly enhanced by integration with molecular diagnostics. PAS and GMS remain the most sensitive and reliable stains for rapid visualization, whereas PCR provides unmatched specificity and species-level identification. The combined use of these modalities represents the most efficient, timely, and clinically relevant strategy for diagnosing fungal infections, ultimately improving patient outcomes and optimizing antifungal stewardship (75).

CONCLUSION

This systematic review demonstrates that histopathological and microbiological methods each play indispensable yet distinct roles in the diagnosis of fungal infections. While conventional Hematoxylin and Eosin (H&E) staining provides essential structural context and allows assessment of inflammatory response and tissue invasion, its diagnostic accuracy is limited by poor visualization of fungal elements. Special stains such as Periodic Acid–Schiff (PAS) and Gomori Methenamine Silver (GMS) significantly enhance the detection of fungi, with GMS emerging as the most sensitive and specific histopathological method across multiple studies (76). Despite these strengths, histopathology alone cannot determine the fungal species or antifungal susceptibility, which are critical for clinical management.

Microbiological culture remains the gold standard for species identification and susceptibility testing, but its diagnostic yield is often low due to slow growth, non-viable organisms, and antifungal pretreatment (77). Molecular methods such as polymerase chain reaction (PCR) have revolutionized fungal diagnostics, offering superior sensitivity and specificity and allowing rapid detection even in culture-negative cases (78). However, the lack of assay standardization and high cost limit their universal implementation, especially in low-resource laboratories (79).

The synthesis of evidence in this review clearly indicates that an integrated diagnostic approach combining histopathological methods (PAS or GMS) with PCR provides the highest overall diagnostic accuracy, achieving pooled sensitivity and specificity of 96% and 94%, respectively. This combination facilitates both morphological confirmation and species-level identification, ensuring early and precise diagnosis that directly impacts therapeutic decisions and patient outcomes (80).

In clinical practice, the most effective strategy for managing suspected fungal infections involves a tiered diagnostic workflow: initial screening through histopathology to detect fungal invasion, followed by confirmatory identification through molecular testing. Such an approach not only enhances diagnostic confidence but also shortens the time to antifungal initiation, which is often a critical determinant of prognosis in invasive fungal infections (81).

Future diagnostic frameworks should aim to standardize molecular testing protocols, promote integration with digital histopathology platforms, and adapt cost-effective diagnostic combinations suitable for resource-constrained environments. Collaborative efforts between clinicians, microbiologists, and pathologists will be essential to establish unified diagnostic algorithms that balance accuracy, turnaround time, and accessibility (82).

In conclusion, histopathology—especially with PAS and GMS staining—remains a cornerstone for the rapid detection of fungal elements in tissue, while PCR serves as a powerful complementary tool for definitive species identification. When combined, these modalities provide the most reliable and clinically meaningful approach for diagnosing fungal infections, optimizing patient care, and guiding antifungal therapy decisions (83).

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