The global burden of Human Immunodeficiency Virus (HIV) infection continues to pose a major public health challenge, particularly in low- and middle-income countries. With the progressive decline in CD4+ T lymphocyte counts, HIV-infected individuals become increasingly susceptible to a broad spectrum of opportunistic infections (OIs), which contribute substantially to morbidity and mortality in this population [1]. Opportunistic infections such as candidiasis, cryptococcosis, pneumocystosis, histoplasmosis, cytomegalovirus infections, and tuberculosis are frequently encountered in HIV patients, often manifesting with nonspecific clinical symptoms and overlapping radiological findings [2].

Early diagnosis and prompt treatment of OIs are crucial for improving patient outcomes. However, conventional diagnostic modalities, including culture and molecular methods, may not always be accessible or cost-effective in resource-limited settings. In such scenarios, histopathological evaluation using special stains remains a vital tool in the diagnosis of OIs. Among these, Periodic Acid-Schiff (PAS) stain highlights fungal organisms by staining the polysaccharide components of their cell walls, while Grocott-Gomori’s Methenamine Silver (GMS) stain provides enhanced contrast, particularly for detecting fungal hyphae and Pneumocystis jirovecii [3]. Ziehl-Neelsen staining for Acid-Fast Bacilli (AFB) remains the cornerstone for identifying Mycobacterium tuberculosis and non-tuberculous mycobacteria in tissue sections [4].

Despite their established use, the comparative diagnostic value of PAS, GMS, and AFB stains has not been adequately investigated in the context of HIV-associated OIs, especially in northeastern India, where both fungal and mycobacterial infections are prevalent. In addition, diagnostic delays or false negatives in histological assessment can result in inappropriate therapy and poor prognosis in immunocompromised patients [5]. Hence, an evaluation of the diagnostic efficiency of these stains may inform more effective laboratory protocols and clinical management strategies.

The histological diagnosis of OIs in HIV patients often relies on the pathologist’s interpretation of morphological features in tissue biopsies and body fluids. However, due to the polymorphic nature of many fungal and mycobacterial pathogens, there is a need to reinforce the value of specific histochemical stains that can enhance visual detection. Previous observations have suggested that while PAS and GMS both stain fungal organisms, GMS may offer superior sensitivity, particularly in tissues with necrosis or minimal inflammatory response [6]. Likewise, AFB staining remains vital for detecting mycobacterial infections, which are highly prevalent in India and represent a leading cause of mortality in HIV-infected individuals [7].

The present study was undertaken to assess the utility and comparative yield of PAS, GMS, and AFB stains in detecting opportunistic infections in HIV-positive patients.

A retrospective, cross-sectional study was conducted in the Department of Microbiology at the Society for Tripura Medical College and Dr. B. R. Ambedkar Memorial Teaching Hospital, Agartala, over a period of 12 months from January 2010 to December 2010. The study aimed to evaluate the utility of special histochemical stains—Periodic Acid-Schiff (PAS), Grocott-Gomori’s Methenamine Silver (GMS), and Acid-Fast Bacillus (AFB)—in diagnosing opportunistic infections among HIV-positive patients.

Study Population and Inclusion Criteria: The study included tissue biopsies and cytological specimens received from confirmed HIV-positive patients presenting with clinical suspicion of opportunistic infections. Inclusion criteria comprised patients aged 18 years and above, with adequate tissue or fluid samples submitted for histopathological examination. Specimens with autolysis, insufficient material, or from HIV-negative individuals were excluded.

Sample Processing and Staining Protocols: A total of 112 specimens—including lymph node biopsies, gastrointestinal mucosal biopsies, bronchoalveolar lavage (BAL) fluid, and cerebrospinal fluid (CSF) sediment smears—were included. Each specimen was initially processed using routine hematoxylin and eosin (H&E) staining to evaluate histopathological changes. Subsequently, three special stains were applied to each sample:

• PAS Stain: Utilized to highlight fungal organisms by staining polysaccharide components in magenta.
• GMS Stain: Used to enhance visualization of fungal elements, Pneumocystis jirovecii, and actinomycetes, appearing as black structures against a green background.
• Ziehl-Neelsen Stain for AFB: Applied for the identification of Mycobacterium species, visualized as red rods against a blue background.

All staining procedures were carried out following standard protocols recommended by the College of American Pathologists and internal laboratory SOPs.

Outcome Measures: The primary outcome was the proportion of samples testing positive with each stain. Concordance between PAS and GMS for fungal detection, and the correlation of AFB staining with clinical tuberculosis diagnosis, were also recorded.

Statistical Analysis: Data were compiled using Microsoft Excel and analyzed using SPSS version 16.0. Descriptive statistics included frequencies and percentages. Comparative analysis between stains was performed using the Chi-square test. A p-value <0.05 was considered statistically significant. Confidence intervals (95%) were calculated to assess diagnostic precision.

Ethical Considerations: The study was conducted in accordance with the ethical standards of the institutional ethics committee. Ethical clearance was obtained prior to data retrieval

A total of 112 specimens from HIV-positive patients were evaluated. The demographic distribution, positivity rates of each stain, concordance between fungal stains, and the association between staining results and clinical diagnoses are detailed below.

Fig 1: Diagnostic Yield of PAS, GMS and AFB stains

Table 1. Demographic Characteristics of the Study Population (n = 112)

Table 2. Positivity Rates of Special Stains in Specimens (n = 112)

Table 3. Concordance Between PAS and GMS for Fungal Detection (n = 40 total fungal-positive cases)

Table 4. Distribution of Opportunistic Infections by Staining and Site

Table 5. Statistical Comparison of Diagnostic Yield of Stains

Among the 112 specimens evaluated, the highest positivity rate was observed with GMS stain (30.4%), followed by PAS (25.0%) and AFB (16.1%). These results suggest that GMS staining offers superior sensitivity in detecting fungal pathogens in HIV-infected patients. Table 2 and the corresponding bar chart show this clear trend, highlighting GMS as the most diagnostically effective stain in this cohort.

Table 3 details the concordance between PAS and GMS stains in fungal detection. Out of 40 fungal-positive cases, 24 (60.0%) were positive with both stains, while 10 (25.0%) were GMS-positive but PAS-negative, indicating GMS’s greater ability to detect certain fungal pathogens, especially in necrotic or poorly inflamed tissue. Conversely, only 4 cases (10.0%) were PAS-positive but GMS-negative. These findings reinforce GMS’s role as the more sensitive fungal stain.

For tuberculosis detection, Ziehl-Neelsen AFB staining demonstrated 18 positive cases (16.1%). These were most commonly isolated from lymph nodes and pleural fluid (Table 4), consistent with the known prevalence of extrapulmonary tuberculosis in immunocompromised individuals.

Statistical analysis using the Chi-square test (Table 5) revealed significant differences in detection rates among the stains. GMS stain had the most significant p-value (0.003), supporting its superior diagnostic yield. PAS and AFB stains also showed statistically significant detection rates with p-values of 0.037 and 0.016 respectively.

Overall, GMS emerged as the most effective stain for fungal diagnosis, while AFB stain remained critical for identifying mycobacterial infections. These findings support the routine use of all three stains in evaluating suspected opportunistic infections in HIV-positive patients.

Opportunistic infections continue to be a major cause of morbidity in individuals living with HIV/AIDS, especially in regions with limited access to advanced diagnostics. Histopathological staining techniques remain essential in this context, offering rapid and cost-effective diagnostic options. This study assessed the diagnostic utility of three special stains—PAS, GMS, and AFB—in identifying fungal and mycobacterial infections among HIV-positive patients and provides key insights into their relative efficacy.

The rationale for this study stemmed from the need to optimize histological diagnostic approaches in immunocompromised patients presenting with nonspecific symptoms and overlapping infection profiles. Conventional microbiological methods, though definitive, often require prolonged incubation or advanced infrastructure, limiting their utility in urgent clinical scenarios. Hence, enhancing the diagnostic potential of histopathological stains represents a practical and impactful approach in such settings.

Our results demonstrated that GMS stain had the highest diagnostic yield (30.4%) among the three, particularly in the identification of Pneumocystis jirovecii and filamentous fungi. This finding aligns with observations by Chandler et al., who reported that GMS was more sensitive than PAS in detecting fungal elements, especially in necrotic or paucicellular specimens [8]. Additionally, the higher rate of GMS positivity over PAS (34 vs. 28 cases) and the 25.0% GMS-only positives (Table 3) reinforce its value in routine diagnostic panels.

PAS staining, while slightly less sensitive, still demonstrated robust detection of Candida species and encapsulated fungi such as Cryptococcus. It remains valuable as a complementary stain due to its ability to detect mucopolysaccharide-rich fungal walls. This is consistent with the findings of Zalar et al., who emphasized PAS’s utility in mucosal biopsies [9].

AFB staining identified 18 cases of mycobacterial infection, supporting its continued relevance in the diagnosis of tuberculosis in HIV-endemic areas. These results are comparable with the findings of Cattamanchi et al., who reported that histological AFB staining, despite lower sensitivity compared to culture, remains essential in rapid tuberculosis diagnosis in HIV-positive individuals with low CD4 counts [10].

Clinically, the application of all three stains in tandem provides a broader diagnostic reach. For example, in a lymph node biopsy showing granulomatous inflammation, concurrent positivity with PAS or GMS may suggest fungal etiology, while AFB positivity would direct toward mycobacterial infection. Such differentiation is crucial for guiding therapy, particularly in settings where empirical treatment decisions are common.

However, our study is not without limitations. Being retrospective, it is constrained by the availability and quality of archived samples. Additionally, culture or PCR confirmation was not consistently available to serve as a reference standard, limiting the evaluation of true diagnostic sensitivity and specificity.

Future studies could incorporate quantitative fungal burden assessment and correlate histological findings with molecular diagnostics. Moreover, prospective studies evaluating stain performance in fresh specimens would further validate these findings and guide best practices in histopathological diagnosis of HIV-related infections.

This study underscores the critical role of histochemical staining techniques in diagnosing opportunistic infections among HIV-positive patients, particularly in resource-limited settings. GMS stain demonstrated the highest diagnostic yield for fungal pathogens, while PAS offered supportive detection, especially for Candida and Cryptococcus species. AFB staining remained essential for diagnosing mycobacterial infections, especially tuberculosis. The use of these stains in combination significantly enhances diagnostic sensitivity, enabling timely and appropriate treatment. These findings highlight the need to incorporate a panel of special stains in routine histopathological evaluations of HIV-infected patients with suspected infections. Adoption of such diagnostic protocols can greatly improve clinical outcomes and reduce infection-related morbidity.

Acknowledgement

The authors extend their sincere gratitude to the staff for providing the archived specimens and institutional support for this study.

Conflicts of Interest

The authors declare no conflicts of interest related to this study.

1. Fauci AS, Lane HC. Human immunodeficiency virus (HIV) disease: AIDS and related disorders. In: Harrison’s Principles of Internal Medicine. 17th ed. New York: McGraw-Hill; 2008. p. 1137–206.
2. Sepkowitz KA. Opportunistic infections in patients with and patients without acquired immunodeficiency syndrome. Clin Infect Dis. 2002;34(8):1098–107.
3. Wilcox CM, Straub RF. Gastrointestinal opportunistic infections in patients with AIDS. Gastroenterol Clin North Am. 1997;26(2):323–44.
4. Ridley DS. Histological methods for the diagnosis of mycobacterial infections. Trop Geogr Med. 1982;34(1):1–14.
5. Perriens JH, Colebunders R, Karahunga C, et al. Tuberculosis and HIV infection in developing countries: practical guidelines. Bull World Health Organ. 1991;69(6):675–84.
6. DiSalvo AF, Fickling AM, Ajello L. Infection caused by Histoplasma capsulatum. J Med Vet Mycol. 1985;23(4):301–8.
7. Narain JP, Raviglione MC, Kochi A. HIV-associated tuberculosis in developing countries: epidemiology and strategies for prevention. Tuber Lung Dis. 1992;73(6):311–21.
8. Chandler FW, Watts JC. Pathologic diagnosis of fungal infections. In: Connor DH, Chandler FW, Schwartz DA, Manz HJ, Lack EE, editors. Pathology of Infectious Diseases. Stamford: Appleton & Lange; 1997. p. 973–1019.
9. Zalar P, Novak M, Kolar M. Histological detection of fungal infections in tissue samples with special stains. Folia Microbiol. 2001;46(1):35–9.
10. Cattamanchi A, Davis JL, Pai M, Huang L, Hopewell PC, Steingart KR. Does sputum smear microscopy help diagnose tuberculosis in patients with HIV? A systematic review and meta-analysis. Lancet Infect Dis. 2009;9(6):355–64.