F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.156469.1 Research Article Articles Spectrum and Antifungal Drug Resistance among Fungal Pathogens Isolated from Prison Inmates in Nairobi, Kenya

[version 1; peer review: 1 not approved]

 Afundi Jackson Larry Conceptualization Data Curation Formal Analysis Investigation Methodology Project Administration Resources Software Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-4444-7328 a 1 Njerawana Sally Conceptualization Data Curation Formal Analysis Investigation Methodology Project Administration Resources Software Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing 2 Chebon Samson Conceptualization Data Curation Formal Analysis Investigation Methodology Project Administration Resources Software Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing 1 Bii Christine Conceptualization Data Curation Formal Analysis Investigation Methodology Project Administration Resources Software Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing 2 Biomedical Sciences, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Nairobi County, Kenya Mycology Division , Center for Microbiology Research, Kenya Medical Research Institute, Nairobi County, Kenya larryjaison@gmail.com

No competing interests were disclosed.

 30 10 2024 2024 13 1301 24 10 2024 Copyright: © 2024 Afundi Jackson L et al. 2024 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background

The emergence of antifungal resistance in fungal pathogens highlights the need for local epidemiological data to guide empirical therapy in clinical settings. Fungal research and anti-fungal drug resistance studies are limited in developing countries; hence, there is a need for burden estimation in low- and middle-income countries. This study aimed to determine the spectrum of fungal pathogens and the anti-fungal resistance profile of fungal pathogens isolated from the respiratory and urinary tracts of prison inmates in Nairobi, Kenya.

 Methods

A cross-sectional study was conducted in which sputum and urine samples were obtained from inmates presenting with symptoms of respiratory and urogenital infections at a prison outpatient clinic. One hundred and sixty-two samples were collected and subjected to fungal investigation using standard protocols. Susceptibility to fluconazole, itraconazole, and voriconazole was assessed using standard broth microdilution. Clinical and sociodemographic data were obtained using a structured questionnaire.

 Results

From the 162, 94 samples were positive for fungal pathogens, with an overall prevalence of 58%. Seventeen (18%) of the isolated fungi were Aspergillus fumigatus, Aspergillus flavus and Histoplasma. There was a statistically significant difference between fungal pathogens isolated from the respiratory and urogenital tracts in both sexes (p<0.05). Antifungal susceptibility testing against itraconazole showed 2 of Aspergillus flavus and A spergillus fumigatus were resistant.

 Conclusion

Mycological agents are significant causes of respiratory and UTI infections among prison inmates, which could be attributed to prison conditions and misdiagnosis as bacterial infections. This highlights the need for specific control measures to reduce exposure to fungal infections in prisons and in the general population.

 Aspergillus spp Histoplasma Antifungal Resistance Prison inmates The author(s) declared that no grants were involved in supporting this work. Introduction

Human fungal infections are increasingly causing health and economic burdens at different echelons, including personal, community, national, and global levels. 1– 4 Despite growing concerns, fungal infections receive very little attention and resources, leading to a scarcity of quality data on fungal disease distribution and antifungal resistance patterns. Similarly, the exact burden of fungal diseases and antifungal resistance is limited; therefore, the response is undermined. 5 Arising from this health concern, 19 fungal pathogens are grouped into three priority categories: critical, high-risk, and medium. The criteria for categorization are based on the average case fatality, number of new cases annually, complications and sequelae, and antifungal resistance. Among the species in the critical group and high category were Cryptococcus neoformans, Aspergllus fumigatus and Candida albicans in the critical group, and Histoplasma spp., Fusarium spp., and Candida glabrata in the high group species. 5

Studies on the prevalence of fungal diseases affecting the pulmonary and urogenital systems of inmates in Kenyan prisons have rarely been documented. However, the prevalence of other infections has also been widely reported. Studies on the prevalence of respiratory diseases caused by fungal pathogens in Kenya’s prison system remain limited. Besides a study at Kamiti Maximum Prison on the prevalence of active pulmonary TB and another on modifiable factors associated with pulmonary TB in inmates from Nakuru Maximum prisons no study has been undertaken on fungal pathogens afflicting inmates in prisons. 6, 7

Histoplasma capsulatum and Cryptococcocus neoformans can cause pulmonary and urogenital infections. 8, 9 The two pathogens have not been elucidated in the two prisons, despite their unique and paradoxically neglected yet highly pathogenic nature. Histoplasma species are not endemic to tropical regions, such as Kenya. The prison microenvironment offers favorable conditions for the proliferation of fungal pathogens missed during routine microbiological diagnosis. Misdiagnosis and inappropriate prescription practices not only prolong illness, but also drive antimicrobial resistance. Furthermore, the management of fungal infections is still highly constrained by the narrow spectrum of antifungal classes of antifungal drugs and the pharmacological toxicity associated with some of these drugs. 10 It is necessary to establish the spectrum and antifungal susceptibility of respiratory and urogenital fungal pathogens among prison inmates.

Urinary tract infections have been studied extensively in diverse communities and among different age groups, but they have not been fully explored in prison inmates, whose health is often neglected. 1 Transmission of respiratory and urogenital infections is enhanced in crowded environments, such as schools, offices, hospitals, and prisons. Prisons worldwide often experience overcrowding, and Kenya is no exception. This situation, coupled with other factors such as delayed diagnosis, often makes prisons a prime area for the spread of respiratory and urogenital infections among inmates. 11 The prison population has a high prevalence of serious, often life-threatening, infections. When prisoners are released back into the community, they carry back new and untreated infectious diseases that pose a threat to community health and add to the burden of disease in the community. Thus, there is compelling interest in society that this vulnerable group receives health protection and treatment for any ill health. 12

Resistance to currently available antifungal agents can develop via acquired mechanisms following exposure to antifungal drugs. The findings of this study highlight the significance of fungal infections in prison that will guide relevant institutions in planning and policy development in such confined settings.

Study site

The study was conducted at a male maximum and female prison institution in Nairobi County. The Maximum prison has a capacity of 1400 prisoners but now houses 1800 and 2500 prisoners. Women’s prison is the largest in Kenya, with a capacity of 700 inmates. The two maximum prisons were selected purposely as the research site because of their geographical location, capacity, and proximity to the prison headquarters, allowing access to records as well as some key informants to participate in the study.

 Study design

This was a cross-sectional study with laboratory investigation. Through purposive sampling, inmates with clinical symptoms of respiratory tract infections and UTI were recruited between January 2023 and March 2023. Sputum and urine samples were collected and shipped to the Mycology Division, Centre for Microbiology Research, Kenya Medical Research Institute (KEMRI) for fungal investigations using standard protocols. The isolates were identified using macro-and microscopic features. Antifungal susceptibility was determined using the broth microdilution method. Sociodemographic information, diagnosis, and intervention were sought using the data collection tool from consenting prison inmates. The study population consisted of adolescent and adult men and women aged 18-76 years old who were crime suspects or convicts at the prison and presented with clinical manifestations of respiratory and urogenital infections.

 Laboratory procedures

Fungal culture and identification

Sputum and urine samples were subjected to direct microscopy and cultured on Sabouraud dextrose agar supplemented with chloramphenicol (16 mg/ml). The inoculated SDA plates were incubated at 35°C for four weeks and examined daily for growth. Yeast isolates were differentiated using the CHROMagar ® Candida and Dalmau techniques. Morphological features, such as hyphae, pseudohyphae, blastospores, chlamydospores, basidiospores, and sporangia, were noted.

Mold identification was performed using standard macroscopic features, such as colony growth rate, surface color, and reverse, texture, and diffusing pigments. Microscopic features of lactophenol cotton blue stain at ×40 magnification such as conidia, shape, and arrangement of phialides, were used. Clarus Histoplasma GM Enzyme Immunoassay (IMMY, Norman Oklahoma) was used to detect Histoplasma antigens in urine samples according to the manufacturer’s instructions. 13 Titres of 1.0 EIA units were interpreted as positive for histoplasma antigen, whereas values less than 1.0 were interpreted as negative.

Antifungal resistance testing

The minimum inhibitory concentrations of fluconazole, itraconazole, and voriconazole were determined using the broth microdilution method. 14 Briefly, 9.6 mg of azoles (Fluconazole, Itraconazole and Voriconazole) was dissolved in 3.0 ml of DMSO to get 1600 μg/ml (Sigma chemicals, USA) stock drug concentration while the required concentration was achieved with further dilution with RPMI. Briefly, the final dilution was achieved by serial dilution where 4.9 ml of RPMI was added to each of the ten tubes. Two hundred microlitres of the test concentration was dispensed into microtiter plates and inoculated with 10 μl of the 0.2 MacFarland concentration of inoculum. The plates were then incubated at 30°C for 72 h. The MIC values of all the drugs were determined visually as the lowest concentrations with no visible growth. The European Committee on Antimicrobial Susceptibility (EUCAST) drug-susceptible control strain ATCC 46645 was used for QC. Antifungal susceptibility was interpreted based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The results were interpreted based on the EUCAST breakpoints for itraconazole (resistant isolates: MIC > 1 mg/L) and voriconazole (resistant isolates: MIC > 2 mg/L). Eucast has no set break points for fluconazole; therefore, in this study, susceptibility testing to fluconazole was based on the Clinical and Laboratory Standard Institute M38-A2 protocol. 15

Data management and analysis

Data were analyzed using SPSS software version 26. Chi-square test and multivariate logistic regression analysis were performed to investigate the correlation between the independent variables and the isolation of fungal isolates. Chi-square test analysis was applied to determine sociodemographic factors and variables associated with fungal infections in patients with respiratory and urogenital-like symptoms. Logistic regression was used to calculate the odds ratios (ORs) with 95% confidence. Statistical significance was set at p<0.05.

 Ethics approval

The study’s Ethical approval was obtained from Institutional Ethics and Review Committee at Jomo Kenyatta University of Agriculture and Technology (Ref No: JKU/ISERC/02316/0735, 22nd September 2022), National Commission for Science Tech & Innovation (Ref No: 659559, 19th October 2022), Kenyatta National Hospital University of Nairobi Ethical Review Committee (Ref No: KNH-ERC/RR/14, 9th February 2023) and Kenya Prison Service (PRIS19/26 VOLII/167, 3rd October 2022).

 Results Prevalence of fungal etiological agents in the two prisons facilities

Of 162 samples investigated from the two prisons, 86 were sputa and 76 were urine samples. A total of 94 (58%) samples were positive for fungi, 19 (20%) for molds, and 75 (80%) for yeasts ( Table 1). Of the 94 positive fungi, 7 (7.4%) tested positive for Aspergillus flavus and 5 (5.3%) were Aspergillus fumigatus. Histoplasma antibodies were detected in 5.3% of the samples. Aspergillus flavus was the predominant filamentous fungus isolated, as shown in Figure 1.

Social demographic and clinical data of inmates who participated.
Variable Number examined N=162 Positive fungal isolates N=94 p value
Mould Yeast
Gender Male 94 12(13%) 46(50%) 1.00
Female 68 7(7%) 75(79%)
Sample Sputum 86 19(20%) 41(44%) <0.001
Urine 76 0 34(36%)
The spectrum of fungi isolated from prison institutions. Antifungal susceptibility pattern of fungal isolates

Twelve isolates of Aspergillus fumigatus and Aspergillus flavus obtained from 162 patients were subjected to antifungal susceptibility testing. Only three (43%) Aspergillus flavus were resistant to itraconazole. Two (40%) isolates of Aspergillus fumigatus were resistant to itraconazole. All Aspergillus fumigatus and Aspergillus flavus isolates were susceptible to voriconazole ( Table 2). Susceptibility to fluconazole indicated that all Aspergillus species were resistant to fluconazole.

Antifungal susceptibility of isolate according to EUCAST.
Fungal isolate (n) Itraconazole Voriconazole
Resistant Susceptible Resistant Susceptible
Aspergillus fumigatus (5) 2(40%) 3(60%) 0 5(100%)
Aspergillus flavus (7) 3(43%) 4(57%) 0 7(100%)

Key: MIC: Minimum Inhibitory concentration. Resistant isolates: MIC > 1 mg/L for Itraconazole, MIC > 2 mg/L for voriconazole.

 Discussion

Findings from this study revealed an overall fungal prevalence among prison inmates of 58%, while the prevalence of Aspergillus flavus and Aspergillus fumigatus, was 7.4% and 5.3%, respectively. This finding indicates the significance of aspergillosis in confined settings, which may increase the burden of communicable diseases in the general population. These findings agree with a previous study reporting Aspergillus spp as the leading fungal pathogen among prisoners. 16

Aspergillus flavus has been reported as an invasive Aspergillus species associated with clinical manifestations, such as cough, chest pain, fever, hemoptysis, weight loss, and dyspnea, observed in the participants enrolled in this study. These symptoms are consistent with those of previous studies and suggest further investigation of chronic pulmonary aspergillosis and co-infection with TB, bacteria, or other pathogens in confined settings.

Five (3.1%) samples tested positive for histoplasma antigen among the prison inmates, confirming the existence of histoplasmosis in Kenya and highlighting its possible role in pulmonary infections. Being the first study to report histoplasmosis infection among Kenyan prisons, this calls for lookout for this priority pathogen in confined settings. Previous studies in Nigeria reported a prevalence of histoplasmosis infection of 8.5% 17 while in Uganda, the prevalence of histoplasma antigens was 1.2%. 18 The distribution of fungal isolates based on sex and anatomical sites showed a significant difference between fungal pathogens and anatomical sites (P< 0.001). There was no association between fungal infections and sex. More fungal pathogens were likely to be isolated from respiratory sites than from urogenital sites. This finding is supported by previous studies that reported an association between fungal isolates and pulmonary related disease. 19

Based on the EUCAST Antifungal Clinical Breakpoint for Aspergillus spp, voriconazole had the lowest MIC against all tested isolates. Aspergillus fumigatus and Aspergillus flavus showed susceptibility to voriconazole, with MICs<2.0 mg/L. A similar study by Sani et al. 20 indicated that all fungal isolates were sensitive to voriconazole.

Some Aspergillus flavus (43%) and Aspergillus fumigatus (29%) isolates were resistant to itraconazole. However, the majority of the patients were susceptible to itraconazole. All A spergillus species tested were resistant to fluconazole.

These findings are consistent with those of Sriramajayam et al., 21 who reported similar widespread antifungal resistance to itraconazole but good antifungal susceptibility to voriconazole.

The findings of this study clearly indicate the need for routine fungal culture and antifungal drug resistance surveillance to improve fungal diagnosis and treatment, particularly in confined settings.

 Conclusion

This study highlights the presence of Histoplasma and other fungal infections associated with respiratory and urogenital infections in prison settings in Kenya. This study also demonstrated that voriconazole was the best antimycotic agent against fungal isolates, but with emerging resistance to itraconazole. We recommend investigation of all presumptive pulmonary TB patients to avoid misdiagnosis and inappropriate use of antibiotics. This calls for diagnostic capacity for fungal infections in prisons and the general population to reduce the burden of infections and antimicrobial resistance.

Ethics approval

The study’s Ethical approval was obtained from Institutional Ethics and Review Committee at Jomo Kenyatta University of Agriculture and Technology (Ref No: JKU/ISERC/02316/0735, 22nd September 2022),National Commission for Science Tech & Innovation (Ref No: 659559, 19th October 2022) Kenyatta National Hospital University of Nairobi Ethical Review Committee (Ref No: KNH-ERC/RR/14, 9th February 2023) and Kenya Prison Service (PRIS19/26 VOLII/167, 3rd October 2022).

 Consent

Written informed consent for the participation of participants was obtained from the participants.

 Data availability Underlying data

Spectrum and Antifungal drug resistance of fungal pathogens, https://doi.org/10.6084/m9.figshare.26964148.v1. 22

Data citation: Jackson, Larry; Njerawana, Sally; Chebon, Samson; Bii, Christine (2024). Data set: Spectrum and antifungal drug resistance of fungal pathogens. figshare. Dataset. https://doi.org/10.6084/m9.figshare.26964148.v1.

This project contains the following underlying data:

Raw Data of samples

Minimum Inhibitory concentrations of the antifungal

Histoplasma antigen results

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 Extended data

Figshare: Extended Data, https://doi.org/10.6084/m9.figshare.27037570.v1. 23

Data citation: Jackson, Larry; Njerawana, Sally; Chebon, Samson; Bii, Christine (2024). Extended Data:Consent form for inmates 3.docx. figshare. Dataset. https://doi.org/10.6084/m9.figshare.27037570.v1

This project contains the following extended data

Participants Consent form

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 Reporting guidelines

Figshare: STROBE checklist of spectrum and antifungal drug resistance among fungal pathogens isolated from prison inmates in Nairobi, Kenya, https://doi.org/10.6084/m9.figshare.27038287.v1. 24

Data Citation: Jackson, Larry; Njerawana, Sally; Chebon, Samson; Bii, Christine (2024). Strobe check list. figshare. Dataset. https://doi.org/10.6084/m9.figshare.27038287.v1.

 References Divine E Ndubuisi N Victor N : Urinary Tract Infection Among Prison Inmates in Afara Jail Umuahia. Int. J. Clin. Urol. 2020;4(1):25. 10.11648/j.ijcu.20200401.16 Guto JA Bii CC Denning DW : Estimated burden of fungal infections in Kenya. J. Infect. Dev. Ctries. 2016;10(8):777–784. 27580321 10.3855/jidc.7614 Oladele RO Denning DW : Burden of serious fungal infection in Nigeria. West Afr. J. Med. 2014;33(2):107–114. 25236826 Kalua K Zimba B Denning DW : Estimated burden of serious fungal infections in Malawi. J. Fungi. 2018;4(2). 29883439 10.3390/jof4020061 PMC6023453 Pfavayi LT Denning DW Baker S : Determining the burden of fungal infections in Zimbabwe. Sci. Rep. 2021;11(1):13213–13240. 34168204 10.1038/s41598-021-92605-1 PMC8225815 D. of C. M. A. T. Univercity of Nairobi: the Prevalence of Active Pulmonary Tuberculosis Among Prisoners At Kamiti. 2012;2012. Amwayi AS Kikuvi GM Muchiri EM : Modifiable factors associated with active pulmonary tuberculosis in a Kenyan prison. East Afr. Med. J. 2010;87(2):43–48. 23057255 10.4314/eamj.v87i2.60596 Barros N Wheat JL Hage C : Pulmonary Histoplasmosis: A Clinical Update. J. Fungi. 2023;9(2):1–21. 36836350 10.3390/jof9020236 PMC9964986 Qurashi S Saleem T Kovalenko I : Cryptococcus neoformans Presenting as a Lung Mass in an Immunocompromised Patient. Am. J. Case Rep. 2022;23:e936968–e936966. 36183161 10.12659/AJCR.936968 PMC9536145 Bongomin F Gago S Oladele RO : Global and multi-national prevalence of fungal diseases—estimate precision. J. Fungi. 2017;3(4). 29371573 10.3390/jof3040057 PMC5753159 Sylverken A : Burden of respiratory viral infections among inmates of a Ghanaian prison. 2019;1–17. 10.21203/rs.2.14139/v1 May B : Good Practices in Managing Infectious Diseases in Prison Settings. 2022; (September). Reference Source Immunoassay E : For the Qualitative Detection of Histoplasma Galactomannan – REF HGM201. The European Committee on Antimicrobial Susceptibility Testing: Breakpoint tables for interpretation of MICs for antifungal agent. Eucast. 2020; (April):8. Reference Source Weinstein MP Lewis JS : The clinical and laboratory standards institute subcommittee on Antimicrobial susceptibility testing: Background, organization, functions, and processes. 2020;58(3). 10.1128/JCM.01864-19 Lilian NC : Airborne Fungi in Prison Indoor Environments of Nsukka and Enugu Metropolis, Nigeria. Annu. Res. Rev. Biol. 2021; (August):124–132. 10.9734/arrb/2021/v36i730405 Oladele RO : Prevalence of Histoplasmosis among Persons with Advanced HIV Disease, Nigeria. Emerg. Infect. Dis. 2022;28(11):2269–2277. 36286009 10.3201/eid2811.220542 PMC9622240 Sekar P : Prevalence of Histoplasma Antigenuria among Outpatient Cohort with Advanced HIV in Kampala, Uganda. J. Fungi. 2023;9(7). 37504745 10.3390/jof9070757 PMC10381727 Moglad E : Molecular Characterization and Antifungal Susceptibility of Aspergillus spp. among Patients with Underlying Lung Diseases. Trop. Med. Infect. Dis. 2022;7(10). 36288015 10.3390/tropicalmed7100274 PMC9612272 Abebe G Kebede W Bonsa Z : Treatment Outcomes and Associated Factors in Tuberculosis Patients at Jimma University Medical Center: A 5-Year Retrospective Study Gemeda. Int. J. Mycobacteriol. 2017;6(3):239–245. 10.4103/ijmy.ijmy Sriramajayam L Kaur R Dhakad MS : Antifungal Resistance Profile of Fungal Isolates from Fungal Rhinosinusitis Patients: A Study from Tertiary Care Hospital. J. Lab. Physicians. 2023;15(04):488–492. 37780889 10.1055/s-0043-1764484 PMC10539057 Jackson L Njerawana S Chebon S : Data set: Spectrum and antifungal drug resistance of fungal pathogens.Dataset. figshare. 2024. 10.6084/m9.figshare.26964148.v1 Jackson L Njerawana S Chebon S Bii C : Extended Data: Consent form for inmates 3.docx.Dataset. figshare. 2024. 10.6084/m9.figshare.27037570.v1 Jackson L Njerawana S Chebon S : Strobe check list.Dataset. figshare. 2024. 10.6084/m9.figshare.27038287.v1 10.5256/f1000research.171787.r422998 Reviewer response for version 1 Kalkanci Ayse 1 Referee https://orcid.org/0000-0003-0961-7325 Gazi University Faculty of Medicine, Gazi, Turkey

Competing interests: No competing interests were disclosed.

 13 10 2025 Copyright: © 2025 Kalkanci A 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

The manuscript addresses an underexplored topic-the occurrence of fungal pathogens and antifungal resistance among prison inmates in Kenya. However, the study suffers from fundamental methodological and conceptual weaknesses. The overall presentation is confusing, the sampling strategy lacks clinical justification, and the data interpretation does not support the authors’ conclusions. The connection between the stated objectives and the presented results is weak, and there is no clear hypothesis explaining why fungal pathogens were studied in this particular population or setting.

2. Major Concerns

2.1. Unclear Sample Type and Clinical Relevance

The most critical flaw is the ambiguity regarding the nature of the respiratory samples. The authors alternately refer to “sputum” and “respiratory tract” samples, but do not specify whether these were true sputum specimens collected under clinical supervision or saliva/oral secretions obtained randomly.

Candida spp. isolation from sputum generally indicates colonization or contamination, not infection. Therefore, the claim that these isolates represent “pathogenic fungi causing respiratory infections” is misleading.

No criteria (e.g., purulence, presence of epithelial cells, neutrophil count, or Gram stain evaluation) are provided to differentiate sputum from saliva.

Without clinical correlation (e.g., radiologic or serologic confirmation), the data cannot support the assertion of “respiratory fungal infections.”

 2.2. Misleading Title and Aim

The title and objective suggest that pathogenic fungi were isolated and antifungal resistance assessed; however, the results show only Candida colonization and environmental molds ( Aspergillus spp.). There is no evidence that these isolates caused true infections. The study thus overstates its clinical impact and does not fulfill the promise of the title.

2.3. Lack of Scientific Rationale

The rationale for investigating fungal infections in a prison environment is not convincing. The authors imply that prisons are “confined environments favorable to fungal spread,” but fungal pathogens such as Candida, Aspergillus, and Histoplasma are not transmitted person-to-person. The inclusion of “prison conditions” as a risk factor is therefore scientifically unsound unless supported by environmental sampling data, which are absent.

2.4. Ethical and Logistical Justification

While several ethical approvals are listed, the study design raises ethical concerns:

There is no explanation of why fungal culture was performed in a population primarily presenting with bacterial symptoms.

The use of prisoners as participants-an inherently vulnerable group-requires strong justification and oversight, which are not adequately described.

 2.5. Methodological Weaknesses

The sample size (n=162) is not statistically justified, and no power calculation is presented.

The description of antifungal susceptibility testing lacks details on species-level identification, inoculum standardization, and replicate reproducibility.

Results combine urine and sputum isolates, creating confusion. The presentation does not separate commensal colonization ( Candida in urine) from pathogenic infection ( Histoplasma or Aspergillus).

Tables and figures are poorly organized and not integrated into the discussion.

 2.6. Superficial Discussion

The Discussion section merely restates results and provides general background.

No attempt is made to differentiate colonization from infection or to compare findings with clinical data.

The authors do not address the fact that Candida in urine or sputum is not diagnostic of invasive disease without corroborating evidence.

The hypothesis that “prison conditions” increase fungal disease burden is speculative and unsupported.

 3. Minor Comments

Several species names are inconsistently italicized.

Reference formatting is inconsistent and requires revision according to journal style.

The term “ Histoplasma antibodies” is used incorrectly; the assay described (IMMY GM EIA) detects antigen, not antibodies.

Statistical analysis (Chi-square and logistic regression) is inappropriate for such small and heterogeneous data.

The manuscript includes numerous grammatical and stylistic errors that affect clarity.

 4. Conclusion

The manuscript in its current form does not provide reliable evidence of fungal infections or antifungal resistance among prison inmates. The isolation of Candida and Aspergillus from unverified sputum and urine samples reflects commensal colonization or environmental contamination, not infection. Furthermore, the lack of a clear hypothesis, weak methodology, and speculative conclusions make it unsuitable for indexing without substantial redesign.

5. Recommendations for the Authors

Clarify the type, quality, and clinical validation of collected samples.

Restrict conclusions to colonization findings unless infection is proven by clinical data.

Revise the title and abstract to reflect actual results.

Reanalyze data separately for urine and sputum samples.

Provide a scientifically sound rationale for selecting prison inmates and justify ethical considerations.

Strengthen the discussion with evidence-based comparisons and remove speculative statements about transmission or prison conditions.

Is the work clearly and accurately presented and does it cite the current literature?

No

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

No

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Mycology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.