Prevalence of Mycoses and Investigation of Fungal Indoor Environment

1. Introduction

Fungal infections are the most common cause of morbidity and mortality in immunocompromised and chronic disease patients 1. This is attributable to the steady increase in opportunistic invasive fungal infections in risk groups 2. It was estimated that a person inhales 5–5000 conidia of opportunistic pathological fungi daily. However, conidia are harmless cells in most individuals, except in immunocompromised patients who are at a higher risk of being affected by fungal infections 3. Patients with impaired immune systems, such as diabetes, asthma, and transplant recipients, are the most susceptible to secondary infections due to prolonged treatment and general immunosuppression 4. The prevalence of asthma varies worldwide, ranging from 1% –20% for both children and adults. The prevalence of asthma in Saudi Arabia, was estimated to be 11.3% 5. The most common fungi found in these patients are Candida spp., Aspergillus spp., Cryptococcus neoformans, and dermatophytes 6. This study focuses on establishing the distribution and characterization of the relationship between indoor environments and fungal abundance across Medina. The detection and characterization of Medina’s environmental fungi in the area are inadequate. This study aims to determine the fungal distribution as well as identify the signature indoor fungi in Medina region and highlight the prevalence of environmental fungal infections in Saudi populations.

1. Materials and MethodsStudy Design

This observational cross-section study assessed the indoor environmental fungi using a questionnaire for collecting demographic data and analysis of dust samples.

• Inclusion and Exclusion Criteria

The inclusion criteria comprised individuals with or without fungal infections, immunocompromised illnesses, and healthy controls. Surveys and samples were excluded from individuals living outside the Medina districts.

• Data and Sample Collection

The role of the questionnaire was to recruit the study subjects and to collect data of their homes. The questions focused on the resident’s previous or current fungal infections, immunosuppression status, pets, and consent for participation. The questionnaire was distributed using snowball sampling to different regions in Medina, collected 1374 responses. Samples were collected from December 2019 to March 2020. The city’s temperature (°C) and humidity (%) were moderate across the months (17.7°C -23.8 °C) and (38%- 28%), respectively. Questionnaires and samples were sorted before analysis to remove any inadequate information and sample. Data were then normalized by random selection to obtain an equal number of samples from each district without bias. A final total of 212 samples were processed after removing insufficient samples and normalization between regions of Medina. The participant vacuum cleaners' content (suction power 250–320 watts) was collected into sterile, well-sealed, and labeled bags. Sample content description was recorded, which included dust, human hair, nails, and cat hair.

• Sample Processing

All sample processing was performed in a class II cabinet for safety reasons and to avoid any contamination. Fungal identification was made using macroscopic and microscopic fungal morphological classification using standard mycological methods (Lactophenol cotton blue staining by the scotch tape and Gram stain method) 7. The sample content was transferred to 10 mL falcon tubes and weighted for normalization (0.7 g). Samples were eluted using 2 mL sterile physiological saline and vortexed for 30 seconds to be cultured on the agar. Agar plates were inoculated in the centre (10 µL) using a platinum loop (Superior Marienfeld Company - Germany) then the inoculation was spread using a glass spreader (Superior Marienfeld Company - Germany) in a circular motion. After incubation at 37 °C for 48 h, all yeasts and moulds colony were counted manually then colony-forming-unit (CFU) was calculated, and two dominant well isolated colonies were selected for purity culture and further investigation.

• Ethical Considerations

This project was approved by the Ethical Committee of Scientific Research at the College of Applied Medical Sciences, Taibah University. Ethical training was organized by the King Abdulaziz City National Committee of Bioethics.

1. Statistical Calculation

All epidemiological, statistical, and laboratory data were calculated using Microsoft Excel 2016. CFU\ml was calculated using (number of colonies × dilution factor) \ volume of sample, prevalence was calculated using (number of people with disease / total number of people), frequency (number of n isolates / total number of isolates) and risk ratio (RR) (Risk of disease in a group of primary interest / Risk of disease in the comparison group).

1. Results The Prevalence of Fungal Infections in Madina

Based on the analysis of the questionnaire from 1374 participants among different regions from Medina, the prevalence and risk of fungal disease who were calculated. The prevalence of fungal infections among the participants was 19.2%. The responses included 133 individuals were identified as risk groups (diabetes, asthma, organ transplant, thyroid disease, irritable bowel syndrome). The prevalence of fungal infections in risk disease was 26.3%.

• The Relationship Between Participants and Fungal Agents

The randomly selected 212 representative samples from each home were analysed considering different variables (Figure 1). The population risk group analysis showed; 25 individuals (12%) risk group which 14 (7%) had diabetes, 8 (4%) had asthma, and 3 (1%) had an organ transplant, thyroid disease, irritable bowel syndrome, and 187 (88 %) healthy individuals. The fungal load among these risk groups was analysed and showed that six of asthmatic patients have Candida spp and one Rhizopus spp. Among diabetic patients, five Candida spp, three non-Candida spp, and one of each Aspergillus spp., Rhizopus spp., and Madurella spp. in their samples. Other risk groups had only two of Candida spp.

Among 212 participants, 18 cases reported to have a past fungal infection, which represented (9%) of the study's population. The data revealed that the percentage of yeast and mould in which they were 134 isolates (60%) and 90 isolates (40%), respectively.

The association between fungal infections and risk group was measured based on the risk group ratio. Therefore, the risk ratio for the risk group was 0.4, indicating that there is no significant association between risk group and fungal infections as the RR is less than 1 (Table1 - A).

The association was carried out between the isolates found during our study from risk groups with past infection and isolates from risk groups with no past infection. The isolates were classified into yeast and mould. The RR then was set to be 0.8, indicating no significant difference between the number of isolates in risk groups with past fungal infections and the isolates from risk groups with no past fungal infections (Table 1 - B).

• Fungal Variability in Medina Indoor Environment

Fungal diversity and distribution across four regions of Medina were identified. Of the 212 homes, 203 (96%) homes showed fungal growth, among them the frequency of each was found to be as shown in (Table 2), whereas 9 (4%) showed no growth.

The analysis of dominant fungal strains showed; Candida spp, were more frequent than non-Candida spp. The highest number of Candida spp isolate was 20 (36%) that is found in the Eastern regions of Medina, whereas the lowest number 9 (16%) was obtained from the Northern region. The highest number of Candida spp was 22 (28%), obtained from the Eastern region, whereas the lowest number 15 (19%) was obtained from the Western region. Based on macroscopic and microscopic examination, each region showed the presence of distinct types of fungi. As for the moulds, 13 genera and 20 species were identified. Aspergillus spp. accounted for the highest count, which was 38 homes (15%); they were found mainly in the Northern region in 13 homes (34%) and least in the Southern region in 7 homes (19%). The second most counted type of moulds was Rhizopus spp., which was found in 20 homes (9%); as they were found mainly in the Western region in 9 homes (45%) and less common in the Eastern region in 3 homes (15%) (Figure 2). Other moulds were also detected, such as Mucor spp. In 7 homes (3%), Neurospora spp. In 4 homes (2%), Penicillium spp. 4 in homes (2%), Epidermophyton spp. In 3 homes (1%), Madurella spp. In 2 homes (1%), Lichtheimia spp in 2 homes (1%), Paecilomyces spp. In 1 home (1%) and unknown fungi with holoarthic/enteroatric conidiogenesis in 9 homes (4%).

• Aspergillus Species in Medina Indoor Environment

Among Aspergillus spp., A. candidus 9 houses (24%) were the most dominant species, followed by A. niger and A. terreus complex in 6 homes (16%), A. flavus complex in 5 homes (13%), while A. fumigatus complex, A. ustus complex, and A. versicolor complex showed a low presence in 2 homes (5%) each; however, A. oryzae and A. glaucus complex in one home (3%) were the lowest to be detected. A. niger was only found in the Northern and Southern regions. Likewise, A. oryzae, A. ustus complex, and A. versicolor complex were only exclusively found in the Eastern and Western regions (Figure 3).

1. Discussion

This study aimed to calculate the prevalence of fungal infections, identifying the signature indoor fungi and determine their distribution in Medina. We hypothesized that this region has signature fungal species that may have further contributed to the public health. As seen in modern societies, people spend 90% of their time indoors, and it is not surprising that factors contributing to poor indoor quality are receiving significant attention from researchers as well as the government 8. Vacuum cleaner dust samples were selected as representative indoor environmental samples, with a heterogeneous mixture of emanation from human subjects, pets, plants, and air. It also contained viable and non-viable fungi, fungal proteins, mycotoxins, and endotoxins 9. The vacuum cleaner was used as sampler for simplicity compared to the air sampling method in regards to, homes privacy, time, and accessibility to a larger sample size. Environmental fungi may be inhaled or ingested due to their adherence, invasion, and colonisation properties 10. We identified Candida and Aspergillus spp as opportunistic fungi, which ranks as top pathogenic fungi for immunocompromised patients 11,12. The dominance of these species in the indoor samples may serve a high risk of infection. Many factors facilitate the occurrence of these fungal infections such as contamination of surfaces, poor hand hygiene, and indoor air pollution. Patients administered immunosuppressive drugs for prolonged periods are at a higher risk for infection 6,13. A recent study highlights an increased incidence of superficial fungal infections in diabetes patients 14. This study included many individuals at risk of fungal infections (patients with diabetes, asthma, organ transplant, chronic diseases, thyroid disease, irritable bowel syndrome, and allergy). In this study, only a few participants had fungal infections; two of them were in the class of risk group. However, population size was not enough to evaluate their exposure risk to fungal disease. The prevalence of fungal infections among all participants in this study showed a result of 19.2%. Participants with risk disease showed a prevalence of 26.3% for fungal infections. However, the study investigation and results were all based on the data obtained from the 212 samples collected from participants and the questionnaire data so we were able to identify only superficial fungal infection without systemic fungal infection; based on this, clinical specimens analysis are recommended in order to identify systemic fungal infection or conduct data collection analysis study from hospitals to obtain patient history whether a previous systemic or superficial fungal infection were acquired or not.

Nine culture plates out of 212 were negative using Sabouraoud Dextrose Agar, which further confirmed the limitation of routine culture not supporting the growth of all types of fungi. Other methods such as Polymerase chain reaction (PCR) or sequencing are best to enhance the detection and allow comprehensive analysis. Many studies highlighted the superiority of PCR over culture in patient samples and environmental studies. Ross et al, found that 25% of negative cultures were found to be positive for dermatophytes using PCR 15. Another study showed, indoor dust samples were examined using mould-specific quantitative PCR (MSQPCR) 16. This study focused on fungal presence in an indoor environment during wintertime when allergic symptoms are higher. Yet, other seasons may show slight variations. Geographical location and climate are important determinants that affect the biodiversity of yeasts and moulds. In this study, the total number of yeast isolates was higher than that of mould isolates. These findings could be explained considering humidity as a factor, as moulds prefer to grow under high humidity. It has been shown that several moulds begin to grow when humidity exceeds 55% 17, while the highest growth was recorded when humidity ranged below 70% 18. During the study conduction, indoor humidity was not recorded in each home. However, the city’s highest humidity recorded between December 2019 and March 2020 was 38%. Although the temperature range during the study was suitable for the growth of both moulds and yeasts, temperature alone as a factor was insufficient to promote higher growth of moulds 19.

This study reported Aspergillus spp. and Rhizopus spp. being the most common mould found in Medina. Several characteristic features enabled Aspergillus spp. to be the most dominant, such as tolerance to high temperatures, pH and other stress conditions 20. Rhizopus spp. is known as spoilage microorganism and is commonly found in agricultural products and food fermentation process 21. Another finding was reported by Aquino et al, in which that the most frequent fungi present in the air filter of vacuum cleaners were environmental contaminants such as Rhizopus spp. and Aspergillus spp. 8.

Among Aspergillus spp., Each district showed varied distribution. A. niger and A. terreus complex were the highest in the North region, A. terreus complex was the highest in the South and West regions, and A. candidus was the highest in the East region. These variations may be due to different factors and influences such as indoor sources (ventilation, building statue like wall cracks, exposed places, and dampness) besides participants' occupation and pet's reservoirs. In this study, we found that the North region ranked with the highest number of household pets. Aspergillus spp. Wide range of thermotolerance increases its dominance among other mould species. For example, temperatures (20-25°C) allowed A. versicolor to grow. As for the other species of Aspergillus, preferred to grow at (37°C). A. niger is known as an outdoor fungus that prefers warm soil, this may indicate their increased transfer from outdoor sources. A. niger was only detected in the Northern and Southern regions which was probably due to the increase of outdoor playgrounds and parks in the Medina region. The abundance of these species in the Northern and Southern regions may also be due to the presence of farms and suitable groundwater providing an optimal growth environment 20. In contrast, A. oryzae, A. ustus complex, and A. versicolor complex are known as indoor fungi that are usually found in damp environments (xerophilic area) and food products. Since Eastern and Western regions have limited farms and plants (outdoor sources), the predominant fungi in these regions were indoor fungi, such as A. oryzae, A. ustus complex, and A. versicolor complex 22,23.

The most common yeast species observed was Candida spp. However, further testing to differentiation between Candida albicans and other Candida spp. could not be completed due to the COVID-19 pandemic and quarantine. Nowadays, environmental isolates and non-pathogenic fungi are becoming a threat. Both aspergillosis and mucormycosis are ranked the highest reported fungal infection in immunocompromised patients 24. Coronavirus disease 2019 (COVID-19) has been associated with a wide range of opportunistic fungal infections. More importantly, aspergillosis has been reported as the main fungal pathogen for co-infection in COVID-19 patients 25.

1. Conclusion

This study evaluated the diversity of the environmental fungal isolates across Medina. We showed that household dust was an important source of exposure to environmental fungi. The results of this study indicate that Aspergillus spp. and Candida spp. were the most dominant species in Medina. However, further efforts are needed to examine the dust samples using other diagnostic methods such as PCR, sequencing to ensure that all fungal strains, even rare and unculturable ones are detectable. Although this study focused on investigating environmental samples, further studies on establishing a correlation between household environments of patients, exposure to fungal strains and health are necessary. This study will increase awareness about the spectrum of fungal species across the Medina region and promote better hygiene practices and periodic decontamination of the households. The findings of this study have several important implications for future practice which help the development of appropriate control strategies to eliminate fungal exposure for immunocompromised, COVID-19 and other patients at risk, further improving public behaviour and health.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or non-profit sectors.

Acknowledgments

We would like to express our thanks to the University of Taibah and the College of Applied Medical Sciences for giving access to the appropriate laboratory and to Ms. Rania Osta for her technical support.

Conflict of Interest

The authors declare no potential conflicts of interest.

Author Contributions

Project conceptualisation, methodology, resources, validation, project administration, and supervision: Aiah Khateb

Formal analysis, investigation, and data curation: Aiah Khateb, Fadwa Mohammed, Fai Alsubhi, Razan Abdullah and Saniyya Alkatib

Writing—original draft preparation: Fadwa Mohammed, Fai Alsubhi, Razan Abdullah and Saniyya Alkatib

Rewriting—review and editing: Aiah Khateb

Visualisation: Fadwa Mohammed and Razan Abdullah

Financial Disclosure Statement: This work was self-funded by the main PI and no specific funding for this work was received. All authors have read and agreed to the publication of this version of the manuscript. All authors declare that no competing interests exist.

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