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Detection of opportunistic fungi from the bronchoalveolar lavage specimens of patients with pulmonary diseases
Sahar Kianipour1, Parvin Dehghan1, Mohammad Emami Ardestani2
1 Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Internal Medicine, School of Medicine, Al-Zahra Hospital, Isfahan University of Medical Sciences, Isfahan, Iran
| Date of Submission | 05-Sep-2022 |
| Date of Acceptance | 29-Oct-2022 |
| Date of Web Publication | 20-Jul-2023 |
Correspondence Address:
Dr. Parvin Dehghan
Department of Medical Mycology and Parasitology, School of Medicine, Isfahan University of Medical Sciences, Isfahan
Iran
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/abr.abr_297_22
Background: Opportunistic fungi are a constantly evolving group of pathogens that become active when the immune system is compromised, begin to multiply, and soon overwhelm the weakened immune system. This study was performed to evaluate the number of opportunistic fungi in bronchoalveolar lavage (BAL) samples of patients with pulmonary diseases.
Materials and Methods: After receiving patients' consent and demographic forms, a total of 120 BAL samples were taken by a pulmonary physician. The etiologic agents were identified by standard morphological and molecular methods. Yeast cells were counted on culture media, and direct smears were precisely examined for the presence of yeasts elements, Pneumocystis, and filamentous fungi.
Results: In this study, 29 (24.1%) patients showed positive direct smears for yeast elements in their BAL samples. The mean colony count of yeasts was 42,000 (CFU/mL) on culture media. Six (5%) species of filamentous fungi, including three (2.5%) isolates of Penicillium species (P. variabile, P. glabrum, and P. thomii), two (1.67%) Aspergillus species (A. flavus and A. fumigatus), 1 case (0.83%) Pseudallescheria boydii were detected. Seven cases (5.83%) of Pneumocystis cysts were observed in the direct smears stained with Giemsa. Identification of all fungi confirmed by molecular or sequencing methods.
Conclusions: Due to the presence of a large number of fungi in the BAL samples and possible physical interference with the selected drugs for treatment, we draw the attention of pulmonologists to this important issue. Rapid diagnosis of fungal infections is essential to optimize treatments and outcomes.
Keywords: Aspergillus, Candida, Penicillium, Pneumocystis, Scedosporium boydii
How to cite this article:
Kianipour S, Dehghan P, Emami Ardestani M. Detection of opportunistic fungi from the bronchoalveolar lavage specimens of patients with pulmonary diseases. Adv Biomed Res 2023;12:176 |
How to cite this URL:
Kianipour S, Dehghan P, Emami Ardestani M. Detection of opportunistic fungi from the bronchoalveolar lavage specimens of patients with pulmonary diseases. Adv Biomed Res [serial online] 2023 [cited 2023 Sep 26];12:176. Available from: https://www.advbiores.net/text.asp?2023/12/1/176/382067 |
| Introduction | |  |
Saprophytic fungal spores are found everywhere in the habitat. The existence of sufficient moisture and nutrients in the respiratory tract provides proper conditions for the growth of fungal spores. Fungal growth and multiplication of spores occur more in immunosuppressed patients than in healthy individuals. So, this condition can provide fungal infections and even invasion of the microorganism into the other organs.[1] The genus Aspergillus is the most important opportunistic filamentous fungi, which causes a wide range of pulmonary diseases. Patients with cystic fibrosis are predisposed to allergic bronchopulmonary aspergillosis (ABPA), and patients with tuberculosis and other pulmonary anatomical defects are predisposed to form fungus balls or colonization of the mold.[2] In addition, the other fungal yeasts or bacteria, which are normal flora in the human body, can overgrowth to be pathogenic in predisposing individuals. This process may lead to infections, colonialization, and biofilm formation in the lungs and thus interfere with targeted drug delivery.[3],[4] Another opportunistic fungal agent in the lung is Pneumocystis, which cannot grow outside the body (in vitro), and its main host is not known, but high-risk patients such as people with HIV (AIDS) and premature infants can cause pneumocystosis. This extracellular organism grows inside the lung alveoli; its lifecycle has an infective eight ascospores ascus, and a cell wall consisting of the major surface glycoprotein can stain by Giemsa.[5],[6],[7] Because of the lack of β-glucans, the trophic form is poorly recognized by phagocytic cells, resulting in insufficient CD4+T cell response. Nowadays, scientists believe this organism is more like fungi than protozoa, although the terms used to describe the organism in tissue may still refer to trophozoites and cysts.[8]
Diagnosis of fungal lesions in the lung depends on the type and quality of sampling. Bronchoalveolar lavage (BAL) is an important adjunct to the diagnosis of pulmonary and disseminated fungal infections. Culture is the gold standard for diagnosis in many instances, but the cytologic and morphologic analysis is often diagnostic, and a biopsy may need to be performed in complicated patients.[8] Serological methods such as the galactomannan antigen test for detecting Aspergillus species antigen and the β-glucan test for Candida species have been applied. On the other hand, many molecular methods have been developed to identify the causative agents of fungal infections.[9],[10] DNA detection related to Pneumocystis in the BAL sample has 91% sensitivity and 96% specificity.[11] Following the increasing prevalence of underlying diseases in recent years with changes in weather conditions or air pollution, the possibility of increasing pulmonary fungal infections due to increasing environmental spore dispersal has been increased in patients with weakened immune systems or anatomical lung failure. Biofilm formation in yeast or colonization with other microorganisms can interfere with drug delivery to the organs or cause drug resistance to other microorganisms. Therefore, it is important to detect the fungal agents in different clinical samples, even if they do not lead to fungal sepsis.
In the present study, BAL samples of people with lung diseases were evaluated for detecting fungal elements (molds, yeasts, or Pneumocystis) by direct examination and culture methods. Also, the number of fungal species in direct smears and culture media was determined using morphological and molecular methods to provide more appropriate treatment.
| Materials and Methods | | |
From October 2016 to May 2017, the BAL samples of 120 patients (62 males and 58 females) with the pulmonary disease were collected after a physical examination by a pulmonologist. All patients' consent forms demographic and predisposing factors registered. The present study has an ethics code number (IR.MUI.REC.1395.3.78). The sample size taken from BAL for each patient was from 100 to 300 cc and was calculated in colony count at a rate of 1 mL.
Direct examination and culture
For microscopic observation, the BAL samples with a volume of 100 to 300 mL were obtained by the physician. Then 10 mL of each sample was centrifuged for 5 min at 5000 rpm. The resulting precipitate was used for culture on either Sabouraud Dextrose Agar (SDA) (Biolife, Italy) or SDA with Chloramphenicol and incubated at 25°C and 35°C. From the remaining sediment, two direct smears were prepared using 10% KOH and Giemsa staining. Both slides were examined for the presence of fungal elements by light microscopy. After 24 to 72 hours of incubation, the culture media were evaluated for the growth of microorganisms. The culture media with mold growth were evaluated for morphology after one week. The colony count was performed for yeast cultures accounting for 1 mL of BAL specimens.
Counting yeast colonies
To find the amount of yeast colony in the sample (CFU/mL), 100 μL of the sample was cultured on SDA as previously reported.[12] If the number of yeasts per milliliter of the BAL sample was more than 10,000 or if observing pseudohypha and blastoconidiain in direct smears, they would be included in the study.[13] Fungal colonies were kept in sterile distilled water until further molecular tests.
DNA extraction of filamentous fungi by phenol-chloroform method
For the DNA extraction of filamentous fungi, 300 μL of glass beads and 300 μL of lysis buffer with a small amount of the fresh detected filamentous colony were added to a 1.5 mL microtube. 300 μL of phenol-chloroform was added to the mixture, and the micro-tubes vortexed for 3 min and centrifuged for 5 min at 5000 rpm. The supernatant was transferred to a new microtube. An equal amount of chloroform was added to the previous suspension and centrifuged at 5000 rpm for 5 min. The supernatant was transferred to the new microtube and added 2.5 times the volume of liquid absolute alcohol and 0.1 volume of 3 M sodium acetate with pH 5.2 to the liquid and placed in the microtube for 30 min in the freezer at −18°C. Then the tubes were centrifuged for 10 min at 10,000 rpm. The supernatant was drained, and 500 μL of 70% alcohol was added to the precipitate and centrifuged for 10 min at 10,000 rpm. The supernatant was drained completely and solved in 50 μL of DW.[14]
PCR reaction for filamentous fungi
The total volume of the reaction in this experiment was 30 μL as follows (premix 2×, 15 μL, ITS1 primer 0.5 μL at 25 picomols, ITS4 primer 0.5 μL at 25 picomols, sterile distilled water at 12 μL, DNA at 2 μL). The temperature cycle included (95°C for 5 min, 94°C for 30 seconds, 55°C for 45 s, 72°C for 45 s (35 cycles), and finally, 72°C was performed for 5 min. The sequence of primers used in the reaction is as follows. These primers amplify the ribosomal DNA fragments of ITS2, 5.8S, and ITS1.
ITS1= (5′TCC GTA GGT GAA CCT TGC GG3′).
ITS4= (5′TCC GCT TAT TGA TAT GC3′).
Negative and positive controls were entered. Electrophoresis with 1% gel agarose was used to observe the amplified bands. For species of the genus Aspergillus, the bandwidth is about 600 bp.[15] The PCR products of all isolated filamentous fungi, which were identified morphologically, were confirmed by the sequencing method.
Additional tests to identify the yeast species
Additional tests were performed to identify yeast species isolated from the lungs and have already been described in detail.[12] Molecular methods were used to confirm the identification of yeasts more accurately after doing morphological tests in different culture media. Briefly, in the molecular method, DNA was extracted by boiling method, and after doing a PCR reaction with universal primers ITS1 and ITS4, the amplified bands were treated using MSP1 enzyme by RFLP - PCR method and electrophoresed. The duplex-PCR method was used to differentiate Candida albicans from Candida dubliniensis.[12]
Complementary tests to identify Pneumocystis
Direct smears were taken from the BAL samples; sediment was fixed with methanol and stained by Giemsa. The prepared smears were thoroughly examined to find the cysts of Pneumocystis.
Nested-PCR reaction to diagnose Pneumocystis
Seven BAL samples that were positive for the presence of Pneumocystis by Giemsa staining were also used to detect the organism by molecular techniques. The molecular method was performed using nested PCR on the mt LSU-rRNA region by Dr. Khodadadi using two pairs of primers.[16] Negative and positive controls (Pneumocystis DNA) were included.
Electrophoresis: Electrophoresis was performed using 1.5% gel agarose. The amplified PCR band should be 332 bp to confirm the presence of Pneumocystis.[16]
| Results | | |
In this study, 120 BAL samples from 120 patients referred to Al-Zahra Hospital in Isfahan from November 2016 to May 2017 were examined. Patients included 62 males and 58 females and ranged in age from 13 to 97 years. The mean age was 57 years. As shown in [Table 1], out of 120 studied BAL specimens, six cases of filamentous fungi and seven cases of Pneumocystis cysts were observed in direct smears. It is noteworthy that only 29 BAL samples containing yeast with a count of more than 10,000 (CFU/mL) were included in the study.[12] Among these samples, there were samples with more than a unique fungal agent. [Table 1] shows the frequency distribution of Pneumocystis and mycelial fungi detected by direct examination or molecular methods of BAL samples in patients with different predisposing factors. | Table 1: Distribution of Pneumocystis and mycelial fungi detected by direct examination or molecular methods of BAL samples in patients with different predisposing factors
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According to the Chi-square test, the relative abundance of yeast fungi in smokers and non-smokers had a significant difference (P = 0.003), and the relative abundance of yeast fungi in cancer and non-cancer patients had a significant difference (P = 0.002) and also the relative frequency of filamentous fungi in rheumatoid arthritis, and non-rheumatoid arthritis was significantly different (P = 0.017).
Results of direct exam and culture of BAL samples
The results of direct exams and culture of BAL samples regarding the isolated candida yeasts have been detailed in the previous report, [Figure 1]. | Figure 1: Bronchoalveolar lavage (BAL) samples of two patients stained with Giemsa (×1000). The image on the right is a BAL sample containing a large number of blastoconidia and the image on the left shows the pseudohyphae and blastoconidia
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In Direct smears of six BAL specimens treated with 10% KOH and stained with Giemsa hyaline septate hyphae were observed [Figure 2]. Also, a very small number of cysts (1–3 cysts) containing eight separate nuclear intracystic bodies were observed in the direct smear stained with Giemsa in seven samples. | Figure 2: Right image shows the bronchoalveolar lavage sample of a patient with filamentous fungal mycelium in a Giemsa-stained slide (×1000) and the left image of the same sample treated with KOH (×400)
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Results of filamentous fungi molecular tests
Six filamentous fungal isolates identified by PCR were detected, and the results were observed on a 1% gel [Figure 3]. | Figure 3: PCR results on 1% gel agarose where M is marker 100 bp, N.C: negative control, 1-A. flavus, 2-A. fumigatus, 3-P. thomii, 4-P. glabrum, 5-P. boydii, 6-P. variable, P.C: positive control. Species identification is adapted after sequencing
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Results of molecular tests for yeasts identification
Based on molecular tests performed to differentiate the isolated yeasts in this study, PCR-RFLP methods, and to differentiate Candida albicans from Candida dubliniensis, the duplex-polymerase chain reaction method was used, and the results were reported in reference.[12] Accordingly, 29 samples containing yeast were identified, and in six of the BAL samples, more than one specific yeast was identified.
Results of molecular tests for Pneumocystis identification
Based on examination of direct smear stained with Giemsa, 1–3 cysts of Pneumocystis were seen in seven BAL samples. Nested-PCR molecular test was performed for these seven samples, but all samples showed negative by nested PCR.
| Discussion | | |
Many fungal spores in the air around us are inhaled daily. Because most people have adequate immunity, despite inhaling a small number of spores, the body defends itself well, and these particles are excreted by macrophages and other immune factors in the airways. However, the same fungal spores can grow and multiply in immunocompromised individuals by escaping from the immune system and, in some cases invading blood vessels.
One of the most important filamentous fungi that can be involved in the lungs is the genus Aspergillus, especially Aspergillus fumigatus.[1]
However, in tropical regions and our country, A. flavus is more isolated from air and soil than other species of Aspergillus.[17] Another species that can opportunistically affect the lungs is Pseudoalcheria boydii. It causes allergic bronchial disease and chronic lung damage that resembles aspergillosis. The spores of his organism are less abundant in the environment than Aspergillus and Penicillium. In the present study, two species of Aspergillus (A. fumigatus and A. flavus) were isolated from the BAL samples of two patients. Both samples showed positive results for Aspergillus in direct examination and cultures. The second case was a 59-year-old male addicted to drugs and cigarettes. Also, Pseudoalcheria boydii was isolated from the BAL specimen of a 59-year-old man with a history of rheumatoid arthritis and taking corticosteroids but unfortunately died before antifungal therapy. Penicillium species are less likely to cause infectious disease and are more likely to cause citrus and other food rot, but fungal spores are rarely involved in allergic diseases and asthma. The reason for observing more fungal elements of Penicillium in the BAL samples in the present study might be the presence of numerous spores of this fungus in the environment of Isfahan city.[17] In the present study, out of 120 BAL samples, six cases (5%) were involved by filamentous fungi; including 3 (2.5%) species of Penicillium (P. variabile, P. glabrum, and P. thomii), 2 (1.67%) species of Aspergillus (A. flavus and A. fumigatus), 1 (0.83%) isolate of Pseudallescheria boydii which confirmed by morphological and sequencing methods.
Pneumocystis cysts were observed in seven samples (5.83%) in direct smears stained with Giemsa. A very small number of one to three cysts were observed in the direct examination of BAL samples. Since the number of cysts in the BAL samples of patients was limited to one to three, it was considered normal flora and was not considered a pathogen. According to studies, most people have antibodies against pneumocystis early in life, and dealing with this microorganism is not far from the mind.[18],[19] This organism has received more attention since the recognition of AIDS disease, and the prevalence of the organism in people with AIDS is much higher.[20] In a study conducted for nine years on AIDS and non-AIDS patients suspected of pneumocystosis, they found a high rate of infection in non-AIDS individuals but with other predisposing factors.[21] In our study, none of the patients had AIDS but had other contributing factors such as cancer, use of antibiotics and corticosteroids, smoking and addiction, or a history of hospitalization. Considering the percentage rate obtained from observing cysts (with eight intracystic bodies) in direct smears stained with Giemsa but a negative nested PCR test for the same samples, observing such a small number of organisms in the BAL samples in the present study can be considered as the normal flora.[22],[23]
As reported in the previous study, the highest frequency of isolated yeasts was related to the Candida albicans/dubliniensis complex, which was differentiated by genotypic and phenotypic methods. In the recent study, C. albicans, similar to other studies, had the highest frequency in pulmonary BAL samples.[24] Studies have shown that the microbial flora of the respiratory tract has a significant positive relationship with the amount of microbial flora in the mouth. In the study of Abharian et al.,[25] which was performed on drug abusers in the Isfahan region, C. albicans, C. dublinensis, and C. glabrata had the highest prevalence in addicts, respectively, and like with the present study, there was significant relationship between opioid drugs and the amount of oral candida flora. Also, in another study in line with our study in Isfahan, which was performed on the mouths of HIV-positive patients, C. albicans had the highest prevalence at 4%, C. glabrata at 26.8%, C. dubliniensis at 17.1%, and other Candida species 12%.[26] In a study conducted by Javaheri et al.[27] on smokers and non-smokers, in line with the present study, the number of isolated organisms was higher in smokers. In our study, the relative frequency of yeast fungi in smokers and non-smokers was significantly different.
In a study conducted in India on people with pulmonary tuberculosis, their lungs were generally affected by different Candida species, mainly C. albicans (50%).[24] In a study on fungal colonization in Babol and Sari patients, the most common isolated fungi were Candida 64.7%, Aspergillus 19.3%, and other filamentous fungi like Penicillium. The increase in the number of filamentous fungal species in the north of Iran is probably related to the humid climate and fungal fauna in that region.[28]
In another study done on patients with cystic fibrosis, the prevalence of A. fumigatus, non-fumigatus spp, Trichospron spp, and Sedosporium spp. have reported 36.3, 26.1, and 2.1%, and 2.3%, respectively, while the prevalence of yeast was 28.5%.[29] One of the strengths of our study was that only samples with a positive direct exam were included in the study because the quantity of opportunistic microorganisms in the organ being tested is very important in interpreting the experiment.
Candida species are considered normal lung flora, and only biopsy specimens are of clinical importance.[30] Considering that all 29 isolates detected from the BAL specimens in the present study had a direct positive exam and also all these specimens had more than 10,000 CFU of Candida on culture media. Therefore, such a large number of yeasts in the lungs, even if they were colonized or formed biofilm, cannot be ignored. In vulvovaginal candidiasis or other skin lesions involved with Candida species, the appearance of pseudo-hypha and blastoconidia in direct exam indicates and causes symptoms such as itching, burning, and sometimes even allergic symptoms. In the lungs, the presence of these fungal agents will not be without clinical symptoms.[31],[32] According to a study done by Morrell et al.,[33] delay in treatment of the disease due to failure to diagnose the causative agent of the disease in invasive candidiasis infections is considered a risk factor for mortality. On the other hand, in the present study, the observation of fungal mycelium of Aspergillus and Pseudallescheria has proven their pathogenicity in the lungs.
| Conclusion | | |
The strength of the present study was performing the yeast colony count, observing and reporting the number of fungal elements in the direct smears, and differentiating etiologic agents by culture and molecular methods. Due to the presence of a large number of fungal elements in the BAL samples and possible interference in the absorption of selected drugs, we draw the attention of pulmonologists to this important issue. Rapid diagnosis of fungal infections is essential to optimize treatments and outcomes.
Ethics approval and consent to participate
All patients' consent forms demographic and predisposing factors registered. The present study has an ethics code number (IR.MUI.REC.1395.3.78).
Acknowledgments
We would like to thank the personnel of the Reference Pulmonary Clinic Center in Alzahra Hospital. Also, appreciate Dr. Khodadadi for performing the Nested-PCR test on seven BAL samples suspected of having Pneumocystis.
Financial support and sponsorship
The article was derived from the thesis (No. 395780). This study was conducted with a grant awarded by the Research Deputy of Isfahan University of Medical Sciences.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1]
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