Abdulrahman et al (2020): DETECTION OF PULMONARY BLASTOMYCOSIS IN PATIENTS Nov 2020 Vol. 23 Issue 19

DETECTION OF PULMONARY BLASTOMYCOSIS IN PATIENTS WITH DIFFERENT RESPIRATORY DISEASES USING CONVENTIONAL AND MOLECULAR METHODS

Zahraa Q. Abdulrahman1*, Azhar A. F. Al-Attraqchi1 , Haider N. D. Al-Taee2, Haider F. Ghazi1 1. College of Medicine, Al-Nahrain University, Iraq

2. Al-Emamein Al-Kadhimein Medical City, Ministry of Health, Iraq

*Corresponding authorE-mail:[email protected]

ABSTRACT Blastomycosis is a rare but important fungal infection cause by . is the most common manifestation and the is almost always the organ initially infected. The performance of blastomycosis diagnosis using microscopic examination and culture are highly dependent on the quality of the clinical specimens and the experience of the laboratory personnel. Moreover, these classical methods have previously shown lower sensitivity in fungal detection compared to molecular methods.Determination the frequency of pulmonary blastomycosis in patients with respiratory symptoms by using conventional (Wet mounting, culture and cytology) and molecular methods (conventional PCR and real time PCR), then comparing between conventional and molecular methods concerning sensitivity and specificity.This study involved 132patients who attended Baghdad Teaching Hospital, Al-Emamein Al- Kadhimein Medical City, Al-Amal National Hospital and Chest and Respiratory Diseases Institute/ Baghdad Medical City who enrolled in this cross sectional study from September(2018) to the end of August (2019). Sputum, bronchial washings, bronchoalveolar lavage (BAL) or pleural fluid were collected from patients with different age groups of both males and females all were diagnosed clinically with pulmonary symptoms.Sample (132) were obtained from patients in dry and clean screw cupped bottles. Fresh samples were divided into two parts, for mycological detection by conventional (Wet mounting, culture and cytology) and molecular methods (conventional PCR and real time PCR).Laboratory diagnosis of blastomycosis been done using different procedures, starting with conventional methods (culture, wet mounting and cytology) followed by two molecular methods. The conventional PCR with ITS1 and ITS4 gene was showed nonspecific result with gel electrophoresis of eighteen positive samples with amplicon size of approximated 600 bp which were recorded as suspectedBlastomycesdermatitidis. It have been recorded that all suspected cases (18/18) were recovered from sputum only. According to wet mount, only one out of eighteen was matches the suspected B.dermatitidis. In case of cytology and PAD culture, none of the eighteen samples were recognized as Blastomyces spp. Concerning qPCR using BAD1 gene, it was seen there was no specific band for Blastomyces hence, these results was negative.This study found no evidence of blastomycosis in samples from Iraqipatients with respiratory symptoms based on BAD1 gene detection using real time PCR and culture, however other tests like wet mountand ITS1-4 based conventional PCR gave false positive results.

Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.232110

Abdulrahman et al (2020): DETECTION OF PULMONARY BLASTOMYCOSIS IN PATIENTS Nov 2020 Vol. 23 Issue 19

Keywords:Blastomycosis, ITS1, ITS4, BAD1, conventional PCR, Real time PCR

How to cite this article: Abdulrahman ZQ, Al-Attraqchi AAF, et al (2020): Detection of pulmonary blastomycosis in patients with different respiratory diseases using conventional and molecular methods, Ann Trop Med & Public Health; 23(S19): SP232110. DOI: http://doi.org/10.36295/ASRO.2020.232110

INTRODUCTION Blastomycosis is a systemic pyogranulomatous infection that arises after inhalation of the conidia of the thermally dimorphic fungus Blastomyces dermatitidis, which is endemic to North America, particularly in the states bordering the Mississippi and Ohio rivers, the Great Lakes, and the St. Lawrence Seaway(1). B. dermatitidis, the etiologic agent of blastomycosis, is a thermally dimorphic fungus that grows as a filamentous in the environment at 25ºC and as budding in human tissue at 37ºC. Infection with B. dermatitidis causes a broad array of clinical manifestations ranging from asymptomatic infection to fulminant sepsis with acute respiratory distress syndrome and death. B. dermatitidis can infect almost any organ in the body, but has a predilection for and skin(2). Blastomycosis is one of the great mimickers in medicine, verrucous cutaneous blastomycosis resembles malignancy and mass-like lung opacities due to B.dermatitidis often are confused with cancer, blastomycosis also clinically indistinguishable from tuberculosis(3). Due to the nonspecific clinical and radiographic manifestations, a delay in the diagnosis as well as starting of therapy for blastomycosis is common. Even in hyperendemic areas, as few as 5% of patients are correctly diagnosed at initial presentation and diagnostic delays longer than 1 month can occur in more than 50% of patients(4–6). Diagnostic performance of microscopic examination and culture are highly dependent on the quality of the clinical specimens and the experience of the laboratory personnel. Moreover, these classical methods have previously shown lower sensitivity in fungal detection compared to molecular methods(7,8). Molecular diagnostic systems have been developed to facilitate the rapid 18S rDNA and ITS-regions are commonly used for diagnostic polymerase chain reaction (PCR) assays(9,10). Since several, up to hundreds of copies are present per genome, targeting these genes guarantees high sensitivity of the PCR assay. However, ribosomal genes are conserved and the primers amplify DNA from several fungal species, genera or even several fungal families. Thus, a limited specificity can be anticipated.As a consequence, PCR products have to be further specified by sequencing(11). Real-time PCR assay targeting BAD1 gene is highly sensitive and specific for the identification of B.dermatitidis in culture and in clinical specimens. As considerable genetic diversity is observed among B.dermatitidis strains, the real-time PCR assay is a valuable diagnostic tool for correct identification of all known haplotypes of B.dermatitidis. Moreover, DNA extraction to final analysis can be completed in 5 h, which makes this a rapid assay, with a turnaround time of less than 1 day. The availability of these two real- time PCR assays is likely to advance rapid identification of B.dermatitidis in clinical laboratories(12). So, this study aimed to investigate the presence of blastomycosis in Iraqi patient by using conventional and molecular methodsand Compare between them concerning sensitivity and specificity.

Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.232110

Abdulrahman et al (2020): DETECTION OF PULMONARY BLASTOMYCOSIS IN PATIENTS Nov 2020 Vol. 23 Issue 19

MATERIALS AND METHODS This study involved 132patients suffering from pulmonary diseases concomitant with pulmonary tuberculosis, lung cancer, pneumonia, chronic obstructive pulmonary disease, uncontrolled diabetes mellitus, hematological malignancy, solid cancer and acute bronchitis who attended Baghdad Teaching Hospital, Al-Emamein Al-Kadhimein Medical City, Al-Amal National Hospital and Chest and Respiratory Diseases Institute/ Baghdad Medical City who enrolled in this cross sectional study from September (2018) to the end of August (2019). Sputum, bronchial washings, bronchoalveolar lavage (BAL) or pleural fluid were collected from patients with different age groups of both males and females all were diagnosed clinically with pulmonary symptoms confirmed by computed tomography (CT) scan and chest x- rays.Sample (123) were obtained from patients in dry and clean screw cupped bottles. Fresh samples were divided into two parts, for mycological detection by conventional (Wet mounting, culture and cytology) and molecular methods (conventional PCR and real time PCR). Specimen collection Sputum specimens were collected from each patient in clean dry containers. These were labeled with case number and name. All specimens were transported to the laboratory for processing and investigations at the same day,each sputum sample was shaken by a vortex mixer for (3–5) minutes for homogenization. The bronchial wash and pleural fluid samples were centrifuged for five minutes, and then the sediment was used for culture on Potato Dextrose Agar (PDA) at 30ºC for 4 weeks and for direct microscopic examination by using lactophenol cotton blue (LPCB) and Periodic acid–Schiff (PAS) then the samples were stored at −20°C for DNA extraction.

Conventional methods for diagnosis of Blastomyces dermatitidis

Lactophenol Cotton Blue Stain (LPCB) (direct examination): Lactophenol cotton blue stain (LPCB) prepared according to the manufacture instructions by Placing 1 drop of the stain on clean slide then a loop full of the sample was taken and mixed, coverslip was applied over staining sample and visualize under the microscope. Cytology by Periodic Acid Schiff (PAS) (direct examination):

Periodic acid Schiff (PAS) prepared according to the manufacture instructions(13).

Culture method

Samples were directly cultured on PDA in anaerobic condition at 30ºc for up to four week. After observe the growth, firstly fungal colonies diagnosed depending on cultural characteristics on PDA that include shape, color, size, then microscopically examined on the slide after staining with lactophenol cotton blue in order to determine the morphology of the isolated fungi(14). Laboratory protocol for manual purification of DNA from respiratory samples.

Sputum and bronchial wash liquefied by combined with equal volume of the DTT solution to yield a final concentration of 50 mg/ml then mixed well by vortex mixer to homogenies(15). Then, fungal DNA was extracted and purified for 100 samples done according to the manufacturer information(16).

Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.232110

Abdulrahman et al (2020): DETECTION OF PULMONARY BLASTOMYCOSIS IN PATIENTS Nov 2020 Vol. 23 Issue 19

Identification of fungi in samples by conventional PCR using ITS1 and ITS4

The nuclear ribosomal ITS1-5.8S-ITS2 region will be amplified with the conventional PCR using ITS1 and ITS4 specific primers. Different product sizes will be identified then used to identify pan fungal DNA in DNA sample the primers sequences were used for the amplification of fungal DNA which were selected according to MARK, et al ITS1"TCCGTAGGTGAACCTGCGG"ITS4"TCCTCCGCTTATTGATATGC" (17).

Identification of Blastomyces dermatitidis by Real Time PCR

Primers The identification of Blastomyces dermatitidis by real time PCR was done using four different designed primer ofBlastomycesdermatitidis (histidine kinase gene, WI-1 adhesin gene, Blastomyces yeast phase specific protein 1 and 18S ribosomal RNA gene) have been taken from NCBI website in FASTA format then designed by IDT website, table (1, 2, 3, 4).

Table 1: The name, Primer sequences of B. dermatitidis histidine kinase (HK) gene:

Gene Name: HK Sequence Tm Amplicon

Forward Primer GGATGTCCAAATGCCTGTTATG 61.668

Reverse Primer GTGCGGTCAAAGCGATAATG 61.669

Product 107

Table 2: The name, Primer sequences ofB.dermatitidisWI-1 adhesin (BAD1) gene: Gene Name: BAD1 Sequence Tm Amplicon

Forward Primer TGATTGGAAGCTCTGGGATAAG 61.813

Reverse Primer GTAGGAGTCCTTGCACCATTTA 61.952

Product 112

Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.232110

Abdulrahman et al (2020): DETECTION OF PULMONARY BLASTOMYCOSIS IN PATIENTS Nov 2020 Vol. 23 Issue 19

Table 3: The name, Primer sequences of B. dermatitidis blastomyces yeast phase-specific protein 1 (bys1) gene: Gene Name: bys1 Sequence Tm Amplicon

Forward Primer CACGAACGACATTCCGCTATAA 62.552

Reverse Primer GAGGGCTTGAGAGTGAGTTTATG 62.477

Product 112

Table 4: The name, Primer sequences of B.dermatitidis18S ribosomal RNA gene Gene Name: 18S Sequence Tm Amplicon

Forward Primer TTGGGTTTAAAGGGTACCTAGAC 61.727

Reverse Primer CTTCGCCGTTATCAGTCCTATT 61.949

Product 136

Positive control

Positive control had been designed for BAD1 primer only with the flowing sequence:

"TCATGAGAAGTATGATTGGAAGCTCTGGGATAAGTGGTGCAAGGACTTCTACAACTGCGAAT GGGACTCTTCTCATGAGAAGTATGATTGGGAGCTCTGGGATAAGTGGTGCAAGGATCCCTACA ACTGCGAATGGGACTCTTCTCATGAGAAGTATGATTGGGAGCTCT"

Standard (positive control) preparation and dilution: According to the standard manufacturer, lyophilized positive control were suspended in nuclease free water (Promega, U.S.A) to reach recommended volume by the protocol (Bioneer, Korea). Nuclease free water was added to reach a standard concentration of 100 pmol/µl, in the stock solution, and then further 10 fold dilution was done with nuclease free water.

Optimization of primers Several trials of thermal cycles were pursued using SYBER Green master mix (SINTOL", Russian, Cat, No M-427) to optimize the PCR reaction to obtain the optimal annealing temperature for all of the primers, while only BAD1 primer had been stable in 60ºC annealing temperature table (5), so the positive control design for BAD1, then the qPCR amplification for 10 fold dilution (1:1, 1:10, 1:100, 1:1000, 1:100000) of positive control, no template control (NTC), buffer control and non-primer control had been done, figure (1) and (2). The cycle threshold (CT) of positive result should be below NTC and above 1:10000 dilution CT.

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Abdulrahman et al (2020): DETECTION OF PULMONARY BLASTOMYCOSIS IN PATIENTS Nov 2020 Vol. 23 Issue 19

Table 5: Real –Time PCR Thermal Profile

Steps Temperature Time Repeats

1- Initial denaturing 95 c 5 min 1

2-Denaturing 95 c 30 sec

40 3-Annealing 60 c 30 sec

4-extension 72 c

95 c 1min 1 60 c 30 sec 5-Malting curve analysis 95 c 30 sec

Figure 1: Data table of BAD1 gene primers, positive control, non-template control (NTC) and sybr negative control optimization results.

Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.232110

Abdulrahman et al (2020): DETECTION OF PULMONARY BLASTOMYCOSIS IN PATIENTS Nov 2020 Vol. 23 Issue 19

Figure 2: Amplification plots at the end of qPCR run for the positive and negative controls.

Statistical analysis

The statistical analysis of this prospective study was performed with the statistical package for social sciences (SPSS) 20.0 was and Microsoft Excel 2013. Numerical data were described as mean and standard deviation, Categorical data were described as count and percentage. Chi-square test or fisher exact test used to estimate the association between variables. The lower level of accepted statistically significant difference is bellow or equal to 0.05 (18).

RESULTS AND DISCUSSION

Concerning the conventional method results that had shown in table (6), there was no isolated B.dermatitidis in culture (PDA) which is the gold standard method for blastomycosis diagnosis, hence the (PDA) is prefect choice to isolate B.dermatitidis as approved by Brown, et al. (2012) that all Blastomyces strain grown on it(19). Also another pathogenic fungal species has been isolated on PDA including Aspergillusspp,Candidaspp,Emmonsiaspp, ScedosporiumsppandTrichosporonspp which were recorded with pulmonary infection, since pulmonary can be found in immunocompromised patients with malignancies and those with chronic obstructive pulmonary disease (COPD), also Emmonsiaspp can cause unusual pulmonary disease named adiaspiromycosis, theScedosporiumspp can cause various infections including pneumonia(20–22).

Trichosporonspp can cause pulmonary infection in immunocompromised patient especially those with hematological malignancies and its isolation further confirmed by giving positive urease test and this

Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.232110

Abdulrahman et al (2020): DETECTION OF PULMONARY BLASTOMYCOSIS IN PATIENTS Nov 2020 Vol. 23 Issue 19

agree with Colombo, et al. (2011) that mention that fungal morphology and urease test act a screening method for (23).

Candidaspp is considered as normal oropharyngeal flora and upper respiratory tract and can cause primary bronchopneumonia in severely debilitated patients with solid tumors(24).

Concerning molecular methods the amplification of internal transcribed spacer (ITS1-4) genes gave an evidence of good fungal DNA extraction method, the amplicon size resulted ITS1-4 amplification was different depending on fungal species. It was found that 18% of sample amplification product with 600 pb which was suspected as Blastomyces dermatitis and other fungal species including Histoplasmacapsulatum, , Coccidioidesimmitis and Candida spp that gave the same amplification product size (600bp) as approved by Mark, et al. (2001), however detection by ITSgene still nonspecific and need to be confirmed by sequencing or by real time PCR(11,25).

Real time PCR using specific Blastomyces gene (BAD1) is more specific and sensitive method for detection of blastomycosis in culture and clinical samples(26).

Table 6: A comparative table display of wet mount, cytology and culture as detective methods for fungi in different samples of respiratory tract

Frequency Percent

Blastomyctes 1 0.76%

Aspergillus 1 0.76%

Emmonsia 1 0.76% Fungi (29) Mucor 1 0.76% Wet mount Candida 12 9.09%

Trichosporon 13 9.85%

Actinomyctes (1) 1 0.76%

Negative (102) 102 77

Aspergillus 1 0.76% Fungi (3) Histoplasma 2 1.52%

Cytology Actinomyctes (1) 1 0.76%

Negative 128 96.97%

Total 132 100%

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Aspergillus 3 2.27%

Candida 35 26.52%

Emmonsia 2 1.52%

PDA Sacaromyces 1 0.76%

Sedospurium 5 3.79%

Trichosporon 11 8.33%

Negative 75 56.82%

Total 132 100

I has been used different procedures for detection of blastomycosis and comparing between them, the conventional methods (culture, wet mounting and cytology) followed by two molecular methods (conventional PCR and real time PCR). The conventional PCR with ITS which is pan fungal gene gave nonspecific results in gel electrophoresis that show amplicon size of 600 pb which was recorded as suspectedBlastomycesdermatitidis as (18%), table (7) concerning this method is moresensitive comparing with conventional ones. Regarding direct microscopical examination by wet mounting, the result shown one sample stained with lactophenol cotton blue appeared with budding yeast consider to be suspected Blastomyces dermatitidis. even though it's still direct examination and need to be confirm by culture as well as cytology preparation and that can be confused with other fungi includingC.neoformans, C.immitis/C.posadasiiorH.capsulatum(27,28), moreover cytology and culture resulted with no evidence of Blastomyces and this result agree with Wagner, et al (2018) who approved that conventional methods have shown lower sensitivity in fungal detection compared to molecular methods regarding to fourteen sample showing negative result in conventional method while 2 of them have been positive in PCR using ITS gene(29).

However, the diagnosis by conventional PCR using ITS gene more sensitive than conventional method still not specific to identify fungal species it has been fallowed with the more specific method like sequencing or Qpcr(29). So, BAD1-qPCR which is the more sensitive and specific method was used for B. dermatitidis and that’s agree with study obtaining by Sidamonidz, et al (2012) who approved that qPCR assay targeting BAD1 gene is highly sensitive and specific as a diagnostic tool of B.dermatitidis in culture and in clinical samples. As considerable genetic diversity is observed among B.dermatitidis strains, real- time PCR assay is a valuable diagnostic tool for correct identification of all known haplotypes of B.dermatitidis. Moreover, DNA extraction to final analysis can be completed in 5 h, which makes this a rapid assay, with a turnaround time of less than 1 day(26).

Table 7:A comparative tablefor diagnosis of different fungi with suspected Blastomyces dermatitidis. Suspected 600bp Total Positive (%) Negative (%)

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BAL 0 (0) 5 (100) 5 Bronchial wash 0 (0) 9 (100) 9 Sample type P.F. 0 (0) 2 (100) 2 Sputum 18 (21.43) 66 (78.57) 84 Blastomyctes 1 (100) 0 (0) 1 Mucor 1 (100) 0 (0) 1 Actinomyctes 0 (0) 1 (100) 1 Wet mount Trichosporon 4 (30.77) 9 (69.23) 13 Candida 1 (14.29) 6 (85.71) 7 Emmonsia 0 (0) 1 (100) 1 Negative 11 (14.47) 65 (85.53) 76 Actinomyctes 1 (100) 0 (0) 1 Cytology Histoplasma 1 (100) 0 (0) 1 Negative 16 (16.33) 82 (83.67) 98 Positive 8 (17.02) 39 (82.98) 47 PDA Negative 10 (18.87) 43 (81.13) 53 Aspergillus 0 (0) 1 (100) 1 Emmonsia 0 (0) 1 (100) 1 Sacaromyces 0 (0) 1 (100) 1 LPCB Sedospurium 1 (20) 4 (80) 5 Trichosporon 2 (18.18) 9 (81.82) 11 Candida 5 (17.24) 24 (82.76) 29 Negative 10 (19.23) 42 (80.77) 52 Positive 0 (0) 0 (0) 0 BAD1 Negative 18 (18) 82 (82) 100

CONCLUSION

For this study and become we obtained no evidenceof Blastomycesdermatitidisout of 132 based on BAD1 gene detection using real time PCR and cultureHowever other tests like other conventional methods and ITS1-4 conventional PCR might give false positive results.

ACKNOWLEDGMENTS

We are the authors grateful to all staff members of the Medical Microbiology Department, Al- Nahrain University/College of Medicine for their help and cooperation. Special thanks for Assistant ProfessorDr. Haider Faisal for making whole statistical analysis of this study and primers designed for this project.

Annals of Tropical Medicine & Public Health http://doi.org/10.36295/ASRO.2020.232110

Abdulrahman et al (2020): DETECTION OF PULMONARY BLASTOMYCOSIS IN PATIENTS Nov 2020 Vol. 23 Issue 19

ETHICAL CLEARANCE

The Research Ethical Committee at scientific research by ethical approval of both environmental and health and higher education and scientific research ministries in Iraq

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

FUNDING: Self-funding

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