Next Generation DNA Sequencing Diagnoses Fungal Etiology in Chronic Respiratory Disease, Clinical Response Supports Clinical Validity

Case Report

Austin J Clin Case Rep. 2021; 8(9): 1229.

Next Generation DNA Sequencing Diagnoses Fungal Etiology in Chronic Respiratory Disease, Clinical Response Supports Clinical Validity

Gordon G and Fry SE*

Fry Laboratories, Scottsdale, AZ, USA

*Corresponding author: Stephen E Fry, Fry Laboratories, LLC 14807 N. 73rd St. #103 Scottsdale, AZ, USA

Received: September 08, 2021; Accepted: October 06, 2021; Published: October 13, 2021

Abstract

A 71-year white male presented to pulmonologists with progressively worsening respiratory problems with a chronic productive cough, asthma symptoms and malaise. The patient was subjected to the traditional treatment of antibiotics, cough suppressants, and inhaled bronchodilator and corticosteroids. After multiple failed combinations of antibiotics, cough suppressants, and inhalers, the patient had a sputum sample, which was analyzed by special stains and targeted Next Generation DNA Sequencing (NGS). Fungal eukaryotic (18S) NGS identified by ‘best match’, Candidae parapsilosis and C. viswanathii and an organism in the Glomerulaceae family, Funnelformis mosseae. The patient was prescribed an antifungal in tandem with an antibiotic, Terbinafine and Doxycycline. The patient rapidly improved to his baseline. This communication communicates the potential of NGS microbial sequencing in pathogen identification in chronic pulmonary disease and improved clinical outcome.

Keywords: Candidae parapsilosis; C. viswanathii; Funneliformis mosseae; DNA Sequencing; Respiratory disease

Introduction

A 71-year-old white male presented to the pulmonologists for having a variety of respiratory problems. Besides having a known history of exercise-induced asthma, the patient reported suffering from allergic rhinitis, had a deviated nasal septum, and consistently produced an above average amount of sputum throughout his life. Over the past year, the patient started to develop increasing respiratory complaints: persistent productive cough, increasing eustachian tube dysfunction, laryngitis, inflammation of the nasal mucosal lining, and a persistent feverish feeling. His primary care physician prescribed Augmentin, which cleared up his symptoms for two weeks. He returned to his symptomatic state with complaint of a cough, sputum, plugged ears, and malaise. The patient received a course of Amoxicillin with symptomatic relief for two weeks and then a sharp remission. The patient switched to another primary care physician who prescribed doxycycline. The patient improved for two weeks and decided he was well enough to travel. Shortly after his trip he became symptomatic again with complaints of cough, significant sputum production, and malaise. These issues worsened and ended up developing into full bronchitis and tracheitis. The patient then consulted a pulmonologist who believed that the patient potentially had acute cocciodomycosis or influenza. The pulmologist emperically prescribed benzonate and phenergan with codine.

Case Presentation

His chest X-ray was normal, sputum cultures were positive for Streptococci spp., Neisseria spp. and Prevotella spp., his Coccidiomycosis serologies were negative. A sinus X-ray or CT was not done. The fungal culture grew Candida spp. While the traditional identification tests were being performed, the patient also had a sputum sample sent to Fry Laboratories for special stains and fungal eukaryotic (18S) microbial sequencing using the RIDITM system [1]. The stain developed in our laboratory preformed on the sputum combines a Hoechst stain and Calcofluor White fungal stain (‘Mosaic’ stain) for identification of DNA containing biofilm and fungal elements [2-4]. The stain resulted in a clear indication of significant DNA positive elements, biofilm, and fungi in the sputum sample. Amplicon sequencing and bioinformatic analysis identified several fungi; Funneliformis mosseae, Candidae parapsilosis and C.viswanathii.

The patient was prescribed terbinafine 250mg/day and doxycycline 100mg/b.i.d. By the 39th day the patient had dramatic improvements of all respiratory symptoms and constitutional complaints. The patient’s respiratory capacity and abilities returned to baseline and his overall energy level and feeling of wellbeing returned to normal as well (Figure 1).

Figure 1: ‘Mosaic’ Stain of sputum, 400X. The ‘Mosaic’ stain is an easy to use combination stain used in our laboratory to simultaneously demonstrate biofilm and the presence of chitin (common to all fungi and some protozoa). Images of the Hoechst stain and then of the Calcofluor white stain are merged electronically to provide the image which include a color-coded interpretation.

    

    
    

    

    Figure 1: ‘Mosaic’ Stain of sputum, 400X. The ‘Mosaic’ stain is an easy to
use combination stain used in our laboratory to simultaneously demonstrate
biofilm and the presence of chitin (common to all fungi and some protozoa).
Images of the Hoechst stain and then of the Calcofluor white stain are merged
electronically to provide the image which include a color-coded interpretation.

Discussion

The patient was most likely colonized in the sinus tract and pulmonary architecture. NGS augments the microscopy and clinical findings and clarifies the infectious process in respiratory disease. Less than 2% of bacteria are cultivable, fungi are also difficult to culture. Of great utility is the ability to rapidly identify fungi, which are usually difficult to grow and require a much longer time for culture. The prominent fungi in the sample Funneliformis mossea (formerly Glomus mosseae) is a common arbuscular mycorrhizal soil fungus, uncultivable, with worldwide distribution. It has been reported in humans by our laboratory [5-7]. The use of Doxycycline augments the fungal activity of terbinafine; doxycycline may play a role in reducing the fungal biofilm allowing the antifungal terbinafine to work more effectively [8,9]. NGS allows the clinician to utilize a technology that has revolutionized human genomics; it is now possible to assess uncultivable microbes, which we are discovering are playing a greater role in human disease.

Acknowledgement

We would like to thank the Southwest Center for Chronic Disease for financial assistance.

References

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  8. Gao Y, Zhang C, Lu C, Liu P, Li Y, Li H, et al. Synergistic effect of doxycycline and fluconazole against Candida albicans biofilms and the impact of calcium channel blockers. FEMS Yeast Res. 2013; 13: 453-462.
  9. Miceli MH, Bernardo SM, Lee SA. In vitro analyses of the combination of highdose doxycycline and antifungal agents against Candida albicans biofilms. Int J Antimicrob Agents. 2009; 34: 326-332.

Citation: Gordon G and Fry SE. Next Generation DNA Sequencing Diagnoses Fungal Etiology in Chronic Respiratory Disease, Clinical Response Supports Clinical Validity. Austin J Clin Case Rep. 2021; 8(9): 1229.