Optimisation of culture protocol for the recovery of fungal pathogens from expectorated sputum of cystic fibrosis patients

Warda Memon *1, Relinda Abellera 1, Sean X. Zhang 1;2, Remedios Marayan 1

Author address: 

1 Johns Hopkins Hospital, Baltimore, United States; 2 Johns Hopkins University School of Medicine, Baltimore, Maryland, United States

Abstract: 

Background: The role that fungi play in patients with Cystic Fibrosis (CF) is not well characterized, largely due to a lack of standardized laboratory guidelines for the optimal recovery of fungi in respiratory tract of CF patients. We previously reported use of selective fungal culture media increased rates of detection of fungi in CF cultures. In this study, we attempted to further determine the impact of incubation temperate of culture media and pre-treatment of expectorated sputum with a mucolytic agent on CF fungal cultures.

Materials/methods: 400 prospective CF expectorated sputa samples were simultaneously tested by two protocols: 1) the current protocol included inoculating sputa on the following media: MacConkey, CNA, blood, chocolate with and without bacitracin, Burkholderia cepacia selective, Mannitol salt & Sabouraud dextrose with Gentamicin (SABG), and incubated at 37°C for 3 days. 2) the study protocol including Phase I [P I] and Phase II [P II]. In P I, 200 sputa were inoculated on SABG, Inhibitory mold with Gentamicin (IMAG) and Brain heart infusion with Gentamicin (BHIG) and incubated at 30°C and 37°C, respectively. In P II, 200 CF sputa were pre-treated with and without a DTT free mucolytic agent, Liquilizer® (Metasystems Group, Inc) followed by repeating the P I protocol (excluding BHIG). All culture plates were observed for seven days.

Results: Total growth rate for the current protocol was 22% (88/400), for P I 38% (76/200), for P II 43% (85/200) using a combination of SABG and IMAG at 30°C and 37°C. The study protocol was statistically better than the current protocol (p<0.05). Pre-treatment of sputa with Liquilizer® did not significantly enhance the yield of fungal organisms. The most common fungal pathogens recovered were Aspergillus (56%), Exophiala (12%), Scedosporium (9%), Trichosporon (6%) and Rasamsonia (5%). The rate of recovery of these fungi was not significantly affected by temperature and media; however, extended incubation time increased the quantity of fungi recovered.

Conclusions: A combination of SABG and IMAG inoculated at both 30°C and 37°C along with a prolonged incubation of 7 days’ time produced the maximum isolation of fungal pathogens, 87% (139/159).

Presenter email address: [email protected]

2020

abstract No: 

5763

Full conference title: 

European Congress of Clinical Microbiology and Infectious Diseases 2020
    • ECCMID 30th (2020)