P395 Clinical utility of semi-nested conventional PCR for diagnosis of mucormycosis in fresh clinical samples
Authors: Choudhary Hansraj , Kanaujia Rimjhim , Singh Shreya , Agnihotri Sourav , Muthu Valliappan , Kaur Harsimran , Ghosh Anup , Chakrabarti Arunaloke , Rudramruthy Shivaprakash M 
Published: September 2022

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM

Objectives
We aim to assess the clinical utility of the semi-nested conventional PCR in smear positive, culture-negative clinical samples for diagnosis of mucormycosis.

Methods
This prospective study was conducted for a period of 3 months (April-June 2021). A total of 218 clinical samples were included from patients in whom smear was positive for aseptate hyphae, but the culture failed to grow within 2 days of incubation or smear was negative but had high suspicion of mucormycosis. Molecular diagnosis was attempted using semi-nested PCR with Mucorales-specific primers targeting 18S region, described previously by Bialek et al.1. Phenol-chloroform based manual DNA extraction protocol was optimized in the mycology laboratory of the department of medical microbiology, PGIMER, Chandigarh. This method was applicable to different types of clinical samples to yield good quality DNA with minimal chances of extraneous contamination (Fig. 1). Amplified PCR products were further sequenced to identify the causative species (Fig. 2).

Results
Among 218 patients with suspected mucormycosis included in this study, the majority were rhino-orbito-cerebral mucormycosis (ROCM), (77.7%, n = 169), followed by pulmonary mucormycosis (19.2%, n = 42), cutaneous (0.02%, n = 4), and gastro-intestinal (GI) mucormycosis (0.01%, n = 3). In 24 samples, the presence of both septate and aseptate hyphae was seen under microscopic examination raising the possibility of mixed infection. On microscopic examination, 90.3% samples (197/218) had aseptate hyphae while the remaining samples were smear-negative but had strong clinical suspicion of mucormycosis. The molecular technique was able to identify causative agent in 154 culture-negative samples (81.9%, 154/188) and 52.4% (11/21) in smear-negative cases. Among 218 patients, only 20 samples show delayed growth of Mucorales, and on comparison with molecular results 100% concordance was observed. However, 10 patients with strong clinical suspicion for mucormycosis were negative by both conventional and molecular methods. The low culture positivity necessities the molecular diagnosis based on in-house semi-nested PCR using above-mentioned primers followed by Sanger sequencing. In the case of 24 mixed infections with aseptate-septate hyphae, Mucorales-specific PCR correctly identified 23/24 (95.8%) Mucorales as a causative agent. The overall turn-around-time from the sample receiving to diagnosis was ˂48 h. Overall, R. arrhizus (85/143, 59.4%) was most commonly associated with ROCM, while R. microsporus (13/38, 34.2%), and R. homothallicus (5/38, 13.1%) were seen mainly with pulmonary mucormycosis. Apophysomyces and Saksenaea genus were associated with GI and cutaneous mucormycosis.

Conclusions
The molecular technique utilizing semi-nested PCR, followed by Sanger sequencing was able to identify Mucorales species 81.9% of culture-negative cases. Optimized manual DNA extraction protocol is suitable for different sample types, with minimum chances of extraneous contamination and offers low cost with a shorter turnaround time (TAT).