AFLP and Antifungal Susceptibility of Rhizopus species from Patients with Mucormycosis, Delhi, India

Shallu Kathuria, Pradeep K. Singh, Ferry Hagen, Jacques F. Meis, Anuradha Chowdhary


Background: Mucormycosis is a highly aggressive fungal infection mostlyinvolving the genus Rhizopus, which show variable susceptibility to antifungals. Herein, we determined the susceptibility and molecular characterization of Rhizopus species.
Methods: Sixty isolates of Rhizopus species obtained from 51 patients during 2004-2013 were included. Isolates were from pulmonary cases (n=44), from rhino-cerebral (n=10), cutaneous (n=2), and disseminated infection (n=4). The isolates were identified by ITS sequencing and were typed by AFLP. Susceptibility was determined using CLSIM27-A3 and for posaconazole (POS) and amphotericin B (AMB) by Etest.
Results: Sequencing identified Rhizopus arrhizus var. delemar (n=25), R. arrhizus var. arrhizus (n=15), R. microsporus (n=17), and R. stolonifer (n=3). ITS phylogenetic tree of R. arrhizus revealed two main clades, representing the varieties delemar (n=25) and arrhizus (n=15). AFLP analysis showed clustering into 5 different genotypes, revealing heterogeneity among the isolates. AMB was the most potent antifungal with MICs ≤1μg/ml. Among azoles, POS exhibited highest activity (GM MIC, 0.4μg/ml) followed by Isavuconazole (GM MIC, 1.27μg/ml). Interestingly, 40% isolates had POS MICs ≥0.5 μg/ml and 8.3% had above ≥2μg/ml, which included 2 isolates of variety delemar, 2 of variety arrhizus and one R. microsporus. Overall 8.3% (5/60) of isolates revealed very high MICs of ISA ranging from 8-16μg/ml, including 4 isolates of R. arrhizus var. delemar and 1 of R. microsporus. Fluconazole, voriconazole and echinocandins demonstrated no or poor activity. Notably, terbinafin was active against R. microsporus but had high MICs against R. arrhizus (MIC90, 32μg/ml). Etest MICs of AMB and POS, revealed a categorical agreement of 87% and 67% respectively with CLSI. The commonest underlying condition was uncontrolled diabetes mellitus. Treatment and outcome records of 42 patients revealed fatalities in 21 cases.
Conclusions: The high mortality and resistance of mucorales to antifungals require proper molecular identification. Furthermore, AFLP clearly revealed marked genotypic diversity suggesting that it could be explored in future studies to examine the relatedness of varieties within R. arrhizus.

abstract No: 

    • ICAAC 54th (2014)