Species distribution and antifungal susceptibilities of clinical isolates of Penicillium and Talaromyces species in China

Highlights

• High species diversity among 111 clinical isolates, with a prevalence of P. oxalicum, T. funiculosus and T. stollii.

• RPB2 provided greater accuracy than benA or ITS for identification of Penicillium and Talaromyces species.

• 89.3% of Penicillium and 98.2% of Talaromyces isolates exhibited growth at 37°C.

• Isolates were mainly recovered from patients with pulmonary disorders (56.8%).

• Antifungal susceptibility profile of T. marneffei differed from that of Penicillium and other Talaromyces species.

ABSTRACT

Morphologically identified Penicillium (n = 103) and Talaromyces marneffei (n = 8) isolates were collected from various clinical sources between 2016 and 2017 at a medical centre in Beijing, China. Identification to species level was confirmed by sequencing of the internal transcribed spacer (ITS) region, β-tubulin gene (benA) and RNA polymerase II second largest subunit (RPB2) gene. Of the 111 isolates, 56 (50.5%) were identified as Penicillium spp. and 55 (49.5%) as Talaromyces spp. Eleven species of Penicillium were detected, of which Penicillium oxalicum was the commonest, accounting for 51.8% (29/56), followed by Penicillium rubens (10.7%; 6/56) and Penicillium citrinum (10.7%; 6/56). Among the 55 Talaromyces isolates, nine species were identified, with Talaromyces funiculosus (36.4%; 20/55), Talaromyces stollii (27.3%; 15/55) and Talaromyces marneffei (14.5%; 8/55) being the most common. Of note, 89.3% (50/56) of the Penicillium isolates and 98.2% (54/55) of the Talaromyces isolates exhibited growth at 37°C. The isolates were mainly recovered from patients with pulmonary disorders (56.8%; 63/111), autoimmune disease (12.6%; 14/111) and AIDS (5.4%; 6/111). The azoles and amphotericin B exhibited potent activity against T. marneffei, while various levels of activity were observed against Penicillium and other Talaromyces species The echinocandins had the lowest MECs (MEC_90, ≤0.12 mg/L) against most Penicillium and Talaromyces species, with the exception of T. marneffei whose MEC₉₀ (4 mg/L) was five or more dilutions higher than that of the other species tested. These data on the species distribution and antifungal susceptibility expand the current clinical knowledge of Penicillium and Talaromyces species.

Introduction

The genus Penicillium belongs to the family Aspergillaceae, order Eurotiales, class Eurotiomycetes, and division Ascomycota. The genus Penicillium comprises at least 483 species that are ubiquitously present in air, soil, various food products and indoor environments [1,2]. Members of Penicillium species are common contaminants of various substrates and are also known as potential producers of numerous bioactive compounds including mycotoxins, antibiotics, enzymes, antioxidants, insecticides and anticancer compounds [1]. Among the Penicillium species, Penicillium marneffei (now Talaromyces marneffei) is the only known dimorphic species, which can cause systemic mycosis (talaromycosis) in humans, mainly in Southeast Asia [3], [4], [5]. Other Penicillium species were previously thought to indicate contamination or harmless colonisation when isolated from clinical specimens. With an increase in the number of individuals with weakened immune systems, the epidemiology of mould infections has changed substantially in recent years [6]. Penicillium species have been increasingly recognised as emerging opportunistic pathogens causing endophthalmitis [7], pneumonia [8], central nervous system (CNS) infection [9] and disseminated disease [10] in immunocompromised patients. Additionally, Penicillium is one of the most common airborne allergenic fungi [11], and exposure to Penicillium species during infancy has been associated with childhood asthma in a birth cohort study [12].

Diagnosis and treatment of invasive Penicillium infection remains challenging. Accurate and rapid identification of Penicillium isolates is of great importance in determining the aetiology of the disease, predicting intrinsic resistance and providing appropriate treatment, especially in high-risk patients [8], [9], [10]. In clinical microbiology laboratories, mould identification has been largely based on typical colony morphology and structures following lactophenol cotton blue staining. Distinguishing between the different Penicillium species with phenotypic data requires highly skilled experts and can be time consuming. Moreover, some isolates display atypical morphology, fail to sporulate or require lengthy incubation [13]. Phenotypic results may be confusing or limited to the family or genus level. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) has been widely used as a fast and accurate identification tool for clinically important fungi [14]. However, use of MALDI-TOF/MS for identifying clinical Penicillium isolates is restricted, mainly due to the limited species coverage in commercial databases [14,15]. Further efforts are needed to continuously update the reference fungal databases by enriching them with more Penicillium isolates.

To address these deficiencies, DNA sequence-based methods are being increasingly employed to identify Penicillium isolates in clinical laboratories. The internal transcribed spacer (ITS) region is the most commonly used gene marker; however, the ITS may not be variable enough to distinguish closely related Penicillium species, whereas the β-tubulin (benA) gene has been proposed as a secondary marker for Penicillium identification [1]. Despite the increasing attention being focused on identification methods for Penicillium, few studies have documented the current epidemiology of these emerging opportunistic pathogens [16]. In the present study, 111 morphologically identified Penicillium isolates originating from various clinical specimens were collected. Species identification was confirmed by sequence analysis of the ITS region, benA gene and RNA polymerase II second largest subunit (RPB2) gene. Furthermore, the antifungal susceptibility profiles of the Penicillium species were also evaluated.