Post-transcriptional control of antifungal resistance in human fungal pathogens.

Global estimates suggest that over 300 million individuals of all ages are affected by serious fungal infections every year, culminating in about 1.7 million deaths. The societal and economic burden on the public health sector due to opportunistic fungal pathogens is quite significant, especially among immunocompromised patients. Despite the high clinical significance of these infectious agents, treatment options are limited with only three major classes of antifungal drugs approved for use. Clinical management of fungal diseases is further compromised by the emergence of antifungal resistant strains. Transcriptional and genetic mechanisms that control drug resistance in human fungal pathogens are well-studied and include drug target alteration, upregulation of drug efflux pumps as well as changes in drug affinity and abundance of target proteins. In this review, we highlight several recently discovered novel post-transcriptional mechanisms that control antifungal resistance, which involve regulation at the translational, post-translational, epigenetic, and mRNA stability levels. The discovery of many of these novel mechanisms has opened new avenues for the development of more effective antifungal treatment strategies and new insights, perspectives, and future directions that will facilitate this process are discussed.

Delineation of the Direct Contribution of Candida auris ERG11 Mutations to Clinical Triazole Resistance.

Resistance to fluconazole is one of clinical characteristics most frequently challenging the treatment of invasive Candida auris infections, and is observed among >90% of all characterized clinical isolates. In this work, the native C. auris ERG11 allele in a previously characterized fluconazole-susceptible clinical isolate was replaced with the ERG11 alleles from three highly fluconazole-resistant clinical isolates (MIC ≥256 mg/L), encoding the amino acid substitutions VF125AL, Y132F, and K143R, using Cas9-ribonucleoprotein (RNP) mediated transformation system. Reciprocally, the ERG11WT allele from the same fluconazole-susceptible clinical isolate, lacking any resistance-associated mutation, was introduced into a previously characterized fluconazole-resistant clinical isolate, replacing the native ERG11K143R allele, using the same methods. The resulting collection of strains was subjected to comprehensive triazole susceptibility testing, and the direct impact each of these clinically-derived ERG11 mutations on triazole MIC was determined. Introduction of each of the three mutant ERG11 alleles was observed to increase fluconazole and voriconazole MIC by 8- to 16-fold. The MIC for the other clinically available triazoles were not significantly impacted by any ERG11 mutation. In the fluconazole-resistant clinical isolate background, correction of the K143R encoding mutation led to a similar 16-fold decrease in fluconazole MIC, and 8-fold decrease in voriconazole MIC, while the MIC of other triazoles were minimally changed. Taken together, these findings demonstrate that mutations in C. auris ERG11 significantly contribute to fluconazole and voriconazole resistance, but alone cannot explain the substantially elevated MIC observed among clinical isolates of C. auris. IMPORTANCE Candida auris is an emerging multidrug-resistant and health care-associated pathogen of urgent clinical concern. The triazoles are the most widely prescribed antifungal agents worldwide and are commonly utilized for the treatment of invasive Candida infections. Greater than 90% of all C. auris clinical isolates are observed to be resistant to fluconazole, and nearly all fluconazole-resistant isolates of C. auris are found to have one of three mutations (encoding VF125AL, Y132F, or K143R) in the gene encoding the target of the triazoles, ERG11. However, the direct contribution of these mutations in ERG11 to fluconazole resistance and the impact these mutations may have the susceptibility of the other triazoles remains unknown. The present study seeks to address this knowledge gap and potentially inform the future application the triazole antifungals for the treatment of infections caused by C. auris.

Molecular and genetic basis of azole antifungal resistance in the opportunistic pathogenic fungus Candida albicans.

Candida albicans is an opportunistic yeast and the major human fungal pathogen in the USA, as well as in many other regions of the world. Infections with C. albicans can range from superficial mucosal and dermatological infections to life-threatening infections of the bloodstream and vital organs. The azole antifungals remain an important mainstay treatment of candidiasis and therefore the investigation and understanding of the evolution, frequency and mechanisms of azole resistance are vital to improving treatment strategies against this organism. Here the organism C. albicans and the genetic changes and molecular bases underlying the currently known resistance mechanisms to the azole antifungal class are reviewed, including up-regulated expression of efflux pumps, changes in the expression and amino acid composition of the azole target Erg11 and alterations to the organism's typical sterol biosynthesis pathways. Additionally, we update what is known about activating mutations in the zinc cluster transcription factor (ZCF) genes regulating many of these resistance mechanisms and review azole import as a potential contributor to azole resistance. Lastly, investigations of azole tolerance in C. albicans and its implicated clinical significance are reviewed.

Genomic perspective of triazole resistance in clinical and environmental Aspergillus fumigatus isolates without cyp51A mutations.

Aspergillus fumigatus is the most common etiologic agent of primarily all clinical manifestations of aspergillosis. A steady increase in the number of azole resistant A. fumigatus (ARAF) isolates from environment and clinical samples leading to therapeutic failures in clinical settings have alarmed the mycologists and clinicians worldwide. Although mutations in azole target cyp51A gene have been implicated in conferring azole resistance in A. fumigatus, recent studies have demonstrated occurrence of azole resistant strains without cyp51A mutations. In this study, next generation sequencing techniques and the expression profiling of transporter genes with single nucleotide polymorphisms (SNPs) in clinical and environmental ARAF isolates with (G54E) and without known cyp51A mutations was undertaken to understand the genetic background and role of transporters in azole resistance. The raw reads of four ARAF strains when mapped to Af293 reference genome (>100X depth) covered at least 93.1% of the reference genome. Among all four strains, a total of 212,711 SNPs was identified with 37,829 were common in at least two isolates. The expression analysis suggested the overexpression of MFS transporter, namely, mfsC in all ARAF isolates. None of the resistant strain showed significant upregulation of cyp51A and cyp51B gene. On the other hand, abcD was upregulated (5-fold) in the isolates with cyp 51A mutation (G54E). The whole genome sequence analysis showed the presence of two previously described amino acid substitutions S269F and F390Y in HMG1 gene in a clinical panazole resistant strain without cyp51A mutations. These mutations have been previously associated with azole resistance in A. fumigatus strains without cyp51A mutations. Further, several punctual mutations and a large-segment deletion among different strains were observed suggesting the involvement of resistance mechanisms other than cyp51A.

Anti-fungal activity of a novel triazole, PC1244, against emerging azole-resistant Aspergillus fumigatus and other species of Aspergillus.

OBJECTIVES: The growing emergence of azole-resistant Aspergillus fumigatus strains worldwide is a major concern for current systemic antifungal treatment. Here we report antifungal activities of a novel inhaled triazole, PC1244, against a collection of multi-azole-resistant A. fumigatus strains. METHODS: MICs of PC1244 were determined for A. fumigatus carrying TR34/L98H (n = 81), TR46/Y121F/T289A (n = 24), M220 (n = 6), G54 (n = 11), TR53 (n = 1), TR463/Y121F/T289A (n = 2), G448S (n = 1), G432C (n = 1) and P216S (n = 1) resistance alleles originating from either India, the Netherlands or France. The effects of PC1244 were confirmed in an in vitro model of the human alveolus and in vivo in temporarily neutropenic, immunocompromised mice. RESULTS: PC1244 exhibited potent inhibition [geometric mean MIC (range), 1.0 mg/L (0.125 to >8 mg/L)] of growth of A. fumigatus strains carrying cyp51A gene mutations, showing much greater potency than voriconazole [15 mg/L (0.5 to >16 mg/L)], and an effect similar to those on other azole-susceptible Aspergillus spp. (Aspergillus flavus, Aspergillus terreus, Aspergillus tubingensis, Aspergillus nidulans, Aspergillus niger, Aspergillus nomius, Aspergillus tamarii) (0.18-1 mg/L). In TR34/L98H and TR46/Y121F/T289A A. fumigatus-infected in vitro human alveolus models, PC1244 achieved superior inhibition (IC50, 0.25 and 0.34 mg/L, respectively) compared with that of voriconazole (IC90, >3 mg/L and >10 mg/L, respectively). In vivo, once-daily intranasal administration of PC1244 (0.56-70 µg/mouse) to the A. fumigatus (AF91 with M220V)-infected mice reduced pulmonary fungal load and serum galactomannan more than intranasal posaconazole. CONCLUSIONS: PC1244 has the potential to become a novel topical treatment of azole-resistant pulmonary aspergillosis.

In vitro combination of voriconazole with micafungin against azole-resistant clinical isolates of Aspergillus fumigatus from different geographical regions.

In vitro interaction of voriconazole with micafungin was evaluated against 33 clinical Aspergillus fumigatus isolates, including azole-resistant (n=31) and -susceptible (n=2) isolates. Interaction was synergistic for only 1 resistant isolate carrying the TR34/L98H mutation. No antagonistic effects were observed for 96.8% of azole-resistant isolates.

A multicentre study of antifungal susceptibility patterns among 350 Candida auris isolates (2009-17) in India: role of the ERG11 and FKS1 genes in azole and echinocandin resistance.

Background: Candida auris has emerged globally as an MDR nosocomial pathogen in ICU patients. Objectives: We studied the antifungal susceptibility of C. auris isolates (n = 350) from 10 hospitals in India collected over a period of 8 years. To investigate azole resistance, ERG11 gene sequencing and expression profiling was conducted. In addition, echinocandin resistance linked to mutations in the C. auris FKS1 gene was analysed. Methods: CLSI antifungal susceptibility testing of six azoles, amphotericin B, three echinocandins, terbinafine, 5-flucytosine and nystatin was conducted. Screening for amino acid substitutions in ERG11 and FKS1 was performed. Results: Overall, 90% of C. auris were fluconazole resistant (MICs 32 to ≥64 mg/L) and 2% and 8% were resistant to echinocandins (≥8 mg/L) and amphotericin B (≥2 mg/L), respectively. ERG11 sequences of C. auris exhibited amino acid substitutions Y132 and K143 in 77% (n = 34/44) of strains that were fluconazole resistant whereas WT genotypes, i.e. without substitutions at these positions, were observed in isolates with low fluconazole MICs (1-2 mg/L) suggesting that these substitutions confer a phenotype of resistance to fluconazole similar to that described for Candida albicans. No significant expression of ERG11 was observed, although expression was inducible in vitro with fluconazole exposure. Echinocandin resistance was linked to a novel mutation S639F in FKS1 hot spot region I. Conclusions: Overall, 25% and 13% of isolates were MDR and multi-azole resistant, respectively. The most common resistance combination was azoles and 5-flucytosine in 14% followed by azoles and amphotericin B in 7% and azoles and echinocandins in 2% of isolates.

Investigation of Multiple Resistance Mechanisms in Voriconazole-Resistant Aspergillus flavus Clinical Isolates from a Chest Hospital Surveillance in Delhi, India.

Invasive and allergic infections by Aspergillus flavus are more common in tropical and subtropical countries. The emergence of voriconazole (VRC) resistance in A. flavus impacts the management of aspergillosis, as azoles are used as the first-line and empirical therapy. We screened 120 molecularly confirmed A. flavus isolates obtained from respiratory and sinonasal specimens in a chest hospital in Delhi, India, for azole resistance using the CLSI broth microdilution (CLSI-BMD) method. Overall, 2.5% (n = 3/120) of A. flavus isolates had VRC MICs above epidemiological cutoff values (>1 µg/ml). The whole-genome sequence analysis of three non-wild-type (WT) A. flavus isolates with high VRC MICs showed polymorphisms in azole target genes (cyp51A, cyp51B, and cyp51C). Further, four novel substitutions (S196F, A324P, N423D, and V465M) encoded in the cyp51C gene were found in a single non-WT isolate which also exhibited overexpression of cyp51 (cyp51A, -B, and -C) genes and transporter genes, namely, MDR1, MDR2, atrF, and mfs1 The homology model of the non-WT isolate suggests that substitutions S196F and N423D exhibited major structural and functional effects on cyp51C drug binding. The substrate (drug) may not be able to bind to binding pocket due to changes in the pocket size or closing down or narrowing of cavities in drug entry channels. Notably, the remaining two VRC-resistant A. flavus isolates, including the one which had a pan-azole resistance phenotype (itraconazole and posaconazole), did not show upregulation of any of the analyzed target genes. These results suggest that multiple target genes and mechanisms could simultaneously contribute to azole resistance in A. flavus.

Azole-Resistant Aspergillosis: Epidemiology, Molecular Mechanisms, and Treatment.

Aspergillus fumigatus remains the most common species in all pulmonary syndromes, followed by Aspergillus flavus which is a common cause of allergic rhinosinusitis, postoperative aspergillosis and fungal keratitis. The manifestations of Aspergillus infections include invasive aspergillosis, chronic pulmonary aspergillosis and bronchitis. Allergic manifestations of inhaled Aspergillus include allergic bronchopulmonary aspergillosis and severe asthma with fungal sensitization. Triazoles are the mainstay of therapy against Aspergillus infections for treatment and prophylaxis. Lately, increased azole resistance in A. fumigatus has become a significant challenge in effective management of aspergillosis. Earlier studies have brought to light the contribution of non-cyp51 mutations along with alterations in cyp51A gene resulting in azole-resistant phenotypes of A. fumigatus. This review highlights the magnitude of azole-resistant aspergillosis and resistance mechanisms implicated in the development of azole-resistant A. fumigatus and address the therapeutic options available.

Molecular bases of antifungal resistance in filamentous fungi.

Invasive mould infections due to Aspergillus spp., Fusarium spp., Scedosporium spp. and other filamentous fungi remain a significant cause of morbidity and mortality in immunocompromised patients. Antifungal therapy is required for successful patient management; however, limited antifungal drugs as well as the emergence of drug resistance pose a challenge to clinicians for effective management of these diseases. The evolution of multidrug-resistant strains owing to selective pressure as well as intrinsically-resistant pathogenic species are a major concern. Thus, it is necessary to gain a better insight into the antifungal resistance mechanisms and their clinical impact. So far, the resistance mechanisms responsible for acquired azole resistance include alteration of the drug target and its overexpression, biofilm formation and efflux pump upregulation. However, mechanisms responsible for polyene and echinocandin resistance are less understood in filamentous moulds, primarily due to incomplete correlation between in vitro susceptibility and clinical response to treatment. The present review gives an insight into the mechanisms implicated in resistance to different classes of antifungal agents both in human and plant pathogenic filamentous fungi. Furthermore, the spectrum of antifungal resistance in these filamentous fungi is highlighted.

Whole Genome-Based Amplified Fragment Length Polymorphism Analysis Reveals Genetic Diversity in Candida africana.

This study aimed at investigating the genetic diversity of a panel of Candida africana strains recovered from vaginal samples in different countries. All fungal strains were heterozygous at the mating-type-like locus and belonged to the genotype A of Candida albicans. Moreover, all examined C. africana strains lack N-acetylglucosamine assimilation and sequence analysis of the HXK1 gene showed a distinctive polymorphism that impair the utilization of this amino sugar in this yeast. Multi-locus sequencing of seven housekeeping genes revealed a substantial genetic homogeneity among the strains, except for the CaMPIb, SYA1 and VPS13 loci which contributed significantly to the classification of our set of C. africana strains into six existing diploid sequence types. Amplified fragment length polymorphism fingerprint analysis yielded greater genotypic heterogeneity among the C. africana strains. Overall the data reported here show that in C. africana genetic diversity occurs and the existence of this intriguing group of C. albicans strains with specific phenotypes associated could be useful for future comparative studies in order to better understand the genetics and evolution of this important human pathogen.

Comparison of EUCAST and CLSI Reference Microdilution MICs of Eight Antifungal Compounds for Candida auris and Associated Tentative Epidemiological Cutoff Values.

Candida auris is an emerging multidrug-resistant yeast. So far, all but two susceptibility testing studies have examined ≤50 isolates, mostly with the CLSI method. We investigated CLSI and EUCAST MICs for 123 C. auris isolates and eight antifungals and evaluated various methods for epidemiological cutoff (ECOFF) determinations. MICs (in milligrams per liter) were determined using CLSI method M27-A3, and the EUCAST E.Def 7.3. ANOVA analysis of variance with Bonferroni's multiple-comparison test and Pearson analysis were used on log2 MICs (significance at P values of <0.05). The percent agreement (within ±0 to ±2 2-fold dilutions) between the methods was calculated. ECOFFs were determined visually, statistically (using the ECOFF Finder program and MicDat1.23 software with 95% to 99% endpoints), and via the derivatization method (dECOFFs). The CLSI and EUCAST MIC distributions were wide, with several peaks for all compounds except amphotericin B, suggesting possible acquired resistance. Modal MIC, geometric MIC, MIC50, and MIC90 values were ≤1 2-fold dilutions apart, and no significant differences were found. The quantitative agreement was best for amphotericin B (80%/97% within ±1/±2 dilutions) and lowest for isavuconazole and anidulafungin (58%/76% to 75% within ±1/±2 dilutions). We found that 90.2%/100% of the isolates were amphotericin B susceptible based on CLSI/EUCAST methods, respectively (i.e., with MICs of ≤1 mg/liter), and 100%/97.6% were fluconazole nonsusceptible by CLSI/EUCAST (MICs > 2). The ECOFFs (in milligrams per liter) were similar across the three different methods for itraconazole (ranges for CLSI/EUCAST, 0.25 to 0.5/0.5 to 1), posaconazole (0.125/0.125 to 0.25), amphotericin B (0.25 to 0.5/1 to 2), micafungin (0.25 to 0.5), and anidulafungin (0.25 to 0.5/0.25 to 1). In contrast, the estimated ECOFFs were dependent on the method applied for voriconazole (1 to 32) and isavuconazole (0.125 to 4). CLSI and EUCAST MICs were remarkably similar and confirmed uniform fluconazole resistance and variable acquired resistance to the other agents.

Arabidopsis CBL interacting protein kinase 3 interacts with ABR1, an APETALA2 domain transcription factor, to regulate ABA responses.

Calcium (Ca2+) plays a vital role as a second messenger in several signaling pathways in plants. The calcineurin B-like proteins (CBLs) represent a family of plant calcium-binding proteins that function in propagating Ca2+ signals by interacting with CBL interacting protein kinases (CIPKs). Phosphorylation of CBL by CIPK is essential for the module to display full activity towards its target protein. Previous genetic analysis showed that the function of CBL9-CIPK3 module was implicated in negatively regulating seed germination and early development. In the present study, we have biochemically investigated the interaction of CBL9-CIPK3 module and our findings show that CBL9 is phosphorylated by CIPK3. Moreover, Abscisic acid repressor 1 (ABR1) is identified as the downstream target of CIPK3 and CIPK3-ABR1 function to regulate ABA responses during seed germination. Our study also indicates that the role of ABR1 is not limited to seed germination but it also regulates the ABA dependent processes in the adult stage of plant development. Combining our results, we conclude that the CBL9-CIPK3-ABR1 pathway functions to regulate seed germination and ABA dependent physiological processes in Arabidopsis.

Diversity and origins of Indian multi-triazole resistant strains of Aspergillus fumigatus.

Aspergillus fumigatus is a widespread opportunistic fungal pathogen causing an alarmingly high mortality rate in immunocompromised patients. Nosocomial infections by drug-resistant A. fumigatus strains are of particular concern, and there is a pressing need to understand the origin, dispersal and long-term evolution of drug resistance in this organism. The objective of this study was to investigate the diversity and putative origins of triazole resistance of A. fumigatus from India. Eighty-nine isolates, including 51 multiple triazole resistant (MTR) isolates and 38 azole-susceptible isolates, were genotyped using multilocus sequence typing (MLST), mating typing and PCR fingerprinting. MLST resolved the 51 MTR isolates into three genotypes, two of which have susceptible counterparts, suggesting that MTR isolates originated multiple times in India. The multiple-origin hypothesis was further supported by the diversity of sequences at the triazole target gene CYP51A among the MTR isolates, and by PCR fingerprints. Interestingly, there is abundant evidence for mating and recombination in natural population of A. fumigatus in India, suggesting that sexual spread of TR34 /L98H, the dominant MTR allele, is possible. Our results call for greater attention to MTR in A. fumigatus and for better management of antifungal drug use.

Draft Genome Sequence of a Fluconazole-Resistant Candida auris Strain from a Candidemia Patient in India.

Candida auris is a multidrug-resistant yeast incriminated in a wide spectrum of invasive infections, especially in intensive care settings. The first draft genome sequence of C. auris, VPCI 479/P/13, from a case with fungemia was sequenced using the Illumina MiSeq platform. The estimated genome size is 12.3 Mb, with 6,675 coding sequences.

Prevalence and mechanism of triazole resistance in Aspergillus fumigatus in a referral chest hospital in Delhi, India and an update of the situation in Asia.

Aspergillus fumigatus causes varied clinical syndromes ranging from colonization to deep infections. The mainstay of therapy of Aspergillus diseases is triazoles but several studies globally highlighted variable prevalence of triazole resistance, which hampers the management of aspergillosis. We studied the prevalence of resistance in clinical A. fumigatus isolates during 4 years in a referral Chest Hospital in Delhi, India and reviewed the scenario in Asia and the Middle East. Aspergillus species (n = 2117) were screened with selective plates for azole resistance. The isolates included 45.4% A. flavus, followed by 32.4% A. fumigatus, 15.6% Aspergillus species and 6.6% A. terreus. Azole resistance was found in only 12 (1.7%) A. fumigatus isolates. These triazole resistant A. fumigatus (TRAF) isolates were subjected to (a) calmodulin and ß tubulin gene sequencing (b) in vitro antifungal susceptibility testing against triazoles using CLSI M38-A2 (c) sequencing of cyp51A gene and real-time PCR assay for detection of mutations and (d) microsatellite typing of the resistant isolates. TRAF harbored TR34/L98H mutation in 10 (83.3%) isolates with a pan-azole resistant phenotype. Among the remaining two TRAF isolates, one had G54E and the other had three non-synonymous point mutations. The majority of patients were diagnosed as invasive aspergillosis followed by allergic bronchopulmonary aspergillosis and chronic pulmonary aspergillosis. The Indian TR34/L98H isolates had a unique genotype and were distinct from the Chinese, Middle East, and European TR34/L98H strains. This resistance mechanism has been linked to the use of fungicide azoles in agricultural practices in Europe as it has been mainly reported from azole naïve patients. Reports published from Asia demonstrate the same environmental resistance mechanism in A. fumigatus isolates from two highly populated countries in Asia, i.e., China and India and also from the neighboring Middle East.

Multidrug-Resistant Candida auris Misidentified as Candida haemulonii: Characterization by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and DNA Sequencing and Its Antifungal Susceptibility Profile Variability by Vitek 2, CLSI Broth Microdilution, and Etest Method.

Candida auris is a multidrug-resistant yeast that causes a wide spectrum of infections, especially in intensive care settings. We investigated C. auris prevalence among 102 clinical isolates previously identified as Candida haemulonii or Candida famata by the Vitek 2 system. Internal transcribed spacer region (ITS) sequencing confirmed 88.2% of the isolates as C. auris, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) easily separated all related species, viz., C. auris (n = 90), C. haemulonii (n = 6), C. haemulonii var. vulnera (n = 1), and Candida duobushaemulonii (n = 5). The in vitro antifungal susceptibility was determined using CLSI broth microdilution (CLSI-BMD), the Vitek 2 antifungal susceptibility test, and the Etest method. C. auris isolates revealed uniformly elevated fluconazole MICs (MIC50, 64 µg/ml), and an alarming percentage of isolates (37%) exhibited elevated caspofungin MICs by CLSI-BMD. Notably, 34% of C. auris isolates had coexisting elevated MICs (≥2 µg/ml) for both fluconazole and voriconazole, and 10% of the isolates had elevated coexisting MICs (≥2 µg/ml) to two additional azoles, i.e., posaconazole and isavuconazole. In contrast to reduced amphotericin B MICs by CLSI-BMD (MIC50, 1 µg/ml) for C. auris, elevated MICs were noted by Vitek 2 (MIC50, 8 µg/ml), which were statistically significant. Candida auris remains an unnoticed pathogen in routine microbiology laboratories, as 90% of the isolates characterized by commercial identification systems are misidentified as C. haemulonii. MALDI-TOF MS proved to be a more robust diagnostic technique for rapid identification of C. auris. Considering that misleading elevated MICs of amphotericin B by the Vitek AST-YS07 card may lead to the selection of inappropriate therapy, a cautionary approach is recommended for laboratories relying on commercial systems for identification and antifungal susceptibility testing of rare yeasts.

Molecular epidemiology and in-vitro antifungal susceptibility of Aspergillus terreus species complex isolates in Delhi, India: evidence of genetic diversity by amplified fragment length polymorphism and microsatellite typing.

Aspergillus terreus is emerging as an etiologic agent of invasive aspergillosis in immunocompromised individuals in several medical centers in the world. Infections due to A. terreus are of concern due to its resistance to amphotericin B, in vivo and in vitro, resulting in poor response to antifungal therapy and high mortality. Herein we examined a large collection of molecularly characterized, geographically diverse A. terreus isolates (n = 140) from clinical and environmental sources in India for the occurrence of cryptic A. terreus species. The population structure of the Indian A. terreus isolates and their association with those outside India was determined using microsatellite based typing (STR) technique and Amplified Fragment Length Polymorphism analysis (AFLP). Additionally, in vitro antifungal susceptibility of A. terreus isolates was determined against 7 antifungals. Sequence analyses of the calmodulin locus identified the recently described cryptic species A. hortai, comprising 1.4% of Aspergillus section Terrei isolates cultured from cases of aspergilloma and probable invasive aspergillosis not reported previously. All the nine markers used for STR typing of A. terreus species complex proved to be highly polymorphic. The presence of high genetic diversity revealing 75 distinct genotypes among 101 Indian A. terreus isolates was similar to the marked heterogeneity noticed in the 47 global A. terreus population exhibiting 38 unique genotypes mainly among isolates from North America and Europe. Also, AFLP analysis showed distinct banding patterns for genotypically diverse A. terreus isolates. Furthermore, no correlation between a particular genotype and amphotericin B susceptibility was observed. Overall, 8% of the A. terreus isolates exhibited low MICs of amphotericin B. All the echinocandins and azoles (voriconazole, posaconazole and isavuconazole) demonstrated high potency against all the isolates. The study emphasizes the need of molecular characterization of A. terreus species complex isolates to better understand the ecology, acquisition and transmission of this species.