Abstract: Background and Objective: Curvularia species not only cause disease in plants but have emerged in the last decade as a human pathogen causing mild, febrile, to life-threatening illness if not well-treated. Because of Curvularia’s interlocking lifestyle on plants, animals and human and increased use of azole fungicides, there is emerging evidence of upsurge in resistance to antifungal drugs, a major public health burden. The objective of this study was to evaluate the genetic diversity of C. lunata from plant origin relative to clinical strains and to profile the current literature on the global emerging antifungal drug resistance associated with Curvularia infections. Materials and Methods: In this study, the glyceraldehyde-3-phosphate dehydrogenase (GPDH) locus was used to illustrate the genetic diversity between C. lunata of clinical and plant origin. Tajima’s X2 test statistics was performed in MEGA6.1 phylogenetic software to investigate the diversity between sequences. Results: The results showed large genetic distance (~0.275±0.041) between lineages of C. lunata of clinical and plant origin. Even though no optimal antifungal therapy for Curvularia infections has been established for elite drugs like triazoles-itraconazole, voriconazole and posaconazole, it is cogently presented herein cases of successful site-specific treatment of infections caused by C. lunata. Conclusion: It is found that C. lunata from plant and clinical origins are genetically diverse and azole-fungicides exert selective pressure that accelerates evolution. Importantly, effective management of Curvularia infections is via combination therapy and regardless of the age and infected organ, treatment that last for at least 3 months is recommended.
INTRODUCTION
Fungal pathogens contribute significantly to global human suffering and the worst hit regions of the world include Brazil (with affected population estimated at 1.7%), Ireland (~1.9%) and the prevalence is believed to be highest in China1,2. In recent years, a sharp increase in morbidity and mortality associated with invasive fungal infections has been observed. Studies revealed over 1.2 billion people worldwide suffered from fungal diseases3,4. Previously, it was shown that 1.5-2 million people died of fungal infections each year, far surpassing those killed by either tuberculosis or malaria5. This high mortality burden is based on lead human fungi such as Cryptococcus neoformans, Aspergillus spp. and Candida spp. Moreover, other fungal infections have witnessed a sharp increase in the last decade such as coccidiomycosis caused by Coccidioides immitis and C. posadasii 6 and species in the genera Cochliobolus, Bipolaris and Curvularia 7. Species in these genera have not only developed the abilities to adapt to host tissues and counterattack the defense mechanisms, but they caused devastating diseases in several plant and animal species8-10. Thus, these fungi are often referred to as cross-kingdom pathogens.
Curvularia lunata (teleomorph sexual state-Cochliobolus lunatus) is the lead causal agent of diseases in both plants and animals within the genera Curvularia. The fungus C. lunata abundantly produces 1-7 celled-conidia during sporulation and survives higher temperature of ~40°C8,9 and principally transmitted via air. Known clinical manifestations include cutaneous and subcutaneous infections, keretomycosis, cerebral phaeohyphomycosis, allergic bronchopulmonary mycosis and endophthalmitis10-12. The level of severity of Curvularia infections varies among patients, making it a public health burden10-12.
Previous studies aiming at identifying aggressive and novel species causing mycotic diseases in the genera Curvularia7,13,14 revealed important genetic diversity. However, a fundamental problem in Curvularia infections is the risk of transferring a genetically evolved isolate from farms (where azole fungicides are used) to humans. Such transferability through human-plant interaction or intake of contaminated air could cause resistance to antifungal drugs since field isolates suffer from fungicide selective pressure and undergo virulence differentiation to adapt to adverse conditions. For instance, eumycetoma caused by C. lunata in farmers in India, South-East Rajasthan and Chandigarh responded poorly to treatment and needed long-term therapies15,16. Most agricultural regions are known for the use of azole fungicides such as metalaxyl, imidazole, propiconazole and tebuconazole formulation that poses selective pressure on cross-kingdom pathogens and impact on antifungal drug resistance5,7. Acquired resistance via azole fungicides exposure and extreme genetic variations poses a critical public health problem because of the ease of resistance build-up to antifungal therapy. Importantly, the current status of Curvularia infections, management and global emerging antifungal drug resistance is comparatively examined in this study.
MATERIALS AND METHODS
Study area and sampling: Based on previous report of black-to-black leaf spot disease of potato (Solanum tuberosum L.) caused by C. lunata17, a routine survey was performed in 5 potato plantations of Burdwan District (23°14’ N, 87°51’ E, altitude 150 m, 102.1 km from Kolkata), West Bengal, India, during the winter month of December to March of 2010, 2011 and 2012. The potato fields often receive two episodes spray of metalaxylmanocozeb fungicide during farming season. Potato leaves showing brown-to-black spot disease were excised and treated with 2% NaClO solution for 2 min and rinsed in sterile water with three changes. The leaf pieces were aseptically plated on V8 agar medium (HiMedia®, Mumbai, India) and incubated at 25°C in the dark. The colonies that developed after 7 days were transferred to fresh V8 agar plates in order to purify the cultures and isolates were morphologically identified based on standard monograph taxonomic keys18. Pathogenicity test was previously done and confirmed aggressive against potato19.
DNA extraction and polymerase chain reaction: The genomic DNA was isolated from fungal isolates grown in potato dextrose broth (PDB; HiMedia®, Mumbai, India). Total genomic DNA was extracted from mycelium mat using UltraCleanTM Microbial DNA isolation kits (Mo Bio Laboratories, Inc., Carlsbad, CA, USA) as described by the manufacturer. The quality and quantity of the DNA was determined using a 1% agarose gel electrophoresis and a nanodrop spectrophotometer (BioSpec-nano, Shimadzu®, Japan), respectively. For genetic diversity studies, specific primers (forward: 5-CGATATGCGGCATATGCA-3; reverse: 5-ACCTACGCATTGCGGAA-3) were designed for glyceraldehyde-3-phosphate dehydrogenase (GPDH) gene using reference C. lunata (GenBank: Gb|X58718) in Integrated DNA Technology (IDT) primer designer software. Amplification of GPDH was performed as follows. The PCR mix contained 11 ng genomic DNA, 5 μL Green GoTaq® reaction buffer (Promega®, Madison, WI, USA), 0.2 mM each of deoxyribonucleoside triphosphate (dNTP), 0.2 μM of each primer and 1.1 U of GoTaq® DNA polymerase in a total reaction volume of 25 μL in triplicates (PCR conditions: 5 min at 95°C, 35 cycles of 1 min at 94°C, 1 min annealing at 53°C, 2 min for extension 72°C and a final 5 min extension at 72°C). The quality of the amplicon was checked by performing agarose gel electrophoresis. The PCR products were purified and sequenced and sequences were assigned to taxa based on 98-100% sequence similarity threshold in DNA Data Base of Japan (DDBJ: AB859034, AB859035, AB859036, AB859037 and AB859038).
In silico mining of DNA databases: The overarching dataset was obtained by using key words such as Cochliobolus, Curvularia, Cochliobolus- and Curvularia- infections for interrogating PubMed database and Google-scholar to retrieve current literature and accession numbers. Herein, both Cochliobolus and Curvularia were used to avoid missing out information following recent nomenclatural changes such as the recommendations of International Commission on the Taxonomy of Fungi (ICTF) that Curvularia should be considered over Pseudocochliobolus and protect Bipolaris Shoemaker 1959 (A) over Cochliobolus Drechsler 1954 (S)20. Using GenBank® BLAST search tool, a studied set of GPDH sequences deposited in the last decade were collected based on the information associated with the sequences such as GC content, length (>200 bp), geographic origin of isolates and trimmed to unique sequence set using ElimDupes (available at http://hcv.lanl.gov/content/sequence/ELIMDUPES/ elimdupes.html). The data set was further cleaned by eliminating sequences with 100% sequence similarity for isolates from the same host and geographical coordinates. A total of 52 unique sequences were aligned in Muscle program21 and the best substitution model parameters were determined based on Akaike Information Criterion, corrected (AICc) and Bayesian Information Criterion (BIC).
Statistical analysis: The diversity analysis and sequence polymorphisms were performed in MEGA6 software22. The overall genetic diversity among C. lunata of plant and clinical origin was computed as previously described23.
Ethical approval and informed consent: No animals were used. Only potato plants and Cochliobolus lunatus strains were used as living organisms. Permission to survey potato farms in Burdwan District, the main potato farming zone in West Bengal, India was granted by the Government of India, Ministry of Science and Technology through the Department of Biotechnology vide program number No.3240223450, for the period of five years starting from 2010 to 2015.
RESULTS AND DISCUSSION
Transferability of plant isolates of C. lunata to human is a high risk factor for resistance based on wide genetic distance: Morphologically, C. lunata produces varied sizes of conidia (Fig. 1a). From the 52 sequences computed, 284 patterns were found out of a total of 732 sites and 360 sites were without polymorphism (49.18%). The estimated average evolutionary divergence over all Curvularia species in the data set was 0.275±0.041 based on T92+G model23. The estimated transition-transversion bias (R) in the data set was 2.78, hallmarked by [A↔T] = 3.303, [C↔A] = 3.303, [A↔G] = 18.392, [T↔C] = 18.392, [C↔G] = 3.303 and [G↔T] = 3.303 at a discrete Gamma distribution ([+G], parameter = 0.1097). While novel Curvularia species isolated from clinical specimens14 formed unique clusters, C. lunata from potato plants failed to cluster with clinical isolates (Fig. 1b). Considering the potato fields from which the C. lunata were located at about 1 km apart from each other, it is concluded that C. lunata are evolving divergently. In recent years, several previously known plant pathogens in the genus Curvularia such as C. lunata, C. brachyspora, C. clavata, C. geniculata, C. inaequalis, C. pallescens, C. senegalensis and C. verruculosa have developed capacities to invade human7,14,24. Among all the Curvularia species, C. lunata have shown high level genome plasticity25. For instance, whole genome sequencing of Curvularia species revealed that C. lunata is the most diverse species, bearing in mind that only ~20% of its genome aligned to the reference Cochliobolus heterostrophus C5 genome, compared to about 75% alignment observed to other Curvularia species25. Importantly, high genetic diversity of C. lunata is also attributed to its cross-mating abilities with other species26,27.
To test equality of evolutionary rate between lineage of C. lunata from plant and clinical origin, Tajima’s test28 for C. lunata (AB859034, AB859035 and AF081394) was performed at X2 test statistic cut-off 0.5 (p = 0.479 with 1 degree of freedom). Unique differences for C. lunata- AB859034 was 5, C. lunata-AB859035 was 3, C. lunata- AF081394 was 204, identical sites for all three sequences was 221 and divergent sites in all three sequences was 6. Thus, rejecting the null hypothesis that lineages of C. lunata from clinical and plant origins undergoes equal rate of evolution (Fig. 1b). This divergent evolution could pose a severe difficulty in management should C. lunata exposed to azoles fungicides jump host (or transferred) from plants to humans. Since it is discerning to assess the probable future burden of the implication of fungicides used for controlling Curvularia plant diseases in parallel with antifungal drug resistance now, rather than when the problem becomes severe.
| Fig. 1(a-b): | Taxonomic placement of C. lunata based on TN92+G substitution model29 and the tree was rooted as previously described19 |
Hypothetical interlocking lifestyles and fungicide pressure on Curvularia species enhances the emergence of resistance: Curvularia species profusely sporulates under adverse condition of high temperatures in or on putative hosts and conidia are easily dispersed in air. Thus, suggesting that Curvularia inoculum might be contracted by a healthy individual via breathing, interaction with infected crops, vegetables and scenario of direct interaction of a healthy individual with turfgrasses or ornamental plants in playgrounds. This is exemplified by footballers and farmers contracting Curvularia diseases caused by C. lunata15,16,30. Introduced in around late-1960’s, the use of azole-fungicides (such as imidazoles, propiconazole and tebuconazole) in farms and turfgrasses in playgrounds acts as selective pressure, creates fungal ecological imbalances, may promote virulence differentiation and in this situation, increases public health risk and burden.
Curvularia species are mostly sensitive to triazoles drugs and the interplay between azole-fungicide and a genetically evolved Curvularia species that is transferred from plant to man could hypothetically lead to resistance development in immunocompromised patients receiving treatment. Fundamentally, azole drugs likewise azole-fungicides inhibit the activity of lanosterol 14α-demethylase and disrupt the production of ergosterol, a major component of cell membrane and impair fungal growth. First and foremost, azoles are highly stable molecules that persist actively in water, soil, fruits and vegetables for months31-34. Thus, patients under long-term azole exposure either from environmental contamination or treatment are prone to develop resistance35. Since Curvularia infections are often times not acute, associated with non-specific symptoms and signs, consequently, many people contract Curvularia diseases unaware and undiagnosed. Considering the worldwide distribution of Curvularia diseases wherever maize, sorghum, millet, sugarcane and rice are farmed and the enormous use of fungicides to manage the diseases, it is tempting to hypothesize that by 2047, Curvularia antifungal drug resistance would be a critical public health problem. This is because by 2047, most Curvularia species infecting humans could have evolved to azoles resistant phenotypes due to azoles fungicide pressure. Taken together, fungicides selective pressure coupled with mutation and recombination event would consequently increases the likelihood of selecting resistance Curvularia species exhibiting resistance to azoles antifungal drugs.
Curvularia species infects immunocompetent and immunocompromised individuals: It has been revealed that common sites of Curvularia infections in humans are the nasal cavity, ocular, skin and nails, respiratory tract, eyes, in that order7,36-39 and in deep tissue such as central nervous system40,41. Although attention has been on a small cohort of fungi genera such as Cryptococcus, Aspergillus, Pneumocystis and Candida, there are increasing reports of Curvularia-mediated phaeohyphomycosis in human and animal10,42. Phaeohyphomycosis refers to cutaneous and systemic diseases caused by melanised fungi that often develop dark-wall-septate mycelia in host tissues. Curvularia lunata efficiently triggers subcutaneous phaeohyphomycosis hallmarked by granulomas in the adipose panniculus10. Eumycetoma caused by C. lunatus in back-shoulder of a female patient that lasted for 12 years was characterized by multiple swellings and discharging wounds43. However, Shinde et al.43 remarked that the rare Curvularia mediated-mycetoma was associated with Gram positive Staphylococcus aureus possibly occurring as opportunistic infections. In this study43 antibiotic therapy did not lead to improvement, but the patient responded well with itraconazole 200 mg twice daily. C. lunata mediated-eumycetoma appears to be common in Africa, India, Mexico and South America16. Recent cases of eumycetoma44-53 caused by C. lunata and treatment regimens are summarized (Table 1). Previously, Rinaldi et al.40 found that in a sample size of 24 patients infected by Curvularia species, only two patients were systemically immunosuppressed. The results indicated that Curvularia species causes diseases both in immunocompetent and immunocompromised individuals at different levels of severity.
Intrinsic resistance and current challenges in the treatment of Curvularia infections: Curvularia species are known to abundantly produce melanin24, coloured metabolites and secretes diverse secretome during infection54. One key attribute of melanin is that it protects the microbes from host defenses, increases resistance to phagocytosis in vitro and in vivo55,56, confer a survival advantage in the environment57 and have the ability to bind amphotericin B and caspofungin58. Thus, melanin make fungal clearance hard in infected tissues. Curvularia species are dark-brown and highly pigmented molds and constitutively secretes melanin during colonisation of putative host, as a result, treatment with current antifungal drugs is challenging.
The optimal antifungal therapy for the treatment of Curvularia infections is still unknown, amphotericin B deoxycholate (which targets ergosterol and disrupting plasma membrane), azoles derivatives viz., miconazole, ketoconazole and itraconazole (which targets ergosterol biosynthesis at 14-α-demethylase) and terbinafine (which inhibits ergosterol synthesis at the level of squalene epoxidase) have actively been used in the treatment of Curvularia infections producing variable results53. Occasional resistance to amphotericin B has been observed in Curvularia spp., Chaeromium spp., Phialemonium spp. and Exophiala spp., which are heavy melanin producing fungi59. Echinocandins has been found clinically irrelevant to heavy melanin producing fungi57, nonetheless, azoles show broader in vitro activity against Curvularia species43,45,46.
| Table 1: | History of C. lunata infections indicate that it affects all age groups and many parts of the body |
Interestingly, voriconazole and itraconazole showed poor activity to most frequently encounter isolates such as Curvularia aeria, Curvularia geniculata/Curvularia senegalensis, C. lunata, Curvularia inaequalis, Curvularia verruculosa and Curvularia borreliae in USA36, suggesting that effective therapy could only be achieve via rigorous trials.
Till date, there is no WHO protocol or consensus on treatment guideline, type of antifungal drugs and treatment regime for Curvularia infections. On the basis that C. lunata indiscriminately infects all parts of the leg, peritoneal cavity, respiratory tract, central nervous system and the eye of human (Table 1) and more so, infections are treated through trials of different antifungal drugs, development of resistance is eminent in the near future. To assert this, Paredes et al.60 used immunosuppressed murine models infected with Curvularia species to test the efficiency of amphotericin B, posanazole and voriconazole and found only the 2 azoles could decrease the fungal load. To achieve immunity suppression, 5-fluorouracil and cyclophosphamide were injected into the mouse. The outcome of amphotericin B resistance in immune-suppressed mouse was in line with Varughese et al.61, who identified an amphotericin-resistant C. lunata that caused peritonitis. Efforts to outwit resistance in C. lunata have begun, notably, the identification of a new cytochrome P450, CYP53A15, largely assumes to be the target for natural antifungal compounds62. Although the incidence of antifungal resistance is low, evidence from Table 1 indicated that combine therapy is the most effective method for the treatment of Curvularia infections.
CONCLUSION
Curvularia species thrived on plant hosts as well as infect immunocompetent and immunocompromised individuals at variable degree of severity. Curvularia lunata from plant and clinical origin are genetically diverse based on GPDH locus. Treatment of Curvularia infection is hard to achieve and appropriate combination therapy last for at least twelve weeks. With the current use of azole-fungicides, it is herein predicted that by the year 2047 most Curvularia species infecting plants and humans may have evolved to azole-resistant phenotypes posing severe difficulties in the management of Curvularia diseases. Finally, strengthening legislation aimed at reducing the use of azole-fungicides could help reduce the selective pressure that contributes to genetic diversity of Curvularia species.
SIGNIFICANCE STATEMENTS
| • | Curvularia species resistance to azole antifungal drugs and fungicides showed global increase in reported cases |
| • | Evidence rationale use of azole-fungicides in farms could reduce selective pressures that drive resistance in Curvularia species to antifungal therapy |
ACKNOWLEDGMENTS
This study was supported by The World Academy of Sciences (TWAS), Trieste, Italy and the Department of Biotechnology, Government of India (DBT/TWAS PG grant No. 3240223450), Alexander von Humbolt (AvH) foundation and National Research Foundation (NRF), South Africa.