Global molecular epidemiology and genetic diversity of Fusarium, a significant emerging group of human opportunists from 1958 to 2015

INTRODUCTION

Fusarium infections are a major challenge with respect to the diagnosis and treatment, especially in neutropenic patients. Disseminated infections may be fatal and are a considerable source of increased healthcare costs. A major area of concern is the intrinsic resistance to a broad range of antifungals,¹ which is a characteristic of Fusarium. During the past decade, the F. solani complex has received special interest because of the increasing numbers of infections worldwide.² More than 300 cases of Fusarium keratitis were associated with contaminated contact lens cleaning solution, causing outbreaks between 2005 and 2007, where members of the F. solani species complex played a major role.³ Furthermore, reservoirs of infectious Fusarium species in hospital environments, especially plumbing and water systems, have been reported.⁴

Although human fusariosis was only recognized since the late 1950s and endemic areas are mostly located in tropical and subtropical countries,⁵ their global significance has only recently come into focus within the past three decades. Etiological agents differ in antifungal susceptibility,⁶ virulence profiles, geographic distribution, ecological niches, life cycle, host and mycotoxin production.⁷ Although agents of fusariosis are mostly environmental,⁸ the potential of nosocomial transmission has recently been raised,⁹ especially with reference to the high mortality rate of ~90% in patients with prolonged and severe neutropenia.¹⁰

The burden of disease has not been established, but numerous clinical case series and case reports provide an estimate of the magnitude of the problem. Most published studies have focused on prevalence in single healthcare centers.^{10, 11, 12, 13, 14, 15, 16} Nucci et al.¹⁷ reported 233 cases from different hospitals on a global scale. Mohammed et al.¹⁸ reported 26 cases from the United States and reviewed 97 cases from the literature, and Horn et al.¹² described 65 cases from the North American Path Alliance Registry. A major problem in comparative studies is the subdivision of the classical species into a series of molecular siblings, which renders the older literature without sequence data uninterpretable. Despite the current clinical importance of the organism, the phylogenetic relationships among species, varieties and geographical groups in Fusarium are currently elusive. Hence, the re-interpretation of these data in the light of modern molecular phylogeny is compulsory.

Molecular phylogenetic studies have led to the description of many Fusarium species with clinical relevance. These include members of the F. solani species complex, namely, F. falciforme, F. keratoplasticum, F. lichenicola, F. petroliphilum, F. pseudensiforme and F. solani (FSSC5), which is also known as Fusisporium solani and Fusarium haplotype ‘6'. The F. oxysporum species complex (FOSC) contains three lineages, which are involved in fusariosis and still have not been formally introduced as taxonomic species. The F. fujikuroi species complex includes F. acutatum, F. ananatum, F. anthophilum, F. andiyazi, F. fujikuroi s.s., F. globosum, F. guttiforme, F. musae, F. napiforme, F. nygamai, F. verticillioides, F. proliferatum, F. ramigenum, F. sacchari, F. subglutinans, F. temperatum and F. thapsinum. Although rare, species of other Fusarium lineages are emerging as potential opportunistic pathogens, for example, in the F. incarnatum-equiseti species complex (FIESC; F. incarnatum and F. equiseti), the F. dimerum species complex (F. dimerum, F. delphiniodes and F. penzigii), the F. chlamydosporum species complex, the F. sambucinum species complex (F. armeniacum, F. brachygibbosum, F. langsethiae and F. sporotrichioides) and the F. tricictum species complex (F. acuminatum and F. flocciferum).¹

Over the past decade, the number of cases of fusariosis has increased worldwide, but there are only a few reports describing the molecular epidemiology; therefore, the aim of the present study is to introduce a hypothetical system that permits the interpretation and use of at least a part of the literature where sequence data are lacking. Pre-molecular publications, which include interpretable case reports and geographical information, were collected. Subsequently, available Fusarium strains that were collected worldwide and deposited during the past century in the CBS-KNAW, Fungal Biodiversity Centre, culture collection Utrecht, The Netherlands, were sequenced and re-identified with current diagnostic technology, which enables the phylogenetic analysis of the human–pathogenic Fusarium species. These data were then compared with published materials and their distribution with the assumption that their distributions in each region had remained unaltered.

MATERIALS AND METHODS

RESULTS

DISCUSSION

The molecular epidemiology of Fusarium was investigated based on the genetic data generated for the RPB2 and TEF1 sequence analysis supplemented with the AFLP fingerprint data. The current study provides information on the locality, sources, species and geographic distribution in countries in which these fungi are common such as Brazil, China, Colombia, India, Mexico and the USA. One hundred and thirty Fusarium isolates, including eight type strains deposited at the CBS-KNAW collection, were included. Overall, there was a good correlation between sequence analysis and AFLP genotypes. To analyze the genetic relationships among the AFLP and sequencing genotypes, we used phylogenetic algorithms, which are commonly used to detect evidence of population subdivision and differentiation.²⁶

The second largest subunit of RNA polymerase (RPB2) and TEF1 showed high resolution with high species-level resolution, distinguishing 29 Fusarium species including the most common clinically important Fusarium species F. falciforme, F. keratoplasticum, F. oxysporum, F. petroliphilum, F. proliferatum and F. verticillioides. Furthermore, these two loci were able to distinguish between other rare Fusarium species that cause a variety of infections (Table 1).

Although human opportunists were highlighted in many studies focusing on specific regions of the world and specific types of infections,^{10, 11, 12, 13, 27, 28, 29, 30, 31, 32, 33, 34} the 127 Fusarium strains from the current study were collected from 26 countries in six continents and included clinical and environmental strains and isolates from cold blooded animals. Of these, Australia, Brazil, India, Mexico and the USA were among the top 10 countries with the highest Fusarium infections based on clinical isolates in the CBS collection. Not surprisingly, 75 of the 127 patients from this study acquired their infection in one of these countries.

Previously, the majority of the clinically relevant Fusarium species were classified as two species complexes that in the past were referred to as a single species, F. oxysporum and F. solani.³⁵ Approximately 80% of human infections are caused by members of both species complexes,³⁶ but a significant share of infections is caused by the following novel species complex members: F. dimerum, F. fujikuroi and F. incarnatum-equiseti. Within the F. solani complex, there are six recognized species and one unnamed lineage (FSSC6) clinically involved in fusariosis (Figure 1). Of these species, F. falciforme (n=14/127 cases; 11%) was the dominant species in our study and mainly isolated from keratitis cases in Brazil, India and Mexico. Recently, Hassan et al.¹³ showed that the majority of keratitis cases (n=46/65 cases; 70.7%) were F. falciforme. This species is emerging as one of the most virulent Fusarium species associated with fusariosis and keratitis.^{15, 36, 37}

In the 2005–2006 mycotic keratitis outbreaks in Southeast Asia and North America that were associated with a contact lens cleaning solution, F. petroliphilum and F. keratoplasticum were the most common species,³⁶ which is consistent with the current study. The AFLP genotypic variability was higher in the environmental species than in the clinical species. A potential explanation is that not all environmental genotypes are sufficiently adapted to the host tissue and are not selected or perhaps a sampling effect is involved. Zhang et al.³⁵ studied the F. solani species complex, specifically those species that cause infections in humans and plants, and concluded that clinical isolates often shared multi-locus haplotypes with isolates from different environmental sources, including hospital locations. An increase of fusariosis among immunosuppressed patients was noted in the bone marrow transplant unit and among patients with superficial infections in a hospital in Rio de Janeiro, Brazil.³⁸ These authors concluded that this increase might be due to airborne conidia circulating in this geographical region. Short et al.³⁶ concluded that there is no evidence that clinical isolates differ from those collected from other sources.

The large diversity of the FOSC is not completely resolved, and it is not yet known whether the species have one or several phylogenetic origins or whether a single species or a species complex is concerned. From a traditional taxonomic point of view, F. oxysporum isolates are differentiated from each other based on the pathogenicity as formae speciales, but this has been shown to be an unreliable approach.⁸ In addition, the species delimitation was for the FOSC, and at least 26 sequence types within the complex were involved in human infections.³⁹ Our FOSC clinical isolates were distributed throughout the complex, although some clustering was found in the clade marked ‘sequence type 33', which is based on TEF1 alone, and this sequence type is considered the most common clade that contains clinical F. oxysporum strains. The remaining species complexes of F. chlamydosporum, F. concolor, F. dimerum and F. incarnatum-equiseti form separate clusters in the highly resolved sequence-based maximum likelihood tree (Figure 1).

The FIESC compromises 28 phylogenetically distinct lineages,³⁴ and only 2 are named and mainly involved in human infections (F. incarnatum and F. equiseti).⁴⁰ Although several members of the FIESC were included in the CDC Fusarium keratitis outbreak investigations, these species have not yet been observed to occur in epidemics or cause outbreaks. Concerning geography, 51 clinical isolates were recovered from the United States, and this revealed that phylogenetically diverse human opportunists are well represented in North America.⁴⁰ In our study, three clinical F. equiseti strains originated from Mexico, and this might suggest that species of this complex are common in this region. The virulence of members of the FIESC has been ascribed to their production of type A and B trichothecene mycotoxins.³⁹

F. dimerum and F. delphinoides belong to the F. dimerum species complex, and both were isolated from superficial and disseminated infections.¹⁵ In our data set, a supported clade of FDSC matching with the AFLP data mainly contained strains from India, and this might suggest a regional prevalence. F. chlamydosporum was reported in disseminated infections in patients with aplastic anemia and lymphocytic lymphoma from the United States^{41, 42} CBS 111770 (F. concolor) is the only clinical strain in the F. concolor species complex, and it was reported in a keratitis case from Spain.⁴³

By comparing AFLP and MLST data, F. falciforme and F. keratoplasticum appear to be widely distributed, at least in Mexico, North America, Europe and India, with dominancy in superficial infections, including keratitis and onychomycosis. F. petroliphilum is the second most diverse species and is also frequently involved in disseminated infections. F. solani sensu stricto, ‘5', which was recently described as Fusisporium (Fusarium) solani (FSSC5),⁷ contains strains such as CBS 135559, CBS 135564 and CBS 135565, which originate from Mexico, and shows significant occurrence in keratitis cases. This species was also recently reported in Asia (India and Qatar).^{13, 16} Given the large distances of identical strains occurring in many different countries, airborne distribution seems likely. However, the presence of F. incarnatum, F. equiseti and F. chlamydosporum in clinical samples from various infections in North America remains puzzling but can perhaps be explained by sampling effects.

As previously noted,⁴⁴ the F. fujikuroi complex contains the highest number of species. In our study, 15 supported clades were recognized in all of the molecular analyses (Figures 1 and 2). Nearly all of the clades have various geographic distributions. Within the F. fujikuroi species complex, F. proliferatum and F. verticillioides were the dominant clinically relevant species, having a global distribution and dominating in disseminated infections. F. sacchari is the second most prevalent species and was often isolated from keratitis restricted to India. Although F. nygamai and F. napiforme are the most multidrug-resistant species within the F. fujikuroi complex,⁴⁵ their presence in human infections is rare. F. acutatum was reported from nail infections in four cases in Qatar, showed a low degree of variability and has been suggested to be clonal.¹⁶ These results emphasize that F. acutatum is an emerging human opportunist, which thus far was only detected in Asia. Sequence analysis of RPB2 and TEF1, and AFLP showed that the strains CBS 119850, CBS 483.94 and CBS 454.97 were nested within the F. fujikuroi complex and close to F. nygamai and F. andiyazi, forming a well-supported monophyletic branch suggestive of a novel species.

Deep fusariosis is rare in healthy individuals; a single brain infection has been reported.⁴⁶ Local infections may occur after a direct inoculation or tissue breakdown by trauma or the entrance of foreign bodies. The treatment of superficial infections is usually successful and requires surgery, the removal of the foreign body and antifungal therapy. The most important risk factors for severe fusariosis are prolonged neutropenia and T-cell immunodeficiency in patients suffering from a hematologic malignancy. Fusarium infections in the majority of these cases were due to neutropenia. Furthermore, in solid organ transplant recipients and cancer patients with neutropenia, infections due to Fusarium spp. increased and led to disseminated infection. Patients develop painful skin lesions, which vary from papules to nodules with or without central necrosis.⁴⁷ In the majority of disseminated infections, secondary skin lesions led to a diagnosis in >50% of the patients and preceded fungemia by ~5 days.⁴⁸ In contrast to aspergillosis, fusariosis frequently shows positive blood cultures because Fusarium conidia are hydrophilic and allow dissemination.⁴⁷ Comparing fusariosis with mucormycosis,⁴⁹ solid tumors and diabetes do not seem to be important risk factors. Only 17 (4.8%) cases were found in patients with solid tumors, and seven infections were reported in patients with diabetes mellitus. No underlying conditions were observed in 20 (5%) of the cases.

Fusarium treatment depends on the site of infection. Surgery with antifungals was used in 80 cases (20.6%). Disseminated fusariosis in immunocompromised patients is usually treated with amphotericin B and voriconazole as the first-line therapy, which is suggested by recent guidelines.⁵⁰ In our literature review, most antifungal therapy was amphotericin B deoxycholate, followed by liposomal amphotericin B and voriconazole. The most commonly used combination is amphotericin B/voriconazole followed by amphotericin B/5-flucytosine. Triple combinations were used in 14 cases with different antifungals.

The major findings of the present study include the following: (i) human-associated fusaria were nested within seven species complexes (that is, F. chlamydosporum, F. concolor, F. dimerum, F. fujikuroi, F. incarnatum-equiseti, F. oxysporum and F. solani), (ii) the three most common species presented in both the clinical and environmental groups are F. falciforme and F. keratoplasticum (members of F. solani species complex) followed by F. oxysporum (FOSC), (iii) most of the reported Fusarium species in this study were shared among the patients and the environment, and this might be due to the colonization of some patients with Fusarium isolates from the environment; hence, there is genetic similarity between the clinical and environmental isolates of the same Fusarium species, and (iv) the species distributions show some evidence of geographical clustering among some of the species studied, although the present study is limited by an over-representation of isolates from Mexico and India.

Supplementary Material