Molecular Epidemiology of Agents of Human Chromoblastomycosis in Brazil with the Description of Two Novel Species.

The human mutilating disease chromoblastomycosis is caused by melanized members of the order Chaetothyriales. To assess population diversity among 123 clinical strains of agents of the disease in Brazil we applied sequencing of the rDNA internal transcribed spacer region, and partial cell division cycle and β-tubulin genes. Strains studied were limited to three clusters divided over the single family Herpotrichiellaceae known to comprise agents of the disease. A Fonsecaea cluster contained the most important agents, among which F. pedrosoi was prevalent with 80% of the total set of strains, followed by 13% for F. monophora, 3% for F. nubica, and a single isolate of F. pugnacius. Additional agents, among which two novel species, were located among members of the genus Rhinocladiella and Cyphellophora, with frequencies of 3% and 1%, respectively.

Introduction

Chromoblastomycosis is a chronic granulomatous infection of the skin caused by melanized fungi. It has a worldwide distribution mainly in tropical and subtropical climate zones, with a preference for humid climates with dense forestation, with Cladophialophora carrionii being the only species that is restricted to semi-arid areas with Cactaceae as main vegetation. Endemic areas are in Japan, Southeast Asia, Australia, Madagascar, as well as South and Central America [1-7]. In Brazil, the infection is observed in all states, with an estimated prevalence of 1/196 thousand inhabitants, but in some hyperendemic regions a considerably higher prevalence is noted [8]. Infection is assumed to occur through accidental inoculation of the fungus via contaminated plant debris, being favored by agricultural activities denoting an occupational nature of the disease. Chromoblastomycosis is one of the most frequent implantation mycoses found among rural populations [2, 8–11].

Clinically, the disease is characterized by pseudoepitheliomatous hyperplasia with epidermal microabscesses and dermal granulomata [12, 13]. The disease has a slow evolution, but finally may result in disfigurement of affected body sites [8]. The initial lesion is noted as a small pink papule at the site of inoculation which gradually enlarges. Development of superficial erythematous plaques with scaly or warty appearance probably takes several months or years. As a result of acanthosis these lesions may develop into large papillomatous and verrucous warts.

Species of the humid climate are particularly members of the genus Fonsecaea, with F. pedrosoi, F. monophora and F. nubica as prevalent agents [14-17]. Recently another species, F. pugnacius was described [18]. Rhinocladiella aquaspersa is an uncommon species of humid as well as of dry climates [19]. Other reported agents such as Phialophora verrucosa and Exophiala dermatitidis [17, 20] are extremely rare and mostly cause other types of infections. Fonsecaea pedrosoi is nearly exclusively isolated from chromoblastomycosis, while F. monophora repeatedly causes brain infection and F. pugnacius combines the two disorders by starting as chromoblastomycosis with cerebral dissemination in a single patient [18]. Species distinction of agents of the disease is clinically significant because of the differences in prognosis of the infection.

The present study evaluates the diversity of agents in endemic areas of Brazil based on multilocus sequence data, clinical aspects, direct mycological examination and culture. An enumeration of currently proven cases with molecular support in endemic areas in Brazil is provided.

Results

A set of 123 clinical strains from cases of chromoblastomycosis from different endemic areas in Brazil was analyzed. Judging from a reference set of partial LSU rDNA sequences of members of Chaetothyriales available at CBS, agents of chromoblastomycosis were polyphyletic within the order, being dispersed in three different clades: jeanselmei-, bantiana- and europaea-clades (Fig 1, arrows).

Fig 1

Phylogeny of a representative selection of species in Chaetothyriales, based on confidently aligned LSU sequences.

Constructed with Maximum likelihood implemented in MEGA 7. Bootstrap values > 80% from 100 resampled datasets are shown with branches. Coloured boxes represent species complexes taken from de Hoog et al. [21], Feng et al. [22], and Vicente et al. [23]. Clades with species causing chromoblastomycosis analysed in this study are indicated with arrows. Type strain in bold.

Multilocus sequence analyses using ITS, and partial BT2 and CDC42 genes were performed for identification and for elucidation of species identities (S1 Fig). The 123 clinical strains clustered in different clades within the Chaetothyriales, being centred around members of the genera Fonsecaea, Rhinocladiella, and Cyphellophora (Table 1). The Brazilian clinical isolates used in this study all were derived from humid climate zones; members of the Cladophialophora carrionii group were not encountered.

Table 1

Strains analysed.

Name	Strain number	Geograph/ Brazil	Chromoblastomycose lesions	ITS; BT2; CDC42; LSU
Fonsecaea monophora	CMRP1290	MA, Northeast	Polymorphic lesions	KR732306; KR732312; KR732318
Fonsecaea monophora	CMRP1329	PR, North	Plaque	KX434631; KX583711
Fonsecaea monophora	CMRP1330	PR, North	Nodular	KX434632
Fonsecaea monophora	CMRP1359	AM, North	NI	KX434633
Fonsecaea monophora	CMRP1360	AM, North	NI	KX434634
Fonsecaea monophora	CMRP1335	MG, Southeast	NI	KX434639
Fonsecaea monophora	CMRP1366	PR, South	Polymorphic lesions	KX434635
Fonsecaea monophora	CMRP1367	RS, South	NI	KX434636
Fonsecaea monophora	CMRP1368	RS, South	NI	KX434637
Fonsecaea monophora	CMRP1369	RS, South	NI	KX434638
Fonsecaea monophora	CMRP1371	PR, South	NI	KX434640
Fonsecaea monophora	CMRP1383	PR, South	NI	KX434641
Fonsecaea monophora	CBS 102242	PR, South	Verrucous	EU938583; EU938549
Fonsecaea monophora	CBS 102243	PR, South	Scarring	EU938579; EU938542
Fonsecaea monophora	CBS 102246	PR, South	NI	AY366928; EU938543
Fonsecaea monophora	CBS 102248	PR, South	NI	AY366926; EU938550
Fonsecaea nubica	IAL 4004	BA, Northeast	Verrucous	KU892412
Fonsecaea nubica	IAL 3994	RO, North	Verrucous	KU881739
Fonsecaea nubica	IAL 3992	MG, Southeast	Nodular and tumoral	KU881735
Fonsecaea nubica	IAL 3999	SP, Southeast	Polymorphic lesions	KU8811742
Fonsecaea pedrosoi	CMRP1342	MA, Northeast	NI	KX434642
Fonsecaea pedrosoi	CMRP1271	MA, Northeast	Polymorphic lesions	KR732301; KR732307; KR732313
Fonsecaea pedrosoi	CMRP1272	MA, Northeast	Nodular and plaque	KR732302; KR732308; KR732314
Fonsecaea pedrosoi	CMRP1273	MA, Northeast	Plaque	KR732303; KR732309; KR732315
Fonsecaea pedrosoi	CMRP1274	MA, Northeast	Nodular and plaque	KR732304; KR732310; KR732316
Fonsecaea pedrosoi	CMRP1275	MA, Northeast	Plaque	KR732305; KR732311; KR732317
Fonsecaea pedrosoi	CMRP1276	MA, Northeast	Scarring, nodular and plaque	KX434643; -; KX583684
Fonsecaea pedrosoi	CMRP1277	MA, Northeast	Plaque	KX434644; KX583712; KX583685
Fonsecaea pedrosoi	CMRP1278	MA, Northeast	Scarring and plaque	KX434645; KX583713; KX583686
Fonsecaea pedrosoi	CMRP1279	MA, Northeast	Plaque	KX434646; -; KX583687
Fonsecaea pedrosoi	CMRP1280	MA, Northeast	Nodular and plaque	KX434647; KX583714; KX583688
Fonsecaea pedrosoi	CMRP1281	MA, Northeast	Nodular and plaque	KX434648
Fonsecaea pedrosoi	CMRP1282	MA, Northeast	Nodular and plaque	KX434649; -; KX583689
Fonsecaea pedrosoi	CMRP1283	MA, Northeast	Nodular and plaque	KX434650; -; KX583690
Fonsecaea pedrosoi	CMRP1284	MA, Northeast	Plaque	KX434651; KX583715; KX583691
Fonsecaea pedrosoi	CMRP1285	MA, Northeast	Plaque	KX434652; -; KX583692
Fonsecaea pedrosoi	CMRP1286	MA, Northeast	Scarring, nodular and plaque	KX434653; KX583716
Fonsecaea pedrosoi	CMRP1288	MA, Northeast	Scarring and plaque	KX434654; KX583717; KX583693
Fonsecaea pedrosoi	CMRP1289	MA, Northeast	Plaque	KX434655; -; KX583694
Name	Strain number	Geograph/ Brazil	Chromoblastomycose lesions	ITS; BT2; CDC42; LSU
Fonsecaea pedrosoi	CMRP1291	MA, Northeast	Plaque	KX434656; KX583718; KX583695
Fonsecaea pedrosoi	CMRP1292	MA, Northeast	Plaque	KX434657; KX583719; KX583696
Fonsecaea pedrosoi	CMRP1293	MA, Northeast	Plaque	KX434658; KX583720; KX583697
Fonsecaea pedrosoi	CMRP1294	MA, Northeast	Scarring and plaque	KX434659; KX583721; KX583698
Fonsecaea pedrosoi	CMRP1295	MA, Northeast	Scarring and plaque	KX434660; -; KX583699
Fonsecaea pedrosoi	CMRP1296	MA, Northeast	Scarring, nodular and plaque	KX434661; -; KX583700
Fonsecaea pedrosoi	CMRP1298	MA, Northeast	Plaque	KX434662; KX583722
Fonsecaea pedrosoi	CMRP1299	MA, Northeast	Plaque	KX434663; KX583723
Fonsecaea pedrosoi	CMRP1300	MA, Northeast	Scarring, nodular and plaque	KX434664
Fonsecaea pedrosoi	CMRP1301	MA, Northeast	Plaque	KX434665; KX583724; KX583701
Fonsecaea pedrosoi	CMRP1302	MA, Northeast	Scarring, nodular and plaque	KX434666; KX583725; KX583702
Fonsecaea pedrosoi	CMRP1303	MA, Northeast	Plaque	KX434667
Fonsecaea pedrosoi	CMRP1304	MA, Northeast	Polymorphic lesions	KX434668; KX583726; KX583703
Fonsecaea pedrosoi	CMRP1305	MA, Northeast	Nodular and plaque	KX434669; KX583727; KX583704
Fonsecaea pedrosoi	CMRP1306	MA, Northeast	Nodular, verrucous and plaque	KX434670; KX583728; KX583705
Fonsecaea pedrosoi	CMRP1308	MA, Northeast	Plaque	KX434671; KX583729; KX583706
Fonsecaea pedrosoi	CMRP1309	MA, Northeast	Scarring and plaque	KX434672; KX583730; KX583707
Fonsecaea pedrosoi	CMRP1310	MA, Northeast	Plaque	KX434673
Fonsecaea pedrosoi	CMRP1311	MA, Northeast	Plaque	KX434674; KX583731
Fonsecaea pedrosoi	CMRP1313	MA, Northeast	Plaque	KX434675; KX583732
Fonsecaea pedrosoi	CMRP1314	MA, Northeast	NI	KX434676; KX583733
Fonsecaea pedrosoi	CMRP1315	MA, Northeast	Nodular, tumoral and plaque	KX434677; KX583734
Fonsecaea pedrosoi	CMRP1316	MA, Northeast	Plaque	KX434678
Fonsecaea pedrosoi	CMRP1318	MA, Northeast	NI	KX434679
Fonsecaea pedrosoi	IAL 3991	BA, Northeast	Verrucous	KU881737
Fonsecaea pedrosoi	IAL 3997	BA, Northeast	Verrucous	KU881741
Fonsecaea pedrosoi	IAL 3996	BA, Northeast	Verrucous	KU881745
Fonsecaea pedrosoi	CMRP1331	PA, North	Nodular	KX434680
Fonsecaea pedrosoi	CMRP1332	PA, North	Nodular	KX434681; KX583735
Fonsecaea pedrosoi	CMRP1333	PA, North	Nodular	KX434682
Fonsecaea pedrosoi	CMRP1334	PA, North	Nodular	KX434683; KX583736
Fonsecaea pedrosoi	CMRP1337	PA, North	Nodular	KX434684
Fonsecaea pedrosoi	CMRP1338	PA, North	Nodular	KX434685; KX583737
Fonsecaea pedrosoi	CMRP1339	PA, North	Nodular	KX434686; KX583738
Fonsecaea pedrosoi	CMRP1340	PA, North	Nodular	KX434687; KX583739
Fonsecaea pedrosoi	CMRP1341	PA, North	Nodular	KX434688; KX583752
Fonsecaea pedrosoi	CMRP1344	PA, North	Nodular	KX434689; KX583740
Fonsecaea pedrosoi	CMRP1345	PA, North	Nodular	KX434690
Fonsecaea pedrosoi	CMRP1346	PA, North	Nodular	KX434691; KX583741
Fonsecaea pedrosoi	CMRP1347	PA, North	Nodular	KX434692
Fonsecaea pedrosoi	CMRP1348	PA, North	Nodular	KX434693; KX583742
Fonsecaea pedrosoi	CMRP1349	PA, North	Nodular	KX434694
Fonsecaea pedrosoi	CMRP1350	PA, North	Nodular	KX434695; KX583743
Fonsecaea pedrosoi	CMRP1351	PA, North	Plaque	KX434696; KX583744
Fonsecaea pedrosoi	CMRP1352	PA, North	Nodular	KX434697; KX583745
Fonsecaea pedrosoi	CMRP1353	PA, North	Nodular	KX434698; KX583746
Fonsecaea pedrosoi	CMRP1354	PA, North	Nodular	KX434699
Fonsecaea pedrosoi	CMRP1355	PA, North	Nodular	KX434700; KX583747
Fonsecaea pedrosoi	CMRP1356	PA, North	Nodular	KX434701
Fonsecaea pedrosoi	CMRP1357	PA, North	Nodular	KX434702; KX583748
Name	Strain number	Geograph/ Brazil	Chromoblastomycose lesions	ITS; BT2; CDC42; LSU
Fonsecaea pedrosoi	CMRP1361	PA, North	Nodular	KX434703
Fonsecaea pedrosoi	IAL 3998	RO, North	Nodular and tumoral	KU881736
Fonsecaea pedrosoi	IAL 4001	RO, North	Verrucous	KU881744
Fonsecaea pedrosoi	IAL 4005	RO, North	Polymorphic lesions	KU892413
Fonsecaea pedrosoi	IAL 4007	RO, North	Verrucous	KU892414
Fonsecaea pedrosoi	CMRP1336	MG, Southeast	NI	KX434709
Fonsecaea pedrosoi	CMRP1362	MS, Southeast	NI	KX434704
Fonsecaea pedrosoi	CMRP1363	MS, Southeast	NI	KX434705
Fonsecaea pedrosoi	CMRP1364	MS, Southeast	NI	KX434706
Fonsecaea pedrosoi	CMRP1365	MS, Southeast	NI	KX434707
Fonsecaea pedrosoi	IAL 3993	MG, Southeast	Nodular and tumoral	KU881738
Fonsecaea pedrosoi	CMRP1372	PR, South	Nodular and verrucous	KX434710
Fonsecaea pedrosoi	CMRP1384	PR, South	Nodular and verrucous	KX434717
Fonsecaea pedrosoi	CMRP1373	PR, South	Scarring	KX434711
Fonsecaea pedrosoi	CMRP1374	PR, South	Scarring and verrucous	KX434712
Fonsecaea pedrosoi	CMRP1375	PR, South	Nodular and verrucous	KX434713
Fonsecaea pedrosoi	CMRP1376	PR, South	Verrucous	KX434714
Fonsecaea pedrosoi	CBS 102245	PR, South	Scarring and verrucous	AY366918; EU938562
Fonsecaea pedrosoi	CBS 102247	PR, South	NI	AY366919; EU938566
Fonsecaea pedrosoi	CMRP1377	PR, South	Verrucous	KX434715
Fonsecaea pedrosoi	CMRP1378	PR, South	Plaque	KX434720
Fonsecaea pedrosoi	CMRP1379	PR, South	Polymorphic lesions	KX434721
Fonsecaea pedrosoi	CMRP1380	PR, South	Nodular and tumoral	KX434716
Fonsecaea pedrosoi	CMRP1381	PR, South	Nodular and tumoral	KX434718
Fonsecaea pedrosoi	CMRP1382	PR, South	NI	KX434719
Fonsecaea pedrosoi	CMRP1370	RS, South	NI	KX434708
Fonsecaea pedrosoi	IAL 4000	NI	Polymorphic lesions	KU881743
Fonsecaea pedrosoi	IAL 4006	NI	Verrucous	KU892409
Fonsecaea pedrosoi	IAL 4002	NI	Verrucous	KU892410
Fonsecaea pugnacius	CBS 139214	MA, Northeast	Plaque and disseminated to brain	KR706553; KR706547; KR706551
Cyphellophora ludoviensis sp. nov.	CMRP1317	MA, Northeast	Nodular and plaque	KX434722; KX583749; -; KX583708
Rhinocladiella tropicalis sp. nov.	CMRP1287	MA, Northeast	Polymorphic lesions	KX434723; KX583750; -; KX583709
Rhinocladiella tropicalis	CMRP1307	MA, Northeast	Plaque	KX434724; KX583751; -; KX583710
Rhinocladiella tropicalis	IMT776	SP, South	NI	KU854928

CMRP, Microbial Collections of Paraná Network- TAXon line; CBS, CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands; IMT, Tropical Medicine Institute, SP, Brazil; IAL, Culture Collection of Adolfo Lutz Institute. BT2, partial beta-tubulin gene; ITS, internal transcribed spacer regions of the rDNA and intervening 5.8S nuclear ribosomal DNA (nrDNA); CDC42, partial cell division cycle gene. Brazil Sates: BA. Bahia; MA. Maranhão; MG. Minas Gerais; MS. Mato Grosso do Sul; PA. Para; RO. Rondônia; PR. Paraná; RS. Rio Grande do Sul; SP. São Paulo; NI. not informed, patients in treatment at SP.

Fonsecaea is nested in the ‘bantiana-clade’ (Table 1) and contains the prevalent agents of the disease. Of the studied isolates, 98 (80%) grouped as Fonsecaea pedrosoi, followed by 16 (13%) isolates of F. monophora, 4 (3%) isolates of F. nubica and one isolate of F. pugnacius. Isolates of the Rhinocladiella group and of phialophora-like species are considered as rare agents of chromoblastomycosis in the Americas. Rhinocladiella was nested in the ‘jeanselmei-clade’ and the phialophora-like agent clustered in the ‘europaea-clade’ (presently known as Cyphellophoraceae); two unnamed species were uncovered (Fig 1).

The analyzed ITS and BT2 regions and a tree resulting from combined loci revealed identical topologies in phylogenetic analyses. The unnamed strains CMRP1287, CMRP1307, IMT776 and CMRP1317 were found to be concordantly positioned in all trees and grouped with remaining members of Rhinocladiella and Cyphellophora causing chromoblastomycosis (Figs 2 and 3). Clinical strain CMRP1317 was located at a significant distance from all reference strains of Cyphellophora, i.e with combined ITS and BT2 data the distance was 23.4%. Strains CMRP1287, CMRP1307 and IMT776 were also located at significant distance from known species in Rhinocladiella (Table 2). Consequently the strains were judged to represent novel taxa in Cyphellophora and Rhinocladiella, respectively.

Fig 2

Multilocus tree of Cyphellophora based on ITS and partial BT2 sequences.

Constructed with maximum likelihood implemented in MEGA 7. Bootstrap values of >80% from 100 resampled data sets are shown with branches. Cladophialophora yegresii and C. carrionii comprised the outgroup. Novel species causing chromoblastomycosis are indicated with red branches. Type strain in bold.

Fig 3

Multilocus tree of Rhinocladiella based on ITS and partial BT2 sequences.

Constructed with maximum likelihood implemented in MEGA 7. Bootstrap values of >80% from 100 resampled data sets are shown with branches. Cladophialophora yegresii and C. carrionii comprised the outgroup. Novel species causing chromoblastomycosis are indicated with red branches. Type strain in bold.

Table 2

Estimates of Evolutionary Divergence of Rhinocladiella tropicalis.

Distance R. aquaspersa to R. phaeophora ITS (4.5) and BT2 (5.6); Lengths of alignments of ITS: 548 bp and BT2: 349 bp. Analyses were conducted using the Kimura 2-parameter model. All ambiguous positions were removed for each sequence pair. Evolutionary analyses were conducted in MEGA 7.

Locus	R. aquaspersa	R. phaeophora	R. fasciculata	R. atrovirens	R. mackenzie	R. anceps	R. basitona	R. similis
ITS	5.1	5.4	11.4	18.6	19.7	20.4	21.7	22.0
BT2	9.7	10.7	----	----	----	----	34.8	36.5

We report the clinical cases caused by the novel Cyphellophora and Rhinocladiella species, which are named below as C. ludoviensis and R. tropicalis, respectively. The species showed optimal development at 30°C and 27°C, respectively, with a wide growth range between 18°C and 37°C and with residual growth at 15°C and 38°C and maximum growth temperature at 37°C; no growth was observed at 40°C (Fig 4).

Fig 4

Cardinal temperatures of strains described.

(A) Cyphellophora ludoviensis with optimal growth temperature at 30°C and maximum at 37°C. (B) Rhinocladiella tropicalis with optimal development at 27°C and maximum at 37°C.

C.M.P.S. Azevedo, R.R. Gomes, V.A. Vicente & de Hoog, sp. nov. ‒ MycoBank MB 817305 (Figs 2 and 5).

Fig 5

Cyphellophora ludoviensis microscopic morphology.

(A) colonies on SGA; (B-E) hyphae with chlamydospores and lateral extensions; (F) anastomosis; (G-H) spirally twisted hyphae; (I) poorly differentiated phialide producing conidia; (J-P) chlamydospores and conidia. Scale bars 10 μm.

Etymology: named after the city where the case was first diagnosed, San Luis in Maranhão State, Brazil.

Holotype: Maranhão State, Brazil, from skin lesion of human patient, dried holotype UPCB 85592 at Department of Botany Herbarium at Federal University of Paraná (UPCB); type strain CMRP1317 = LMICRO356 = CBM47. Additional information listed in Table 1.

Description of CMRP51317 after two weeks incubation on MEA at 28°C: Colonies moderately expanding, greyish olive to olivaceus black, with olivaceus black reverse. Hyphae pale olivaceous brown, 1.2–6 μm wide, septate every 9–25 μm, occasionally bearing scarce phialides and conidia. Phialides poorly differentiated, producing sub-spherical, hyaline conidia, 1.8−2.5 μm. Creeping hyphae producing lateral outgrowths which become septate, pale brown conidiophores 1.5−2.0 μm wide and with frequent anastomoses; chlamydospores developing intercalarily or terminally on hyphae. Chlamydospores ovoidal, brown, 4.5−6.5 × 4.5 μm, with irregularly thickened walls. Thickened terminal hyphae and spirally twisted hyphae frequently present. Teleomorph unknown. Cardinal temperatures: minimum 18°C, optimum 30°C, maximum 37°C, with residual growth at 15°C and 38°C.

Case report: Patient, a 57-year-old Caucasian male from Icatu, Brazil, was diagnosed in 2008 with lower limb injuries with nodular appearance and plaque. Moderate disease progressed with 3 years of evolution; muriform cells were observed in tissue (Fig 6A and 6B). Patient was treated orally with itraconazole (200 mg/day), leading to improvement of the lesions during treatment within three months. Patient abandoned treatment after 12 months and returned in 2011 with recurred lesions spreading throughout the lower limb, following a lymphatic path, with secondary infections, presenting verrucous type injuries, ulceration and crusting at the surface. The fungus was re-isolated from the recurred lesions. The patient was treated using itraconazole (200 mg/day) combined with cryosurgery.

Fig 6

Clinical case pictures.

(A, B) Nodular and verrucous lesions and muriform cells from skin tissue biopsy of arm lesion caused by Cyphellophora ludoviensis (CMRP1317); (C-E) polymorphic infiltrative plaque lesions caused by different strains of Rhinocladiella tropicalis affecting the legs, (C) with nodular and cicatricial and (E) verrucous lesions; (D-F) muriform cells from skin tissue biopsy of lesions caused by R. tropicalis strains CMRP1287 and CMRP1307, respectively.

Notes: The strain clustered with members of Chaetothyriales variously classified in Cyphellophora or Phialophora. Several of these have been reported from mild infections of human skin but without presence of muriform cells. Cyphellophora ludoviensis is the only species in the ‘europaea-clade’ (Cyphellophoraceae) where muriform cells were observed in the lesions, and is distant from remaining species causing this disease, as well as from the generic type of Phialophora, P. verrucosa.

C.M.P.S. Azevedo, R.R. Gomes, V.A. Vicente & de Hoog, sp. nov. ‒ MycoBank MB 817306 (Figs 3 and 7).

Fig 7

Rhinocladiella tropicalis, microscopic morphology.

(A) Colonies on SGA; (B, C) twisted hyphae and conidia; (D-G) conidiophores with conidia produced in sympodial order; (H-O) conidial apparatus with conidia. Scale bars 10 μm.

Etymology: Named after its occurrence in tropical South America.

Holotype: Maranhão State, Brazil, from skin lesion of human patient, dried holotype UPCB 85593 at Department of Botany Herbarium at Federal University of Paraná (UPCB); type strain CMRP1287 = LMICRO326 = CBM17. Additional material examined listed in Table 1.

Description of CMRP1287 after two weeks incubation on MEA at 28°C: Colonies growing moderate rapidly, velvety, elevated, olivaceus black with dark reverse. Mycelium partly immersed but mainly aerial, composed of branched hyphae which are pale brown, occasionally reddish brown, 1.5–2.5 μm wide, regularly septate every 7–18 μm, with integrated conidiogenous cells which are somewhat differentiated from vegetative hyphae. Conidiophores erect, straight, thick-walled, brown to dark brown, up to 19 μm high, bearing small, pigmented denticles on which conidia are produced in sympodial order. Conidiogenous cells terminal or lateral, often becoming intercalary, cylindrical in the apical part with numerous flat scars. Conidia one-celled, hyaline to pale brown, ellipsoidal to clavate, 2.5−5.0 × 1.5−2.5 μm; scars slightly prominent, approx. 0.4 μm diam, pigmented. Conidia usually 1-septate, 5−8 × 1.5−3.0 μm. Budding cells developing from single conidia may be present. Teleomorph unknown. Cardinal temperatures: minimum 18°C, optimum 27°C, maximum 37°C, with residual growth at 15°C.

Case reports: Patient infected by R. tropicalis CMRP1287 was a 65-year-old caucasian male from Pinheiro, Maranhão, Brazil, diagnosed in 2004 with lower limb injuries showing polymorphic lesions with plaques and nodular and cicatrized lesions. Muriform cells were observed in tissue (Fig 6C and 6D). The infection evolved during a 6-year period. Treatment was started with itraconazole (200 mg/day), irregularly until 2012; subsequently regular treatment was installed but with low response. From then onwards additional cryosurgery with liquid nitrogen was applied once a week. After 4 years of therapy still some lesions with a fibrotic aspect and with few murifom cells were observed. Patient infected by R. tropicalis CMRP1307 strain was a 78-year-old afrodescendant male, from Icatu, Maranhão, Brazil, diagnosed in 2002 with lower limb injuries showing polymorphic lesions with plaques and infiltration and muriform cells in tissue (Fig 6E and 6F). The diseased showed moderate development over a 3-year period of evolution. Patient was treated with oral itraconazole (200 mg/day), initiated in 2010. Some treatment interruptions occurred because of the distance to the patient’s living place and the low response to itraconazole, with worsening of the symptoms.

Both patients are still under treatment in 2016. The patient infected by R. tropicalis CMRP1287 is currently treated using itraconazole (200 mg/day) combined with cryosurgery and the patient infected by R. tropicalis CMRP1307 uses the antifungals itraconazole (400 mg/day) and terbinafin (250 mg twice daily) in combination.

During our studies we noticed strain IMT766, isolated in 1970 from a case of chromoblastomycosis and deposited in the Institute of Tropical Medicine, São Paulo, Brazil, and supplementary material from a patient infected by strain CBS 132913, a 63-year-old male construction worker from Venezuela with an asymptomatic and localized skin lesion of the hand with a scaly, crusted, dull-red appearance, friable with hemorrhagic dots [19].

The response to treatment has been evaluated by clinical, mycological and histopathological criteria. A complete clinical response is being accompanied assuming for cure criteria after two years follow up without recurrence, with complete healing of the lesions and disappearance of itching and local pain. The patients must also be monitored by three to four consecutive biopsies to access the mycological and histopathological criteria of cure. A mycological response will be achieved after no observation of fungal elements upon direct examination and failure to isolate the causal agent from tissue fragments.

Notes: With LSU rDNA the novel species clusters close to Rhinocladiella aquaspersa and R. phaeophora in the ‘jeanselmei clade’ in Herpotrichiellaceae (Fig 1). Members of this cluster are morphologically outstanding by stiff, erect conidiophores packed with sympodial, non-catenate conidia. With ITS (Fig 3), distances of 5.1% were noted between R. tropicalis and R. aquaspersa, and 5.4% with R. phaeophora (Table 2), underlining that a complex of sibling species is concerned.

Discussion

In this study, we describe two novel species of black fungi causing chromoblastomycosis infections. Among a set of 123 strains from cases of this disease, representatives of three genera were recognized, i.e. Fonsecaea, Rhinocladiella, and Cyphellophora. No member of Cladophialophora was encountered, which is explained by climatic conditions, as C. carrionii is prevalent in arid environmental conditions. In Brazil C. carrionii is rarely reported [24, 25]. The prevalent species in humid tropical climates of South America remains Fonsecaea pedrosoi, followed by F. monophora [2], while the latter species is predominant in southern China [26]. All four species of genus Fonsecaea related to chromoblastomycosis were found in the Brazilian endemic area. Judging from literature data, F. pedrosoi and F. nubica seem to be pathogens that are strictly associated with chromoblastomycosis, while F. monophora and F. pugnacius show some degree of neurotropism eventually leading to dissemination to the brain and other organs [14, 27].

The genus Cyphellophora is characterized by phialides producing sickle-shaped, septate conidia [28]. The group clusters with some phialidic species with small, one-celled conidia. Although the generic type species of Phialophora, P. verrucosa clusters in the ‘carrionii-clade’ distant from Cyphellophora, Feng et al. [22] classified them in Phialophora on morphological criteria. Later Réblová et al. [29] took phylogeny as the leading principle and reclassified all species of the ‘europaea-clade’ in Cyphellophora, the only genus of the newly established family Cyphellophoraceae. Since the taxonomy of Chaetothyriales is in a flux with numerous species to be added, we judge establishment of categories above the species level premature. Currently, phylogenetic data do not match with any other criterion established thus far in Chaetothyriales [21], and therefore phylogenetic genera and families in this order inevitably will remain counterintuitive.

Cyphellophora ludoviensis is not effective to produce conidia on common mycological media; it was placed in the genus based on DNA sequence analyses. Members of the genus Cyphellophora colonize different habitats including, in addition to humans, plant debris, ant nests and abiotic substrates [22]. Our species is genetically close to C. capiguarae, described as living in association with ants (Atta capiguara), and to C. oxyspora from decaying leaves [30, 31]. Other members of Cyphellophora originate from mild cutaneous infections in humans, mostly from skin and nails [22]. Cyphellophora europaea in particular has been encountered globally as an agent of mild skin disease and onychomycosis [31] and was noted co-occurring with dermatophytes, mainly Trichophyton rubrum affecting the skin of diabetic patients [32]. Environmental strains of this species were found in indoor wet cells, such as bathrooms and washing machines [33, 34]. Cyphellophora ludoviensis is the first species in the Cyphellophoraceae that had muriform cells in tissue and showed acanthosis rather than necrosis; on the basis of these features the infection was classified as chromoblastomycosis.

The new species Rhinocladiella tropicalis is a cryptic species close to R. phaeophora and R. aquaspersa. Rhinocladiella phaeophora was known from a single strain recovered from maize field soil in Colombia [35]. It has recently been reported from a case of human chromoblastomycosis, but the sequence of this strain was not available for comparison [36]. This report nevertheless suggests that all members of the cluster consistently are able to cause chromoblastomycosis when inoculated into human skin. Rhinocladiella aquaspersa is a classical agent of chromoblastomycosis, nearly all cases having been reported from the American continent [19], but the majority of historical clinical cases have not been verified by sequence data. Strain CBS 132913 was originally reported as R. aquaspersa but was found to be 100% identical with R. tropicalis. The three species are phenotypically and clinically similar, but their sequence diversity interferes with molecular recognition of R. aquaspersa as a single species. According to Chen et al. [37] the term ‘species complex’ could be applied used to indicate closely related species do not seem to differ in clinically relevant parameters. The present rhinocladiella-like lineages have sufficient molecular distance to be recognized as species, but additional studies of antifungal susceptibility, clinical course, virulence and physiology are needed to verify significance of distinction of the three agents as individual species in hospital routine.

Chromoblastomycosis is clinically highly variable, with six different clinical types [8, 38]. Despite the polymorphic nature of lesions, common factor at the patient side is the absence of necrosis and often even hyper-growth of dermal tissues, which distinguishes the disease from its clinical counterpart, phaeohyphomycosis. At the fungal side, the invasive form is the muriform cell, which is likely to be the cause of the growth-promoting dermal response. As such the disease is polyphyletic within the Chaetothyriales, but has not or extremely rarely been observed outside this order. In both cases caused by these two new species here described it was encountered polymorphic lesions frequently related to clinical cases of chromoblastomycosis [8].

In the State of Maranhão, Brazil, five chaetothyrialean agents of chromoblastomycosis are endemic. A potential source of infection has been suggested [39] to be the harvest of babassu coconuts from a wild palm tree (Orbignya phalerata). A large part of the local rural population is involved in the collection of nuts to extract babassu oil, an important component for local and international beauty product manufacturers. Members of Chaetothyriales are indeed enriched on babassu shell fragments, which are considered a risk factor for developing chromoblastomycosis after trauma sustained at work [39, 40]. A direct link between shells and agents of the disease has however not unambiguously been established. In other Brazilian regions the number of new cases of chromoblastomycosis is decreasing [8, 41, 42]. This is thought to reflect changes in agricultural practices, the extensive use of agricultural antifungals, especially azole derivatives, and the progressive mechanization of plantations resulting in a reduction of the risk of occupational exposure [8, 43, 44].

Our results showed that C. ludoviensis and R. tropicalis had their optimal development at 30 and 27°C, respectively, the maximum growth temperature of all strains analyzed being at 37°C. The chronic nature of the infection corresponds with borderline survival of the fungus in tissue. Epidermal temperatures are usual below 37°C, allowing infection by fungi that barely support this temperature. This agrees with the clinical observation that members of the Cyphellophoraceae (‘europaea-clade’), which have their maximum growth at 36°C, cause only mild, superficial infections, having a very low degree of invasive ability and virulence [22].

According to the World Health Organization (WHO) [45] the Neglected Tropical Diseases include a series of endemic diseases that prevail in tropical or subtropical areas worldwide. The prevalence of causative microbes is linked to poverty and disadvantage. However, fungal diseases were still not included in this list, except mycetoma, another implantation mycosis [46]. Chromoblastomycosis has been reported in the literature as an overlooked disease [11]. Its global burden is comparable to or greater than that of mycetoma. Considering to its global distribution, its impact on the impoverished, and its refractoriness, it also should be considered a true neglected disease as defined by WHO.

Materials and methods

Strains studied

Strains analyzed comprised 123 clinical isolates (Table 1) from different cases and several endemic areas in Brazil, with viable cultures for molecular epidemiology studies and with detailed registration data deposited in medical file systems of the institutions involved in this study. All the clinical strains were deposited at Microbial Collections of Paraná Network- TAXon line at Federal University of Paraná, the register numbers, others nomenclature references and additional information were informed in the Table 1. The Holotype number was provided from Department of Botany Herbarium at Federal University of Paraná (UPCB), TAXon line collections network (http://taxonline.nerdweb.com.br/), register number at http://www.splink.org.br/.

This work was approved by the Research Ethics Committee-CEP-HUUFMA (University Hospital of the Federal University of Maranhão), according to Brazilian Resolution -Approval number: 1.276.342. All samples were anonymized. The set was supplemented with reference strains from the Centraalbureau voor Schimmelcultures (CBS/KNAW) Fungal Biodiversity Centre, Utrecht, Netherlands and the strain previously described as R. aquaspersa CBS 132913 was included in the phylogenetic analysis. Stock cultures were maintained in slants of 2% malt extract agar (MEA) and oatmeal agar (OA) at 24°C. For morphological studies, MEA and potato dextrose agar (PDA) slide cultures were prepared and mounted in aniline blue.

Physiology

Cardinal growth temperatures were determined on 2% MEA. Plates were incubated in the dark for 3 weeks at a 3–36°C temperature range with intervals of 3°C; growth was also recorded at 14, 37, 38, 39 and 40°C. Growth rates per species were obtained by calculation of the average growth of all isolates proven to belong to that species, including the respective standard deviations. Results were plotted with temperature (°C) versus colony diameter (mm) as parameters. Optimum range (= average ± standard deviation) and maximum growth temperatures were determined using the type strains of each species with three replicates averages of three measurements were calculated. Different temperatures were tested, at 28°C was observed the largest number of structures to Chyphellophora and it was used to describe the species morphology.

DNA extraction and amplification

DNA extraction and quality tests were performed using glass beads (Sigma G9143) according to protocols described previously [23] and when it was required, the purification of DNA was undertaken using the UltraClean™ Microbial DNA Kit (MO Bio, Carlsbad, CA, USA) according to manufacturer’s instructions. Colonies were cultivated on Sabouraud’s glucose agar (SGA).

The partial large subunit of the nuclear ribosomal RNA gene (LSU) was amplified using primers NL1 and LR5 [22] for phylogenetic assessment. Three gene regions were chosen for species delimitation: rDNA Internal Transcribed Spacer (ITS), and the partial genes cell division cycle gene (CDC42) and β-tubulin (BT2). ITS amplicons were generated with primers V9G and LS266 [47, 48] and were sequenced with primers ITS1 and ITS4. CDC42 amplification and sequencing was generated with cdc42w and cdc42f [49] and BT2 amplification and sequencing was generated with Bt-2a and T2 [50]. PCR was performed in a 12.5 μL volume of a reaction mixture containing 1× PCR buffer, 2.0 mM MgCl2, 25 μM dNTPs, 0.5 μM of each forward and reverse primers, 1 U of BioTaq DNA polymerase, and 10 ng of genomic DNA.

Amplification was performed in an ABI PRISM 2720 (Applied Biosystems, Foster City, USA) thermocycler as follows: 95°C for 4 min, followed by 35 cycles consisting of 95°C for 45s, 52°C for 30s and 72°C for 2 min, and a delay at 72°C for 7 min. Annealing temperatures were changed to 52°C, 55°C and 58°C for ITS, CDC42 and BT2 respectively. Amplicons were cleaned with Exonuclease I and Shrimp Alkaline Phosphatase (SAP) according to manufacturer’s instructions. Amplicons were sequenced with a BigDye Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions, reactions were purified with Sephadex G-50 fine (GE Healthcare Bio-Sciences, Uppsala, Sweden) and sequences were analysed on an ABI Prism 3700 DNA Sequencer (Perkin-Elmer, Norwalk, Foster City, CA, USA).

Phylogenetic analysis

Consensus sequences of the ITS region, BT2, CDC42, and the LSU were visually inspected using MEGA v.7 software [51]. The alignment of obtained sequences was performed using the online MAFFT interface [52]. The genes ITS, CDC42 and BT2 were first analyzed separately and for analysis of multilocus (S1 Fig). We did the LSU analyses to assess the phylogenetic position of the species analyzed in this study. The phylogenetic analyses of the small subunit (SSU) and LSU groups previously recognized in the Herpotrichiellaceae by de Hoog et al. [21], Feng et al. [22] and Vicente et al. [23] were taken as a basis for clade delimitation. Trees were constructed with 100 bootstrap replicates using the Maximum Likelihood Implemented in Mega v. 7 software [51], with the best evolutionary model to this dataset. Conflicts were estimated using the partition homogeneity test available in PAUP* v. 4.Ob10 [53]. To elucidated and explore a more detailed the clustering unnamed species, their sequences were compared to those deposited at GenBank and the CBS-KNAW sequence data sets.

Multilocus tree of Fonsecaea and Cladophialophora based on confidently aligned ITS and partial BT2 and CDC42 sequences.

Constructed with maximum likelihood implemented in MEGA 7.0. Bootstrap values of <80% from 1,000 resampled data sets are shown with branches. Cladophialophora yegresii (CBS 114406, CBS 114407 and CBS 114405) and C. carrionii (CBS 161.54, CBS 406.96, CBS 165.54 and CBS 108.97) comprised the outgroup. Fonsecaea species causing chromoblastomycosis are indicated in red. Type strain in bold.

(EPS)

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