|Year : 2017 | Volume
| Issue : 2 | Page : 269-273
Molecular identification and phenotypic characterisation of Sporothrix globosa from clinical cases of Eastern Assam, North-east India
Reema Nath, Pinky Lahon, Longmindar Timung
Department of Microbiology, Assam Medical College and Hospital, Dibrugarh, Assam, India
|Date of Web Publication||5-Jul-2017|
Department of Microbiology, Assam Medical College and Hospital, Dibrugarh - 786 001, Assam
Source of Support: None, Conflict of Interest: None
Sporotrichosis is known to be endemic in the state of Assam, North-east India, which is situated in the Sub-Himalayan region. This disease is an acute or chronic infection caused by Sporothrix schenckii species complex which currently includes several species of clinical relevance such as Sporothrix brasiliensis, Sporothrix globosa, Sporothrix schenckii sensu stricto, Sporothrix albicans, Sporothrix mexicana, Sporothrix pallida and Sporothrix luriei. S. globosa is the prevalent species in India. Eight culture-positive patients were diagnosed from suspected consecutive cases of two lymphocutaneous and six fixed cutaneous forms over a period of 4 years in a clinical mycology laboratory of a tertiary care centre in Eastern Assam. Phenotypic speciation was inconclusive using the criteria of Marimon et al. because of atypical growth pattern shown by the isolates. Our isolates showed good growth at 37°C ranging from 6 to 27 mm; four of the isolates showed growth of 11–27 mm unlike S. globosa strains reported earlier. Molecular identification was done by sequencing both the internal transcribed spacer (ITS) region and the calmodulin (CAL) protein encoding gene (partial). All the isolates were identified as S. globosa. Molecular confirmation of species using ITS region and CAL protein encoding gene (partial) is necessary for isolates of S. globosa showing atypical biopatterns.
Keywords: Assam, ITS region, North-east India, Sporothrix globosa, Sporothrix schenckii species complex
|How to cite this article:|
Nath R, Lahon P, Timung L. Molecular identification and phenotypic characterisation of Sporothrix globosa from clinical cases of Eastern Assam, North-east India. Indian J Med Microbiol 2017;35:269-73
|How to cite this URL:|
Nath R, Lahon P, Timung L. Molecular identification and phenotypic characterisation of Sporothrix globosa from clinical cases of Eastern Assam, North-east India. Indian J Med Microbiol [serial online] 2017 [cited 2017 Sep 26];35:269-73. Available from: http://www.ijmm.org/text.asp?2017/35/2/269/209580
| ~ Introduction|| |
Sporotrichosis is caused by the dimorphic fungus Sporothrix schenckii sensu lato, which is commonly seen in tropical and subtropical areas of the world. This disease is known to be endemic in Assam, North-east India, which is situated in the Sub-Himalayan region., It is bounded by the foothills of the Himalayas to the north and another lower range of hills and mountains to the south. Assam, with an area of 78,438 km 2, extends from 89° 42' E to 96° E longitude and 24° 8 N to 28° 2 N latitude. This region experiences heavy rainfall and humidity. We isolated eight strains of Sporothrix from suspected cases of sporotrichosis within a period of 4 years from June 2012 to May 2016 in a clinical mycology laboratory of the region. Phenotypic speciation of the isolates was inconclusive due to atypical growth characteristics of the isolates. We confirmed all the isolates as Sporothrix globosa after molecular characterisation.
| ~ Materials and Methods|| |
Eight isolates from different parts of Eastern Assam were examined in this study. All isolates were previously identified as Sporothrix schenckii species complex in the mycology laboratory of this tertiary care hospital by means of morphological studies, ability to convert to yeast phase at 37°C. Culture was done as part of standard patient care, and Institutional Ethical approval was taken for their use. They were preserved in distilled water (Castellani's method) as well as in glycerol and recovered by growth on Sabouraud dextrose agar with chloramphenicol (150 μg/ml) at room temperature for 7 days.
Isolates were phenotypically characterised according to already published protocol of Marimon et al. The key phenotypic features of Marimon et al. for recognising this species were the globose or subglobose sessile-pigmented conidia, no growth 37°C, growth in potato dextrose agar (PDA) at 30°C not exceeding 50 mm in 21 days and assimilation of sucrose. Raffinose is not assimilated by this species.
Macroscopic features of colonies were studied by culturing isolates on PDA (Hi-Media, India) plates; incubation was done at room temperature, 30°C and 37°C in the dark in duplicates. Petri dish More Detailses were inoculated according to the protocol followed by Camacho et al. Colony diameters were measured at 14 and 21 days in duplicate, and the mean of the diameters of growths were recorded. Spore morphology in corn meal agar was determined as described by Marimon et al. Inoculums' size used to subculture PDA plates was standardised when colony size is compared in phenotypic identification of species.
Carbohydrate assimilation tests were performed for sucrose and raffinose in duplicate in 96-well microplates as described by Camacho et al. and were read at 5 and 10 days of incubation at 25°C.
Total genomic DNA was extracted from the growth in yeast extract, peptone and dextrose broth following already published protocol. Using degenerated primers CL1 and CL2A, partial amplification of calmodulin (CAL) locus was obtained by amplifying a region corresponding to 3 through 5 of CAL gene. Internal transcribed spacer (ITS) (ITS1, 5.8S r RNA, ITS 2) region of genomic DNA was amplified using universal primers ITS1 and ITS4 as previously described. Polymerase chain reaction (PCR) products were purified and sent for sequencing on both strands to Xcelris Labs, Ahmedabad, India, with the same primers used for PCR. Assembly of the sequences was done with CodonCode Aligner. Homology searches were made on GenBank database using BLAST. Five of our ITS sequences were deposited in the GenBank database under the accession numbers KT380137–KT380140 and KT779228.
The isolates were deposited in National Pathogenic Fungi Collection Centre at Postgraduate Institute of Medical Education and Research at Chandigarh, India.
Similarly, CAL-related and ITS-related sequences of Sporothrix were retrieved from GenBank and included in the analysis [Figure 2] and [Figure 3]. The saprophytic fungus Ophiostomataceae was included in the analysis as an outgroup. The multiple sequence analysis was performed with Clustal W algorithm implemented in MEGA 6 software. Phylogenetic analysis was also performed in MEGA 6 as described earlier.,,
|Figure 2: Phylogenetic tree constructed with partial calmodulin gene sequences encoding calmodulin–encoding gene. The phylogenetic tree was built with MEGA 6.1, by maximum likelihood method based on the Kimura 2-parameter model. Number close to branches represents Bootstraps support values based on 1000 bootstrap replications. GenBank accessions indicated in branch labels|
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|Figure 3: Phylogenetic tree inferred from internal transcribed spacer sequences; built by MEGA 6.1, by the maximum likelihood method based on Tamura-Nei model. Number close to the branches represents Bootstraps support values based on 1000 bootstrap replications. GenBank accessions numbers are indicated in branch labels|
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| ~ Results|| |
[Table 1] shows the particulars of the patients as well as phenotypic characterisation results. All the phenotypic characters were consistent with the criteria of Marimon et al. for S. globosa except the growth at 37°C. Four of the eight isolates have shown growth of 6–8 mm. Four isolates yielded growth of 11–27 mm at 37°C. The growth at 30°C was 50 mm or less which was consistent with the criteria of Marimon et al. for S. globosa [Figure 1]. Sucrose assimilation was positive and raffinose assimilation was negative in all the isolates. The morphology of the sessile conidia was either globose or subglobose, 2.2–3.4 μm in length and 2–3 μm in width. Phylogenetic analysis of the isolates was performed using amplified fragment from the CAL locus [Figure 2]. The complete alignment included 19 sequences, 8 from this study and others retrieved from GenBank from previous studies., The sequences clustered with isolates from previous studies AM398994 (FMR 9020, Clinical, Japan), JF811338 (CBS 132924 Ss49, clinical, Brazil) and AM399002 (KMU4208, environmental, China). A second phylogenetic analysis was done with amplified products using universal primers ITS1 and ITS4 [Figure 3]. All the strains of this study clustered with S. globosa strains JX 997717 (LC2430, China), KJ 999896 (C 8775, clinical, Venezuela), KJ 999878 (C0329, clinical, Andes, Venezuela), JX 997695 (LC2440, China), KP 017084 (CBS 120340) and KP017085 (CBS 129719).
|Table 1: The particulars of patients, type of lesions, phenotypic characterization are summarized|
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| ~ Discussion|| |
Sporotrichosis is an endemic disease in the north-eastern states of India and has been reported time to time from states of Assam, Manipur, Sikkim and Nagaland.,,,, Clinical diagnosis of sporotrichosis is often made by clinicians of this endemic region based on clinical features and histopathology reports which show chronic granulomatous reaction in the subcutaneous tissue with or without the presence of the yeast form. Most of the cases get treated in the peripheral hospitals before mycological confirmation is done due to nonavailability of fungal culture facility in the peripheral areas. This may be the reason for less number of culture isolates in this study. We isolated only eight strains of Sporothrix spp. in the clinical mycology laboratory of this tertiary care hospital over a period of 4 years. The age of the patients in our study ranged from 20 to 59 years. Male to female ratio was 1:1. Both lymphocutaneous and fixed cutaneous forms were found. The isolates of our study were confirmed as S. globosa after molecular characterisation. The key phenotypic characters shown by the isolates which satisfied Marimon's criteria were globose to subglobose conidia, assimilation of sucrose without assimilation of raffinose and growth of 35–50 mm diameter on PDA at 30°C. However, unlike Marimon's criteria of restricted or no growth of this species at 37°C, all our isolates produced good growth of 6–27 mm. Four of the eight isolates have shown growth of 6–8 mm at 37°C [Table 1]. This finding is similar to the report of Yu et al. from North-east China where growth up to 9 mm diameter was found. Growth up to 7 mm at 37°C has been reported from Brazil by Oliveira et al. Other four isolates of our study showed good growth of 11–23 mm at 37°C. Although several exceptions to the Marimon's accepted criteria were found in the literature, growth exceeding 9 mm were not reported earlier. The size of growth at 37°C was not related to the type of clinical disease. This finding is similar to that of Yu et al. Phenotypic characterisation of the Assam strains showing growth at 37°C was not reported earlier.
Our study shows S. globosa to be the prevalent species in Assam. S. globosa is prevalent in Asia and Europe (56% and 28%, respectively) followed by the United states of America (11%). Genetic variation in S. globosa was reported to be low. Camacho et al. in the study from South America had reported this species only in fixed cutaneous type of clinical disease. Lymphocutaneous and disseminated sporotrichosis was reported to be caused by S. schenckii sensu stricto in that study. However, in China and India, S. globosa is the predominant species responsible for both the fixed cutaneous and lymphocutaneous forms.,
To the best of our knowledge, this is the first report of Eastern Assam strains where we report the growth characteristics of the isolates.
| ~ Conclusion|| |
This study shows that S. globosa is a predominant species in Assam, North-east India. Restricted growth at 37°C which is one of the established criteria for identification of this species was not found to be useful in phenotypic characterisation of the isolates. Other Marimon's phenotypic criteria were fulfilled by the isolates. Molecular confirmation of species is necessary for isolates of S. globosa showing atypical biopatterns.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]