Cryptococcosis is characterized by a chronic course and is usually caused by the ubiquitous yeast Cryptococcus neoformans, predominantly by its variety C. neoformans var. grubii, and in endemic areas by Cryptococcus gattii. Other Cryptococcus species are rarely reported to cause infections and most of them are known to be low or non-pathogenic .
In the present case, Cryptococcus magnus was isolated from a 23-year-old female patient suffering from a vulvovaginal disease, and the outcome of this uncommon yeast was examined.
C. magnus can clearly be distinguished from other Cryptococcus spp. by ribosomal DNA sequence analysis [2, 3]. C. magnus has been isolated from certain plants, and it is detectable in soil, air, and even on arctic glaciers. It has also been detected in goat milk, murine intestinal tract, feline ear canal, and human respiratory tract . In most of these reports, the identification of C. magnus was confirmed by molecular methods.
Herein, we present a case of vulvovaginitis due to C. magnus in a 23-year-old Iranian woman and discuss the biology of this agent. To the best of our knowledge, this is the first report on C. magnus vaginal infection.
A 23-year-old married female patient presented to one of the hospitals of Damavand city, Tehran, Iran, due to severe itching and vaginal irritation. Vaginal examination revealed thick, curdle-like, white-colored discharge, edema, and intense pruritus of the vulva. The vagina and labia were erythematous. She was using an intra-uterine device (IUD) as a contraceptive method.
Due to the suspected diagnosis of vulvovaginal candidiasis, fresh samples of vaginal discharge were sent for mycological examination to the Division of Mycology, School of Medicine, Shahid Beheshti University of Tehran, Iran. Vaginal discharge was sampled by using a speculum and sterile swab. The swab was transported to the laboratory into normal saline. Two specimens were obtained under sterile conditions, one for microscopic examination and the other for fungal culture. A slide was prepared for Methylene blue staining. The vaginal swab was inoculated on Sabouraud Dextrose Agar (SDA; Merck, Germany)  and incubated at 30°C for 24 h. The produced cream-colored colonies were slightly mucoid, smooth, highly glossy, and slim in texture that were indistinguishable from Candida spp. colony. After three days, the cream color changed to pink (Figure 1).
Microscopic examination of the culture after 48 h showed round to oval yeast, single or pairs of cells without true hyphae or pseudohyphae and without capsule in Indian ink (Figure 2).
Genomic DNA was extracted from culture using glass bead method . Then, the ITS regions of rDNA gene of isolates was amplified by the universal fungal primers, ITS1 (5´-TCCGTAGGTGAACCTGCGG-3´) and ITS4 (5´-TCCTCCGCTTATTGATATGC-3´) . The PCR product was applied for the accurate identification of isolate (Bioneer, Korea). For confirmation of species identity, the obtained sequences were compared with similar sequences in the open access NCBI database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Alignment of the obtained sequence in BLAST revealed a 100% identity with the type strain of Filobasidium magnum, which is indicated with sequence ID: MG786767.1 and high homology (99%) with Cryptococcus magnus with sequence ID: GU237052.1. The sequences were in GenBank under accession number ‘‘MG786767-1.’’
Antifungal susceptibility tests were performed by broth micro dilution method as described in Clinical and Laboratory Standards Institute  guidelines, document M27-S3 . C. parapsilosis type strain (ATCC 22019) was used for quality control in all antifungal susceptibility tests. Tests were performed in 96-well round-bottom microtiter plates. Drug concentration ranges were 0.03 to 64 μg/ml for itraconazole (ITC), fluconazole (FLC), ketoconazole (KTC), and amphotericin B (AMB). Cell suspensions were prepared in RPMI 1640 medium (Invitrogen, USA) and were adjusted to give a final inoculum concentration of about 0.5 × 103 to 2.5 × 103 cfu/ml. The plates were incubated at 35°C for 48 h. Minimum inhibitory concentration (MIC) was then determined and compared with a drug-free control. All the tests were performed in duplicate [8, 9].
The MIC values for ITC, KTC, AMB, and FLC were 0.031 μg/ml, 0.031 μg/ml, 0.062 μg/ml, and 0.062 μg/ml respectively, revealing the sensitivity of the mentioned causative agent. The patient received treatment with topical ketoconazole ointment, but there were no signs of recovery after seven days. Then, the patient was finally treated successfully using 200 mg daily of oral ketoconazole in 10 days. No further clinical signs of vaginal infection were observed after six months. Relapse of the infection was not revealed in two, four, and six-months follow up of the patient. No clinical manifestations were shown at the involved site.
The Ethics Committee of Shahid Beheshti University of Medical Sciences in Iran approved this study (ethics committee code: IR.SBMU.MSPPP.REC.1395.3).
Khan et al. reported the isolation of C. magnus from nasal specimens of acute lymphoblastic leukemia patients. This species was resistant to caspofungin, anidulafungin, 5-flucytosine, and itraconazole, but it was susceptible to amphotericin B, posaconazole, and voriconazole by E-test .
Kano et al.  from Japan isolated this yeast from the ear canal of a clinically healthy cat that had a history of otitis externa. C. magnus was attributed to Aspergillus fumigatus, and then it was successfully cured through antifungal treatment for 25 days.
In contrast to the report by Kano et al., Poth et al. described a severe case of C. magnus infection in an immunocompetent cat. The left foreleg was completely amputated and the cervical lymph node was excised in this male domestic cat due to suspicion of a tumor. Based on histopathological and mycological investigations, response to treatment with 10 mg/kg of fluconazole for five weeks was satisfactory .
Generally, the identification of yeasts is limited in histological preparation. Candida spp. often show budding yeasts, pseudohyphae, and true hyphae in tissue. C. neoformans can be identified based on its capsule . C. magnus may look like Histoplasma capsulatum in form and size by using routine microscopic techniques. Thus, a reliable method is required for the final identification of yeasts followed by mycological studies and confirmation by PCR and sequencing, especially for unusual pathogens.
To the best of our knowledge, the case described here is the first report of vulvovaginitis due to C. magnus. In the present case, after MIC approval, treatment was initiated with topical ketoconazole ointment, but there were no signs of recovery after seven days. Therefore, 200 mg of oral ketoconazole was administered daily for 10 days, which resulted in successful treatment. Relapse of the infection was not revealed in two-, four-, and six-month follow up of the patient. At the involved site, no clinical manifestations were observed. Although Cryptococcus spp. rarely cause vulvovaginitis, there have been some articles on this issue (Table 1).
|NO||Age/year||Location||No. Total/ case samples||Agent||Clinical presentation||Examination||Treatment||Case characteristics||Reference|
|1||NI/ 1985||USA||805/1||Cryptococcus. ungulaticus||Vulvovaginal complaints||Culture||NI||NI|||
|2||60/1987||USA||Case report||Cryptococcus. neoformans||Cutaneous lesion||Culture and Biopsy||NI||Renal transplant recipient|||
|3||72/1993||NI||Case report||Cryptococcus. spp||Colon cancer||Culture||FLC||Colon cancer|||
|4||20/2005||Malaysia||Case report||Cryptococcus. neoformans||Vulvovaginal complaints||Biopsy||Oral FLC||NI|||
|5||NI/2009||Saudi Arabia||1000/1||Cryptococcus. neoformans||NI||Culture and API20C kit||NI||NI|||
|6||23/2018||Iran||Case report||Cryptococcus. magnus||severe itching and vaginal irritation||Culture and sequencing||KCZ||immunocompetent||Current case|
|NI =Not indicated; spp,, species; FLU, fluconazole; ketoconazole, KCZ|
C. magnus was found to have the ability to cause vulvovaginitis. This report could be of clinical significance and helpful for the management of similar cases. The case presented here is the first report of successful detection and treatment of vulvovaginal infection with C. magnus.
- Poth T, Seibold M, Werckenthin C, Hermanns W. First report of a Cryptococcus magnus infection in a cat. Med Mycol. 2010; 48(7):1000-4.
- Randhawa HS, Paliwal DK. Survey of Cryptococcus neoformans in the respiratory tract of patients with bronchopulmonary disorders and in the air. Sabouraudia. 1979; 17(4):399-404.
- Emami S, Foroumadi A, Falahati M, Lotfali E, Rajabalian S, Ebrahimi SA. 2-Hydroxyphenacyl azoles and related azolium derivatives as antifungal agents. Bioorg Med Chem Lett. 2008; 18(1):141-6.
- Nagornaya SS, Babich TV, Podgorsky VS, Beharav A, Nevo E, Wasser SP. Yeast interslope divergence in soils and plants of" Evolution Canyon";, Lower Nahal Oren, Mount Carmel, Israel. Israel J Plant Sci. 2003; 51(1):55-7.
- Pfaller MA, Houston A, Coffmann S. Application of CHROMagar Candida for rapid screening of clinical specimens for Candida albicans, Candida tropicalis, Candida krusei, and Candida (Torulopsis) glabrata. J Clin Microbiol. 1996; 34(1):58-61.
- Makimura K, Tamura Y, Mochizuki T, Hasegawa A, Tajiri Y, Hanazawa R. Phylogenetic classification and species identification of dermatophyte strains based on DNA sequences of nuclear ribosomal internal transcribed spacer 1 regions. J Clin Microbiol. 1999; 37(4):920-4.
- Ghahri M, Mirhendi H, Zomorodian K, Kondori N. Identification and antifungal susceptibility patterns of Candida strains isolated from blood specimens in Iran. Arch Clin Infect Dis. 2013; 8:3.
- CLSI C. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard. CLSI: Wayne, Pa, USA; 2008.
- Lotfali E, Kordbacheh P, Mirhendi H, Zaini F, Ghajari A, Mohammadi R. Antifungal susceptibility analysis of clinical isolates of Candida parapsilosis in Iran. Iran J Public Health. 2016; 45(3):322.
- Pusey PL, Stockwell VO, Mazzola M. Epiphytic bacteria and yeasts on apple blossoms and their potential as antagonists of Erwinia amylovora. Phytopathology. 2009; 99(5):571-81.
- Khan Z, Mokaddas E, Ahmad S, Burhamah MH. Isolation of Cryptococcus magnus and Cryptococcus chernovii from nasal cavities of pediatric patients with acute lymphoblastic leukemia. Med Mycol. 2011; 49(4):439-43.
- Kano R, Hosaka S, Hasegawa A. First isolation of Cryptococcus magnus from a cat. Mycopathologia. 2004; 157(3):263-4.
- Greene CE. Cryptococcosis. Infection diseases in the dog and the cat. WB Saunders: Philadelphia; 1990.
- Chayakulkeeree M, Perfect JR. Cryptococcosis. Infect Dis Clin North Am. 2006; 20(3):507-44.
- Jurina K, Maaß S, Aupperle H, Grevel V. Meningoenzephalitis infolge einer systemischen Kryptokokkose bei einem Hund. Tierärztliche Praxis K. 2003; 31(3):156-61.
- Horowitz BJ, Edelstein SW, Lippman L. Candida tropicalis vulvovaginitis. Obstet Gynecol. 1985; 66(2):229-32.
- Blocher KS, Weeks JA, Noble RC. Cutaneous cryptococcal infection presenting as vulvar lesion. Genitourin Med. 1987; 63(5):341-3.
- Chen CK, Chang DY, Chang SC, Lee EF, Huang SC, Chow SN. Cryptococcal infection of the vagina. Int J Gynecol Obstet. 1994; 44(2):192.
- Ranganathan S, Moosa F, Kamarulzaman A, Looi LM. MRI and CT findings of cryptococcalvaginitis. Br J Radiol. 2005; 78(928):353-4.
- El-Din AZ, Habib F, Abd-Allah N, Khorshid O. Mycotic vulvovaginitis: epidemiology, pathogenesis and profile of antifungal agents. J Taibah Univ Med Sci. 2009; 4(2):123-36.