Cryptococcus gattii genotype AFLP 6 / VGII meningoencephalitis in an immunocompetent Filipino male in Kuwait : activation of a dormant infection

Correspondence Ziauddin Khan zkhan@hsc.edu.kw Department of Microbiology, Faculty of Medicine, Kuwait University, Safat, Kuwait Department of Laboratories, Farwaniya Hospital, Kuwait Department of Medicine, Farwaniya Hospital, Kuwait Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands


Introduction
Cryptococcosis is the most common fatal mycosis worldwide (Park et al., 2011).It is principally caused by Cryptococcus neoformans var.grubii and var.neoformans (serotypes A, D and AD) and C. gattii (serotypes B and C) (Bovers et al., 2008).The reproductive cycle of Cryptococcus involves both asexual (budding) and sexual (basidiospores) states, both of which are pathogenic to humans (Velagapudi et al., 2009).Whilst both species are capable of infecting immunocompetent and immunocompromised individuals, there are differences between them with respect to ecological niche, host susceptibility, virulence attributes, susceptibility to antifungal agents and response to antifungal therapy (Byrnes et al., 2011;Hagen et al., 2012;Illnait-Zaragozı ét al., 2013;Sorrell et al., 2011).Using a variety of molecular typing tools, it has been demonstrated that C. neoformans/ Cryptococcus gattii species represent a complex of distinct genotypes, which differ in their geographical distribution and virulence potential (Hagen et al., 2012).C. gattii occurs predominantly in tropical/subtropical regions and represents five major molecular types: AFLP4/VGI, AFLP6/ VGII, AFLP5/VGIII, AFLP7/VGIV and AFLP10/VGIV.Of these, genotype AFLP6/VGII, like its sibling genotype AFLP4/VGI, appears to be the most virulent and infects individuals that have no identifiable immune deficiency (Hagen et al., 2012;Ma et al., 2009).Here, we describe a case of C. gattii meningoencephalitis in a Filipino male who came to Kuwait 3 years ago in normal health.

Case report
A 34-year-old Filipino male was admitted at the Accident and Emergency Department in Farwaniya Hospital, Kuwait, on 21 December 2012 with a history of progressive generalized headache, nausea and repeated episodes of vomiting without blood or bile for the last 3 days.The severity of the headache increased in the occipital region accompanied by drowsiness and an inability to walk.He was healthy prior to his present illness.He came to Kuwait 3 years before and worked as a baker.He was leading a healthy lifestyle with no promiscuous behaviour and had no history of recreational drug use.On examination, his oral temperature was 36 uC, pulse 46 beats min 21 , blood pressure 120/78 mmHg, oxygen saturation by pulse oximetry 95% and respiratory rate 20 min 21 .He had mild neck stiffness with positive Kernig's sign.His Glasgow coma scale score was 13/15.Power in the right upper and lower limbs was 3/5 and in the left upper and lower limbs was 4/5.Fundoscopic examination showed normal findings, with no papilloedema.Further physical examination showed normal findings.
His blood count showed white blood cells of 13.1|10 9 l 21 , neutrophils 76.4%, lymphocytes 10.9% and platelets 274|10 9 l 21 .His serum levels for urea, electrolytes, creatinine, sodium and potassium were 8.2, 3.9, 82, 138 and 4.2 mmol l 21 , respectively.His C-reactive protein level was 6 mg l 21 and liver function parameters were within the normal range.Radiological examination of his chest and head did not reveal any abnormalities.He received empirical ceftriaxone (2 g day 21 ) and vancomycin (15 mg kg 21 ) every 12 h and acyclovir (10 mg kg 21 ) three times per day intravenously, and a lumber puncture was performed.Indian ink examination of cerebrospinal fluid (CSF) showed budding yeast cells with a thick capsule, which was confirmed by culture.The isolate was identified as C. neoformans by VITEK2 (bioMe ´rieux).A latex agglutination test performed on CSF and serum samples was positive for cryptococcal polysaccharide.The biochemical and cytological findings of the first CSF sample were as follows: glucose 3.2 mmol l 21 (random blood glucose taken at the same time was 8.0 mmol l 21 ), protein 485 mg l 21 , white blood cells 40 mm 23 with 60% lymphocytes and 40% polymorphonuclear cells, and red blood cells v5 mm 23 .
The isolate was sent to the Mycology Reference Laboratory (Faculty of Medicine) for determination of MICs for different antifungal agents.Whilst the isolate appeared to be susceptible to amphotericin B (MIC 0.012 mg ml 21 ), voriconazole (MIC 0.047 mg ml 21 ) and posaconazole and flucytosine (both MIC 0.094 mg ml 21 ), it showed reduced susceptibility to fluconazole (24 mg ml 21 ) by Etest as well as by VITEK antifungal susceptibility card, prompting further characterization.
The patient was started on liposomal amphotericin B (Abelcet; 1 mg kg 21 ) and fluconazole 800 mg (due to the non-availability of flucytosine), followed by 400 mg intravenously, once daily.This combination was used for 2 weeks.As the isolate showed reduced susceptibility to fluconazole, it was replaced with voriconazole (200 mg twice daily, intravenously) with continued administration of Abelcet (1 mg kg 21 , once daily).After 5 days of treatment, the patient became afebrile and his condition improved; however, he still complained of a continued headache over the frontal, left temporal and occipital regions.
CSF examination repeated on day 23 of treatment was positive for C. gattii by microscopy and culture.A latex agglutination test of serum showed a titre of 1 : 1024.Consequently, on day 35, the dosage of Abelcet was increased to 5 mg kg 21 and then reduced to 3 mg kg 21 after 1 week of therapy due to evidence of renal toxicity.After 5 days of treatment, his headache subsided and he became asymptomatic.He left the hospital against medical advice.
However, after 3 months, the patient was readmitted with similar symptoms with papilloedema, grade 4. Again, CSF examination was positive for microscopy, culture and polysaccharide antigen with a titre of 1 : 512.Abelcet (3 mg kg 21 ) was started with flucytosine (100 mg kg 21 , daily).After 4 weeks of combination therapy, the CSF culture became negative, whilst Indian ink examination still showed scanty encapsulated yeast cells.This was accompanied by a fall in cryptococcal antigen titre in the CSF (from 1 : 512 to 1 : 32).The patient left the hospital against medical advice and was prescribed oral voriconazole (200 mg, twice daily) for 3 months.Five months later, he returned to the hospital with the symptom of persistent headache.Two specimens of CSF obtained 5 days apart failed to yield positive cultures for C. gattii, although scanty, encapsulated yeast cells were visible in Indian ink preparations.The cryptococcal antigen titres in serum and CSF samples were 1 : 128 and 1 : 16, respectively.Urine cultures obtained without prostate massage were negative.He received intravenous voriconazole (6 mg kg 21 , every 12 h) on the day of admission, followed by 4 mg kg 21 (every 12 h) for 1 week.He left the hospital against medical advice on an oral maintenance dose of voriconazole (200 mg, twice daily) for 3 months.
The isolates were positive for urease activity and produced dark brown colonies on Niger seed agar at 30 uC.The isolate showed good growth on canavanine/glycine/ bromothymol blue medium, turning the colour of the medium from yellow to blue, suggesting its identity as C. gattii.The molecular identification as C. gattii genotype AFLP6/VGII was performed by amplified fragment length polymorphism (AFLP) fingerprinting as described previously (Hagen et al., 2010).Mating-type analysis of the STE12a and STE12a alleles indicated that the isolate was mating-type a (Hagen et al., 2012).Multilocus sequencing typing (MLST) of seven nuclear loci -CAP59, IGS1, LAC1, PLB1, SOD1, TEF1 and URA5 -and subsequent phylogenetic analysis was performed as previously described (Hagen et al., 2012(Hagen et al., , 2014;;Meyer et al., 2009).This phylogenetic comparative analysis revealed that our C. gattii isolate was genetically closely related to isolates obtained from the Vancouver Island outbreak and from Australia (Hagen et al., 2012) (Fig. 1), thus representing a sequence type that is known to have a global distribution pattern (Hagen et al., 2012(Hagen et al., , 2013)).

Discussion
The case described here has three noteworthy features: first, to the best of our knowledge, this is the first time an infection due to C. gattii has been recognized in Kuwait; secondly, the infection occurred in a healthy individual who had no known immunodeficiency; and thirdly, the infection manifested itself after 3 years of continuous stay in Kuwait.All these observations are consistent with the emerging epidemiology of C. gattii infection.Historically, C. gattii is considered to have a somewhat restricted geographical distribution in tropical and subtropical regions, but recent studies have indicated that the species has extended its ecological niche to new climatological areas, which include Canada, north-western USA and Europe (Byrnes et al., 2011;Chowdhary et al., 2012, Datta et al., 2009;Hagen et al., 2012Hagen et al., , 2013;;Harris et al.,  (Hagen et al., 2012(Hagen et al., , 2014) ) using the concatenated nuclear loci CAP59, GPD1, IGS1, LAC1, PLB1, TEF1 and URA5 as described previously (Hagen et al., 2012;Meyer et al., 2009).Bootstrap values ¢75 are indicated next to branches.The Kuwaiti C. gattii isolate CBS12984 is indicated in red and the cluster to which it belongs is highlighted in grey.
2013; Illnait-Zaragozı ´et al., 2013).Among the five major genotypes of C. gattii, AFLP4/VGI and AFLP6/VGII are frequently associated with infections in immunocompetent individuals, whereas AFLP5/VGIII and AFLP7/VGIV more often infect immunosuppressed patients including individuals with human immunodeficiency virus/AIDS (Hagen et al., 2010(Hagen et al., , 2012)).The factors that determine differences in the virulence or host susceptibility between the two species (C.gattii and C. neoformans) and even between the genotypes are yet to be fully elucidated (Hagen et al., 2012;Rolston, 2013).The ongoing outbreak of C. gattii infections among immunocompetent humans and animals that occurred in Vancouver Island of British Colombia, Canada, and in the Pacific north-west of the USA was found to be caused by AFLP6/VGII (Byrnes et al., 2011;Hagen et al., 2013).Further molecular studies have revealed that about 90% of the isolates recovered from the infected cases belonged to subgenotype AFLP6A/ VGIIa (Hagen et al., 2012(Hagen et al., , 2013;;Kidd et al., 2004).This genotype was found to be more virulent than genotype AFLP6B/VGIIb (Hagen et al., 2013;Ngamskulrungroj et al., 2011).Based on a 10-locus MLST analysis, the isolate from our patient was found to belong to a sequence type that has a global distribution pattern (Hagen et al., 2013).Therefore, it was not possible to pinpoint the origin of the obtained C. gattii isolate to a geographical region.
It is probable that our patient acquired the infection while he was in the Philippines, which is within the endemic region of C. gattii (Chen et al., 2012;Hagen et al., 2012Hagen et al., , 2013)).Apparently, his C. gattii infection remained dormant for several years, a phenomenon that has been described in several previous reports in humans (Dromer et al. 1992;Garcia-Hermoso et al., 1999;Georgi et al., 2009;Hagen et al., 2012Hagen et al., , 2013) ) and animals (Castrodale et al., 2013;Illnait-Zaragozı ´et al., 2011).A large population study of 57 isolates from European patients by MLST revealed that 24 (60%) of the patients had probably acquired the infection outside Europe (Hagen et al. 2012).Twenty-nine (51%) isolates belonged to genotype AFLP4/ VGI and 23 (40%) to genotype AFLP6/VGII.Similarly, MLST of 26 clinical C. gattii isolates from Asian patients revealed that AFLP4/VGI (73%) and AFLP6/VGII (19%) were the two major genotypes associated with human infections (Hagen et al., 2012).Whilst it is difficult to ascertain the precise incubation period for C. gattii infection, it has been estimated that symptoms may appear between 6 weeks to 13 months after exposure (Castrodale et al., 2013;Johannson et al., 2012;Lindberg et al., 2007;MacDougall & Fyfe, 2006).Consistent with these reports, C. gattii isolate in our patient remained dormant for several years before becoming clinically apparent.So far, all the human cases of cryptococcosis diagnosed in the Kuwait Mycology Reference Laboratory during the last 20 years have been caused by C. neoformans var.grubii (Khayhan et al., 2013).Our attempts to isolate C. neoformans or C. gattii from environmental sources (pigeon excreta and plant debris) have not been successful.It seems that climatological conditions prevailing in Kuwait may not be congenial for long-term survival of this organism in nature.
Clinically, there are no specific signs and symptoms that distinguish infection caused by C. gattii and C. neoformans.In addition, the therapeutic guidelines for the treatment of cryptococcal meningitis caused by the two species are similar, comprising induction, consolidation and suppressive regimens (Perfect et al., 2010).Unfortunately, our patient was initially not treated optimally.First, there was a delay in procuring flucytosine, and secondly, the patient was initially started on suboptimal dosage of Abelcet (1 mg kg 21 ) with fluconazole, which failed to eradicate the organism from the CSF.As the C. gattii isolate showed reduced susceptibility to fluconazole, it was replaced with voriconazole.The patient showed symptomatic improvement only when the dose of Abelcet was increased to 3 mg kg 21 , although the culture of CSF was still positive.Finally, Abelcet when combined with flucytosine resulted in negative CSF cultures, although a few cryptococcal cells were still visible in Indian ink examinations.Due to reduced susceptibility of the strain to fluconazole (MIC 16-32 mg ml 21 ), an initial combination either with voriconazole or posaconazole might have been preferable in the absence of flucytosine.However, both of these drugs have been used only rarely in induction or eradication therapy of cryptrococcal meningitis, and clinical experience is limited (Chen et al., 2013).
Infections with C. gattii are known to run a protracted course of illness, often accompanied by serious neurological complications (Chen et al., 2012;Illnait-Zaragozı ´et al., 2013;Mitchell et al., 1995;Speed & Dunt, 1995).However, no neurological mass lesions, such as development of cryptococcoma(s), were observed in our patient.Additionally, several studies have shown that C. gattii isolates are significantly less susceptible to azoles than the isolates of C. neoformans and that azole susceptibility varies significantly among C. gattii genotypes (Hagen et al., 2010;Khan et al., 2007;Trilles et al., 2012).As indicated above, our isolate showed reduced susceptibility to fluconazole.It is unclear whether differences in antifungal susceptibilities between the two species should warrant a change in therapeutic approach (Rolston, 2013;Trilles et al., 2012).In a recent study, Lockhart et al. (2012) reported that VGII isolates were less susceptible to fluconazole (geometric mean value 8.6 mg ml 21 ) than other molecular types of C. gattii and proposed a combined epidemiological cut-off value of 32 mg ml 21 for all genotypes of the species.Similar results were obtained in an international study involving large number of C. gattii isolates from many countries (Espinel-Ingroff et al., 2012).However, regardless of the species involved, combined use of amphotericin B with flucytosine remains the mainstay of induction therapy for cryptococcal meningitis worldwide (Chen et al., 2013;Perfect et al., 2010).As seen in our patient, the restricted accessibility of flucy- tosine may pose management challenges, particularly if the isolate is resistant to fluconazole, an issue that has been highlighted recently (Loyse et al., 2013).
In conclusion, a case of meningoencephalitis caused by C. gattii genotype AFLP6/VGII in an immunocompetent Filipino male, has been described.In all probability, the patient acquired the C. gattii infection in his home country, which remained dormant for several years.To the best of our knowledge, this is the first report of isolation of C. gattii in Kuwait, reinforcing its expanding role in human infections.