Case Report

Disseminated cryptococcosis and fluconazole resistant oral candidiasis in apatient with acquired immunodeficiency syndrome (AIDS)

Rajendra J. Kothavadel, chetan M. oberaiz, Arvind G. Valand3, Mehroo H. panthakia

l,Microbiology Laboratory WQA, E?COR, 10065, Edmonton, AB, TSJ 381, Canada' Department of Dermatology, Sir J.J. Group of Hospitals and Grant Medical College, Mumbai, tndia"DeparTment of-Pathology, Sir J.J. Group of Hospitals and Grant Medicat Coltege,-hiumoai, lndia'Deparlment of lmmunocytobiology, Sir H.N. Hospitat and MedicatResearch Centre, Mumibai, tnaia

AbstractDisserninated cryptococcosis and recurrent oral candidiasis was presented in a-heterosexual AIDS patient. Candida tropicalis (C.tropicalis)was isolated from the oral pseudomembranous plaques and Cryptococcus neofonnans (C. neofoimons) was isolated frorn rnaculopapulailesions on body parts (face, hands and chest) and body fluids (urine, exjectorated sputum, and cerebrospinal fluid). In vinlo'druEsusceptibility testing on the yeast isolates demonstrated resistance to fluconazole acquired by C. tropicalis whicir was a suggestive possibleroot cause ofrecurrent oral candidiasis in this patient.

Key words: HrV; AIDS; cryptococcosis; candidiasis; immunocomprornised; fluconazole

J Infect Dev Ctries 2010;4(10):674-678.

(Received 5 Decernber 2009 - Accepted 28 June 2010)

Copyright O 2010 Kothavade et al. This is an open-access article distributed under the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Gase ReportA 34-year-old heterosexual HIV-I positive male

developed mucopurulent productive cough, recurrentoral plaques, occasional syncope, and neurologicalsymptoms that included headache and dizziness.Physical examination revealed discrete erythematousmaculopapular lesions on his face (Figure l), neck,chest, and both hands. There were not any significantenlargement of the cervical lymph nodes, and oralexamination revealed pseu{o-membranous plaques(Figure i). He was previously treated wiih anti-fungal drugs (fluconazole and amphotericin-B),primary anti-tuberculous drugs (isoniazid, rifampin,ethambutol, and streptomycin), and an antigiardialdrug (tinidazole) for giardiasis, caused by, Giardialamblia. The CD4+ lymphocyte count for this patientwas 40 cells/pl with a CD4+/CD8+ ratio of l:72.Despite a history of multiple unprotected sexualexposures, the patient tested negative for venerealdisease.

The skin biopsies and body fluids, such as,cerebrospinal fluid (CSF), urine, and mucopurulentexpectorated sputum, showed encapsulated yeasts inIndia-ink-wet-mount preparation. The cultures foracid-fast bacilli (AFB) on Lowenstein-Jenson and

non-selective Middle-brook 7Hl2 agar media werenegative. Periodic-acid-schiff and Grocott-Gomori-methylamine-silver-stained smears were negative forPneumocystis carinii. Serum and CSF tested positivefor capsular Cryptococcal polysaccharide antigenusing the latex agglutination test with a titer ofl:1015. Skin sections revealed gelatinous troma(Figure 2) frlled with numerous encapsulated yeastcells (Figure 3). Biopsied specimens of skin and otherbody fluids,(CSF, urine, and sputum) yielded thegrowth of C. neoformans on Sabouraud's dextroseagar (SDA) medium. The resultaut mucoid-cream-colored colonies were negative for germ tube andpositive for urease test. Colonies failed to grow onCyclohexamide-supplemented SDA. Colonic growthat 37"C on plain SDA was weakly positive.Microscopic examination of the Gram-stainpreparation from a portion of scraped oral lesionsshowed Gram-positive yeasts and pseudo-hyphalforms. The remaining portion the scraped oral lesionswere inoculated on SDA which then showed a typicalgro'nrth of C. tropicals. Identification of C. tropicaliswas further confirmed by the germ tube test;morphological characteristics were determined oncornmeal tween-S0-agar and Vitek-32 and ApI 20C

Kothavade et al - Disseminated cryptococcosis and candidiasis in AIDS

AUX identification systems (bioM6rieux, Craponne,France). Drug susceptibility testing to fluconazole,ketoconazole, clotrimazole, posaconazole, andamphotericin-B was performed using the CLSImicro-dilution method i1] The in-vitro drugsusceptibility study revealed fluconazole resistance toC. tropicalis only; whereas C. neofoftnans werefound to be susceptible to all antifungal agents. Labinvestigations for further clinical management couldnot be followed as the patient died on the third day ofhis hospitalization.

DiscussionThe two species of genus Cryptococcus (C.

neoforntans and C. gatti) are pathogenic andresponsible for the life-threatening infections inimmunocompromised patients, particularly those thatare diagnosed with HIV/AIDS l2-3). C. albicans andother species of Candida, such as, C. tropicalis, areresponsible for causing oral and systemic candidiasisin immunocompromised patients. Succeedinginfection occurs due ts high to moderately virulentstrains of Candida against the intensity of theimmune response generated by the host cells.Nonpathogenic Candida may colonize inimmunocompromised hosts, such as, neutropenic andHIV-AIDS patients. The colonization of avirulentCandida species does occur due to poor cell mediatedimmune response, mounted by the compromisedhosts [4].

Figure 1: Erythematous maculopapular lesions on the faceand curdy coating on the tongue (oral candidiasis)

I In.fect Det Ctries 2010:. 4(10):674-618.

Figure 2: Histopathology of a skin biopsy fromerythematous maculopapular lesions (tissue sectionsstained with Hematoxyllin and Eosin) shows (100x) a

gelatinous stroma - (no inflammatory or granulomatousreactions).

Development of Cryptococcal infection isdetermined by a cell-mediated immune responseagainst an encapsulated yeast population and itsvirulence. Immunocompetent patients withcryptococcosis usually demonstrate granulomatousinflammatory reactions (infected sites) to

'encapsulated yeasts or antigens [5]. However, in thispatient, there were no granulomatous reactions orinflammatory t'esponses seen in any tissue sections;instead, there were extensive disseminatedgelatinous-appearing lesions and large number ofencapsulated yeasts. Comparable findings are seen inother full-blown AIDS patients as well [6,7,8].

In this case study, the CD4+ cell count was anideal marker to assess the immunological status ofthe patient because from the T lymphocytepopulation CD4+ and CD8+ cells are the keyelements in mounting immune resporuie agailstpathogenic organisms [9]. However, it has beenobserved from the in-vilro studies on humatr T cellsthat CD4+ plays a significant role in mediating CD8+proliferation against C. neoformans [0]. The HIVinfected patients are more prone to Cryptococcalinfection when their CD4+ counts drop below 50-100celVmm [11,12]. In vitro studies on human T cells,particularly CD4+ and CD8+ cells against C.neoformans, have demonstrated interleukin andtransferrin receptor expression with subsequentproliferative response [10]. The production of IFN-gamma from CD8+ cells have been found ininactivating encapsulated yeasts of C. neoformanswithin macrophages [13]. Hence CD8+ cells inimmune response may be functioning independent of

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CD4+ cells. Some experimental studies suggest, thatappropriate CD4+ signaling could allow CDS+ cellsto respond against C. neoforamans. Further studiesare required in human T cells to explore the potentialrole of interleukin-2 and IFN-gamma in resolving theambiguity of CD4+-CD8+ linked response against C.neoformans infection. Inadequate T cell(CD4+/CD8+) response against C. neoformansinfection leads to the death ofpatients suffering fromAIDS [4,10]. C. neoformairs is the most commonfatal mycosis in AIDS patients, but is less common innon-cell-mediated immunodeficiency [15,16], whichsuggest that CMI is an important immune mechanismin the clearance of C. neoformans.

Figure 3: Histopathology of Skin biopsy (Erythematousmaculopapular lesions): tissue sections stained withPeriodic acid-schiffs stain under 400x shou,s intenselypinked muco-polysaccharide capsule of C. neofotmans ingelatinous stroma.

In the present case report, it appears that the lowCD4+ lymphocyte count supports the opportunisticcondition to establish mixed fungal infections causedby C. neoformans and C. tropicalis. Unlike C.albicans, which can be occasionally found as acommensal, C. tropicalis has almost always beenassociated with the development of fungal infectionsin immunocompromised patients [17]. Some reportsfrom India suggests that C. tropicalis is the mostprevalent yeast species responsible for causinginfection in HIV/AIDS patients [18]. However, in thepresent case study, fluconazole-resistant C. tropicaliswas isolated from the oral-pseudomembranouslesions, which was an uncommon finding in theIndian subcontinent. Such observations might havebeen overlooked due to a lack of drug susceptibilitystudies or tracking of recurrent Candida infections rnimmunocompromised or immunocompetent patientsin India.

J lnJbct Det' Ctries 20t0;4(10):674-678.

The extensive use of fluconazole in the clinicalmanagement of AIDS and in other high-risk patientscould be the basic cause of increasing prevalence ofCandida species other than C. albicans. Among thesespecies C. tropicalis is predominant in some parts ofthe world U9,20,21,22\ As a result, C. tropicalishasbeen emerging in both immunocompromised andimmuno-competent patients; hence, it has beensuggested that the use of fluconazole in a high-riskgroup ofpatients should be avoided [19]. Irrespectiveof host immune response and other predisposingfactors, a pathogenicity of C. tropicalis, might beanother contributing factor for its mucosalcolonization in the buccal cavity. Similarly,colonization in the gastrointestinal tract can lead tothe penetration of sub-mucosal layers. Some clinicalstudies have shown that C. tropicalrs is more virulentthan C. albicans in neutropenic and non-neutropenicpatients [23,24]. The purified tropiase from C.tropicalis (a novel acid proteinase) demonstratedhemorrhagic activity and its ability to increasecapillary pemreability [25]. In-spite of the recurrentinfections due to C. tropicalis in AIDS patients, therewas no evidence of its dissemination. However,dissemination did occur due to encapsulated yeasts ofC. neoformars. The dominant mechanism of C.neoformans over C. tropicalis inimhunocompromised patients is still not clearlyunderstood.

Antifungal drug intolerance is certainly a majorchallenge in the clinical management of AIDS andother neutropenic and non-neutropenic patients (withseveral underlying subclinical conditions) 124,261.An interesting clinico-mycological scenario in thisreport is that C. neoformans isolates were susceptibleto fluconazole; however, C. tropicalis was found tobe resistant. Such clinico-mycological aspects mustbe carefully considered before switching to anyantifungal therapeutic options. In addition, C.tropicalis has often been reported as resistant tofluconazole 1271. The plausible mechanisms offluconazole resistance have been extensively studiedin the strains of C. albicans isolated from patientswith HIV-AIDS-related oropharyngeal candidiasis[4]. Based on the literature suryey, we believe thatthis could be the fust case report from lndiapresented with disseminated cryptococcosis andrecurrent oral candidiasis due to fluconazole-resistantC. tropicalis.

Development of fluconazole resistance in C.tropicalis could be due to several mechanisms.Nevertheless, some plausible mechanisms [28] could

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include, escalation of emanation (efflux) of the azolecompounds due to (i) over'expression of effluxpumps, such as the adenine tri-phosphate bindingcassette (ABC) transporter and major facilitatorsuperfamily (t\GS) membrane transporters; (ii) up-regulation of the ERG11 gene, coding for the azoletarget lanosterol 14 (c)-demethylase; (iii) ERGLIsequence related point mutation and (iv) decreasedaffinity of azoles for their target-induced azoleresistance. The acquired azole resistance could be theresult of either one of these mechanisms or coupledwith other steps in a harmonized manner 1291. hgeneral, azole resistance in C. tropicalis suggests thatover-expression of CtERgl I, associated withmissense mutation in this gene, and seemed to beresponsible for acquired azole resistance.

The intracranial pressure due to the heavy load ofencapsulated yeasts and extensive colonization offluconazole-resistant C. tropicalis (or intolerance toantifungal agents) could be the major cause of thedeath of this patieut. Epidemiological studies suggestthat, increasing prevalence of C. tropicaffs related tocandidiasis in immunocompromised patients is due tothe underlying subclinical conditions and subsequentantifungal agents associated toxicity; in addition tothe genetic relatedness of the Candida species130,31,327. Although, morbidity and mortality ratesdue to infections caused by C. tropicalls have beenreported more often, than those attributed to C.albicans [33]; it would be worth comparing thisscenario in the cases of AIDS with disseminatedcryptococcosis.

Comparing the susceptibility patterns of oralisolates during the highly active anti-retroviraltherapy (HAART) era with those from 1994, suggestthat most cases of HfV-associated oropharyngealcandidiasis observed during the HAART era weie -

caused by azole-susceptible stains. However, thereversion of isolates from azole-resistant to azole-susceptible was related to strain substitution [34].Ever since the introduction of HAART, incidences ofopportunistic infections such &s, cerebraltoxoplasmosis, Pneumocystis carinii pneumonia andothers have been declining. However, the prevalenceof Candidial esophegitis remained unchanged [34].These findings suggests that HAART does not play a

significant role in minimizing any fluconazole drugresistance in Candida species and still remains achallenging problem for its long-term therapeuticapplication in HlV-infected or immunocompromisedpatients.

J ln/bct Der Ctlles 2010:.1(10):674-678.

ConclusionThe major gap in the pathogenicity of C.

neoformans is the limited understanding of itsdominant mechanism over C. n-opicalis in AiDSpatients. Secondly, the interlinked cellularmechanisms between CD4+ and CDS+ cells againstC. neoformans infection remain unclear. Hence, someadditional studies need to be performed on theexpression of IL-z receptors and IFN-gammaproduction by human T cells (T cells - derived fromthe various clinical stages of HIV infection; may befrom sero-rectaive to full blown AIDS stage).

Although, fluconazole is still the first line oftreatment in a number of cases of invasivecandidiasis, its therapeutic application should berestricted to selected high-risk patients in order tominimize the risk of emergence of azole-resistantstrains of Candida. Consistent use of standard, rapidand reliable methods for species identification inconjunction with drug susceptibility testing isadvocated; particularly, for the cases of recurrentcandidiasis. This approach would allow systematictracking of emerging Candida species, other than C.albicans in various clinical conditions. Additionally,its application in monitoring drug-resistant strains orchanging patterns of Candida species, other than C.albicans in clinical settings may support clinicians indetermining appropriate therapeutic agents foreffective clinical management. The therapeutic use ofHAART, in combination with granulocyte colony-stimulating factor (G-CSF) cytokine in HlV-infectedpatients may be useful in the management ofconcomitant infections caused by fluconazole-resistant strains of C. tropicalis.

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Corresponding authorDr. R. J. KothavadeMicrobiology Laboratory -WQAEPCOR10065, Jasper AveEdmonton, AB, T5J 3BlCanada

Tel/Fax (780) 412-7617Email: rkothava@epcor.ca

Conflict of interests: No conflict of inrerests is declared.

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