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Case Report

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

Rajendra J. Kothavade', chetan M. oberai2, Arvind G. Valand3, Mehroo H. panthakia

1^Microbiology Laboratory WQA, EPCOR, 10065, Edmonton, AB, TSJ 381, Canada'"Depaftment of Dermatology, Sir J.J. Group of Hospitats and Grant Medical Coltege, Mumbai, lndia",Department of Pathology, Sir J.J. Group of Hospitals and Grant Medical Cottege, Mumbai, tndia'Department of lmmunocytobiology, S/rH.N. Hospital and Medical Research Centre, Mumbai, tndia

AbstractDissen-rinated cryptococcosis and recurrent oral candidiasis was presented in a-heterosexual AIDS patient. Candida tr.opicalis (C.tropicalis)was isolatcd from the oral pseudornembranous plaques and Cryptococcus neoforntans (C. neoformans) was isolated frorn praculopapularIcsions otr body parts (face, hands and chest) and body fluids (urine, expectorated sputuln, and cerebrospinal fluid). In vitio'drtgsr'rsceptibility testing on the yeast isolates demonstrated resistance to fluconazole acquired by C. tt"opicalis which was a suggestive possibleroot cause ofrecurrent oral candidiasis in this patient.

Key words: I{lV; AiDS; cryptococcosis; catrdidiasis; immunocornpromised; fluconazole

J lnfecr Dev Crries 2010:_4(10):674-678.

(Rcceived 5 December 2009 - Accepted 28 .lune 2010)

Copyright O 2010 Kothavade et al. This is zur open-access article distributed under the Creative Comrnons Attribution License, whichperrrits unrestricted use, distribution, aud reproduction ir any rnedium. provided the original work is properly cited.

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

developed mucopurulent productive cough, recunentoral plaqucs, occasional syncope, and neurologicalsyrnptoms that included headache and dizziness.Physical examination revealed discrete erythematousmaculopapular lesions on his face (Figure 1), neck,chest, and both hands. There were not any significantenlargement of the cervical lymph nodes, and oralexamination revealed pseudo-rnembranous plaques(Figure 1). He was previously treated with anti-fungal drugs (fluconazole and arnphotericin-B),prirnary anti-tuberculous drugs (isoniazid, rifampin,ethambutol, and streptomycin), and an antigiardialdrug (tinidazole) for giardiasis, caused by, Giardialamblia. The CD4+ lyrnphocyte count for this patientwas 40 cells/pl with a CD4+/CD8+ rutio of t:72.Despite a history of rnultiple 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 Lowerrstein-Jenson and

nbn-selective Middle-brook 7H12 agar media werenegative. Periodic-acid-schiff and Grocott-Gomori-methylarnine-silver-stained smears were negative forPneumoc)tstis carinii. Serum and CSF tested positivefor capsular Cryptococcal polysaccharide antigenusing the latex agglutination test with a titer of1 : 1 0 1 5. Skin sections revealed gelatinous troma(Figure 2) filled 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 resultant mucoid-cream-colored colonies were negative for germ tube andpositive for urease test. Colonies failed to grow onCyclohexarnide-supplemented SDA. Colonic growthat 37oC on plain SDA was weakly positive.Microscopic exarnination of the Gram-stainpreparation fi'om 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 typicalgrowth 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 Apl 20C

Kothavade e/ al - Disseminated cryptococcosis and candidiasis in AIDS

AUX identification systeurs (bioMdrieux, Craponne,France). Drug susceptibility testing to fluconazole,ketoconazole, clotrirnazole, posaconazole, andarnphotericin-B was perfonned using the CLSIrricro-dilution method t1]. The in-ttitro drugsusceptibility study revealed fluconazole resistance toC. tropicalis only; whereas C. neoformans werefound to be susceptible to all antifungal agents. Labinvestigations for further clinical managernent couldnot be followed as the patient died on the third day ofhis hospitalization.

DiscussionThe two species of genus Cryptococcus (C.

neofonnans and C. gatti) are pathogenic andresponsible for the life-threatening infections inimmunocompromised patients, particularly those thatale diagnosed with HIV/AIDS 12-31. C. albicans and,other species of Candida, such as, C. tropicalis, areresponsible for causing oral and systemic candidiasisin immunocompromised patients. Succeedinginfection occurs {ue to high to moderately virulentstrains of Candida against the intensity of theimrnune lesponse generated by the host cells.Nonpathogenic Candidq lnay colonize inirnrrunocorrpromised hosts, such as, neutropenic andHIV-AIDS patients. The colonization of avirulentCandida species does occur due to poor cell rnediatedirnrnune response, uounted by the compromisedhosts [4].

Figure I: Erythernatous maculopapular lesions on the faceand curdy coating on the tongue (oral candidiasis)

J htfect Dev Ctries 2010.,4(10):674-678.

Figure 2: Histopathology of a skin biopsy fromerythernatous maculopapular lesions (tissue sectionsstained with Hematoxyllin and Eosin) shows (100x) agelatinous 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 dernonstrate granulomatousinflammatory reactions (infected sites) toencapsulated yeasts or antigens [5]. However, in thispatient, there were no granulomatous reactions or

- inflammatory responses 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 16,7,91.

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 key

. elements in mounting immune response- againitpathogenic organisms [9]. However, it has beenobserved from the in-vitro studies on human T cellsthat CD4+ plays a significant role in mediating CDg+proliferation against C. neoformam [10]. The HIVinfected patients are rrore prone to Cryptococcalinfection when their CD4+ counts drop below 50-100cell/rnm l1l,l2l. 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 CDg+ cells inimmune response may be functioning independent of

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Kothavade er al - Disseminated cryptococcosis and candidiasis in AIDS

CD4+ cells. Some experimental studies suggest, thatappropriate CD4+ 5ignaling could allow CD8+ cellsto respond against C. neoforaruans. Further studiesare required in human T cells to explore the potentialrole of interleukin-2 and IFN-garnma in resolving thearnbiguity of CD4+-CD8+ Iinked response against C.neoformans infection. Inadequate T cell(CD4+/CD8+) response against C. neoformansinfection leads to the death ofpatients suffering fi.omAIDS [14,10]. C. neoformans is the most commonfatal mycosis in AIDS patients, but is less common innon-cell-mediated irnmunodeficiency [15,16], whichsuggest that CMI is an imporlant irnrnune mechanismin the clearance of C. neoformans.

Figure 3: Histopathology of Skin biopsy (Erythematousmaculopapular lesions): tissue sections stained withPeriodic acid-schifls stain under 400x shorvs intensely

- pinked muco-polysaccharide capsule of C. neoformans ingelatinous stroma.

In the present case report, it appears that the lowCD4+ lymphoc)'te count supports the opportunistic

- _condition to establish. mixed fungal infections causedby C. neoformans and C. tropicalis. Unlike C.albicans, which can be occasionally found as acornrnensal, C. tropicalis has almost always beenassociated with the development of fungal infectionsin imrnunocompromised patients [17]. Sorne reportsfrorn 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-pseudolnembranouslesions, 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 inirnmunocompromised or imrnunocompetent patientsin India.

J lnfect Dev Ctries 2010, 4(t0):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. *opicalis is predominant in some parts ofthe world U9,20,21,221. As a result, C. tropicalis hasbeen 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 mucosalcoloirization in the buccal cavity. Similarly,colonization in the gastrointestinal tract can lead tothe penetration of sub-mucosal layers. Some clinicalstudies have shown that C. tropicalis is more virulentthan C. albicans in neutropenic and non-neutropenicpatients 123,241. The purified tropiase from C.tropicalis (a novel acid proteinase) demonstratedhemorrhagic activity and its ability to increasecapillary permeability [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. neoformans. The dominant mechanism of C.negformans over C. tropicalis inimmunocomprornised 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 repofted 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 survey, we believe thatthis could be the first case report from Indiapresented 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, solne plausible mechanisms [28] could

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include, escalation of emanation (efflux) of the azolecornpounds due to (i) over-expression of effluxpumps, such as the adenine tri-phosphate bindingcassette (ABC) transporter and major facilitatorsuperfamily (MFS) membrane transporters; (ii) up-regulation of the ERGI1 gene, coding for the azoletarget lanosterol 14 (cr)-demethylase; (iii) ERGllsequence related point rnutation and (iv) decreased

affinity 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 129). Ingeneral, azole resistance in C. tropicalis suggests thatover-expression of CtERgi 1, 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 rnajor cause of thedeath of this patient. Epiderniological studies suggestthat, increasing prevalence of C. tropicalis related tocandidiasis in immunocompromised patients is due tothe underlying subclinical conditions and subsequentantifungal agents associated toxicity; in addition to ...

the genetic relatedness of the Candida species

L30,31,32]. A1though, morbidity and mortality ratesdue to infections caused by C. nopicalrs 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 HlV-associated oropharyngealcandidiasis observed during the HAART era werecaused by azole-susceptible strains. 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 BS, cerebraltoxoplasmosis, Pneumocystis carinii pneumonia andothers have been declining. However, the prevalenceof Candidial esophegitis remained unchanged [34].These findings suggests that HAART does not play asignificant 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 lnfect Dev Ctries 20101 4(10):674-678.

GonclusionThe major gap in the pathogenicity of C.

neoformans is the limited understanding of itsdominant mechanism over C. tropicalis in AIDSpatients. Secondly, the interlinked cellularmechanisms between CD4+ and CD8+ cells againstC. neoformans infection remain unclear. Hence, someadditional studies need to be performed on theexpression of lL-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 bereshicted 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.qlbicans 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 indeterrnining 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 381CanadaTelffax (780) 412-7617Email: [email protected]

Gonflict of interests: No conflict of interests is declared.

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