FEMS Immunology and Medical Microbiology 41 (2004) 27–34

www.fems-microbiology.org

Anti-retroviral therapy with protease inhibitors decreasesvirulence enzyme expression in vivo by Candida albicanswithout selection of avirulent fungus strains or decreasing

their anti-mycotic susceptibility

Flavia De Bernardis a,*, Evelina Tacconelli b, Francesca Mondello a, Adriana Cataldo b,Silvia Arancia a, Roberto Cauda b, Antonio Cassone a

a Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanit�a, Viale Regina Elena 299, Rome 00161, Italyb Department of Infectious Diseases, Catholic University, Rome, Italy

Received 1 September 2003; received in revised form 22 December 2003; accepted 24 December 2003

First published online 15 January 2004

Abstract

Highly active anti-retroviral therapies (HAART) with human immunodeficiency virus (HIV) protease inhibitors (PIs) or non-

nucleoside reverse-transcriptase inhibitors (NNRTI) were compared for their effect on prevalence, aspartyl proteinase (Sap) pro-

duction and the biotypes and anti-mycotic sequential susceptibility of Candida spp. isolates from the oral cavity in a longitudinal

prospective study. HAART-PI, but not HAART-NNRTI strongly inhibited Sap expression in the oral cavity without exerting any

consistent effect on the role of Candida spp. isolation or selection of low virulence or anti-mycotic resistant fungus biotype. More

importantly, the sequential isolates of Candida albicans from HAART-PI, but not those from suspended HAART-NNRTI, showed

an increased Sap production in vitro. While further demonstrating that HIV-PI inhibit Sap expressions, our results do not support

the view that the mentioned inhibition could eliminate Candida or its selection of the oral cavity.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Candida; HIV; Proteinase inhibitors; Proteinase secretion; Biotypes

1. Introduction

Oropharyngeal candidiasis (OC) was originally a

frequent opportunistic infection in human immunode-

ficiency virus (HIV)-positive subjects before the ad-

vent of highly active anti-retroviral therapy (HAART)

[1,2]. The most common agent causing mucosal candi-

diasis is Candida albicans, which has the ability to se-crete aspartyl proteinases (Sap), a principal family of

virulence enzymes, facilitating the invasion of mucosal

tissue [3,4]. This is a relevant aspect of the oral pathol-

ogy caused by this fungus that has been demon-

* Corresponding author. Tel.: +39-06-49902809; fax: +39-06-

49387112.

E-mail address: fdberna@iss.it (F. De Bernardis).

0928-8244/$22.00 � 2004 Federation of European Microbiological Societies

doi:10.1016/j.femsim.2003.12.006

strated by the repeated observations of the strains of

C. albicans of particularly high Sap production. These

were more frequently isolated from the oral and vaginal

cavities of HIV-positve rather than from HIV-negative

subjects [5,6]. A distinctive set of Sap genes has been

found to express the fungus in the oral and vaginal

cavity [7].

As mentioned above, HAART including HIV pro-teinase inhibitors (HAART-PI) have caused a marked

decline of OC and other opportunistic infections [8–11].

We and others have demonstrated that this therapy

exerts an early immune reconstitution-independent ben-

eficial effect against OC, which is entirely due to the PI

capacity to inhibit Sap enzymes of Candida secretory

aspartyl proteinase (Sap) family that belongs to the

same family as HIV-proteinase [12–15]. There is now

. Published by Elsevier B.V. All rights reserved.

28 F. De Bernardis et al. / FEMS Immunology and Medical Microbiology 41 (2004) 27–34

further evidence that PI could directly affect some other

opportunistic agents of disease common in HIV-positive

subjects [16]. However, previous studies did not clarify

whether the anti-retroviral therapy with PI had a per-

sistent effect on the fungus infection or carriage in theoral cavity. They did not confirm whether the therapy

caused the selection of different fungal strains with dif-

ferent Sap-producing abilities or had any impact on

fungus susceptibility to triazole anti-mycotics. In this

study, we took advantage of comparing a longitudinal,

comparative study of subjects who received HAART-PI

with another group who were under a therapeutic regi-

men with a nonnucleoside reverse-transcriptase inhibi-tor (NNRTI) utilized as a control. Thus, we have

longitudinally assessed the prevalence of fungal isolates

as well as the biotypes of C. albicans isolated from each

patient, together with the Sap production ability of each

isolate from each subject. In specific reports on the as-

sociation between Sap production level and anti-mycotic

susceptibility [5,17], we also tested the susceptibility of

all C. albicans isolates to fluconazole and itraconazole.The results clearly demonstrate that PI-containing

HAART regimens strongly inhibit the expression of Sap

activity in the oral cavity without selecting the low-Sap

producer fungus biotypes or affecting Candida preva-

lence and anti-mycotic susceptibility.

2. Subjects and methods

2.1. Study setting

The clinical investigation was performed at the

Catholic University Hospital, a 1700-bed tertiary care

centre located in Rome, Italy, as detailed elsewhere

[15,18]. Seventy HIV-infected subjects were considered

that were naive for anti-retroviral therapy and whowere admitted as outpatients from July 1st through

December 31st 1999. Among this group, 35 subjects

began HAART-PI (Group 1) and 35 received HAART-

NNRTI (Group 2). Therapy start time was designated

as 0 and all subjects were monitored for a period of 180

days. Clinical details and general clinical parameters

recorded from these patients are given elsewhere

[12,15,18].

2.2. Parameters evaluated

With the consent of each enrolled subject in the

study, the following data were obtained: the age, sex,

HIV-risk behavior; any active intravenous drug abuse;

the stage of HIV infection; previous manifestations of

HIV infection; date of AIDS-defining illness; presence ofOC and history of previous OC episodes; concurrent

opportunistic infections; risk factors for OC anti-

microbial therapy; type and use of prophylaxis for

Pneumocystis carinii pneumonia and toxoplasmosis;

and any other medications (anti-tuberculous agents,

anti-virals, corticosteroids, anti-acids); the number of

circulating CD4þT (/mm3) cells and of peripheral poly-

morphonuclear (PMN) cells (/mm3) and HIV-viremiawith a detection limit below 50 copies/ml. The analyzed

risk factors for OC included neutropenia (PMNs <1000/

mm), alcoholism, cirrhosis, diabetes, neoplastic diseases

and chronic renal failure.

The first episode of microbiologically documented

diagnosis of OC was routinely made of the typical signs

and symptoms. All OC were clinically categorized into

three types: pseudomembranous, erythematous and hy-perplastic forms, as detailed elsewhere [18].

2.3. Yeast isolation and identification

Oropharyngeal samples were taken with cotton-

tipped swabs and kept in sterile physiological saline,

before transferring to the laboratory where they were

streaked on plates of Sabouraud–dextrose agar (BBL,Baltimore, MD) with added chloramphenicol (50

lgml�1), and Mycosel agar, before immersion into en-

richment broth. All cultures were incubated for 48–72 h

at 30 �C. All isolates were identified on the basis of their

morphology and profiles in the API 20C system (Bio-

Merieux, Marcy l�Etoile, France).

2.4. Sap detection in vitro and in salivar fluids

All isolates were tested for their ability to grow and

produce a clear zone of hydrolysis in bovine serum al-

bumin (BSA) agar, as detailed elsewhere [4,15].

Sap antigen secretion in BSA broth was also assayed

by ELISA [4,15]. Briefly, the isolates were precultured in

YPD broth (1% yeast extract; 2% peptone and 2% glu-

cose), the cultures were centrifuged and the cellularpellet was washed and incubated (106 cells in 25 ml of

YPD broth plus 0.2% BSA) at 30 �C with slight agita-

tion. After 40 h of incubation, the samples were centri-

fuged at 3400g for 10 min and the supernatants were

treated with 0.5 ml of 50% trichloroacetic acid (TCA),

incubated for 30 min in ice and centrifuged at 3400g for

10 min. The pellets were washed twice with 95% ethanol,

dissolved in 1% (w/v) sodium dodecyl sulfate (SDS),diluted in 0.2 M sodium carbonate buffer (pH 9.6) and

applied to the MicroTest plates. Purified rabbit anti-Sap

serum [12,19] was added at a 1:100 dilution and incu-

bated for 2 h at 37 �C. The second antibody was phos-

phatase-conjugated goat anti-rabbit immunoglobulin G

(1:1000 dilution, Sigma. St. Louis, MO.) The reaction

was detected with phosphatase substrate nitrophenol

phosphate (Sigma). Sap amount was calculated from astandard curve (1–110 ng) determined with a highly

purified Sap preparation [12,19] as coating antigen. The

plates were read with a Titer K Multiscan (Labsystem

F. De Bernardis et al. / FEMS Immunology and Medical Microbiology 41 (2004) 27–34 29

Helsinki, Finland) at 405 nm blanked against air. The

specificity of the ELISA for proteinase detection was

confirmed by Western blotting [19].

ELISA was also used for Sap detection in the saliva

of patients, according to a procedure fully describedelsewhere [15].

2.5. Anti-mycotic susceptibility test

A recently modified, improved method of anti-

mycotic susceptibility assays in vitro, derived from the

standardized NCCLS method [20,21] was used

throughout this study for the determination of Candidaisolate susceptibility to fluconazole and itraconazole.

Stock solution of fluconazole (Pfizer Inc., New York,

USA) was prepared in sterile distilled water. Stock so-

lution of itraconazole (Janssens Pharmaceutica, Beerse,

Belgium) was prepared by heating at 75 �C for 45 min in

polyethylene glycol 400.

The yeast isolates were grown on Sabouraud dextrose

agar for 48 h at 35 �C. The inoculum suspension wasprepared by picking five colonies of at least 1 mm in

diameter and suspending them in 5 ml of sterile distilled

water. The cell density of the suspension was adjusted

spectrophotometrically to a final transmission of 85% at

530 nm. The working suspension was made by a 1:50

dilution in sterile distilled water followed by a 1:20

dilution in media to obtain a 2� final suspension. In-

oculum sizes were confirmed by the enumeration ofcolony-forming units (CFU) on Sabouraud dextrose

agar. The medium for susceptibility testing was RPMI

1640 media (American Biorganics, Inc., Niagara Falls,

NJ) added with LL-glutamine and buffered at pH 7.0 with

0.165 M morpholinepropanesulphonic acid. Testing was

performed in sterile, flat-bottom 96-well microtiter

plates. Drugs were prepared at 10� the strength of the

final concentration, and these mother solutions werediluted 1:5 with RPMI 1640 to obtain 2� the final

concentrations. Volumes of 50 ll of the 2� drug dilu-

tions were dispensed into wells; two wells in each row

were used, one as a growth control and the other as a

sterility check. The microtiter plates were stored at )70�C until use. On the day of the test, 50 ll of the yeast

suspension were added to each well.

The microtiter plates were incubated at 35 �C andread at 24 and 48 h with the aid of a reading mirror after

shaking the growth samples in each well and compared

these with that of the growth control (drug-free) well.

The MIC was defined according to previously estab-

lished criteria [19,20].

2.6. Karyotype analysis by pulsed field gel electrophoresis

Cells of C. albicans were grown to a stationary phase

overnight at 28 �C in YPD medium (glucose 2%; yeast

extract 1% and Bacto-Peptone 2%; Difco, Detroit, MI).

The cells were packed using centrifugation (3000 rpm

for 5 min) and washed in 1.2 M sorbitol solution con-

taining 20 mM EDTA, pH 8.0. As described above, in a

solution of EDTA–sorbitol, the pellet was re-suspended

in a cell concentration of 109 ml�1, but this time con-taining 20 mM mercaptoethanol and incubated for 15

min at 37 �C. The samples were embedded in a low-

melting-point agar (L.M.) (BioRad, Richmond, VA).

The spheroplast lysis method, as described by Vollrath

and Davis [22], was used to prepare the chromosomal

DNA. The cell/agarose L.M. mixture was transferred to

plug molds. Solidified pellets were removed from the

molds and placed in 1.2 M sorbitol solution containing20 mM EDTA, 10 mM Tris–HCl, pH 7.5, 100 ll zy-moliasi 100 T (100,000 Ug�1) (1 mgml�1) (Seikagaku

Corporation, Tokyo, Japan). After a 2-h incubation at

37 �C, the plugs were incubated in 1% SDS solution

containing 10 mM EDTA, 10 mM Tris–HCl, pH 7.5,

and left overnight at 37 �C. The pellets were stored in

1% sarcosyl solution containing 100 mM EDTA, 10 mM

Tris–HCl, pH 7.5, at 4 �C.The DNA samples in agarose inserts were resolved by

contour clamped homogenous field electrophoresis

(CHEF). CHEF analysis was performed with the

CHEF-DRII apparatus (BioRad). The operating con-

ditions included three consecutive runs on each gel (14.5

by 20.5 cm, 1-cm thick 1% agarose; BioRad) containing

the agarose inserts of DNA immersed in a running

buffer (50 mM Tris–HCl, 50 mM boric acid and 1.5 mMEDTA, pH 8.2). Saccharomyces cerevisiae (BioRad) was

used as a standard. The parameters for each run were

150 V, 14 �C, 24 h with 90-s, 120-s and 180-s switches.

Gels were then stained with ethidium bromide (0.5

lgml�1; 30 min), de-stained and photographed under

ultraviolet light.

2.7. Statistics

The comparisons of the mean values were assessed by

ANOVA and Student�s t-test with Bonferroni�s correc-

tion. Nonparametric tests were used for frequency as-

sessment (v2 or Fisher method, as appropriate).

3. Results

3.1. Prevalence and distribution of Candida species

isolated from HIVþ subjects receiving HAART-PI or

HAART-NNRTI

Seventy patients were monitored for a presence of

fungus in the oral cavity for a total of 180 days follow-

up. As shown in Table 1, Candida spp. were isolatedfrom 95 (64%) and 70 (52%) samples from 30 out of 35–

27 out of 35 HAART-PI and HAART-NNRTI patients,

respectively. In both subject groups, C. albicans was

Table 1

Prevalence and distribution of yeast species isolated from HIV-positive subjects receiving HAART-PI or HAART-NNRTI

Treatment Subjects no. Positive no. Samples no. Positive no. No. Isolates species

HAART-PI 35 30 150 95 86 C. albicans�

4 C. glabrata

1 C. tropicalis��

1 C.guilliermondii

1 C. krusei

1 Saccharomyces spp.

1 C. famata

HAART-NNRTI 35 27 135 70 54 C. albicans

7 C. tropicalis

1 C. lipolytica

1 C. guilliermondii

5 C. famata

2 C. kefyr

* Significantly more prevalent in HAART-PI patients.** Significantly more prevalent in HAART-NNRTI patients.

P < 0:05v2 two tailed; P < 0:05 Fisher�s exact test.

30 F. De Bernardis et al. / FEMS Immunology and Medical Microbiology 41 (2004) 27–34

largely the most prevalent species, however, it was more

frequently isolated from samples of subjects under

HAART-PI (P < 0:05, two-tailed). The second most

frequent isolate was C. tropicalis (Table 1). Overall, thetwo Sap-producing species (C. albicans and C. tropicalis)

accounted for about 90% of all Candida spp. isolates in

our patients.

3.2. Sap secretion

All isolates of C. albicans and C. tropicalis were

qualitatively confirmed to be proteolytic on BSA-agar[4,19]. This essentially qualitative test did not reveal

gross differences in BSA degradation between the iso-

lates from HAART-PI and those from HAART-

NNRTI subjects (data not shown). All isolates had se-

creted the Sap antigen in the culture medium (tested

only for C. albicans). By this latter quantitative method,

however, a clear trend of increasing gradient of Sap

production among the sequential isolates from the samepatient under treatment with HAART-PI was noticed.

In some subjects, the increased Sap production ability

was already apparent with the initial isolates, in partic-

ular at 7 or 14 days from the initiation of therapy. At the

end of follow-up (day 180), Sap production by the iso-

lates from HAART-PI subjects was significantly higher

than Sap production by the initial isolates [351� 202

versus 100� 66, P < 0:05]. This trend was common inall the subjects examined, which included all whose

isolates were also examined for karyotype (see below)

but not those with a positive Candida isolation (Table 2).

In contrast, the increase in Sap production was not so

evident in HAART-NNRTI subjects, since the consec-

utive isolates of half of these did not show an increase

and in all others the increase was not very significant as

in the HAART-PI subjects (Table 2). Overall, therewas no significant statistical difference between Sap

production of the isolates on day 0 and those on day 90

or 180 for this category of subjects.

The amount of Sap in the saliva of each subject of

both groups was redetermined see also [15]. On day 0,data confirmed that Sap was present in the saliva of all

subjects from whom C. albicans was isolated, and that

quantity significantly declined becoming undetectable in

HAART-PI subjects from the second week of treatment

(as average, from >300 ngml�1 at day 0–100 ngml�1 on

day 14 and <20 ngml�1 on day 30, respectively). In

contrast, the amount of proteinase was not markedly

changed in the saliva samples taken from the patientswith HAART-NNRTI therapy (data not shown, [22]).

3.3. Strain biotyping

The data above demonstrated a preserved carriage of

the fungus in the oral cavity and, in the absence of the

disease [22], a decreased amount of saliva Sap in the

HAART-PI subjects. Interestingly, for these subjects, C.albicans isolates tended to produce increased Sap

quantities in vitro. One possible explanation of the latter

data could be intra therapy replacement of the initial

strain of C. albicans by other strains with increased

potential for Sap production. To obtain an insight into

this aspect, we analyzed the electrophoretic karyotype of

C. albicans isolates from HAART-PI subjects which

showed the best definite trend to increased Sap pro-duction (37 isolates from 11 subjects). As expected, the

complete chromosomal pattern of C. albicans isolates

was unique for each initial (day 0) isolate, consisting of

7–9 distinct bands ranging from more than 3.0 to less

than 1.0 Mbp. Nevertheless, in all but two patients (both

belonging to the HAART-NNRTI) all sequential iso-

lates showed the same electrophoretic karyotype sug-

gesting there was persistent oral colonization by thesame initial strain. As an example, Fig. 1(a) and (b)

Table

2

Sapamounta

producedin

vitro

bytheconsecutiveisolatesfrom

inHIV

-positivesubject

receivingPI-orNNRTI-HAART

HAART-PIondays

HAART-N

NRTIondays

Patientno.

07

14

30

60

180

Patientno.

014

30

90

180

253

–53

186

166

200

16

––

66

83

80

5180

346

346

––

466

17

166

170

170

170

–

6180

180

213

520

680

653

23

80

–80

–121

753

53

83

––

–32

80

83

106

121

121

10

180

166

–166

186

246

33

–66

83

83

–

18

46

146

––

––

46

106

–168

––

26

17

83

83

93

83

193

67

66

66

61

66

–

35

83

66

83

83

160

–69

80

106

106

206

–

53

61

66

173

166

173

–86

160

180

170

––

60

66

66

146

146

146

––

––

––

–

75

––

293

333

––

––

––

––

78

186

266

––

––

––

––

––

Mean�SD

100.4�66.2

143.8�98.7

163�102

211�146

227�202

351�202b

105�41

111�51

112�45

118�58

107�22

aIn

ngml�

1byELISA.

bP<

0:05,byStudent�s

t-test.

Fig. 1. (a) Ethidium bromide-stained agarose gels showing karyotypes

of C. albicans generated by pulsed-field gel electrophoresis (PFGE).

Lanes 1–5 are the karyotypes of five sequential isolates from patient

no. 6, while lanes 6–9 are four sequential isolates from patient no. 60.

Both patients were treated with PI-HAART. Lane M, marker (S. ce-

revisiae chromosome size standard, fragment sizes in kilobases). (b)

Ethidium bromide-stained agarose gels showing karyotypes of C. al-

bicans generated by pulsed-field gel electrophoresis (PFGE). Lanes 1–5

are the karyotypes of five sequential isolates from patient no. 10. The

patient was treated with HAART-PI. Lane M, marker (S. cerevisiae

chromosome size standard, fragment sizes in kilobases).

F. De Bernardis et al. / FEMS Immunology and Medical Microbiology 41 (2004) 27–34 31

shows the karyotype of sequential isolates from three

patients under HAART-PI, while Fig. 2 shows the

electrophoretic karyotype of sequential isolates from

three subjects under HAART-NNRTI.

Overall, these findings strongly support the notion

that biotype replacement was not the reason for in-

creased Sap production by the isolates of C. albicans

during HAART-PI.

3.4. Anti-mycotic susceptibility

We also tested for any possible variation in the sus-

ceptibility to triazole anti-mycotic agents for the con-

secutive isolates of C. albicans during HAART, also in

view of previous reports of an association between

Fig. 2. Ethidium bromide-stained agarose gels showing karyotypes of

C. albicans generated by pulsed-field gel electrophoresis (PFGE).

Lanes 1–4 are the karyotypes of four sequential isolates from patient

no. 32, lanes 5 and 6 are two sequential isolates from patient no. 23,

while lanes 7 and 8 are two sequential isolates from patient no. 46. All

patients were treated with HAART-NNRTI. Lane M, marker (S. ce-

revisiae chromosome size standard, fragment sizes in kilobases).

32 F. De Bernardis et al. / FEMS Immunology and Medical Microbiology 41 (2004) 27–34

increased Sap production and refractoriness to some

anti-mycotics as for instance, fluconazole [17].All the 85 isolates of C. albicans from the 35 patients

receiving HAART-PI were tested. They were all found

susceptible to fluconazole (MIC range 0.125–32 lgml�1

and itraconazole (MIC range 0.078–0.5 lgml�1). In

particular, no evidence of susceptibility changes in

consecutive fungus isolates were found, and no relation

was either found between MIC level and Sap production

in vitro (data not shown).

4. Discussion

We have previously demonstrated that HIV protein-

ase inhibitors (PIs) exert an early, immune reconstitu-

tion-independent clinical benefit against oral candidiasis

in HIV-positive subjects. This effect is mediated by theinhibition of C. albicans secretory aspartyl proteinase

(Sap), a family of a critical virulence factor for mucosal

candidiasis [4,6,7,12,14,23,24]. From these initial ob-

servations, a wealth of data has been generated con-

firming and extending these PI unexpected effects on

other agents of opportunistic infections [16], also pro-

voking the active search of anti-fungal PI [25]. The anti-

candidal benefit of HAART-PI was entirely due to PIhas recently been ascertained by the comparison with a

controlled group of subjects under HAART with PI-

sparing regimens [15].

We have now extended these studies in the same co-

horts of previously examined HIV-positive subjects [15]

with the specific aim of examining whether HAART-PIfavored the elimination of the fungus from the oral

cavity or it was substituted with less virulent specie or

strain. Sap appears to play a role in fungus adherence to

epithelial cells [26], an effect which should in turn create

a serious obstacle to Candida colonization. Moreover,

we speculated whether the therapy caused variations in

the anti-mycotic susceptibility of the oral fungus iso-

lates, which were shown by others to be affected in theirsusceptibility to some anti-fungals from an elevated Sap

production [17,27,28].

In order to solve these questions, we identified all

sequential oral yeast isolates during the whole follow-up

period of 180 days and determined their capacity to

produce Sap in vitro. We also determined their suscep-

tibility to some of the most common anti-fungal agents,

such as fluconazole and itraconazole, from which es-tablished standards of susceptibility testing exist [21].

Finally we re-determined the amount of oral Sap during

the whole follow-up period to confirm the clear-cut

difference of this parameter for subjects under PI- and

those under HAART-NNRTI [15,18].

We isolated Candida spp. from the oral cavity of the

large majority (from 80% to 90%) of all HAART-treated

subjects, irrespective of treatment regimen. Althoughthere were differences in the prevalence of Candida spp.

between the two groups of patients, the two Sap-

producing species (C. albicans and C. tropicalis) ac-

counted overall for more than 90% of all isolates and

were consistently isolated throughout the follow-up

period. More importantly, without exception, all se-

quential isolates from each positive subject under HA-

ART-PI had identical electrophoretic karyotype andidentical susceptibility to fluconazole and itraconazole.

Conversely, the data clearly confirmed, from the early

initiation of HAART-PI, but not HAART-NNRTI,

that no Sap could be detected in the oral cavity.

Consequently this data strongly suggests that PI may

have a quantitative yet certainly not a qualitative effect

on colonization of the oral cavity. It is also clear that PI

do not induce a replacement of strains incapable ofcausing disease or more susceptible to triazole anti-

mycotics. Although not ruling out completely the pres-

ence of subtle genetic changes in the sequential strains,

the electrophoretic karyotype is rather generally taken

as a method which can reliably define Candida biotype

[29]. (In fact, only two isolates showed an apparent

strain replacement: both belonged to NNRTI-group and

did not show significant increase in Sap production.This is in keeping with an observation made by Taylor

et al. [29] who showed that C. albicans isolates from the

oral cavity of HIV-positive patients under HAART

F. De Bernardis et al. / FEMS Immunology and Medical Microbiology 41 (2004) 27–34 33

regimen had no decrease in virulence, compared to ref-

erence strains, for experimental mucosal infection. Un-

fortunately however, Sap quantities or activity by these

isolates were not determined in the above study [29].

Besides host immunereconstitution, the beneficialclinical effect of PI appears to be entirely due to their

capacity to inhibit Sap production in vivo, as they po-

tently do with in vitro [12,23,24]. The above interpre-

tation appears still more plausible if we consider an

unexpected and intriguing finding of this investigation.

That is, the fact that the sequential C. albicans isolates

from HAART-PI subjects we examined do not only

produce Sap in vitro but have a clear trend to producemore Sap in vitro as long as the subject is treated. At the

end of treatment, the average Sap production by the

isolates on day 180 (351� 87 ngml�1; means� SE) was

significantly higher than Sap production first day (day 0)

isolates (100� 21 ngml�1; means� SE). Although this

did not occur in the isolates from HAART-NNRTI

subjects, it is unlikely that PI are the indirect cause of

this phenomenon. In several studies it was shown thatoral and vaginal isolates of C. albicans from HIV-posi-

tive subjects to be higher Sap producers in vitro than the

isolates of HIV-negative subjects, in the pre-HAART

era [4–6].

It is not clear why the same strain or biotype of C.

albicans could be induced to produce more Sap in vitro

when isolated from subjects undergoing treatment with

PI, in a situation where Sap production is inhibited invivo. In the pre-HAART era, this phenomenon has been

attributed to Sap induction by HIV envelope proteins

[30], a fact that seems to be unlikely with a therapy

causing rapid loss of viremia such as the present-day

HAART. Nor can it be attributed to anti-mycotic usage

before entering HAART and acquired resistance to

them, since, as shown here, all isolates were susceptible

to both fluconazole and itraconazole. It is possible thatduring oral colonization and persistence, the strain of

Candida is induced in vivo. In the presence of PI, it alters

the type of secreted Sap, which could be more reactive

with our anti-serum in the ELISA test. For this, we used

a polyclonal reagent raised against a purified Sap2

preparation. Because of the strong homologies within

Sap family, it is expected that a polyclonal reagent

would contain antibody specificity against the majorityof Saps, but clearly not to the same extent or reactivity.

If so, our data anticipate that during therapy with PI C.

albicans could change the type of secreted Sap. Most of

all, the type of Sap produced on BSA medium in vitro

may be different from that (or those) secreted in the oral

cavity [7]. At any rate, this change should not impact on

the clinical benefit of PI against oral candidosis which is

evident in all subjects regardless Sap production abilityin vitro by the fungus isolate.

There have been interesting reports suggesting a

correlation between high Sap production and decreased

susceptibility to some azoles by C. albicans isolates from

HIV-positive subjects, in the pre-HAART era [5,17]. We

could not confirm these reports as there was no sus-

ceptibility decrease to both anti-fungal during either or

HAART-PI or HAART-NNRTI. Importantly, the iso-lates on day 0 were, as a whole, all susceptible to

fluconazole and itraconazole, in contrast to the isolates

of C. albicans studied by Ollert et al. [5] which were

resistant to these drugs. It appears that a population of

triazole-susceptible isolates, although persisting in the

oral cavity and gaining some increase in Sap production,

is not going to change its pattern of drug susceptibility

during 180 days of PI-HAART.

Acknowledgements

We are grateful to Daniela Adriani and Antonietta

Girolamo for their technical assistance. Thanks are ex-

tended to Dr. Giusi Mandarino for helping in the

preparation of the manuscript.This work was supported in part by National AIDS

Research Program under ISS contract 50E.

References

[1] Holmberg, K. and Meyer, R.D. (1986) Fungal infections in

patients with AIDS and AIDS-related complex. Scand. J. Infect.

Dis. 18, 179–185.

[2] Klein, R.S., Harris, C.A., Butkus Small, C., Moll, B., Lesser, M.

and Friendland, G.H. (1984) Oral candidiasis in high risk patients

as the initial manifestation of the acquired immunodeficiency

syndrome. N. Engl. J. Med. 311, 354–357.

[3] Cutler, J.E. (1991) Putative virulence factors of Candida albicans.

Ann. Rev. Microbiol. 45, 187–218.

[4] De Bernardis, F., Boccanera, M., Rainaldi, L., Guerra, C.E.,

Quinti, I. and Cassone, A. (1992) The secretion of aspartyl

proteinase, a virulence enzyme, by isolates of Candida albicans

from the oral cavity of HIV-infected subjects. Eur. J. Epidemiol. 8,

362–367.

[5] Ollert, M.W., Wende, C., Gorlich, M., McMullan-Vogel, C.G.,

Borg von-Zepelin, M., Vogel, C.W. and Korting, H.C. (1995)

Increased expression of Candida albicans secretory proteinase, a

putative virulence factor in isolates from human immunodefi-

ciency virus-positive patients. J. Clin. Microbiol. 33, 2543–2549.

[6] De Bernardis, F., Mondello, F., Scaravelli, G., Pach�ı, A.,

Girolamo, A., Agatensi, L. and Cassone, A. (1999) High aspartyl

proteinase production and vaginitis in human immunodeficiency

virus-infected women. J. Clin. Microbiol. 37, 1376–1380.

[7] Naglick, J.R., Rodgers, C.A., Shirlaw, P.J., Dobbie, J.L.,

Fernandez-Naglick, l., Greenspan, D., Agabian, N. and Challa-

combe, S.J. (2003) Differential expression of Candida albicans

secreted aspartyl proteinase and phospholipase B genes in human

correlates with active oral and vaginal infections. J. Infect. Dis.

188, 469–479.

[8] Deeks, S.G., Smith, M., Holodniy, I. and Kahn, J.O. (1997) HIV-

1 protease inhibitors. A review for clinicians. JAMA 277, 145–155.

[9] Palella, F.J., Delaney, K.M., Moorman, A.C., Loveless, M.O.,

Fuhrer, J., Satten, G.A., Aschman, D.J. and Holmberg, S.D.

(1998) Declining morbidity and mortality among patients with

advanced human immunodeficiency virus infection. New Eng. J.

Med. 338, 853–860.

34 F. De Bernardis et al. / FEMS Immunology and Medical Microbiology 41 (2004) 27–34

[10] Sepkowitz, K.A. (1998) Effect of HAART on natural history of

AIDS-relate opportunistic disorders. Lancet 351, 228–230.

[11] Brodt, H.R., Kamps, B.S., Gute, P., Knupp, B., Staszewski, S.

and Helm, E.B. (1997) Change incidence of AIDS-defining illness

in the era of antiretroviral therapy. AIDS 11, 1731–1738.

[12] Cassone, A., De Bernardis, F., Torosantucci, A., Tacconelli, E.,

Tumbarello, M. and Cauda, R. (1999) In vitro and vivo

anticandidal activity of human immunodeficiency virus protease

inhibitors. J. Infect. Dis. 180, 448–453.

[13] Hube, B. (1996) Candida albicans secreted aspartyl proteinase.

Curr. Top. Med. Mycol. 7, 55–69.

[14] De Bernardis, F., Arancia, S., Morelli, L., Hube, B., Sanglard, D.

and Schaefer, W. (1999) Evidence that members of the secretory

aspartyl proteinases gene family (SAP), in particolar SAP2, are

virulence factors for Candida vaginitis. J. Infect. Dis. 179, 201–208.

[15] Cassone, A., Tacconelli, E., De Bernardis, F., Tumbarello, M.,

Torosantucci, A. and Chiani, P. (2002) Antiretroviral therapy

with protease inhibitors has an early, immune reconstitution-

independent beneficial effect on Candida virulence and oral

candidiasis in human immunodeficiency virus-infected subjects.

J. Infect. Dis. 185, 188–195.

[16] Atzori, C., Angeli, E., Mainini, A., Agostoni, F., Micheli, V. and

Cargnel, A. (2000) In vitro activity of human immunodeficiency

virus protease inhibitors against Pneumocystis carinii. J. Infect.

Dis. 181, 1629–1634.

[17] Wu, T., Wright, K., Hurst, S.F. and Morrison, C.J. (2000)

Enhanced extracellular production of aspartyl proteinase, a

virulence factor by Candida albicans isolates following growth in

subinhibitory concentration of fluconazole. Antimicrob. Agents

Chemother. 44, 1200–1208.

[18] Cauda, R., Tacconelli, E., Tumbarello, M., Morace, G., De

Bernardis, F., Torosantucci, A. and Cassone, A. (1999) Role of

protease inhibitors in preventing recurrent oral candidosis in

patients with HIV infection: a prospective case-control study.

J. Acquir. Immun. Defic. Syndr. Hum. Retrovirol. 21, 20–25.

[19] De Bernardis, F., Agatensi, L., Ross, I.K., Emerson, G.W.,

Lorenzini, R., Sullivan, P.A. and Cassone, A. (1990) Evidence for

a role for secreted aspartate proteinase of Candida albicans in

vulvovaginal candidiasis. J. Infect. Dis. 161, 1276–1283.

[20] Barchiesi, F.A., Colombo, L., McGough, D.A., Fothergill, A.W.

and Rinaldi, M.G. (1994) In vitro activity of itraconazole

against fluconazole-susceptible and -resistant Candida albicans

isolates from oral cavities of patients infected with human

immunodeficiency virus. Antimicrob. Agents Chemother. 38,

1530–1533.

[21] National Committee for Clinical Laboratory Standard (1997).

Reference method for broth dilution antifungal susceptibility

testing of yeasts; Approved Standard M27-A, NCCLS, Wayne,

PA.

[22] Vollrath, D. and Davis, R.W. (1987) Resolution of DNA

molecules greater than 5 megabases by contour clamped homog-

enous electric fields. Nucleic Acids Res. 15, 7865–7876.

[23] Korting, H.C., Schaller, M., Eder, G., Hamm, G., Bohmer, U.

and Hube, B. (1999) Effects of the human immunodeficiency virus

(HIV) proteinase inhibitors saquinavir and indinavir on in vitro

activities of secreted aspartyl proteinases of Candida albicans

isolates from HIV-infected patients. Antimicrob. Agents Che-

moth. Aug., 2038–2042.

[24] Munro, C.A. and Hube, B. (2002) Antifungal therapy at the

HAART of viral therapy. Trends Microb. Opin. 10, 173–177.

[25] Skrbec, D. and Romeo, D. (2002) Inhibition of Candida albicans

secreted aspartic protease by a novel series of peptidomimetics,

also active on the HIV-1 protease. Biochem. Biophys. Res.

Commun. 297, 1350–1353.

[26] Bektic, J., Lell, C.P., Fuchs, A., Stoiber, H., Speth, C., Lass-Florl,

C., Borg-von Zepelin, M., Dierich, M.P. and Wurzner, R. (2001)

HIV protease inhibitors attenuate adherence of Candida albicans

to epithelial cells in vitro. FEMS, Immun. Med. Microb. 31,

65–71.

[27] Schaller, M., Krnjac, N., Niewerth, M., Hamm, G., Hube, B. and

Korting, H. (2003) Effect of antimycotic agents on the activity of

aspartyl proteinases secreted by Candida albicans. J. Med. Microb.

52, 247–249.

[28] Stewart, K. and Abad-Zapatero, C. (2001) Candida proteases and

their inhibition: prospects for antifungal therapy. Curr. Med.

Chem. 8, 941–948.

[29] Taylor, B.N., Fischenbaum, C., Saavedra, M., Slaviinsky III, J.,

Swoboda, R., Wozniak, K., Arribas, A., Powderly, W. and Fidel

Jr., P.L. (2000) In vivo virulence of Candida albicans isolates

causing mucosal infections in people infected with the human

immunodeficiency virus. J. Inf. Dis. 182, 955–959.

[30] Gruber, A., Speth, C., Lukasser-Vogl, E., Zangerle, R., Borg-von

Zepelin, M., Dierich, M.P. and Wurzner, R. (1999) Human

immunodeficiency virus type 1 protease inhibitor attenuates

Candida albicans virulence properties in vitro. Immunopharma-

cology 41, 227–234.