archives of oral biology 59 (2014) 775–782
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Salivary lactoferrin in HIV-infected children: Correlation with Candida albicans carriage, oral manifestations, HIV infection and its antifungal activity Thais Pinto Alves a, Ana Clara Duarte Correa Simo˜es a, Rosangela Maria de Arau´jo Soares b, Daniela Sales Alviano Moreno b, Maristela Barbosa Portela c,*, Gloria Fernanda Barbosa de Arau´jo Castro a a Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil b Department of General Microbiology, Instituto de Microbiologia Professor Paulo de Go´es, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil c Department of Clinics and Pediatric Dentistry, School of Dentistry, Universidade Federal Fluminense/Nitero´i, Rio de Janeiro, Brazil
article info
abstract
Article history:
Objectives: This study aimed to evaluate the concentration of lactoferrin in the saliva of HIV
Accepted 14 April 2014
infected and healthy children and analyze the associations between lactoferrin levels, Candida sp. colonization, oral manifestations and medical data. Also, the antifungal ability
Keywords:
of lactoferrin to inhibit the growth of Candida albicans isolated from saliva of these children
Salivary antimicrobial protein
was investigated in vitro.
Lactoferrin
Subjects and methods: Saliva was collected from 70 HIV-infected and 50 healthy children,
HIV-infected children
followed by oral manifestation assessments. The salivary lactoferrin was quantified using
Fungal colonization
an ELISA Kit1. The salivary samples were cultured and the Candida spp. colonies counted
Candida albicans
and then identified by sugar assimilation and fermentation. The antifungal activity of lactoferrin was analyzed in vitro with 10 isolates of C. albicans from each group. Results: The HIV infected children (mean age 9.8 2.8) had higher lactoferrin levels (median 6.13 mg/ml (3.58–7.89)) and were colonized three times more by Candida sp. than the control group (mean age 9.4 2.4) (median 5.74 mg/ml (3.12–6.86)) ( p = 0.003). Statistical associations were found considering the salivary lactoferrin levels and Candida sp. and oral manifestations between the groups. No associations between lactoferrin concentrations and oral manifestations, immunosuppression, presence of AIDS and use of HAART were observed in the HIV group. The percentage of dead C. albicans due to lactoferrin was inversely proportional to C. albicans cell density for both groups ( p < 0.001). Conclusions: HIV-infected children have higher concentrations of lactoferrin and it was associated with Candida sp. colonization but no association with medical data was found. Also, both groups showed similar lactoferrin antifungal activity. # 2014 Published by Elsevier Ltd.
* Corresponding author. Tel.: +55 21 25622098/+55 21 31061231; fax: +55 21 25622098. E-mail address: [email protected] (M.B. Portela). http://dx.doi.org/10.1016/j.archoralbio.2014.04.010 0003–9969/# 2014 Published by Elsevier Ltd.
776
1.
archives of oral biology 59 (2014) 775–782
Introduction
The oral cavity is a unique environment and a primary target for opportunistic infections in HIV-infected children, particularly oral candidiasis, mainly caused by the pathogen Candida albicans.1 In fact, more than 72% of HIV-infected children develop oral candidiasis during the course of their illness2,3 and this infection is considered an important marker of the HIV disease and its progression3,4 because its prevalence is correlated to the severity of the immunological dysfunction.2 Saliva plays an important role in physical–chemical barriers as well as the immune defence of the oral mucosal surfaces,5 and contains a complex set of antimicrobial proteins including lactoferrin – an iron – binding cationic glycoprotein of 80 kDa.6 This protein has been shown to have a number of biological functions, including antimicrobial and immunomodulatory effects in vitro and in vivo7. It also appears to differentially modulate the carriage of various Candida species in the oral cavity.8 The multiple fungistatic mechanisms of lactoferrin make this protein a promising compound for combination therapy, and lactoferrin is itself very important for pediatric patients especially immunosuppresed patients, such as HIV Infected children.5,7 Although the properties of salivary lactoferrin have been thoroughly investigated7 there is a lack of studies into the association between lactoferrin salivary levels and HIV infected children. Hence the main aim of this study was to evaluate the concentration of lactoferrin in the saliva of HIV infected and healthy children, and analyze the associations between lactoferrin levels, Candida sp. colonization, and medical data. Also, the antifungal ability of lactoferrin to inhibit the growth of C. albicans isolated from saliva of these HIV infected children was investigated in vitro.
2.
Material and methods
2.1.
Subjects
This study was a cross sectional, descriptive and analytical study. There were a total of 120 subjects in this study, 70 HIVinfected children (HIV group) and 50 healthy children (control group), of both genders, aged between 3 and 13 years old, selected by convenience over a 12-month period. The HIV group was selected from a group of HIV-infected children attending the Pediatric AIDS Outpatient Clinic at the Universidade Federal do Rio de Janeiro (Brazil) and followed up at the Pediatric Dentistry Department of the same institution. The HIV-infected children had a definitive diagnosis for HIV infection according to criteria established by the Centers for Disease Control and Prevention.9 The control group was made up of patients from the Pediatric Dentistry Department who were clinically healthy, without any signs of immunosuppression and no history of any systemic disease. Children who were or had been under antifungal therapy in the last three months and those who were using oropharyngeal antimicrobial drugs were excluded from this study. The following data were collected from the children’s medical records: personal information and medical data that included: diagnosis of
AIDS, antiretroviral therapy and the use of antifungal or any other medication; and results from laboratory tests (CD4 and CD8 count and viral load) (the closest ones to sample collection). We considered the presence of AIDS when a patient had a CD4 count of 50) Notes: NS = not significant. Mann–Whitney test. b Qui-square test. a
HIV group (N = 70)
Control group (N = 50)
p Value
6.13 (3.58–7.89)
5.74 (3.12–6.86)
0.0002a
43 (61.4%) 27 (38.6%) 4.50 (0–1880) HIV group (N = 43)
17 (34%) 33 (66%) 0 (0–584) Control group (N = 17)
0.0003b
34 (48.6%) 5 (17.1%) 8 (11.14%) 1 (1.4%) 1 (1.4%) 1 (1.4%)
15 (30%) 8 (16%) 0 0 0 0
0.04b NSb –
11 (25.6%) 16 (37.2%) 16 (37.2%)
5 (29.4%) 7 (41.2%) 5 (29.4%)
NSb
0.003a p Value
779
archives of oral biology 59 (2014) 775–782
Table 3 – Association between lactoferrin concentration and Candida spp., oral manifestation and medical data of HIVinfected children (HIV group) and health children (Control group). Lactoferrin concentration
Candida sp. Candida sp. colonization: Yes No Candida sp. growth classification*: Light (10) Moderate/strong(>10) Oral manifestations Actual presence**: Yes No Candidiasis Yes No Caries disease Presence of caries Yes No
HIV group (N = 70)
Control group (N = 50)
p Value
6.12 (3.58–7.89) a 6.15 (4.68–7,77) a
5.77 (3.48–6,51) b 5.50 (3.12–6.86) b
0.05 0.002
6.02 (3.58–7.26) c 6.13 (4.94–7.88) c
6.03 (5.27–6.24) d 5.77 (3.48–6.51) d
NS 0.05
6.20 (3.58–7.88) e 6.01 (4.76–7.73) e
5.74 (3.48–6.49) f 5.75 (3.126.86) f
0.008 0.02
6.19 (3.58–7.88) g 6.06 (4.68– 7.88) g
6.16 (5.19–6.51) h 5.63 (3.12–6.86) h
NS 0.004
5.97 (5.45–6.76) i 6.28 (3.58– 7.88) i HIV group (N = 70)
5.74 (3.12–6.86)
Medical data Immunological classification (CD4%): Absent (>25%) Moderate (15–24%) Severe ( 10–102 colonies CFU/ml. ** Oral manifestations include the presence of one or more oral manifestations such as: Oral candidiasis, Herpes simplex lesions, linear gingival erythema and Parotid enlargement (CDC Criteria).9
In order to determine the anticandidal potency of lactoferrin the death percentage of C. albicans by lactoferrin was determined. The results of the killing assays demonstrated that the death percentage of C. albicans by lactoferrin was inversely proportional to its cell density for the HIV group (r = 0.79, p < 0.001) as well as for the control group (r = 0.94, p < 0.001). Both groups showed similar antifungal activity of lactoferrin but the concentration used (100 ml/ml) did not reach 100% cell death (Fig. 1). Lactoferrin was able to kill various clinical isolates of C. albicans at concentrations less than 1 106 cell/ml, in both groups.
4.
Discussion
Salivary anti-microbial proteins are considered to be an important part of the non-immune host defence system in preventing colonization and infection of the oral cavity by oral microorganisms. These protective effects of saliva are evidenced by the microbial overgrowth found in patients with salivary deficiencies.15 Earlier studies have already shown that intrinsic components of the host defence system
such as histatins, cystatins, immunoglobulins and several saliva proteins, like lysozyme and lactoferrin are capable of killing various Candida species.16,17 We focused our attention on lactoferrin as this protein has demonstrated its ability to inhibit the growth of several bacteria and Candida species, which is the etiological agent of opportunistic infections with predictive values for HIV infection.18 To the best of our knowledge, the current study is the first to investigate the relationship between the lactoferrin levels and Candida sp. colonization of HIV-infected children, as well as the first in vitro study to determine of the capability of lactoferrin to inhibit the growth of C. albicans isolated from the saliva of the same HIV patients. The concentration of salivary lactoferrin in unstimulated parotid saliva in the adult population varies from 7 to 20 mg/ ml19,20 but decreases upon stimulation. This wide range occurs due to the individual characteristics of the groups and methods used with no significant differences in levels in whole and parotid saliva. Van Der Strate et al.21 determined that the lactoferrin concentration in saliva was in the range of 10 mg/ml in HIV-infected adults and it was not significantly different in healthy individuals. The values we found in
780
archives of oral biology 59 (2014) 775–782
Table 4 – Personal and medical data from the 10 HIVinfected children and the 10 health children whose isolates were used in the Lactoferrin killing assay experiment.
Personal data Age (years) Medical data Presence of AIDSa
Oral manifestations Actual presenceb: Yes No Candidiasis Yes No Caries disease Presence of caries Yes No Mean dmft Mean DMFT
HIV group (N = 10)
Control group (N = 10)
9.11 2.31 HIV group (N = 10)
8.90 2.60
Yes—1 (10%) No—9 (90%) HIV group (N = 10)
Control group (N = 10)
4 (40%) 6 (60%)
2 (20%) 8 (80%)
3 (30%) 7 (70%)
1 (10%) 9 (90%)
0 10 (100%) 0.2 0.42 1.4 1.26
0 10 (100%) 1.6 1.17 0.4 0.51
a
CD4 < 15%. Oral manifestations include the presence of one or more oral manifestations as: oral candidiasis, Herpes simplex lesions, linear gingival erythema and Parotid enlargement (CDC criteria9).
b
children were lower than those cited for adults and the average lactoferrin concentration we detected was significantly higher in HIV-infected children compared with healthy children. There are no values considered normal for lactoferrin levels in children, so there is no cut-off-point or normal range. Moreover, to date, no studies on quantification of lactoferrin in children have been found in the literature, thus we are not able to make any comparisons with our results. Alterations in saliva secretion and composition during infection by HIV may alter the defence mechanisms of the host, and influence adherence and colonization by Candida.22 There are reports that indicate alterations in major salivary
gland functions following HIV-infection with effects on both the salivary composition and output (flow rate), which could influence lactoferrin levels.23 The children in our study had no difficulty during the saliva collection and the subjects with salivary flow less than 0.5 ml/min were not included in our sample. Evidence has shown that increased salivary lactoferrin production is most probably a response to the elevated stimulus provided by the HIV-infection and by the opportunistic pathogens that accompany the progression of the disease.2 Although this suggests that the secretory immune system was responding directly to the challenge presented by the Candida colonization, the association between salivary anti-microbial proteins and oral candidiasis remains controversial.2,24 Our microbiological analyses demonstrated a significantly higher prevalence of Candida colonization in HIV-infected children compared with the controls, however no association was found between lactoferrin levels and fungal colonization within the HIV group showing that colonized or not by Candida, in this group, did not influence the levels of lactoferrin, even when there were high amounts of Candida (Moderate/Strong growth). Lourenc¸o et al.25 related no correlation between the concentration of salivary lactoferrin and the quantification of Candida sp. and also between the lactoferrin concentration and systemic condition of HIV-infected adults, corroborating our results. In addition, Van Der Strate et al.21 observed that, in a group of 15 HIV-infected subjects, the titres of Candida present in the oral cavity were unaffected by salivary lactoferrin concentration. Drawing a parallel with studies that observed other salivary proteins, Pomarico et al.2 reported positive correlations between salivary IgA levels and Candida sp. isolated from saliva of HIV-infected children. Also, Jainkittivong et al.26 demonstrated that the levels of mucin concentration were higher in the HIV-infected subjects than in the controls. In light of the pivotal role of CD4+ cells in the maturation of the mucosal immune system, and the fact that HIV-infection leads to a decrease in the numbers of such cells, secretory immunity, including salivary proteins, would be expected to be compromised with the progression of HIV-infection.24 Therefore, similar to our study, HIV-infected children appear
Fig. 1 – Killing assay of lactoferrin (100 mg/ml) against C. albicans isolated from HIV-infected children (HIV-group) and health children (Control group).
archives of oral biology 59 (2014) 775–782
to be able to maintain their capacity to mount a mucosal immune response to opportunistic microorganisms.24,27 It is interesting to note that our levels of lactoferrin were always higher in HIV infected children except when the number of Candida CFU was low in these children, and also when the healthy patients had oral candidiasis. This observation further supports the hypothesis that the production of lactoferrin by the immune system and its presence in saliva is directly relate to a compensatory response in both groups. It would seem that, in general, HIV-infected children maintain an intact immune response capacity to a number of antigens presented to the mucosal immune system. As already suggested by Castro et al.,24 it is possible that the level of CD4+ helper activity, although reduced, is still sufficient to induce normal mucosal responses to oral microorganisms in HIV-infected individuals, at least until the later stages of disease. According to the literature, lactoferrin has an antifungal capacity but there is no consensus with regard to its inhibitory potency in terms of cell density and lactoferrin concentration against Candida species.19,21 We showed that lactoferrin was able to kill over 50% of the cell growth of C. albicans at concentrations less than 1 106 cell/ml with 100 mg/ml of lactoferrin in both groups. Also, the death percentage of C. albicans by lactoferrin was inversely proportional to its cell density in both groups The antifungal capacity of salivary proteins was reported by Miller et al.,14 when they studied the antifungal activity of the Histatin-5; however it is known that this potential is not related to one single phenomenon.28 At the same time, the action mechanism of our fungal isolates has not yet been investigated and is therefore a limitation of our study. In addition, the varying experimental conditions, such as differences in medium composition, pH, incubation temperature, incubation time, and end point criteria, as well as the variable use of human versus bovine lactoferrin and of iron-free lactoferrin (apo-lactoferrin) versus iron-containing lactoferrin, makes it difficult to compare the results of the present study with those reported earlier.29 A number of researchers have reported that Candida species from HIV-infected patients with recurrent episodes of oral thrush are significantly less genetically diverse than commensal strains from healthy individuals.30,31 This implies that strains derived from the same parental stock may persist through recurrent infections in these immunocompromised patients. Others have reported similar findings on comparing serotypes from immunologically compromised patients and healthy individuals,32 thus corroborating with our study where both groups showed similar antifungal activity of lactoferrin despite not attaining 100% cell death. Lin et al.33 reported that salivary yeast carriage rates and concentration were comparable in HIV-infected and healthy individuals and that the salivary anticandidal activities were not altered. Further studies into the association between lactoferrin levels and oral fungal colonization are ongoing in our laboratories. Recognizing the various factors and conditions that play a role in oral candidal colonization and the progression of colonization with the infection will greatly contribute to our understanding of fungal pathogenesis and the contributing host factors. In our investigation, crucial information can have important clinical implications in the
781
identification and the design of novel therapeutic strategies aimed at the prevention and/or treatment of fungal infections in these patients. Particularly, the anticandidal property coupled with its lack of toxicity to human cells can make lactoferrin a promising therapeutic agent for the treatment or prevention of oral candidiasis in immunocompromised patients. In conclusion, our study shows that the HIV-infected children in our study had higher concentrations of lactoferrin and it was associated with Candida sp. colonization but no association with medical data was found. Also, both groups showed similar lactoferrin antifungal activity. These present data give us a tantalizing glimpse of the adaptive responses of oral Candida species to innate antimicrobial defences in saliva, such as lactoferrin in HIV-infected and healthy children. This phenomenon and results from further works can help elucidate the true role of these oral secretions related to oral Candida colonization in HIV-infected children.
Funding None.
Competing interests None declared.
Ethical approval Not required.
Acknowledgements The authors would like to thank CAPES and FAPERJ (Brazil) for the financial support.
references
1. Portela MB, Souza IP, Costa EM, Hagler AN, Soares RM, Santos AL. Differential recovery of Candida species from subgingival sites in human immunodeficiency virus positive and healthy children from Rio de Janeiro, Brazil. J Clin Microbiol 2004;42:5925–7. 2. Pomarico L, Cerqueira DF, De Araujo Soares RM, De Souza IPR, De Araujo Castro GFB, Socransky S, Haffajee A, Teles RP. Associations among the use of highly active antiretroviral therapy, oral candidiasis, oral Candida species and salivary immunoglobulin A in HIV-infected children. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;108(2):203–10. 3. Cerqueira DF, Portela MB, Pomarico L, Sares RMA, Souza IPR, Castro GF. Oral Candida colonization and its relation with predisposing factors in HIV-infected children and their uninfected siblings in Brazil: the era of highly active antiretroviral therapy. J Oral Pathol Med 2010;39:188–94. 4. Domaneschi C, Massarente DB, de Freitas RS, de Sousa Marques HH, Paula CR, Migliari DA, Antunes JL. Oral
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colonization by Candida species in AIDS pediatric patients. Oral Dis 2011;17:393–8. Fa´bia´n TK, Hermann P, Beck A, Feje´rdy P, Fa´bia´n G. Salivary defense proteins: their network and role in innate and acquired oral immunity. Int J Mol Sci 2012;13:4295–320. Wiesner J, Vilcinskas A. Antimicrobial peptides: the ancient arm of the human immune system. Virulence 2010;1:440–64. Gonza´lez-Cha´vez SA, Are´valo-Gallegos S, Rasco´n-Cruz Q. Lactoferrin structure, function and applications. Int J Antimicrob Agents 2009;33(4):301. Farnaud S, Evans RW. Lactoferrin—multifunctional protein with antimicrobial properties. Mol Immunol 2003;40:395–405. Center for Disease Control and Prevention—CDC. Revised classification system for human immunodeficiency virus infection in children less than 13 years of age. MMWR 1994;43:1–19. Shannon IL, Suddick RP, Dowd Jr FJ. Saliva: composition and secretion. Monogr Oral Sci 1974;2:1–103. Hibino K, Samaranayake LP, Hagg U, Wong RWK, Kee W. The role of salivary factors in persistent oral carriage of Candida in humans. Arch Oral Biol 2009;54:678–83. Lamey PJ, Darwazeh A, Fisher BM, Samaranayake LP, MacFarlane TW, Frier BM. Secretor status, candidal carriage and candidal infection in patients with diabetes miellitus. J Oral Pathol 1988;17:354–7. Andre´s MT, Viejo-Dı´az M, Fierro JJ. Human lactoferrin induces apoptosis-like cell death in Candida albicans: critical role of K+-channel-mediated K+ efflux. Antimicrob Agents Chemother 2008;52(11):4081–8. Meiller TF, Hube B, Schild L, Shirtliff ME, Scheper MA, Winkler R, Ton A, Jabra-Rizk MA. A novel immune evasion strategy of Candida albicans: proteolytic cleavage of a salivary antimicrobial peptide. PLoS One 2009;4(4):e50339. Back-Britto GN, Mota AJ, Vasconcellos TC, Querido SMR, Jorge AOC, Reis ASM, Balducci I, Koga-Ito CY. Frequency of Candida spp. in the oral cavity of brazilian HIV-positive patients and correlation with CD4 cell counts and viral load. Mycopathologia 2009;167:81–7. Van Nieuw Amerongen A, Bolscher JG, Veerman EC. Salivary proteins: protective and diagnostic value in cariology? Caries Res 2004;38(3):247–53. Jenssen H, Hancock RE. Antimicrobial properties of lactoferrin. Biochimie 2009;91:19–29. Kuipers ME, Beljaars L, Van Beek N, De Vries HG, Heegsma J, Van Den Berq JJ, Meijer DK, Swart PJ. Conditions influencing the in vitro antifungal activity of lactoferrin combined with antimycotics against clinical isolates of Candida. APMIS 2002;110:290–8. Rudney JD, Kajander KC, Smith QT. Correlations between human salivary levels of lysozyme, lactoferrin, salivary peroxidase and secretory immunoglobulin A with different
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Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/aob
Salivary lactoferrin in HIV-infected children: Correlation with Candida albicans carriage, oral manifestations, HIV infection and its antifungal activity Thais Pinto Alves a, Ana Clara Duarte Correa Simo˜es a, Rosangela Maria de Arau´jo Soares b, Daniela Sales Alviano Moreno b, Maristela Barbosa Portela c,*, Gloria Fernanda Barbosa de Arau´jo Castro a a Department of Pediatric Dentistry and Orthodontics, School of Dentistry, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil b Department of General Microbiology, Instituto de Microbiologia Professor Paulo de Go´es, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil c Department of Clinics and Pediatric Dentistry, School of Dentistry, Universidade Federal Fluminense/Nitero´i, Rio de Janeiro, Brazil
article info
abstract
Article history:
Objectives: This study aimed to evaluate the concentration of lactoferrin in the saliva of HIV
Accepted 14 April 2014
infected and healthy children and analyze the associations between lactoferrin levels, Candida sp. colonization, oral manifestations and medical data. Also, the antifungal ability
Keywords:
of lactoferrin to inhibit the growth of Candida albicans isolated from saliva of these children
Salivary antimicrobial protein
was investigated in vitro.
Lactoferrin
Subjects and methods: Saliva was collected from 70 HIV-infected and 50 healthy children,
HIV-infected children
followed by oral manifestation assessments. The salivary lactoferrin was quantified using
Fungal colonization
an ELISA Kit1. The salivary samples were cultured and the Candida spp. colonies counted
Candida albicans
and then identified by sugar assimilation and fermentation. The antifungal activity of lactoferrin was analyzed in vitro with 10 isolates of C. albicans from each group. Results: The HIV infected children (mean age 9.8 2.8) had higher lactoferrin levels (median 6.13 mg/ml (3.58–7.89)) and were colonized three times more by Candida sp. than the control group (mean age 9.4 2.4) (median 5.74 mg/ml (3.12–6.86)) ( p = 0.003). Statistical associations were found considering the salivary lactoferrin levels and Candida sp. and oral manifestations between the groups. No associations between lactoferrin concentrations and oral manifestations, immunosuppression, presence of AIDS and use of HAART were observed in the HIV group. The percentage of dead C. albicans due to lactoferrin was inversely proportional to C. albicans cell density for both groups ( p < 0.001). Conclusions: HIV-infected children have higher concentrations of lactoferrin and it was associated with Candida sp. colonization but no association with medical data was found. Also, both groups showed similar lactoferrin antifungal activity. # 2014 Published by Elsevier Ltd.
* Corresponding author. Tel.: +55 21 25622098/+55 21 31061231; fax: +55 21 25622098. E-mail address: [email protected] (M.B. Portela). http://dx.doi.org/10.1016/j.archoralbio.2014.04.010 0003–9969/# 2014 Published by Elsevier Ltd.
776
1.
archives of oral biology 59 (2014) 775–782
Introduction
The oral cavity is a unique environment and a primary target for opportunistic infections in HIV-infected children, particularly oral candidiasis, mainly caused by the pathogen Candida albicans.1 In fact, more than 72% of HIV-infected children develop oral candidiasis during the course of their illness2,3 and this infection is considered an important marker of the HIV disease and its progression3,4 because its prevalence is correlated to the severity of the immunological dysfunction.2 Saliva plays an important role in physical–chemical barriers as well as the immune defence of the oral mucosal surfaces,5 and contains a complex set of antimicrobial proteins including lactoferrin – an iron – binding cationic glycoprotein of 80 kDa.6 This protein has been shown to have a number of biological functions, including antimicrobial and immunomodulatory effects in vitro and in vivo7. It also appears to differentially modulate the carriage of various Candida species in the oral cavity.8 The multiple fungistatic mechanisms of lactoferrin make this protein a promising compound for combination therapy, and lactoferrin is itself very important for pediatric patients especially immunosuppresed patients, such as HIV Infected children.5,7 Although the properties of salivary lactoferrin have been thoroughly investigated7 there is a lack of studies into the association between lactoferrin salivary levels and HIV infected children. Hence the main aim of this study was to evaluate the concentration of lactoferrin in the saliva of HIV infected and healthy children, and analyze the associations between lactoferrin levels, Candida sp. colonization, and medical data. Also, the antifungal ability of lactoferrin to inhibit the growth of C. albicans isolated from saliva of these HIV infected children was investigated in vitro.
2.
Material and methods
2.1.
Subjects
This study was a cross sectional, descriptive and analytical study. There were a total of 120 subjects in this study, 70 HIVinfected children (HIV group) and 50 healthy children (control group), of both genders, aged between 3 and 13 years old, selected by convenience over a 12-month period. The HIV group was selected from a group of HIV-infected children attending the Pediatric AIDS Outpatient Clinic at the Universidade Federal do Rio de Janeiro (Brazil) and followed up at the Pediatric Dentistry Department of the same institution. The HIV-infected children had a definitive diagnosis for HIV infection according to criteria established by the Centers for Disease Control and Prevention.9 The control group was made up of patients from the Pediatric Dentistry Department who were clinically healthy, without any signs of immunosuppression and no history of any systemic disease. Children who were or had been under antifungal therapy in the last three months and those who were using oropharyngeal antimicrobial drugs were excluded from this study. The following data were collected from the children’s medical records: personal information and medical data that included: diagnosis of
AIDS, antiretroviral therapy and the use of antifungal or any other medication; and results from laboratory tests (CD4 and CD8 count and viral load) (the closest ones to sample collection). We considered the presence of AIDS when a patient had a CD4 count of 50) Notes: NS = not significant. Mann–Whitney test. b Qui-square test. a
HIV group (N = 70)
Control group (N = 50)
p Value
6.13 (3.58–7.89)
5.74 (3.12–6.86)
0.0002a
43 (61.4%) 27 (38.6%) 4.50 (0–1880) HIV group (N = 43)
17 (34%) 33 (66%) 0 (0–584) Control group (N = 17)
0.0003b
34 (48.6%) 5 (17.1%) 8 (11.14%) 1 (1.4%) 1 (1.4%) 1 (1.4%)
15 (30%) 8 (16%) 0 0 0 0
0.04b NSb –
11 (25.6%) 16 (37.2%) 16 (37.2%)
5 (29.4%) 7 (41.2%) 5 (29.4%)
NSb
0.003a p Value
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archives of oral biology 59 (2014) 775–782
Table 3 – Association between lactoferrin concentration and Candida spp., oral manifestation and medical data of HIVinfected children (HIV group) and health children (Control group). Lactoferrin concentration
Candida sp. Candida sp. colonization: Yes No Candida sp. growth classification*: Light (10) Moderate/strong(>10) Oral manifestations Actual presence**: Yes No Candidiasis Yes No Caries disease Presence of caries Yes No
HIV group (N = 70)
Control group (N = 50)
p Value
6.12 (3.58–7.89) a 6.15 (4.68–7,77) a
5.77 (3.48–6,51) b 5.50 (3.12–6.86) b
0.05 0.002
6.02 (3.58–7.26) c 6.13 (4.94–7.88) c
6.03 (5.27–6.24) d 5.77 (3.48–6.51) d
NS 0.05
6.20 (3.58–7.88) e 6.01 (4.76–7.73) e
5.74 (3.48–6.49) f 5.75 (3.126.86) f
0.008 0.02
6.19 (3.58–7.88) g 6.06 (4.68– 7.88) g
6.16 (5.19–6.51) h 5.63 (3.12–6.86) h
NS 0.004
5.97 (5.45–6.76) i 6.28 (3.58– 7.88) i HIV group (N = 70)
5.74 (3.12–6.86)
Medical data Immunological classification (CD4%): Absent (>25%) Moderate (15–24%) Severe ( 10–102 colonies CFU/ml. ** Oral manifestations include the presence of one or more oral manifestations such as: Oral candidiasis, Herpes simplex lesions, linear gingival erythema and Parotid enlargement (CDC Criteria).9
In order to determine the anticandidal potency of lactoferrin the death percentage of C. albicans by lactoferrin was determined. The results of the killing assays demonstrated that the death percentage of C. albicans by lactoferrin was inversely proportional to its cell density for the HIV group (r = 0.79, p < 0.001) as well as for the control group (r = 0.94, p < 0.001). Both groups showed similar antifungal activity of lactoferrin but the concentration used (100 ml/ml) did not reach 100% cell death (Fig. 1). Lactoferrin was able to kill various clinical isolates of C. albicans at concentrations less than 1 106 cell/ml, in both groups.
4.
Discussion
Salivary anti-microbial proteins are considered to be an important part of the non-immune host defence system in preventing colonization and infection of the oral cavity by oral microorganisms. These protective effects of saliva are evidenced by the microbial overgrowth found in patients with salivary deficiencies.15 Earlier studies have already shown that intrinsic components of the host defence system
such as histatins, cystatins, immunoglobulins and several saliva proteins, like lysozyme and lactoferrin are capable of killing various Candida species.16,17 We focused our attention on lactoferrin as this protein has demonstrated its ability to inhibit the growth of several bacteria and Candida species, which is the etiological agent of opportunistic infections with predictive values for HIV infection.18 To the best of our knowledge, the current study is the first to investigate the relationship between the lactoferrin levels and Candida sp. colonization of HIV-infected children, as well as the first in vitro study to determine of the capability of lactoferrin to inhibit the growth of C. albicans isolated from the saliva of the same HIV patients. The concentration of salivary lactoferrin in unstimulated parotid saliva in the adult population varies from 7 to 20 mg/ ml19,20 but decreases upon stimulation. This wide range occurs due to the individual characteristics of the groups and methods used with no significant differences in levels in whole and parotid saliva. Van Der Strate et al.21 determined that the lactoferrin concentration in saliva was in the range of 10 mg/ml in HIV-infected adults and it was not significantly different in healthy individuals. The values we found in
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Table 4 – Personal and medical data from the 10 HIVinfected children and the 10 health children whose isolates were used in the Lactoferrin killing assay experiment.
Personal data Age (years) Medical data Presence of AIDSa
Oral manifestations Actual presenceb: Yes No Candidiasis Yes No Caries disease Presence of caries Yes No Mean dmft Mean DMFT
HIV group (N = 10)
Control group (N = 10)
9.11 2.31 HIV group (N = 10)
8.90 2.60
Yes—1 (10%) No—9 (90%) HIV group (N = 10)
Control group (N = 10)
4 (40%) 6 (60%)
2 (20%) 8 (80%)
3 (30%) 7 (70%)
1 (10%) 9 (90%)
0 10 (100%) 0.2 0.42 1.4 1.26
0 10 (100%) 1.6 1.17 0.4 0.51
a
CD4 < 15%. Oral manifestations include the presence of one or more oral manifestations as: oral candidiasis, Herpes simplex lesions, linear gingival erythema and Parotid enlargement (CDC criteria9).
b
children were lower than those cited for adults and the average lactoferrin concentration we detected was significantly higher in HIV-infected children compared with healthy children. There are no values considered normal for lactoferrin levels in children, so there is no cut-off-point or normal range. Moreover, to date, no studies on quantification of lactoferrin in children have been found in the literature, thus we are not able to make any comparisons with our results. Alterations in saliva secretion and composition during infection by HIV may alter the defence mechanisms of the host, and influence adherence and colonization by Candida.22 There are reports that indicate alterations in major salivary
gland functions following HIV-infection with effects on both the salivary composition and output (flow rate), which could influence lactoferrin levels.23 The children in our study had no difficulty during the saliva collection and the subjects with salivary flow less than 0.5 ml/min were not included in our sample. Evidence has shown that increased salivary lactoferrin production is most probably a response to the elevated stimulus provided by the HIV-infection and by the opportunistic pathogens that accompany the progression of the disease.2 Although this suggests that the secretory immune system was responding directly to the challenge presented by the Candida colonization, the association between salivary anti-microbial proteins and oral candidiasis remains controversial.2,24 Our microbiological analyses demonstrated a significantly higher prevalence of Candida colonization in HIV-infected children compared with the controls, however no association was found between lactoferrin levels and fungal colonization within the HIV group showing that colonized or not by Candida, in this group, did not influence the levels of lactoferrin, even when there were high amounts of Candida (Moderate/Strong growth). Lourenc¸o et al.25 related no correlation between the concentration of salivary lactoferrin and the quantification of Candida sp. and also between the lactoferrin concentration and systemic condition of HIV-infected adults, corroborating our results. In addition, Van Der Strate et al.21 observed that, in a group of 15 HIV-infected subjects, the titres of Candida present in the oral cavity were unaffected by salivary lactoferrin concentration. Drawing a parallel with studies that observed other salivary proteins, Pomarico et al.2 reported positive correlations between salivary IgA levels and Candida sp. isolated from saliva of HIV-infected children. Also, Jainkittivong et al.26 demonstrated that the levels of mucin concentration were higher in the HIV-infected subjects than in the controls. In light of the pivotal role of CD4+ cells in the maturation of the mucosal immune system, and the fact that HIV-infection leads to a decrease in the numbers of such cells, secretory immunity, including salivary proteins, would be expected to be compromised with the progression of HIV-infection.24 Therefore, similar to our study, HIV-infected children appear
Fig. 1 – Killing assay of lactoferrin (100 mg/ml) against C. albicans isolated from HIV-infected children (HIV-group) and health children (Control group).
archives of oral biology 59 (2014) 775–782
to be able to maintain their capacity to mount a mucosal immune response to opportunistic microorganisms.24,27 It is interesting to note that our levels of lactoferrin were always higher in HIV infected children except when the number of Candida CFU was low in these children, and also when the healthy patients had oral candidiasis. This observation further supports the hypothesis that the production of lactoferrin by the immune system and its presence in saliva is directly relate to a compensatory response in both groups. It would seem that, in general, HIV-infected children maintain an intact immune response capacity to a number of antigens presented to the mucosal immune system. As already suggested by Castro et al.,24 it is possible that the level of CD4+ helper activity, although reduced, is still sufficient to induce normal mucosal responses to oral microorganisms in HIV-infected individuals, at least until the later stages of disease. According to the literature, lactoferrin has an antifungal capacity but there is no consensus with regard to its inhibitory potency in terms of cell density and lactoferrin concentration against Candida species.19,21 We showed that lactoferrin was able to kill over 50% of the cell growth of C. albicans at concentrations less than 1 106 cell/ml with 100 mg/ml of lactoferrin in both groups. Also, the death percentage of C. albicans by lactoferrin was inversely proportional to its cell density in both groups The antifungal capacity of salivary proteins was reported by Miller et al.,14 when they studied the antifungal activity of the Histatin-5; however it is known that this potential is not related to one single phenomenon.28 At the same time, the action mechanism of our fungal isolates has not yet been investigated and is therefore a limitation of our study. In addition, the varying experimental conditions, such as differences in medium composition, pH, incubation temperature, incubation time, and end point criteria, as well as the variable use of human versus bovine lactoferrin and of iron-free lactoferrin (apo-lactoferrin) versus iron-containing lactoferrin, makes it difficult to compare the results of the present study with those reported earlier.29 A number of researchers have reported that Candida species from HIV-infected patients with recurrent episodes of oral thrush are significantly less genetically diverse than commensal strains from healthy individuals.30,31 This implies that strains derived from the same parental stock may persist through recurrent infections in these immunocompromised patients. Others have reported similar findings on comparing serotypes from immunologically compromised patients and healthy individuals,32 thus corroborating with our study where both groups showed similar antifungal activity of lactoferrin despite not attaining 100% cell death. Lin et al.33 reported that salivary yeast carriage rates and concentration were comparable in HIV-infected and healthy individuals and that the salivary anticandidal activities were not altered. Further studies into the association between lactoferrin levels and oral fungal colonization are ongoing in our laboratories. Recognizing the various factors and conditions that play a role in oral candidal colonization and the progression of colonization with the infection will greatly contribute to our understanding of fungal pathogenesis and the contributing host factors. In our investigation, crucial information can have important clinical implications in the
781
identification and the design of novel therapeutic strategies aimed at the prevention and/or treatment of fungal infections in these patients. Particularly, the anticandidal property coupled with its lack of toxicity to human cells can make lactoferrin a promising therapeutic agent for the treatment or prevention of oral candidiasis in immunocompromised patients. In conclusion, our study shows that the HIV-infected children in our study had higher concentrations of lactoferrin and it was associated with Candida sp. colonization but no association with medical data was found. Also, both groups showed similar lactoferrin antifungal activity. These present data give us a tantalizing glimpse of the adaptive responses of oral Candida species to innate antimicrobial defences in saliva, such as lactoferrin in HIV-infected and healthy children. This phenomenon and results from further works can help elucidate the true role of these oral secretions related to oral Candida colonization in HIV-infected children.
Funding None.
Competing interests None declared.
Ethical approval Not required.
Acknowledgements The authors would like to thank CAPES and FAPERJ (Brazil) for the financial support.
references
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