Viral coinfection in the oral cavity of HIV-infected children : relation among HIV viral load , CD 4 + T lymphocyte count and detection of EBV , CMV and HSV

229 ABSTRACT: Viral coinfection in the oral cavity associated to HIV infection was evaluated in 180 children from birth to 13 years of age of both sexes. The oral examinations were performed at the Pediatric AIDS Outpatient Clinic, São Lucas Hospital and Clinic Hospital, both in Porto Alegre, Brazil and at the School of Dental Medicine, University Hospital Center, State University of New York at Stony Brook, USA. The aim of this study was to identify the presence of viral infections in the oral cavity. PCR technique was used to determine opportunistic viral infections caused by CMV, EBV, and HSV in mucosal swabs. A high frequency of viral infection was detected in the oral cavity of HIV-infected children determined by the PCR technique. HIV-infected children with viruses had a favorable CD4+T lymphocyte count and unfavorable viral load. DESCRIPTORS: AIDS-related opportunistic infections; Herpes simplex virus; Cytomegalovirus; Epstein-Barr virus; Viral load; CD4-positive T-lymphocytes.


INTRODUCTION
Children and adults infected with the Human Immunodeficiency Virus (HIV) are prone to develop opportunistic viral infections in the oral mucosa mainly by the Herpesviridae family members such as the Cytomegalovirus (CMV), the Epstein-Barr Virus (EBV), and the Herpes Simplex Virus (HSV),

Subjects
HIV-infected children between zero and 13 years of age were investigated.This study included 180 subjects: 143 Brazilians (Pediatric AIDS Outpatient Clinic, São Lucas Hospital, Pontifical Catholic University of Rio Grande do Sul, and Clinic Hospital, Federal University of Rio Grande do Sul -both in Porto Alegre, Brazil); and 37 Americans (Pediatric Infectious Disease Clinic, University Hospital Center, School of Dental Medicine, State University of New York at Stony Brook, NY, USA).All patients had CD4 + T lymphocyte count (expressed in % value/cytometric technique) and HIV viral load (expressed in log 10 value/NASBA method) determined.

Oral samples
Oral swabs were collected after obtaining a signed informed consent from the children's parents or guardians.Oral swabs (saliva and epithelium cells) were collected with sterile cotton tipped applicators 14 (Citmed, Citronelle, AL, USA) from the buccal mucosa bilaterally, regardless of the presence of oral lesions.Specimens were placed in test tubes (Eppendorf AG, Hamburg, Germany) containing 500 µl of sterile phosphate buffered saline (PBS -Merck KGaA, Darmstadt, Germany).DNA was extracted by the organic method (phenol: chloroform:IAA -Invitrogen Corporation, Carlsbad, CA, USA) 22 .

PCR assay
DNA extracted from the buccal swabs was used to amplify HSV (type 1 and type 2) 18 , CMV 12 , and EBV 2 by two-step (semi-nested and nested) PCR amplifications.All specimens were tested for the presence of the β-globin gene to ensure the presence of DNA.All PCR reactions had positive (stored DNA obtained from a patient with HSV, CMV or EBV disease) and negative controls (stored DNA obtained from a patient without HSV, CMV or EBV disease) and a reaction mix.Some samples were lost during the laboratory process.The primer sequences used in the PCR reactions are shown in Table 1.
CMV was amplified with CMV-1/CMV-2 in the first and CMV-1/CMV-4 in the second round.EBV was amplified with EBV-1/EBV-2 in the first round and EBV-3/EBV-4 in the second.HSV was amplified with primers HSV-1/HSV-2 in the first round and HSV-3/HSV-4 in the second round.The final products included a 220 bp fragment of the Major all of which are important etiologic agents of morbidity 14,19,23 .Coinfections of HSV and CMV 19 ; and CMV and EBV 23 have been reported in oral ulcers of HIV-infected patients.Despite the unknown pathogenesis of these coinfections 20 , the detection of more than one virus in the oral mucosa of HIV-infected patients may have important clinical implications and, therefore, requires further investigation.
Many authors have investigated bacterial, fungal and viral infections in oral lesions of HIVinfected patients utilizing the Polymerase Chain Reaction (PCR) technique 6,13,22 .This technique offers several advantages over other methods.It requires a low quantity of biological material 16 and can detect the viral presence on "early" infections 24 .PCR detection of HSV, EBV, and CMV is highly sensitive and specific, which can aid in the prevention of clinical manifestations of virus-associated oral lesions through the selection of the appropriate therapy.
Many tests are used to evaluate the status of the immune system of HIV-infected patients, specially the CD4 + T lymphocyte count and the HIV viral load 5 .The CD4 + T lymphocyte count provides an estimation of the immune system status of the HIV-infected individual, and reflects the previous history of the disease 20 .The CD4 + T-lymphocyte count also indicates the necessity for prophylaxis for opportunist infections and helps to evaluate initial antiretroviral therapy or treatment failure 5,8,20 .Children without evidence of immunodeficiency have CD4 + T lymphocyte count around 25% and many authors have related the oral lesions in HIV-infected patients to low CD4 + T lymphocyte count 10,15,17 .
In addition to the immune system abnormalities, HIV-infected Brazilian children can be affected by the lack of appropriate caregiver supervision 4 .The low education level of caregivers of the HIVinfected Brazilian children can be related to the poor compliance with the antiretroviral treatment, thus affecting the child's health 4,5 .
The aim of this research was to detect the presence of some viruses (EBV, CMV and HSV) in the oral cavity of HIV+ children by the PCR technique and study the relation among these virus types with the HIV viral load and CD4 + T lymphocyte count.

METHODS
This research was approved by the hospitals' Research Ethics Committees.
Immediate Early Antigen (MIE) gene from CMV; a 209 bp product of the EBNA-1 gene from EBV and a 142 bp fragment of the D gene of HSV.
The PCR reaction mix contained 0.4 µM of the appropriate primer (Invitrogen Corporation, Carlsbad, CA, USA), 1 X PCR Buffer (Invitrogen Corporation, Carlsbad, CA, USA), 200 µM of each dNTP (Invitrogen Corporation, Carlsbad, CA, USA), 1.25 units of Taq DNA polymerase (Invitrogen Corporation, Carlsbad, CA, USA) and 1.5 mM (HSV), 2.0 mM (CMV) or 2.5 mM (EBV) of MgCl 2 (Invitrogen Corporation, Carlsbad, CA, USA) in a final volume of 50 µl.Viral DNA, human DNA and reaction controls were included in each run.DNA amplification was performed in an automated thermal cycler (MJ Research, Waltham, MA, USA).Reactions were brought to 95°C for 10 min, followed by thirty cycles consisting of a denaturing step for 30 s at 94°C, annealing step for 30 s at 50°C (CMV), 60°C (EBV), 50°C (HSV first round) or 60°C (HSV second round), and an extension step for 30 s at 72°C.A final extension step at 72°C was carried out for 5 min.A total of 5 µl of the first round product was used in the second round of amplifications.Aliquots of 15 µl of the PCR product were analyzed on 2% agarose gel (Merck KGaA, Darmstadt, Germany) containing 0.5 g/ml of ethidium bromide (Merck KGaA, Darmstadt, Germany) and visualized under ultraviolet light (Bio-Rad Laboratories Inc., Hercules, CA, USA).The PCR sensitivities were the same in children from Brazil and from the USA: 0.5 pg for CMV; 50 ng for EBV; 5 × 10 -5 pg for HSV.

Viral detection by PCR
HSV, CMV and EBV results obtained analyzing the Brazilian and American children are presented in Table 2.According to the Chi-squared test, there were no differences in CMV infection between Brazilian and American children (p = 0.110); there were more Brazilian children infected with EBV and HSV than American children (p = 0.001).
Figures 1, 2, and 3 show the reaction products of the nested PCR for CMV, EBV and HSV after electrophoresis (Bio-Rad Laboratories Inc.,  *The primer sequences were obtained from Krajden et al. 12 (1996) (CMV); Cinque et al. 2 (1993) (EBV) and Powell et al. 18 (1990) (HSV).CMV: Cytomegalovirus; EBV: Epstein-Barr Virus; HSV: Herpes Simplex Virus.Hercules, CA, USA) in 2% TAE-agarose gel (Merck KGaA, Darmstadt, Germany) containing 0.5 g/ml of ethidium bromide and visualized under ultraviolet light.Table 3 shows the results of the Sensitivity Test for CMV, EBV and HSV. Figure 4 shows an example of PCR sensitivity (CMV) test.Although there is a difference in the sample size between Brazilian and American patients, when comparing the data obtained from the two countries, no statistical differences were observed between the number of Brazilian and American children with viral infections in the oral cavity (Chi-squared test, p = 0.659).

Relationship between viral infection in the oral cavity; CD4 + T lymphocyte count and HIV viral load
The average ± standard deviation of CD4 + T lymphocyte count (%) and HIV viral load (log 10 ) were determined to evaluate a possible relationship between viral infection in the oral cavity and the general health status of the patient.Table 4 shows that the mean CD4 + T lymphocyte count for all patients infected with viruses (HSV, CMV and/or EBV) were higher than 25%, thus suggesting no evidence of immunossuppression 20 .There was no statistical difference between mean CD4 + T lymphocyte count of patients with different virus types in their oral cavity as shown by the Variance Analysis (ANOVA) test.
The mean HIV viral load of all patients infected with viruses (HSV, CMV and/or EBV) was higher than 1,000 copies of HIV/ml (Table 5).There were no statistical differences among the mean viral load of patients with different viruses in the oral cavity according to the Variance Analysis (ANOVA).6 shows the frequency of HSV, CMV and EBV in the oral cavity of our study cohort.It was observed that the number of Brazilian children (29.37%) without viruses was similar to the number of American children (45.95%), according to the Chi-squared test (p = 0.372).Sixty-three Brazilian children (44.06%) and 17 American children (45.95%) had at least one virus type present.Seven American (18.92%) and 35 Brazilian children (24.48%) had two different virus types at the same time.Only 3 Brazilian children (2.10%) had HSV, CMV and EBV oral coinfection.

DISCUSSION
Since CMV viruses are endemic in children populations from developing countries 1,24 , a higher number of CMV infections was expected in Brazil.The development of the antiviral therapy could be responsible for the control of CMV infection in HIVinfected individuals 9 .Since 1984, CMV-related ulcers in HIV-infected children had been reported 1,6,9 .The pathology of CMV in oral ulcerations remains unknown 19 .
EBV is the etiologic agent of an illness that can affect children and young adults, called Infectious Mononucleosis 3,24 .Approximately 90% of the global population has acquired EBV in a non-symptomatic way 24 and the virus remains latent in the lymph nodes and pharynx cells.In immunodeficient patients, Infectious Mononucleosis could be severe and the EBV recurrence can cause lymphoma 21 , Kaposi's Sarcoma 24 and hairy leukoplakia 7,8 .
HSV infection is considered the most frequent viral infection in HIV-infected patients.The early HSV infection and its high frequency in HIV-infected children correlates with the rapid evolution of the disease, suggesting a worsening prognosis for the patient.In immunodeficient patients, as well as in HIV-infected patients, HSV infection can be severe, clinically atypical, more painful and with a long-term duration 11 .
Twenty-five percent of CD4 + T lymphocyte count means no evidence of immunosuppression.One thousand copies of HIV/ml (log 10 = 3.0) is considered a significant viral load 20 .The absence of clinical manifestations in the HIV-infected children of this study was possibly due to the presence of their high CD4 + T lymphocyte count.The children who had viruses detected in their oral cavities had more than 1,585 copies of HIV/ml (log 10 = 3.2), which is considered a high viral load 20 .
Before the AIDS era, Herpesviridae coinfection in human tissue was rare.Presently, this is not the case.Coinfection with HSV and CMV has been described in many anatomic sites, such as the nervous system, skin, esophageal area, and lips.CMV infection often presents severe, painful and long-term duration oral ulcerations 19 .Viral and fungal coinfection has been also observed, in addition to viral coinfection with CMV and EBV in oral ulcers of HIV-infected patients 23 .

CONCLUSION
A high frequency of viral infection was detected in the oral cavity of HIV-infected children determined by the PCR technique.EBV was the virus most commonly found, followed by HSV and CMV.HIV-infected children with viruses had a favorable CD4 + T lymphocyte count and unfavorable viral load.Multiple viral coinfections were also observed in the oral cavity of our cohort.Early viral infection was detected in the oral cavity of the patients, despite the absence of clinical manifestations.
Considering the complexity of the viral infection therapy in HIV-infected patients, it is very important to identify the opportunistic agents present in the oral cavity as soon as possible.

TABLE 3 -
Results of the Sensitivity test for CMV, EBV and ; EBV: Epstein-Barr Virus; HSV: Herpes Simplex Virus.

TABLE 1 -
Primer sequences* used in the semi-nested and nested PCR technique.

TABLE 2 -
Frequency (f) and percentage (%) of Brazilian and American children as detected by PCR.

TABLE 6 -
Frequency (f) and percentage (%) of Brazilian and American children with virus coinfection.

TABLE 4 -
Relationship between virus type and CD4 + T lymphocyte counts (in % unit) of Brazilian and American children (average ± standard deviation).

TABLE 5 -
Relationship between virus type and viral load (in log 10 unit) of Brazilian and American children (average ± standard deviation).