Human Herpesvirus 8 in Perinatally HIV-infected Children with Interstitial Lung Disease

Human Herpesvirus 8 in Perinatally HIV-infected Children with Interstitial Lung Disease Abstract Introduction Human herpesvirus 8 (HHV-8) is associated with the pathogenesis of Kaposi Sarcoma and interstitial pneumonitis in adults. This study aims to evaluate association between HHV-8 and interstitial lung disease in HIV-infected children. Methods HIV-infected children with interstitial pneumonitis underwent lung biopsies in a tertiary hospital and were investigated for HHV-8, Epstein-Barr virus (EBV) and cytomegalovirus (CMV) using polymerase chain reaction (PCR) and immunohistochemistry in lung tissue. Peripheral blood PCR was also performed for HHV-8. Results From six patients included, PCR for HHV-8 was positive in lung samples in four children and in peripheral blood in one. PCR for EBV and CMV and immunohistochemical study for HHV-8, EBV and CMV in lung were negative in all patients. Conclusion No previous cases of HHV-8-associated interstitial pneumonitis was described in HIV-infected children. An immunological disorder and an infectious agent might influence development of the lymphoid interstitial pneumonitis. HHV-8 may be this infectious trigger. human herpesvirus 8, interstitial lung disease, HIV, child INTRODUCTION Human herpesvirus 8 (HHV-8) was first described in 1994 and related to the pathogenesis of Kaposi's sarcoma [1]. This virus was subsequently linked to Multicentric Castleman's disease, body-cavity lymphoma, as well as other types of non-Hodgkin’s Lymphoma [2–7]. HHV-8 is transmitted primarily via the sexual route. In addition, the vertical, salivary, transfusion and transplantation routes have been described as alternative modes of transmission [8–14]. Detection of HHV-8 in lung tissue was reported in few adults with interstitial pneumonitis, with and without associated HIV infection [15, 16]. In children with HIV infection, lymphoid interstitial pneumonitis (LIP) and follicular bronchitis/bronchiolitis (FBB) are different parts of the spectrum of the same non-neoplastic, inflammatory, lymphoproliferative disorder, differing mainly in the location of the lymphocyte infiltrate (peribronchiolar vs. alveolar) [17, 18]. These manifestations represent up to 40% of interstitial lung diseases and are less commonly seen in HIV-infected adults [19, 20]. Pulmonary lymphoid hyperplasia complex or LIP is an AIDS-defining condition, considered a ‘B’ clinical criterion by the Center for Disease Control and Prevention classification and is related to intermediate progression of HIV infection and associated with bronchiectasis [21–23]. The etiology and pathogenesis of this pneumonitis are not completely understood and have been associated with Epstein-Barr virus (EBV), both in HIV-positive and -negative patients, but not found in all cases [24–26]. The aim of this study is to evaluate association between HHV-8 and interstitial lung disease in HIV-infected children. MATERIALS AND METHODS This study is a case series describing results from virology and histopathology tests and clinical course from all children vertically infected with HIV who underwent open lung biopsy for diagnostic purposes in a tertiary hospital during the past two decades. The Clinics Hospital associated with the Ribeirão Preto Medical School is the largest public hospital that provides medical assistance for HIV-infected children and adolescents in the city of Ribeirão Preto and surrounding cities. This hospital covers a large area in the north and northeast of Sao Paulo, which has an estimated population of around 1 600 000 people [27]. Criteria for biopsy was the presence with chronic cough of insidious origin, dyspnea on exertion and ground-glass opacities on thorax computed tomography (CT) scan. Peripheral blood samples were obtained for the detection of HHV-8 by polymerase chain reaction (PCR). Lung biopsies were analyzed for the presence of HHV-8, EBV and cytomegalovirus (CMV) using PCR and immunohistochemical staining. PCR for HHV-8, EBV and CMV was performed with DNA extracted from lung and blood tissue. DNA was extracted from tissues with the QIAamp DNA tissue kit (Qiagen) according to manufacturer instructions, and eluted in 50 μl TAE. A total volume of 50 μl of PCR mixture was used to extract HHV-8 DNA from peripheral blood. The mixture contained 0.2 mM of each dNTP, 25 pmol of each sense and antisense primers (5ʹ-AGCCGAAAGGATTCCACCAT-3ʹ and 5ʹ-TCCGTGTTGTCTACGTCCAG-3ʹ), 50 mM KCl, 1.5 mM MgCl2, 20 mM Tris–HCl, pH 8.4, 2.5 U Taq DNA Polymerase (Invitrogen, New York, USA). Ten microliters of DNA was extracted from each sample. PCR mixtures were incubated at 94°C for 1 min, followed by 35 cycles of 1 min at 94°C, 1 min at 58°C and 1 min at 72°C. After the last cycle, samples were incubated for 10 min at 72°C to allow complete extension of the amplicons to amplify a 233 bp sequence. The nested PCR amplification mixture contained 1 μl of the first PCR mixture and the same PCR reagents described above, except that 25 pmol of each sense and anti-sense internal primers (5ʹ-TTCCACCATTGTGCTCGAAT-3ʹ and 5ʹ-TACGTCCAGACGATATGTGC-3ʹ) were used. The mixture was subjected to 94°C for 1 min, followed by 35 cycles of 1 min at 94°C, 1 min a 60°C, 1 min at 72°C and a final extension of 10 min at 72°C. Ten microliters of each reaction mixture were analyzed by 2% agarose gel electrophoresis. The amplicon bands of 211 bp were visualized using ethidium bromide staining. As a positive control, β-globin was amplified by PCR from each specimen using primers GL1 and GR2. To avoid contamination of specimens with viral DNA, standard precautions concerning spatial separation of pre- and post-PCR steps and aliquoting reagents were followed strictly. A set of negative controls (water and a negative clinical sample) was included during all steps of DNA isolation and amplification. For EBV and CMV detection, sense and antisense primers were, respectively, 5ʹ-CTTTAGAGGCGAATGGGCGCCA-3ʹ and 5ʹ-TCCAGGGCCTTCACTTCGGTCT-3ʹ and gB1 5ʹ-GAAACGCGCGGCAATCGG-3ʹ and gB2 5ʹ-TGGAACTGGAACGTTTGGC-3ʹ. Internal primers used in the nested PCR confirmatory of CMV genome amplification by PCR were gBn1 5ʹ-GCGCCGTTGATCCACACACC-3ʹ and gBn2 5ʹ-TACGCTGCAGTTCACCCCAG-3ʹ, as described elsewhere [28]. Immunostaining was performed using the mouse monoclonal antibody against HHV-8 (clone 13B10, dilution 1:20, Cell Marque, Rocklin, California, USA), mouse monoclonal antibody against EBV (clone CS.1-4, dilution 1:100, Dako, USA) and mouse monoclonal antibody against CMV (clone CCH2 + DDG9, dilution 1:100, Dako, USA) with a labeled streptavidin-biotin peroxidase system. Antigen retrieval was effected by pressure cooking in EDTA buffer, pH 8.0, for 3 min and 10 s. A fragment of known Kaposi sarcoma tissue verified by molecular study to be positive for HHV-8 was mounted on every test slide as a positive control. This study was approved by institutional review board of Clinics Hospital, Ribeirão Preto Medical School, University of São Paulo. RESULTS All six children who met clinical and radiological criteria mentioned above underwent open lung biopsy and were included. All biopsies were performed from 2004 onwards. Two girls and four boys were included, with four of them living in the same charity institution dedicated to the care of children infected or orphaned by HIV. The age at biopsy ranged from 6 to 16 years and lymphocyte CD4 count ranged from 141 to 474 cells/mm3. Table 1 lists the clinical and laboratorial features of the six patients. In all six children, ground-glass opacities and bronchiectasis were found at thorax CT scan, as shown in Fig. 1. Table 1 Clinical features in patients with interstitial lung disease Patients Gender Race Agea (years/ months) CD4 (%)a (cells/mm3) Clinical findings Home HAARTa,b Time since first ARVa Age of onset of respiratory signs and symptoms Pneumo- cystosis age Comorbiditiesa Outcome at the end of follow-up Patient 1 Female Black 8 400 (20) Cough, chronic dyspnea Institution ZDVc/DDId/ LPV/re 7 years 3 months 3 months Pulmonary tuberculosis Alive Patient 2 Female White 8/6 193 (10) Cough, chronic dyspnea Institution D4Tf/3TCg/ LPV/r 4 years 5 years 4 months No Died Patient 3 Male Black 6/8 94 (0.1) Cough, chronic dyspnea Institution D4T/3TC/ EFZh 5 years and 10 months 8 months No Bilateral lower limbs spasticity related to HIV encephalopathy Alive Patient 4 Male Black 16/3 141 (9) Cough, chronic dyspnea Institution TDFi/3TC/ LPV/r 6 years 12 years Unknown Chronic Hepatitis C infection (Child-Pugh B) Alive Patient 5 Male White 9/1 474 (11) Cough, chronic dyspnea Relatives D4T/3TC/ LPV/r 6 years and 2 months 1 year and 8 months 5 months No Alive Patient 6 Male Black 7/11 252 (22) Cough, chronic dyspnea Relatives 3TC/DDI/ LPV/r 4 years 5 years 8 months Bilateral lower limbs spasticity related to HIV encephalopathy Alive Patients Gender Race Agea (years/ months) CD4 (%)a (cells/mm3) Clinical findings Home HAARTa,b Time since first ARVa Age of onset of respiratory signs and symptoms Pneumo- cystosis age Comorbiditiesa Outcome at the end of follow-up Patient 1 Female Black 8 400 (20) Cough, chronic dyspnea Institution ZDVc/DDId/ LPV/re 7 years 3 months 3 months Pulmonary tuberculosis Alive Patient 2 Female White 8/6 193 (10) Cough, chronic dyspnea Institution D4Tf/3TCg/ LPV/r 4 years 5 years 4 months No Died Patient 3 Male Black 6/8 94 (0.1) Cough, chronic dyspnea Institution D4T/3TC/ EFZh 5 years and 10 months 8 months No Bilateral lower limbs spasticity related to HIV encephalopathy Alive Patient 4 Male Black 16/3 141 (9) Cough, chronic dyspnea Institution TDFi/3TC/ LPV/r 6 years 12 years Unknown Chronic Hepatitis C infection (Child-Pugh B) Alive Patient 5 Male White 9/1 474 (11) Cough, chronic dyspnea Relatives D4T/3TC/ LPV/r 6 years and 2 months 1 year and 8 months 5 months No Alive Patient 6 Male Black 7/11 252 (22) Cough, chronic dyspnea Relatives 3TC/DDI/ LPV/r 4 years 5 years 8 months Bilateral lower limbs spasticity related to HIV encephalopathy Alive a Data when lung biopsies were performed. b HAART = highly active antiretroviral therapy. c ZDV = Zidovudine. d DDI = Didanosine. e LPV/r = Lopinavir/ritonavir. f D4T = Stavudine. g 3TC = Lamivudine. h EFZ = Efavirenz. i TDF = Tenofovir. Table 1 Clinical features in patients with interstitial lung disease Patients Gender Race Agea (years/ months) CD4 (%)a (cells/mm3) Clinical findings Home HAARTa,b Time since first ARVa Age of onset of respiratory signs and symptoms Pneumo- cystosis age Comorbiditiesa Outcome at the end of follow-up Patient 1 Female Black 8 400 (20) Cough, chronic dyspnea Institution ZDVc/DDId/ LPV/re 7 years 3 months 3 months Pulmonary tuberculosis Alive Patient 2 Female White 8/6 193 (10) Cough, chronic dyspnea Institution D4Tf/3TCg/ LPV/r 4 years 5 years 4 months No Died Patient 3 Male Black 6/8 94 (0.1) Cough, chronic dyspnea Institution D4T/3TC/ EFZh 5 years and 10 months 8 months No Bilateral lower limbs spasticity related to HIV encephalopathy Alive Patient 4 Male Black 16/3 141 (9) Cough, chronic dyspnea Institution TDFi/3TC/ LPV/r 6 years 12 years Unknown Chronic Hepatitis C infection (Child-Pugh B) Alive Patient 5 Male White 9/1 474 (11) Cough, chronic dyspnea Relatives D4T/3TC/ LPV/r 6 years and 2 months 1 year and 8 months 5 months No Alive Patient 6 Male Black 7/11 252 (22) Cough, chronic dyspnea Relatives 3TC/DDI/ LPV/r 4 years 5 years 8 months Bilateral lower limbs spasticity related to HIV encephalopathy Alive Patients Gender Race Agea (years/ months) CD4 (%)a (cells/mm3) Clinical findings Home HAARTa,b Time since first ARVa Age of onset of respiratory signs and symptoms Pneumo- cystosis age Comorbiditiesa Outcome at the end of follow-up Patient 1 Female Black 8 400 (20) Cough, chronic dyspnea Institution ZDVc/DDId/ LPV/re 7 years 3 months 3 months Pulmonary tuberculosis Alive Patient 2 Female White 8/6 193 (10) Cough, chronic dyspnea Institution D4Tf/3TCg/ LPV/r 4 years 5 years 4 months No Died Patient 3 Male Black 6/8 94 (0.1) Cough, chronic dyspnea Institution D4T/3TC/ EFZh 5 years and 10 months 8 months No Bilateral lower limbs spasticity related to HIV encephalopathy Alive Patient 4 Male Black 16/3 141 (9) Cough, chronic dyspnea Institution TDFi/3TC/ LPV/r 6 years 12 years Unknown Chronic Hepatitis C infection (Child-Pugh B) Alive Patient 5 Male White 9/1 474 (11) Cough, chronic dyspnea Relatives D4T/3TC/ LPV/r 6 years and 2 months 1 year and 8 months 5 months No Alive Patient 6 Male Black 7/11 252 (22) Cough, chronic dyspnea Relatives 3TC/DDI/ LPV/r 4 years 5 years 8 months Bilateral lower limbs spasticity related to HIV encephalopathy Alive a Data when lung biopsies were performed. b HAART = highly active antiretroviral therapy. c ZDV = Zidovudine. d DDI = Didanosine. e LPV/r = Lopinavir/ritonavir. f D4T = Stavudine. g 3TC = Lamivudine. h EFZ = Efavirenz. i TDF = Tenofovir. Fig. 1. View largeDownload slide Ground glass opacities and bronchiectasis in thorax CT scan. Fig. 1. View largeDownload slide Ground glass opacities and bronchiectasis in thorax CT scan. Histological examination revealed FBB in four patients (Fig. 2), LIP in one and obliterans bronchiolitis in one. Kaposi’s sarcoma was excluded in all samples. In one patient (Patient 1 from Tables 1 and 2), acid-fast bacilli were also found through Ziehl-Neelsen stain, and the clinical picture improved significantly after tuberculosis treatment. PCR for HHV-8 was positive in four patients in lung tissue and in peripheral blood of another patient. As shown in Table 1, all patients in which PCR was positive in lung tissue were living together. Immunohistochemical study for HHV-8, EBV and CMV in the lung biopsy was negative in all six patients, as well as PCR for EBV and CMV. In all of these negative tests, a positive control was performed successfully. The procedure caused no complications in five patients. One patient presented a surgical wound infection by methicillin-resistant Staphylococcus aureus. Table 2 presents the combined histological and virological results for each patient. Table 2 Histological and virological findings Patients Histological diagnosis HHV-8a lung HHV-8a peripheral blood Immunohistochemistry for HHV-8 Patient 1 Obliterans bronchiolitis Positive Negative Negative Patient 2 Lymphocytic interstitial pneumonia Positive Negative Negative Patient 3 Follicular bronchiolitis Positive Negative Negative Patient 4 Follicular bronchiolitis Positive Negative Negative Patient 5 Follicular bronchiolitis Negative Negative Negative Patient 6 Follicular bronchiolitis Negative Positive Negative Patients Histological diagnosis HHV-8a lung HHV-8a peripheral blood Immunohistochemistry for HHV-8 Patient 1 Obliterans bronchiolitis Positive Negative Negative Patient 2 Lymphocytic interstitial pneumonia Positive Negative Negative Patient 3 Follicular bronchiolitis Positive Negative Negative Patient 4 Follicular bronchiolitis Positive Negative Negative Patient 5 Follicular bronchiolitis Negative Negative Negative Patient 6 Follicular bronchiolitis Negative Positive Negative a Study performed using PCR. Table 2 Histological and virological findings Patients Histological diagnosis HHV-8a lung HHV-8a peripheral blood Immunohistochemistry for HHV-8 Patient 1 Obliterans bronchiolitis Positive Negative Negative Patient 2 Lymphocytic interstitial pneumonia Positive Negative Negative Patient 3 Follicular bronchiolitis Positive Negative Negative Patient 4 Follicular bronchiolitis Positive Negative Negative Patient 5 Follicular bronchiolitis Negative Negative Negative Patient 6 Follicular bronchiolitis Negative Positive Negative Patients Histological diagnosis HHV-8a lung HHV-8a peripheral blood Immunohistochemistry for HHV-8 Patient 1 Obliterans bronchiolitis Positive Negative Negative Patient 2 Lymphocytic interstitial pneumonia Positive Negative Negative Patient 3 Follicular bronchiolitis Positive Negative Negative Patient 4 Follicular bronchiolitis Positive Negative Negative Patient 5 Follicular bronchiolitis Negative Negative Negative Patient 6 Follicular bronchiolitis Negative Positive Negative a Study performed using PCR. Fig. 2. View largeDownload slide Follicular bronchiolitis (hematoxylin and eosin, magnification 50×). Note lymphocytic infiltrate with peribronchial distribution. Fig. 2. View largeDownload slide Follicular bronchiolitis (hematoxylin and eosin, magnification 50×). Note lymphocytic infiltrate with peribronchial distribution. One patient (Patient 5, whose PCR were negative both in lung and in peripheral blood) died 2 years after biopsy owing to progressive respiratory failure. All other five patients were followed until 18 years of age (mean follow-up 8.5 years). At the end of follow-up, all but one patient (Patient 2) had a HIV viral load <40 copies/ml and all five patients had mild respiratory signs and symptoms, presenting only intermittent episodes of cough and increase in respiratory secretions. DISCUSSION As far as we know, this is the first study relating HHV-8 to interstitial lung disease in children with HIV. In this sample, HHV-8 might be a viral stimulus leading some of these patients to develop LIP or FBB, instead of EBV, whose search in lung tissue, as well as CMV, was negative in all cases. Many studies have attempted to establish a viral etiologic agent for LIP, but there is no single agent consistently related to all cases [25]. In more than half of the cases, EBV DNA is found in lung samples, indicating a possible role of this virus in LIP pathogenesis in some cases [25, 29]. Interestingly, HHV-8 and EBV are both members of subfamily Gammaherpesvirinae, both viruses are related to oncogenesis and possibly other similar feature shared may be the potential to stimulate the development of these forms of lymphoproliferative interstitial pneumonitis. Another important aspect is the presence of an underlying disease in virtually all cases of LIP, especially immune disorders, such as HIV, Sjögren syndrome and systemic lupus erythematosus [29–31]. According to the initial description by Liebow and Carrington, these features suggest that LIP may be a response to several viral stimuli in patients with underlying immune disorders [32]. Several factors might have contributed to high prevalence found in this study. First, prevalence of HHV-8 appears to vary widely. Further, lower socioeconomic status and HIV status were associated with higher prevalence of HHV-8 [33]. Another possible contributing factor is the fact that four patients were living in close contact, leading to horizontal transmission, which is described in children [9]. Most data about prevalence of HHV-8 in Brazil in both HIV-positive and -negative population are available from adults [34–39]. However, two Brazilian studies reported higher prevalence in HIV-infected children [40, 41]. The patient who presented a different histological diagnosis, obliterans bronchiolitis, had, in addition to HHV-8, acid-fast bacilli in pulmonary tissue with an important response to tuberculosis treatment, suggesting a different pathogenesis compared with the remaining five children, although the clinical and radiological findings were identical in all six cases, permitting no diagnostic distinction before the biopsies were performed. Four children presented infection with Pneumocystis jirovecii during infancy, but its implication in the pathogenesis of interstitial disease is not known. The benign clinical course in five of our patients is in agreement with evidence suggesting that prognosis of HIV-related interstitial lung disease depends on underlying disease control [42]. This study has some limitations. First, differently from cases reported in adults, these four patients with positive pulmonary HHV-8 PCR did not show positive immunohistochemical staining. Hence, there is not a direct evidence of viral replication at the time of diagnosis. However, HHV-8 DNA was present in lung tissue, possibly indicating a past infection that could have influenced the development of LIP/FBB. As HHV-8 was not found in the blood of these four patients, at the time of biopsy, the infection was probably a localized pulmonary disease without ongoing viremia. Only one patient had positive HHV-8 PCR only in peripheral blood and perhaps this infection may have influenced a proliferative lung disorder. It is not possible to accurately explain the reason why no EBV was found in any of our patients. Second, lack of data about lung function, absence of bronchoalveolar lavage studies and lack of control tests for HHV-8 in other tissues might also be limitations of our study. Last, as all patients with positive PCR in lung tissue were living in the same institution, generalization of our findings might be limited. Conversely, the report of a complete sample and a long follow-up of patients who met inclusion criteria and underwent open lung biopsy during more than one decade in a large tertiary hospital make bias less likely and strengthen our findings. Finally, findings from this study suggest the possible association between HHV-8 and LIP pathogenesis. Nevertheless, further studies are necessary to reinforce this possibility. ACKNOWLEDGEMENTS Special thanks to Mrs Amanda Campos Scotta for technical assistance in the preparation of the images. FUNDING This study received no financial support. REFERENCES 1 Chang Y , Cesarman E , Pessin MS , et al. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma . Science 1994 ; 266 : 1865 – 9 . Google Scholar CrossRef Search ADS PubMed 2 Deloose ST , Smit LA , Pals FT , et al. High incidence of Kaposi sarcoma-associated herpesvirus infection in HIV-related solid immunoblastic/plasmablastic diffuse large B-cell lymphoma . 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Abstract

Abstract Introduction Human herpesvirus 8 (HHV-8) is associated with the pathogenesis of Kaposi Sarcoma and interstitial pneumonitis in adults. This study aims to evaluate association between HHV-8 and interstitial lung disease in HIV-infected children. Methods HIV-infected children with interstitial pneumonitis underwent lung biopsies in a tertiary hospital and were investigated for HHV-8, Epstein-Barr virus (EBV) and cytomegalovirus (CMV) using polymerase chain reaction (PCR) and immunohistochemistry in lung tissue. Peripheral blood PCR was also performed for HHV-8. Results From six patients included, PCR for HHV-8 was positive in lung samples in four children and in peripheral blood in one. PCR for EBV and CMV and immunohistochemical study for HHV-8, EBV and CMV in lung were negative in all patients. Conclusion No previous cases of HHV-8-associated interstitial pneumonitis was described in HIV-infected children. An immunological disorder and an infectious agent might influence development of the lymphoid interstitial pneumonitis. HHV-8 may be this infectious trigger. human herpesvirus 8, interstitial lung disease, HIV, child INTRODUCTION Human herpesvirus 8 (HHV-8) was first described in 1994 and related to the pathogenesis of Kaposi's sarcoma [1]. This virus was subsequently linked to Multicentric Castleman's disease, body-cavity lymphoma, as well as other types of non-Hodgkin’s Lymphoma [2–7]. HHV-8 is transmitted primarily via the sexual route. In addition, the vertical, salivary, transfusion and transplantation routes have been described as alternative modes of transmission [8–14]. Detection of HHV-8 in lung tissue was reported in few adults with interstitial pneumonitis, with and without associated HIV infection [15, 16]. In children with HIV infection, lymphoid interstitial pneumonitis (LIP) and follicular bronchitis/bronchiolitis (FBB) are different parts of the spectrum of the same non-neoplastic, inflammatory, lymphoproliferative disorder, differing mainly in the location of the lymphocyte infiltrate (peribronchiolar vs. alveolar) [17, 18]. These manifestations represent up to 40% of interstitial lung diseases and are less commonly seen in HIV-infected adults [19, 20]. Pulmonary lymphoid hyperplasia complex or LIP is an AIDS-defining condition, considered a ‘B’ clinical criterion by the Center for Disease Control and Prevention classification and is related to intermediate progression of HIV infection and associated with bronchiectasis [21–23]. The etiology and pathogenesis of this pneumonitis are not completely understood and have been associated with Epstein-Barr virus (EBV), both in HIV-positive and -negative patients, but not found in all cases [24–26]. The aim of this study is to evaluate association between HHV-8 and interstitial lung disease in HIV-infected children. MATERIALS AND METHODS This study is a case series describing results from virology and histopathology tests and clinical course from all children vertically infected with HIV who underwent open lung biopsy for diagnostic purposes in a tertiary hospital during the past two decades. The Clinics Hospital associated with the Ribeirão Preto Medical School is the largest public hospital that provides medical assistance for HIV-infected children and adolescents in the city of Ribeirão Preto and surrounding cities. This hospital covers a large area in the north and northeast of Sao Paulo, which has an estimated population of around 1 600 000 people [27]. Criteria for biopsy was the presence with chronic cough of insidious origin, dyspnea on exertion and ground-glass opacities on thorax computed tomography (CT) scan. Peripheral blood samples were obtained for the detection of HHV-8 by polymerase chain reaction (PCR). Lung biopsies were analyzed for the presence of HHV-8, EBV and cytomegalovirus (CMV) using PCR and immunohistochemical staining. PCR for HHV-8, EBV and CMV was performed with DNA extracted from lung and blood tissue. DNA was extracted from tissues with the QIAamp DNA tissue kit (Qiagen) according to manufacturer instructions, and eluted in 50 μl TAE. A total volume of 50 μl of PCR mixture was used to extract HHV-8 DNA from peripheral blood. The mixture contained 0.2 mM of each dNTP, 25 pmol of each sense and antisense primers (5ʹ-AGCCGAAAGGATTCCACCAT-3ʹ and 5ʹ-TCCGTGTTGTCTACGTCCAG-3ʹ), 50 mM KCl, 1.5 mM MgCl2, 20 mM Tris–HCl, pH 8.4, 2.5 U Taq DNA Polymerase (Invitrogen, New York, USA). Ten microliters of DNA was extracted from each sample. PCR mixtures were incubated at 94°C for 1 min, followed by 35 cycles of 1 min at 94°C, 1 min at 58°C and 1 min at 72°C. After the last cycle, samples were incubated for 10 min at 72°C to allow complete extension of the amplicons to amplify a 233 bp sequence. The nested PCR amplification mixture contained 1 μl of the first PCR mixture and the same PCR reagents described above, except that 25 pmol of each sense and anti-sense internal primers (5ʹ-TTCCACCATTGTGCTCGAAT-3ʹ and 5ʹ-TACGTCCAGACGATATGTGC-3ʹ) were used. The mixture was subjected to 94°C for 1 min, followed by 35 cycles of 1 min at 94°C, 1 min a 60°C, 1 min at 72°C and a final extension of 10 min at 72°C. Ten microliters of each reaction mixture were analyzed by 2% agarose gel electrophoresis. The amplicon bands of 211 bp were visualized using ethidium bromide staining. As a positive control, β-globin was amplified by PCR from each specimen using primers GL1 and GR2. To avoid contamination of specimens with viral DNA, standard precautions concerning spatial separation of pre- and post-PCR steps and aliquoting reagents were followed strictly. A set of negative controls (water and a negative clinical sample) was included during all steps of DNA isolation and amplification. For EBV and CMV detection, sense and antisense primers were, respectively, 5ʹ-CTTTAGAGGCGAATGGGCGCCA-3ʹ and 5ʹ-TCCAGGGCCTTCACTTCGGTCT-3ʹ and gB1 5ʹ-GAAACGCGCGGCAATCGG-3ʹ and gB2 5ʹ-TGGAACTGGAACGTTTGGC-3ʹ. Internal primers used in the nested PCR confirmatory of CMV genome amplification by PCR were gBn1 5ʹ-GCGCCGTTGATCCACACACC-3ʹ and gBn2 5ʹ-TACGCTGCAGTTCACCCCAG-3ʹ, as described elsewhere [28]. Immunostaining was performed using the mouse monoclonal antibody against HHV-8 (clone 13B10, dilution 1:20, Cell Marque, Rocklin, California, USA), mouse monoclonal antibody against EBV (clone CS.1-4, dilution 1:100, Dako, USA) and mouse monoclonal antibody against CMV (clone CCH2 + DDG9, dilution 1:100, Dako, USA) with a labeled streptavidin-biotin peroxidase system. Antigen retrieval was effected by pressure cooking in EDTA buffer, pH 8.0, for 3 min and 10 s. A fragment of known Kaposi sarcoma tissue verified by molecular study to be positive for HHV-8 was mounted on every test slide as a positive control. This study was approved by institutional review board of Clinics Hospital, Ribeirão Preto Medical School, University of São Paulo. RESULTS All six children who met clinical and radiological criteria mentioned above underwent open lung biopsy and were included. All biopsies were performed from 2004 onwards. Two girls and four boys were included, with four of them living in the same charity institution dedicated to the care of children infected or orphaned by HIV. The age at biopsy ranged from 6 to 16 years and lymphocyte CD4 count ranged from 141 to 474 cells/mm3. Table 1 lists the clinical and laboratorial features of the six patients. In all six children, ground-glass opacities and bronchiectasis were found at thorax CT scan, as shown in Fig. 1. Table 1 Clinical features in patients with interstitial lung disease Patients Gender Race Agea (years/ months) CD4 (%)a (cells/mm3) Clinical findings Home HAARTa,b Time since first ARVa Age of onset of respiratory signs and symptoms Pneumo- cystosis age Comorbiditiesa Outcome at the end of follow-up Patient 1 Female Black 8 400 (20) Cough, chronic dyspnea Institution ZDVc/DDId/ LPV/re 7 years 3 months 3 months Pulmonary tuberculosis Alive Patient 2 Female White 8/6 193 (10) Cough, chronic dyspnea Institution D4Tf/3TCg/ LPV/r 4 years 5 years 4 months No Died Patient 3 Male Black 6/8 94 (0.1) Cough, chronic dyspnea Institution D4T/3TC/ EFZh 5 years and 10 months 8 months No Bilateral lower limbs spasticity related to HIV encephalopathy Alive Patient 4 Male Black 16/3 141 (9) Cough, chronic dyspnea Institution TDFi/3TC/ LPV/r 6 years 12 years Unknown Chronic Hepatitis C infection (Child-Pugh B) Alive Patient 5 Male White 9/1 474 (11) Cough, chronic dyspnea Relatives D4T/3TC/ LPV/r 6 years and 2 months 1 year and 8 months 5 months No Alive Patient 6 Male Black 7/11 252 (22) Cough, chronic dyspnea Relatives 3TC/DDI/ LPV/r 4 years 5 years 8 months Bilateral lower limbs spasticity related to HIV encephalopathy Alive Patients Gender Race Agea (years/ months) CD4 (%)a (cells/mm3) Clinical findings Home HAARTa,b Time since first ARVa Age of onset of respiratory signs and symptoms Pneumo- cystosis age Comorbiditiesa Outcome at the end of follow-up Patient 1 Female Black 8 400 (20) Cough, chronic dyspnea Institution ZDVc/DDId/ LPV/re 7 years 3 months 3 months Pulmonary tuberculosis Alive Patient 2 Female White 8/6 193 (10) Cough, chronic dyspnea Institution D4Tf/3TCg/ LPV/r 4 years 5 years 4 months No Died Patient 3 Male Black 6/8 94 (0.1) Cough, chronic dyspnea Institution D4T/3TC/ EFZh 5 years and 10 months 8 months No Bilateral lower limbs spasticity related to HIV encephalopathy Alive Patient 4 Male Black 16/3 141 (9) Cough, chronic dyspnea Institution TDFi/3TC/ LPV/r 6 years 12 years Unknown Chronic Hepatitis C infection (Child-Pugh B) Alive Patient 5 Male White 9/1 474 (11) Cough, chronic dyspnea Relatives D4T/3TC/ LPV/r 6 years and 2 months 1 year and 8 months 5 months No Alive Patient 6 Male Black 7/11 252 (22) Cough, chronic dyspnea Relatives 3TC/DDI/ LPV/r 4 years 5 years 8 months Bilateral lower limbs spasticity related to HIV encephalopathy Alive a Data when lung biopsies were performed. b HAART = highly active antiretroviral therapy. c ZDV = Zidovudine. d DDI = Didanosine. e LPV/r = Lopinavir/ritonavir. f D4T = Stavudine. g 3TC = Lamivudine. h EFZ = Efavirenz. i TDF = Tenofovir. Table 1 Clinical features in patients with interstitial lung disease Patients Gender Race Agea (years/ months) CD4 (%)a (cells/mm3) Clinical findings Home HAARTa,b Time since first ARVa Age of onset of respiratory signs and symptoms Pneumo- cystosis age Comorbiditiesa Outcome at the end of follow-up Patient 1 Female Black 8 400 (20) Cough, chronic dyspnea Institution ZDVc/DDId/ LPV/re 7 years 3 months 3 months Pulmonary tuberculosis Alive Patient 2 Female White 8/6 193 (10) Cough, chronic dyspnea Institution D4Tf/3TCg/ LPV/r 4 years 5 years 4 months No Died Patient 3 Male Black 6/8 94 (0.1) Cough, chronic dyspnea Institution D4T/3TC/ EFZh 5 years and 10 months 8 months No Bilateral lower limbs spasticity related to HIV encephalopathy Alive Patient 4 Male Black 16/3 141 (9) Cough, chronic dyspnea Institution TDFi/3TC/ LPV/r 6 years 12 years Unknown Chronic Hepatitis C infection (Child-Pugh B) Alive Patient 5 Male White 9/1 474 (11) Cough, chronic dyspnea Relatives D4T/3TC/ LPV/r 6 years and 2 months 1 year and 8 months 5 months No Alive Patient 6 Male Black 7/11 252 (22) Cough, chronic dyspnea Relatives 3TC/DDI/ LPV/r 4 years 5 years 8 months Bilateral lower limbs spasticity related to HIV encephalopathy Alive Patients Gender Race Agea (years/ months) CD4 (%)a (cells/mm3) Clinical findings Home HAARTa,b Time since first ARVa Age of onset of respiratory signs and symptoms Pneumo- cystosis age Comorbiditiesa Outcome at the end of follow-up Patient 1 Female Black 8 400 (20) Cough, chronic dyspnea Institution ZDVc/DDId/ LPV/re 7 years 3 months 3 months Pulmonary tuberculosis Alive Patient 2 Female White 8/6 193 (10) Cough, chronic dyspnea Institution D4Tf/3TCg/ LPV/r 4 years 5 years 4 months No Died Patient 3 Male Black 6/8 94 (0.1) Cough, chronic dyspnea Institution D4T/3TC/ EFZh 5 years and 10 months 8 months No Bilateral lower limbs spasticity related to HIV encephalopathy Alive Patient 4 Male Black 16/3 141 (9) Cough, chronic dyspnea Institution TDFi/3TC/ LPV/r 6 years 12 years Unknown Chronic Hepatitis C infection (Child-Pugh B) Alive Patient 5 Male White 9/1 474 (11) Cough, chronic dyspnea Relatives D4T/3TC/ LPV/r 6 years and 2 months 1 year and 8 months 5 months No Alive Patient 6 Male Black 7/11 252 (22) Cough, chronic dyspnea Relatives 3TC/DDI/ LPV/r 4 years 5 years 8 months Bilateral lower limbs spasticity related to HIV encephalopathy Alive a Data when lung biopsies were performed. b HAART = highly active antiretroviral therapy. c ZDV = Zidovudine. d DDI = Didanosine. e LPV/r = Lopinavir/ritonavir. f D4T = Stavudine. g 3TC = Lamivudine. h EFZ = Efavirenz. i TDF = Tenofovir. Fig. 1. View largeDownload slide Ground glass opacities and bronchiectasis in thorax CT scan. Fig. 1. View largeDownload slide Ground glass opacities and bronchiectasis in thorax CT scan. Histological examination revealed FBB in four patients (Fig. 2), LIP in one and obliterans bronchiolitis in one. Kaposi’s sarcoma was excluded in all samples. In one patient (Patient 1 from Tables 1 and 2), acid-fast bacilli were also found through Ziehl-Neelsen stain, and the clinical picture improved significantly after tuberculosis treatment. PCR for HHV-8 was positive in four patients in lung tissue and in peripheral blood of another patient. As shown in Table 1, all patients in which PCR was positive in lung tissue were living together. Immunohistochemical study for HHV-8, EBV and CMV in the lung biopsy was negative in all six patients, as well as PCR for EBV and CMV. In all of these negative tests, a positive control was performed successfully. The procedure caused no complications in five patients. One patient presented a surgical wound infection by methicillin-resistant Staphylococcus aureus. Table 2 presents the combined histological and virological results for each patient. Table 2 Histological and virological findings Patients Histological diagnosis HHV-8a lung HHV-8a peripheral blood Immunohistochemistry for HHV-8 Patient 1 Obliterans bronchiolitis Positive Negative Negative Patient 2 Lymphocytic interstitial pneumonia Positive Negative Negative Patient 3 Follicular bronchiolitis Positive Negative Negative Patient 4 Follicular bronchiolitis Positive Negative Negative Patient 5 Follicular bronchiolitis Negative Negative Negative Patient 6 Follicular bronchiolitis Negative Positive Negative Patients Histological diagnosis HHV-8a lung HHV-8a peripheral blood Immunohistochemistry for HHV-8 Patient 1 Obliterans bronchiolitis Positive Negative Negative Patient 2 Lymphocytic interstitial pneumonia Positive Negative Negative Patient 3 Follicular bronchiolitis Positive Negative Negative Patient 4 Follicular bronchiolitis Positive Negative Negative Patient 5 Follicular bronchiolitis Negative Negative Negative Patient 6 Follicular bronchiolitis Negative Positive Negative a Study performed using PCR. Table 2 Histological and virological findings Patients Histological diagnosis HHV-8a lung HHV-8a peripheral blood Immunohistochemistry for HHV-8 Patient 1 Obliterans bronchiolitis Positive Negative Negative Patient 2 Lymphocytic interstitial pneumonia Positive Negative Negative Patient 3 Follicular bronchiolitis Positive Negative Negative Patient 4 Follicular bronchiolitis Positive Negative Negative Patient 5 Follicular bronchiolitis Negative Negative Negative Patient 6 Follicular bronchiolitis Negative Positive Negative Patients Histological diagnosis HHV-8a lung HHV-8a peripheral blood Immunohistochemistry for HHV-8 Patient 1 Obliterans bronchiolitis Positive Negative Negative Patient 2 Lymphocytic interstitial pneumonia Positive Negative Negative Patient 3 Follicular bronchiolitis Positive Negative Negative Patient 4 Follicular bronchiolitis Positive Negative Negative Patient 5 Follicular bronchiolitis Negative Negative Negative Patient 6 Follicular bronchiolitis Negative Positive Negative a Study performed using PCR. Fig. 2. View largeDownload slide Follicular bronchiolitis (hematoxylin and eosin, magnification 50×). Note lymphocytic infiltrate with peribronchial distribution. Fig. 2. View largeDownload slide Follicular bronchiolitis (hematoxylin and eosin, magnification 50×). Note lymphocytic infiltrate with peribronchial distribution. One patient (Patient 5, whose PCR were negative both in lung and in peripheral blood) died 2 years after biopsy owing to progressive respiratory failure. All other five patients were followed until 18 years of age (mean follow-up 8.5 years). At the end of follow-up, all but one patient (Patient 2) had a HIV viral load <40 copies/ml and all five patients had mild respiratory signs and symptoms, presenting only intermittent episodes of cough and increase in respiratory secretions. DISCUSSION As far as we know, this is the first study relating HHV-8 to interstitial lung disease in children with HIV. In this sample, HHV-8 might be a viral stimulus leading some of these patients to develop LIP or FBB, instead of EBV, whose search in lung tissue, as well as CMV, was negative in all cases. Many studies have attempted to establish a viral etiologic agent for LIP, but there is no single agent consistently related to all cases [25]. In more than half of the cases, EBV DNA is found in lung samples, indicating a possible role of this virus in LIP pathogenesis in some cases [25, 29]. Interestingly, HHV-8 and EBV are both members of subfamily Gammaherpesvirinae, both viruses are related to oncogenesis and possibly other similar feature shared may be the potential to stimulate the development of these forms of lymphoproliferative interstitial pneumonitis. Another important aspect is the presence of an underlying disease in virtually all cases of LIP, especially immune disorders, such as HIV, Sjögren syndrome and systemic lupus erythematosus [29–31]. According to the initial description by Liebow and Carrington, these features suggest that LIP may be a response to several viral stimuli in patients with underlying immune disorders [32]. Several factors might have contributed to high prevalence found in this study. First, prevalence of HHV-8 appears to vary widely. Further, lower socioeconomic status and HIV status were associated with higher prevalence of HHV-8 [33]. Another possible contributing factor is the fact that four patients were living in close contact, leading to horizontal transmission, which is described in children [9]. Most data about prevalence of HHV-8 in Brazil in both HIV-positive and -negative population are available from adults [34–39]. However, two Brazilian studies reported higher prevalence in HIV-infected children [40, 41]. The patient who presented a different histological diagnosis, obliterans bronchiolitis, had, in addition to HHV-8, acid-fast bacilli in pulmonary tissue with an important response to tuberculosis treatment, suggesting a different pathogenesis compared with the remaining five children, although the clinical and radiological findings were identical in all six cases, permitting no diagnostic distinction before the biopsies were performed. Four children presented infection with Pneumocystis jirovecii during infancy, but its implication in the pathogenesis of interstitial disease is not known. The benign clinical course in five of our patients is in agreement with evidence suggesting that prognosis of HIV-related interstitial lung disease depends on underlying disease control [42]. This study has some limitations. First, differently from cases reported in adults, these four patients with positive pulmonary HHV-8 PCR did not show positive immunohistochemical staining. Hence, there is not a direct evidence of viral replication at the time of diagnosis. However, HHV-8 DNA was present in lung tissue, possibly indicating a past infection that could have influenced the development of LIP/FBB. As HHV-8 was not found in the blood of these four patients, at the time of biopsy, the infection was probably a localized pulmonary disease without ongoing viremia. Only one patient had positive HHV-8 PCR only in peripheral blood and perhaps this infection may have influenced a proliferative lung disorder. It is not possible to accurately explain the reason why no EBV was found in any of our patients. Second, lack of data about lung function, absence of bronchoalveolar lavage studies and lack of control tests for HHV-8 in other tissues might also be limitations of our study. Last, as all patients with positive PCR in lung tissue were living in the same institution, generalization of our findings might be limited. 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Journal of Tropical PediatricsOxford University Press

Published: Nov 20, 2017

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