Indian J Pediatr DOI 10.1007/s12098-013-1286-2
ORIGINAL ARTICLE
Serotype Determination of Adenoviruses in Children with Respiratory Infection Yusuf Ghasemi & Manoochehr Makvandi & Ali Reza Samarbafzadeh & Ahmad Nejati & Saeed Najafifard & Niloofar Neisi & Mojtaba Rasti & Kambiz Ahmadi & Ahmad Shamsizadeh & Roya Nikfar
Received: 1 June 2013 / Accepted: 23 October 2013 # Dr. K C Chaudhuri Foundation 2014
Abstract Objective To determine adenovirus serotypes among children with acute respiratory infection (ARI) in Khoozestan province, Iran during years 2010–2011. Methods One hundred sixty three nasopharyngeal swabs were collected from children between 1 and 15 y who were hospitalized for the acute respiratory infection. The viral DNA was extracted from the nasopharyngeal swabs and adenoviruses were detected by Nested PCR. Positive PCR samples were sequenced in order to confirm the adenovirus serotypes. Results Out of 163 samples, 30 (18.4 %) were positive for adenoviruses by nested PCR. Twenty two PCR products were sequenced and recognized as Ad5 and Ad2 serotypes including 19 (86.3 %) Ad5 and 3 (13.7 %) Ad2. Conclusions This study reveals that adenoviruses with Ad5 predominance are important cause of respiratory tract infection in children. M. Makvandi : A. R. Samarbafzadeh : A. Shamsizadeh Health Research Institute, Infectious and Tropical Disease Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Y. Ghasemi : M. Makvandi : A. R. Samarbafzadeh : A. Nejati : S. Najafifard : N. Neisi : M. Rasti Department of Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran K. Ahmadi Department of Statistics, School of Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran A. Shamsizadeh : R. Nikfar Pediatric Infectious Disease Section, Abozar Children’s Hospital, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran M. Makvandi (*) Golestan Boulevard, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran e-mail: [email protected]
Keywords Lower respiratory tract infection . Adenoviruses . Polymerase chain reaction . Sequencing . Serotype determination . Respiratory infection
Introduction Human adenoviruses are important ubiquitous pathogens responsible for a variety of illnesses, including acute respiratory infection (ARI), pharyngoconjunctival fever, hemorrhagic cystitis, hepatitis, nephritis, myocarditis, and meningoencephalitis, gastroenteritis and nosocomial infections [1–3]. Human adenoviruses are icosahedral, non-enveloped, double stranded viruses, 70–90 nm in diameter. They belong to adenoviridae family and genus mastadenovirus [4]. Human adenoviruses are classified into 54 serotypes and 7 species (A-G) on the basis of serology, whole genome sequencing and phylogenic analysis. The adenovirus serotypes most frequently associated with respiratory infection are classified as subspecies B1 (Ad3, Ad7, and Ad21), species C (Ad1, Ad2, Ad5, and Ad6) and species E (Ad4) [5–7]. Ad3, Ad7 and, less frequently, Ad 21, are involved in severe infections associated with outbreaks [8, 9]. Adenovirus infection has worldwide distribution; serological studies showed that Ad 2 and 5 are more common in Europe, Asia and United state, Ad3 and Ad7 have been related with outbreaks as reported from Japan, Korea, UK, US, South America and China [10, 11]. Clinical manifestations of Ad-associated respiratory disease range from febrile respiratory illness (FRI) to pharyngitis and pneumonia and may depend on the Ad serotype, the age of the patient, and other factors [12]. Adenoviruses are responsible for 5–10 % of respiratory infection, especially among children and young adults. Acute respiratory infection is one of the most important cause of morbidity and mortality in children under 5 y old in developing countries [13]. Major respiratory infection is due to group B, C and E adenoviruses. Sometimes adenoviral
Indian J Pediatr
respiratory infection clinically is not differentiable from influenza, parainfluenza, RSV and bacterial respiratory infection [14, 15]. Children and adults with severe respiratory infection mainly are infected with Ad3 and Ad7 serotypes. Other serotype of species B (Ad11, Ad14, Ad16, Ad21, Ad34 and Ad35) rarely cause acute respiratory infections [16, 17]. Member of species C (Ad1, Ad2, Ad5, Ad6) are responsible for 59 % of adenoviral infections [6]. Serotype 4 is one of the main cause of acute respiratory infections in military personnel [17]. The purpose of this study was to determine the relative frequency of adenovirus’s infection in children less than 15 y with signs of acute respiratory infections in Khuzestan province south-west of Iran. The adenovirus’s serotypes were determined by sequencing.
Material and Methods The present cross sectional study was carried out on hospitalized children with acute respiratory infection (ARIs) in the Khoozestan province during Oct 2010 through Sep 2011 (Fig. 1). One hundred sixty three children were randomly registered in the study. The patients aged between 1-15 y with mean age of 6.25±4.5 y. All the 163 patients were negative for influenza A and B viruses by the Real Time PCR. The physicians collected history from each ARI patient and noted clinical signs and symptoms including cough, runny nose, fever, difficult breathing, rales and wheezing. The nasopharyngeal samples were collected from each patient and kept in 2 mL microtube containing transport medium M199. DNA was extracted from 200 μL of the each sample using High Pure Viral Nucleic Acid Kit (Roche Diagnostic Systems Inc., Mannheim, Germany) according to the manufacturer’s instruction. The specific adenovirus primers consisting of ADH-01 (ACTACAAYATTGGCTACCAGG) and ADH-02 (CAAAACATAAAGAAGKGTGGGC) with 440 bp PCR product and ADH-I1 (AACTTCCAGCCCATGAGCMG) and ADH-I2 (CTCAAAAGTCATGTCBAGCGC) with 330 bp PCR product which are conserved for adenoviruses hexon were used for nested PCR [18]. The first round of PCR was performed with ADH-01 and ADH-02 primers in 50 μL reaction containing 45 μL of reaction mixture (50 μM KCl, 20 μM Tris – HCl (pH 8.4), 1.5 Mm Magnesium Chloride,
Fig. 1 The distribution of collected samples during months of 2010– 2011 and the number of positive cases in the different months of year
0.2 mM each deoxynucleoside triphosphate, 0.5 μM each primer, 1 U Taq DNA Polymerase (Roche Diagnostic Systems Inc., Mannheim, Germany) and 5 μL of the extracted DNA. The tubes containing reaction mixture were placed in thermal cycler (Techne TC-512, UK) with following program: an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 64 °C for 45 s, and extension at 72 °C for 45 s. The final step was extension at 72 °C for 5 min. The second round of PCR were performed by using 1 μL of first round of PCR products with reaction mixture containing the ADH-I1 and ADH-I2 primers with the same PCR condition that mentioned for the first round. The results of the final products (330 bp) were observed by ethidium bromide staining in 2 % agarose gel (Fig. 2). Of those 30 patients positive for adenoviruses, 22 PCR product were sequenced for determination of adenoviruses serotypes, using the PRISM 377 automatic DNA sequencer (AppliedQ6 Biosystems). The phylogenic tree was drawn based on “hexon” region of adenovirus by neighbor-joining method and Kimura-two parameter distance model using MEGA software version 5.1 (Fig. 3). Adenoviruses reference sequences used for comparisons with sequences from this study were obtained from GenBank (Table 1). Data were analyzed by chi square test using SPSS software version 20; P 0.05), there was no any significant relationship between adenovirus infection and different age groups. The index results indicate that most of the
Table 2 Adenovirus 2 and 5 distribution between different age groups Age groups
Ad5 no (%)
Ad2 no (%)
Total no (%)
Under 5 y 6–10 y 11–15 y
11(84.6) 3 (75) 5 (100)
2 (15.4) 1 (25) 0 (0)
13 (59.1) 4 (18.1) 5 (22.8)
Indian J Pediatr Table 3 Adenovirus’s serotype distribution in different countries
Author’s name
Place
Number of Ad2 and Ad5 cases
Year
Serotypes
Cases with adenoviral infection
Gao et al. [19] Moura et al. [21] Metzgar et al. [11] Hong et al. [22] Biere et al. [20] Present study
China Brazil Egypt South Korea Germany Iran
Ad2 (10), Ad5 (6) Ad2 (2), Ad5 (1) Ad2 (3), Ad5 (2) Ad2 (13) Ad2 (29), Ad5 (11) Ad2 (3), Ad5 (19)
2012 2007 2005 2001 2010 2010
3,7,1,2,5,6,57,4 1,2,5,7 3,7,16,11,1,2,5 2,3,7 1,2,3,5,6 2,5
171 11 88 87 101 30
adenoviral infections occurred in autumn, but there was not any significant relationship between season and adenoviral infection (P =0.603). The results of sequencing were analyzed by Mega software version 5.1 and were registered in NCBI gene bank with following accessible gene code: JX164000.1 to JX164014 for adenovirus 5 and JX163993.1 to JX163999.1 for adenovirus 2. The adenovirus serotype 5 with 19 cases was predominant while only 3 cases were confirmed for adenovirus serotype 2.
Discussion The adenovirus serotypes 5 and 2 were detected in children with acute respiratory infection and adenovirus serotype 5 was recognized as predominant serotype in Khoozestan province. In this study 22 PCR products were sequenced, phylogenic tree was drawn and compared with same serotypes from different parts of the world. The results revealed that Ad5 serotypes found in the study were similar to Ad5 serotypes detected from different parts of the world, while Ad2 serotypes show new evolutionary branch that had much evolutionary distance with other detected Ad2 serotypes in Malaysia, Germany, Japan, Korea, Paraguay (Fig. 3). The prevalence of adenovirus serotype 2 and 5 among the patients with respiratory infection reported in China, Germany, Egypt and Brazil are as follows, 5.8 %, 28.7 %, 3.4 %, 18.1 % for Ad2 and 3.5 %, 10.9 %, 2.2 % and 9 % for Ad5 respectively [1, 19–21]. Details have been shown in Table 3. In the present study most of the detected adenoviruses were classified in adenovirus group C that is comparable with a study that has been conducted in Brazil [21] but the outbreaks of Ad7 and Ad3 (group B1) with severe respiratory infection were reported from China and Egypt [11, 19]. Based on these reports, differences between the index results and mentioned studies is because of adenovirus worldwide prevalence and its different geographical distribution pattern. There was no evidence of other adenoviral outbreaks in Khoozestan province but it requires more investigations each year. The result of sequencing including 19(86.3 %) Ad5 and 3 (13.7 %) Ad2 is similar to the studies reported from other countries like Canada and
Brazil [23–26]. The prevalence of adenovirus (6.5 %) in children with respiratory infection has been reported in Iran [27]. Certain adenovirus serotypes including 4,6,7,14 and 21 have been reported in children as well as adults with acute respiratory infection [16, 17]. The relative frequency of adenoviral infection in 3 different age groups were 19.1 % (group 1), 18.4 % (group 2) and 16.7 % (group 3). There was no significant relationship between adenoviral infection and different age groups (P =1). Echavarria et al., reported that relative frequency of adenoviral infection among children between 6 mo and 4 y was 9.4 % [23] and Winter et al., reported that relative frequency of adenoviral infection in children was 10 % [28]. The higher relative frequency of adenoviral respiratory infection (18.4 %) in the present study might be due to low sanitation measurement. There was no significant relationship between adenoviral infection and gender (P =0.269). Based on authors knowledge, this is the first report about serotype determination of respiratory adenoviruses in their province.
Conclusions The present study reveals that adenoviruses with predominant Ad5 are important cause of respiratory tract infection in children. PCR offers a rapid method for detecting common respiratory viruses. Acknowledgments This study was done as part of thesis with 89108 registration number in Health research institute, Infectious and Tropical Diseases Research Center, Jundishapur University of Medical Science, Ahvaz, Iran. The authors are thankful to Director of Infectious and Tropical Disease Research Center, Ahwaz Jundishapur University of Medical Sciences, Ahwaz, Iran for the financial support to carry out this project. Contributions YG: Did experiments and manuscript preparation; MM: Study design, funding support and manuscript preparation; ARS: Manuscript preparation; AN, NN and SN: Did experiments; MR: Data analysis and manuscript preparation; KA: Statistical analysis; AS and RN: Clinical consultation. MM will act as guarantor for this paper. Conflict of Interest None.
Indian J Pediatr Role of Funding Source Infectious and Tropical Disease Research Center, Ahwaz Jundishapur University of Medical Sciences, Ahwaz, Iran.
15.
16.
References 1. Elizabet GR, Dan HB. Adenoviruses. In: Mandell GL, Bennet JE, Dolin R, eds. Principles and Practice of Infectious Diseases. Philadephia: Churchill Livingstome; 2010. pp. 2077–33. 2. Vaideeswar P, Bavdekar SB, Jadhav SM, Balan R, Pandit SP. Necrotizing adenoviral pneumonia: Manifestation of nosocomial infection in pediatric intensive care unit. Indian J Pediatr. 2008;75:1171–4. 3. Yeolekar LR, Damle RG, Kamat AN, Khude MR, Simha V, Pandit AN. Respiratory viruses in acute respiratory tract infections in Western India. Indian J Pediatr. 2008;75:341–5. 4. Berk AJ. Adenoviridae the viruses and their replicarion. In: Knipe DM, Howley PM, eds. Fields Virology. Philadelphia: Lippincott Williams & Wilkins; 2007. pp. 2355–94. 5. Ishiko H, Aoki K. Spread of epidemic keratoconjunctivitis due to a novel serotype of human adenovirus in Japan. J Clin Microbiol. 2009;47:2678–9. 6. Saffold JM, Harrach B, Ganac RD, Gozum MA, Wilfred PD, Riedel B, et al. New adenovirus species found in a patient presenting with gastroenteritis. J Virol. 2007;81:5978–84. 7. Walsh MP, Chintakuntlawar A, Robinson CM, Madisch I, Harrach B, Hudson NR, et al. Evidence of molecular evolution driven by recombination events influencing tropism in a novel human adenovirus that causes epidemic keratoconjunctivitis. PLoS One. 2009;4:e5635. 8. Kim YJ, Hong JY, Lee HJ, Shin SH, Kim YK, Inada T, et al. Genome type analysis of adenovirus types 3 and 7 isolated during successive outbreaks of lower respiratory tract infections in children. J Clin Microbiol. 2003;41:4594–9. 9. Vogels R, Zuijdgeest D, van Rijnsoever R, Hartkoorn E, Damen I, de Bethune MP, et al. Replication deficient human adenovirus type 35 vectors for gene transfer and vaccination: Efficient human cell infection and bypass of preexisting adenovirus immunity. J Virol. 2003;77:8263–71. 10. Yu B, Wang Z, Dong J, Wang C, Gu L, Sun C, et al. A serological survey of human adenovirus serotype 2 and 5 circulating pediatric populations in Changchun, China, 2011. Virol J. 2012;9:287–92. 11. Metzgar D, Osuna M, Yingst S, Rakha M, Earhart K, Elyan D, et al. PCR analysis of egyptian respiratory adenovirus isolates, including identification of species, serotypes, and coinfections. J Clin Microbiol. 2005;43:5743–52. 12. Shimitz H, Wigand R, Heinrich W. Worldwide epidemiology of human adenovirus infection. Am J Epidemiol. 1983;117:455–66. 13. Williams BG, Gouws E, Boschi-Pinto C, Bryce J, Dye C. Estimates of world-wide distribution of child deaths from acute respiratory infections. Lancet Infect Dis. 2002;2:25–32. 14. Burman LA, Trollfors B, Anderson B, Henrichsen J, Juto P, Kallings I, et al. Diagnosis of pneumonia by cultures, bacterial and viral
17. 18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
antigen detection tests, and serology with special refence to antibodies against pneumococcal antigens. J Infect Dis. 1991;163:1087–93. Cherry JD. Adenoviruses. In: Feigin-Cherry’s Textbook of Pediatric Infectious Disease. Philadelphia: Saunders company; 1998. pp. 1666–8. Brandt CD, Kim HW, Vargosko AJ, Jeffries BC, Arrobio JO, Rindge B, et al. Infections in 18,000 infants and children in a controlled study of respiratory tract disease. Adenovirus pathogenicity in relation to serologic type and illness syndrome. Am J Epidemiol. 1969;90:484–500. Rubin BA. Clinical picture and epidemiology of adenovirus infections (a review). Acta Microbiol Hung. 1993;40:303–23. Pauschinger M, Bowles NE, Fuentes-Garcia J, Pham V, Kuhl U, Schwimmbeck PL, et al. Detection of adenoviral genome in the myocardium of adult patients with idiopathic left ventricular dysfunction. Circulation. 1999;99:1348–54. Gao XQ, Jin Y, Xie ZP, Gao HC, Xie LY, Zhang J, et al. The epidemiological study of adenovirus in children with respiratory tract infections in Nanjing area from 2010 to 2011. Bing Du Xue Bao. 2012;28:531–5. Biere B, Schweiger B. Human adenoviruses in respiratory infections: Sequencing of the hexon hypervariable region reveals high sequence variability. J Clin Virol. 2010;47:366–71. Moura FE, Mesquita JR, Portes SA, Ramos EA, Siqueira MM. Antigenic and genomic characterization of adenovirus associated to respiratory infections in children living in Northeast Brazil. Mem Inst Oswaldo Cruz. 2007;102:937–41. Hong JY, Lee HJ, Piedra PA, Choi EH, Park KH, Koh YY, et al. Lower respiratory tract infections due to adenovirus in hospitalized Korean children: Epidemiology, clinical features, and prognosis. Clin Infect Dis. 2001;32:1423–9. Echavarria M, Maldonado D, Elbert G, Videla C, Rappaport R, Carballal G. Use of PCR To demonstrate presence of adenovirus species B, C, or F as well as coinfection with two adenovirus species in children with flu-like symptoms. J Clin Microbiol. 2006;44:625–7. Alharb S, Caeseele PV, Consunji-Araneta R, Zoubeidi T, Fanella S, Souid AK, et al. Epidemiology of severe pediatric adenovirus lower respiratory tract infections in Manitoba, Canada, 1991–2005. BMC Infect Dis. 2012;12:55. Luiz LN, Leite JP, Yokosawa J, Carneiro BM, Pereira FE, Oliveira TF, et al. Molecular characterization of adenoviruses from children presenting with acute respiratory disease in Uberlândia, Minas Gerais, Brazil, and detection of an isolate genetically related to feline adenovirus. Mem Inst Oswaldo Cruz. 2010;105:712–6. Pumariega T, Savón C, Muné M, Cancio R, Gonzalez G, Valdivia A, et al. Isolation and identification of adenovirus in hospitalized children under five years with acute respiratory disease in Havana. Cuba Mem Inst Oswaldo Cruz. 2000;95:859–61. Barati M, Talebi-Taher M, Noorbakhsh S, Ebrahimi Taj F, Tabatabaei A. Diagnosis of the adenovirus, RSV, and influenza virus by rapid detection test (immunochromatography) in children with acute respiratory infection. Iranian J Pediatr Soc. 2010;2:79–83. Winter GF, Hallam NF, Hargreaves FD, Molyneaux PJ, Burns SM, Inglis JM. Respiratory viruses in a hospitalized pediatrics in Edinburgh (1985–1994). J Infect Dis. 1996;33:207–11.
ORIGINAL ARTICLE
Serotype Determination of Adenoviruses in Children with Respiratory Infection Yusuf Ghasemi & Manoochehr Makvandi & Ali Reza Samarbafzadeh & Ahmad Nejati & Saeed Najafifard & Niloofar Neisi & Mojtaba Rasti & Kambiz Ahmadi & Ahmad Shamsizadeh & Roya Nikfar
Received: 1 June 2013 / Accepted: 23 October 2013 # Dr. K C Chaudhuri Foundation 2014
Abstract Objective To determine adenovirus serotypes among children with acute respiratory infection (ARI) in Khoozestan province, Iran during years 2010–2011. Methods One hundred sixty three nasopharyngeal swabs were collected from children between 1 and 15 y who were hospitalized for the acute respiratory infection. The viral DNA was extracted from the nasopharyngeal swabs and adenoviruses were detected by Nested PCR. Positive PCR samples were sequenced in order to confirm the adenovirus serotypes. Results Out of 163 samples, 30 (18.4 %) were positive for adenoviruses by nested PCR. Twenty two PCR products were sequenced and recognized as Ad5 and Ad2 serotypes including 19 (86.3 %) Ad5 and 3 (13.7 %) Ad2. Conclusions This study reveals that adenoviruses with Ad5 predominance are important cause of respiratory tract infection in children. M. Makvandi : A. R. Samarbafzadeh : A. Shamsizadeh Health Research Institute, Infectious and Tropical Disease Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Y. Ghasemi : M. Makvandi : A. R. Samarbafzadeh : A. Nejati : S. Najafifard : N. Neisi : M. Rasti Department of Virology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran K. Ahmadi Department of Statistics, School of Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran A. Shamsizadeh : R. Nikfar Pediatric Infectious Disease Section, Abozar Children’s Hospital, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran M. Makvandi (*) Golestan Boulevard, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran e-mail: [email protected]
Keywords Lower respiratory tract infection . Adenoviruses . Polymerase chain reaction . Sequencing . Serotype determination . Respiratory infection
Introduction Human adenoviruses are important ubiquitous pathogens responsible for a variety of illnesses, including acute respiratory infection (ARI), pharyngoconjunctival fever, hemorrhagic cystitis, hepatitis, nephritis, myocarditis, and meningoencephalitis, gastroenteritis and nosocomial infections [1–3]. Human adenoviruses are icosahedral, non-enveloped, double stranded viruses, 70–90 nm in diameter. They belong to adenoviridae family and genus mastadenovirus [4]. Human adenoviruses are classified into 54 serotypes and 7 species (A-G) on the basis of serology, whole genome sequencing and phylogenic analysis. The adenovirus serotypes most frequently associated with respiratory infection are classified as subspecies B1 (Ad3, Ad7, and Ad21), species C (Ad1, Ad2, Ad5, and Ad6) and species E (Ad4) [5–7]. Ad3, Ad7 and, less frequently, Ad 21, are involved in severe infections associated with outbreaks [8, 9]. Adenovirus infection has worldwide distribution; serological studies showed that Ad 2 and 5 are more common in Europe, Asia and United state, Ad3 and Ad7 have been related with outbreaks as reported from Japan, Korea, UK, US, South America and China [10, 11]. Clinical manifestations of Ad-associated respiratory disease range from febrile respiratory illness (FRI) to pharyngitis and pneumonia and may depend on the Ad serotype, the age of the patient, and other factors [12]. Adenoviruses are responsible for 5–10 % of respiratory infection, especially among children and young adults. Acute respiratory infection is one of the most important cause of morbidity and mortality in children under 5 y old in developing countries [13]. Major respiratory infection is due to group B, C and E adenoviruses. Sometimes adenoviral
Indian J Pediatr
respiratory infection clinically is not differentiable from influenza, parainfluenza, RSV and bacterial respiratory infection [14, 15]. Children and adults with severe respiratory infection mainly are infected with Ad3 and Ad7 serotypes. Other serotype of species B (Ad11, Ad14, Ad16, Ad21, Ad34 and Ad35) rarely cause acute respiratory infections [16, 17]. Member of species C (Ad1, Ad2, Ad5, Ad6) are responsible for 59 % of adenoviral infections [6]. Serotype 4 is one of the main cause of acute respiratory infections in military personnel [17]. The purpose of this study was to determine the relative frequency of adenovirus’s infection in children less than 15 y with signs of acute respiratory infections in Khuzestan province south-west of Iran. The adenovirus’s serotypes were determined by sequencing.
Material and Methods The present cross sectional study was carried out on hospitalized children with acute respiratory infection (ARIs) in the Khoozestan province during Oct 2010 through Sep 2011 (Fig. 1). One hundred sixty three children were randomly registered in the study. The patients aged between 1-15 y with mean age of 6.25±4.5 y. All the 163 patients were negative for influenza A and B viruses by the Real Time PCR. The physicians collected history from each ARI patient and noted clinical signs and symptoms including cough, runny nose, fever, difficult breathing, rales and wheezing. The nasopharyngeal samples were collected from each patient and kept in 2 mL microtube containing transport medium M199. DNA was extracted from 200 μL of the each sample using High Pure Viral Nucleic Acid Kit (Roche Diagnostic Systems Inc., Mannheim, Germany) according to the manufacturer’s instruction. The specific adenovirus primers consisting of ADH-01 (ACTACAAYATTGGCTACCAGG) and ADH-02 (CAAAACATAAAGAAGKGTGGGC) with 440 bp PCR product and ADH-I1 (AACTTCCAGCCCATGAGCMG) and ADH-I2 (CTCAAAAGTCATGTCBAGCGC) with 330 bp PCR product which are conserved for adenoviruses hexon were used for nested PCR [18]. The first round of PCR was performed with ADH-01 and ADH-02 primers in 50 μL reaction containing 45 μL of reaction mixture (50 μM KCl, 20 μM Tris – HCl (pH 8.4), 1.5 Mm Magnesium Chloride,
Fig. 1 The distribution of collected samples during months of 2010– 2011 and the number of positive cases in the different months of year
0.2 mM each deoxynucleoside triphosphate, 0.5 μM each primer, 1 U Taq DNA Polymerase (Roche Diagnostic Systems Inc., Mannheim, Germany) and 5 μL of the extracted DNA. The tubes containing reaction mixture were placed in thermal cycler (Techne TC-512, UK) with following program: an initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 64 °C for 45 s, and extension at 72 °C for 45 s. The final step was extension at 72 °C for 5 min. The second round of PCR were performed by using 1 μL of first round of PCR products with reaction mixture containing the ADH-I1 and ADH-I2 primers with the same PCR condition that mentioned for the first round. The results of the final products (330 bp) were observed by ethidium bromide staining in 2 % agarose gel (Fig. 2). Of those 30 patients positive for adenoviruses, 22 PCR product were sequenced for determination of adenoviruses serotypes, using the PRISM 377 automatic DNA sequencer (AppliedQ6 Biosystems). The phylogenic tree was drawn based on “hexon” region of adenovirus by neighbor-joining method and Kimura-two parameter distance model using MEGA software version 5.1 (Fig. 3). Adenoviruses reference sequences used for comparisons with sequences from this study were obtained from GenBank (Table 1). Data were analyzed by chi square test using SPSS software version 20; P 0.05), there was no any significant relationship between adenovirus infection and different age groups. The index results indicate that most of the
Table 2 Adenovirus 2 and 5 distribution between different age groups Age groups
Ad5 no (%)
Ad2 no (%)
Total no (%)
Under 5 y 6–10 y 11–15 y
11(84.6) 3 (75) 5 (100)
2 (15.4) 1 (25) 0 (0)
13 (59.1) 4 (18.1) 5 (22.8)
Indian J Pediatr Table 3 Adenovirus’s serotype distribution in different countries
Author’s name
Place
Number of Ad2 and Ad5 cases
Year
Serotypes
Cases with adenoviral infection
Gao et al. [19] Moura et al. [21] Metzgar et al. [11] Hong et al. [22] Biere et al. [20] Present study
China Brazil Egypt South Korea Germany Iran
Ad2 (10), Ad5 (6) Ad2 (2), Ad5 (1) Ad2 (3), Ad5 (2) Ad2 (13) Ad2 (29), Ad5 (11) Ad2 (3), Ad5 (19)
2012 2007 2005 2001 2010 2010
3,7,1,2,5,6,57,4 1,2,5,7 3,7,16,11,1,2,5 2,3,7 1,2,3,5,6 2,5
171 11 88 87 101 30
adenoviral infections occurred in autumn, but there was not any significant relationship between season and adenoviral infection (P =0.603). The results of sequencing were analyzed by Mega software version 5.1 and were registered in NCBI gene bank with following accessible gene code: JX164000.1 to JX164014 for adenovirus 5 and JX163993.1 to JX163999.1 for adenovirus 2. The adenovirus serotype 5 with 19 cases was predominant while only 3 cases were confirmed for adenovirus serotype 2.
Discussion The adenovirus serotypes 5 and 2 were detected in children with acute respiratory infection and adenovirus serotype 5 was recognized as predominant serotype in Khoozestan province. In this study 22 PCR products were sequenced, phylogenic tree was drawn and compared with same serotypes from different parts of the world. The results revealed that Ad5 serotypes found in the study were similar to Ad5 serotypes detected from different parts of the world, while Ad2 serotypes show new evolutionary branch that had much evolutionary distance with other detected Ad2 serotypes in Malaysia, Germany, Japan, Korea, Paraguay (Fig. 3). The prevalence of adenovirus serotype 2 and 5 among the patients with respiratory infection reported in China, Germany, Egypt and Brazil are as follows, 5.8 %, 28.7 %, 3.4 %, 18.1 % for Ad2 and 3.5 %, 10.9 %, 2.2 % and 9 % for Ad5 respectively [1, 19–21]. Details have been shown in Table 3. In the present study most of the detected adenoviruses were classified in adenovirus group C that is comparable with a study that has been conducted in Brazil [21] but the outbreaks of Ad7 and Ad3 (group B1) with severe respiratory infection were reported from China and Egypt [11, 19]. Based on these reports, differences between the index results and mentioned studies is because of adenovirus worldwide prevalence and its different geographical distribution pattern. There was no evidence of other adenoviral outbreaks in Khoozestan province but it requires more investigations each year. The result of sequencing including 19(86.3 %) Ad5 and 3 (13.7 %) Ad2 is similar to the studies reported from other countries like Canada and
Brazil [23–26]. The prevalence of adenovirus (6.5 %) in children with respiratory infection has been reported in Iran [27]. Certain adenovirus serotypes including 4,6,7,14 and 21 have been reported in children as well as adults with acute respiratory infection [16, 17]. The relative frequency of adenoviral infection in 3 different age groups were 19.1 % (group 1), 18.4 % (group 2) and 16.7 % (group 3). There was no significant relationship between adenoviral infection and different age groups (P =1). Echavarria et al., reported that relative frequency of adenoviral infection among children between 6 mo and 4 y was 9.4 % [23] and Winter et al., reported that relative frequency of adenoviral infection in children was 10 % [28]. The higher relative frequency of adenoviral respiratory infection (18.4 %) in the present study might be due to low sanitation measurement. There was no significant relationship between adenoviral infection and gender (P =0.269). Based on authors knowledge, this is the first report about serotype determination of respiratory adenoviruses in their province.
Conclusions The present study reveals that adenoviruses with predominant Ad5 are important cause of respiratory tract infection in children. PCR offers a rapid method for detecting common respiratory viruses. Acknowledgments This study was done as part of thesis with 89108 registration number in Health research institute, Infectious and Tropical Diseases Research Center, Jundishapur University of Medical Science, Ahvaz, Iran. The authors are thankful to Director of Infectious and Tropical Disease Research Center, Ahwaz Jundishapur University of Medical Sciences, Ahwaz, Iran for the financial support to carry out this project. Contributions YG: Did experiments and manuscript preparation; MM: Study design, funding support and manuscript preparation; ARS: Manuscript preparation; AN, NN and SN: Did experiments; MR: Data analysis and manuscript preparation; KA: Statistical analysis; AS and RN: Clinical consultation. MM will act as guarantor for this paper. Conflict of Interest None.
Indian J Pediatr Role of Funding Source Infectious and Tropical Disease Research Center, Ahwaz Jundishapur University of Medical Sciences, Ahwaz, Iran.
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16.
References 1. Elizabet GR, Dan HB. Adenoviruses. In: Mandell GL, Bennet JE, Dolin R, eds. Principles and Practice of Infectious Diseases. Philadephia: Churchill Livingstome; 2010. pp. 2077–33. 2. Vaideeswar P, Bavdekar SB, Jadhav SM, Balan R, Pandit SP. Necrotizing adenoviral pneumonia: Manifestation of nosocomial infection in pediatric intensive care unit. Indian J Pediatr. 2008;75:1171–4. 3. Yeolekar LR, Damle RG, Kamat AN, Khude MR, Simha V, Pandit AN. Respiratory viruses in acute respiratory tract infections in Western India. Indian J Pediatr. 2008;75:341–5. 4. Berk AJ. Adenoviridae the viruses and their replicarion. In: Knipe DM, Howley PM, eds. Fields Virology. Philadelphia: Lippincott Williams & Wilkins; 2007. pp. 2355–94. 5. Ishiko H, Aoki K. Spread of epidemic keratoconjunctivitis due to a novel serotype of human adenovirus in Japan. J Clin Microbiol. 2009;47:2678–9. 6. Saffold JM, Harrach B, Ganac RD, Gozum MA, Wilfred PD, Riedel B, et al. New adenovirus species found in a patient presenting with gastroenteritis. J Virol. 2007;81:5978–84. 7. Walsh MP, Chintakuntlawar A, Robinson CM, Madisch I, Harrach B, Hudson NR, et al. Evidence of molecular evolution driven by recombination events influencing tropism in a novel human adenovirus that causes epidemic keratoconjunctivitis. PLoS One. 2009;4:e5635. 8. Kim YJ, Hong JY, Lee HJ, Shin SH, Kim YK, Inada T, et al. Genome type analysis of adenovirus types 3 and 7 isolated during successive outbreaks of lower respiratory tract infections in children. J Clin Microbiol. 2003;41:4594–9. 9. Vogels R, Zuijdgeest D, van Rijnsoever R, Hartkoorn E, Damen I, de Bethune MP, et al. Replication deficient human adenovirus type 35 vectors for gene transfer and vaccination: Efficient human cell infection and bypass of preexisting adenovirus immunity. J Virol. 2003;77:8263–71. 10. Yu B, Wang Z, Dong J, Wang C, Gu L, Sun C, et al. A serological survey of human adenovirus serotype 2 and 5 circulating pediatric populations in Changchun, China, 2011. Virol J. 2012;9:287–92. 11. Metzgar D, Osuna M, Yingst S, Rakha M, Earhart K, Elyan D, et al. PCR analysis of egyptian respiratory adenovirus isolates, including identification of species, serotypes, and coinfections. J Clin Microbiol. 2005;43:5743–52. 12. Shimitz H, Wigand R, Heinrich W. Worldwide epidemiology of human adenovirus infection. Am J Epidemiol. 1983;117:455–66. 13. Williams BG, Gouws E, Boschi-Pinto C, Bryce J, Dye C. Estimates of world-wide distribution of child deaths from acute respiratory infections. Lancet Infect Dis. 2002;2:25–32. 14. Burman LA, Trollfors B, Anderson B, Henrichsen J, Juto P, Kallings I, et al. Diagnosis of pneumonia by cultures, bacterial and viral
17. 18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
antigen detection tests, and serology with special refence to antibodies against pneumococcal antigens. J Infect Dis. 1991;163:1087–93. Cherry JD. Adenoviruses. In: Feigin-Cherry’s Textbook of Pediatric Infectious Disease. Philadelphia: Saunders company; 1998. pp. 1666–8. Brandt CD, Kim HW, Vargosko AJ, Jeffries BC, Arrobio JO, Rindge B, et al. Infections in 18,000 infants and children in a controlled study of respiratory tract disease. Adenovirus pathogenicity in relation to serologic type and illness syndrome. Am J Epidemiol. 1969;90:484–500. Rubin BA. Clinical picture and epidemiology of adenovirus infections (a review). Acta Microbiol Hung. 1993;40:303–23. Pauschinger M, Bowles NE, Fuentes-Garcia J, Pham V, Kuhl U, Schwimmbeck PL, et al. Detection of adenoviral genome in the myocardium of adult patients with idiopathic left ventricular dysfunction. Circulation. 1999;99:1348–54. Gao XQ, Jin Y, Xie ZP, Gao HC, Xie LY, Zhang J, et al. The epidemiological study of adenovirus in children with respiratory tract infections in Nanjing area from 2010 to 2011. Bing Du Xue Bao. 2012;28:531–5. Biere B, Schweiger B. Human adenoviruses in respiratory infections: Sequencing of the hexon hypervariable region reveals high sequence variability. J Clin Virol. 2010;47:366–71. Moura FE, Mesquita JR, Portes SA, Ramos EA, Siqueira MM. Antigenic and genomic characterization of adenovirus associated to respiratory infections in children living in Northeast Brazil. Mem Inst Oswaldo Cruz. 2007;102:937–41. Hong JY, Lee HJ, Piedra PA, Choi EH, Park KH, Koh YY, et al. Lower respiratory tract infections due to adenovirus in hospitalized Korean children: Epidemiology, clinical features, and prognosis. Clin Infect Dis. 2001;32:1423–9. Echavarria M, Maldonado D, Elbert G, Videla C, Rappaport R, Carballal G. Use of PCR To demonstrate presence of adenovirus species B, C, or F as well as coinfection with two adenovirus species in children with flu-like symptoms. J Clin Microbiol. 2006;44:625–7. Alharb S, Caeseele PV, Consunji-Araneta R, Zoubeidi T, Fanella S, Souid AK, et al. Epidemiology of severe pediatric adenovirus lower respiratory tract infections in Manitoba, Canada, 1991–2005. BMC Infect Dis. 2012;12:55. Luiz LN, Leite JP, Yokosawa J, Carneiro BM, Pereira FE, Oliveira TF, et al. Molecular characterization of adenoviruses from children presenting with acute respiratory disease in Uberlândia, Minas Gerais, Brazil, and detection of an isolate genetically related to feline adenovirus. Mem Inst Oswaldo Cruz. 2010;105:712–6. Pumariega T, Savón C, Muné M, Cancio R, Gonzalez G, Valdivia A, et al. Isolation and identification of adenovirus in hospitalized children under five years with acute respiratory disease in Havana. Cuba Mem Inst Oswaldo Cruz. 2000;95:859–61. Barati M, Talebi-Taher M, Noorbakhsh S, Ebrahimi Taj F, Tabatabaei A. Diagnosis of the adenovirus, RSV, and influenza virus by rapid detection test (immunochromatography) in children with acute respiratory infection. Iranian J Pediatr Soc. 2010;2:79–83. Winter GF, Hallam NF, Hargreaves FD, Molyneaux PJ, Burns SM, Inglis JM. Respiratory viruses in a hospitalized pediatrics in Edinburgh (1985–1994). J Infect Dis. 1996;33:207–11.
