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Human metapneumovirus in Jordan: prevalence and clinical symptoms in hospitalized pediatric patients and molecular virus characterization
Diagnostic Microbiology and Infectious Disease 74 (2012) 288–291
Contents lists available at SciVerse ScienceDirect
Diagnostic Microbiology and Infectious Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d i a g m i c r o b i o
Virology
Human metapneumovirus in Jordan: prevalence and clinical symptoms in hospitalized pediatric patients and molecular virus characterization☆ Lina M. Qaisy a, Mamdoh M. Meqdam b,⁎, Asem Alkhateeb a, Abdallah Al-Shorman c, Hiyam O. AL-Rousan a, Mohammed S. Al-Mogbel b a b c
Department of Applied Biology, Jordan University of Science and Technology, Jordan College of Health Sciences, University of Ha'il, P.O. Box 2440, Ha'il, Saudi Arabia Department of Pediatrics, Princess Rahma Hospital, Irbid, Jordan
a r t i c l e
i n f o
Article history: Received 1 April 2012 Accepted 12 July 2012 Available online 6 September 2012 Keywords: hMPV RT-PCR Sequencing
a b s t r a c t Respiratory viral infections account for significant morbidity and mortality especially in young children worldwide. Human metapneumovirus (hMPV) causes illnesses ranging from mild respiratory problems to bronchiolitis and severe pneumonia. From January to December 2007, 220 nasopharyngeal aspirates were collected from children younger ≤13 years old hospitalized with lower respiratory tract infection to detect hMPV by revese transcription–polymerase chain reaction and to clone and sequence the hMPV-positive samples. Human metapneumovirus was detected in 28 (12.7%) specimens with a median age of 7 months (range 1.3 to 24 months). Human metapneumovirus type A and type B were detected in 26 (93%) and 8 (28.6%) of specimens, respectively. Coinfection with hMPV type A and type B was detected in 6 (21.4%) specimens positive for hMPV. The major clinical diagnosis of hMPV-positive patients was bronchiolitis (75%). Human metapneumovirus and hMPV type B were found to be significantly associated with bronchiolitis (P = 0.03 and 0.01, respectively). Human metapneumovirus and hMPV type A were found to be significantly associated with pneumonia (P = 0.004 and 0.002, respectively). The main symptoms in patients infected with hMPV were cough (92.9%), fever (82.1%), and wheezing (78.6%), with a significant association of hMPV type A with fever (P = 0.018). Human metapneumovirus was seasonally distributed; most infections with hMPV were reported in the late winter and early spring. The peak of hMPV incidence was in February (10/28; 35.7%). Sequencing of purified plasmid DNA was performed in forward and reverse direction to confirm the results of hMPV-positive samples which scored 97% identity to hMPV type A genome isolate NL/17/00 and showing C-T variation that had no effect on the amino acid sequence F(Phe)–F(Phe). © 2012 Elsevier Inc. All rights reserved.
1. Introduction Respiratory viral infections account for significant morbidity and mortality especially in young children worldwide. Despite improved sensitivity of diagnostic techniques, the cause of a significant portion of lower respiratory tract infections has still eluded identification (Boivin et al., 2003; Esper et al., 2003). Unidentified human viruses may exist, and acute and chronic diseases may be caused by such unidentified viruses. A new respiratory virus, human metapneumovirus (hMPV), was first isolated in 2001 from nasopharyngeal aspirates obtained from young children in the Netherlands with lower respiratory tract infections with clinical observations encompassing illnesses ranging
☆ Financial support for this research was provided by Jordan University of Science and Technology (grant no. 191/2007). ⁎ Corresponding author. Tel.: +966-6-5434660(Office), +966-533-630-658(Mobile); fax: +966-6-5317027. E-mail addresses: [email protected], [email protected] (M.M. Meqdam). 0732-8893/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2012.07.004
from mild respiratory problems (fever, cough, pulmonary symptoms, rhinitis) to bronchiolitis and severe pneumonia (Boivin et al., 2003; van den Hoogen et al., 2001). Human metapneumovirus is classified in the family Paramyxoviridae, subfamily Pneumovirinae, and genus Metapneumovirus (Domachowske, 2003). Several studies isolated hMPV from children with respiratory tract infections worldwide, with an incidence rate ranging from 1.5% to 41% (Ali et al., 2010; Al-Turab et al., 2011; Banerjee et al., 2011; Falsey et al., 2003; Kuypers et al., 2004; Mahalingam et al., 2006; Nissen et al., 2002; Noyola et al., 2005), and the rate of coinfection with other respiratory viruses with high rates made it difficult to clarify the effect of co-detection of hMPV on other respiratory viruses or vice versa (Al Hajjar et al., 2011; García et al., 2006; Talavera and Dorantes, 2007). Few studies have focused on the presence of hMPV in individuals without respiratory symptoms (Falsey et al., 2006), thus leaving open the possibility that hMPV might also be present in healthy, asymptomatic individuals. The clinical symptoms observed in patients infected with hMPV were fever, cough, tachypnea, dyspnea,
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wheezing, stridor, rhinitis/rhinorrhea, sore throat, myalgia, headache, and rash, conjunctivitis, pharyngitis, respiratory crackles/râles, bronchiolitis, and severe pneumonia (Boivin et al., 2003; Mahalingam et al., 2006; Noyola et al., 2005). This study was aimed at investigating the prevalence of hMPV in Jordanian children ≤13 years old hospitalized with lower respiratory tract infection (LRTI) along with its clinical symptoms, the seasonal distribution of hMPV, and cloning and DNA sequencing of hMPVpositive samples.
2. Materials and methods 2.1. Patients A total of 220 nasopharyngeal aspirate (NPA) specimens were collected from children, one sample from each child, aged ≤13 years, hospitalized with LRTI at the Princess Rahma Hospital in North Jordan during the period between January 2007 and December 2007. All information concerning patients, including clinical data, age, sex, length of hospitalization, use of antibiotics, was collected using a special form and under the supervision of a consultant pediatrician participating in this research. This study was approved by the ethical committee of the hospital, and informed consent was obtained from parents. Patients with congenital and persistent respiratory disease were not included in this study. Bronchiolitis is defined as the inflammation of the bronchioles, the smallest air passages of the lungs, while pneumonia is defined as the inflammation of the microscopic air sacs.
2.2. Clinical specimens Each sample was processed in the same day of collection. If immediate processing of the sample was not possible, the sample was stored at −70 °C pending processing. Specimens were tested for the presence of hMPV by revese transcription–polymerase chain reaction (RT- PCR) (iCycler, Bio-Rad, USA). Viral nucleic acids were extracted from patients' NPA using the High Pure Viral Nucleic Acid Kit (Roche, Mannheim, Germany) according to the protocol described by the manufacturer. The nucleic acid was eluted in 50 μL of buffer and stored at −70 °C until analyzed.
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2.4. Gel electrophoresis Five microliters of each PCR product was mixed with 2 μL 6× loading dye (Promega, USA), then loaded into wells of 3% agarose gel. Then, 2 μL of a 50- or 100-bp ladder (Promega) was loaded into the gel 1× Tris borate EDTA (TBE) running buffer which was used in the gel electrophoresis under a constant voltage (80 V) for 2 h. Five microliters of ethidium bromide (Bio-Rad), an intercalating dye, was added to the agarose gel to stain DNA and visualized under a UV transluminator provided with the gel documentation system (BioRad). The PCR product size (69 bp for type A and 74 bp for type B) was verified by matching with known bands of the DNA ladder. 2.5. Polymerase chain reaction assay for determining the two major types of hMPV A and B The amplification of hMPV type A and B was carried out as separate reactions for all positive samples of hMPV using a master mix, which contained Tag DNA polymerase, MgCl2, and dNTPs. Polymerase chain reactions were carried out in a total volume of 25 μL and contained 12.5 μL master mix (Promega), 9.9 μL of nuclease free water, 0.8 μL (200 nM) of each hMPV forward and reverse for hMPV type A or B, and 1 μL of cDNA. Each run included negative control with all PCR mix, and a positive control for hMPV. The amplification protocol was carried as described by Kuypers et al. (2004) except for the initial cDNA synthesis step. The PCR product size was verified by gel electrophoresis. 2.6. Sequencing of hMPV-positive samples by cloning the PCR products The PCR products of the positive NPA specimens were ligated to pJET1.2/blunt cloning vector (Fermentas) and transformed into competent E. coli cells. Plasmids were purified using the GeneJET™ Plasmid Miniprep Kit (Fermentas) and sequenced in forward and reverse directions by primers included in the Clone JET™ PCR Cloning Kit. Sequencing of the purified plasmid DNA was done using the BigDye Terminator Cycle Sequencing Kit version 3.1 (Qiagen) and the ABI 310 DNA sequencer (Applied Biosystems, USA). The sequences of samples were analyzed using ChromasPro Software version 1.34 (available at: http://www.technelysium.com.au/ChromasPro.html). Blast N (available at: http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used for comparison of the obtained sequences to nucleotide sequence databases.
2.3. Detection of hMPV
2.7. Statistical analysis
The amplification of hMPV type A and B (iCycler) was carried out using the Qiagen One-Step RT-PCR Kit (Qiagen, Hilden, Germany) according to the protocol described by the manufacturer. Multiplex RT-PCR reactions were carried out in a total volume of 25 μL 5× Qiagen One-Step RT-PCR buffer (containing 12.5 μmol/L of MgCl2), 5 μL 5× Qsolution, 1 μL Qiagen One-Step RT-PCR Enzyme Mix, 1 μL dNTP mix (containing 10 μmol/L of each dNTP), and 0.8 μL (200 nmol/L) of each primer. For hMPV type A the forward primer (GCC GTT AGC TTC AGT CAA TTC AA), and the reverse primer (TCC AGC ATT GTC TGA AAA TTG C) and for hMPV type B the forward primer (GCT GTC AGC TTC AGT CAA TTC AA), and the reverse primer (GTT ATC CCT GCA TTG TCT GAA AAC T) in one reaction mix, 4.8 μL RNase-free water, and 5 μL of extracted RNA. Each run included negative control; all PCR mix components except DNA and RNA, and positive control for hMPV (supplied by Dr Eric C.J. Claas, Department of Medical Microbiology, Leiden University Medical Center, Netherlands). Amplification parameters were carried out according to Kuypers et al. (2004) as follows: initial cDNA synthesis for 30 min at 48 °C followed by 10 min at 95 °C, and 40 cycles were performed with denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min, and extension at 72 °C for 30 s.
Data were collected and analyzed using SPSS version 15 (SPSS, Chicago, IL, USA). Fisher's Exact Test was used to test the correlation between discrete variables. Alpha was taken as 0.05 in all analyses. 3. Results Two hundred and twenty NPA specimens were collected from children aged ≤13 years who were hospitalized with lower respiratory tract infections from January to December, 2007. The median age of children was 6 months (range 0.5–156 months). The male-to-female ratio was 1.78:1. Human metapneumovirus was detected in 28 (12.7%) specimens. The male-to-female ratio was 3:1 with a median age of 7 months (range 1.3–24 months). Human metapneumovirus type A was detected in 26 (93%), and hMPV type B was detected in 8 (28.6%) hMPV-positive specimens. Co-infection with hMPV type A and type B was detected in 6 (21.4%) specimens positive for hMPV. The age distribution of the hMPV-positive patients is shown in Fig. 1. hMPV was detected in 22 (78.6%) children less than 1 year old; 5 (17.9%) in each group of 0 to b3 months and 9 to b12 months, 8 (28.6%) in 3 to b6
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Fig. 2 shows the monthly distribution of hMPV infection. Most infections with hMPV were reported from January to March, November, and December. The peak of hMPV incidence was in February with 10 (35.7%) patients infected, followed in December with 6 (21.4%) patients and in January and March each with 5 (17.9%) patients. Sequencing of purified plasmid DNA was performed in the forward and reverse direction to confirm the results of hMPV-positive samples which scored 97% identity to hMPV type A genome isolate NL/17/00 and showing C-T variation that had no effect on the amino acid sequence F(Phe)–F(Phe). The pair-wise alignment was performed in the forward direction and showed the C-T variation at codon 12. This variation does not affect the amino acid sequence F(TTC)–F(TTT). Alignment sequence was also performed in the reverse direction and showed the G-A variation at codon 12 that confirmed the C-T variation in the forward direction. This variation in hMPV F gene has been reported many times in GenBank in EU857545, EU857544, and AF371337 isolates. Fig. 1. Etiologic agent with different age group of children positive for hMPV.
months, 4 (14.3%) in 6 to b9 months, and 6 (21.4%) in children more than 1 year old; 1 (3.6%) in 12 to b15 months, 2 (7.1%) in each group of 15 to b18 months and 21 to b24 moths, and 1 (3.6%) in N2 years. Clinical diagnosis and frequency of signs and symptoms among children with hMPV are shown in Table 1. The major clinical diagnosis of hMPV-positive patients were bronchiolitis 21(75%) and pneumonia 5(17.9%). hMPV and hMPV type B were significantly associated with bronchiolitis (P = 0.03 and 0.01, respectively). hMPV and hMPV type A were significantly associated with pneumonia (P = 0.004 and 0.002, respectively). The main symptoms in patients infected with hMPV were cough (92.9%), fever (82.1%), and wheezing (78.6%), where hMPV type A was significantly associated with fever (P = 0.018). The mean of hospital period for 28 children positive for hMPV was 4.5 days and the median was 4 days (range 2 to 9 days) (data not shown). Twenty-seven (96.4%) patients positive for hMPV had taken antibiotics (data not shown).
Table 1 The clinical diagnosis, signs, and symptoms in 28 children positive for hMPV. Clinical diagnosis
No. of positive hMPVs (%)
No. of positive hMPV A (%)
No. of positive hMPV B (%)
Bronchiolitis Bronchiolitis and wheezing chest Wheezing chest Pneumonia Other respiratory infection Total Signs and symptoms Cough Fever Wheezing Tachypnea Retraction Crepitation Vomiting Cyanosis Grunting Diarrhea Conjunctivitis Othera Total
21 (75)⁎ 1 (3.6)
19 (73.1) 1 (3.8)
8 (100)⁎ 0 (0)
1 (3.6) 5 (17.9)⁎⁎ 0 (0) 28
1 (3.8) 5 (19.2)⁎⁎ 0 (0) 26
0 (0) 0 (0) 0 (0) 8
26 (92.9) 23 (82.1) 22 (78.6) 11 (39.3) 14 (50) 6 (21.4) 7 (25) 7 (25) 1 (3.6) 2 (7.1) 0 (0) 10 (35.7) 28
24 (29.3) 23 (88.5)⁎⁎⁎ 22 (84.6) 10 (38.5) 12 (46.1) 6 (23.1) 7 (27) 5 (19.2) 1 (3.8) 2 (7.9) 0 (0) 10 (38.5) 26
7 (87.5) 5 (62.5) 5 (62.5) 5 (62.5) 7 (87.5) 0 (0) 3 (37.5) 2 (25) 1 (12.5) 1 (12.5) 0 (0) 2 (25) 8
a Decreased air entry, difficult breathing, irritability and poor feeding, lymphatic enlarge, shortness of breath, and rapid breathing. ⁎ Significant difference between bronchiolitis and infection with hMPV and hMPV type B (P = 0.026 and 0.01, respectively). ⁎⁎ Significant difference between pneumonia and patients infected with hMPV and hMPV type A (P = 0.004 and 0.002, respectively). ⁎⁎⁎ Significant difference between fever and infection with hMPV type A (P = 0.018).
4. Discussion Several studies have demonstrated that hMPV was detected in children suffering from URTI and LRTI. In this study, hMPV was detected in 28 (12.7%) hospitalized children suffering from LRTI. The prevalence of hMPV reported in different parts of the world using both conventional and real-time RT-PCR methods varies between 1.5% and 41% (Ali et al., 2010; Al-Turab et al., 2011; Banerjee et al., 2011; Falsey et al., 2003; Kuypers et al., 2004; Mahalingam et al., 2006; Nissen et al., 2002; Noyola et al., 2005). In the Middle East, there was a variation in the prevalence of hMPV; in Jordan, the prevalence of hMPV was 6% in Amman and 2.5% in Zarka (Ali et al., 2010; Kaplan et al., 2008); 5.4% in Kuwait (Al-Turab et al., 2011), 8.3% in Saudi Arabia (Al Hajjar et al., 2011); 10.8% in Israel (Regev et al., 2006); and 11% in Yemen (Al-Sonboli et al., 2006). The data on the incidence of hMPV infections from other countries are inconsistent due to different calculation methods, year-to-year variation within a given region, and geographical locations (Serafino et al., 2004). The method of collection and processing of specimens and the time of specimens collection may have also played a role in this difference. Our study indicated that both lineages A and B are represented in our population and indicated a predominant circulation of hMPV genotype A; hMPV type A was detected in 26 (93%) and hMPV type B in 8 (28.6%) of hMPV-positive samples. Mixed genotypic infection was detected in 6 (21.4%) of hMPV-positive samples. A similar finding was reported in Italy (Sarasini et al., 2006). The predominant circulation of hMPV genotype A has been reported in many countries: in Mexico by Noyola et al. (2005) and in Israel by Regev et al. (2006). However, a shift to a predominant B genotype has been reported in Italy (Sarasini et al., 2006) and in the USA (Agapov et al., 2006). The major clinical features associated with hMPV infection were bronchiolitis and pneumonia (Falsey et al., 2006; Kaplan et al., 2008). Patients infected with hMPV showed a significant difference in contracting bronchiolitis and pneumonia (P = 0.001 and 0.03,
Fig. 2. Monthly distribution of children positive for hMPV.
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respectively) (Bharaj et al., 2009). The common clinical diagnosis was bronchiolitis (57.1%) (P b 0.05) and pneumonia (25%) (Caracciolo et al., 2008). In this study, bronchiolitis (75%) and pneumonia (17.9%) were recorded as the major clinical syndromes associated with hMPVpositive patients. Furthermore, 73% of patients infected with hMPV type A had bronchiolitis and 19.2% had pneumonia, and all patients infected with hMPV type B had bronchiolitis. Patients infected with hMPV and hMPV type B showed a significance difference in contracting bronchiolitis (P = 0.03 and 0.01, respectively) compared to patients with no bronchiolitis. Patients infected with hMPV and hMPV type A showed a highly significant difference in contracting pneumonia (P = 0.004 and 0.002, respectively). The major clinical signs and symptoms associated with hMPV infection were cough, fever, and wheezing (Al-Turab et al., 2011; Mahalingam et al., 2006). In this study, the major clinical symptoms and signs noted in children with hMPV were cough (92.9%), fever (82.1%), and wheezing (78.6%). Statistical analysis showed a significant difference between fever and infection with hMPV type A (P = 0.018). In the present study, oral and intravenous antibiotics were given excessively (94.1%) to infected patients (data not shown). Unavailability of routine laboratory diagnosis of viral illness in Jordan may be the cause of the high percentage of antibiotic therapy among our patients. To avoid secondary bacterial infection, pediatric physicians in Jordan gave antibiotics to their patients (Nasrallah et al., 2002). The seasonal pattern of hMPV was reported in different countries worldwide with most cases reported during the winter and early spring months. In Mexico, the months with the highest activity of hMPV were February and March (Noyola et al., 2005). In North America, the highest numbers of hMPV cases were detected between January and April (Esper et al., 2003; van den Hoogen et al., 2002). In Israel, increased hMPV incidence was observed during February/March for the 2002 to 2003 season (Regev et al., 2006). The present study demonstrated that hMPV infections were mostly detected during the winter months and early spring; from January to March, November, and December, the peak of hMPV incidence was in February (55.6%). This seasonal pattern is consistent with previous studies. The NL/17/00 reference strain for hMPV type A genome isolate had been reported in several countries worldwide (Kuypers et al., 2004; van den Hoogen et al., 2002). The F gene sequences show a very high degree of conservation (Esper et al., 2003; Monica et al., 2006; van den Hoogen et al., 2002). In this study, sequencing of purified plasmid DNA was performed to confirm the results of hMPV-positive samples. Sequencing was performed in the forward and reverse directions. Alignment sequence of specimens with F gene in hMPV type A genome isolate NL/17/00 scored an identity equal to 97% and showed a C-T variation that had no effect on the amino acid sequence F(Phe)–F(Phe). This variation in hMPV F gene has been reported many times in GenBank (EU857545, EU857544, and AF371337) and demonstrated in several studies (van den Hoogen et al., 2001; Yang et al., 2009), but it is reported for the first time in Jordan. Acknowledgments The authors acknowledge Dr Eric C.J. Claas (Department of Medical Microbiology, Leiden University Medical Centre, Netherlands) for providing the positive control of hMPV. The authors also acknowledge Mr Manny C. Ritchie for the critical review of the manuscript.
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Contents lists available at SciVerse ScienceDirect
Diagnostic Microbiology and Infectious Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d i a g m i c r o b i o
Virology
Human metapneumovirus in Jordan: prevalence and clinical symptoms in hospitalized pediatric patients and molecular virus characterization☆ Lina M. Qaisy a, Mamdoh M. Meqdam b,⁎, Asem Alkhateeb a, Abdallah Al-Shorman c, Hiyam O. AL-Rousan a, Mohammed S. Al-Mogbel b a b c
Department of Applied Biology, Jordan University of Science and Technology, Jordan College of Health Sciences, University of Ha'il, P.O. Box 2440, Ha'il, Saudi Arabia Department of Pediatrics, Princess Rahma Hospital, Irbid, Jordan
a r t i c l e
i n f o
Article history: Received 1 April 2012 Accepted 12 July 2012 Available online 6 September 2012 Keywords: hMPV RT-PCR Sequencing
a b s t r a c t Respiratory viral infections account for significant morbidity and mortality especially in young children worldwide. Human metapneumovirus (hMPV) causes illnesses ranging from mild respiratory problems to bronchiolitis and severe pneumonia. From January to December 2007, 220 nasopharyngeal aspirates were collected from children younger ≤13 years old hospitalized with lower respiratory tract infection to detect hMPV by revese transcription–polymerase chain reaction and to clone and sequence the hMPV-positive samples. Human metapneumovirus was detected in 28 (12.7%) specimens with a median age of 7 months (range 1.3 to 24 months). Human metapneumovirus type A and type B were detected in 26 (93%) and 8 (28.6%) of specimens, respectively. Coinfection with hMPV type A and type B was detected in 6 (21.4%) specimens positive for hMPV. The major clinical diagnosis of hMPV-positive patients was bronchiolitis (75%). Human metapneumovirus and hMPV type B were found to be significantly associated with bronchiolitis (P = 0.03 and 0.01, respectively). Human metapneumovirus and hMPV type A were found to be significantly associated with pneumonia (P = 0.004 and 0.002, respectively). The main symptoms in patients infected with hMPV were cough (92.9%), fever (82.1%), and wheezing (78.6%), with a significant association of hMPV type A with fever (P = 0.018). Human metapneumovirus was seasonally distributed; most infections with hMPV were reported in the late winter and early spring. The peak of hMPV incidence was in February (10/28; 35.7%). Sequencing of purified plasmid DNA was performed in forward and reverse direction to confirm the results of hMPV-positive samples which scored 97% identity to hMPV type A genome isolate NL/17/00 and showing C-T variation that had no effect on the amino acid sequence F(Phe)–F(Phe). © 2012 Elsevier Inc. All rights reserved.
1. Introduction Respiratory viral infections account for significant morbidity and mortality especially in young children worldwide. Despite improved sensitivity of diagnostic techniques, the cause of a significant portion of lower respiratory tract infections has still eluded identification (Boivin et al., 2003; Esper et al., 2003). Unidentified human viruses may exist, and acute and chronic diseases may be caused by such unidentified viruses. A new respiratory virus, human metapneumovirus (hMPV), was first isolated in 2001 from nasopharyngeal aspirates obtained from young children in the Netherlands with lower respiratory tract infections with clinical observations encompassing illnesses ranging
☆ Financial support for this research was provided by Jordan University of Science and Technology (grant no. 191/2007). ⁎ Corresponding author. Tel.: +966-6-5434660(Office), +966-533-630-658(Mobile); fax: +966-6-5317027. E-mail addresses: [email protected], [email protected] (M.M. Meqdam). 0732-8893/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2012.07.004
from mild respiratory problems (fever, cough, pulmonary symptoms, rhinitis) to bronchiolitis and severe pneumonia (Boivin et al., 2003; van den Hoogen et al., 2001). Human metapneumovirus is classified in the family Paramyxoviridae, subfamily Pneumovirinae, and genus Metapneumovirus (Domachowske, 2003). Several studies isolated hMPV from children with respiratory tract infections worldwide, with an incidence rate ranging from 1.5% to 41% (Ali et al., 2010; Al-Turab et al., 2011; Banerjee et al., 2011; Falsey et al., 2003; Kuypers et al., 2004; Mahalingam et al., 2006; Nissen et al., 2002; Noyola et al., 2005), and the rate of coinfection with other respiratory viruses with high rates made it difficult to clarify the effect of co-detection of hMPV on other respiratory viruses or vice versa (Al Hajjar et al., 2011; García et al., 2006; Talavera and Dorantes, 2007). Few studies have focused on the presence of hMPV in individuals without respiratory symptoms (Falsey et al., 2006), thus leaving open the possibility that hMPV might also be present in healthy, asymptomatic individuals. The clinical symptoms observed in patients infected with hMPV were fever, cough, tachypnea, dyspnea,
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wheezing, stridor, rhinitis/rhinorrhea, sore throat, myalgia, headache, and rash, conjunctivitis, pharyngitis, respiratory crackles/râles, bronchiolitis, and severe pneumonia (Boivin et al., 2003; Mahalingam et al., 2006; Noyola et al., 2005). This study was aimed at investigating the prevalence of hMPV in Jordanian children ≤13 years old hospitalized with lower respiratory tract infection (LRTI) along with its clinical symptoms, the seasonal distribution of hMPV, and cloning and DNA sequencing of hMPVpositive samples.
2. Materials and methods 2.1. Patients A total of 220 nasopharyngeal aspirate (NPA) specimens were collected from children, one sample from each child, aged ≤13 years, hospitalized with LRTI at the Princess Rahma Hospital in North Jordan during the period between January 2007 and December 2007. All information concerning patients, including clinical data, age, sex, length of hospitalization, use of antibiotics, was collected using a special form and under the supervision of a consultant pediatrician participating in this research. This study was approved by the ethical committee of the hospital, and informed consent was obtained from parents. Patients with congenital and persistent respiratory disease were not included in this study. Bronchiolitis is defined as the inflammation of the bronchioles, the smallest air passages of the lungs, while pneumonia is defined as the inflammation of the microscopic air sacs.
2.2. Clinical specimens Each sample was processed in the same day of collection. If immediate processing of the sample was not possible, the sample was stored at −70 °C pending processing. Specimens were tested for the presence of hMPV by revese transcription–polymerase chain reaction (RT- PCR) (iCycler, Bio-Rad, USA). Viral nucleic acids were extracted from patients' NPA using the High Pure Viral Nucleic Acid Kit (Roche, Mannheim, Germany) according to the protocol described by the manufacturer. The nucleic acid was eluted in 50 μL of buffer and stored at −70 °C until analyzed.
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2.4. Gel electrophoresis Five microliters of each PCR product was mixed with 2 μL 6× loading dye (Promega, USA), then loaded into wells of 3% agarose gel. Then, 2 μL of a 50- or 100-bp ladder (Promega) was loaded into the gel 1× Tris borate EDTA (TBE) running buffer which was used in the gel electrophoresis under a constant voltage (80 V) for 2 h. Five microliters of ethidium bromide (Bio-Rad), an intercalating dye, was added to the agarose gel to stain DNA and visualized under a UV transluminator provided with the gel documentation system (BioRad). The PCR product size (69 bp for type A and 74 bp for type B) was verified by matching with known bands of the DNA ladder. 2.5. Polymerase chain reaction assay for determining the two major types of hMPV A and B The amplification of hMPV type A and B was carried out as separate reactions for all positive samples of hMPV using a master mix, which contained Tag DNA polymerase, MgCl2, and dNTPs. Polymerase chain reactions were carried out in a total volume of 25 μL and contained 12.5 μL master mix (Promega), 9.9 μL of nuclease free water, 0.8 μL (200 nM) of each hMPV forward and reverse for hMPV type A or B, and 1 μL of cDNA. Each run included negative control with all PCR mix, and a positive control for hMPV. The amplification protocol was carried as described by Kuypers et al. (2004) except for the initial cDNA synthesis step. The PCR product size was verified by gel electrophoresis. 2.6. Sequencing of hMPV-positive samples by cloning the PCR products The PCR products of the positive NPA specimens were ligated to pJET1.2/blunt cloning vector (Fermentas) and transformed into competent E. coli cells. Plasmids were purified using the GeneJET™ Plasmid Miniprep Kit (Fermentas) and sequenced in forward and reverse directions by primers included in the Clone JET™ PCR Cloning Kit. Sequencing of the purified plasmid DNA was done using the BigDye Terminator Cycle Sequencing Kit version 3.1 (Qiagen) and the ABI 310 DNA sequencer (Applied Biosystems, USA). The sequences of samples were analyzed using ChromasPro Software version 1.34 (available at: http://www.technelysium.com.au/ChromasPro.html). Blast N (available at: http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used for comparison of the obtained sequences to nucleotide sequence databases.
2.3. Detection of hMPV
2.7. Statistical analysis
The amplification of hMPV type A and B (iCycler) was carried out using the Qiagen One-Step RT-PCR Kit (Qiagen, Hilden, Germany) according to the protocol described by the manufacturer. Multiplex RT-PCR reactions were carried out in a total volume of 25 μL 5× Qiagen One-Step RT-PCR buffer (containing 12.5 μmol/L of MgCl2), 5 μL 5× Qsolution, 1 μL Qiagen One-Step RT-PCR Enzyme Mix, 1 μL dNTP mix (containing 10 μmol/L of each dNTP), and 0.8 μL (200 nmol/L) of each primer. For hMPV type A the forward primer (GCC GTT AGC TTC AGT CAA TTC AA), and the reverse primer (TCC AGC ATT GTC TGA AAA TTG C) and for hMPV type B the forward primer (GCT GTC AGC TTC AGT CAA TTC AA), and the reverse primer (GTT ATC CCT GCA TTG TCT GAA AAC T) in one reaction mix, 4.8 μL RNase-free water, and 5 μL of extracted RNA. Each run included negative control; all PCR mix components except DNA and RNA, and positive control for hMPV (supplied by Dr Eric C.J. Claas, Department of Medical Microbiology, Leiden University Medical Center, Netherlands). Amplification parameters were carried out according to Kuypers et al. (2004) as follows: initial cDNA synthesis for 30 min at 48 °C followed by 10 min at 95 °C, and 40 cycles were performed with denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min, and extension at 72 °C for 30 s.
Data were collected and analyzed using SPSS version 15 (SPSS, Chicago, IL, USA). Fisher's Exact Test was used to test the correlation between discrete variables. Alpha was taken as 0.05 in all analyses. 3. Results Two hundred and twenty NPA specimens were collected from children aged ≤13 years who were hospitalized with lower respiratory tract infections from January to December, 2007. The median age of children was 6 months (range 0.5–156 months). The male-to-female ratio was 1.78:1. Human metapneumovirus was detected in 28 (12.7%) specimens. The male-to-female ratio was 3:1 with a median age of 7 months (range 1.3–24 months). Human metapneumovirus type A was detected in 26 (93%), and hMPV type B was detected in 8 (28.6%) hMPV-positive specimens. Co-infection with hMPV type A and type B was detected in 6 (21.4%) specimens positive for hMPV. The age distribution of the hMPV-positive patients is shown in Fig. 1. hMPV was detected in 22 (78.6%) children less than 1 year old; 5 (17.9%) in each group of 0 to b3 months and 9 to b12 months, 8 (28.6%) in 3 to b6
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Fig. 2 shows the monthly distribution of hMPV infection. Most infections with hMPV were reported from January to March, November, and December. The peak of hMPV incidence was in February with 10 (35.7%) patients infected, followed in December with 6 (21.4%) patients and in January and March each with 5 (17.9%) patients. Sequencing of purified plasmid DNA was performed in the forward and reverse direction to confirm the results of hMPV-positive samples which scored 97% identity to hMPV type A genome isolate NL/17/00 and showing C-T variation that had no effect on the amino acid sequence F(Phe)–F(Phe). The pair-wise alignment was performed in the forward direction and showed the C-T variation at codon 12. This variation does not affect the amino acid sequence F(TTC)–F(TTT). Alignment sequence was also performed in the reverse direction and showed the G-A variation at codon 12 that confirmed the C-T variation in the forward direction. This variation in hMPV F gene has been reported many times in GenBank in EU857545, EU857544, and AF371337 isolates. Fig. 1. Etiologic agent with different age group of children positive for hMPV.
months, 4 (14.3%) in 6 to b9 months, and 6 (21.4%) in children more than 1 year old; 1 (3.6%) in 12 to b15 months, 2 (7.1%) in each group of 15 to b18 months and 21 to b24 moths, and 1 (3.6%) in N2 years. Clinical diagnosis and frequency of signs and symptoms among children with hMPV are shown in Table 1. The major clinical diagnosis of hMPV-positive patients were bronchiolitis 21(75%) and pneumonia 5(17.9%). hMPV and hMPV type B were significantly associated with bronchiolitis (P = 0.03 and 0.01, respectively). hMPV and hMPV type A were significantly associated with pneumonia (P = 0.004 and 0.002, respectively). The main symptoms in patients infected with hMPV were cough (92.9%), fever (82.1%), and wheezing (78.6%), where hMPV type A was significantly associated with fever (P = 0.018). The mean of hospital period for 28 children positive for hMPV was 4.5 days and the median was 4 days (range 2 to 9 days) (data not shown). Twenty-seven (96.4%) patients positive for hMPV had taken antibiotics (data not shown).
Table 1 The clinical diagnosis, signs, and symptoms in 28 children positive for hMPV. Clinical diagnosis
No. of positive hMPVs (%)
No. of positive hMPV A (%)
No. of positive hMPV B (%)
Bronchiolitis Bronchiolitis and wheezing chest Wheezing chest Pneumonia Other respiratory infection Total Signs and symptoms Cough Fever Wheezing Tachypnea Retraction Crepitation Vomiting Cyanosis Grunting Diarrhea Conjunctivitis Othera Total
21 (75)⁎ 1 (3.6)
19 (73.1) 1 (3.8)
8 (100)⁎ 0 (0)
1 (3.6) 5 (17.9)⁎⁎ 0 (0) 28
1 (3.8) 5 (19.2)⁎⁎ 0 (0) 26
0 (0) 0 (0) 0 (0) 8
26 (92.9) 23 (82.1) 22 (78.6) 11 (39.3) 14 (50) 6 (21.4) 7 (25) 7 (25) 1 (3.6) 2 (7.1) 0 (0) 10 (35.7) 28
24 (29.3) 23 (88.5)⁎⁎⁎ 22 (84.6) 10 (38.5) 12 (46.1) 6 (23.1) 7 (27) 5 (19.2) 1 (3.8) 2 (7.9) 0 (0) 10 (38.5) 26
7 (87.5) 5 (62.5) 5 (62.5) 5 (62.5) 7 (87.5) 0 (0) 3 (37.5) 2 (25) 1 (12.5) 1 (12.5) 0 (0) 2 (25) 8
a Decreased air entry, difficult breathing, irritability and poor feeding, lymphatic enlarge, shortness of breath, and rapid breathing. ⁎ Significant difference between bronchiolitis and infection with hMPV and hMPV type B (P = 0.026 and 0.01, respectively). ⁎⁎ Significant difference between pneumonia and patients infected with hMPV and hMPV type A (P = 0.004 and 0.002, respectively). ⁎⁎⁎ Significant difference between fever and infection with hMPV type A (P = 0.018).
4. Discussion Several studies have demonstrated that hMPV was detected in children suffering from URTI and LRTI. In this study, hMPV was detected in 28 (12.7%) hospitalized children suffering from LRTI. The prevalence of hMPV reported in different parts of the world using both conventional and real-time RT-PCR methods varies between 1.5% and 41% (Ali et al., 2010; Al-Turab et al., 2011; Banerjee et al., 2011; Falsey et al., 2003; Kuypers et al., 2004; Mahalingam et al., 2006; Nissen et al., 2002; Noyola et al., 2005). In the Middle East, there was a variation in the prevalence of hMPV; in Jordan, the prevalence of hMPV was 6% in Amman and 2.5% in Zarka (Ali et al., 2010; Kaplan et al., 2008); 5.4% in Kuwait (Al-Turab et al., 2011), 8.3% in Saudi Arabia (Al Hajjar et al., 2011); 10.8% in Israel (Regev et al., 2006); and 11% in Yemen (Al-Sonboli et al., 2006). The data on the incidence of hMPV infections from other countries are inconsistent due to different calculation methods, year-to-year variation within a given region, and geographical locations (Serafino et al., 2004). The method of collection and processing of specimens and the time of specimens collection may have also played a role in this difference. Our study indicated that both lineages A and B are represented in our population and indicated a predominant circulation of hMPV genotype A; hMPV type A was detected in 26 (93%) and hMPV type B in 8 (28.6%) of hMPV-positive samples. Mixed genotypic infection was detected in 6 (21.4%) of hMPV-positive samples. A similar finding was reported in Italy (Sarasini et al., 2006). The predominant circulation of hMPV genotype A has been reported in many countries: in Mexico by Noyola et al. (2005) and in Israel by Regev et al. (2006). However, a shift to a predominant B genotype has been reported in Italy (Sarasini et al., 2006) and in the USA (Agapov et al., 2006). The major clinical features associated with hMPV infection were bronchiolitis and pneumonia (Falsey et al., 2006; Kaplan et al., 2008). Patients infected with hMPV showed a significant difference in contracting bronchiolitis and pneumonia (P = 0.001 and 0.03,
Fig. 2. Monthly distribution of children positive for hMPV.
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respectively) (Bharaj et al., 2009). The common clinical diagnosis was bronchiolitis (57.1%) (P b 0.05) and pneumonia (25%) (Caracciolo et al., 2008). In this study, bronchiolitis (75%) and pneumonia (17.9%) were recorded as the major clinical syndromes associated with hMPVpositive patients. Furthermore, 73% of patients infected with hMPV type A had bronchiolitis and 19.2% had pneumonia, and all patients infected with hMPV type B had bronchiolitis. Patients infected with hMPV and hMPV type B showed a significance difference in contracting bronchiolitis (P = 0.03 and 0.01, respectively) compared to patients with no bronchiolitis. Patients infected with hMPV and hMPV type A showed a highly significant difference in contracting pneumonia (P = 0.004 and 0.002, respectively). The major clinical signs and symptoms associated with hMPV infection were cough, fever, and wheezing (Al-Turab et al., 2011; Mahalingam et al., 2006). In this study, the major clinical symptoms and signs noted in children with hMPV were cough (92.9%), fever (82.1%), and wheezing (78.6%). Statistical analysis showed a significant difference between fever and infection with hMPV type A (P = 0.018). In the present study, oral and intravenous antibiotics were given excessively (94.1%) to infected patients (data not shown). Unavailability of routine laboratory diagnosis of viral illness in Jordan may be the cause of the high percentage of antibiotic therapy among our patients. To avoid secondary bacterial infection, pediatric physicians in Jordan gave antibiotics to their patients (Nasrallah et al., 2002). The seasonal pattern of hMPV was reported in different countries worldwide with most cases reported during the winter and early spring months. In Mexico, the months with the highest activity of hMPV were February and March (Noyola et al., 2005). In North America, the highest numbers of hMPV cases were detected between January and April (Esper et al., 2003; van den Hoogen et al., 2002). In Israel, increased hMPV incidence was observed during February/March for the 2002 to 2003 season (Regev et al., 2006). The present study demonstrated that hMPV infections were mostly detected during the winter months and early spring; from January to March, November, and December, the peak of hMPV incidence was in February (55.6%). This seasonal pattern is consistent with previous studies. The NL/17/00 reference strain for hMPV type A genome isolate had been reported in several countries worldwide (Kuypers et al., 2004; van den Hoogen et al., 2002). The F gene sequences show a very high degree of conservation (Esper et al., 2003; Monica et al., 2006; van den Hoogen et al., 2002). In this study, sequencing of purified plasmid DNA was performed to confirm the results of hMPV-positive samples. Sequencing was performed in the forward and reverse directions. Alignment sequence of specimens with F gene in hMPV type A genome isolate NL/17/00 scored an identity equal to 97% and showed a C-T variation that had no effect on the amino acid sequence F(Phe)–F(Phe). This variation in hMPV F gene has been reported many times in GenBank (EU857545, EU857544, and AF371337) and demonstrated in several studies (van den Hoogen et al., 2001; Yang et al., 2009), but it is reported for the first time in Jordan. Acknowledgments The authors acknowledge Dr Eric C.J. Claas (Department of Medical Microbiology, Leiden University Medical Centre, Netherlands) for providing the positive control of hMPV. The authors also acknowledge Mr Manny C. Ritchie for the critical review of the manuscript.
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