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Indian Journal of Medical Microbiology, (2015) 33(1): 43-50
Original Article
Surveillance of Acute Respiratory Infections in Mumbai during 2011-12 *RD Chavan, ST Kothari, K Zunjarrao, AS Chowdhary
Abstract Purpose: Acute respiratory infections (ARIs) are a leading cause of morbidity and mortality in individuals aged less than 5 years. ARI often leads to hospitalisation, and it has been indicated that causative viral and bacterial infections go undetermined and results in the occurrence of resistant strains. The objective of the study was to assess the prevalence of various viral and bacterial infections in patients with ARIs. Materials and Methods: Two hundred samples were collected from July 2011 to July 2012 with patients suffering from ARI. Viral and bacterial infections were determined by real time reverse transcriptase polymerase chain reaction. Results: Influenza-like illness (ILI) consisted of 109 patients and ARI consisted of 91 patients. Pandemic influenza A H1N1 was the major viral infection with 21 (19.2%) patients in ILI as compared with 16 (17.4%) patients in ARI. Respiratory syncytial virus (RSV) was found to be 1 (0.9%) in ILI and ARI. Viral co-infections were 16 (14.4%) in ILI and 4 (4.37%) in ARI where pandemic influenza A H1N1 and influenza type B were major contributors. In bacterial infections, Streptococcus pneumoniae with 11 (10.9%) cases were predominant in both the groups. Bacterial co-infection accounted for only 1 (1.09%) case in both the groups but the most significant finding was the viral-bacterial co-infection in which Haemophilus influenzae was the major co-infecting bacteria with the influenza viruses with 4 (4.36%) cases as compared with Streptotoccus pneumoniae. Conclusion: This data indicate the need to undertake continued surveillance that will help to better define the circulation of respiratory viruses along with the bacterial infections. Key words: Acute respiratory infection, influenza-like illness, pandemic influenza A H1N1, respiratory syncytial virus, viral-bacterial co-infection
Introduction Acute respiratory tract infections (ARIs) are a leading cause of morbidity and mortality in children worldwide[1] accounting for about 30% of all childhood deaths in the developing world.[2] Viruses account for 50-90% of acute lower respiratory tract infections (ALRIs) in young children[3] with respiratory syncytial virus (RSV), parainfluenza viruses (PIVs), influenza viruses A and B and human metapneumoviruses (hMPV) being the most commonly identified.[4-6] ARIs are classified as upper respiratory tract infections (URIs) or lower respiratory tract infections (LRIs). ARIs are the most common causes of both illness and mortality in children under five, who average *Corresponding author (email: ) Departments of Virology and Immunology (RDC, STK, KZ, ASC), Haffkine Institute for Training, Research and Testing, Parel, Mumbai, India Received: 27-07-2013 Accepted: 02-04-2014 Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.148376
three to six episodes of ARIs annually regardless of where they live or what their economic situation is.[7] Approximately 75% of antibiotics are prescribed for ARI, and many of these prescriptions are unnecessary. This unwarranted use of antibiotics is not only very expensive but has also contributed to rapid increase in antimicrobial resistance to bacteria causing respiratory infections. The proportion of mild to severe disease varies between high- and low-income countries, and because of differences in specific aetiologies and risk factors, the severity of LRIs in children below five is worse in developing countries, resulting in a higher case-fatality rate. Although medical care can, to some extent, mitigate both severity and fatality, many severe LRIs do not respond to therapy, largely because of the lack of highly effective antiviral drugs. Some 10.8 million children die each year due to LRIs.[8] Estimates indicate that in 2000, of the 1.9 million deaths due to ARIs, 70% of children belonged to Africa and Southeast Asia.[9] The World Health Organisation (WHO) estimates that 2 million children under five die of pneumonia each year.[10] URIs are the most common infectious diseases, usually with a viral aetiology. Rhinoviruses account for 25-30% of URIs; RSVs, parainfluenza and influenza viruses, hMPV and adenoviruses for 25-35%; Corona viruses for 10%; and unidentified viruses for the remainder.[11] Because most URIs are self-limiting, their complications are more important than the infections. Acute viral infections predispose children to bacterial infections of the sinuses and
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middle ear,[12] and aspiration of infected secretions and cells can result in LRIs.
The samples were divided in three aliquots and stored in -80°C till further processing.
Viral infection is assumed to precede bacterial infections and may generate the environment for bacterial super-infections.[13,14] There is also evidence that viral infections make the lower respiratory tract more susceptible for fungal infections.[15] These findings indicate the need to develop new point of care tools for the diagnosis of viral infections and the differentiation from bacterial infections or the diagnosis of viral/bacterial co-infections. The argument that viral infections are difficult to treat still applies for most viruses; however, there are newly developed drugs, especially monoclonal antibodies that are virus specific and can be used to treat or even prevent viral infections.[16]
Viral RNA extraction
Haffkine Institute is a sentinel surveillance centre under the National Center for Disease Control (NCDC) for the detection of influenza viruses among the population in Mumbai. The aim of the study was to investigate other viral and bacterial infections in patients with ARIs, which generally go undetermined.
Amplification of viral genome
Materials and Methods A total of 200 nasal swabs/throat swabs were collected from July 2011 to July 2012, which comprised of 114 males and 86 females from outpatient department (OPD) and inpatient department (IPD) from hospitals like B. J. Wadia Hospital for Children, L. H. Hiranandani Hospital, King Edward Memorial Hospital, Mumbai. Written informed consent was obtained from the patients or their parents/ guardians. The study was approved by the Ethical Committee of the hospitals and Institutional Ethics Committee of Haffkine Institute. Patients presenting with ARI having the following case definition were included in the study:[17] Influenza-like Illness (ILI): • Inclusion Criteria: A person with sudden onset of fever >38°C and cough or sore throat • Exclusion Criteria: Absence of any other clinical diagnosis. Acute Respiratory Infection: • Inclusion Criteria: Cough, sore throat, shortness of breath; coryza, and a clinical judgment that illness is due to an infection. The ILI case definition is generally intended for use in outpatient treatment centres and the ARI definitions are for inpatient hospital settings. The ARI definition aims to capture both the influenza-related pneumonias and influenza-related exacerbations of chronic illnesses such as asthma or heart disease. Clinical samples were collected in viral transport medium (VTM), in a triple layer packing, in a styrofoam box containing ice packs and transported to the laboratory.
The viral RNA was extracted from clinical samples using the spin column based QIAmp Viral RNA Mini Kit (Qiagen GmbH, Hielden, Germany) as per manufacturer’s instructions. Bacterial DNA extraction The total genomic DNA was extracted from clinical samples using spin column based HiPuraTM96 Bacterial Genomic DNA Purification Kit (HiMedia, India) as per the manufacturer’s instructions.
Primer and Probes for pandemic influenza A H1N1,[13] seasonal influenza A[13] and influenza type B[15] were provided by NCDC, New Delhi, India, under Integrated Disease Surveillance Programme. RSV,[19] Streptococcus Primers for hMPV,[18] pneumoniae, Haemophilus influenzae,[20] Staphylococcus aureus[21] and Klebsiella pneumoniae[22] were synthesised as illustrated in Table 1. The Real time reverse transcriptase polymerase chain reaction (rRT-PCR) for pandemic influenza A H1N1, seasonal influenza A and influenza Type B was performed as described in an earlier protocol[23] on a Step One real time PCR Instrument (ABI) with an rRT-PCR containing 2× PCR master mix and SuperscriptTM III RT/Platinum Taqman Mix (Enzyme Mix) from Invitrogen (CA, USA). A master mix of 20 μl was prepared in a PCR plate and 5 μl of RNA template was added. Amplification was carried out as given in Table 2: PCR-based identification for hMPV and RSV was carried out with cDNA generated by using BluePrintTM First Strand Synthesis kit (DSS TakaraBio India Pvt. Ltd.) RSV amplification conditions comprised of initial denaturation at 94°C for 3 min, followed by 35 cycling stages at 94°C for 60 s, 50°C for 60 s and 72°C for 60 s, with a final extension at 72°C for 10 min. Similarly, hMPV amplification conditions comprised of initial denaturation at 95°C for 3 min, followed by 35 cycling stages at 95°C for 60 s, 52°C for 45 s and 72°C for 60 s, with a final extension at 72°C for 7 min. After PCR, the amplified product was examined by electrophoresis on an agarose gel containing SYBR safe DNA gel stain (Invitrogen). The results were compared with a DNA molecular weight marker. Amplification of bacterial genome Genomic DNA extracted from the clinical samples (5 μl) was added to 20 μl master mix, which contained Fermentas
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Virus Seasonal influenza A Pandemic influenza A H1N1 Influenza type B Rnase P hMPV RSV Streptococcus pneumoniae Haemophilu s influenzae Staphylococcus aureus Klebsiella pneumoniae
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Chavan, et al.: Acute respiratory infections
Table 1: Sequence of Primer and Probes used in the study Primers and probes Sequence (5ʹto 3ʹ) Forward GAC CRA TCC TGT CAC CTC TGA C Reverse AGG GCA TTY TGG ACA AAK CGT CTA Probe TGC AGT CCT CGC TCA CTG GGC ACG Forward GCA CGG TCA GCA CTT ATY CTR AG Reverse GTG RGC TGG GTT TTC ATT TGG TC Probe CYA CTG CAA GCC CAT ACA CAC AAG CAG GCA Forward TCC TCA AYT CAC TCT TCG AGC G Reverse CGG TGC TCT TGA CCA AAT TGG Probe CCA ATT CGA GCA GCT GAA ACT GCG GTG Forward AGA TTT GGA CCT GCG AGC G Reverse GAG CGG CTG TCT CCA CAA GT Probe TTC TGA CCT GAA GGC TCT GCG CG Forward GTC AGC TTC AGT CAA TTC AAC AGA Reverse CAG TCT TTT TCA TTT GGG TAG TAA Forward CTG TCA TCC AGC AAA TAC AC Reverse ACC ATA GGC ATT CAT AAA CAA TC Forward CGG ACT ACC GCC TTT ATA TCG Reverse GTT TCA ATC GTC AAG CCG TT Forward TCC TAA GAA GAG CTC AGA GAT Reverse TGA TCC AAC CGC AGG TTC C Forward AAT CTT TGT CGG TAC ACG ATA TTC TTC ACG Reverse CGT AAT GAG ATT TCA GTA GAT AAT ACA ACA Forward GCG TGG CGG TAG ATC TAA GTCATA Reverse TTC AGC TCC GCC ACA AAG GTA
Reference 13 13 15 18 19 20 21 21 22 23
RSV: Respiratory syncytial virus, hMPV: Human metapneumoviruses
Table 2: Cycling conditions for real time reverse transcriptase polymerase chain reaction PCR conditions Temperature/time duration Reverse transcription 50°C/30 min Taq Inhibitor activation 95°C/10 min 40 cycles of amplification 95°C/15 s 55°C/30 s PCR: Polymerase chain reaction
PCR Master Mix 2X (Thermo Scientific) and the 0.3 μmolar of S. pneumoniae primers, 0.6 μmolar of S. aureus primer, 0.25 μmolar of H. influenzae primers and 0.7 μmolar of K. pneumoniae primers. S. aureus amplification conditions comprised of initial denaturation at 94°C for 3 min, followed by 30 cycling stages at 94°C at 30 s, 57.8°C at 30 s, 72°C at 1 min with a final extension at 72°C for 4 min. S. pneumoniae amplification conditions comprised of initial denaturation at 94°C for 10 min, followed by 40 cycling stages at 94°C at 30 s, 56.5°C at 30 s, 72°C at 1 min with a final extension at 72°C for 7 min. H. influenzae amplification condition comprised of initial denaturation at 94°C for 10 min, followed by 40
cycling stages at 94°C at 30 s 56.2°C at 30 s, 72°C at 1 min with a final extension at 72°C for 7 min. K. pneumonia amplification conditions comprised of initial denaturation at 95°C for 5 min, followed by 30 cycling stages at 96°C at 60 s, 54.9°C at 30 s, 72°C at 1 min with a final extension at 72°C for 10 min. The amplified samples were examined by electrophoresis on an agarose gel containing SYBR safe DNA gel stain (Invitrogen). The results were compared with a 100 bp DNA molecular weight marker. In every PCR run, one positive and one negative control for each one of the four investigated bacteria were included. Statistical analysis Results presented in the tables were reported as percentage positives. Data was subjected to Graph Pad Prism v5.04 and v6.0,[24] two way analysis of variance (ANOVA) was carried out for symptom with respect to infection with P < 0.001 was considered to be significant. Results The samples (N = 200) collected for detection of respiratory infections were broadly categorised into ILI
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and ARI, wherein 109 patients were suffering from ILI and 91 patients with ARI.
adolescence group (6-20 years) were 11%, whereas the middle aged groups (21-40 years) comprised of 34% [Figure 1].
As can be observed from Table 3, the patients with ILI, viral co-infection was found to be 6.3% positive for influenza A and B, whereas 8.1% positivity for pandemic influenza A H1N1 and influenza type B, as compared to other viral infections, which were predominant. In case of bacterial infections, S. pneumoniae was the major bacterial infection and only a single case of bacterial co-infection with 0.9% positivity was observed. The most significant finding was viral-bacterial co-infection with 1.8% positivity for pandemic influenza A H1N1 and H. influenzae and 0.9% positivity for pandemic influenza A H1N1 with influenza Type B and S. pneumonia [Table 3].
Month-wise distribution In Graph 2, to understand the seasonality and trend of circulating influenza and bacterial strains, the month-wise percentage distribution was plotted over the total number of samples positive for influenza, bacterial infection along with viral-bacterial co-infection for the year July 2011 to July 2012. A minor peak in the influenza positivity percentage was observed in the monsoon season (July 2011-September 2011) and bacterial positivity percentage rise during the winter season (July 2011-September 2011) and bacterial percentage positivity rise during the winter seasons (December 2011-January 2012). A major finding was for the viral bacterial co-infection, which was observed during the
As can be observed from Table 4, patients with ARI, pandemic influenza A H1N1was predominant with 17.4% positivity as compared with other viral infections. In viral co-infection, seasonal influenza A and influenza type B accounted for 2.19% positivity. In case of bacterial infections, S. pneumoniae was found to be the major bacterial infection. The most significant finding was the viral-bacterial co-infection in which H. influenzae was the major co-infecting bacteria with the influenza viruses as compared with S. pneumonia [Table 4]. Age-wise distribution As is illustrated in Graph 1, 30% of the patients who were admitted due to respiratory infection belonged to the paediatric group, which was found to be the most vulnerable to infections as compared with other age groups. The patients of
Figure 1: Age-wise distribution of patients representing respiratory symptoms
Table 3: Viral and bacterial infections in patients with influenza like illness Male Percentage positivity Females Percentage positivity
Infections (ILI) Viral infections Seasonal influenza A Influenza type B Pandemic influenza A H1N1 RSV hMPV Viral co-infections Seasonal influenza A+influenza type B Pandemic influenza A H1N1+influenza type B Bacterial infections Streptococcus pneumoniae Haemophilus influenzae Staphylococcus aureus Klebsiella pneumoniae Bacterial co-infections Streptococcus pneumoniae+Haemophilus influenzae Viral bacterial co-infections Pandemic influenza A H1N1+Haemophilus influenzae Pandemic influenza A H1N1+influenza type B+Streptococcus pneumoniae
8 7 12 1 0
7.3 6.4 11 0.9 0
0 6 9 0 0
0 5.5 8.2 0 0
4 5
3.6 4.5
3 4
2.7 3.6
5 1 0 0
4.5 0.9 0 0
0 1 0 0
0 0.9 0 0
1
0.9
0
0
1 0
0.9 0
1 1
0.9 0.9
ILI: Influenza-like illness, RSV: Respiratory syncytial virus, hMPV: Human metapneumoviruses www.ijmm.org
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Discussion
monsoons of year 2012 (June-July) that coincided with the influenza positives [Figure 2].
ARIs are responsible for 3.9 million deaths. It is estimated that Bangladesh, India, Indonesia and Nepal together account for 40% of the global ARI mortality.[25] On an average, children aged below 5 years suffer about five episodes of ARI per child per year, thus accounting about 238 million cases. ARI is responsible for about 30-50% of visits to health facilities and for about 20-40% of admissions to hospitals. It is also a leading cause of deafness as sequelae of acute otitis media.[26]
Symptomatic and infection positivity As observed from Figure 3, a 2 way ANOVA was carried to compare symptoms with infections. Symptoms like temperature (axilla and oral), cough and sore throat were found to be consistent in different types of influenza viruses as compared with shortness of breathing and vomiting, which varied. In bacterial infections caused by S. pneumoniae and H. influenzae cough was the major symptom. [Figure 3].
We report here that the percentage positivity for pandemic influenza A H1N1 is about 19.2% in patients with
Figure 2: Month-wise distribution for Influenza and Bacterial positives along with co-infection
Figure 3: Graph of Symptoms with respect to different Infections
Table 4: Viral and bacterial infections in patients with acute respiratory infection Male Percentage positivity Females Percentage positivity
Infections (ARI) Viral infections Seasonal influenza A Influenza type B Pandemic influenza A H1N1 RSV hMPV Viral co-infections Seasonal influenza A+influenza type B Pandemic influenza A H1N1+influenza type B Bacterial infections Streptococcus pneumoniae Haemophilus influenzae Staphylococcus aureus Klebsiella pneumoniae Bacterial co-infections Streptococcus pneumoniae+Haemophilus influenzae Viral bacterial co-infections Pandemic influenza A H1N1+Haemophilus influenzae Influenza type B+Haemophilus influenzae Seasonal influenza+Haemophilus influenzae Seasonal influenza A+Streptococcus pneumoniae RSV+Streptococcus pneumoniae
3 6 10 0 0
3.2 6.5 10.9 0 0
4 5 6 1 0
4.3 5.4 6.5 1.09 0
0 1
0 1.09
2 1
2.19 1.09
4 0 0 0
4.3 0 0 0
2 0 1 0
2.1 0 1.09 0
1
1.09
0
0
1 0 0 1 1
1.09 0 0 1.09 1.09
0 1 1 0 0
0 1.09 1.09 0 0
ARI: Acute respiratory infections, RSV: Respiratory syncytial virus, hMPV: Human metapneumoviruses www.ijmm.org
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ILI as compared with 17.4% in patients with ARIs. The most significant finding in the study was influenza type B seen to be co-circulating strain with 11.9% positivity in contrast to seasonal influenza A where only 8% and 7.5% positivity was observed in ILI and ARI, respectively. As reported by a surveillance study carried out in Delhi,[27] pandemic influenza A H1N1 and influenza type B accounted for almost equal distribution in 2007, with predominance of influenza type B in 2008. Pandemic influenza A accounted for 50% infections both in 2009 and 2010, whereas influenza A (H3N2) accounted for 35% infections in 2009 and influenza type B for 44% infections in 2010. Previous reports of prevalence studies carried out at Haffkine Institute[28] demonstrated the screening of 100 children for influenza type A and influenza type B and it was noted that 11 were positive for influenza type A and only 5 were positive for influenza type B. However, they did not carry out any screening for other respiratory viruses like RSV and hMPV neither for any viral nor bacterial co-infections. RSV was the most common identified viral pathogen in children aged below 5 years in Delhi from April 2005 to Mach 2007 where 301 nasopharyngeal aspirates were collected showing the symptoms of ALRIs and the incidence of RSV positives was 20.26%.[19] With such a high incidence of RSV infection prevailing in Delhi, we carried out the screening of the samples for RSV in addition to influenza viruses. The positivity for RSV accounted for 0.9% and 1.09% in ILI and ARI, respectively. A preliminary study carried out in Pune[25] during the 2-month period, which included 9 children and 18 adults, hMPV RNA was detected in 19.2% of the children, while it was undetected in adults. Hence it may be speculated that hMPV may well exist in the Indian population. These facts suggest the importance of screening of hMPV, therefore, we screened all samples for hMPV and none of the patients were found to be positive in the study group we selected. Bacterial pneumonia complicating viral influenza was reported during the pandemic of 1918 and multiple subsequent epidemic and inter-epidemic periods.[29] The most common pathogens were S. pneumoniae, S. aureus, H. influenzae and occasionally other Gram-negative bacilli.[30] During the 2006-2007 influenza season, cases of severe community-acquired pneumonia due to methicillin-resistant Staphylococcus aureus (MRSA) were reported with 33% mortality.[31] These show an increase in the morbidity and mortality when influenza was associated with another underlying respiratory infection, usually of bacterial origin. The mechanisms by which bacteria act synergistically with influenza virus includes increased
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binding and invasion of bacteria along with viral replication and modification of the host inflammatory response. The interactions between influenza virus and bacteria have most thoroughly been established with S. pneumoniae, where the viral neuraminidase of influenza virus cleaves the sialic acid to release new viral particles from host cells, which results the damage of the epithelial layer of the airways and expose the binding sites necessary for adherence of the pneumococcus.[32] In one of the studies,[33] the author stated co-infection with influenza and bacterial pathogens occurred more frequently in pandemic as compared with seasonal influenza periods. Of these, S. pneumoniae was the most common cause of bacterial co-infection with influenza and accounted for 40.8% and 16.6% of bacterial co-infections during pandemic and seasonal periods, respectively. These results suggest that bacterial pathogens will play a key role in many countries, as the influenza pandemic moves forward. The increasing evidence of bacterial infection along with the influenza viruses impelled us to screen all the samples for bacterial infections. We observed that S. pneumoniae and H. influenzae were the major bacterial infections encountered in patients with respiratory infections. The incidence for viral-bacterial co-infection could also be clearly observed in both ILI and ARI with positivity accounting for 0.9% and 1.09% respectively. In the present study, month-wise distribution was also observed for the viral and bacterial positivity and it displayed that influenza positivity peaked during the monsoon seasons (July 2011 and June 2012-July 2012). The bacterial infections were predominant during winter season (December 2011 and January 2012). The co-infection between the bacterial and viral pathogens was observed during the monsoons of June 2012 and July 2012. We report here that seasonal peaks of influenza activity in India are consistent with data from surrounding countries in the region, where peaks of influenza activities coincide with rainy seasons.[34,35] In some tropical regions; there is high background influenza activity throughout the year with distinct peaks appearing during monsoon or cooler months.[36] While the exact mechanisms leading to variation in influenza seasonality are not clear, attempts to correlate fluctuation in meteorological variables have shown relationship with influenza positivity during the rainy season in the tropics.[37] Multi-site influenza surveillance from different geographic regions in India has also revealed a positive correlation between the rainy season and rates of influenza virus isolation. In the clinical presentation of symptoms versus infection where a 2 way ANOVA was carried out; significant findings (P < 0.01) were observed for cough and sore throat in pandemic influenza A in comparison to nasal catarrh, shortness of breathing and vomiting for bacterial infections.
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Conclusion
9.
Taken together, our data provides evidence about significance of bacterial infection, which needs to be carried out along with viral infections in patients presenting with respiratory symptoms. This study highlights the existence of viral-bacterial co-infection, which also holds an importance in cases of antimicrobial resistance so that appropriate drugs could be prescribed to encounter such infections. Together with other studies, our data imply that the diagnosis of viruses and bacteria in ILI and ARI patients should be performed on daily basis. This would help in patient management, treatment and prevention of viral and bacterial infection. We are aware that the available diagnostic tools for point of care diagnosis are not optimal and we are also aware that virus therapy has to be further improved. We hope that the findings of our study can alert clinicians regarding the risk of viral and bacterial infection in patients and stimulate the development of diagnostic tools as well as therapeutic strategies. The need of the hour is to undertake continued surveillance globally, which will help to better define the circulation of influenza viruses along with the bacterial infections, as well as to determine optimal periods to implement vaccination programmes among the priority population.
10. 11. 12. 13.
14.
15.
16.
17.
Acknowledgment The authors thank Dr. Arun Kumar, Manipal Center for Virus Research, Manipal University for providing the RSV and hMPV.
18.
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How to cite this article: Chavan RD, Kothari ST, Zunjarrao K, Chowdhary AS. Surveillance of Acute Respiratory Infections in Mumbai during 2011-12. Indian J Med Microbiol 2015;33:43-50. Source of Support: The complete research was financially supported by Haffkine Institute for Training, Research and Testing, Mumbai, Conflict of Interest: In reference to the above, I as a corresponding author, submit the manuscript for publication in the Indian Journal of Medical Microbiology. This manuscript has not been published or considered for publication by any other journal or elsewhere. I declare no conflict of interest between the authors and accept all the terms and conditions displayed in http://www.ijmm.org/contributors.asp to the best of my knowledge.
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Indian Journal of Medical Microbiology, (2015) 33(1): 43-50
Original Article
Surveillance of Acute Respiratory Infections in Mumbai during 2011-12 *RD Chavan, ST Kothari, K Zunjarrao, AS Chowdhary
Abstract Purpose: Acute respiratory infections (ARIs) are a leading cause of morbidity and mortality in individuals aged less than 5 years. ARI often leads to hospitalisation, and it has been indicated that causative viral and bacterial infections go undetermined and results in the occurrence of resistant strains. The objective of the study was to assess the prevalence of various viral and bacterial infections in patients with ARIs. Materials and Methods: Two hundred samples were collected from July 2011 to July 2012 with patients suffering from ARI. Viral and bacterial infections were determined by real time reverse transcriptase polymerase chain reaction. Results: Influenza-like illness (ILI) consisted of 109 patients and ARI consisted of 91 patients. Pandemic influenza A H1N1 was the major viral infection with 21 (19.2%) patients in ILI as compared with 16 (17.4%) patients in ARI. Respiratory syncytial virus (RSV) was found to be 1 (0.9%) in ILI and ARI. Viral co-infections were 16 (14.4%) in ILI and 4 (4.37%) in ARI where pandemic influenza A H1N1 and influenza type B were major contributors. In bacterial infections, Streptococcus pneumoniae with 11 (10.9%) cases were predominant in both the groups. Bacterial co-infection accounted for only 1 (1.09%) case in both the groups but the most significant finding was the viral-bacterial co-infection in which Haemophilus influenzae was the major co-infecting bacteria with the influenza viruses with 4 (4.36%) cases as compared with Streptotoccus pneumoniae. Conclusion: This data indicate the need to undertake continued surveillance that will help to better define the circulation of respiratory viruses along with the bacterial infections. Key words: Acute respiratory infection, influenza-like illness, pandemic influenza A H1N1, respiratory syncytial virus, viral-bacterial co-infection
Introduction Acute respiratory tract infections (ARIs) are a leading cause of morbidity and mortality in children worldwide[1] accounting for about 30% of all childhood deaths in the developing world.[2] Viruses account for 50-90% of acute lower respiratory tract infections (ALRIs) in young children[3] with respiratory syncytial virus (RSV), parainfluenza viruses (PIVs), influenza viruses A and B and human metapneumoviruses (hMPV) being the most commonly identified.[4-6] ARIs are classified as upper respiratory tract infections (URIs) or lower respiratory tract infections (LRIs). ARIs are the most common causes of both illness and mortality in children under five, who average *Corresponding author (email: ) Departments of Virology and Immunology (RDC, STK, KZ, ASC), Haffkine Institute for Training, Research and Testing, Parel, Mumbai, India Received: 27-07-2013 Accepted: 02-04-2014 Access this article online Quick Response Code:
Website: www.ijmm.org PMID: *** DOI: 10.4103/0255-0857.148376
three to six episodes of ARIs annually regardless of where they live or what their economic situation is.[7] Approximately 75% of antibiotics are prescribed for ARI, and many of these prescriptions are unnecessary. This unwarranted use of antibiotics is not only very expensive but has also contributed to rapid increase in antimicrobial resistance to bacteria causing respiratory infections. The proportion of mild to severe disease varies between high- and low-income countries, and because of differences in specific aetiologies and risk factors, the severity of LRIs in children below five is worse in developing countries, resulting in a higher case-fatality rate. Although medical care can, to some extent, mitigate both severity and fatality, many severe LRIs do not respond to therapy, largely because of the lack of highly effective antiviral drugs. Some 10.8 million children die each year due to LRIs.[8] Estimates indicate that in 2000, of the 1.9 million deaths due to ARIs, 70% of children belonged to Africa and Southeast Asia.[9] The World Health Organisation (WHO) estimates that 2 million children under five die of pneumonia each year.[10] URIs are the most common infectious diseases, usually with a viral aetiology. Rhinoviruses account for 25-30% of URIs; RSVs, parainfluenza and influenza viruses, hMPV and adenoviruses for 25-35%; Corona viruses for 10%; and unidentified viruses for the remainder.[11] Because most URIs are self-limiting, their complications are more important than the infections. Acute viral infections predispose children to bacterial infections of the sinuses and
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middle ear,[12] and aspiration of infected secretions and cells can result in LRIs.
The samples were divided in three aliquots and stored in -80°C till further processing.
Viral infection is assumed to precede bacterial infections and may generate the environment for bacterial super-infections.[13,14] There is also evidence that viral infections make the lower respiratory tract more susceptible for fungal infections.[15] These findings indicate the need to develop new point of care tools for the diagnosis of viral infections and the differentiation from bacterial infections or the diagnosis of viral/bacterial co-infections. The argument that viral infections are difficult to treat still applies for most viruses; however, there are newly developed drugs, especially monoclonal antibodies that are virus specific and can be used to treat or even prevent viral infections.[16]
Viral RNA extraction
Haffkine Institute is a sentinel surveillance centre under the National Center for Disease Control (NCDC) for the detection of influenza viruses among the population in Mumbai. The aim of the study was to investigate other viral and bacterial infections in patients with ARIs, which generally go undetermined.
Amplification of viral genome
Materials and Methods A total of 200 nasal swabs/throat swabs were collected from July 2011 to July 2012, which comprised of 114 males and 86 females from outpatient department (OPD) and inpatient department (IPD) from hospitals like B. J. Wadia Hospital for Children, L. H. Hiranandani Hospital, King Edward Memorial Hospital, Mumbai. Written informed consent was obtained from the patients or their parents/ guardians. The study was approved by the Ethical Committee of the hospitals and Institutional Ethics Committee of Haffkine Institute. Patients presenting with ARI having the following case definition were included in the study:[17] Influenza-like Illness (ILI): • Inclusion Criteria: A person with sudden onset of fever >38°C and cough or sore throat • Exclusion Criteria: Absence of any other clinical diagnosis. Acute Respiratory Infection: • Inclusion Criteria: Cough, sore throat, shortness of breath; coryza, and a clinical judgment that illness is due to an infection. The ILI case definition is generally intended for use in outpatient treatment centres and the ARI definitions are for inpatient hospital settings. The ARI definition aims to capture both the influenza-related pneumonias and influenza-related exacerbations of chronic illnesses such as asthma or heart disease. Clinical samples were collected in viral transport medium (VTM), in a triple layer packing, in a styrofoam box containing ice packs and transported to the laboratory.
The viral RNA was extracted from clinical samples using the spin column based QIAmp Viral RNA Mini Kit (Qiagen GmbH, Hielden, Germany) as per manufacturer’s instructions. Bacterial DNA extraction The total genomic DNA was extracted from clinical samples using spin column based HiPuraTM96 Bacterial Genomic DNA Purification Kit (HiMedia, India) as per the manufacturer’s instructions.
Primer and Probes for pandemic influenza A H1N1,[13] seasonal influenza A[13] and influenza type B[15] were provided by NCDC, New Delhi, India, under Integrated Disease Surveillance Programme. RSV,[19] Streptococcus Primers for hMPV,[18] pneumoniae, Haemophilus influenzae,[20] Staphylococcus aureus[21] and Klebsiella pneumoniae[22] were synthesised as illustrated in Table 1. The Real time reverse transcriptase polymerase chain reaction (rRT-PCR) for pandemic influenza A H1N1, seasonal influenza A and influenza Type B was performed as described in an earlier protocol[23] on a Step One real time PCR Instrument (ABI) with an rRT-PCR containing 2× PCR master mix and SuperscriptTM III RT/Platinum Taqman Mix (Enzyme Mix) from Invitrogen (CA, USA). A master mix of 20 μl was prepared in a PCR plate and 5 μl of RNA template was added. Amplification was carried out as given in Table 2: PCR-based identification for hMPV and RSV was carried out with cDNA generated by using BluePrintTM First Strand Synthesis kit (DSS TakaraBio India Pvt. Ltd.) RSV amplification conditions comprised of initial denaturation at 94°C for 3 min, followed by 35 cycling stages at 94°C for 60 s, 50°C for 60 s and 72°C for 60 s, with a final extension at 72°C for 10 min. Similarly, hMPV amplification conditions comprised of initial denaturation at 95°C for 3 min, followed by 35 cycling stages at 95°C for 60 s, 52°C for 45 s and 72°C for 60 s, with a final extension at 72°C for 7 min. After PCR, the amplified product was examined by electrophoresis on an agarose gel containing SYBR safe DNA gel stain (Invitrogen). The results were compared with a DNA molecular weight marker. Amplification of bacterial genome Genomic DNA extracted from the clinical samples (5 μl) was added to 20 μl master mix, which contained Fermentas
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Virus Seasonal influenza A Pandemic influenza A H1N1 Influenza type B Rnase P hMPV RSV Streptococcus pneumoniae Haemophilu s influenzae Staphylococcus aureus Klebsiella pneumoniae
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Table 1: Sequence of Primer and Probes used in the study Primers and probes Sequence (5ʹto 3ʹ) Forward GAC CRA TCC TGT CAC CTC TGA C Reverse AGG GCA TTY TGG ACA AAK CGT CTA Probe TGC AGT CCT CGC TCA CTG GGC ACG Forward GCA CGG TCA GCA CTT ATY CTR AG Reverse GTG RGC TGG GTT TTC ATT TGG TC Probe CYA CTG CAA GCC CAT ACA CAC AAG CAG GCA Forward TCC TCA AYT CAC TCT TCG AGC G Reverse CGG TGC TCT TGA CCA AAT TGG Probe CCA ATT CGA GCA GCT GAA ACT GCG GTG Forward AGA TTT GGA CCT GCG AGC G Reverse GAG CGG CTG TCT CCA CAA GT Probe TTC TGA CCT GAA GGC TCT GCG CG Forward GTC AGC TTC AGT CAA TTC AAC AGA Reverse CAG TCT TTT TCA TTT GGG TAG TAA Forward CTG TCA TCC AGC AAA TAC AC Reverse ACC ATA GGC ATT CAT AAA CAA TC Forward CGG ACT ACC GCC TTT ATA TCG Reverse GTT TCA ATC GTC AAG CCG TT Forward TCC TAA GAA GAG CTC AGA GAT Reverse TGA TCC AAC CGC AGG TTC C Forward AAT CTT TGT CGG TAC ACG ATA TTC TTC ACG Reverse CGT AAT GAG ATT TCA GTA GAT AAT ACA ACA Forward GCG TGG CGG TAG ATC TAA GTCATA Reverse TTC AGC TCC GCC ACA AAG GTA
Reference 13 13 15 18 19 20 21 21 22 23
RSV: Respiratory syncytial virus, hMPV: Human metapneumoviruses
Table 2: Cycling conditions for real time reverse transcriptase polymerase chain reaction PCR conditions Temperature/time duration Reverse transcription 50°C/30 min Taq Inhibitor activation 95°C/10 min 40 cycles of amplification 95°C/15 s 55°C/30 s PCR: Polymerase chain reaction
PCR Master Mix 2X (Thermo Scientific) and the 0.3 μmolar of S. pneumoniae primers, 0.6 μmolar of S. aureus primer, 0.25 μmolar of H. influenzae primers and 0.7 μmolar of K. pneumoniae primers. S. aureus amplification conditions comprised of initial denaturation at 94°C for 3 min, followed by 30 cycling stages at 94°C at 30 s, 57.8°C at 30 s, 72°C at 1 min with a final extension at 72°C for 4 min. S. pneumoniae amplification conditions comprised of initial denaturation at 94°C for 10 min, followed by 40 cycling stages at 94°C at 30 s, 56.5°C at 30 s, 72°C at 1 min with a final extension at 72°C for 7 min. H. influenzae amplification condition comprised of initial denaturation at 94°C for 10 min, followed by 40
cycling stages at 94°C at 30 s 56.2°C at 30 s, 72°C at 1 min with a final extension at 72°C for 7 min. K. pneumonia amplification conditions comprised of initial denaturation at 95°C for 5 min, followed by 30 cycling stages at 96°C at 60 s, 54.9°C at 30 s, 72°C at 1 min with a final extension at 72°C for 10 min. The amplified samples were examined by electrophoresis on an agarose gel containing SYBR safe DNA gel stain (Invitrogen). The results were compared with a 100 bp DNA molecular weight marker. In every PCR run, one positive and one negative control for each one of the four investigated bacteria were included. Statistical analysis Results presented in the tables were reported as percentage positives. Data was subjected to Graph Pad Prism v5.04 and v6.0,[24] two way analysis of variance (ANOVA) was carried out for symptom with respect to infection with P < 0.001 was considered to be significant. Results The samples (N = 200) collected for detection of respiratory infections were broadly categorised into ILI
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and ARI, wherein 109 patients were suffering from ILI and 91 patients with ARI.
adolescence group (6-20 years) were 11%, whereas the middle aged groups (21-40 years) comprised of 34% [Figure 1].
As can be observed from Table 3, the patients with ILI, viral co-infection was found to be 6.3% positive for influenza A and B, whereas 8.1% positivity for pandemic influenza A H1N1 and influenza type B, as compared to other viral infections, which were predominant. In case of bacterial infections, S. pneumoniae was the major bacterial infection and only a single case of bacterial co-infection with 0.9% positivity was observed. The most significant finding was viral-bacterial co-infection with 1.8% positivity for pandemic influenza A H1N1 and H. influenzae and 0.9% positivity for pandemic influenza A H1N1 with influenza Type B and S. pneumonia [Table 3].
Month-wise distribution In Graph 2, to understand the seasonality and trend of circulating influenza and bacterial strains, the month-wise percentage distribution was plotted over the total number of samples positive for influenza, bacterial infection along with viral-bacterial co-infection for the year July 2011 to July 2012. A minor peak in the influenza positivity percentage was observed in the monsoon season (July 2011-September 2011) and bacterial positivity percentage rise during the winter season (July 2011-September 2011) and bacterial percentage positivity rise during the winter seasons (December 2011-January 2012). A major finding was for the viral bacterial co-infection, which was observed during the
As can be observed from Table 4, patients with ARI, pandemic influenza A H1N1was predominant with 17.4% positivity as compared with other viral infections. In viral co-infection, seasonal influenza A and influenza type B accounted for 2.19% positivity. In case of bacterial infections, S. pneumoniae was found to be the major bacterial infection. The most significant finding was the viral-bacterial co-infection in which H. influenzae was the major co-infecting bacteria with the influenza viruses as compared with S. pneumonia [Table 4]. Age-wise distribution As is illustrated in Graph 1, 30% of the patients who were admitted due to respiratory infection belonged to the paediatric group, which was found to be the most vulnerable to infections as compared with other age groups. The patients of
Figure 1: Age-wise distribution of patients representing respiratory symptoms
Table 3: Viral and bacterial infections in patients with influenza like illness Male Percentage positivity Females Percentage positivity
Infections (ILI) Viral infections Seasonal influenza A Influenza type B Pandemic influenza A H1N1 RSV hMPV Viral co-infections Seasonal influenza A+influenza type B Pandemic influenza A H1N1+influenza type B Bacterial infections Streptococcus pneumoniae Haemophilus influenzae Staphylococcus aureus Klebsiella pneumoniae Bacterial co-infections Streptococcus pneumoniae+Haemophilus influenzae Viral bacterial co-infections Pandemic influenza A H1N1+Haemophilus influenzae Pandemic influenza A H1N1+influenza type B+Streptococcus pneumoniae
8 7 12 1 0
7.3 6.4 11 0.9 0
0 6 9 0 0
0 5.5 8.2 0 0
4 5
3.6 4.5
3 4
2.7 3.6
5 1 0 0
4.5 0.9 0 0
0 1 0 0
0 0.9 0 0
1
0.9
0
0
1 0
0.9 0
1 1
0.9 0.9
ILI: Influenza-like illness, RSV: Respiratory syncytial virus, hMPV: Human metapneumoviruses www.ijmm.org
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Discussion
monsoons of year 2012 (June-July) that coincided with the influenza positives [Figure 2].
ARIs are responsible for 3.9 million deaths. It is estimated that Bangladesh, India, Indonesia and Nepal together account for 40% of the global ARI mortality.[25] On an average, children aged below 5 years suffer about five episodes of ARI per child per year, thus accounting about 238 million cases. ARI is responsible for about 30-50% of visits to health facilities and for about 20-40% of admissions to hospitals. It is also a leading cause of deafness as sequelae of acute otitis media.[26]
Symptomatic and infection positivity As observed from Figure 3, a 2 way ANOVA was carried to compare symptoms with infections. Symptoms like temperature (axilla and oral), cough and sore throat were found to be consistent in different types of influenza viruses as compared with shortness of breathing and vomiting, which varied. In bacterial infections caused by S. pneumoniae and H. influenzae cough was the major symptom. [Figure 3].
We report here that the percentage positivity for pandemic influenza A H1N1 is about 19.2% in patients with
Figure 2: Month-wise distribution for Influenza and Bacterial positives along with co-infection
Figure 3: Graph of Symptoms with respect to different Infections
Table 4: Viral and bacterial infections in patients with acute respiratory infection Male Percentage positivity Females Percentage positivity
Infections (ARI) Viral infections Seasonal influenza A Influenza type B Pandemic influenza A H1N1 RSV hMPV Viral co-infections Seasonal influenza A+influenza type B Pandemic influenza A H1N1+influenza type B Bacterial infections Streptococcus pneumoniae Haemophilus influenzae Staphylococcus aureus Klebsiella pneumoniae Bacterial co-infections Streptococcus pneumoniae+Haemophilus influenzae Viral bacterial co-infections Pandemic influenza A H1N1+Haemophilus influenzae Influenza type B+Haemophilus influenzae Seasonal influenza+Haemophilus influenzae Seasonal influenza A+Streptococcus pneumoniae RSV+Streptococcus pneumoniae
3 6 10 0 0
3.2 6.5 10.9 0 0
4 5 6 1 0
4.3 5.4 6.5 1.09 0
0 1
0 1.09
2 1
2.19 1.09
4 0 0 0
4.3 0 0 0
2 0 1 0
2.1 0 1.09 0
1
1.09
0
0
1 0 0 1 1
1.09 0 0 1.09 1.09
0 1 1 0 0
0 1.09 1.09 0 0
ARI: Acute respiratory infections, RSV: Respiratory syncytial virus, hMPV: Human metapneumoviruses www.ijmm.org
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ILI as compared with 17.4% in patients with ARIs. The most significant finding in the study was influenza type B seen to be co-circulating strain with 11.9% positivity in contrast to seasonal influenza A where only 8% and 7.5% positivity was observed in ILI and ARI, respectively. As reported by a surveillance study carried out in Delhi,[27] pandemic influenza A H1N1 and influenza type B accounted for almost equal distribution in 2007, with predominance of influenza type B in 2008. Pandemic influenza A accounted for 50% infections both in 2009 and 2010, whereas influenza A (H3N2) accounted for 35% infections in 2009 and influenza type B for 44% infections in 2010. Previous reports of prevalence studies carried out at Haffkine Institute[28] demonstrated the screening of 100 children for influenza type A and influenza type B and it was noted that 11 were positive for influenza type A and only 5 were positive for influenza type B. However, they did not carry out any screening for other respiratory viruses like RSV and hMPV neither for any viral nor bacterial co-infections. RSV was the most common identified viral pathogen in children aged below 5 years in Delhi from April 2005 to Mach 2007 where 301 nasopharyngeal aspirates were collected showing the symptoms of ALRIs and the incidence of RSV positives was 20.26%.[19] With such a high incidence of RSV infection prevailing in Delhi, we carried out the screening of the samples for RSV in addition to influenza viruses. The positivity for RSV accounted for 0.9% and 1.09% in ILI and ARI, respectively. A preliminary study carried out in Pune[25] during the 2-month period, which included 9 children and 18 adults, hMPV RNA was detected in 19.2% of the children, while it was undetected in adults. Hence it may be speculated that hMPV may well exist in the Indian population. These facts suggest the importance of screening of hMPV, therefore, we screened all samples for hMPV and none of the patients were found to be positive in the study group we selected. Bacterial pneumonia complicating viral influenza was reported during the pandemic of 1918 and multiple subsequent epidemic and inter-epidemic periods.[29] The most common pathogens were S. pneumoniae, S. aureus, H. influenzae and occasionally other Gram-negative bacilli.[30] During the 2006-2007 influenza season, cases of severe community-acquired pneumonia due to methicillin-resistant Staphylococcus aureus (MRSA) were reported with 33% mortality.[31] These show an increase in the morbidity and mortality when influenza was associated with another underlying respiratory infection, usually of bacterial origin. The mechanisms by which bacteria act synergistically with influenza virus includes increased
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binding and invasion of bacteria along with viral replication and modification of the host inflammatory response. The interactions between influenza virus and bacteria have most thoroughly been established with S. pneumoniae, where the viral neuraminidase of influenza virus cleaves the sialic acid to release new viral particles from host cells, which results the damage of the epithelial layer of the airways and expose the binding sites necessary for adherence of the pneumococcus.[32] In one of the studies,[33] the author stated co-infection with influenza and bacterial pathogens occurred more frequently in pandemic as compared with seasonal influenza periods. Of these, S. pneumoniae was the most common cause of bacterial co-infection with influenza and accounted for 40.8% and 16.6% of bacterial co-infections during pandemic and seasonal periods, respectively. These results suggest that bacterial pathogens will play a key role in many countries, as the influenza pandemic moves forward. The increasing evidence of bacterial infection along with the influenza viruses impelled us to screen all the samples for bacterial infections. We observed that S. pneumoniae and H. influenzae were the major bacterial infections encountered in patients with respiratory infections. The incidence for viral-bacterial co-infection could also be clearly observed in both ILI and ARI with positivity accounting for 0.9% and 1.09% respectively. In the present study, month-wise distribution was also observed for the viral and bacterial positivity and it displayed that influenza positivity peaked during the monsoon seasons (July 2011 and June 2012-July 2012). The bacterial infections were predominant during winter season (December 2011 and January 2012). The co-infection between the bacterial and viral pathogens was observed during the monsoons of June 2012 and July 2012. We report here that seasonal peaks of influenza activity in India are consistent with data from surrounding countries in the region, where peaks of influenza activities coincide with rainy seasons.[34,35] In some tropical regions; there is high background influenza activity throughout the year with distinct peaks appearing during monsoon or cooler months.[36] While the exact mechanisms leading to variation in influenza seasonality are not clear, attempts to correlate fluctuation in meteorological variables have shown relationship with influenza positivity during the rainy season in the tropics.[37] Multi-site influenza surveillance from different geographic regions in India has also revealed a positive correlation between the rainy season and rates of influenza virus isolation. In the clinical presentation of symptoms versus infection where a 2 way ANOVA was carried out; significant findings (P < 0.01) were observed for cough and sore throat in pandemic influenza A in comparison to nasal catarrh, shortness of breathing and vomiting for bacterial infections.
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Conclusion
9.
Taken together, our data provides evidence about significance of bacterial infection, which needs to be carried out along with viral infections in patients presenting with respiratory symptoms. This study highlights the existence of viral-bacterial co-infection, which also holds an importance in cases of antimicrobial resistance so that appropriate drugs could be prescribed to encounter such infections. Together with other studies, our data imply that the diagnosis of viruses and bacteria in ILI and ARI patients should be performed on daily basis. This would help in patient management, treatment and prevention of viral and bacterial infection. We are aware that the available diagnostic tools for point of care diagnosis are not optimal and we are also aware that virus therapy has to be further improved. We hope that the findings of our study can alert clinicians regarding the risk of viral and bacterial infection in patients and stimulate the development of diagnostic tools as well as therapeutic strategies. The need of the hour is to undertake continued surveillance globally, which will help to better define the circulation of influenza viruses along with the bacterial infections, as well as to determine optimal periods to implement vaccination programmes among the priority population.
10. 11. 12. 13.
14.
15.
16.
17.
Acknowledgment The authors thank Dr. Arun Kumar, Manipal Center for Virus Research, Manipal University for providing the RSV and hMPV.
18.
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How to cite this article: Chavan RD, Kothari ST, Zunjarrao K, Chowdhary AS. Surveillance of Acute Respiratory Infections in Mumbai during 2011-12. Indian J Med Microbiol 2015;33:43-50. Source of Support: The complete research was financially supported by Haffkine Institute for Training, Research and Testing, Mumbai, Conflict of Interest: In reference to the above, I as a corresponding author, submit the manuscript for publication in the Indian Journal of Medical Microbiology. This manuscript has not been published or considered for publication by any other journal or elsewhere. I declare no conflict of interest between the authors and accept all the terms and conditions displayed in http://www.ijmm.org/contributors.asp to the best of my knowledge.
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