Induced Sputum as a Diagnostic Tool in Pneumonia in Under Five Children—A Hospital-based Study

Induced Sputum as a Diagnostic Tool in Pneumonia in Under Five Children—A Hospital-based Study Abstract Objective The objective of this articlewas to study the success, tolerability of sputum induction and the bacterial isolates of induced sputum in children aged <5 years. Methods The cross-sectional study included 120 hospitalized children aged 1–59 months meeting WHO criteria for pneumonia. Sputum induction was performed using hypertonic (3%) saline. Results Mean age of the subjects was 19.5 months (2–59 months). Overall success of sputum induction was 53.3% and highest (64.28%) in 37–59 months age group. Adverse events such as tachypnea, hypoxemia (SpO2 <90) and vomiting were observed in 41.6, 17.5 and 15.8%, respectively. A potential pathogen was isolated in 45 (70.3%) of 64 cases with good quality sputum. Klebsiella pneumoniae was the commonest (38.2%) followed by Streptococcus pneumoniae (14.8%) and others. Conclusion Sputum induction in young children is safe and feasible in Indian settings. While the success was limited, bacterial yield was high. Klebsiella pneumoniae, success, bacterial isolates INTRODUCTION According to WHO, around 43 million new cases of pneumonia (23% of world’s total) occur in India with an incidence of 0.37 episodes per child-year. Predicted deaths because of pneumonia in India are ∼4 08 000 annually [1]. Most pneumonias respond to empirical therapy, and causative organism is seldom identified. Etiological diagnosis of childhood pneumonia is challenging because of the difficulty in obtaining specimen from the site of infection and the lack of gold standard. Chest radiographs, nonspecific inflammatory markers and nasopharyngeal aspirates have been used as diagnostic tools, but the results have been inconsistent. Though pulmonary aspirate and bronchoalveolar lavage are considered ideal in determining the etiology of pneumonia, they have many limitations and not feasible in all settings [2, 3]. Sputum has the advantage that the specimen comes directly from the site of infection, and contamination aside indicates microbiological flora in the diseased lung [4]. Microbiologic examination of sputum is often performed to determine the etiology of lower respiratory tract infections in adults [5]. In children, sputum induction is used as a diagnostic tool in settings with high prevalence of tuberculosis and in cystic fibrosis [3, 6, 7]. It has also proven useful in hospitalized children with community acquired pneumonia [6]. Despite its advantages, sputum induction is not a standard procedure in infants and young children. Infants and children tend to swallow sputum, thus making sputum collection difficult. Recently published results of PERCH (Pneumonia Etiology Research for Child Heath), the first large-scale study of its kind involving seven countries (nine sites) has thrown light on the safety, utility and quality assessment of induced sputum in children aged 1–59 months in resource-constraint settings [8–10] Reported success for obtaining good quality sputum in children varies considerably [5, 11, 12]. Sputum induction is generally considered safe with satisfactory success in children aged >6 years [13]. There is no study describing the success and safety of sputum induction in young children from India. The purpose of this study was to evaluate the success and tolerability of sputum induction and the bacterial isolates in children aged <5 years, as they are at greater risk of severe pneumonia. METHODS The present cross-sectional study was conducted from November 2013 to May 2015 at a tertiary care teaching hospital in Western India after institutional ethics committee clearance. Sample size calculation for success was done based on the success (76.5%) reported by Hammitt et al. [14] and for bacterial yield study by Nantanda et al. [15] (54%) was used. With a significance level of 5% and power of 90%, calculated minimum sample sizes were 95.43 and 69.06, respectively, for success and bacterial yield. Hence, in the study, 120 observations were considered. Children aged 1–59 months meeting WHO criteria for pneumonia and severe pneumonia were included [16]. Exclusion criteria were room air oxygen saturation <92%, patients requiring oxygen >8 l/min, platelets <25 000 per cumm, children with major congenital malformations and children with history of seizures, severe bronchospasm and severe pneumonia. After obtaining informed consent oxygen saturation at room air was measured. Sputum induction was carried out within 24 h by a skilled physiotherapist along with the research physician. All samples were collected by same set of operators. Sputum induction was performed according to European respiratory society task force recommendation [13, 17]. Various prerequisites with regard to this procedure are equipment, personal, bronchodilator treatment and monitoring of the subjects. Equipment used in the study included ultrasonic nebulizer with oxygen flow, suction apparatus, multipara monitor and mucus extractor. Subjects were kept nil oral for 2–3 h before the procedure. They were pretreated with salbutamol inhalation (2.5 mg salbutamol in 2 ml of normal saline) to prevent bronchospasm. This was followed by 3% saline nebulization using ultrasonic nebulizer with oxygen flow for 5 min at a time for 15 min to induce cough. Chest vibrations and percussions were given, and child was positioned for postural drainage. Sputum was obtained by oropharyngeal suctioning using the catheter of mucus extractor passed through an appropriate size airway. The specimen was collected directly in to the sterile mucus extractor container. Suctioning through an airway was done to minimize contamination. Samples were transported within 2 h of collection to the microbiology laboratory for processing. Slides for gram staining were prepared according to standard methods. The sputum sample was inoculated on to blood agar, chocolate agar and McConkey agar. Antibiotic sensitivity was determined according to the Clinical Laboratory Standards Institute (CLSI) guidelines. Sample quality was assessed by the number of squamous epithelial cells and leucocytes on the basis of standard metrics designed by Bartlett, Murray and Washington [18]. Sputum was labeled good quality when there were <10 squamous epithelial cells and >25 leucocytes per low-power field. Only good quality samples were processed for microbiological testing. Respiratory rate (RR) and oxygen saturation (SpO2) were continuously monitored during procedure till 1-h post-procedure. Subjects were carefully observed for persistent cough, increase in wheeze, change in sensorium, vomiting or clinical worsening [4, 13]. A drop in oxygen saturation <90% was considered hypoxemia in the study. Criterion for stopping the procedure was drop in saturation <88% for >60 s and not picking up with supplemental oxygen. Tachypnea was defined as per WHO criteria. The success of the procedure was defined as (i) obtaining at least 1 ml of sputum, (ii) obtaining good quality sputum and (iii) completing the induction procedure without interruption. STATISTICAL ANALYSIS Proportions and percentages were obtained for qualitative data, whereas mean and SD were obtained for quantitative data. Chi-square test was used to check the association of parameters. p-value of <0.05 was considered significant. Paired t-test was used to compare baseline and post-procedure parameters. Odds ratio of occurrence of adverse events was calculated. Analysis was done using Microsoft Excel and SPSS-22. RESULTS Of 120 subjects, 64 infants (53.3%) formed the largest group followed by 42 children between 13 and 36 months (40.1%) and 14 children between 37 and 59 months (11.6%). Males constituted 65.8%. The mean age was 19.5 months (range 2–59 months). Mean duration of symptoms was 6.1 days. Most 74 (61.6%) reported antibiotic use and 52 (43.3%) had hospitalization before sputum collection. Mean duration of hospitalization was 3.7 days (range 1–8 days) and that of antibiotic use 4.1 days (range 1–10 days). In the study, 80 (66.6%) subjects were of pneumonia and 40 (33.3%) were of severe pneumonia. Good quality sputum was obtained in 64 (53.3%) cases (Fig. 1). The success was highest (64.28%) in children aged 37–59 months followed by 59.3% in 1–12 months, and 40.4% in 13–36 months. Baseline tachypnea was present in 42 (35%), but overall 50 (41.6%) subjects had tachypnea post-procedure. Drop in oxygen saturation <90% was observed in 17.5% and vomiting in 15.8% (Table 1). The lowest recorded saturation was 86%. The odds (95% confidence interval) of occurrence of hypoxemia was 6.913 (1.913, 24.976) times greater and that of tachypnea 2.11 (1.0025, 4.445) times greater in infants as compared with children >12 months. The increased RR lasted only for around 10 min and drop in O2 saturation for <1 min. Hypoxemia corresponded with the time of suctioning. All parameters started normalizing once suctioning was done and returned to near baseline values within 0.5 h (Table 2). None required increased oxygen, bronchodilator therapy or premature discontinuation of the procedure. The lowest collected sputum volume was 1 ml and highest was 5 ml. Duration of the procedure ranged from 15 to 20 min. Table 1 Adverse events associated with sputum induction in children aged <5 years in a tertiary care hospital setting Age in months (N) TachypneaaN (%) Hypoxemia N (%) Vomiting N (%) 1–12 (64) 32 (50) 18 (21.1) 9 (14.06) 13–36 (42) 15 (35.7) 2 (0.47) 8 (19.04) 37–59 (14) 3 (21.4) 1 (0.7) 2 (14.28) Total (120) 50 (41.6) 21 (17.5) 19 (15.8) Age in months (N) TachypneaaN (%) Hypoxemia N (%) Vomiting N (%) 1–12 (64) 32 (50) 18 (21.1) 9 (14.06) 13–36 (42) 15 (35.7) 2 (0.47) 8 (19.04) 37–59 (14) 3 (21.4) 1 (0.7) 2 (14.28) Total (120) 50 (41.6) 21 (17.5) 19 (15.8) a Tachypnea: <2 months ≥ 60/min, 2–12 months ≥ 50/min, 1–5 years ≥ 40/min. Table 1 Adverse events associated with sputum induction in children aged <5 years in a tertiary care hospital setting Age in months (N) TachypneaaN (%) Hypoxemia N (%) Vomiting N (%) 1–12 (64) 32 (50) 18 (21.1) 9 (14.06) 13–36 (42) 15 (35.7) 2 (0.47) 8 (19.04) 37–59 (14) 3 (21.4) 1 (0.7) 2 (14.28) Total (120) 50 (41.6) 21 (17.5) 19 (15.8) Age in months (N) TachypneaaN (%) Hypoxemia N (%) Vomiting N (%) 1–12 (64) 32 (50) 18 (21.1) 9 (14.06) 13–36 (42) 15 (35.7) 2 (0.47) 8 (19.04) 37–59 (14) 3 (21.4) 1 (0.7) 2 (14.28) Total (120) 50 (41.6) 21 (17.5) 19 (15.8) a Tachypnea: <2 months ≥ 60/min, 2–12 months ≥ 50/min, 1–5 years ≥ 40/min. Table 2 Comparison of baseline and post-procedure RR and SpO2 during sputum induction Age (months) RR Oxygen saturation Baseline Immediate post-procedure 30 min post- procedure 1-h post- procedure Baseline Immediate post-procedure 30-min post- procedure 1-h post- procedure 1–12 45.93 (12.4) 57.2 (14.99) 47.09 (12.88) 46.25 (12.38) 95.71 (1.85) 90.6 (2.27) 96.34 (1.38) 97.14 (1.32) 13–36 38.6 (7.08) 48.42 (8.96) 39.57 (6.96) 39.09 (7.68) 96 (1.68) 91.54 (1.7) 96.42 (1.15) 96.80 (1.38) 37–59 33 (4.42) 42 (4.71) 34.57 (5.23) 34.28 (5.12) 96.35 (2.06) 91.5 (2.14) 91.5 (2.14) 96.59 (1.85) Age (months) RR Oxygen saturation Baseline Immediate post-procedure 30 min post- procedure 1-h post- procedure Baseline Immediate post-procedure 30-min post- procedure 1-h post- procedure 1–12 45.93 (12.4) 57.2 (14.99) 47.09 (12.88) 46.25 (12.38) 95.71 (1.85) 90.6 (2.27) 96.34 (1.38) 97.14 (1.32) 13–36 38.6 (7.08) 48.42 (8.96) 39.57 (6.96) 39.09 (7.68) 96 (1.68) 91.54 (1.7) 96.42 (1.15) 96.80 (1.38) 37–59 33 (4.42) 42 (4.71) 34.57 (5.23) 34.28 (5.12) 96.35 (2.06) 91.5 (2.14) 91.5 (2.14) 96.59 (1.85) Note: Data are presented in mean (SD). Comparison of mean between baseline and immediate post-procedure values by paired t-test showed p < 0.001 in all age groups. Table 2 Comparison of baseline and post-procedure RR and SpO2 during sputum induction Age (months) RR Oxygen saturation Baseline Immediate post-procedure 30 min post- procedure 1-h post- procedure Baseline Immediate post-procedure 30-min post- procedure 1-h post- procedure 1–12 45.93 (12.4) 57.2 (14.99) 47.09 (12.88) 46.25 (12.38) 95.71 (1.85) 90.6 (2.27) 96.34 (1.38) 97.14 (1.32) 13–36 38.6 (7.08) 48.42 (8.96) 39.57 (6.96) 39.09 (7.68) 96 (1.68) 91.54 (1.7) 96.42 (1.15) 96.80 (1.38) 37–59 33 (4.42) 42 (4.71) 34.57 (5.23) 34.28 (5.12) 96.35 (2.06) 91.5 (2.14) 91.5 (2.14) 96.59 (1.85) Age (months) RR Oxygen saturation Baseline Immediate post-procedure 30 min post- procedure 1-h post- procedure Baseline Immediate post-procedure 30-min post- procedure 1-h post- procedure 1–12 45.93 (12.4) 57.2 (14.99) 47.09 (12.88) 46.25 (12.38) 95.71 (1.85) 90.6 (2.27) 96.34 (1.38) 97.14 (1.32) 13–36 38.6 (7.08) 48.42 (8.96) 39.57 (6.96) 39.09 (7.68) 96 (1.68) 91.54 (1.7) 96.42 (1.15) 96.80 (1.38) 37–59 33 (4.42) 42 (4.71) 34.57 (5.23) 34.28 (5.12) 96.35 (2.06) 91.5 (2.14) 91.5 (2.14) 96.59 (1.85) Note: Data are presented in mean (SD). Comparison of mean between baseline and immediate post-procedure values by paired t-test showed p < 0.001 in all age groups. Fig. 1. View largeDownload slide Study flow chart. Fig. 1. View largeDownload slide Study flow chart. Of the 64 cases with good quality sputum, 45 (70.3%) yielded a potential pathogen. Two cases had mixed organisms. Most common pathogen isolated was Klebsiella pneumoniae (38.2%) followed by Streptococcus pneumoniae (14.8%) and others (Table 3). All cases from which Acinetobactor and Enterococci were isolated had prior hospitalization. Klebsiella was isolated from six of seven cases with pleural effusion. Table 3 Bacterial isolates from induced sputum Bacterial isolates Frequency Percentage Klebsiella pneumoniae 18 38.2 Streptococcus pneumoniae 7 14.8 Pseudomonas aeruginosa 6 12.7 Escherichia coli 6 12.7 Staphylococcus aureus 4 8.5 Acinetobacter spp. 4 8.5 Enterococcus spp. 2 4.2 Total 47 100 Bacterial isolates Frequency Percentage Klebsiella pneumoniae 18 38.2 Streptococcus pneumoniae 7 14.8 Pseudomonas aeruginosa 6 12.7 Escherichia coli 6 12.7 Staphylococcus aureus 4 8.5 Acinetobacter spp. 4 8.5 Enterococcus spp. 2 4.2 Total 47 100 Table 3 Bacterial isolates from induced sputum Bacterial isolates Frequency Percentage Klebsiella pneumoniae 18 38.2 Streptococcus pneumoniae 7 14.8 Pseudomonas aeruginosa 6 12.7 Escherichia coli 6 12.7 Staphylococcus aureus 4 8.5 Acinetobacter spp. 4 8.5 Enterococcus spp. 2 4.2 Total 47 100 Bacterial isolates Frequency Percentage Klebsiella pneumoniae 18 38.2 Streptococcus pneumoniae 7 14.8 Pseudomonas aeruginosa 6 12.7 Escherichia coli 6 12.7 Staphylococcus aureus 4 8.5 Acinetobacter spp. 4 8.5 Enterococcus spp. 2 4.2 Total 47 100 DISCUSSION We attempted to study the success, tolerability and bacterial isolates of induced sputum, as it is a standard practice only in some clinical sites. Duration of inhalation, type and output of nebulizer and concentration of hypertonic saline might influence the success and composition of collected specimen [12, 17, 19]. Shorter induction of 15 min and 3% hypertonic saline were used in the study, as it was found optimal based on earlier studies [17]. Although we obtained adequate sputum samples from all cases, only 53.3% were of good quality. The presence of <10 squamous epithelial cells and >25 polymorphonuclear leucocytes per low power field is indicative of high-quality expectorated sputum in adults. However, PERCH study has concluded that the presence of <10 squamous cells per low power field is the best measure of induced sputum quality in young children [9]. In the present study, success was highest in the 37–59 months age group and lowest in the 13–36 months group. The difficulty in getting the subject’s co-operation and making them follow the instructions in the latter group could possibly explain this finding. The success in the study was lower than most of the previous reports. The reported success of sputum induction in children varies from 26 to 100% [5, 6, 14, 15, 19, 20]. Studies from Finland and Kenya have reported success of 75.2 and 76.5%, respectively. In contrast, only 26.3% sputum samples were of good quality in a pilot study for the PERCH project from New Caledonia, despite the fact that sputum was one of the crucial specimens recommended in the study [11]. This clearly shows that standardization of the procedure and training of the personal is critical in obtaining comparable results. The differences in the age group studied and technique and criteria used are likely to be responsible for this wide variability observed between different studies. Transient hypoxemia was observed in 17.5% and was significantly more frequent (21%) in infants. Though none required supplemental oxygen, the potential of the procedure to cause hypoxemia especially in infants should not be overlooked. Subjects with resting hypoxemia or those requiring high supplemental oxygen are not candidates for sputum induction. The PERCH study also reported hypoxemia as the most common (9 of 13 cases) serious adverse event (SAE). Despite the low (0.34%) frequency of SAEs, they recommended vigilant monitoring for up to 2 h post-procedure [8]. The adverse effects in the study were tolerable with acceptable risks as reported by previous investigators [4, 8, 13]. This was a preliminary study in our unit, and it seems feasible now to repeat it with better success. We observed high (70.3%) bacterial yield from good quality sputum (Fig. 1). The bacterial isolation from induced sputum using culture varies from 17 to 79% [10, 12, 14, 15, 19]. There was a striking surge of K. pneumoniae in the study. Similar organism profile as in the present study was reported from India by Taneja et al. [21] in their study on nasopharyngeal aspirates. Studies from Uganda and Nigeria have also reported predominance of gram negative isolates [15, 22]. The spectrum of pathogens varies between regions and settings with bacterial pneumonia more prevalent in poor communities and low-income countries. The predominance of necrotizing pathogens in present study as against conventional S. pneumoniae and Haemophilusinfluenzae can at least partly be explained by the hospital-based nature of the study and inclusion of subjects with prior antibiotic exposure. The widely prevalent practice of antibiotic overuse and misuse coupled with malnutrition may be responsible for the emergence of necrotizing pathogens in developing countries [22]. Non-isolation of H. influenzae in the study could arguably be attributed to the laboratory inadequacies. H. influenzae has fastidious growth requirements and need special laboratory techniques. Prior antibiotic use and Hib vaccination also affect isolation of the organism [23]. At the time of the study, Hib conjugate vaccine had just been introduced in the national immunization program, and there was still lower coverage with pneumococcal conjugate vaccine. Currently, pneumococcal vaccine is given only through private sectors in India. Although all children tolerated the procedure, they generally found sputum collection unpleasant. Sputum induction requires technical support, expertise and substantial amount of time. Hence, the procedure is of value only in hospital settings. The limitations in obtaining good quality specimen probably preclude its use in all clinical sites. Even with meticulous technique, contamination from upper airways is common, and bacterial culture of induced sputum must be interpreted carefully to distinguish between infection and colonization. An organism isolated from sputum could either mean that the sample is contaminated with upper respiratory secretions or an invasive infection caused by a colonizing pathogen, which is well known in respiratory tract infections. One of the key observations in PERCH study is that they did not find any clear association between positive sputum culture results and pneumonia status. Hence, routine use of induced sputum as a diagnostic tool is not recommended. Nevertheless, if interpreted carefully, induced sputum could be relevant in the etiological diagnosis of childhood pneumonia, which fails to respond to appropriate antibiotics. Our observation suggests that the procedure is likely to be more successful in children >3 years. Our study has limited external validity, as it is from a single a referral center. In conclusion, sputum induction in young children can be performed safely in hospital settings and has acceptable risks. The procedure should be conducted by trained staff who are experienced in identification and management of adverse events. Although the success was modest, bacterial yield was good. Hence, the importance of good technique to yield high-quality specimens. Large multicenter studies to evaluate different induction methods, influence of age on success and the repeatability of induced sputum are suggested. REFERENCES 1 Rudan I , Boschi Pinto C , Biloglav Z , et al. Epidemiology and etiology of childhood pneumonia . Bull World Health Org 2008 ; 86 : 408 – 16 . Google Scholar CrossRef Search ADS PubMed 2 Blau H , Linnane B , Carzino R , et al. Induced sputum compared to broncho-alveolar lavage in young, non-expectorating cystic fibrosis children . J Cyst Fibros 2014 ; 13 : 106 – 10 . Google Scholar CrossRef Search ADS PubMed 3 García-Elorriaga G , Rey DPG. Basic concepts on community-acquired bacterial pneumonia in pediatrics . J Pediatric Infect Dis 2016 ; 1 : 3 . 4 Grant L , Hammitt L , Murdoch D , et al. Procedure for collection of induced sputum specimens from children . Clin Infect Dis 2012 ; 54 : 140 – 5 . Google Scholar CrossRef Search ADS 5 Lahiti E , Peltola V , Waris M , et al. Induced sputum in the diagnosis of childhood community acquired pneumonia . Thorax 2009 ; 64 : 252 – 7 . Google Scholar CrossRef Search ADS PubMed 6 Honikinen M , Lahiti E , Osterback R , et al. Viruses and bacteria in sputum samples of children with community-acquired pneumonia . Clic Microbiol Infect 2012 ; 18 : 300 – 7 . Google Scholar CrossRef Search ADS 7 Hammitt L , Murdoch D , Scott J , et al. Specimen collection for diagnosis of pediatric pneumonia . Clin Infect Dis 2012 ; 54 : S132 – 9 . Google Scholar CrossRef Search ADS PubMed 8 DeLuca AN , Hammitt LL , Kim J , et al. Safety of induced sputum collection in children hospitalized with severe or very severe pneumonia . Clin Infect Dis 2017 ; 64(Suppl 3) : S301 – 8 . Google Scholar CrossRef Search ADS 9 Murdoch DR , Morpeth SC , Hammitt LL , et al. Microscopic analysis and quality assessment of induced sputum from children with pneumonia in the PERCH study . Clin Infect Dis 2017 ; 64(Suppl 3) : S271 – 9 . Google Scholar CrossRef Search ADS 10 Murdoch DR , Morpeth SC , Hammitt LL , et al. The diagnostic utility of induced sputum microscopy and culture in childhood pneumonia . Clin Infect Dis 2017 ; 64(Suppl 3) : S280 – 8 . Google Scholar CrossRef Search ADS 11 Mermond S , Zurawski V , D’Ortenzio E , et al. Lower respiratory infections among hospitalized children in New Caledonia: a pilot study for the pneumonia etiology research for child health project . Clin Infect Dis 2012 ; 54 : S180 – 9 . Google Scholar CrossRef Search ADS PubMed 12 Zar HJ , Tannenbaum E , Hanslo D , et al. Sputum induction as a diagnostic tool for community acquired pneumonia in infants and young children from a high HIV prevalence area . Pediatr Pulmonol 2003 ; 36 : 58 – 62 . Google Scholar CrossRef Search ADS PubMed 13 Gibson P , Grootendorst D , Henry R , et al. Sputum induction in children—report of working group 6 . Eur Respir J 2002 ; 20 : 44S – 6S . Google Scholar CrossRef Search ADS 14 Hammit L , Kazunga S , Morpeth S , et al. A preliminary study of pneumonia etiology among hospitalized children in Kenya . Clin Infect Dis 2012 ; 54 : 190 – 9 . Google Scholar CrossRef Search ADS 15 Nantanda R , Hildenwall H , Peterson S , et al. Bacterial aetiology and outcome in children with severe pneumonia in Uganda . Ann Trop Pediatr 2008 ; 28 : 253 – 60 . Google Scholar CrossRef Search ADS 16 Revised WHO Classification and Treatment of Pneumonia in Children at Health Facilities: Evidence Summaries . Geneva : World Health Organization , 2014 . http://www.ncbi.nlm.nih.gov/books/NBK264162. 17 Paggiaro P , Chanez P , Holz O , et al. Sputum induction – report of working group 1 . Eur Respir J 2002 ; 20 : 3S – 8S . 18 Winn W , Allen S , Janda W , et al. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology . 6th edn. Philadelphia: Lippincott Williams and Wilkins, 2006 , 83 – 84 . 19 Jasin M , Setyanto D , Hadinegoro S , et al. Efficacy of sputum induction from lower respiratory tract in children . Pediatr Indones 2015 ; 55 : 101 – 8 . 20 Chen CJ , Lin PY , Tsai MH , et al. Etiology of community acquired pneumonia in hospitalized children in Northern Taiwan . Pediatr Infect Dis J 2001 ; 31 : 196 – 201 . Google Scholar CrossRef Search ADS 21 Taneja J , Malik A , Malik A , et al. Acute lower respiratory tract infections in children . Indian Pediatr 2009 ; 46 : 509 – 11 . Google Scholar PubMed 22 Johnson AW , Osinusi K , Aderele WI , et al. Etiologic agents and outcome determinants of community acquired pneumonia in urban children: a hospital based study . J Natl Med Assoc 2008 ; 100 : 370 – 85 . Google Scholar CrossRef Search ADS PubMed 23 Tille P. Bailey and Scott’s Diagnostic Microbiology . 13th edn. St. Louis : Mosby Inc , 2013 , 403 – 9 . © The Author(s) [2018]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Tropical Pediatrics Oxford University Press

Induced Sputum as a Diagnostic Tool in Pneumonia in Under Five Children—A Hospital-based Study

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Abstract

Abstract Objective The objective of this articlewas to study the success, tolerability of sputum induction and the bacterial isolates of induced sputum in children aged <5 years. Methods The cross-sectional study included 120 hospitalized children aged 1–59 months meeting WHO criteria for pneumonia. Sputum induction was performed using hypertonic (3%) saline. Results Mean age of the subjects was 19.5 months (2–59 months). Overall success of sputum induction was 53.3% and highest (64.28%) in 37–59 months age group. Adverse events such as tachypnea, hypoxemia (SpO2 <90) and vomiting were observed in 41.6, 17.5 and 15.8%, respectively. A potential pathogen was isolated in 45 (70.3%) of 64 cases with good quality sputum. Klebsiella pneumoniae was the commonest (38.2%) followed by Streptococcus pneumoniae (14.8%) and others. Conclusion Sputum induction in young children is safe and feasible in Indian settings. While the success was limited, bacterial yield was high. Klebsiella pneumoniae, success, bacterial isolates INTRODUCTION According to WHO, around 43 million new cases of pneumonia (23% of world’s total) occur in India with an incidence of 0.37 episodes per child-year. Predicted deaths because of pneumonia in India are ∼4 08 000 annually [1]. Most pneumonias respond to empirical therapy, and causative organism is seldom identified. Etiological diagnosis of childhood pneumonia is challenging because of the difficulty in obtaining specimen from the site of infection and the lack of gold standard. Chest radiographs, nonspecific inflammatory markers and nasopharyngeal aspirates have been used as diagnostic tools, but the results have been inconsistent. Though pulmonary aspirate and bronchoalveolar lavage are considered ideal in determining the etiology of pneumonia, they have many limitations and not feasible in all settings [2, 3]. Sputum has the advantage that the specimen comes directly from the site of infection, and contamination aside indicates microbiological flora in the diseased lung [4]. Microbiologic examination of sputum is often performed to determine the etiology of lower respiratory tract infections in adults [5]. In children, sputum induction is used as a diagnostic tool in settings with high prevalence of tuberculosis and in cystic fibrosis [3, 6, 7]. It has also proven useful in hospitalized children with community acquired pneumonia [6]. Despite its advantages, sputum induction is not a standard procedure in infants and young children. Infants and children tend to swallow sputum, thus making sputum collection difficult. Recently published results of PERCH (Pneumonia Etiology Research for Child Heath), the first large-scale study of its kind involving seven countries (nine sites) has thrown light on the safety, utility and quality assessment of induced sputum in children aged 1–59 months in resource-constraint settings [8–10] Reported success for obtaining good quality sputum in children varies considerably [5, 11, 12]. Sputum induction is generally considered safe with satisfactory success in children aged >6 years [13]. There is no study describing the success and safety of sputum induction in young children from India. The purpose of this study was to evaluate the success and tolerability of sputum induction and the bacterial isolates in children aged <5 years, as they are at greater risk of severe pneumonia. METHODS The present cross-sectional study was conducted from November 2013 to May 2015 at a tertiary care teaching hospital in Western India after institutional ethics committee clearance. Sample size calculation for success was done based on the success (76.5%) reported by Hammitt et al. [14] and for bacterial yield study by Nantanda et al. [15] (54%) was used. With a significance level of 5% and power of 90%, calculated minimum sample sizes were 95.43 and 69.06, respectively, for success and bacterial yield. Hence, in the study, 120 observations were considered. Children aged 1–59 months meeting WHO criteria for pneumonia and severe pneumonia were included [16]. Exclusion criteria were room air oxygen saturation <92%, patients requiring oxygen >8 l/min, platelets <25 000 per cumm, children with major congenital malformations and children with history of seizures, severe bronchospasm and severe pneumonia. After obtaining informed consent oxygen saturation at room air was measured. Sputum induction was carried out within 24 h by a skilled physiotherapist along with the research physician. All samples were collected by same set of operators. Sputum induction was performed according to European respiratory society task force recommendation [13, 17]. Various prerequisites with regard to this procedure are equipment, personal, bronchodilator treatment and monitoring of the subjects. Equipment used in the study included ultrasonic nebulizer with oxygen flow, suction apparatus, multipara monitor and mucus extractor. Subjects were kept nil oral for 2–3 h before the procedure. They were pretreated with salbutamol inhalation (2.5 mg salbutamol in 2 ml of normal saline) to prevent bronchospasm. This was followed by 3% saline nebulization using ultrasonic nebulizer with oxygen flow for 5 min at a time for 15 min to induce cough. Chest vibrations and percussions were given, and child was positioned for postural drainage. Sputum was obtained by oropharyngeal suctioning using the catheter of mucus extractor passed through an appropriate size airway. The specimen was collected directly in to the sterile mucus extractor container. Suctioning through an airway was done to minimize contamination. Samples were transported within 2 h of collection to the microbiology laboratory for processing. Slides for gram staining were prepared according to standard methods. The sputum sample was inoculated on to blood agar, chocolate agar and McConkey agar. Antibiotic sensitivity was determined according to the Clinical Laboratory Standards Institute (CLSI) guidelines. Sample quality was assessed by the number of squamous epithelial cells and leucocytes on the basis of standard metrics designed by Bartlett, Murray and Washington [18]. Sputum was labeled good quality when there were <10 squamous epithelial cells and >25 leucocytes per low-power field. Only good quality samples were processed for microbiological testing. Respiratory rate (RR) and oxygen saturation (SpO2) were continuously monitored during procedure till 1-h post-procedure. Subjects were carefully observed for persistent cough, increase in wheeze, change in sensorium, vomiting or clinical worsening [4, 13]. A drop in oxygen saturation <90% was considered hypoxemia in the study. Criterion for stopping the procedure was drop in saturation <88% for >60 s and not picking up with supplemental oxygen. Tachypnea was defined as per WHO criteria. The success of the procedure was defined as (i) obtaining at least 1 ml of sputum, (ii) obtaining good quality sputum and (iii) completing the induction procedure without interruption. STATISTICAL ANALYSIS Proportions and percentages were obtained for qualitative data, whereas mean and SD were obtained for quantitative data. Chi-square test was used to check the association of parameters. p-value of <0.05 was considered significant. Paired t-test was used to compare baseline and post-procedure parameters. Odds ratio of occurrence of adverse events was calculated. Analysis was done using Microsoft Excel and SPSS-22. RESULTS Of 120 subjects, 64 infants (53.3%) formed the largest group followed by 42 children between 13 and 36 months (40.1%) and 14 children between 37 and 59 months (11.6%). Males constituted 65.8%. The mean age was 19.5 months (range 2–59 months). Mean duration of symptoms was 6.1 days. Most 74 (61.6%) reported antibiotic use and 52 (43.3%) had hospitalization before sputum collection. Mean duration of hospitalization was 3.7 days (range 1–8 days) and that of antibiotic use 4.1 days (range 1–10 days). In the study, 80 (66.6%) subjects were of pneumonia and 40 (33.3%) were of severe pneumonia. Good quality sputum was obtained in 64 (53.3%) cases (Fig. 1). The success was highest (64.28%) in children aged 37–59 months followed by 59.3% in 1–12 months, and 40.4% in 13–36 months. Baseline tachypnea was present in 42 (35%), but overall 50 (41.6%) subjects had tachypnea post-procedure. Drop in oxygen saturation <90% was observed in 17.5% and vomiting in 15.8% (Table 1). The lowest recorded saturation was 86%. The odds (95% confidence interval) of occurrence of hypoxemia was 6.913 (1.913, 24.976) times greater and that of tachypnea 2.11 (1.0025, 4.445) times greater in infants as compared with children >12 months. The increased RR lasted only for around 10 min and drop in O2 saturation for <1 min. Hypoxemia corresponded with the time of suctioning. All parameters started normalizing once suctioning was done and returned to near baseline values within 0.5 h (Table 2). None required increased oxygen, bronchodilator therapy or premature discontinuation of the procedure. The lowest collected sputum volume was 1 ml and highest was 5 ml. Duration of the procedure ranged from 15 to 20 min. Table 1 Adverse events associated with sputum induction in children aged <5 years in a tertiary care hospital setting Age in months (N) TachypneaaN (%) Hypoxemia N (%) Vomiting N (%) 1–12 (64) 32 (50) 18 (21.1) 9 (14.06) 13–36 (42) 15 (35.7) 2 (0.47) 8 (19.04) 37–59 (14) 3 (21.4) 1 (0.7) 2 (14.28) Total (120) 50 (41.6) 21 (17.5) 19 (15.8) Age in months (N) TachypneaaN (%) Hypoxemia N (%) Vomiting N (%) 1–12 (64) 32 (50) 18 (21.1) 9 (14.06) 13–36 (42) 15 (35.7) 2 (0.47) 8 (19.04) 37–59 (14) 3 (21.4) 1 (0.7) 2 (14.28) Total (120) 50 (41.6) 21 (17.5) 19 (15.8) a Tachypnea: <2 months ≥ 60/min, 2–12 months ≥ 50/min, 1–5 years ≥ 40/min. Table 1 Adverse events associated with sputum induction in children aged <5 years in a tertiary care hospital setting Age in months (N) TachypneaaN (%) Hypoxemia N (%) Vomiting N (%) 1–12 (64) 32 (50) 18 (21.1) 9 (14.06) 13–36 (42) 15 (35.7) 2 (0.47) 8 (19.04) 37–59 (14) 3 (21.4) 1 (0.7) 2 (14.28) Total (120) 50 (41.6) 21 (17.5) 19 (15.8) Age in months (N) TachypneaaN (%) Hypoxemia N (%) Vomiting N (%) 1–12 (64) 32 (50) 18 (21.1) 9 (14.06) 13–36 (42) 15 (35.7) 2 (0.47) 8 (19.04) 37–59 (14) 3 (21.4) 1 (0.7) 2 (14.28) Total (120) 50 (41.6) 21 (17.5) 19 (15.8) a Tachypnea: <2 months ≥ 60/min, 2–12 months ≥ 50/min, 1–5 years ≥ 40/min. Table 2 Comparison of baseline and post-procedure RR and SpO2 during sputum induction Age (months) RR Oxygen saturation Baseline Immediate post-procedure 30 min post- procedure 1-h post- procedure Baseline Immediate post-procedure 30-min post- procedure 1-h post- procedure 1–12 45.93 (12.4) 57.2 (14.99) 47.09 (12.88) 46.25 (12.38) 95.71 (1.85) 90.6 (2.27) 96.34 (1.38) 97.14 (1.32) 13–36 38.6 (7.08) 48.42 (8.96) 39.57 (6.96) 39.09 (7.68) 96 (1.68) 91.54 (1.7) 96.42 (1.15) 96.80 (1.38) 37–59 33 (4.42) 42 (4.71) 34.57 (5.23) 34.28 (5.12) 96.35 (2.06) 91.5 (2.14) 91.5 (2.14) 96.59 (1.85) Age (months) RR Oxygen saturation Baseline Immediate post-procedure 30 min post- procedure 1-h post- procedure Baseline Immediate post-procedure 30-min post- procedure 1-h post- procedure 1–12 45.93 (12.4) 57.2 (14.99) 47.09 (12.88) 46.25 (12.38) 95.71 (1.85) 90.6 (2.27) 96.34 (1.38) 97.14 (1.32) 13–36 38.6 (7.08) 48.42 (8.96) 39.57 (6.96) 39.09 (7.68) 96 (1.68) 91.54 (1.7) 96.42 (1.15) 96.80 (1.38) 37–59 33 (4.42) 42 (4.71) 34.57 (5.23) 34.28 (5.12) 96.35 (2.06) 91.5 (2.14) 91.5 (2.14) 96.59 (1.85) Note: Data are presented in mean (SD). Comparison of mean between baseline and immediate post-procedure values by paired t-test showed p < 0.001 in all age groups. Table 2 Comparison of baseline and post-procedure RR and SpO2 during sputum induction Age (months) RR Oxygen saturation Baseline Immediate post-procedure 30 min post- procedure 1-h post- procedure Baseline Immediate post-procedure 30-min post- procedure 1-h post- procedure 1–12 45.93 (12.4) 57.2 (14.99) 47.09 (12.88) 46.25 (12.38) 95.71 (1.85) 90.6 (2.27) 96.34 (1.38) 97.14 (1.32) 13–36 38.6 (7.08) 48.42 (8.96) 39.57 (6.96) 39.09 (7.68) 96 (1.68) 91.54 (1.7) 96.42 (1.15) 96.80 (1.38) 37–59 33 (4.42) 42 (4.71) 34.57 (5.23) 34.28 (5.12) 96.35 (2.06) 91.5 (2.14) 91.5 (2.14) 96.59 (1.85) Age (months) RR Oxygen saturation Baseline Immediate post-procedure 30 min post- procedure 1-h post- procedure Baseline Immediate post-procedure 30-min post- procedure 1-h post- procedure 1–12 45.93 (12.4) 57.2 (14.99) 47.09 (12.88) 46.25 (12.38) 95.71 (1.85) 90.6 (2.27) 96.34 (1.38) 97.14 (1.32) 13–36 38.6 (7.08) 48.42 (8.96) 39.57 (6.96) 39.09 (7.68) 96 (1.68) 91.54 (1.7) 96.42 (1.15) 96.80 (1.38) 37–59 33 (4.42) 42 (4.71) 34.57 (5.23) 34.28 (5.12) 96.35 (2.06) 91.5 (2.14) 91.5 (2.14) 96.59 (1.85) Note: Data are presented in mean (SD). Comparison of mean between baseline and immediate post-procedure values by paired t-test showed p < 0.001 in all age groups. Fig. 1. View largeDownload slide Study flow chart. Fig. 1. View largeDownload slide Study flow chart. Of the 64 cases with good quality sputum, 45 (70.3%) yielded a potential pathogen. Two cases had mixed organisms. Most common pathogen isolated was Klebsiella pneumoniae (38.2%) followed by Streptococcus pneumoniae (14.8%) and others (Table 3). All cases from which Acinetobactor and Enterococci were isolated had prior hospitalization. Klebsiella was isolated from six of seven cases with pleural effusion. Table 3 Bacterial isolates from induced sputum Bacterial isolates Frequency Percentage Klebsiella pneumoniae 18 38.2 Streptococcus pneumoniae 7 14.8 Pseudomonas aeruginosa 6 12.7 Escherichia coli 6 12.7 Staphylococcus aureus 4 8.5 Acinetobacter spp. 4 8.5 Enterococcus spp. 2 4.2 Total 47 100 Bacterial isolates Frequency Percentage Klebsiella pneumoniae 18 38.2 Streptococcus pneumoniae 7 14.8 Pseudomonas aeruginosa 6 12.7 Escherichia coli 6 12.7 Staphylococcus aureus 4 8.5 Acinetobacter spp. 4 8.5 Enterococcus spp. 2 4.2 Total 47 100 Table 3 Bacterial isolates from induced sputum Bacterial isolates Frequency Percentage Klebsiella pneumoniae 18 38.2 Streptococcus pneumoniae 7 14.8 Pseudomonas aeruginosa 6 12.7 Escherichia coli 6 12.7 Staphylococcus aureus 4 8.5 Acinetobacter spp. 4 8.5 Enterococcus spp. 2 4.2 Total 47 100 Bacterial isolates Frequency Percentage Klebsiella pneumoniae 18 38.2 Streptococcus pneumoniae 7 14.8 Pseudomonas aeruginosa 6 12.7 Escherichia coli 6 12.7 Staphylococcus aureus 4 8.5 Acinetobacter spp. 4 8.5 Enterococcus spp. 2 4.2 Total 47 100 DISCUSSION We attempted to study the success, tolerability and bacterial isolates of induced sputum, as it is a standard practice only in some clinical sites. Duration of inhalation, type and output of nebulizer and concentration of hypertonic saline might influence the success and composition of collected specimen [12, 17, 19]. Shorter induction of 15 min and 3% hypertonic saline were used in the study, as it was found optimal based on earlier studies [17]. Although we obtained adequate sputum samples from all cases, only 53.3% were of good quality. The presence of <10 squamous epithelial cells and >25 polymorphonuclear leucocytes per low power field is indicative of high-quality expectorated sputum in adults. However, PERCH study has concluded that the presence of <10 squamous cells per low power field is the best measure of induced sputum quality in young children [9]. In the present study, success was highest in the 37–59 months age group and lowest in the 13–36 months group. The difficulty in getting the subject’s co-operation and making them follow the instructions in the latter group could possibly explain this finding. The success in the study was lower than most of the previous reports. The reported success of sputum induction in children varies from 26 to 100% [5, 6, 14, 15, 19, 20]. Studies from Finland and Kenya have reported success of 75.2 and 76.5%, respectively. In contrast, only 26.3% sputum samples were of good quality in a pilot study for the PERCH project from New Caledonia, despite the fact that sputum was one of the crucial specimens recommended in the study [11]. This clearly shows that standardization of the procedure and training of the personal is critical in obtaining comparable results. The differences in the age group studied and technique and criteria used are likely to be responsible for this wide variability observed between different studies. Transient hypoxemia was observed in 17.5% and was significantly more frequent (21%) in infants. Though none required supplemental oxygen, the potential of the procedure to cause hypoxemia especially in infants should not be overlooked. Subjects with resting hypoxemia or those requiring high supplemental oxygen are not candidates for sputum induction. The PERCH study also reported hypoxemia as the most common (9 of 13 cases) serious adverse event (SAE). Despite the low (0.34%) frequency of SAEs, they recommended vigilant monitoring for up to 2 h post-procedure [8]. The adverse effects in the study were tolerable with acceptable risks as reported by previous investigators [4, 8, 13]. This was a preliminary study in our unit, and it seems feasible now to repeat it with better success. We observed high (70.3%) bacterial yield from good quality sputum (Fig. 1). The bacterial isolation from induced sputum using culture varies from 17 to 79% [10, 12, 14, 15, 19]. There was a striking surge of K. pneumoniae in the study. Similar organism profile as in the present study was reported from India by Taneja et al. [21] in their study on nasopharyngeal aspirates. Studies from Uganda and Nigeria have also reported predominance of gram negative isolates [15, 22]. The spectrum of pathogens varies between regions and settings with bacterial pneumonia more prevalent in poor communities and low-income countries. The predominance of necrotizing pathogens in present study as against conventional S. pneumoniae and Haemophilusinfluenzae can at least partly be explained by the hospital-based nature of the study and inclusion of subjects with prior antibiotic exposure. The widely prevalent practice of antibiotic overuse and misuse coupled with malnutrition may be responsible for the emergence of necrotizing pathogens in developing countries [22]. Non-isolation of H. influenzae in the study could arguably be attributed to the laboratory inadequacies. H. influenzae has fastidious growth requirements and need special laboratory techniques. Prior antibiotic use and Hib vaccination also affect isolation of the organism [23]. At the time of the study, Hib conjugate vaccine had just been introduced in the national immunization program, and there was still lower coverage with pneumococcal conjugate vaccine. Currently, pneumococcal vaccine is given only through private sectors in India. Although all children tolerated the procedure, they generally found sputum collection unpleasant. Sputum induction requires technical support, expertise and substantial amount of time. Hence, the procedure is of value only in hospital settings. The limitations in obtaining good quality specimen probably preclude its use in all clinical sites. Even with meticulous technique, contamination from upper airways is common, and bacterial culture of induced sputum must be interpreted carefully to distinguish between infection and colonization. An organism isolated from sputum could either mean that the sample is contaminated with upper respiratory secretions or an invasive infection caused by a colonizing pathogen, which is well known in respiratory tract infections. One of the key observations in PERCH study is that they did not find any clear association between positive sputum culture results and pneumonia status. Hence, routine use of induced sputum as a diagnostic tool is not recommended. Nevertheless, if interpreted carefully, induced sputum could be relevant in the etiological diagnosis of childhood pneumonia, which fails to respond to appropriate antibiotics. Our observation suggests that the procedure is likely to be more successful in children >3 years. Our study has limited external validity, as it is from a single a referral center. In conclusion, sputum induction in young children can be performed safely in hospital settings and has acceptable risks. The procedure should be conducted by trained staff who are experienced in identification and management of adverse events. Although the success was modest, bacterial yield was good. Hence, the importance of good technique to yield high-quality specimens. Large multicenter studies to evaluate different induction methods, influence of age on success and the repeatability of induced sputum are suggested. REFERENCES 1 Rudan I , Boschi Pinto C , Biloglav Z , et al. Epidemiology and etiology of childhood pneumonia . Bull World Health Org 2008 ; 86 : 408 – 16 . Google Scholar CrossRef Search ADS PubMed 2 Blau H , Linnane B , Carzino R , et al. Induced sputum compared to broncho-alveolar lavage in young, non-expectorating cystic fibrosis children . J Cyst Fibros 2014 ; 13 : 106 – 10 . Google Scholar CrossRef Search ADS PubMed 3 García-Elorriaga G , Rey DPG. Basic concepts on community-acquired bacterial pneumonia in pediatrics . 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Google Scholar CrossRef Search ADS 9 Murdoch DR , Morpeth SC , Hammitt LL , et al. Microscopic analysis and quality assessment of induced sputum from children with pneumonia in the PERCH study . Clin Infect Dis 2017 ; 64(Suppl 3) : S271 – 9 . Google Scholar CrossRef Search ADS 10 Murdoch DR , Morpeth SC , Hammitt LL , et al. The diagnostic utility of induced sputum microscopy and culture in childhood pneumonia . Clin Infect Dis 2017 ; 64(Suppl 3) : S280 – 8 . Google Scholar CrossRef Search ADS 11 Mermond S , Zurawski V , D’Ortenzio E , et al. Lower respiratory infections among hospitalized children in New Caledonia: a pilot study for the pneumonia etiology research for child health project . Clin Infect Dis 2012 ; 54 : S180 – 9 . Google Scholar CrossRef Search ADS PubMed 12 Zar HJ , Tannenbaum E , Hanslo D , et al. Sputum induction as a diagnostic tool for community acquired pneumonia in infants and young children from a high HIV prevalence area . 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Eur Respir J 2002 ; 20 : 3S – 8S . 18 Winn W , Allen S , Janda W , et al. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology . 6th edn. Philadelphia: Lippincott Williams and Wilkins, 2006 , 83 – 84 . 19 Jasin M , Setyanto D , Hadinegoro S , et al. Efficacy of sputum induction from lower respiratory tract in children . Pediatr Indones 2015 ; 55 : 101 – 8 . 20 Chen CJ , Lin PY , Tsai MH , et al. Etiology of community acquired pneumonia in hospitalized children in Northern Taiwan . Pediatr Infect Dis J 2001 ; 31 : 196 – 201 . Google Scholar CrossRef Search ADS 21 Taneja J , Malik A , Malik A , et al. Acute lower respiratory tract infections in children . Indian Pediatr 2009 ; 46 : 509 – 11 . Google Scholar PubMed 22 Johnson AW , Osinusi K , Aderele WI , et al. Etiologic agents and outcome determinants of community acquired pneumonia in urban children: a hospital based study . J Natl Med Assoc 2008 ; 100 : 370 – 85 . Google Scholar CrossRef Search ADS PubMed 23 Tille P. Bailey and Scott’s Diagnostic Microbiology . 13th edn. St. Louis : Mosby Inc , 2013 , 403 – 9 . © The Author(s) [2018]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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Journal of Tropical PediatricsOxford University Press

Published: Feb 5, 2018

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