Antibiotic use and prescription and its effects on Enterobacteriaceae in the gut in children with mild respiratory infections in Ho Chi Minh City, Vietnam. A prospective observational outpatient study.

BACKGROUND AND OBJECTIVES: Treatment guidelines do not recommend antibiotic use for acute respiratory infections (ARI), except for streptococcal pharyngitis/tonsillitis and pneumonia. However, antibiotics are prescribed frequently for children with ARI, often in absence of evidence for bacterial infection. The objectives of this study were 1) to assess the appropriateness of antibiotic prescriptions for mild ARI in paediatric outpatients in relation to available guidelines and detected pathogens, 2) to assess antibiotic use on presentation using questionnaires and detection in urine 3) to assess the carriage rates and proportions of resistant intestinal Enterobacteriaceae before, during and after consultation.
MATERIALS AND METHODS: Patients were prospectively enrolled in Children's Hospital 1, Ho Chi Minh City, Vietnam and diagnoses, prescribed therapy and outcome were recorded on first visit and on follow-up after 7 days. Respiratory bacterial and viral pathogens were detected using molecular assays. Antibiotic use before presentation was assessed using questionnaires and urine HPLC. The impact of antibiotic usage on intestinal Enterobacteriaceae was assessed with semi-quantitative culture on agar with and without antibiotics on presentation and after 7 and 28 days.
RESULTS: A total of 563 patients were enrolled between February 2009 and February 2010. Antibiotics were prescribed for all except 2 of 563 patients. The majority were 2nd and 3rd generation oral cephalosporins and amoxicillin with or without clavulanic acid. Respiratory viruses were detected in respiratory specimens of 72.5% of patients. Antibiotic use was considered inappropriate in 90.1% and 67.5%, based on guidelines and detected pathogens, respectively. On presentation parents reported antibiotic use for 22% of patients, 41% of parents did not know and 37% denied antibiotic use. Among these three groups, six commonly used antibiotics were detected with HPLC in patients' urine in 49%, 40% and 14%, respectively. Temporary selection of 3rd generation cephalosporin resistant intestinal Enterobacteriaceae during antibiotic use was observed, with co-selection of resistance to aminoglycosides and fluoroquinolones.
CONCLUSIONS: We report overuse and overprescription of antibiotics for uncomplicated ARI with selection of resistant intestinal Enterobacteriaceae, posing a risk for community transmission and persistence in a setting of a highly granular healthcare system and unrestricted access to antibiotics through private pharmacies. REGISTRATION: This study was registered at the International Standard Randomised Controlled Trials Number registry under number ISRCTN32862422: http://www.isrctn.com/ISRCTN32862422.

Introduction

Antimicrobial resistance is a major threat to global health, sustainable development and the global economy [1-3]. Globally, the number of deaths related to antimicrobial resistance is predicted to increase from 700,000 currently to 10 million by 2050 without intervention [1]. In the US and the EU, 23,000 and 25,000 deaths secondary to drug-resistant infections are estimated to occur annually, respectively [4, 5]. These numbers are 3–5 times higher (relative to population size) in Thailand, a middle-income country where antibiotic use is relatively well controlled [6]. Figures are likely to be higher in settings with unrestricted access, a highly granular healthcare system and lack of antibiotic stewardship programmes like Vietnam, where antibiotic usage and resistance rates are among the highest in Asia [7, 8].

Antibiotics are commonly used or prescribed for mild acute respiratory infections (ARI) in both children and adults in primary care. In the UK, for instance, a quarter of the population visit their general practitioner for ARI annually, accounting for 60% of total GP antibiotic prescribing [9, 10]. In Vietnam, the 14th most populous country globally and with a population of 90 million and a high infectious diseases burden, antibiotics are readily available over-the-counter without prescription, including in community health posts and from private pharmacies [11]. Previous work has shown that 78% of antibiotics for human use were dispensed in the 60,000 private pharmacies, of which around 90% without prescription, and that the most common indication was for ARI (80%) [12]. In a cross-sectional study in northern Vietnam 75% of children under 5 had received antibiotics for an ARI in the preceding month, the large majority dispensed without prescription in private pharmacies [13]. However, aetiological studies show that ARIs are mostly caused by viruses and clinical trials have shown no efficacy of antibiotics in treating conditions like acute bronchitis, bronchiolitis and (naso)pharyngitis [14-17].

To inform policy and potential interventions, we aimed to quantify real life antibiotic prescription rates compared with what was recommended from clinical guidelines and in relation to detected pathogens in patients presenting with ARI at an outpatient clinic at a large tertiary care paediatric hospital (Children’s Hospital 1) in Ho Chi Minh City, Vietnam. We also assessed antibiotic use on presentation using questionnaires and HPLC detection of 6 frequently prescribed antibiotics in urine (S1 Table), and the immediate impact of antibiotic usage on selection of resistant intestinal Enterobacteriaceae using semi-quantitative culture.

Materials and methods

Patients and samples

Patients were enrolled at the respiratory infection examination rooms at the outpatient clinic of Children’s hospital 1 (CH1) in Ho Chi Minh City, Vietnam. CH1 is a large tertiary referral centre with an outpatient clinic with 54 examination rooms where 1,600,000 patients are seen annually. At the 2 respiratory infection examination rooms, 45,000 patients are seen annually. Patients are referred to the respiratory infection examination rooms when they register at the outpatient department by nursing staff when presenting with respiratory symptoms as the chief complaint. Patients were eligible for enrolment if they were under 16 years of age, had a clinical diagnosis of ARI (defined as having an acute onset, within the last 5 days, of at least one of the following symptoms as the chief complaint: cough, sore throat, runny nose or nasal congestion), had no underlying illness (except asthma), were not admitted to hospital, and agreed to return for a follow up visit after 1 week. After interim analysis of the first 100 patients a strong increment in the proportions of resistant counted colonies was shown. A study amendment was made and approved and the remaining patients to be enrolled were asked to return after 4 weeks for an additional rectal swab to assess the duration of this effect. Patients with a clinical diagnosis of sinusitis or acute otitis media were not enrolled. Written informed consent was obtained from parents or legal guardians. Eligible patients were recruited by a study doctor during a one-hour period on Mondays, Tuesdays, Wednesdays and Thursdays, with weekly enrolment restricted to a maximum of 10 to 12 patients. Apart from follow-up visits for study purposes, it was common for patients to return a number of times in the first week after first presentation as part of standard outpatient care.

Demographic, clinical and antibiotic prescription and usage data were collected in case report forms (CRF). Attending physicians assessed and recorded clinical diagnosis and outcome. At enrolment combined nose and throat swabs (NTS) in Viral Transport Medium, a rectal swab in normal saline and a urine sample were collected. At follow-up visits after 1 week and, in a subset of patients, after 4 weeks an additional rectal swab was collected.

Based on systematic reviews, local hospital guidelines and international guidelines antibiotics were considered inappropriate based on clinical diagnosis if prescribed for a clinical diagnosis of uncomplicated ARI other than pneumonia or streptococcal pharyngitis/tonsillitis, and based on clinical diagnosis and molecular diagnostic results if in addition to the clinical diagnosis a virus was detected (and no bacteria) [14–16, 18–20].

Ethics approval

Protocols and amendments were reviewed and approved by the Institutional Review Boards of Children’s Hospital 1 and the Health Service of Ho Chi Minh City, and by the University of Oxford Tropical Research Ethics Committee (OxTREC).

Molecular diagnostics

The following bacterial and viral pathogens were detected using inhouse molecular diagnostics: Streptococcus pneumoniae, Haemophilus influenzae type b, Mycoplasma pneumoniae, Legionella pneumophila, Chlamydophila pneumoniae and psittaci, Bordetella pertussis and parapertussis, Influenza virus A-B, Parainfluenza virus 1–4, Respiratory Syncytial Virus A-B, human metapneumovirus A-B, rhinoviruses, enteroviruses, parechoviruses, coronaviruses, bocaviruses and adenoviruses. All assays were published previously, except the L. pneumophila assay [21-28]. For L. pneumophila an inhouse validated assay, developed and used in the Amsterdam University Medical Centres, was used. Nucleic acids were extracted using an automated commercial Guanidinium Thiocyanate (GuSCN) based method on the EasyMag (Easy MAG 2.0, bioMérieux, Marcy l’Étoile, France) or MagnaPure 96 (Roche, Mannheim, Germany) platform. Viral RNA was reversely transcribed using Superscript III reverse transcriptase (Invitrogen, Carlsbad, California, USA) and random hexamers (Roche). Real-time PCR was performed on a LC480 II Thermocycler (Roche). Primers were manufactured by Sigma Proligo (Singapore). Probes containing Minor Groove Binders (MGB) were manufactured by Applied Biosystems Inc. (Foster City, CA, USA), probes containing LCRED 610, 670 or CYAN500 fluorophores or with incorporated Locked Nucleic Acid (LNA) residues were manufactured by Tib Molbiol (Berlin, Germany), and probes containing only HEX or FAM by Sigma Proligo. Equine Arteritis Virus and Phocid Herpes Virus were used as non-competitive internal controls in PCRs with RNA and DNA targets, respectively [29, 30]. Testing was not part of standard of care, was conducted batch-wise in a research laboratory and results were not reported to the treating physicians.

Measurement of antibiotic levels in urine

Antibiotic levels in urine were determined using validated in-house protocols, as described earlier [31]. The most frequently used antibiotics in the hospital and sold in the pharmacy of Children’s Hospital 1 are listed in S1 Table. Briefly, urine concentrations of six frequently used antibiotics (cephadroxil, cephalexin, cefaclor, cefixime, amoxicillin and cefuroxime) were assessed using in-house validated High-Performance Liquid Chromatography (HPLC) protocols. Briefly, HPLC was done on a Lachrom Elite–Hitachi HPLC controlled by EZChrom v3.18 software (Merck–Hitachi Japan). Solid phase extraction was done using Isolute 96 fixed well plates (Biotaga AB, Uppsala, Sweden). Separation was done through a 5μm LichroCart 240x4.6mm Purosphere RP-18/5μm 4x4 RP-18e for cephadroxil, cefaclor, cephalexin and cefixime (limit of detection: 0.08mg/L; lower limit of quantification [LLOQ]: 0.3mg/L) and through a 5μm 125x4mm RP-8/5μm 4x4 RP-8e for amoxicillin and cefuroxime (LOD: 0.1mg/L and 0.05mg/L, respectively; LLOQ: 0.2mg/L) [18].

Semi-quantitative culture of Enterobacteriaceae

Rectal swabs were kept at 4°C and processed within 24 hours of collection. Swabs were suspended in saline and serially diluted. Dilutions were cultured on MacConkey agar and the dilution yielding between 20–200 colonies was made again the next day from the original specimen for culture on MacConkey plates containing tetracycline (4 mg/L), amoxicillin (8 mg/L), amoxicillin-clavulanic acid (8–4 mg/L), ceftazidime (2 mg/L), ciprofloxacin (1 mg/L), trimethoprim/sulfamethoxazole (2/38 mg/L), gentamicin (4 mg/L), meropenem (4 mg/L) and a control plate without antibiotics. The concentrations used were selected based on the CLSI criteria for intermediate susceptibility of these antibiotics for Enterobacteriaceae. Pan-susceptible and resistant ATCC isolates were used as internal quality controls for each batch of plates. Lactose fermenting Enterobacteriaceae were identified as large circular smooth pink colonies by experienced clinical microbiology laboratory technicians and were counted. For each antibiotic the proportion of resistant Enterobacteriaceae was determined as the ratio of bacterial counts on plates with and without different antibiotics. Only pink (lactose fermenting) colonies were counted and throughout this manuscript when we refer to Enterobacteriaceae counts, lactose fermenting Enterobacteriaceae are meant. Bacterial counts (+1) were logarithmically transformed for display in graphs and regression was used to calculate the position of lines representing the mean for each collection timepoint. This semi-quantitative method was designed to be a simple and elegant way to show relative selection / enrichment of resistant easily culturable relevant human pathogens during appropriate or inappropriate use of antibiotics for an infection in a different body compartment.

Statistical analysis

All variables of interest were summarised by group (age, clinical diagnosis, single infection or co-infection). For descriptive statistics, prevalence and percentage were used for categorical variables while mean and standard deviation or median and interquartile range (IQR) were used for normally or non-normally distributed continuous data, respectively. Comparisons of epidemiological and clinical characteristics among age groups or infection groups (no pathogen, one viral infection, one bacterial infection and co-infection) were analysed using Fisher’s exact test for categorical data and the Kruskal-Wallis test for continuous data. Differences of proportions of resistant Enterobacteriaceae colonies between days 1 and 8, and days 8 and week 4 were assessed using the Wilcoxon matched pairs signed-rank test. In order to assess concordance of antibiotic use as determined by parent interviews and urine tests, a Kappa measure of agreement was used. All statistical tests were performed as two-tailed tests at 5% significance levels. SPSS version 20 was used for all analyses.

Results

Demographic and clinical data

Between February 2009 and February 2010 563 patients were enrolled, 545 returned for a follow-up visit at day 8, 13 were followed-up by telephone and 5 were lost to follow-up. Fig 1 shows a flowchart of enrolment and follow-up. Demographics, clinical diagnoses and outcome are displayed in Table 1. Patients had a median age of 1.96 years (IQR 1.05–3.18) and 95.2% of patients were aged 5 years or below. The male: female ratio was 1.28:1. Complete or partial recovery at day 8 according to the attending physician was reported for 138/563 (24.5%) and 361/563 (64.1%) patients, respectively. In contrast, 59/563 (10.5%) patients did not recover: 6.4% remained clinically unchanged, 2.8% worsened and 1.2% were admitted to hospital. By proportion of fully recovered patients at day 8, upper respiratory infections had a better outcome than lower respiratory tract infections (34% [53/155] vs. 21% [81/391]) and, similarly, bronchitis had a worse outcome than other clinical diagnoses (20% [53/262]).

Fig 1

Enrolment flowchart.

Flowchart of enrolment and follow-up of 563 patients with acute respiratory infection enrolled at the outpatient department of Children’s Hospital 1, Ho Chi Minh City, Vietnam.

Table 1

Patient baseline, diagnosis and outcome data.

Characteristicsa	ARI patients (n = 563)
Median age in years (IQR)	1.96 (1.05–3.18)
≤ 1 year, n (%)	137 (24.3)
1-≤ 2 years, n (%)	148 (26.3)
2- ≤ 5 years, n (%)	251 (44.6)
>5 years, n (%)	27 (4.8)
Male, n (%)	316 (56.1)
Clinical diagnosisb
asthma	17 (3.0)
bronchitis	262 (46.5)
bronchiolitis	122 (21.7)
pneumonia	7 (1.2)
nasopharyngitis	105 (18.7)
pharyngitis	42 (7.5)
tonsillitis	7 (1.2)
laryngotracheobronchitis	1 (0.2)
Outcomec
Complete recovery	138 (24.5)
Partial recovery	361 (64.1)
Unchanged	36 (6.4)
Worsened	16 (2.8)
Admitted	7 (1.2)
Unknown	5 (0.9)

a Baseline characteristics of 563 patients with acute respiratory infection enrolled at the outpatient department of Children’s Hospital 1, Ho Chi Minh City, Vietnam

b Clinical diagnosis of 563 patients with acute respiratory infection enrolled at the outpatient department of Children’s Hospital 1, Ho Chi Minh City, Vietnam

c Outcome recorded at day 8 of 563 patients with acute respiratory infection enrolled at the outpatient department of Children’s Hospital 1, Ho Chi Minh City, Vietnam.

Prescription of antibiotics and other medications

Antibiotics were prescribed for all but two patients (561/563, 99.6%), with a median duration of 6 days (IQR: 4–7). The most commonly prescribed antibiotics at presentation were amoxicillin-clavulanic acid (45.6%), cefuroxime axetil (22.0%), cefixime (11.4%), cefaclor (8.2%), erythromycin (3.7%), amoxicillin (3.0%), and cefpodoxime (2.3%). First choice antibiotics for ambulatory pneumonia treatment as recommended by the 2009 hospital guidelines, i.e. amoxicillin or co-trimoxazole, were infrequently prescribed (3.0 and 0.4%, respectively).

In addition to antibiotics, cough syrup (394/563, 70.0%), bronchodilators (324/563, 57.6%), antipyretics (150/563, 26.6%), antihistamines (66/563, 11.7%), mucolytics (62/563, 11.0%) and steroids (58/563, 10.3%) were also prescribed.

Pathogen detection rates and associations

Detection of viral and bacterial pathogens using molecular assays is shown in Table 2. S. pneumoniae and H. influenzae type b were detected in 553/563 (98.4%) and 69/563 (12.3%) NTS samples from patients (Table 2). Results for these two bacteria were not included in further analyses (see discussion).

Table 2

Pathogens detected in respiratory samples.

	ARI patients
	(n = 563)
Viruses
Influenza virus A, n(%)	39 (6.9)
Influenza virus B, n(%)	5 (0.9)
Enterovirus A-D, n(%)	59 (10.5)
Adenovirus, n(%)	52 (9.2)
Rhinovirus A-C, n(%)	152 (27.0)
RSV A/B, n(%)	54 (9.6)
Human metapneumovirus, n(%)	41 (7.3)
Parainfluenza virus 1, n(%)	10 (1.8)
Parainfluenza virus 2, n(%)	11 (2.0)
Parainfluenza virus 3, n(%)	46 (8.2)
Parainfluenza virus 4, n(%)	19 (3.4)
Coronaviruses, n(%)	21 (3.7)
Human parechovirus, n(%)	4 (0.7)
Human bocavirus, n(%)	19 (3.4)
Bacteria
Haemophilus influenzae, n(%)	69 (12.3)
Streptococcus pneumoniae, n(%)	553 (98.4)
Bordetella pertussis, n(%)	4 (0.7)
Bordetella parapertussis, n(%)	11 (2.0)
Legionella pneumophila, n(%)	0 (0)
Mycoplasma pneumoniae, n(%)	25 (4.4)
Chlamydophila pneumoniae, n(%)	3 (0.5)
Chlamydophila psitacci, n(%)	0 (0)
Any pathogen positive, n(%)	426 (75.6)
Single viral infection, n(%)	294 (52.2)
Single bacterial infection, n(%)	16 (2.8)
Co infection, n(%)	116 (20.6)
No pathogen, n(%)	137 (24.3)

Viral and bacterial pathogens detected by real-time multiplex or single (RT-)PCR in pooled nasal and pharyngeal swabs taken at enrolment among 563 patients with acute respiratory infection enrolled at the outpatient department of Children’s Hospital 1, Ho Chi Minh City, Vietnam.

Viruses were detected in 410/563 (72.8%) NTS samples, 294/410 (71.7%) were single infections and 116/410 (28.3%) were co-infections with either viruses or bacteria. Atypical bacteria and Bordetella spp. were detected in 43/563 (7.6%) samples, in most of which (27/43, 63%) viruses were also detected. Most commonly detected viruses were rhinoviruses (27%, 152/563) and enteroviruses (10.5%, 95/563). M. pneumoniae was the most frequently detected atypical bacterium (4.4%, 25/563).

Among patients older than five, a pathogen was detected in only 40% (11/27), as opposed to 80–90% in the <1, 1 and 2–5 age groups.

Detections by month of viral and atypical bacterial pathogens are shown in S1 Fig. Detection rates of pathogens varied from month to month during the year and clear seasonal patterns were observed for several viruses. Influenza virus A, Respiratory Syncytial Virus (RSV) and human metapneumovirus (hMPV) were mostly seen during and around the rainy season (June to November) with hMPV peaking after RSV, Parainfluenzavirus 3 (PIV3) was mostly detected from November to June. Enteroviruses and Rhinoviruses were detected throughout the year, with highest detection rates for Enteroviruses in November and for Rhinoviruses in March.

Significant associations between specific pathogens and clinical diagnosis or outcome were not observed, but co-infections and infections with multiple viruses were associated with a worse outcome.

Among the detected viruses, several are known to also be frequently detected among asymptomatic children, whereas influenza virus A and B, RSV, hMPV and PIV3 have stronger associations with disease and outbreaks of respiratory infections. These 4 were detected in 186/563 (33.0%) patients (influenza A 39, B 5, RSV 54, hMPV 41, PIV3 46, RSV + hMPV 1).

Appropriateness of antibiotics according to guidelines and pathogens

We retrospectively classified antibiotic use as inappropriate based on clinical diagnosis and (RT-)PCR results. We used two definitions for inappropriate antibiotic use: 1) based on clinical diagnosis, i.e. antibiotic use in patients who were not diagnosed with pneumonia or pharyngitis/tonsillitis and 2) based on a combination of clinical and laboratory diagnosis in patients whose respiratory samples were positive for at least one of 14 viruses and negative for 6 bacterial pathogens tested by PCR (excluding S. pneumoniae and H. influenzae).

Only 7 (1.2%) patients had a clinical diagnosis of pneumonia and 49 (8.7%) patients had a diagnosis of pharyngitis or tonsillitis (Table 1). The remaining ninety percent (90.1%, 507/563) of patients received antibiotics inappropriately according to definition 1. First choice antibiotics for ambulatory pneumonia treatment as recommended by the 2009 hospital guidelines, i.e. amoxicillin or co-trimoxazole, were rarely prescribed (3.0 and 0.4%, respectively). Among these 507 patients, 380 had a virus and no atypical bacteria detected, and therefore, according to definition 2, 67.5% (380/563) of patients received antibiotics inappropriately.

Antibiotic use on presentation by questionnaire and by detection in urine by HPLC

Results from parent interviews on antibiotic use and HPLC detection in urine collected at presentation are shown in Tables 3 and 4. From 553/563 (98.2%) patients a urine sample was collected at presentation. Antibiotic use during the two days prior to enrolment was reported by 123/553 (22.2%) of parents, 206/553 (37%) reported no antibiotic use and 224/553 (40.5%) did not know. Among patients from whom a urine sample was collected, 32% (178/553) had a positive result for one of 6 frequently prescribed antibiotics detected by HPLC in urine; 2 different antibiotics were detected in 14 patients and 3 in 4 patients (Table 3). Cefixime, a third-generation oral cephalosporin, was the most frequently detected antibiotic.

Table 3

Antibiotic use assessed by questionnaire and HPLC.

	HPLC	(%)	Questionnaire	(%)	of which HPLC positive
	(total number = 553)		(total number = 553)
Any antibiotic (patients)	178	32.2	123	22.2	60
1 antibiotic	160	28.9	119	21.5
2 antibiotics	14	2.5	4	0.7
3 antibiotics	4	0.7
Total (antibiotics)	200		127
Per antibioitc
Amoxicillin (with or without clavulanic acid)	52	9.4	48	8.7	22
Cefaclor	28	5.1	37	6.7	12
Cefadroxil	34	6.1	3	0.5	3
Cefixime	54	9.8	14	2.5	8
Cefuroxime	10	1.8	16	2.9	4
Cephalexin	22	4.0	1	0.2	1
Other			8	0.2
Total (antibiotics)	200		127		60
Negative (below the level of detection)	375	67.8
Denied use			206	37.3	29
Unknown			224	40.5	89

Antibiotic use among 553 patients with acute respiratory infection prior to enrolment at the outpatient department of Children’s Hospital 1, Ho Chi Minh City, Vietnam as assessed by questionnaire on enrolment and by HPLC of urine collected on enrolment.

Table 4

Kappa score.

Kappa	0.37	questionnaire
		+	-
HPLC	+	60	29	89
	-	63	177	240
		123	206	329

Kappa score of antibiotic use as assessed by questionnaire on enrolment and by HPLC of urine collected on enrolment.

Among those who confirmed or denied antibiotic use, there was agreement with HPLC results in 237/329 (72%; kappa score 0.37 [0.21–0.40: fair agreement], Table 4). Antibiotics were detected in urine of 29/206 (14%) patients whose parents denied use, and in 60/123 (49%) patients for whom use was reported, with the measured antibiotic corresponding to the reported antibiotic in 50/60 (83%) of cases. Use of the 6 tested antibiotics was reported in 56 of the remaining 63 (89%) cases that tested negative. In patients for whom prior use was unknown, antibiotics were detected in 89/224 (40%). The sensitivity of HPLC to detect 6 antibiotics in urine from patients whose parents reported use of these same 6 antibiotics, was 49%.

Effect of antibiotics on intestinal Enterobacteriaceae

We determined the proportion of patients carrying resistant Enterobacteriaceae and the fraction of antibiotic resistant intestinal Enterobacteriaceae by semi-quantitative culture of rectal swabs taken on day 1 (n = 563) and 8 (n = 542) on 9 MacConkey agar plates each: with and different antibiotics (tetracycline, amoxicillin, amoxicillin-clavulanic acid, ceftazidime, ciprofloxacin, trimethoprim/sulfamethoxazole, gentamicin, meropenem). Thirty-five of 40 consecutive invited patients came back to hospital after 4 weeks for re-assessment of intestinal Enterobacteriaceae, after interim analysis of the first 100 patients had shown strong increments of the proportion of patients carrying resistant bacteria and the proportions of resistant bacteria in their samples (Fig 2). Proportions of patients and proportions of counted resistant colonies are shown in Table 5. McNemar test and Wilcoxon matched pairs signed-rank tests were used to assess statistical significance of different proportions of patients and resistant colonies, respectively. Log transformed colony counts at 3 different timepoints on plates with and without 4 different antibiotics (amoxicillin, ceftazidime, gentamicin, and ciprofloxacin) are shown in Fig 2. Regression was used to calculate the position of lines representing the mean.

Fig 2

Gut Enterobacteriaceae grown on plates with and without antibiotics.

Logarithmically transformed colony counts of Enterobacteriaceae on MacConkey agar with (y-axis) and without (x-axis, MC) antibiotics from rectal swabs taken on day 1 (n = 563, blue), day 8 (n = 542, red) and day 29 (n = 35, black) from patients with acute respiratory infection enrolled at the outpatient department of Children’s Hospital 1, Ho Chi Minh City, Vietnam. Red, blue and black lines represent the mean as determined by regression analysis.

Table 5

Proportions of patients with resistant Enterobacteriaceae and proportions of resistant Enterobacteriaceae at different timepoints.

Proportion of children carrying resistant bacteria	Day 1 (n = 563)	Day 8 (n = 542)	Day 28 (n = 35)	p1 (Day 1 vs 8)	p2 (Day 1 vs 28)
Amoxicillin, n (%)	513 (91.1)	516 (95.2)	28 (80.0)	0.008	0.5
Amoxicillin—Clavulanic acid, n (%)	504 (89.5)	513 (94.7)	28 (80.0)	0.001	1
Ceftazidime, n (%)	379 (67.3)	446 (82.3)	13 (37.1)	<0.001	0.2
Ciprofloxacin, n (%)	322 (57.2)	361 (66.6)	20 (57.1)	<0.001	0.2
Gentamicin, n (%)	333 (59.1)	377 (69.6)	19 (54.3)	<0.001	0.3
Tetracycline, n (%)	516 (91.7)	498 (91.9)	29 (82.9)	1	0.5
Cotrimoxazole, n (%)	524 (93.1)	513 (94.6)	25 (71.4)	0.4	0.3
Meropenem, n (%)	4 (0.7)	2 (0.4)	0 (0)	0.7	NA
Proportion of resistant bacteria
Amoxicillin, n (%)	0.84	0.94	0.79	<0.001	0.06
Amoxicillin—Clavulanic acid, n (%)	0.82	0.92	0.82	<0.001	0.87
Ceftazidime, n (%)	0.50	0.70	0.29	<0.001	0.005
Ciprofloxacin, n (%)	0.40	0.50	0.44	<0.001	0.1
Gentamicin, n (%)	0.41	0.52	0.44	<0.001	0.7
Tetracycline, n (%)	0.82	0.84	0.83	0.36	0.28
Cotrimoxazole, n (%)	0.85	0.89	0.68	0.054	0.02
Meropenem, n (%)	0.004	0.002	0	0.5	0.3

The proportion of patients from whom antibiotic resistant Enterobacteriaceae were cultured on 8 plates containing different antibiotics and the proportion of counted resistant Enterobacteriaceae colonies (defined as the ratios of the number of colonies on 8 different plates containing 8 different antibiotics divided by the number of counted colonies on 1 plate without antibiotics) among 563, 542 and 35 patients with acute respiratory infection enrolled at the outpatient department of Children’s Hospital 1, Ho Chi Minh City, Vietnam at day 1, day and day 28 of enrolment. McNemar test and Wilcoxon matched pairs signed-rank tests were used to assess statistical significance of different proportions of patients and resistant colonies, respectively.

For most antibiotics, the proportions of patients carrying resistant bacteria were already high at day 1. These proportions further increased significantly at day 8 for amoxicillin, amoxicillin with clavulanic acid, ceftazidime, ciprofloxacin and gentamicin, and restored to baseline proportions or below after 4 weeks. Similarly, the proportions of resistant intestinal Enterobacteriaceae colonies increased for these 5 antibiotics and returned to day 1 levels after 4 weeks (Table 5 and Fig 2). For ceftazidime and co-trimoxazole the proportions of resistant Enterobacteriaceae colonies were significantly lower after 4 weeks compared to day 1, which may be explained by antibiotic use before presentation. Indeed, when excluding patients who were already using antibiotics on day 1 (assessed by questionnaire or HPLC), proportions of resistant Enterobacteriaceae colonies after 4 weeks were similar to those at day 1.

Discussion

According to systematic reviews and current international and local guidelines, including those in place at the recruiting hospital, only clinical diagnoses of pneumonia and streptococcal pharyngitis/tonsillitis are indications for prescription of antibiotics in outpatients with ARI [14–16, 18–20]. Antibiotics have not been proven effective and therefore are not recommended for other diagnoses unless there are bacterial super-infections, but these have been reported to occur in only around 5–8% of children with ARI [32, 33]. In our study, pneumonia and pharyngitis/tonsillitis were diagnosed in only 1.2% (n = 7) and 8.7% (n = 49) of patients presenting with ARI, but antibiotics were prescribed in all but two patients enrolled. This means that more than 90% of patients inappropriately received antibiotics for clinical diagnoses, which do not require antibiotic treatment according to treatment guidelines. Using more stringent criteria, based on detection of viral and bacterial pathogens (although tested retrospectively in this study), nearly 70% of patients were inappropriately treated with antibiotics, i.e. when only a viral pathogen was detected. Moreover, the choice of antibiotics rarely followed hospital guidelines, but instead broad-spectrum agents, such as amoxicillin-clavulanic acid and second or third generation cephalosporins were prescribed which are not recommended for ARIs.

In addition to high rates of inappropriate antibiotic prescription by attending physicians, antibiotics were also frequently used prior to presentation. Interviewed parents reported antibiotic use before presentation in 22% of children, while 41% of parents did not know whether antibiotics were used. As determined by HPLC detection of antibiotics in urine, the rate of prior use was 32% with fair agreement between reported and detected antibiotics (Table 4). HPLC may provide a more accurate and objective detection rate of antibiotic use than the questionnaire, although sensitivity is limited and HPLC only detects antibiotics several hours after use. Antibiotics were detected by HPLC in only 49% of samples from patients whose parents reported antibiotic use. If we extrapolate this, we can then also assume that the antibiotic use among patients whose parents didn’t report or weren’t sure about antibiotic use is in reality also approximately twice as high as detected with HPLC: 28% and 80%, respectively, or a total of 65% of patients who had used antibiotics before presentation. These high reported, measured and estimated rates of antibiotic use reflect the over-the-counter availability and widespread use of antibiotics in Vietnam.

High rates of antibiotic use in outpatient children with ARI have been reported before, particularly in Asian countries, with rates ranging from 30% in the UK to around 80% in China and Korea [34-36]. Although differences may be partially explained by the use of different definitions and differences in study populations, they support our observed high level of antibiotic overuse in Vietnamese children. A high rate of antibiotic consumption in Vietnamese children was also suggested by a community survey in northern Vietnam where 73% of interviewed parents indicated that their children between 1–5 years old had used antibiotics in the preceding month [13].

That high usage of antibiotics is not without consequences was shown by the selection of antibiotic-resistant Enterobacteriaceae in stool samples of our patients. The proportions of patients carrying resistant bacteria as well as the proportions of resistant Enterobacteriaceae colonies in stools of individual patients to commonly used antibiotics were already high at presentation but showed further significant increases at day 8. This included Enterobacteriaceae resistant to aminoglycosides or quinolones, representing drugs that were not frequently prescribed for ARI, presumably reflecting the presence of multiple resistance genes on mobile genetic elements such as plasmids. The significant increase in resistant bacteria under antibiotic pressure may lead to increased transfer of resistance genes to other commensal or pathogenic bacteria, and to person-to-person spread in the community through faecal-oral transmission. After withdrawing antibiotics, we observed restoration to baseline values of proportions of resistant Enterobacteriaceae colonies for all antibiotics tested. Proportions of Enterobacteriaceae colonies resistant to ceftazidime and co-trimoxazole were restored to below baseline values, which may be explained by antibiotic use before presentation. While these observations suggest that immediate effects of antibiotic treatment on selection of resistant bacteria are only short-lived, long-term persistence of selected resistance genes has been shown in other studies [37-42]. Given the widespread use of antibiotics in Vietnam, the selection, persistence and transmission of resistant bacteria likely occurs on a large scale at community levels, as is also indicated by the high prevalence of resistance at presentation in our outpatients.

We acknowledge there were a number of limitations to this work, e.g. clinical diagnoses were not assessed and recorded in a systematic and standardized way. Although we acknowledge this may be seen as a limitation, our work reflects the real life day-to-day practice in the busy outpatient clinics of a paediatric hospital and representative high antibiotic prescription rates. Results presented here may not be generalisable to countries where there is no over-the-counter availability of antibiotics. Other limitations may have been:

Attending physicians recorded clinical diagnoses at their discretion without the use of exact case definitions. Theoretically, doctors may have over-diagnosed conditions as pneumonia to justify antibiotic prescriptions, although this did not seem to have occurred.

We did not record any laboratory values that are markers of bacterial infection like white blood cell count and C-reactive protein. This is not routinely done in the outpatient clinics where patients were recruited and results would have influenced the observational nature of this study.

The CRF we used did not specifically record (warning signs of) complications as mentioned in some guidelines, e.g. NICE criteria for complications or Centor criteria for streptococcal pharyngitis [43, 44].

We used the respiratory examination rooms for recruitment where the relatively more severe cases of respiratory infections are seen. This may have selected for a patient population more likely to receive antibiotics.

Common childhood bacterial pneumonia pathogens S. pneumoniae and H. influenzae type b were not optimally diagnosed in this study. Respiratory infection with these pathogens should be assessed using wet and Gram-staining and bacterial culture of sputum, which was not performed here. Molecular testing results were not taken into account when describing aetiology as these results were considered to include colonisation, and in the case of S. pneumoniae potentially cross-reactivity. High carriage rates of S. pneumoniae up to 80% have been reported in children in the region, but we didn’t find report of rates as high as found in our cohort [45, 46]. Our PCR assay for S. pneumoniae targeted the pneumolysin gene [21]. This gene is a virulence factor and expressed by almost all isolates of S. pneumoniae. However, recent reports also showed that other species of Streptococcus can also express this gene [47, 48]. Our assay was developed for use in diagnosis of central nervous system infections in otherwise sterile cerebrospinal fluid. It is not unlikely that when used in respiratory swabs cross-reactivity with other oral streptococci may occur. We further justified disregarding these results because we detected these bacteria at similar rates in NTS samples from a cohort of healthy children (unpublished data). The bacterial load, as expressed by Cp value for S. pneumoniae was significantly higher in patients than in healthy children, however, this did not correlate with disease, disease severity, or whether other pathogens were detected. We hypothesized that this higher load, which has also been observed in other studies, was caused by increased secretions and shedding of (colonized) nasopharyngeal epithelial cells and results for these two bacteria were not included in this analysis [49].

We show that in a setting of unrestricted access to antibiotics and non-adherence to clinical guidelines there is an alarmingly high inappropriate use of antibiotics for children with mild self-limiting illness, before visiting the outpatient clinic and prescribed by doctors in the outpatient clinic. This situation is similar in many low- and middle-income countries across Asia and Africa [50]. We also show that taking antibiotics exerts strong but reversible selective pressure on intestinal Enterobacteriaeceae.

Physicians report that they prescribe antibiotics because of a lack of diagnostic tools to help discriminate between bacterial and viral infections. Laboratory capacity building, including classical microbiology and molecular testing for viruses could help provide this information for more severe patients and inpatients within hours to days, which is too slow for outpatients. Point-of-care testing for 4 common viruses associated with ARI (Influenza virus A and B, RSV, hMPV and PIV-3) might have prevented antibiotic prescription in 186/563 (33%) of our patients, assuming good sensitivity and adherence. Likewise, use of a rapid CRP test could help distinguish viral from bacterial infections and reduce antibiotic prescription rates significantly, as was recently shown in a study in northern Vietnam [51].

In 2013, Vietnam established a National Action Plan for AMR of which, similar to the WHO Global Action Plan, key components are to raise public awareness, to strengthen and improve national surveillance on antibiotic use and susceptibility, to ensure adequate supply of quality antibiotics, to promote the proper and safe use of antibiotics in humans, livestock, poultry and aquaculture and to promote infection control [52]. Based on our observations, and more recent surveillance studies on antimicrobial resistance in Vietnam, we propose that Vietnam’s strategy for AMR should, besides enhancement of laboratory support, include a focus on intensive and continued medical training (CME) in rational antibiotic use for medical students and certified medical staff including physicians and development and implementation of antibiotic stewardship programmes and treatment guidelines at all levels of the healthcare system, similar to but more extensive than what published in a recent study [53]. It is crucial to involve commercial pharmacies in this plan, as 80% of antibiotics for human use are dispensed in the 60.000 pharmacies in Vietnam (of which 90% without prescription) and these are dependent on antibiotic sales for 25% of their revenue [12]. The general Vietnamese public should be engaged through educational programmes through national media and in the communities. At government level, existing legislation on prescription-only drugs should be enforced and expanded to assure that antibiotics can no longer be purchased without a doctor’s consultation.

Pathogen detection by month.

Detections by month of viral and atypical bacterial pathogens detected by real-time multiplex or single (RT-)PCR in pooled nasal and pharyngeal swabs taken at enrolment among 563 patients with acute respiratory infection enrolled at the outpatient department of Children’s Hospital 1, Ho Chi Minh City, Vietnam. X-axis: month; Y-axis: number of positive cases per pathogen. FluA: Influenza virus A; FluB: Influenza virus B; RSV A/B: Respiratory Syncytial Virus A and B; PIV1-4: Human parainfluenza viruses 1–4; hRV: Human Rhinovirus; EV: Enterovirus A-D; CoV: Human Coronavirus; BoV: Human Bocavirus; MPV: Human Metapneumovirus; PeV: Human Parechovirus; AdV: Adenovirus; MP: Mycoplasma pneumoniae; CPn: Chlamydophila pneumoniae; CPs: Chlamydophila psitacci; LP: Legionella pneumophila; BPt: Bordetella pertussis; BPp: Bordetella parapertussis.

(TIF)

Click here for additional data file.

Prescribed antibiotics.

(a) List of most frequently prescribed antibiotics in the outpatient department in 2007 of Children’s Hospital 1, Ho Chi Minh City, Vietnam, (b) List of most frequently sold antibiotics for outpatient use in 2007 in the hospital pharmacy of Children’s Hospital 1, Ho Chi Minh City, Vietnam.

(XLSX)

Click here for additional data file.

(XLSX)

Click here for additional data file.

17 Jan 2020

PONE-D-19-28321

Antibiotic use and prescription and its effects on intestinal flora in children with mild respiratory infections in Ho Chi Minh City, Vietnam. A prospective observational outpatient study.

PLOS ONE

Dear Dr. van Doorn,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we have decided that your manuscript does not meet our criteria for publication and must therefore be rejected.

I am sorry that we cannot be more positive on this occasion, but hope that you appreciate the reasons for this decision.

Yours sincerely,

Mehreen Arshad, M.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The authors of this manuscript attempt to determine the overuse of antibiotics in children with URI. While the premise of this is interesting this study does not add new information to this research field. The objective of this study is also unclear. They have looked at resistant organisms very superficially. The 28 day follow up is only done on 35 kids without an explanation on why such few children were enrolled and how they were chosen. The authors have made several comments without showing any supporting data. No details are given on how the molecular diagnostics were done. Specific comments are below:

Introduction

Line 38: data from UK seems out of place, would recommend using Asian countries or those of similar economic status

Methods:

Line 62: how was the follow up subset decided upon? Was there a selection criterion of some sort?

Line 66: was every patient enrolled in that 1 hr? Were they randomized in any way?

Line 89: Was the testing done a standard of care? Is this a commercial assay?

Results:

Line 126: This should be in a flow chart

Line 127-129: This sentence should either be deleted or the data shown.

Line 146-148: It is hard to believe that almost all children were colonized with S. pneumoniae, which then brings in to question the validity of the entire assay. No control data is shown either.

Line 168: It is unclear why the urine HPLC was done. It would have made more sense if the authors stated the objectives clearly in the beginning.

The discussion is too wordy, and comes across as a policy paper in some places.

Figure 1: the x and y axis does not make sense. What is this a log of? CFU/ml? What does MC stand for?

There is no discussion of the supplementary figure or table in the main text.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript prepared by Quang Minh et al presented a report on unnecessary usage of antibiotics in the Vietnamese population. The author summarized data collection on 563 children and evaluated the appropriateness of antibiotic use on presentation and proportion of resistant enterobacteriaceae in the gut flora before, during and after antibiotic prescription.

My major critique of the manuscript is the presentation and analyses of data do not align with the objectives of the manuscript. All objectives should be clearly mentioned in the introduction section. The table and figures are not explained well in the results section, which creates a problem for a reader to understand the objectives and actual summary of the results. I would suggest to rewrite the result and discussion section.

Regression analysis is not a suitable analysis for showing effect of antibiotics on normal flora. Authors has not discussed which colonies they picked and no details were given on bacterial identification. Data is not substantial to support the semi-quantitative quantification of normal flora.

Figure S1 is about the seasonality of pathogens , I am confused, there is no discussion about seasonality. It was discussed out of context in line # 151. Among pathogens detected Strep. Pneumonia (98%), H.influenzae (12.3%) and rhinovirus (27%), somehow the discussion on rhinovirus was left out.

Similarly, table 4 is hard to understand. The author should explain a little bit about the fraction of bacteria and antibiotics.

Add a sentence for a rationale of semi-quantitative detection and how well it represents normal intestinal flora? It was not mentioned whether antibiotics were added in media or it was measured through disk diffusion?

Molecular diagnostics: the details of methods should be given, with the name of the platform, principle of test and details of kits used.

Overall, this manuscript requires language editing as sentence structure is not correct at various places and grammatical mistakes should be corrected.

various sentences donot make sense for example:

line 180-182 212-213, 221-222

Specific comments:

Abstract:

Objective: line# 3-5 needs to be rephrased

The word moleculary should be changed to molecular detection

Line#38: The term GP stands for..?

Line #131-139: data is not shown in any table,

Line# 161: worse outcome should be defined if it is hospital discharge then analyses should be changed accordingly.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

- - - - -

For journal use only: PONEDEC3

18 Mar 2020

We have pasted the editor’s and reviewer’s comments and indicated our replies to them by “>”

The authors of this manuscript attempt to determine the overuse of antibiotics in children with URI. While the premise of this is interesting this study does not add new information to this research field.

>We strongly believe that the systematic and comprehensive way in which we assessed several aspects of outpatient treatment of mild respiratory infections and the impact of antibiotic use adds to the field. While the high rates of antibiotic prescription for ARI in Vietnam are not new, assessing of pre-presentation antibiotic use and the comparison between reported and measured use (using HPLC in urine) certainly is in this setting. Furthermore, the semi-quantitative assessment of selection of resistant flora from longitudinal samples showing the negative impact of unnecessary antibiotic use using standard culture methods has to our knowledge not been done before and shows a very consistent and highly significant difference between resistant fractions among samples on presentation and after antibiotic use among >500 children against 7 out of 8 tested drugs, that was restored in a subset of patients after 28 days.

The objective of this study is also unclear.

>We have more explicitly clarified and expanded further on the objectives that were listed in abstract (line 5-9) and introduction (line 52-58)

They have looked at resistant organisms very superficially.

>We have expanded on the methods (lines 122-137, 147-149), interpretation and analysis (lines 233-254 and legends Figure 2 and table 4) to make clearer what was done and that this was not superficial. Semi-quantitative culture was used to assess the proportions of resistant colonies of lactose fermenting Enterobacteriaceae to 8 different drugs by culturing rectal swabs on MacConkey agar with and without antibiotics. More than 1100 faecal specimens were cultured and counted on 9 plates each.

The 28 day follow up is only done on 35 kids without an explanation on why such few children were enrolled and how they were chosen.

>This additional sample was collected from 35 of the last 40 patients who were enrolled into the study. This amendment to the protocol was made after a very strong selective effect was seen after interim analysis of the first 100 patients. Added to the manuscript in lines 70-73, 237-240)

The authors have made several comments without showing any supporting data.

>The editor did not indicate the instances where we made comments without supporting data. We have gone through the manuscript and think we have addressed these. There are now two instances left where we say “data not shown” or “unpublished data”. One concerns a discussion section of relative loads of S. pneumoniae and H. influenzae in samples from our study patients vs healthy volunteers form an unpublished study. We do not think including these data will contribute to the discussion or the justification of why these data weren’t used in the analysis of our study. The second concerns the exact data of switching regimens during different visits to the outpatient clinic. Although we don’t show the interpretation and analysis of these data, they are included in the raw dataset we uploaded.

No details are given on how the molecular diagnostics were done.

>References for the assays were given, we have published the use of these assays previously. We have added information from these previous publications and expanded on the described methods (line 92-110).

Specific comments are below:

Introduction

Line 38: data from UK seems out of place, would recommend using Asian countries or those of similar economic status

>Added examples from Vietnam in lines 41-51

Methods:

Line 62: how was the follow up subset decided upon? Was there a selection criterion of some sort?

>See above

Line 66: was every patient enrolled in that 1 hr? Were they randomized in any way?

>No randomisation was done. One study doctor was present at the outpatient clinic during this hour and enrolled the first eligible patient from the waiting list, helped the clinic doctors with consent and CRF completion and then moved on to enrol the next eligible patient etc.

Line 89: Was the testing done as standard of care? Is this a commercial assay?

>No and no, added to the manuscript (lines 92-93, 109-110)

Results:

Line 126: This should be in a flow chart

>Added as Figure 1

Line 127-129: This sentence should either be deleted or the data shown.

>Data were added (lines 161-164)

Line 146-148: It is hard to believe that almost all children were colonized with S. pneumoniae, which then brings in to question the validity of the entire assay. No control data is shown either.

>We have not used these data for analysis and have added more specific comments / justification / explanation on this in the discussion (lines 325-344).

Line 168: It is unclear why the urine HPLC was done. It would have made more sense if the authors stated the objectives clearly in the beginning.

>This is now stated more clearly in abstract (line 5-9) and introduction (line 52-58)

The discussion is too wordy, and comes across as a policy paper in some places.

>We have reviewed the discussion. We do not think it is too wordy for a journal without word limit. We think the policy parts (assuming the editor means lines 358-373) contain relevant context and contextual recommendations for the readers

Figure 1: the x and y axis does not make sense. What is this a log of? CFU/ml? What does MC stand for?

> MC stands for MacConkey agar, i.e. agar without antibiotics added, logs are of colony counts. This was stated in the legends. We have tried to show this clearer in both legends to figure 2 and methods (lines 135-137).

There is no discussion of the supplementary figure or table in the main text.

>Lines referring to this table and figure were moved / added to make their purpose and what is shown more clear (lines 55-58 and 189-194)

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript prepared by Quang Minh et al presented a report on unnecessary usage of antibiotics in the Vietnamese population. The author summarized data collection on 563 children and evaluated the appropriateness of antibiotic use on presentation and proportion of resistant enterobacteriaceae in the gut flora before, during and after antibiotic prescription.

My major critique of the manuscript is the presentation and analyses of data do not align with the objectives of the manuscript. All objectives should be clearly mentioned in the introduction section.

>Added in abstract (line 5-9) and introduction (line 52-58)

The table and figures are not explained well in the results section, which creates a problem for a reader to understand the objectives and actual summary of the results. I would suggest to rewrite the result and discussion section.

>We acknowledge this wasn’t done very well and has been changed, see also replies to editors comments. All figures are better referenced in the body of text, legends have been expanded where needed and parts of legends have been copied in the text for better reading and easier understanding.

Regression analysis is not a suitable analysis for showing effect of antibiotics on normal flora.

>The reviewer is of the opinion that something was done incorrectly in their view, without explanation or alternative. Statistical analysis to show difference between proportions of counts of resistant vs all cultured colonies was done using Wilcoxon matched pairs signed-rank test (see lines 147-149, 240-243 and legends to table 4) and regression was only used for visualisation of a mean line (lines 135-137, 243-245, legends to figure 2). We have made this clearer. We have also consulted a second statistician who confirmed the suitability of these analyses both for visual and statistical purposes. They commented the following on the use of the Wilcoxon matched pairs signed-rank test: “The low power of non-parametric tests in general reinforces the strength of these highly significant results.”

Authors has not discussed which colonies they picked and no details were given on bacterial identification.

>Added. Colonies were not picked, but counted. Colonies were not IDd but counted as Enterobacteriaceae based on shape, texture and colour (lines 130-135)

Data is not substantial to support the semi-quantitative quantification of normal flora.

> The reviewer is of the opinion that something was done incorrectly in their view, without explanation or alternative.

We have added more information on methods, analysis and interpretation and believe that both the data and the consistent and significant differences between timepoints and drugs are sufficiently robust and substantial.

Figure S1 is about the seasonality of pathogens , I am confused, there is no discussion about seasonality.

>Apologies, this sentence was cut out without making corrections to the figure. Text was restored and figure is referenced (lines 189-194)

It was discussed out of context in line # 151.

>See above. Apologies.

Among pathogens detected Strep. Pneumonia (98%), H.influenzae (12.3%) and rhinovirus (27%), somehow the discussion on rhinovirus was left out.

>We discussed why we did not include the bacteria in the analysis (lines 178-181, 322-341). We did not discuss all viral families separately, but have assessed appropriateness of antibiotic use taking viral PCR results into consideration for all viruses + atypical bacteria (lines 202-207)

Similarly, table 4 is hard to understand. The author should explain a little bit about the fraction of bacteria and antibiotics.

>We have tried to further clarify this, see lines 122-137, legends to figure 2 and table 4)

Add a sentence for a rationale of semi-quantitative detection and how well it represents normal intestinal flora?

>Added, lines 137-140

It was not mentioned whether antibiotics were added in media or it was measured through disk diffusion?

>Antibiotics were added to the media. We described this more clearly (lines 122-137 and legends to figure 2 and table 4)

Molecular diagnostics: the details of methods should be given, with the name of the platform, principle of test and details of kits used.

>Added in lines 92-110

Overall, this manuscript requires language editing as sentence structure is not correct at various places and grammatical mistakes should be corrected.

>We thank the author for this comment and suggestion and have done our best to check and improve the manuscript for correct use of the English language

various sentences donot make sense for example:

line 180-182 212-213, 221-222

>we have changed these and other sentences and hope they make sense to the reviewer now

Specific comments:

Abstract:

Objective: line# 3-5 needs to be rephrased

>Done (lines 3-7)

The word moleculary should be changed to molecular detection

>Changed “molecularly” to “using molecular assays” (line 10)

Line#38: The term GP stands for..?

>Clarified. GP stands for General Practitioner (line 40-41)

Line #131-139: data is not shown in any table,

>Correct, we do not feel repeating these numbers in a table would add significantly to the manuscript

Line# 161: worse outcome should be defined if it is hospital discharge then analyses should be changed accordingly.

>Outcome was defined and recorded by the outpatient physicians (line 158-161). Unclear what the reviewer means with hospital discharge in the context of outpatient care. Hospital admission was a (rarely) recorded outcome (1.2%, line 161).

Submitted filename: Appeal.docx

Click here for additional data file.

5 Jun 2020

PONE-D-19-28321R1

Antibiotic use and prescription and its effects on intestinal flora in children with mild respiratory infections in Ho Chi Minh City, Vietnam. A prospective observational outpatient study.

PLOS ONE

Dear Dr. van Doorn,

Thank you for submitting your manuscript to PLOS ONE. In response to your request, your revised manscript was sent to 2 additional peer reviewers and I have now received feedback from both of them. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please address the additional comments and in addition, it seems that Table 3b is missing from the manuscript.

Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact.

==============================

Please submit your revised manuscript by Jul 20 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Jane Foster, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

1.) Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2.) We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. "Validation of the HPLC detection methods of antibiotics in urine was described in 10.1002/bmc.4699 (als referenced in manuscript). The total percentage of positive samples (aggregated and without clinical metadata an d questionnaire results) was mentioned in this manuscript" Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript.

3.) In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

4.) Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary).

We note that you have included affiliation numbers 1 - 10 however affiliations 8 does not have an author linked to it. Please amend affiliation 8 to link an author to it or remove if added in error.

5.) We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

6.) Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables should be uploaded as separate "Supporting Information" files.

7.) PRTC Notes I'm not sure if those comments also need to be added or it has already been sent back to author before.

8). Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

Please also clarify whether your study was specifically reviewed and approved by your IRB.

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Antibiotic resistance is a global concern with one of the major contributors being the over-prescription of antibiotics. The current study shines a light on this issue by focusing on antibiotic use and prescription in children with mild respiratory infections in Ho Chi Minh City, Vietnam. To address this issue, the authors enrolled patients and recorded diagnoses, prescribed therapy and outcome at visit 1 and on follow-up after 7 days. Respiratory bacterial and viral pathogens were also detected using molecular assays. Antibiotic use before presentation was assessed using questionnaires and urine HPLC. The main finding reported is the overuse and over-prescription of antibiotics for uncomplicated respiratory infections.

This is an important topic and while the results are not surprising, they are still interesting and worth reporting. I have the following additional queries and recommendations:

(1) The extent to which these findings can be generalised to jurisdictions where antibiotics are available on prescription only needs to be considered more in the discussion. While the over-prescription aspect might be common, over the counter use varies quite a lot.

(2) I agree with the authors that an important part of this study is the measurement of antibiotics in urine. However, the sensitivity of the HPLC assay is very low at 49% and argues against the interpretation provided in the discussion that it may provide a more accurate and higher detection rate of antibiotic use than the questionnaire.

(3) Following on from this point, the authors also make a somewhat flawed leap in logic by stating that they can assume on the basis of this low sensitivity that antibiotic use is twice as high as detected in patients where no antibiotic use was reported. This needs to be removed from the discussion.

(4) The use of the term ‘gut flora’ is problematic as this description has been superseded by ‘gut microbiota’. In any case, since they have only assessed specific members in a targeted way, the analysis falls short of the current gold standards for a global assessment of the microbiota. This makes the title a little misleading and I suggest removing the reference to the gut flora altogether as it will create expectations that are not met with the analysis.

Reviewer #3: The authors of this paper have nicely showed the misuse of antibiotics in uncomplicated ARI in Vietnamese children. They have also determined the presence of different common respiratory viral and bacterial pathogens; as well as went on to determine the presence of antibiotics in their urine. Lastly they have also established the association of “selection of antibiotic-resistant members of Enterobacteriaceae in gut” with antibiotic use. I think that their data will further highlight the irrational overuse of antibiotics in ARI and its effect on the resistance development and selection in microbiota.

I am suggesting some minor edits to further improve the reader’s experience of this paper.

1. Though mentioned in abstract and results but please also mention the sample collection period in methods section.

2. Line 165-176 and lines 200-213. Under both these subheadings, though you have nicely defined results in the text form but would be nice if authors can also present their data in form of tables or figures, either in the paper or in supplementary information.

3. Lines 190-191. Please mention the whole virus name before using abbreviations. Similarly in the associated-figure also mention viral abbreviations.

4. Figure-2 was incorrectly marked as “Figure-1”.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

11 Oct 2020

Academic Editor

Point by point reply (marked with ">")

1.) Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

>I have reformatted tables and figures and affiliations according to these requirements

2.) We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. "Validation of the HPLC detection methods of antibiotics in urine was described in 10.1002/bmc.4699 (als referenced in manuscript). The total percentage of positive samples (aggregated and without clinical metadata an d questionnaire results) was mentioned in this manuscript" Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript.

>We have added a paragraph on this in the cover letter:

Some data presented in this study have been published previously in a technical paper describing validation of the methods. This paper was peer reviewed and published in Biomedical Chromatography 34:e4699. This paper describes development of HPLC methods to detect antibiotics in urine and analysis of samples collected in our study. The paper deals almost entirely with technical and validation aspects of the methods and only has one paragraph and one figure (pasted in cover letter / response to reviewers files) where clinical evaluation and results on clinical samples are presented but only in an aggregated manner and without the context of clinical metadata and questionnaire results:

“The urine samples were collected from 563 pediatric patients under 16 years of age (50% of patients were less than 2 years old, and 95% of patients under 5 years old). The validated methods were successfully applied to determine the six β‐lactams in urine samples (10 patients with severe ARIs were anuria). Figure 2 presents the results of a qualitative measurement of the β‐lactams in clinical samples. Among of the tested β‐lactams, CFI was detected at the highest rate (54/553–9.8%), followed by amoxicillin (52/553–9.4%), while CFU was the least common identified medication, at only 1.8% (10/553).”

We reference the methods and their validation in the methods section of our paper and we present the results in context of their clinical metadata and the questionnaire results and therefore we believe this does not constitute double publication.

3.) In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

>We have now uploaded our dataset with the submission

4.) Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary).

We note that you have included affiliation numbers 1 - 10 however affiliations 8 does not have an author linked to it. Please amend affiliation 8 to link an author to it or remove if added in error.

>Thank you for spotting this, this has been corrected

5.) We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

>The two occasions where we mentioned “data not shown” have been corrected. In the first instance about antibiotic regimen change we deleted the sentence, in the second instance on Streptococci and Haemophilus we deleted “(data not shown)” as the full data set has been uploaded now.

6.) Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables should be uploaded as separate "Supporting Information" files.

>This has been corrected

7.) PRTC Notes I'm not sure if those comments also need to be added or it has already been sent back to author before.

>I have assumed there was nothing to address here

8). Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

Please also clarify whether your study was specifically reviewed and approved by your IRB.

>This was all already included in the methods section, subsections “Patients and samples” and “Ethics approval”.

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Antibiotic resistance is a global concern with one of the major contributors being the over-prescription of antibiotics. The current study shines a light on this issue by focusing on antibiotic use and prescription in children with mild respiratory infections in Ho Chi Minh City, Vietnam. To address this issue, the authors enrolled patients and recorded diagnoses, prescribed therapy and outcome at visit 1 and on follow-up after 7 days. Respiratory bacterial and viral pathogens were also detected using molecular assays. Antibiotic use before presentation was assessed using questionnaires and urine HPLC. The main finding reported is the overuse and over-prescription of antibiotics for uncomplicated respiratory infections.

This is an important topic and while the results are not surprising, they are still interesting and worth reporting. I have the following additional queries and recommendations:

(1) The extent to which these findings can be generalised to jurisdictions where antibiotics are available on prescription only needs to be considered more in the discussion. While the over-prescription aspect might be common, over the counter use varies quite a lot.

>We have added a sentence to the Limitations section on this

(2) I agree with the authors that an important part of this study is the measurement of antibiotics in urine. However, the sensitivity of the HPLC assay is very low at 49% and argues against the interpretation provided in the discussion that it may provide a more accurate and higher detection rate of antibiotic use than the questionnaire.

>We have rephrased this sentence

(3) Following on from this point, the authors also make a somewhat flawed leap in logic by stating that they can assume on the basis of this low sensitivity that antibiotic use is twice as high as detected in patients where no antibiotic use was reported. This needs to be removed from the discussion.

>We think that with the rephrasing of the previous sentence, this assumption / interpretation can still be made albeit with caution

(4) The use of the term ‘gut flora’ is problematic as this description has been superseded by ‘gut microbiota’. In any case, since they have only assessed specific members in a targeted way, the analysis falls short of the current gold standards for a global assessment of the microbiota. This makes the title a little misleading and I suggest removing the reference to the gut flora altogether as it will create expectations that are not met with the analysis.

>We have removed this from the manuscript and instead refer to this work using “intestinal Enterobacteriaceae”

Reviewer #3: The authors of this paper have nicely showed the misuse of antibiotics in uncomplicated ARI in Vietnamese children. They have also determined the presence of different common respiratory viral and bacterial pathogens; as well as went on to determine the presence of antibiotics in their urine. Lastly they have also established the association of “selection of antibiotic-resistant members of Enterobacteriaceae in gut” with antibiotic use. I think that their data will further highlight the irrational overuse of antibiotics in ARI and its effect on the resistance development and selection in microbiota.

I am suggesting some minor edits to further improve the reader’s experience of this paper.

1. Though mentioned in abstract and results but please also mention the sample collection period in methods section.

>Sorry to disagree here, but I don’t think the dates of the actual delivery of methods should be in the methods, as it is a result in my opinion. Happy to add if the editor / reviewer insists, but I don’t think it belongs in methods.

2. Line 165-176 and lines 200-213. Under both these subheadings, though you have nicely defined results in the text form but would be nice if authors can also present their data in form of tables or figures, either in the paper or in supplementary information.

>We have now included all figures/numbers from these sections in the tables

3. Lines 190-191. Please mention the whole virus name before using abbreviations. Similarly in the associated-figure also mention viral abbreviations.

>This has been corrected

4. Figure-2 was incorrectly marked as “Figure-1”.

>Apologies, this has been corrected

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

21 Oct 2020

Antibiotic use and prescription and its effects on Enterobacteriaceae in the gut in children with mild respiratory infections in Ho Chi Minh City, Vietnam. A prospective observational outpatient study.

PONE-D-19-28321R2

Dear Dr. van Doorn,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jane Foster, PhD

Academic Editor

PLOS ONE

23 Oct 2020

PONE-D-19-28321R2

Antibiotic use and prescription and its effects on Enterobacteriaceae in the gut in children with mild respiratory infections in Ho Chi Minh City, Vietnam. A prospective observational outpatient study.

Dear Dr. van Doorn:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Jane Foster

Academic Editor

PLOS ONE