Epidemiology, etiology, and diagnosis of health care acquired pneumonia including ventilator-associated pneumonia in Nepal.

Epidemiologic data regarding health care acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP) from Nepal are negligible. We conducted a prospective observational cohort study in the intensive care unit (ICU) of a major tertiary hospital in Nepal between April 2016 and March 2018, to calculate the incidence of VAP, and to describe clinical variables, microbiological etiology, and outcomes. Four hundred and thirty-eight patients were enrolled in the study. Demographic data, medical history, antimicrobial administration record, chest X-ray, biochemical, microbiological and haematological results, acute physiology and chronic health evaluation II score and the sequential organ failure assessment scores were recorded. Categorical variables were expressed as count and percentage and analyzed using the Fisher's exact test. Continuous variables were expressed as median and interquartile range and analyzed using Kruskal-Wallis rank sum test and the pairwise Wilcoxon rank-sum test. 46.8% (205/438) of the patients required intubation. Pneumonia was common in both intubated (94.14%; 193/205) and non-intubated (52.36%; 122/233) patients. Pneumonia developed among intubated patients in the ICU had longer days of stay in the ICU (median of 10, IQR 5-15, P< 0.001) when compared to non-intubated patients with pneumonia (median of 4, IQR 3-6, P< 0.001). The incidence rate of VAP was 20% (41/205) and incidence density was 16.45 cases per 1,000ventilator days. Mortality was significantly higher in patients with pneumonia requiring intubation (44.6%, 86/193) than patients with pneumonia not requiring intubation (10.7%, 13/122, p<0.001, Fisher's exact test). Gram negative bacteria such as Klebsiella and Acinetobacter species were the dominant organisms from both VAP and non-VAP categories. Multi-drug resistance was highly prevalent in bacterial isolates associated with VAP (90%; 99/110) and non-VAP categories (81.5%; 106/130). HAP including VAP remains to be the most prevalent hospital-acquired infections (HAIs) at Patan hospital. A local study of etiological agents and outcomes of HAP and VAP are required for setting more appropriate guidelines for management of such diseases.

Introduction

Pneumonia is clinically defined as the presence of a new lung infiltrate with evidence that the infiltrate is triggered by an infectious agent such as, the new onset of fever, purulent sputum, or leukocytosis [1]. Healthcare acquired pneumonia (HAP) is an infection of the pulmonary parenchyma that develops>48 hours of admission to a health care facility and is commonly caused by pathogens that circulate in hospital settings [2]. In clinical practice, HAP is suspected when a patient presents with fever, impaired oxygenation, and suppurative secretions [3]. HAP is an important infectious disease worldwide and is associated with high morbidity, mortality, and additional health system expenditure [4]. In the US, the prevalence of HAP has been estimated to be1.6% of all hospital admissions, representing a rate of 3.63 cases per 1,000 patient-days [5]. Epidemiologic data regarding HAP in Asia are scarce; however, the incidence of HAP is predicted to be high across Asia and especially problematic in intensive care units (ICUs), where the proportion of ICU-acquired respiratory infections ranges from 9% to 23% of admissions [6].

Ventilator-associated pneumonia (VAP) is a subtype of HAP that develops in ICU patients who have been mechanically ventilated for at least 48 hour [2, 7, 8]. VAP remains one of the most common infections in patients requiring invasive mechanical ventilation and is the leading cause of ICU mortality [2, 7]. The reported prevalence of VAP vary from 5 to 40% of ventilated patients depending on country, ICU type, and criteria used to diagnose VAP [9]. In high-income countries, a combination of surveillance, education, and tailored intervention and prevention bundles have led to a major reduction in VAP [10]. However, even with the implementation of such programs, VAP is still commonly reported in the US [7]. In Asia there are limited data on incidence of VAP, the causative pathogens, and their antimicrobial susceptibility profiles [6, 11].A meta-analysis which encompassed 88 studies from 22 Asian countries from 2008 to 2018 indicated that the pooled incidence density of VAP in low-middle-income countries (LMICs) (18.5 per 1,000 ventilator-days) was more than twice that in high-income countries (9.0 per 1,000 ventilator-days) [12].

VAP has received little attention in LMICs until relatively recently [13]. In a low income country, like Nepal, where the incidence of infectious disease is high and strategies for control and prevention are weak, the opportunity for nosocomial infection is significantly higher [11, 14–16].This problem is further exacerbated by antimicrobial resistance (AMR) in organisms such as Acinetobacter baumannii and Klebsiella pneumoniae, which are responsible for a large proportion of nosocomial infections and commonly multi-drug resistant (MDR) [17, 18].

A delayed diagnosis and delay in initiating appropriate therapy in VAP may be associated with poor outcomes [2, 19–21]. Therefore, an early and accurate diagnosis is fundamental in the management of patients with VAP. In order to develop effective therapeutic strategies to optimize the use of antimicrobial agents we need a better understanding of the local pathogens causing. Therefore, we performed a prospective study to describe some epidemiological features of HAP among patients admitted to the ICU of major tertiary hospital in Kathmandu, Nepal. We measured the incidence rate of VAP, investigated the antimicrobial susceptibility profiles of the etiological agents, and compared clinical profiles associated with HAP/VAP mortality.

Materials and methods

Ethics approval and consent to participate

This study was approved by Nepal Health Research Council (NHRC) (Reference number 11/2016,Date: 11 March 2016) and Oxford Tropical Research Ethics Committee (OxTREC 32–16, Date:19 October 2016). Adult patients admitted in the ICU or next-of-kin of the patient were approached for written informed consent to participate in this study.

Setting and study design

This was a prospective observational cohort study conducted in the ICU of Patan hospital between April 2016 and March 2018. Patan hospital is a 450-bed tertiary care referral teaching hospital with 15 ICU beds, located in the Lalitpur Metropolitan area of the Kathmandu valley in Nepal.

Study structure

All adult patients, ≥ 18 years of age admitted to the ICU were eligible to participate in the study. Adult patients admitted in the ICU or next-of-kin of the patient were approached for written informed consent to participate. Patients who denied consent and under the 18 years of age were not included in the study. Upon recruitment, demographic data, medical history, antimicrobial administration record, chest X-ray or other imaging findings, biochemical, microbiological and haematological results, and clinical parameters were recorded in a case report form (CRF). The acute physiology and chronic health evaluation (APACHE) II score and the sequential organ failure assessment (SOFA) score were recorded from the biochemical findings of the day of admission. Daily observation of the individual was conducted and CRF completed until an outcome of discharge, death, transfer to another ward or development of VAP.

VAP was defined by following the modified US Centers for Disease Control and Prevention criteria which requires to fulfill radiographic, systemic, and pulmonary criteria [16, 22–24] “Fig 1”.

Fig 1

Flow diagram for VAP definition.

Firstly, a deterioration in ventilation following a period of stability defined according to positive end expiratory pressure (PEEP): ≥ 2 days of stable or decreasing daily minimum PEEP followed by a rise in daily minimum PEEP of ≥ 2.5 cm H2O, sustained ≥ 2 calendar days; or FiO2: ≥2 days of stable or decreasing daily minimum fraction of inspired oxygen(FiO2) followed by a rise in daily minimum FiO2 ≥ 0.15 points, sustained ≥ 2 calendar days. Secondly, systemic signs of fever >38°C or <36°C or white blood cell count >12×109/L or <4×10 9/L were required. Final criteria was an increased/new purulent tracheal aspirate (TA) samples/tracheobronchial secretions or ≥25 neutrophils per low power field (10 objective) on Gram stain of tracheal aspirate and either new and persistent infiltrates, consolidation, or cavitation as read by two study physicians on chest X-ray, or the decision to commence new antibiotic therapy.

The day when the patient fulfilled the criteria of VAP was taken as day 0 of VAP diagnosis. These VAP confirmed patients were followed up on day 3, day 7, and day14. During these visits comparable clinical information was collected via hospital records. The final follow up was conducted on day 30 by phone if the patient was discharged, or in person if the patient was still in the hospital. Final diagnosis or working diagnosis if still under admission were recorded in the patient CRF.

Sample collection for microbiological culture

Respiratory samples [either tracheal aspirates (TA), bronchoalveolar lavage (BAL), or sputum] and blood samples were obtained from enrolled patients for microbiological culture. A respiratory sample of either TA or BAL was obtained from all patients before the diagnosis of VAP. The decision for BAL or TA samples were at the discretion of the treating physician.

Collection of TA sample

TA samples were collected as previously described, following local standard operating procedures [25]. Specimens were transported to the microbiology laboratory, and processed within 2 hours of collection. The tracheal aspirate specimens were examined by Gram staining, and the aspirate fluid was diluted 1:1 with Sputasol (Oxoid) and incubated at 37°C, with periodic agitation, until liquefaction. The sample was diluted (1:1, 10−1 and 10−2) using maximum recovery diluent (Oxoid), and 20 ml 1:1 diluent was inoculated onto blood agar and chocolate agar plates. Additionally, 20μl of the 10−1 and 10−2 dilutions was plated onto MacConkey media and blood agar base (Mast diagnostics, UK). Inoculated media were incubated at 37°Cand examined after 24 and 48 h of incubation. The threshold used to discriminate between infection and colonization was ≥1x105 colony forming unit (CFU)/ ml-1 (i.e., 20 colonies on either media from the 10−2 dilution). Colonies above this threshold were identified using biochemical tests following standard operating protocol of Patan Hospital. In the interpretation of results, each colony corresponded to 20,000 CFU/ml, and it was considered to be TA positive when the count was ≥105CFU/ml [26].

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed using the Kirby Bauer disc diffusion method. The inhibitory zone sizes were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) 2018 guidelines. Mueller–Hinton agar and antimicrobial discs were purchased from Mast Diagnostics, UK. Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used as controls for these assays. The antimicrobials tested against Acinetobacter spp., Pseudomonas spp., and the Enterobacteriaceae were amikacin (30 mg), piperacillin/tazobactam (100/10 mg), imipenem (10 mg), ofloxacin (5 mg), and ceftriaxone (30 mg). An isolate was defined as MDR when it was non-susceptible to at least one agent in ≥3 antimicrobial categories[CLSI guidelines (2018)] [27].Gram positive organisms were tested against co-trimoxazole (1.25/ 23.75 mg), penicillin (10 mg), gentamicin (10 mg), erythromycin(15mg) and oxacillin (1 mg).

Statistical analysis

Data recorded onto a case record form were entered into a CliRes database system protecting participant information. Verification was done by double entry. Data analysis was performed in R Software (version 3.2). Categorical variables were expressed as count and percentage and analyzed using the Fisher’s exact test. Continuous variables were expressed as median and interquartile range and analyzed using Kruskal-Wallis rank sum test and the pairwise Wilcoxon rank—sum test. Each variable with a p- value < 0.05 was considered a significant variable. VAP incidence was calculated as follows: (Number of cases with VAP/Total number of patients who received MVx100) = VAP rate per 100 patients. VAP incidence density was calculated as follows: (Number of cases with VAP/Number of ventilator days) x 1000 = VAP per 1,000 ventilator days. Flow diagram of the study enrollment procedure and categorization into five categories is shown in “Fig 2”.

Fig 2

Flow diagram of the study enrollment procedure and categorization into five categories.

Patients enrolled in the study were into five groups depending upon intubation, pneumonia development, and VAP development: These were: I-P-V- (not intubated, no pneumonia), I-P+V- (not intubated, but pneumonia developed), I+P-V- (intubated but no pneumonia), I+P+V-(intubated and pneumonia developed, but VAP not confirmed), and I+P+V+(VAP confirmed). The flow diagram of the study enrollment procedure and categorization into five categories is shown in “Fig 2”.

Results

Baseline characteristics

Four hundred and thirty-eight patients between April 2016 and March 2018were hospitalized in the ICU and enrolled in the study. The patients were between the ages of 18 and 95 years and 48.9% (214) were male and 51.1% (224) were female “Table 1”.

Table 1

Baseline characteristics of patients enrolled in the study.

Variables	I-P-V- (111)	I-P+V- (122)	I+P-V- (12)	I+P+V- (152)	I+P+V+ (41)
Age (years)	48 (30–62)	62.5 (48.5–72)	35.5 (23.3–50.3)	57.5 (35–72)	59.5 (42.8–76.3)
Weight (Kilogram)	59.5 (50–65)	57 (49.5–64)	60 (50.5–70.5)	58 (50–65)	58 (49–62)
M:F	57:54	53:69	7:5	77:75	20:21
SOFA	3 (2–5)	3 (2–5)	6.5 (4–10)	8 (5–11)	10 (7–11)
APACHE	10 (6–14)	11 (7.3–14)	12 (6.8–15.3)	18 (13–24)	17 (13–22)
Comorbidity	60 (54.1%)	90 (73.8%)	7 (58.3%)	91 (59.9%)	27 (65.9%)
Hospital previous 90 days	22 (19.8%)	19 (15.6%)	3 (25%)	38 (25%)	7 (17.1%)
Antibiotics in last 90 days	11 (9.9%)	28 (23%)	3 (25%)	54 (35.5%)	4 (9.8%)

Values given are median (IQR) or count (percent).

I-P-V- (Non intubated, no pneumonia), I-P+V- (non- intubated, pneumonia developed), I+P-V- (intubated, no pneumonia), I+P+V- (intubated, pneumonia but not VAP also called non–VAP), I+P+V+ (intubated, pneumonia and VAP confirmed).

The total numbers of participants in each of the five categories were I-P-V- = 111, I-P+V- = 122, I+P-V- = 12, I+P+V- = 152, and I+P+V+ = 41, who had median ages of 48, 62.5, 35.5, 57.5, and 59.5years, respectively. More than 50% of participants in all five categories had co-morbidities at the time of admission in the ICU with >15% of the participants in each category having had a history of hospital admission in the past 90 days with antimicrobial use ranging from 9.8% (4/41 in the I+P+V+ group) to 35.5% (54/152 in the I+P+V- group). The median SOFA scores ranged from 3 to 10, with the highest scores being observed in the VAP group (median of 10, IQR 7–11). The median APACHE II score ranged from 10 to 18; the highest score was observed in the non-VAP group 18 (IQR 13–24).

Association of different variables with intubation and pneumonia

In total, 29.7% (130/438) of patients had a diagnosis of pneumonia when admitted in the ICU and 46.8% (205/438) of patients required intubation. The most common requirement for intubation was failure to oxygenate (24.4%; 50/205), followed by failure to maintain or protect the airway (99%; 39/205). Pneumonia was common in both the intubated (94.14%; 193/205) and the non-intubated (52.36%; 122/233) patients. Pneumonia in intubated patients in the ICU was associated with longer days of stay (median of 10, IQR 5–15, p< 0.001) when compared to non-intubated patients with pneumonia (median of 4, IQR 3–6, p< 0.001). SOFA and APACHE II scores were also significantly higher among the patients that then on-intubated patients (p<0.05; Kruscal-wallis rank sum test) “Table 2”.

Table 2

Kruskal-wallis rank sum test for the variables associated with intubation and pneumonia.

Variables	IntPosPneum	NonIntPosPneum	p—value
	I+P+V(V+and V-)	I-P+V-
	(193)	(122)
Days in Hospital	11 (7–17)	5 (3–7)	<0.001
Days in ICU	10 (5–15)	4 (3–6)	<0.001
Days in Intubation	9 (5–15)	NA	NA
FiO2	50 (40–80)	36 (29–41)	<0.001
PaO2	79 (48.2–114)	68.1 (53.7–88)	0.056
Temperature	98 (97.2–99.2)	98 (97.2–98.4)	0.027
SOFA	8 (5–11)	3 (2–5)	<0.001
APACHE	18 (13–23)	11 (7.25–14)	<0.001
Mortality	86/193 (44.6%)	13/122 (10.7%)	<0.001

Values given are median (IQR) or count (percent). Kruskal-Wallis rank sum test for median (IQR). Fisher’s Exact Test for count (percent). I+P+ (intubated, pneumonia) and I-P+ (non- intubated, pneumonia developed).

Incidence density of VAP

Out of 205 patients requiring mechanical ventilation during their stay in ICU, 41 patients were diagnosed with VAP (20%), equating with a total incidence density of 16.45 cases per 1,000 ventilator days.

Factors associated with VAP confirmed cases

We aimed to identify factors associated with VAP in the VAP confirmed group (I+P+V+) “Table 3”. VAP was significantly associated with the duration of stay in the hospital (median 18 days, IQR 11–27, p<0.001), the duration of stay in ICU (median16 days, IQR 10–25, p<0.001), number of days of intubation (median 17 days, IQR 11–27, p<0.001), fraction of inspired oxygen(FiO2) (median 60, IQR 40–100, p<0.001), APACHE II score (median 17, IQR 13–22 p<0.001), SOFA score (median 10, IQR 7–11, p<0.001) and PaO2 (median 70.6, IQR 52.3–102, p<0.04).

Table 3

Kruskal-Wallis rank sum test for the variables associated with different categories.

	I-P-V- (n = 111)	I-P+V- (n = 122)	I+P-V- (n = 12)	I+P+V- (n = 152)	I+P+V+ (n = 41)	p—value
Days in Hospital	4 (3–6)	5 (3–7)	5 (2.75–7)	10 (7–14)	18 (11–27)	<0.001
Days in ICU	3 (2–6)	4 (3–6)	4.5 (2.75–6)	9 (5–12)	16 (10–25)	<0.001
Days in Intubation	NA	NA	4 (2.75–7.25)	7.5 (4–11)	17 (11–27)	<0.001
FiO2	29 (21–34)	36 (29–41)	40 (30–41)	50 (40–70)	60 (40–100)	<0.001
PaO2	85 (65.8–111.4)	68.1 (53.7–88)	103 (64–150.5)	82.5 (48.1–119)	70.6 (52.3–102)	0.041
Temperature	98 (97–98.7)	98 (97.2–98.4)	98.2 (97–99.2)	98 (97.1–99.15)	98.6 (98–99.2)	0.085
SOFA	3 (2–5)	3 (2–5)	6.5 (4–10)	8 (5–11)	10 (7–11)	<0.001
APACHE	10 (6–14)	11 (7.25–14)	12 (6.75–15.25)	18 (13–24)	17 (13–22)	<0.001
Mortality	4/109(3.7%)	13/122(10.7%)	3/12 (25%)	62/155 (40%)	24/41 (58.5%)	<0.001

Values given are median (IQR) or count (percent). Kruskal-Wallis rank sum test for median (IQR).

Fisher’s Exact Test for count (percent).

A pairwise Wilcoxon signed rank test was performed among the five defined categories to identify significance between the groups “Fig 3”. The number of days of hospital stay, the number of days of ICU stay, APACHE II score, and SOFA score were all significantly higher in the I+P+V+ and I+P+V- groups than in the I-P-V- group (p< 0.001).

Fig 3

Pairwise Wilcoxon signed rank test performed among the five defined categories to identify significance between the groups.

I = intubated, P = pneumonia, V = VAP. P-value from pairwise wilcox-test with bonferroni correction.

Mortality

Mortality was significantly higher in patients with pneumonia requiring intubation than patients with pneumonia not requiring intubation (44.6% (86/193) vs. 10.7%, (13/122)p<0.001, Fisher’s exact test) “Table 2”. Between the groups, the highest mortality (58.5%; 24/41) was observed among VAP patients (I+P+V+) followed by non-VAP (I+P+V-) patients (40.8%,62/152); and lowest mortality (3.7%; 4/109) was observed among patients who neither required intubation nor had pneumonia during their stay in the ICU (I-P-V-)(p< 0.001; Kruskal wallis test) “Table 3”.

Microbiology of VAP and non- VAP

A total of 110 samples from those with confirmed VAP(I+P+V+) and 130 samples from those without-VAP category (includes all categories except I+P+V+) were subjected to microbiological cultured. The majority of these samples were TA samples; 81/110 in VAP category and 63/130 in non-VAP category.

Gram negative bacteria were the dominant organisms from both VAP and non-VAP patients “Table 4”, “Fig 4”). Klebsiella species was the most common bacteria associated with VAP (n = 36,32.7%) followed by Acinetobacter species (n = 35, 31.8%).Acinetobacter species was the predominant organism (n = 48, 36.9%) isolated from those without-VAP, followed by Klebsiella species (n = 28, 21.5%).Klebsiella species was more likely to be isolated from VAP patients (OR1.76, 95%CI 0.96–3.3,p value 0.05).

Table 4

Etiology of VAP and non-VAP specimens.

Isolates	VAP (n = 110)	Non-VAP (n = 130)	OR (95%CI)	p-value
Acinetobacter spp	35 (31.8%)	48 (36.9%)	0.8 (0.45–1.4)	0.42
Klebsiella spp	36 (32.7%)	28 (21.5%)	1.76 (0.96–3.3)	0.05
Pseudomonas spp	14 (12.7%)	17 (13.1%)	0.97 (0.42–2.21)	1
E. coli	11 (10%)	18 (13.8%)	0.69 (0.28–1.64)	0.43
Enterobacter spp	5 (4.5%)	8 (6.2%)	0.73 (0.18–2.61)	0.78
Coagulase negative Staphylococcus (CoNS)	7 (6.4%)	3 (2.3%)	2.9 (0.6–17.6)	0.19
S. aureus	0 (0%)	5 (3.8%)	NA	NA

Fig 4

Etiology of VAP and non-VAP specimen.

MDR in VAP and non-VAP categories

MDR was prevalent in all bacterial isolates from both VAP and non-VAP categories. The distribution of MDR isolates from various VAP and non-VAP samples are presented in “Fig 5”. 90% (n = 99/110) of the isolates from various VAP samples and 81.5% (n = 106/130) of the non-VAP isolates were MDR. The data was suggestive of association of MDR with the VAP isolates but this was none significant (OR 2.03, 95%CI 0.90–4.85, p 0.07).

Fig 5

MDR isolates in VAP and non-VAP specimen.

Discussion

The data on HAP from prospective studies are scant notably from LMIC setting. Our study showed that HAP was common in our ICU setting regardless of intubation requirement [94.14% (193/205) among intubated and 52.36% (122/233) among non-intubated patients] indicating that these pneumonia cases may be a common HAI at Patan hospital. In addition to this, at least 16% of the patients from all the categories have had a visit to the hospital in the past 90 days and 50% of the patients had co-morbidities “Table 1” mainly chronic respiratory illness such as chronic obstructive pulmonary disorder. As a result, antibiotic usage was also common. Pneumonia as HAP was also common in a Malaysian study where,21% of HAP infections were pneumonia [6].

The complex interplay between the endotracheal tube, presence of risk factors, virulence of the invading bacteria and host immunity largely determine the development of VAP [28].The diagnosis of VAP is traditionally based on clinical symptoms and radiographic criteria that require further bacteriological confirmation. However, it has been demonstrated that these criteria are not sensitive or specific [8]. There is no gold standard for the diagnosis of VAP however, the qualitative method of culturing the tracheobronchial aspirate samples is said to be better at differentiating colonization and actual infection.

Despite recent advances in microbiological tools, the epidemiology and diagnostic criteria for VAP are still controversial, complicating the interpretation of treatment, prevention, and outcomes studies [9]. Data on rates of VAP, the common associated pathogens, and their antimicrobial susceptibility profiles from Asia are limited [12]. Pooled incidence density of VAP was 18.5 per 1000 ventilator days in high in Asian LMIC countries [12]. This finding is similar to our study where we observed the incidence rate of VAP of 20% (n = 41/205) with a total of incidence density of 16.45 per 1000 ventilator days. However, some other Asian countries have reported a lower VAP incidence (9.9%) and VAP density (8.7/1000 ventilator days) [16]. Meta- analysis study from mainland China reported that the cumulative incidence of VAP was 23.8% [29]. In contrast, studies from India have reported the higher incidence density of VAP of 39.6% to 40.1% [30, 31].Such reported incidences vary widely from 5 to 40% depending on the setting and diagnostic criteria [9] indicating that the incidence rates vary not only between countries but also among different settings within a country. In high-income countries, a combination of surveillance, education, and tailored intervention and prevention bundles have led to a reduction in the incidence of VAP [32].

Gram negative bacteria were the dominant organisms from both VAP and non-VAP categories. Klebsiella species was the most common bacteria associated with VAP followed by Acinetobacter species Among non-VAP category, Acinetobacter species was the predominant organism followed by Klebsiella species (n = 28, 21.5%). Klebsiella species was more likely to be isolated from VAP category). We did not find major differences in the etiologic agents of VAP and non-VAP organism and their antimicrobial susceptibility profiles. However, there was a suggestive association of MDR with VAP isolates. Similar findings have been reported from other studies where the leading pathogens are A. baumannii, P. aeruginosa and K. pneumoniae [33, 34]. In a large meta-analysis of 88 studies analyzing VAP in adults in Asia, it was revealed that A. baumannii was the most common organism in the LMIC group and the proportion due to this organism gradually reduced as income levels increased, and S. aureus and P. aeruginosa were the most common in the high income country group [12].Studies on VAP from other Asian countries also have reported A. baumannii to be the most common isolate [35].

One of the differences between our data and reports from Western countries was the proportion of gram- negative and gram-positive bacterial causes of VAP. We found a much lower proportion of gram-positive organisms as a causative agents of VAP and non VAP [35, 36].

ICUs often have the highest levels of infections due to antimicrobial resistant pathogens as a result of the environment that is under constant pressure with high antimicrobial usage due to the presence of severely ill patients. Etiologic agents of VAP are generally associated with pathogens with high levels of antimicrobial resistance, resulting in the need to treat with broad-spectrum antibiotics, which further drives antibiotic resistance [12].

Early onset VAP is usually attributed to antibiotic sensitive pathogens whereas late onset VAP is more likely caused by MDR bacteria and emerges after 4 days of intubation [37, 38]. However, this scenario seems to be different in the LMIC settings. This study revealed that MDR isolates were slightly higher in VAP than in non-VAP categories. Although significant association was not observed, there was an indication of association of VAP with MDR organisms. This further highlights the need to have infection control protocol guidelines in order to control such HAIs. s Guidelines for VAP prevention, including hand washing, elevation of the head of the bed, oral care with chlorhexidine, optimized endotracheal tube cuff pressure, respiratory circuit manipulation, and weaning protocols to early extubation were established in our hospital. These are cost effective control and preventive measures of VAP. Strict compliance, staff training, and regular monitoring of implementation of such guidelines will be effective in the prevention of VAP.

Mortality attributable to HAP is estimated between 5 and 13% [39].Even in HAP, generally considered to be less severe than VAP, serious complications occur in approximately 50% of patients [40].Mortality was significantly higher in patients with pneumonia requiring intubation (44.6%, 86/193) than patients with pneumonia without intubation (10.7%, 13/122, p< 0.001). Highest mortality of 58.5% (24/41) was observed among VAP patients (I+P+V+) followed by non VAP (I+P+V-) patients (40.8%,62/152) and lowest mortality of 3.7% (4/109) was observed among patients who neither required intubation nor had pneumonia during their stay in the ICU (I-P-V-) category. The mortality in our ICU due to VAP was still lower than reported in other studies where it was as high as 68.4% [31].

Development of pneumonia increased the number of days of intubation. Intubated patients with pneumonia had to spend a median of 7.5(4–11) (I+P+V-) to 17(11–27) (I+P+V+) days being intubated as in comparison to those without pneumonia (I+P-V-) {4(2.75 to 7.25)}. In addition, VAP confirmed patients spent a median of16 days in ICU ranging from 10 to 27 days.24.6% (50 / 203) patients required intubation due to the reduction in exchange of oxygen (low PaO2), followed by cognitive impairment and airway obstruction (19.2%, 39/203).

Conclusion

Pneumonia was one of the common infections in our ICU setting. Pneumonia developed among intubated patients in the ICU had longer days of stay in the ICU when compared to non-intubated patients with pneumonia. We found high VAP incidence in this study and highest mortality was observed among VAP patients followed by non VAP (I+P+V-) patients. MDR Gram negative bacteria were the dominant organisms from both VAP and non-VAP categories.

HAP including VAP remains to be the most prevalent HAIs at Patan hospital. One of the limitations of this study was that it was conducted at a single hospital. Surveillance studies on HAIs at various hospitals within the country are required in identifying the etiological agents. Antimicrobial susceptibility profiles of the etiological agents and outcomes of HAP and VAP would be beneficial for setting more appropriate guidelines for management of such diseases. In addition, countries like Nepal lack proper protocols of infection control and implementation for minimizing such infections in the hospital. Therefore, a suitable surveillance programs should be implemented, analyzing differences in VAP rates between different ICUs, and evaluating potential therapeutic approaches, and prevention strategies.

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4 Aug 2021

PONE-D-21-22640

Epidemiology, etiology, and diagnosis of health care acquired pneumonia including ventilator-associated pneumonia in Nepal

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Please submit your revised manuscript by Sep 18 2021 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

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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

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2. In your ethics statement, please specify the date of your ethics approval.

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.

[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: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #2: 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

Reviewer #2: 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: All abbreviations in the text must be spelled correctly (first in full).

English grammar needs to be revised.

CFU / ml must be spelled correctly in the text.

The sample was collected between 2016-2018, but the methods used CLSI 2016, this is a problem.

What is the meaning of CONS(coagulase-negative staphylococci)? Please refer to it in Table 5.

In the first paragraph of the discussion, it is better to first explain about health care acquired pneumonia.

Reviewer #2: The study is very interesting ; results and conclusion are very useful mainly in this part of world with low resources setting. Authors have clearly discussed pneumonia among patients in ICU; compared according to intervention done. Simliarly, VAP incidence, mortality, etiological agents , Antimicrobial susceptibility tests and other clinical profiles were the items thoroughly discussed in the study. The author also suggests different control measures and preventive strategies. Overall, the paper is good quality but some shortcomings need to be addressed

In abstract

Only add about the main aim of study not all and present in the results serially.

Introduction

The authors have put excellent effort to justify the rationale and background behind the problem.

Methods

The authors have done well in describing methodology under different headings. It would be great if you could further clarify inclusion and exclusion criteria.

Results

The authors have documented the results well mainly for association of intubation and pneumonia.

Discussion

It is good that you have tried to compare data of Nepal mainly VAP density with other Asian countries and LMICs. It would be nice to elaborate causes for contrasting results among these countries??

Why contrasting findings were seen among your study and other western countries study. What might be the likely cause for low numbers of gram positive organism?

As Nepal is poor in economy, please add some cost-effective control and preventive measures of HAP and VAP.

As the outcomes are relevant , possible description of risk factors of VAP in country like Nepal can also be included.

Are ventilator care bundles used in your study site?

Please add any strengths and limitations above conclusion in your study if any.

Minor issues

Spacing not done in some places like line no. 58

Referencing no. Should be after full-stop.

In table 1, write unit of weight , age

Page 266 to 230 n"=" is missing

And also Page 277 to 280 ; and other lines.

Brackets are only open or only closed some where.

Table 4 and 5 can be merged into a single table.

Correct typographical errors.

Thank you!

**********

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

Reviewer #2: 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.

Submitted filename: vap.docx

Click here for additional data file.

4 Oct 2021

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

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Answer: Correction made

2. In your ethics statement, please specify the date of your ethics approval.

Answer: Date added : NHRC approval date: 11March 2016 and OxTREC approval date was 19 Oct 2018. The study was started after obtaining the approval from Nepal health Research Council (NHRC).

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.

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

Answer: Dataset is submitted as S-Dataset_xlsx. relevant URLs, DOIs, or accession numbers within your revised cover letter.

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: Yes

Reviewer #2: Yes

________________________________________

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

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

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

Reviewer #2: 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

Reviewer #2: 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: All abbreviations in the text must be spelled correctly (first in full).

English grammar needs to be revised.

CFU / ml must be spelled correctly in the text.

Answer: Corrected on the line 150 CFU/ml. The full form of CFU has been added to abbreviation list.

The sample was collected between 2016-2018, but the methods used CLSI 2016, this is a problem.

Answer: we corrected it to 2018. We checked the interpretation of the antibiotics that we tested against 2016 and 2018 CLSI guidelines and confirm that both have the same interpretation criteria for the antibiotics that we tested in this study.

What is the meaning of CONS(coagulase-negative staphylococci)? Please refer to it in Table 5.

Answer: CONS has been corrected as CoNS. The full form is coagulase negative Staphyloccus. Full form is added in the table 5.

In the first paragraph of the discussion, it is better to first explain about health care acquired pneumonia.

Answer: The data on HAP from prospective studies are scant notably from LMIC setting. This is added in the discussion

Reviewer #2: The study is very interesting ; results and conclusion are very useful mainly in this part of world with low resources setting. Authors have clearly discussed pneumonia among patients in ICU; compared according to intervention done. Simliarly, VAP incidence, mortality, etiological agents , Antimicrobial susceptibility tests and other clinical profiles were the items thoroughly discussed in the study. The author also suggests different control measures and preventive strategies. Overall, the paper is good quality but some shortcomings need to be addressed

In abstract

Only add about the main aim of study not all and present in the results serially.

Answer: We included only the main aim of the study in the abstract. This was followed by the summary of the statistical analysis.

Introduction

The authors have put excellent effort to justify the rationale and background behind the problem.

Methods

The authors have done well in describing methodology under different headings. It would be great if you could further clarify inclusion and exclusion criteria.

Answer: There were no exclusion criteria per se. One line was added in the study structure section: Patients who denied consent and patients under the age of 18 years of age were not included in the study.

Results

The authors have documented the results well mainly for association of intubation and pneumonia.

Discussion

It is good that you have tried to compare data of Nepal mainly VAP density with other Asian countries and LMICs. It would be nice to elaborate causes for contrasting results among these countries??

Answer: In high-income countries, a combination of surveillance, education, and tailored intervention and prevention bundles have led to a reduction in the incidence of VAP

Why contrasting findings were seen among your study and other western countries study. What might be the likely cause for low numbers of gram positive organism?

Answer: We are unable to provide explanation based on the findings from the study. There could be multidimensional factors ranging from patient care management protocols, antibiotic stewardship program, VAP bundle implementation, and continuous training and surveillance system.

As Nepal is poor in economy, please add some cost-effective control and preventive measures of HAP and VAP.

Answer: Guidelines for VAP prevention, including hand washing, elevation of the head of the bed, oral care with chlorhexidine, optimized endotracheal tube cuff pressure, respiratory circuit manipulation, and weaning protocols to early extubation were established in our hospital. These are cost effective control and preventive measures of VAP. Strict compliance, staff training, and regular monitoring of implementation of such guidelines will be effective in the prevention of VAP. This is added in the discussion

As the outcomes are relevant , possible description of risk factors of VAP in country like Nepal can also be included.

Answer: We have analysed data by grouping them into categories of intubation required and non intubated group and further divided into five categories into VAP development. We measured various variables and their association to each group.

Are ventilator care bundles used in your study site?

Answer: Guidelines for VAP prevention, including hand washing, elevation of the head of the bed, oral care with chlorhexidine, optimized endotracheal tube cuff pressure, respiratory circuit manipulation, and weaning protocols to early extubation were established in our hospital. These are cost effective control and preventive measures of VAP. Strict compliance, staff training, and regular monitoring of implementation of such guidelines will be effective in the prevention of VAP.

Please add any strengths and limitations above conclusion in your study if any.

Answer: These lines in the conclusion section explains the strength of this study.

Pneumonia was one of the common infections in our ICU setting. Pneumonia developed among intubated patients in the ICU had longer days of stay in the ICU when compared to non-intubated patients with pneumonia. We found high VAP incidence in this study and highest mortality was observed among VAP patients followed by non VAP (I+P+V-) patients. MDR Gram negative bacteria were the dominant organisms from both VAP and non-VAP categories.

One of the limitations of this study was that it was conducted at a single hospital. Surveillance studies on HAIs at various hospitals within the country are required in identifying the etiological agents. Antimicrobial susceptibility profiles of the etiological agents and outcomes of HAP and VAP would be beneficial for setting more appropriate guidelines for management of such diseases.

Minor issues

Spacing not done in some places like line no. 58

Answer: Corrected

Referencing no. Should be after full-stop.

Answer: we referred to the Plos one formatting style.

In table 1, write unit of weight , age

Answer: Corrections were made. Added kilogram as a unit of weight and Years along with age in table 1.

Page 266 to 230 n"=" is missing

And also Page 277 to 280 ; and other lines.

Answer: Corrected throughout the manuscript.

Brackets are only open or only closed some where.

Answer: Checked throughout the manuscript and corrected

Table 4 and 5 can be merged into a single table.

Answer: We have removed table 4 as this information is depicted in Fig 5. We are keeping Table 5 as it is (without merging it to table 4).

Correct typographical errors.

Answer: Done

Thank you!

________________________________________

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

Reviewer #2: 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.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

25 Oct 2021

Epidemiology, etiology, and diagnosis of health care acquired pneumonia including ventilator-associated pneumonia in Nepal

PONE-D-21-22640R1

Dear Dr. Karkey,

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,

Monica Cartelle Gestal, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

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

**********

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

Reviewer #2: 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

**********

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

**********

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: The authors have address all the queries. Congratulations! on publication og article. This article address major issue in developing countries like Nepal.

**********

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: Yes: Sanjeev Kharel

8 Nov 2021

PONE-D-21-22640R1

Epidemiology, etiology, and diagnosis of health care acquired pneumonia including ventilator-associated pneumonia in Nepal

Dear Dr. Karkey:

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. Monica Cartelle Gestal

Academic Editor

PLOS ONE