Mixed Methods Pilot Study of Sharing Behaviors among Waterpipe Smokers of Rural Lao PDR: Implications for Infectious Disease Transmission
2013

Robyn Martin
Department of Epidemiology and Biostatistics
School of Public Health
Loma Linda University
92350Loma LindaCAUSA

Department of Global Health
School of Public Health
Loma Linda University
92350Loma LindaCAUSA

Sahar D Safaee firstname.lastname@example.com.
Department of Epidemiology and Biostatistics
School of Public Health
Loma Linda University
92350Loma LindaCAUSA

Department of Global Health
School of Public Health
Loma Linda University
92350Loma LindaCAUSA

Khamphithoun Somsamouth
Center for Information and Education for Health
Ministry of Health
VientianeLao PDR

Boualoy Mounivong firstname.lastname@example.com.
Center for Information and Education for Health
Ministry of Health
VientianeLao PDR

Ryan Sinclair firstname.lastname@example.com
Department of Environmental Health
School of Public Health
Loma Linda University
92350Loma LindaCAUSA

Shweta Bansal firstname.lastname@example.com
Department of Biology
Georgetown University
20057WashingtonDCUSA

Fogarty International Center
National Institutes of Health
20892BethesdaMDUSA

Pramil N Singh
Department of Epidemiology and Biostatistics
School of Public Health
Loma Linda University
92350Loma LindaCAUSA

Department of Global Health
School of Public Health
Loma Linda University
92350Loma LindaCAUSA

Center for Health Research
School of Public Health
Loma Linda University
92350Loma LindaCAUSA

International Journal of Environmental Research and Public Health

Mixed Methods Pilot Study of Sharing Behaviors among Waterpipe Smokers of Rural Lao PDR: Implications for Infectious Disease Transmission

Int. J. Environ. Res. Public Health
10201310.3390/ijerph10062120Received: 11 March 2013; in revised form: 10 May 2013 / Accepted: 12 May 2013 /* Author to whom correspondence should be addressed; E-Mail: firstname.lastname@example.com;infectious diseasewaterpipemode of transmissionperson-to-personcommunicable
To date, the sharing behaviors associated with the homemade tobacco waterpipe used in rural areas of the Western Pacific Region have not been studied. Evidence from studies of manufactured waterpipes raises the possibility of infectious disease transmission due to waterpipe sharing. The objective of our pilot study in rural Lao People's Democratic Republic (PDR) was to identify and measure the prevalence of waterpipe sharing behaviors. We first conducted ethnographic studies to investigate waterpipe-smoking behaviors. These findings were then used to develop an interviewer-administered household survey that was used in a sampling of waterpipe smokers from three villages of the Luang NamthaOPEN ACCESSprovince of Lao PDR (n = 43). Sampled waterpipe smokers were predominantly male (90.7%), older (mean age 49, SD 13.79), married (95.4%), farmers (78.6%), and had completed no primary education. Pipes were primarily made from bamboo (92.9%). Almost all (97.6%) smokers were willing to share their pipe with others. At the last time they smoked, smokers shared a pipe with at least one other person (1.2 ± 0.5 persons). During the past week, they had shared a pipe with five other persons (5.2 ± 3.8 persons). The high prevalence of sharing behaviors among waterpipe smokers in rural Southeast Asia raises the possibility that this behavior provides important and unmeasured social network pathways for the transmission of infectious agents.

Introduction

The World Health Organization has reported that the Western Pacific Region has the highest global rate of cigarette smoking, with nearly two-thirds of men being current smokers [1]. Although the public health impact of cigarette smoking has been quantified in this region, the contribution of non-cigarette forms of tobacco use is only beginning to be studied and in some cases is virtually unknown. Throughout Southeast Asia, several ethnic groups (i.e., Akha, Hmong, Khamu) living in communities as large as 90,000 have long smoked tobacco using homemade bamboo waterpipes [2]. In 2010, findings from the Global Adult Tobacco Survey of Vietnam indicated that there were an estimated 4.1 million tobacco waterpipe smokers in the nation [3]. Similar to cigarettes, recent findings also link pipe tobacco use to poverty in Vietnam [4]. In a 2006 national survey of Cambodia, the prevalence of tobacco pipe use among ethnic minorities living on the Lao-Cambodia border exceeded 30% in men and 48% in women [5]. Globally, there is a perceived "safety" associated with waterpipe smoking based on the belief that relative to cigarette smoking, the water filtration of the smoke in the pipe removes harmful toxicants [6]. Contrary to these popular beliefs, recent studies have shown that waterpipe smoking is associated with lung disease, cardiovascular disease [7], malignancy [8][9][10], adverse birth outcomes [8,11,12], and increased susceptibility to infectious agents [13,14].

When considering the global health burden associated with tobacco waterpipe smoking, the common practice of multiple users sharing a waterpipe during a single session of smoking [15][16][17] is noteworthy in its potential creation of communicable disease health hazards. For the manufactured waterpipes long used in the Eastern Mediterranean Region, and now an emerging behavior in the West, the most common (90% of users) form of waterpipe smoking is a shared behavior that usually occurs through multiple hoses connected to the same source of filtered smoke [17]. In contrast, in rural areas of the Western Pacific Region, a homemade, cylindrical waterpipe ( Figure 1) is used to smoke tobacco and the design does not include charcoal for burning the tobacco [2,18]. To date, the sharing behaviors of homemade waterpipe users in the Western Pacific have not been characterized in detail in the health literature. Current data suggest that the use of a common mouthpiece or smoke convection path (i.e., through a hose or bamboo pipe) by manufactured waterpipe users increases the risk of acquiring herpes simplex virus (HSV-1), Epstein Barr [19], Helicobactor pylori [14], hepatitis B, various respiratory infections, including bacterial meningitis [13], and periodontal conditions such as oral candida [15,20]. There have also been confirmed cases of tuberculosis spread through communal use of a waterpipe [21]. This adds to the evidence from South Asia and the surrounding regions linking tobacco smoking to an important burden of tuberculosis infections [13,21,22].

In our present study, we focus on the homemade tobacco waterpipes of Lao PDR that tend to be made of bamboo, and less commonly of adapted pieces of plumbing pipe (PVC) or metal ( Figure 1). The long tubular shape of the Laotian waterpipe provides ideal conditions for biofilm growth and the smoking mechanism of the waterpipe creates many potential pathogen-transmission pathways. When waterpipe smokers contaminate or come into contact with contaminated waterpipes or smoking materials, a potential exposure-pathway to fungal, bacterial, and viral pathogens is created between waterpipe smokers (See Figure 2). Due to poor access to modern sanitation, this population is particularly susceptible to transmission of infectious agents. In order to better understand the risk associated with the communal sharing practices of waterpipe smokers in the Western Pacific Region, it is imperative to first understand to what extent tobacco smokers are sharing waterpipes.

In this mixed methods pilot study of waterpipe smokers in rural Lao PDR our aims were as follows: (1) to identify behavior patterns associated with waterpipe use (i.e., sharing, ownership); (2) to determine for each waterpipe user the number of persons who used the waterpipe they last smoked during the past 24 h and past 7 days; (3) to determine for each waterpipe user the concept of ownership of the pipe (by a household, by an individual) as well as willingness to share the waterpipe with others.

Methods

Study Population

During July 2011, as part of the validation study of a national tobacco survey [23], we worked with the Ministry of Health to select five villages from a rural district of the Luang Nam Tha province that were known to have a high prevalence of waterpipe smokers. In one of the villages, key informant interviews of five male waterpipe smokers (selected by systematic household sampling) were conducted by a Lao-speaking public health professional. This village was exclusively used to collect qualitative data. In the remaining four villages, we worked with multilingual interviewers to complete a systematic household sampling of waterpipe smokers and collect quantitative data via a paper-based survey. In each of the four villages, households had been enumerated as part of previous large-scale survey efforts and all enumerated households were eligible for selection. Study personnel (Ministry of Health interviewers, local tribal governance) were used to select all waterpipe smokers (all enumerated households were asked whether waterpipe smoking was occurring) from the first three villages and in the fourth village selection continued until a sampling target of more than 40 subjects was achieved. The selection criteria for all villages studied was that subjects were current waterpipe smokers aged 18 years and older who allowed investigators to examine the waterpipe they last smoked.

We had an overall response rate of 98% with a final sample of 43 subjects (four women, 39 men). Ministry of Health interviewers conducted the survey and there is a cultural tendency of the Lao to be compliant to government health surveys. Similar response rates have been found in large national surveys in Lao PDR (i.e., Laos Reproductive Health Survey) [24]. Informed consent was obtained and ethics approval was obtained from the Ethics Committee of the Ministry of Health Vientiane Capital and the Institutional Review Board of Loma Linda University.

Qualitative Methods

A windshield survey of each village was conducted to obtain information about the environment around the villages. Windshield surveys are used as a qualitative research method in which researchers travel through a community to directly observe and assess factors that can contribute to community health. These observations can then be used to formulate interview or survey questions, make observations about environmental risk factors, and determine access to resources that promote community health (i.e., fresh water, health clinics, etc.) [25]. From these surveys we observed type of waterpipe smoked, substances smoked in the pipes, types of houses in the village, potential water sources, and distance of the village from the main road. Key informant interviews were also used to gather qualitative data that provided a better understanding of waterpipe smoking behaviors in the community. Key informant questions were developed using information from secondary data-sources and through potential pathways that may increase risk of spreading disease. Questions focused on the type of pipe smoked, what substances the individual smoked, description of a typical smoking session, frequency of smoking, water source used for pipe, storage practices of smoked materials, eating habits, and personal hygiene. During each key informant interview, notes were taken for later analysis. After all interviews had been conducted, raw qualitative data was analyzed for themes using the open coding method. These themes were turned into codes, or labels, and placed into a codebook. These codes were then used to identify topics for further study in the survey. Several of the themes that were identified through the coding process were used to edit existing or create new questions for the survey.

Survey Design

The survey was designed based on: (1) the findings of a validation study where survey measures of tobacco use were compared to the findings from salivary cotinine and carbon monoxide testing [18];

(2) items adapted from the Global Adult Tobacco Survey (GATS) [3]; (3) a literature review of potential infectious disease transmission behaviors among users of smoked tobacco, and (4) a qualitative study described in the previous section.

The survey included items on demographics, tobacco (smoked, smokeless), behaviors associated with waterpipe use, environmental tobacco exposure and other environmental exposures, household and personal exposures, and current health status. The final survey was translated and back translated (between English and Lao) and checked for consistency by a Laotian public health professional. For data entry, a third party survey contractor fluent in Lao was used to complete double entry of the paper-based survey data into electronic format.

Three local interviewers (one Ministry level and two district level) conducted the surveys in each village. The Ministry level interviewer had extensive experience in demographic and health surveys and trained the provincial interviewers. Multi-lingual assistants from the subject's villages were also used for subjects who could not understand the Lao language.

Statistical Analysis

Descriptive analyses were done on demographic, waterpipe type and usage behaviors, environmental exposure variables, and chronic symptoms. The confidence intervals for the number of shared users were determined using a non-parametric bootstrapping method (bias-corrected and accelerated) to account for small sample size and non-normal distributions. All analyses were performed using SAS version 9.3 (Cary, NC, USA) and SPSS version 20 (Armonk, NY, USA).

Results

Qualitative Study

Our findings from five key informant interviews of male waterpipe users from one village, and windshield surveys from five villages are described.

Waterpipe Tobacco Characteristics and Storage

During five key informant interviews of male waterpipe users, two interviewees reported using loose tobacco in their waterpipes, but also reported their impression that the use of loose tobacco was declining due to the availability of manufactured cigarettes. The remaining three reported that they used commercial cigarettes in their tobacco waterpipes. Some of the interviewees loaded the waterpipe with crumbled cigarettes (excluding the filter). Others loaded the pipe by placing the entire cigarette, filter side down, in the bowl (stem on the side of the cylindrical pipe) of the waterpipe. When the entire cigarette was placed in the bowl, the smoker would hold the cigarette in the bowl of the waterpipe until they had completed their smoking session.

The two interviewees who used loose tobacco in their waterpipes stored their dried tobacco in a plastic bag or container due to humidity. They stated a belief that storage in such containers would prevent fungal growth in the tobacco that when smoked is "bad for your lung". During windshield surveys of the five villages studied, we (R.M., S.D.S) also noticed that some men stored their loose tobacco in wood or metal boxes. Cigarettes were almost always stored in the packaging they were purchased in.

Across the five villages, three types of homemade waterpipes were seen in varying frequencies: bamboo, adapted PVC pipe, and adapted metal piping. Metal waterpipes tended to have been more labor intensive in their construction and were considered by the community to be more valuable.

Sharing Behaviors

All five interviewees indicated that tobacco waterpipes in their region are frequently shared and waterpipe smoking is more often a communal activity than a solitary practice in their village. In fact, one of the five interviewees did not own a pipe, but did smoke a tobacco waterpipe daily by always borrowing a waterpipe. The interviewees indicated that, both personally and in the community, most waterpipe smokers had more than one pipe at home, and at least one in their hut in the field. Although our study did not specifically ask about the sharing of smoking materials (e.g., tobacco), we observed that ingredients were shared when an individual had their own pipe available to smoke but was missing ingredients. In this case, the individual borrowed smoking materials to smoke with their own pipe instead of using another pipe that was already loaded with ingredients. This also highlights the fact that waterpipes are shared for convenience, but an individual's own pipe is preferred when available.

Quantitative Study

As shown in Table 1, the demographics of the sample indicate that the waterpipe smokers were predominantly male (90.7%), older (mean age 49, SD 13.79), married (95.4%), and farmers (78.57%) that had completed no primary education. In this sample, we found that almost all (97.1%) waterpipe users smoked daily. Findings from NATSC 2011 and national prevalence survey data being analyzed from Lao under the Fogarty/NIH also indicated that 1% or less of smokers in the region are less than daily smokers [26].

Although a wider variety of waterpipe types were observed during windshield surveys of the region, the quantitative survey findings shown in Table 2 reveal that the waterpipe smokers we studied primarily use homemade bamboo waterpipes (92.86%) as compared to metal (4.76%) and PVC pipe (2.38%). Images of the pipes actually used by some of the subjects are provided in Figure 1.

In this sample (n = 43), all waterpipe smokers claimed ownership of the pipe they last smoked, but most (97.6%) also indicated that they were willing to share their pipe with others (Table 3). In Table 3, we report that at the last time they smoked a waterpipe, each smoker had shared the pipe with at least one other person (1.21 ± 0.54 persons). During the past seven days, each smoker had shared a pipe with approximately 5 other people (5.24 ± 3.82).

Discussion

To date, our pilot study is the first to provide detailed qualitative and quantitative data on the pipe sharing practices of tobacco waterpipe smokers from rural regions of the Western Pacific Region. In a sample of 43 male and female waterpipe smokers of rural Lao PDR, our major findings are as follows:

(1) most waterpipe smokers (97.6%) were willing to share their pipe with others, (2) at the last smoking session, smokers shared a pipe with at least one other person (1.21 ± 0.54 persons) (3) on a weekly basis, waterpipe smokers had shared a pipe with, on average, five other persons (5.24 ± 3.82 persons). The very high prevalence of sharing waterpipes in this sample raises the possibility that this behavior represents a significant route for transmission of the infectious disease pathogens that are highly prevalent in Lao PDR and the surrounding region (i.e., tuberculosis, typhoid).

Evidence of Infectious Disease Transmission due to Sharing Among Waterpipe Users

The use and sharing of waterpipes have the potential to transmit bacterial and viral pathogens through airborne, droplet, fomite and fecal-oral routes of transmission. In Figure 2, we highlight some of these transmission pathways. The evidence, thus far, linking infectious disease transmission to waterpipe smoking is limited to case studies and small samples of those smoking manufactured waterpipes, but is convincing. Findings from samples that tested an association include: (1) a pulmonary tuberculosis cluster [21] found among young Australian adults (n = 149) where sharing a "bong" (a waterpipe with very similar construction to the Lao waterpipes) was associated with a two-fold increase in the likelihood of TB transmission (OR 2.22, 95% CI 0.96-5.17), and (2) a study of tuberculosis index patients and their household contacts (n = 112) in Storstroem County, Denmark found the same tuberculosis risk rates for household contacts of the index cases and shared waterpipe contacts of any infected case [27]. Additionally, there was the case report of a Russian student who was diagnosed with extremely drug resistant tuberculosis that was linked to a hookah lounge exposure before immigration to the United States [28].

When considering the possibility of a transmission pathway between the sharing of waterpipes and infectious disease, it should be noted that some of the findings may relate to fungal growth and other microbial contamination of the tobacco as the true point source rather than waterpipe itself. These microbial contaminants can become airborne or transfer from the tobacco to the surfaces of the waterpipe, fingers, or clothing, offering further routes of pathogen transmission. Such fungal growth in the tobacco used in these pipes includes Aspergillus fumigatus, Penicillium marneffei, Fusarium proliferatum, and several other microbiological contaminants [29][30][31]. A study performed in 2008 showed that tobacco smokers are also at increased risk of respiratory infection due to immunosuppression by several pathogens including Streptococcus pneumonia, Neisseria meningitides, Haemophilus influenza and Legionella pneumophila [13]. When comparing these findings to our pilot data from Lao PDR, we note that the design of the homemade waterpipe allows no exchange of pipe tips and involves greater contact between mouth, skin, and the point of inhalation than the more widely studied Eastern Mediterranean waterpipe (i.e., "hookah"). Efforts to directly link the transmission of tuberculosis, herpes, typhoid and infectious pathogens through this behavior are needed in regional studies.

Implications for the Infectious Disease Burden in the Western Pacific Region

The Western Pacific Region is distinctive in having one of the highest global rates of both smoking and tuberculosis. A recent analysis by Basu et al. indicated that the Millennium Development Goals for TB control are delayed by at least two decades due to the high prevalence of smoked tobacco in the Western Pacific Region [32]. In Lao PDR, respiratory disease is still one of the leading causes of death in children under the age of five [33]. Recent studies linking the correlation between tobacco smoking and increased morbidity and mortality from respiratory infections show how dangerous waterpipe smoking can be in regions with high prevalence of infectious diseases [13,22]. Within these epidemics, our findings from Lao raise the possibility of transmission of infectious disease from smoker to smoker through social networks based on sharing (Figure 2). When waterpipes are shared, pathogens may spread from one host to another person through interactions with the pipe or smoking materials. The waterpipe sharing behavior also creates a social context in which airborne or droplet transmission due to proximity may occur with contacts not otherwise a part of an individual's social network. For many years, infectious disease epidemiology has relied on social network modeling to track, control, and prevent the spread of infectious agents. Recent research on the mathematical modeling of these social networks has shown that an understanding of pathogen spread on local scales can lead to a better understanding of infection dynamics on larger spatial scales [34][35][36][37]. Tracking the sharing behaviors of waterpipe smokers may allow public health professionals to develop strategies to reduce infectious disease spread while decreasing the prevalence of tobacco smoking.

Limitations

Due to the small sample size (n = 43) of this pilot study of adults in the Luang Nam Tha Province, findings should be viewed as a reference for generating hypotheses to be investigated in large representative samples. We have focused on how sharing of a tobacco waterpipe may increase the potential for transmission of infections. Other factors that may increase risk of pathogen transmission include: tobacco handling and storage, cleaning of the waterpipe, hygiene practices, and environmental factors such as access to clean water, exposure to livestock, and availability of modern sanitation.

It is also relevant that we have only described the effects of tobacco in the waterpipes and did not find evidence of other additives during key informant interviews. During environmental sampling of the quantitative study sample (n = 43), however, we noticed that some subjects did add bamboo to the tobacco before smoking it. When asked about the reason for the added bamboo, one subject stated that "the tobacco was too strong and irritating to the lungs" when smoked alone, and the bamboo was added to make the smoke less irritating.

Conclusions

The findings from a mixed methods pilot study of waterpipe smokes in rural Lao PDR identify an extremely high prevalence of sharing behaviors that can potentially transmit infectious disease pathogens. Further efforts to identify the mode of transmission and measure the public health burden of this behavior are needed in this region where high rates of tobacco and infectious respiratory disease are occurring.

Conflict of Interests

The authors declare no conflict of interest.

Figure 1 .
1Homemade waterpipes used by study subjects. (a) PVC Pipe Waterpipe (b) Bamboo Waterpipe (c) Metal Piping Waterpipe

Figure 2 .
2(a) Hypothetical network of potential disease-causing contacts in a rural Lao community, highlighting the possibility of additional links in an individual's social network due to waterpipe smoking and sharing behaviors. (b) Possible pathways for the transmission of viral and bacterial pathogens due to waterpipe sharing behaviors.

Table 1 .
1Demographics of 43 tobacco waterpipe smokers of rural Lao PDR (Luang Namtha Province, 2010).Table 2. Prevalence of Waterpipe Types.N (%)

Table 3 .
3Waterpipe Sharing Practices.N (%)

© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
AcknowledgementsThe authors are grateful for the assistance of Scott Rawson of the Adventist Development and Relief Agency in Lao. We are also grateful to the people of Luang Nam Tha district for their time and willingness to participate in our study. Funding: This study was supported by grant R03 TWOO7345-01 from Fogarty/NIH (PI: Pramil Singh). Ethic Approval: Ethics review and approval was obtained by the Institutional Review Board of Loma Linda University and the Ethics Committee on Research, Ministry of Health, Vientiane Capital, Lao PDR. Participant Consent: Obtained.
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Investigation of association between maternal 25-OH vitamin D serum levels and neonatal early onset sepsis in newborns by evaluating key factors

Maryam Saboute
Rahman Yavar
Mandana Kashaki
Fatemeh Kazemi Khaledi
Nasrin Khalesi
Farzaneh Rohani
Investigation of association between maternal 25-OH vitamin D serum levels and neonatal early onset sepsis in newborns by evaluating key factors
10.1186/s12944-019-1095-3R E S E A R C H Open Access
Background: The goal of this study was to evaluate the relationship between maternal 25-OH Vitamin D serum levels and neonatal early-onset sepsis in newborns by the effective factors. Methods: A case-control study was done and 64 neonates hospitalized in Akbar Abadi Hospital (Tehran-Iran; 2016) and their mothers were enrolled. The case group consisted of 32 NICU term hospitalized neonates due to neonatal early-onset sepsis. Thirty-two term newborns that referred to hospital for rule out hyperbilirubinemia during the first 72 h of life were also considered as the control. Results: Sixty-four mothers with mean age 28.76 ± 6.60 years and mean gestational age 39.64 ± 1.62 weeks entered the study. There was a significant correlation between sepsis and older age of mothers and low Apgar score (P-value = 0.02, 0.01 respectively). The maternal vitamin D serum level was reversely correlated with neonatal sepsis occurrence (P-value = 0.03). There was a significant correlation between maternal vitamin D supplement intake during pregnancy and lower risk for neonatal sepsis (P-value = 0.003). Conclusion: The level of maternal serum Vitamin D was inversely correlated with neonatal sepsis occurrence and intake of vitamin D supplement during pregnancy could decrease the risk of early neonatal sepsis.

Background

Neonatal early-onset sepsis (NEOS) defines as systemic bacteremia with positive blood or cerebrospinal fluid culture in the first 7 days of neonate's life. It is a serious complication with relatively low incidence (0.5-1.2 cases per 1,000 live births) and high morbidity/mortality rates [1][2][3]. Several maternal and neonatal causes including the colonization of organisms in the genitourinary tract, invasive procedures during pregnancy, prolonged rupture of membranes, instrumental delivery, prematurity, low birth weight, neonatal anomalies, and low Apgar scores are involved in the etiology of NEOS. Group B Streptococcus, Escherichia coli, and Listeria monocytogenes are also reported as the most frequent pathogens in NEOS [4,5]. Different studies demonstrated that low 25-hydroxyvitamin D3 (25 (OH) D) or calcidiol, levels are linked to low birth weight and gestational age. Moreover, low maternal 25 (OH) D levels effect on the Apgar score of the newborn [6].

Recent studies have indicated a correlation between maternal serum vitamin D status and health consequences for neonates, it has been indicated that maternal 25OHD is markedly associated with offspring birth weight, where vitamin D controlling gestational age plays a key role in mediating the effect of 25 (OH) D on birth weight. There has been a growing body of evidence that indicated gestational 25 (OH) D insufficiency can be associated with adverse pregnancy outcomes [6]. Increasing evidence from a meta-analysis of randomized controlled trials has helped to elucidate the role of D supplementation on maternal and neonatal results, where at pregnancy time the vitamin D supplementation appear to be related to a higher circulating 25(OH) D levels, birth length and birth weight. Vitamin D deficiency may be involved in the pathogenesis of sepsis and sepsis-related disseminated intravascular coagulation (DIC) [7]. Vitamin D has also been found to have some neonatal protective properties against infectious diseases by regulating of the innate-adaptive immune system, anti-inflammatory effects, increased monocyte responses, and enhanced mucosal barrier as well as by endothelial function [8][9][10]. There is close and deep relationship between maternal and fetal vitamin D [11].

Vitamin D deficiency is a worldwide complication with prevalence ranging from 18 to 84% that is more dependent upon geographic region, ethnicity, type of clothing and dietary intake. The highest prevalence rate (60-80%) is reported among high-risk population such as pregnant women, subjects with low dietary vitamin D intake, pre-pregnancy obese women and individuals with limited sun exposure [12,13]. Various studies have shown that the difference in free vitamin D has an effect on DBP levels, but the effect of vitamin D supplementation has not been established [11].

Although there are a few studies that assessed correlations among maternal serum vitamin D status and neonatal sepsis. Another approach to try to clarify the effect of vitamin D supplementation is to design better-designed randomized controlled trials assessing clinically relevant outcomes. it seems that more approaches are needed in this field. There is a growing of evidence that sufficient vitamin D-supply of mother and child can play a pivotal role in preventing birth complications [11]. It is noteworthy that free 25(OH) D measuring has been recently demonstrated to be favorable laboratory parameter for monitoring vitamin D during pregnancy [14]. Therefore, measurement of free 25 (OH) D should be considered for different concentrations of DBP, including pregnancy. It should be taken into consideration that the 25 (OH) D measurements are relatively cost-effective, timely and robust [11,14].

Our results can provide some informative data related such correlations among Iranian mothers with a specific type of clothing, ethnicity, geographic region and high intake of vitamin D [12,13,15,16]. Therefore, the present study was planned to investigate the relationship between maternal 25-OH Vitamin D serum levels and neonatal early-onset sepsis in newborns by the effective factors.

Materials and methods

Study design

A case-control study was carried out in the NICU of Akbar-Abadi Hospital affiliated to Iran University of Medical Sciences (Tehran-Iran) from 2016 to 2017.

Sixty-four term neonates and their mothers have entered the study.

Patients

Neonatal early-onset sepsis (case group; NEOS) consisted of 32 terms NICU hospitalized neonates as suspected sepsis in the patients' group. EOS diagnosis was considered based on clinical and laboratory findings during the first 72 h of life to have a high potential NEOS according to the criteria defined by Soliman Gamal et al., 2017, such as temperature instability, heart rate dysrhythmia, poor feeding or intolerance, weight loss, abdominal distension, and necrotizing enterocolitis, and respiratory clinical signs containing distress, supplemental oxygen, mechanical ventilation requirement, apnea, tachycardia or bradycardia, and hypoxemia, as well as positive blood culture, elevates C-reactive protein (CRP) and hematologic cell blood count findings [17].

Controls

In the control group, thirty-two healthy neonates with no prenatal risk factor for NEOS were enrolled. Whole neonates were subjected to complete history taking containing gestational age, maternal age, sex, type of delivery, maternal vitamin D concentration, birth weight, Apgar scores, the birth season and neonatal activity (doing or not doing well). Term newborns that referred to the hospital for ruling out hyperbilirubinemia during the first 72 h of life were also considered as the control group. Both groups were adjusted regarding their ages. Moreover, all neonates had breastfeeding during the time of the study.

Inclusion criteria were normal birth weight (> 2500 g), term birth and suspected with sepsis. It should be taken in to account that sepsis is a bacterial infection were confirmed by a positive culture before the baby is 1 month old.

Exclusion criteria were included; Maternal poor bad obstetric history correlated to neonatal sepsis including PROM, Chorioamnionitis, congenital anomalies, insulin dependent diabetes mellitus (IDDM), maternal systemic and chronic disease, hematologic disorders, use of medication, twin pregnancy, drug abuse, as well as mother's death before assessing of vitamin D serum level.

Demographic characteristics and clinical data of neonates and mothers including maternal age, level of education, geographic region, perinatal complications, history of using supplement during pregnancy, neonatal birth weight, gestational age, sex, first minute Apgar score, the season of birth were recorded in some checklists.

Laboratory evaluation

For determining maternal vitamin D status of both groups, 5 ml of mother's blood was collected, labeled and afterward sent to the laboratory to measure 25-OH vitamin D serum level by ELISA Kit (bioMérieux, France). In addition, vitamin D insufficiency was defined as a serum concentration of vitamin D < 30 ng/ml [18].

Based on an investigation by certinkaya et al. [19], the mean vitamin D serum concentrations among EOS suspected and unsuspected newborns were determined as 21.7 ± 6.5 and 27.1 ng/ml (CI = 8.7), respectively. Using the following formula and proposed sample size of 32 for each group, our study had a power of 80% and an alpha error of 0.05.
n ¼ Z 1-α=2 ð Þ þ Z 1-β ð Þ À Á 2 sd 1 2 þ sd 2 2 À Á d 2

Statistical analysis

The statistical analyses were performed using software package SPSS version 18. Quantitative and qualitative variables were reported by mean ± SD and percent, respectively. Student t-test and Mann-Whitney test were utilized to compare among groups for comparing quantitative data. Furthermore, Chi-square and Fischer exact test were used to analyzing the correlations between qualitative variables. Additionally, One-way ANOVA and logistic regression analyses were performed to analyze the correlations between variables. The level of significance was considered as P < 0.05.

Results

Sixty-four mothers with a mean age of 28.76 ± 6.60 years and mean gestational age of 39.64 ± 1.62 weeks have entered the study. Forty-one mothers had a normal vaginal delivery and 23 had a cesarean section. Among all enrolled mothers, 16 subjects exhibited perinatal complications including hypothyroidism and diabetes, while 23 did not receive vitamin D supplement during pregnancy (20 mothers consumed vitamin D supplement for more than 3 months). The mean maternal serum vitamin D was determined as 25.72 ± 12.37 (Min: 2, Max: 51.8), (Table 1). Of all 64 neonates, 30 subjects were male and 34 were female. The mean birth weight was 3002.96 ± 521.74 g. The lowest and highest Apgar score at first minute were determined to be 6 and 9, respectively.

Our results revealed that the mean maternal age in the case group was significantly higher than the control group; older mothers had a greater chance of having a neonate with EOS (p = 0.004). Neonatal sepsis was more common in males when compared with females (P = 0.003). A significant correlation was also observed between neonatal sepsis and low first minute Apgar score (P = 0.003). The level of maternal serum vitamin D was reversely correlated with sepsis occurrence (P = 0.03). Furthermore, there was a significant correlation between maternal vitamin D supplement intake during pregnancy and lower risk for neonatal sepsis (P value = 0.003). An increasing body of evidence suggested that maternal complications like hypothyroidism and gestational diabetes could increase the risk of neonatal sepsis (P = 0.02). Although vitamin D insufficiency was less common among mothers who spent their pregnancy during spring and summer as compared to those with a fall and winter pregnancy, the difference of vitamin D status between groups was not found to be significant (P = 0.09). Type of delivery gestational age and neonatal birth weight were not also significant determining factors for neonatal sepsis (P > 0.05).

We used the binary logistic regression to determine interactions between variables. Sepsis was considered as dependent variable, while maternal age, serum Vitamin D level, gestational age, first minute Apgar score and perinatal complications were considered as independent factors (Table 2) Among all evaluated factors, older mothers and low Apgar score affect the incidence of neonatal sepsis (p = 0.02; p = 0.01). The detail of vitamin D and other variables displayed in the Fig. 1 Prediction value for related risk factors used to estimate the coefficient of determination based on the linear regression model. Based on Cox and Snell model, 63.7% of neonatal sepsis could be predicted by aforementioned variables The regression coefficient was confirmed based on the Cox and Snell mod as 0.637, where indicates that the change of the response variable (sepsis) is presented by predictor variables.

Discussion

In the present study, we evaluated the correlation between maternal serum vitamin D and neonatal earlyonset sepsis. Based on the results presented herein, the level of maternal serum vitamin D was inversely correlated with neonatal sepsis occurrence. It seems that vitamin D has protective effects against neonatal sepsis.

Vitamin D has been found to play a key role in increasing defense against bacterial and viral agents by triggering the production of the antimicrobial peptide and has been found to trigger its immunomodulatory role by suppressing inflammatory cytokines such as interleukin-6 (IL-6), [20]. 1a,25(OH)2D is capable of increasing the antimicrobial properties of monocytes and macrophages as well as play a role in increasing chemotaxis and the phagocytic properties of macrophages. Moreover, can be involved in the mitigation of adaptive immunity, which is likely to yield benefits in autoimmune afflictions. A systematic review and meta-analysis by Haan et al. suggested an association between vitamin D deficiency with infection rate, sepsis, and neonatal mortality rate [12]. Although we did not consider the neonatal vitamin D status, other studies suggested that there is a positive correlation among mother and umbilical cord blood vitamin D concentration [21,22]. Moreover, our previous study has shown a strong positive correlation between maternal serum vitamin D and neonate's birth weight [23]. It is also supposed that low birth weight newborns are at an increased risk of developing neonatal morbidities like NEOS. In accordance to our results, Yang et al. demonstrated that mothers and their neonates with NEOS had lower 25-OHD levels in comparison with their counterparts in the control group, leading to the neonatal earlyonset sepsis risk for full-term infants [24].

Gamal et al. also confirmed a significant negative association between neonatal-maternal 25-OH vitamin D serum levels and sepsis markers in 50 neonates with NEOS [25].

Our findings revealed that at pregnancy time the vitamin D supplementation could decrease the hazard of early neonatal sepsis. In accordance to our results, an investigation by Quraishi et al. indicated that a single bolus dose of 400,000 IU vitamin D in patients with sepsis could quickly increase serum 25OHD concentration, where was capable of modulating the expression level of pro-inflammatory cytokines such as IL-1β and IL-6, resulting in improved clinical outcomes [26]. On the other hand, maternal vitamin D level has been found to be potentially linked to a decreased risk of neonatal infectious diseases including respiratory infections and sepsis [27].

Regarding the maternal demographic factors, we found that the mean maternal age in the case group was significantly higher than the control group. It is supposed that risks of neonatal morbidity are more common among infants of older mothers, but this finding was not confirmed by other investigations; it has been indicated that maternal age < 20 can be considered as a risk factor for early-onset sepsis, while maternal age is not considered as a determining factors linked to the occurrence of NEOS in neonates [28,29].

It the current study, we assessed the association between NEOS occurrence and some neonatal demographic factors. Based on the findings presented herein, a significant  Fig. 1 The detail of vitamin D level in both sepsis and non-sepsis groups: a Vitamin D levels the sepsis and non-sepsis group b Normal distribution of vitamin D levels the sepsis and non-sepsis group correlation was observed between neonatal sepsis and low Apgar score. In comparison, other studies suggested different first minute Apgar scores (< 7/5, 4 or 6) as an independent risk factor which was capable of increasing the risk of NEOS [30][31][32]. Moreover, our findings revealed that neonatal sepsis was significantly more common in males than females. As s previous study indicated, the male/female sex ratio has been found to be 1.3:1 among 344 neonates suffering from sepsis [33]. On the other hand, another study from Iran by Afsharpaiman et al. have demonstrated that although EOS was more common among males (24;54.5%) than females (20;45.5%), where there have been no significant differences between the two gender [34].

Overall, ample evidence has indicated that Vitamin D is capable of showing an impressive physiologic enhancement in maternal circulation. High prevalence rates of vitamin. D deficiency has been depicted previously in pregnant women, where can lead to negative outcomes for the mother and subsequently can affect infants' shortterm and long-term health. There is strong evidence that a number of mechanisms are potentially involved in observed correlations including immunomodulatory antiinflammatory and metabolic effects of vitamin D; additionally, it can potentially play a pivotal role in epigenetic modifications of vitamin D -related genes. Whilst there is often controversial evidence that vitamin D has not been introduced as therapeutic modalities to selectively prevent pregnancy complications. However, it seems that maternal and neonatal supplies of vitamin D can provide a confirmatory approach to elucidate the events that contribute to the prevention of birth complications [11].

Our study should be considered in the light of some limitation, as the study could not evaluate our case group regarding blood culture and type of pathogen responsible sepsis and also the sample size was small. Another limitation of the study was the lack of evaluation of the possible relationship between maternal serum vitamin D concentrations with late-onset sepsis, where further studies are suggested in the near future. Therefore, further interventional randomized controlled trials and observational investigations could provide informative and beneficial data in order to prevent birth complications.

Moreover, further studies with larger sample size are strongly encouraged. Further development will need the establishment of evidence-based guidelines and novel markers for vitamin D supplementation [11].

Conclusion

Our data suggested that the level of maternal serum Vitamin D was inversely correlated with neonatal sepsis occurrence and intake of vitamin D supplement during pregnancy could decrease the risk of early neonatal sepsis.

Abbreviations 25 OHD: 25-hydroxyvitamin D3; CRP: C-reactive protein; DIC: Disseminated intravascular coagulation; IDDM: Insulin dependent diabetes mellitus; IL-6: Interleukin-6; NEOS: Neonatal early-onset sepsis; NICU: Neonatal Intensive Care Unit

Table 1
1Basic demographic characteristics of the mothers and their newbornsCharacteristic (n = 64)

Maternal age
28.76 ± 6.60

Educational Level
Diploma and upper: 37.5

Lower diploma: 62.5

Maternal Vitamin D concentration (nmol/L)
25.72 ± 12.37

Gestational age
39.64 ± 1.62

Birthweight
3002.96 ± 521.74

Type of delivery

NVD a
64.06

C/S b
35.93

a

vaginal delivery

b

cesarean section

Table 2
2Descriptive data of mother-infant according to suspected sepsis and non-septic subjectsData of mothers and newborn (n = 64)
Suspected sepsis
Non-septic sepsis
p-value

Maternal age
Skewed: − 0.851 ± 2
Skewed: 0.802 ± 2
0.004;

Kurtosis: −0.214 ± 2
Kurtosis: 0.154 ± 2
OR = 2.12

First minute Apgar score (mean ± SD)
8.5 ± 8.80
8.93 ± 0.24
0.003; OR = 1.19

Type of delivery (%):

NVD
50
78.13
0.32

C/S
50
21.88

Birth weight
Skewed: 0.731 ± 2
Skewed: 0.861 ± 2
0.249

Kurtosis: 0.094 ± 2
Kurtosis: 0.090 ± 2

Maternal Vitamin D
Skewed: −0.901 ± 2
Skewed: −0.930 ± 2
0.034; OR = 2.92

concentration (nmol/L)
Kurtosis: −0.103 ± 2
Kurtosis: − 0.089 ± 2

Sex (%)
Male: 46.87
Female: 53.13
0.003; OR = 3.27

Gestational age
3.39
1.71
0.078

A

B

AcknowledgementsNoAuthors' contributions MS, RY, MK, FKK, NKH and FR conceived the study, interpreted the data, and critically revised the reports and obtained clinical information from study participants. NKH participated in writing and revising the manuscript. All authors read and approved the final manuscript.FundingThe authors received no specific funding for this work.Availability of data and materialsThe datasets used and analyzed during the current study are available from the corresponding author on reasonable request.Ethics approval and consent to participateThe present study was provided from the medical student thesis (ID number: 2398). Ethics approval was obtained from the institutional review board of Iran University of Medical Sciences according to Helsinki declaration.Consent for publicationAll participants' parents gave written consent before enrollment.Competing interestsThe authors declare no conflict of interest.
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