The prevalence and influencing factors of the oropharyngeal carriage of Haemophilus influenzae in healthy children in a high-altitude area of China: A cross-sectional study

Abstract

Haemophilus influenzae is a common commensal organism of the human upper respiratory tract and an important cause of human disease. No data on H influenzae carriage rate has been carried out on the Qinghai-Tibet Plateau of China. This study aims to present the H influenzae carriage rate and influencing factors of H influenzae in healthy children <15 years of age in Qinghai Province, an area located on the Qinghai-Tibet Plateau in China. Oropharyngeal swabs for the detection of H influenzae DNA were collected between September and October 2019. Taqman real-time polymerase chain reaction was used to detect the nucleic acids from the oropharyngeal swabs. Self-designed questionnaires were used to investigate the related information among this group of children. A number of 284 children were enrolled in this study. The carriage rate of H influenzae was 44.7%. The carriage rate in cities was 47.5%, in rural areas was 21.9%, and in pastoral areas was 52.8%. The carriage rate was found to be higher among children of minority ethnic groups than those of Han ethnicity (55.6% vs 38.1%). H influenzae carriage rate was influenced by tobacco smoke exposure (adjusted odds ratio [aOR] = 2.31, 95% CI [confidence interval]: 1.14–4.70), having siblings <5 years of age (aOR = 2.36, 95% CI: 1.21–4.59), respiratory infections during the last 30 days (aOR = 2.37, 95% CI: 1.11–5.06), and parent/guardian education level (aOR = 0.08, 95% CI: 0.02–0.27). H influenzae was highly prevalent in healthy children in Qinghai Province, especially among children of minority ethnicities and those living in pastoral areas. Tobacco smoke exposure, having siblings <5 years of age, and respiratory infections during the last 30 days were risk factors for H influenzae carriage. Parents or guardians having education levels of college or higher was a protective factor for H influenzae carriage. It is of critical importance that the government take effective measures to reduce the carriage rate and the occurrence of H influenzae related diseases in susceptible populations.

1. Introduction

Haemophilus influenzae (H influenzae) is a gram-negative coccobacillus including encapsulated strains and unencapsulated strains. Based on H influenzae encapsulated strains can be separated into 6 serotypes (a–f).^[1] Pharyngeal carriers are the only reservoir and the transmission vector of invasive disease.^[2] The primary diseases caused by H influenzae are childhood pneumonia, meningitis, and bacteremia. H influenzae is commonly found in the upper respiratory tract of healthy children and adults and is one of the main pathogens that cause community-acquired respiratory tract infections during childhood.^[2] One study has shown that the overall nasopharyngeal carriage rate of H influenzae is 26.3% among children <5 years old with acute respiratory tract infection (ARI).^[3] H influenzae meningitis accounts for 30% to 50% of bacterial meningitis. H influenzae b (Hib) is the most common cause of bacterial pneumonia, following streptococcus pneumonia among children.^[4] Children who survive Hib meningitis may develop permanent neurological disabilities, including brain damage, hearing loss, and mental retardation, accounting for 40% of the cases.^[5] In 2000, Hib caused approximately 8.13 million cases of serious disease and 371,000 deaths in children aged 1 to 59 months worldwide.^[6] In addition, approximately 59,000 deaths due to Hib pneumonia in children <5 years old occurred worldwide in 2015.^[7]

H influenzae related diseases are difficult to diagnose, therefore, blood cultures, cerebrospinal fluid, or other aseptic body fluid cultures are needed for diagnosis. However, the laboratory findings may be negative due to prior to the use of antibiotics. Moreover, the outcome was affected by increasing drug resistance of the bacteria. Widespread vaccination is proven to be the most effective way to prevent H influenzae related diseases.^[8] As of December 2016, 191 countries have incorporated Hib vaccination into their National Immunization Programs and this strategy has effectively controlled the occurrence of related diseases.^[8,9] In China, however, the Hib vaccine has not yet been introduced into the National Expanded Program on Immunization.^[10] A survey carried out by the Chinese Center for Disease Control and Prevention shows that the coverage rate of Hib vaccination in China is low, especially in the high-altitude areas of western China.^[11,12] Thus, monitoring H influenzae carriage rate can offer an early warning for the occurrence of invasive diseases, which is urgently needed in China.

Investigation on the carriage status of H influenzae among healthy populations has been carried out in China^[13,14] and most studies were carried out in Central and Eastern China. There has been little research on H influenzae carriage on the Qinghai-Tibet Plateau of China. A previous study reported that a high-altitude hypoxic environment affects the colonization of bacteria.^[15] The Qinghai-Tibet Plateau is an area with ethnic minorities gathering located in the underdeveloped region of Western China. With limited health resources, the task of preventing and controlling infectious diseases in this region is very heavy. Thus, this study aims to investigate the status of H influenzae carriage and its influencing factors in healthy people under the age of 15 years in the Plateau and to provide useful data for health care policy makers to take effective measures to protect the susceptible population from H influenzae related diseases.

2. Materials and Methods

2.1. Participants

In the present study, a questionnaire survey and laboratory tests were conducted between September and October 2019 in Qinghai Province. Qinghai Province is located in the northeastern Qinghai-Tibet Plateau, Western China. According to the geographical location and residents’ living environment, Qinghai Province can be divided into 3 regions: urban, rural area, and pastoral regions. Pastoral region is an area with natural grassland and residents live on raising draught animals and their production of livestock. Agricultural area refers to the area where farming is the main way of living. Urban regions are areas predominantly by nonagricultural industries, especially the service industry.^[16] Qinghai Province covers 721,000 km² and, as of 2019, has a population of approximately 5,980,000 and a per capita disposable income of ¥10,220 (approximately 1525.85 United States dollar). The annual average temperature in all regions of Qinghai Province is between −5.1°C and 9.0°C (22.8°F–48.2°F). The minority nationalities account for 47.71% of the population in Qinghai and mainly include Tibetan, Hui, and Tu ethnicities, and others.

2.2. Data collection

A multistage stratified design was used in this survey. This is consistent of recruited healthy children under 15 years of age in Chengxi district (city), Ledu district (agricultural district), Gui-de, and Wulan County (pastoral regions) as our research subjects. The respondents in 0- to 3-year-old group were recruited from among children who visited community health centers or vaccination clinics. The parents/guardian of the children were approached by a centers for disease control staff member in the clinic waiting area, and an informed written consent was obtained from the parents/guardians. The 4- to 14-year-old children were recruited from day-care centers and schools as our research subjects. Parents/guardians were informed in advance by the school medical staff to come to the school on the day of the survey. The parents/guardians were informed about all oropharyngeal swab collections in written form, and collections were only carried out with the consent of the parents/guardians. Children were excluded if they met at least one of the following criteria: took antibiotics within 15 days of enrollment; had a long-term infection (such as chronic otitis media or chronic sinusitis); had coagulopathy; had a body temperature over 38°C at the time of enrollment; or the oropharyngeal swab was unable to collect. According to our multistage stratified design, a total of 310 children were recruited for oropharyngeal swab collection. Based on the exclusion criteria and excluded subjects with missing data, 26 subjects were excluded from the analysis, and 284 subjects were enrolled in this study. All subjects’ parents/guardians provided written informed consent prior to participation.

2.3. Questionnaire

The prevalence of H influenzae carriage varies with age, socioeconomic factors, vaccine coverage, and environmental factors (overcrowded housing, environmental smoke exposure, and indoor air). These factors were completely considered in our research. A self-designed questionnaire was used. The parents/guardians of the children whose oropharyngeal swabs were collected were asked to complete the questionnaire at the survey site. The information provided included 4 parts: the basic demographic characteristics of the children, including gender, age, ethnicity, and average family monthly income; related information of the parents/guardians, such as the identification of the primary caregiver, occupation, and their education level; the environmental conditions of the children, such as their attendance of school or day-care centers, the period of time they attended school or day-care centers, whether they had siblings under 5 years old, whether they lived with smokers, and household size; and health status of children, such as whether they had been vaccinated with Hib vaccine and whether that had any respiratory infections within the last 30 days.

2.4. Laboratory testing

2.4.1. Swab specimen collection and transportation.

The participants were asked not to drink or eat before swabbing, then professionals from the centers for disease control of Qinghai Province swabbed each participant’s lateral and posterior pharyngeal wall with a disinfectant oropharyngeal swab. The oropharyngeal swab was from Yocon Biotechnology Co., Ltd (Beijing, China). The specimens were stored at 4°C to 8°C in a precooled transfer box and were transported to the laboratory.

2.4.2. Laboratory methods.

2.4.2.1. Reagents and instruments.

Using Taqman real-time polymerase chain reaction (PCR) technique was utilized to detect the carriage status. Quantitative real-time PCR was performed using Bio-Rad CFX96 Touch Deep Well Real-time PCR detection system (Bio-Rad Laboratories, CA). Reagent kits included bacterial DNA exact reagent (MagMAX(TM)-96 (No. AMB 1836-5, ABI, USA), negative and positive controls, and H influenzae gene amplification primers and probes. Target gene is hpd. Fluorescence signal was collected using FAM channel, these can be found in the manufacturer’s instructions. The reagent kits and fluorescent quantitative detection reagents were purchased from MABSKY BIO-TECH CO., LTD (Shenzhen, China). All reagents utilized were within the validity period.

2.4.2.2. Sample collection and analysis.

Reagent kits at −15°C were taken out from the refrigerator and balanced at room temperature (20–25°C) later. After solving completely, the reagents were mixed by vibrating and centrifuged at low speed for 10 seconds.

DNA of specimens from the oropharyngeal swabs were extracted by using the centrifugal column method. Each PCR was performed in 25 μL volumes and PCR mixture contained HI & SP PCR fluid 19.5 μL, HI & SP mixed enzyme 0.5 μL. Sample DNA fluid was 5 μL. Five microliter of positive and negative controls were added, respectively. The amplification protocol of PCR briefly included the following steps: Uracil-N-glycosylase (UNG) at 50°C for 2 minutes. The reaction was performed with an initial step of 95°C for 3 minutes and then 40 cycles of 95°C for 5 seconds, 55°C, and 60 seconds. The threshold cycle (Ct) value was automatically analyzed with fluorescence monitoring. In each set of experiments, negative as well as positive controls were included. The results were judged by laboratory technicians, reviewing officers, and administered officers were in charge of according to the instruments of the reagent kits. In the case of the Ct value was ≤34.5 associated with the exponential growth of the PCR product during the log-linear phase, the result was positive. Provided that the Ct value ranged from 34.5 to 37.5, the sample was considered as suspected positive and needed to retest. If the value still ranged 34.5 to 37.5 associated with the exponential growth of the PCR products during the log-linear phase, the sample was considered positive, otherwise the sample was considered negative. If the Ct value was >37.5 or no Ct value, the result was judged as negative. The minimum test limit was 10³ copies/mL.

2.5. Statistical analysis

SPSS Statistics Version 21.0 (IBM Corp., Armonk, NY) software was used for statistical analysis. Characteristics of the overall respondents were described using percentages for categorical data. Univariate logistic regression analyses were performed to explore the relationship between demographic characteristics and other variables and H influenzae carriage. Multivariate logistic regression analyses were used to identify the influencing factors of H influenzae. Adjusted odds ratios (aORs) were presented using a 95% confidence interval (CI), with a P < .05 being considered statistically significant. Large CI indicated a low level of precision of the OR, whereas a small CI indicated a higher precision of the OR.

3. Results

3.1. Respondents’ characteristics of the study

A total of 284 children were enrolled in this study, and 129 (45.4%) of them were males. The mean age of the respondents was 7.50 ± 4.23 years of age. Overall, 56.7% of the respondents were from pastoral areas, and 46.1% of the respondents had monthly family incomes of <1500 yuan (216.9 United States dollar). Most children’s primary caregivers were their mothers, accounting for 61.6%, and predominantly farmers (63.4%). The education level of most of the parents/guardians was junior or lower (44.4%). The vast majority of children (81.3%) attended either school or kindergarten, and 50.2% stayed there for over 8 hours every day. A total of 49.3% of children lived with smokers, and 65.1% of the children had an average household size <30 m². A total of 123 children have siblings <5 years of age and live at home. Overall, 28.9% of the children had respiratory infections during the last 30 days, and 73.2% of children had not been vaccinated with the Hib vaccine (Table 1).

Table 1.

Univariate analysis of influencing factors of Haemophilus influenzae carriage with different characteristics.

Characteristic	Categories	Total sample (n = 284)	Proportion (%)	Number of H influenzae carriage (n = 127)	Carriage rate (%)	OR (95% CI)	P value
Gender	Male	129	45.4	57	44.2	Ref.
	Female	155	54.6	70	45.2	1.040 (0.650–1.660)	.869
Age (yr)	<2	49	17.3	13	26.5	Ref.
	≥2 and <5	49	17.3	23	46.9	2.450 (1.050–5.713)	.038
	≥5 and <8	59	20.8	23	39.0	1.769 (0.778–4.026)	.174
	≥8 and <11	50	17.6	28	56.0	3.524 (1.514–8.205)	.003
	≥11 and ≤14	77	27.0	40	51.9	2.994 (1.378–6.504)	.006
Ethnicity	Han	176	62.0	67	38.1	Ref.
	Minority	108	38.0	60	55.6	2.034 (1.250–3.308)	.004
Region	City	59	20.8	28	47.5	Ref.
	Agricultural area	64	22.5	14	21.9	0.310 (0.142–0.678)	.003
	Pastoral area	161	56.7	85	52.8	1.238 (0.681–2.250)	.483
Family monthly income	<1500	131	46.1	59	45.0	Ref.
	1500–3000	79	27.8	43	54.4	1.458 (0.832–2.554)	.188
	>3000	74	26.1	25	33.8	0.623 (0.344–1.126)	.117
Parents/guardians	Father	85	29.9	46	54.1	Ref.
	Mother	175	61.6	71	40.6	0.579 (0.343–0.976)	.040
	Grandparents	24	8.5	10	41.7	0.606 (0.242–1.515)	.284
Parents/guardians occupation	Farmer	180	63.4	83	46.1	Ref.
	Medical staff	18	6.3	7	38.9	0.744 (0.276–2.005)	.558
	Civil servants	22	7.7	13	59.1	1.688 (0.687–4.148)	.254
	No job	52	18.3	22	42.3	0.857 (0.459–1.598)	.628
	Others	12	4.2	2	16.7	0.234 (0.050–1.097)	.065
Parents/guardians’ education level	Junior or lower	126	44.4	65	51.6	Ref.
	Senior high school and equivalent	84	29.5	44	52.4	1.032 (0.594–1.794)	.910
	College or higher	74	26.1	18	24.3	0.302 (0.160–0.570)	.001
Attending school or day-care centers	No	53	18.7	18	34.0	Ref.
	Yes	231	81.3	109	47.2	1.737 (0.930–3.244)	.083
Duration of attending school or kindergarten	≤8 h	115	49.8	50	43.5	Ref.
	>8	116	50.2	59	50.9	1.346 (0.802–2.259)	.261
Tobacco smoke exposure	No	144	50.7	64	44.4	Ref.
	Yes	140	49.3	63	45.0	1.023 (0.641–1.633)	.925
Average household size (m2)	<30	185	65.1	76	41.1	Ref.
	30–59	87	30.6	45	51.7	1.537 (0.920–2.565)	.100
	>60	12	4.2	6	50.0	1.434 (0.446–4.616)	.545
Sibling under 5 yr of age	No	161	56.7	59	36.6	Ref.
	Yes	123	43.3	68	55.3	2.137 (1.324–3.450)	.002
Respiratory infections during the last 30 days	No	202	71.1	81	40.1	Ref.
	Yes	82	28.9	46	56.1	1.909 (1.136–3.208)	.015
≥1 dose Hib vaccine	No	208	73.2	96	46.2	Ref.
	Yes	76	26.8	31	40.8	0.804 (0.472–1.369)	.421

CI = confidence interval, H influenzae = Haemophilus influenzae, OR = odds ratio, Ref. = reference.

3.2. The carriage rate of H influenzae

Among the enrolled children, H influenzae carriage was detected in 127 children, and the positive rate was 44.7%, 47.5% in cities, 21.9% in agricultural areas, and 52.8% in pastoral areas, respectively. The carriage rate was higher among children of minority ethnicity groups than among those of Han ethnicity (55.6% vs 38.1%). There were significant differences in age, ethnicity, region, family monthly income, parent/guardian education level, having siblings <5 years of age, and respiratory infections during the last 30 days in the H influenzae carriage rate (P < .05) (Table 1).

3.3. Influencing factors of H influenzae carriage

The results from multivariate logistic regression analyses showed that tobacco smoke exposure (aOR = 2.31, 95% CI: 1.14–4.70), having siblings <5 years of age (aOR = 2.36, 95% CI: 1.213–4.591), and respiratory infections during the last 30 days (aOR = 2.37, 95% CI: 1.11–5.06) were independent risk factors for H influenzae carriage in children (P < .05). An inverse association was found between the parent/guardian education level (college and higher) and H influenzae carriage (aOR = 0.08, 95% CI: 0.02–0.27) (Table 2).

Table 2.

Multivariate logistic regression analyses of influencing factors of Haemophilus influenzae carriage.

Covariate	aOR (95% CI)	P value
Gender (ref.: female)
Male	1.274 (0.659–2.464)	.471
Ethnicity (ref.: Han)
Minority	1.866 (0.928–3.754)	.080
Age (ref.: <2)
≥2 and <5	6.357 (0.486–83.153)	.159
≥5 and <8	6.523 (0.511–83.327)	.149
≥8 and <11	6.188 (0.485–78.959)	.161
≥11 and <14	6.572 (0.532–81.139)	.142
Region (ref.: city)
Agricultural area	0.454 (0.144–1.431)	.178
Pastoral area	1.138 (0.447–2.897)	.786
Family monthly income (ref.: <1500)
1500–3000	2.136 (0.966–4.724)	.061
>3000	1.129 (0.457–2.794)	.792
Primary parents/guardians (ref.: father)
Mother	2.242 (0.510–9.860)	.285
Grandparents	1.487 (0.359–6.152)	.584
Occupation (ref.: farmer)
Medical staff	1.583 (0.345–7.269)	.555
Civil servants	1.386 (0.376–5.112)	.624
No job	1.59 (0.605–4.181)	.347
Others	1.541 (0.194–12.214)	.682
Education (ref.: junior and lower)
Senior high school and equivalent	0.702 (0.301–1.635)	.412
College and higher	0.080 (0.024–0.269)	<.001
Smoking in home (ref.: no)
Yes	2.310 (1.135–4.699)	.021
Period of time attending school or kindergarten (ref.: ≤8 h)
>8	0.819 (0.393–1.704)	.593
Sibling under 5 yr of age (ref.: no)
Yes	2.360 (1.213–4.591)	.011
Household size (ref.: <30)
30–59	1.512 (0.742–3.080)	.255
>60	1.012 (0.234–4.374)	.987
≥1 dose Hib vaccine (ref.: no)
Yes	0.775 (0.373–1.611)	.495
Respiratory infections during the last 30 days (ref.: no)
Yes	2.369 (1.11–5.056)	.026

aOR = adjusted odds ratio, CI = confidence interval, Ref. = reference.

4. Discussion

In our study, the H influenzae carriage rate among healthy children <15 years old in Qinghai Province was 44.7%, which is higher than those in eastern China (11.3%–19.6%)^[2,17] and central China (1.7%).^[18]

A number of potential factors affecting the high prevalence of H influenzae aforementioned were found in this study. Firstly, a previous study demonstrated that widespread use of Hib vaccines can reduce the H influenzae carriage rate.^[19] In our survey, only 26.8% of children had been obtained Hib vaccine, therefore, it is suggested that improvement of vaccination of Hib should be critical to prevent H influenzae related diseases. Secondly, there are fewer health resources in Western China. Health education at schools did not meet the needs of students. Yet West region is not as sound as East and Middle regions’ popularity ratings regarding infectious disease prevention.^[20,21] Thirdly, Qinghai is located in Qinghai-Tibet Plateau, it is colder than that of Eastern and Middle regions of China, the inclement weather may drive people to be longer indoor stay and to expose to indoor pollution.^[22] These reasons may contribute to some diseases’ occurrence, such as H influenzae related diseases among individuals.

In our study, the carriage rate of H influenzae was the highest in pastoral areas, followed by the city, and the lowest was agricultural areas. Firstly, in pastoral areas, due to the low availability of medical care, many residents cannot make full use of healthcare resources, resulting in a high carriage rate of H influenzae.^[23] Additionally, lack of information was considered one of the reasons of not being vaccinated with Hib vaccine. Vaccination knowledge and attitudes are important influencing factors on the vaccination rate. Moreover, inhaling cold air may weaken the nonspecific immune function of the respiratory mucosa, reduce cilia activity, and affect the elimination of secretions.^[23] These factors are conducive to the successful colonization of H influenzae in the respiratory tract mucosa. This may, therefore, the government should provide policy support to meet the vaccination needs of residents in pastoral areas. Additional publicity and education of health care personnel are necessary. The inclusion of Hib vaccination in the National Immunization Program could help improve the coverage of Hib vaccination. The results revealed that there is a lower carriage rate in rural areas. The explanation for this phenomenon may be that rural area usually locate in the eastern monsoon region with a comfortable natural environment and the residents own self-built houses with larger space.^[24] While in cities, where a large number of individuals are inhabiting a confined space thus increasing the likelihood acquiring the condition, assembly occupancies such as schools, markets, and hospitals may lead to higher occurrence of respiratory carriage of the organism.^[25]

The carriage rate of H influenzae was higher among children of minority ethnicity than among children of Han ethnicity (55.6% vs 38.1%). Many ethnic minorities have low levels of education in China^[26] and some ethnic minorities do not even understand Chinese. Therefore, health care personnel should enhance personalized publicity and education for ethnic minorities, such as printing brochures in minority languages and recording publicity videos in minority languages. In addition, people from religious institutions can be used for publicity to strengthen the publicity’s effect.

Our study found that children whose parents or guardians had education levels of college or higher were less likely to carry H influenzae. Individuals with higher education levels are more likely to have knowledge and awareness in disease prevention.^[27] These parents pay more attention to their children’s health issues and ensure their children with more medical security. This result is similar to the studies from Japan and Ethiopia.^[28,29] Our study also revealed that tobacco smoke exposure was a risk factor for H influenzae carriage, similar to the findings in a study that evaluated children aged 7 to 12 years in Turkey.^[30] Smoking can lead to increased bacteria in human oropharynx, increase pathogenic bacteria, and change the amount of bacteria, which can cause microecological disorders in the respiratory tract.^[31] Therefore, parents/guardians should pay more attention to children who are exposed to tobacco smoke in their environment. This study showed that having siblings <5 years old was also a risk factor for H influenzae carriage. This finding is consistent with a study from India^[32] and can be explained by the fact that children living in large families and having close contact with a number of siblings increase the chances of acquisition of H influenzae. Our study found that respiratory infections during the last 30 days were significantly associated with H influenzae carriage. This result was consistent with a previous study in Gambian.^[33]

A number of limitations may be highlighted in this study. First, the conclusion in the present study is limited to Qinghai Province and may not be extended to the whole high-altitude area. Additionally, the carriage rate of H influenzae in the oropharynx may vary in different seasons. Thus, further surveys should be conducted in each season. Different tests (or methods) are needed to investigate and compare carriage rates in different contexts. Due to the majority of children who obtained Hib vaccine did not complete the 4-dose-scheduled vaccine in the present survey, the population were classified as vaccinated and nonvaccinated groups. The results only suggested that there were no significant differences between nonvaccinated and not enough vaccinated individuals. The results could not confirm the effect of vaccination.

5. Conclusion

The findings of this survey indicated that the overall carriage rate of H influenzae was higher in Qinghai, especially among children of minority ethnicities and those living in pastoral areas. To have a close look at aspects preceding, a higher prevalence of these conditions, such as Hib vaccine vaccination coverage, health education, education popularization, improvement of housing, prevention of respiratory infections, and avoidance of tobacco smoke exposure, is imperative to reduce the carriage rate and recurrence of H influenzae disease in susceptible populations.

Acknowledgments

We thank Prof Biao Guo, Prof Guijun Ning, Prof Lili Xu, and Prof He-xi-ge Saiyin for their helpful comments on earlier draft.

Author contributions

Conceptualization: Kezhong A, Puren Li, WenSheng Ba.

Data curation: Zhen Li.

Formal analysis: Chang Fu, Zhen Li.

Investigation: Shaohui Ma, Xiaolei Tang, Xueqin Yang, Zengping Hao, Zhen Li.

Writing – original draft: Chang Fu, Zhen Li.

Writing – review & editing: Chang Fu, Kezhong A, Zhen Li.