Haemophilus influenzae serotype b seroprevalence in central Lao PDR before and after vaccine introduction

VPD laboratory staff; Vilaysone Khounvisith, Antony Black.

Collaborating institutions; Luxembourg Institute of Health, Luxembourg; Lao-Oxford Mahosot Hospital Wellcome Trust Research Unit, Laos; Murdoch Children’s Research Institute, Australia; National Centre for Epidemiology and Population Health, Australia; The University of Melbourne, Australia.

Communications; Manuscript published in Plos One, September 2022.


Haemophilus influenzae type b (Hib) causes pneumonia and meningitis almost exclusively in children under 5 years of age. Before widespread vaccination in 2000, Hib was responsible for at least 8.13 million cases of serious disease in children (<5 years) and 371 000 deaths globally. Vaccination has dramatically reduced invasive Hib disease worldwide. The pentavalent vaccine DTPw- HepB-Hib (Diphtheria-Tetanus-Pertussis (whole cell)- Hepatitis B-Haemophilus influenzae type b), containing purified capsular Hib polysaccharide (PRP) conjugated to the tetanus toxoid (carrier protein), was introduced in Lao PDR in 2009, replacing the DTPw-HepB vaccine. DTPw-HepB-Hib is scheduled at 6, 10 and 14 weeks of age. In 2018, the coverage with the DTPw-HepB-Hib vaccine was 84% in Lao PDR. Immunity against Hib can be determined by measuring antibody levels against the PRP immunogenic component of Hib conjugate vaccines.

There are few data on the level of immunity against Hib or the burden of disease in the general population in Lao PDR. Two serosurveys conducted in Bolikhamxay province found that 66.4% and 71.7% of children 8 to 28 months old had long-term Hib antibody protection (>1μg/ml) in 2013/14 and 2017 respectively.

To contribute to the understanding of the epidemiology of Hib in Lao PDR and the protection levels before and after the introduction of the vaccination, we tested serum samples from existing cohorts of vaccine age-eligible children and unvaccinated adolescents in the Lao PDR for antibodies against PRP. We also investigated possible predictors for long-term protection.


Serum samples from 296 adolescents born before vaccine introduction and from 1017 children under 5 years (vaccinated and unvaccinated) were tested for anti-Hib antibodies by ELISA. Bivariate analyses were performed to investigate factors associated with longterm protection.

Cohort 1: Unvaccinated adolescents. Serum samples of 296 students from Bolikhamxay province and Vientiane Capital, collected in 2018 in the framework of another study were selected from a total of 779 students. All participants in this study were born before 2008, before Hib vaccine was introduced into the national immunization program and therefore were most likely not vaccinated against Hib. However, we cannot exclude the possibility that parents paid for the vaccination outside of the Lao PDR as this vaccine has been available on the private market in Thailand for many years. Students between the age of 11 and 18 years were selectively randomized for the same age and sex ratios in both provinces. Socio-economic data (i.e. district, age, sex, ethnicity, place of birth and number of household members) were collected using a standardized questionnaire. The study was approved by the Lao National Ethics Committee (Reference number 022/NECHR) and the Institutional Review Board of the Institut Pasteur du Laos (Reference number 9). All parents/guardians signed an informed consent form.

Cohort 2: Fully vaccinated children. This cohort consisted of 761 children from Vientiane (n = 178), Bolikhamxay (n = 228) and Khammouane (n = 355) provinces aged 9 to 50 months, recruited in the context of a previous study in 2013/14. All children had records of three doses of the pentavalent vaccine, as confirmed by reviewing the vaccination log books at the health care facilities. A subset (n = 140) of the anti-Hib data from Bolikhamxay was reported in a previous study; the anti- Hib data from the entire cohort from Bolikhamxay (n = 228) were not reported before nor in conjunction with the other cohorts presented here. Socio-economic and health related data (i.e. district, age, sex, place of birth and date of vaccination etc.) were collected using a standardized questionnaire. In addition, the nutritional status of the children was determined by mid-upper arm circumference, weight for height, height for age, weight for age and body mass index z scores as described in the previous publication. The study was approved by the Lao National Ethics Committee (NECHR2013-860). Informed consent was obtained from all parents of the children.

Cohort 3: Vaccine age-eligible acute respiratory infection contacts. 256 children (<5 years of age) living in the Vientiane Capital were purposively selected as contacts of children hospitalized with acute respiratory infection (ARI) between 2013 and 2016. A contact was defined as any child under the age of 5 coming into contact with the case in the preceding two weeks of hospitalization with ARI. The vaccination status of the children was assessed by reviewing the vaccination records in the parent-held mother child handbook (MCH) or via immunisation registers at the respective health centre. Participant information (i.e. age, sex, self-reported ethnicity, vaccination status, date of vaccination) was collected using a questionnaire. Ethics approval was received from the Royal Children’s Hospital (RCH) Human Research Ethics Committee (33177B; MCRI), Oxford Tropical Research Ethics Committee (1050–13; LOMWRU), WPRO Ethics Research Committee (2013.30.LAO.2.EPI), the Lao National Ethics Committee (2013–057) and the Human Research Ethics Committee (2016/770; ANU). Informed consent was obtained from all parents of the children.


Participants’ characteristics

In total, 1313 participants from the three cohorts were included. The majority of the participants in cohorts 1 and 3 were of Tai-Kadai ethnicity, which is the main ethnic group in Lao PDR. Information on ethnicity was not available for cohort 2. Serum samples of all three cohorts were collected in provinces located in central Lao PDR. Since all adolescents (cohort 1) were born before 2008, it was assumed they would not have received routine Hib vaccination. All children included in cohort 2 and half (52.3%) of the ARI contacts (cohort 3) had written records of a full course of the DTPw-HepB-Hib vaccine.

Prevalence of anti-Hib IgG

The vast majority of the participants in cohorts 1, 2 and 3 showed evidence of short- (42.7%) and long-term (56.1%) protection against Hib. Almost all (95.9%) of the unvaccinated adolescents (cohort 1) had an anti-Hib IgG titer >0.15μg/ml indicating at least short-term protection against Hib and almost half (45.6%) had anti-Hib IgG titers >1.0 μg/ml corresponding to long-term protection.

58.9% of the vaccinated children in cohort 2, all born after the introduction of the pentavalent vaccine, showed long-term protection. Long-term protection varied from 54.9% to 62.9% between the provinces and from 56.9% to 80% between the age groups. In ARI contacts (cohort 3), long-term protection rates ranged between 25.0% for unvaccinated to 67.2% for fully vaccinated children. None of the participants in this cohort had anti-Hib IgG titers below 0.15 μg/ml.

Factors associated with long-term protection

Since nearly every participant in our study showed at least short-term protection, we could not assess associations between variables and short-term protection in either of the cohorts and instead focused on long-term protection as outcome.

Despite some visible differences in the proportion of participants with long-term protection between age groups at the different locations, there was no statistical difference between the respective youngest and older age groups in any of the cohorts. However, the proportion of long-term protection was higher in the cohort of children <5 years (cohort 2 and 3; n = 1017) compared to the adolescents (cohort 1; n = 296) (59.1% vs 45.6%; p<0.0001).


Our findings indicate that the circulation of Hib was high in Lao PDR before the introduction of the vaccine and continues to be high in unvaccinated children. Indeed, after vaccine introduction, all vaccinated children, but also all others showed serological markers of vaccination/ past infection and protection. Thus, robust surveillance and systematic reporting of invasive Hib cases are required to determine the current burden of disease despite vaccination.