Measurement of pneumococcal IgG antibody, carriage and infection ...

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Measurement of pneumococcal IgG antibody, carriage and infection with Streptococcus pneumoniae in children under five years of age with acute respiratory infection in Niger Dan Dano Ibrahim a,∗ , Sani Ousmane a , Kamayé Moumouni b , Ali Elhadji Mahamane a a b

Centre de Recherche Médicale et Sanitaire (CERMES),634 Boulevard de la Nation YN034, BP 10 887, Niamey, Niger Hôpital National de Niamey (HNN), Service de pédiatrie A, BP 238, Niamey, Niger

a r t i c l e

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Article history: Received 26 December 2016 Received in revised form 2 May 2017 Accepted 30 May 2017 Keywords: Pneumococcal antibody Carriage Respiratory infection Children under five years

a b s t r a c t Pneumonia is the major cause of mortality in children under five years. A total of 751 children aged 1–59 months admitted for acute respiratory infection were included in this study. Pneumococcal serum IgG antibody was determined by ELISA. Carriage of Streptococcus pneumoniae was determined by molecular analyses of nasopharyngeal swabbings, and the rapid urinary diagnostic ® test Binax NOW Sp was used for detection of pneumococcal antigen in urine. A total of 224 (29.8%) children had vaccination record books, and among them, 186 (83%) were vaccinated with the 23-Valent Pneumococcal Polysaccharide Vaccine (PPSV-23), 7 (3%) were vaccinated with the 13-Valent Pneumococcal Conjugate Vaccine (PCV-13), and 31 (14%) had not been vaccinated. IgG levels against pneumococcal polysaccharide were ≥1.3 ␮g/mL in most of the children (99.4%). The carriage ®

rate of S. pneumoniae was 39%, and the Binax NOW Sp test was positive in 26% of children. There was no significant variation between the means of IgG concentrations against pneumococcal polysaccharides as related to vaccination status, age and nasopharyngeal carriage of S. pneumoniae. However, there was a weak positive correlation between age and level of IgG (r = 0.08; p = 0.021), and there was a significant variation (p = 0.038) of the IgG level according to presence of S. pneumoniae antigen in urine. The presence of S. pneumoniae antigen in urine is significantly (p ≤ 0.01) higher in children with nasopharyngeal carriage of S. pneumoniae (40%) than non-carriers (20%). This study shows that S. pneumoniae has high circulation in children under the age of five years either through infection or carriage. © 2017 The Authors. Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

1 Introduction Pneumonia is the major cause of mortality in children under five years, particularly in low- and middle-income countries [1]. According to the World Health Organization pneumonia killed 920 136 children under the age of 5 years in 2015, accounting for 16% of all deaths of children under five years old. Pneumonia affects children and families everywhere but is most prevalent in South Asia and sub-Saharan Africa [2]. Most deaths from severe pneumonia cases are caused by Streptococcus pneumoniae. S. pneumoniae is a commensal bacterium of human respiratory tract asymptomatically carried by many people. An important

feature is that pneumococcal diseases will not occur without preceding nasopharyngeal (NP) colonization with a homologous strain [3]. Cross-sectional studies have shown that, at any given time, approximately 20–50% of healthy children harbor at least one serotype of this bacterium in the rhinopharynx, whereas longitudinal studies have shown that almost all children can be shown to carry this bacterial species [4,5]. In Niger, the 13-Valent Pneumococcal Conjugate Vaccine (PCV-13) was introduced in routine vaccination program in July 2014; however, the 23-Valent Pneumococcal Polysaccharide Vaccine (PPSV-23) was already available but is not part of the routine national childhood vaccination program. We assessed the role of S. pneumoniae in respiratory infection in children under five years of age through measurement of blood serum pneumococcal IgG antibodies, carriage and antigen detection in urine.

∗ Corresponding author. Fax: +227 20 75 31 80. E-mail addresses: [email protected], [email protected] (D.D. Ibrahim). http://dx.doi.org/10.1016/j.jiph.2017.05.007 1876-0341/© 2017 The Authors. Published by Elsevier Limited on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Ibrahim DD, et al. Measurement of pneumococcal IgG antibody, carriage and infection with Streptococcus pneumoniae in children under five years of age with acute respiratory infection in Niger. J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2017.05.007

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2 Methods

Table 1 Demographic characteristics and pneumococcal vaccination status.

2.1 Patients

Parameter

This study is a prospective study conducted in Niger from January 2015 to June 2017 in the pediatric departments of the National Hospital of Niamey and that of Lamordé. Children aged 1–59 months with signs of respiratory infection and hospitalized in one of the pediatric departments of either hospital were enrolled. Children under the age of one month or over the age of five years, and children suffering from heart disease or chronic respiratory infection were excluded.

Total number of children Male Female Gender not mentioned Mean age of children (month) Number of patients with vaccination record book Mean age of children with vaccination record book (month) Number and % of children non vaccinated Number and % of vaccinated children with only PPSV-23 Number and % of children vaccinated with PCV-13

2.2 Ethical considerations This study was approved by the National Consultative Ethics Committee of Niger by decision No. 0016/2013/CCNE of 30 October 2013 and informed consent from parents of children were requested before inclusion. 2.3 Clinical samples Blood and urine were collected by the treating medical staff and safely sent to the Medical Research and Health Center (CERMES) within 4 h in a thermos with an ice pack for processing and laboratory diagnosis. Serum samples were stored at −20 ◦ C before analyses. 2.4 Nasopharyngeal swabbing Nasopharyngeal swabbing was performed with the kit Nasopharyngeal flocked swabs UTMTM 350C, Copan Diagnostics, Murrieta, USA. Swabs were inserted along the nasal septum just above the floor of the passage to the nasopharynx until resistance is met. The swab was rotated gently against the nasopharyngeal mucosa for 10 − 15 seconds and then gently removed. After the swab was removed from the patient, it was placed all the way to the bottom of the tube of UTMTM transport medium. 2.5 Urinary S. pneumoniae antigen detection Urine samples were used for detection of S. pneumoniae antigen ® with the Binax NOW S. pneumoniae Antigen Card (Ref: 710-000). This is a rapid assay for the qualitative detection of S. pneumoniae antigen in the urine of patients with pneumonia and in the cerebral spinal fluid (CSF) of patients with meningitis. 2.6 Pneumococcal IgG antibody assays ELISA was used to determine blood serum concentration of IgG antibody against pneumococcal capsular polysaccharide. ELISA microtiter plates (Immulon 2HB, Thermo Fisher Scientific Ref: 3455) were coated with a mix of methylated human serum albumin (mHSA) at a final concentration of 5 ␮g/mL and purified S. pneumoniae polysaccharide antigen. Concentrations of IgG against pneumococcal polysaccharide above 1.3 ␮g/mL were considered protective [6]. 2.7 RT-PCR for detection of S. pneumoniae Nasopharyngeal carriage of S. pneumoniae was determined by a multiplex real time PCR with the FTD Respiratory pathogens 21 plus kit (Ref: FTD-2+.1-32. Fast-Track Diagnostics Luxembourg Sarl). Only S. pneumoniae results were considered in this study.

751 411 (54.7%) 332 (44.2%) 8 (1.1%) 13.2 ± 12.6 224 (29.8%) 9.7 ± 8.2 31 (14%) 186 (83%) 7 (3%)

Table 2 Mean IgG antibody responses measured by ELISA against pneumococcal polysaccharide according to vaccination status for children with vaccination record book.

Mean IgG concentration ␮g/mL (standard deviation)

Vaccination with PPSV-23, n = 186

Vaccination with PCV-13, n = 7

Non vaccinated, n = 31

74.7 ± 42.4

92.2 ± 59.5

75.2 ± 46.0

2.8 Statistical analysis Statistical analyses were performed using IBM SPSS Statistics version 20 software and one-way analysis of variance (ANOVA) and Pearson’s r-correlation were used for comparison between groups, and chi-square analysis was used for proportion comparison. 3 Results 3.1 Population characteristics A total of 751 children with a mean age of 13.24 ± 12.65 months were included in this study. Only 224 (29.8%) children had vaccination record books, among whom 193 children were vaccinated either with the PPSV-23 (n = 186; 83%) or the PCV-13 (n = 7; 3%). The demographic and vaccination status of the included children are summarized in Table 1. 3.2 Antibody concentration according to vaccination status There was no significant variation between the mean concentrations of IgG against pneumococcal polysaccharide according to vaccination status. Although the mean concentration of IgG was higher in children vaccinated with the conjugate vaccine, the difference was not significant (Table 2). Among the 7 children vaccinated with the conjugate vaccine PCV-13, 4 children received the 2 doses. 3.3 Antibody concentration according to age The mean IgG level against pneumococcal polysaccharide was ≥1.3 ␮g/L in most of the children. Only 4 children out of 688 children (0.6%) had IgG levels against pneumococcal polysaccharide below 1.3 ␮g/mL, and among these children 3 had positive Binax ® NOW Sp tests. There is no significant variation (p = 0.075) in the mean levels of IgG antibody against pneumococcal polysaccharide according to age group (Table 3). However, there is a weak positive correlation between age and levels of IgG (r = 0.08; p = 0.021). 3.4 Proportion of carriage and infection with S. pneumoniae The nasopharyngeal carriage rate of S. pneumoniae obtained in this study is 39% (239/612 children) and the rate of S. pneumoniae


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Table 5 Distribution of IgG concentration according to carriage and infection with S. pneu® moniae based on Binax NOW Sp test.

Age (month)

Number

Mean IgG level (␮g/mL)

Parameters

1–6 7–12 13–24 25–36 37–59 Total

274 174 145 55 26 674

70.5 ± 42.9 65.9 ± 39.3 75.1 ± 40.2 68.7 ± 37.4 88.2 ± 60.8 70.9 ± 42.0

Carriage of S. pneumoniae Infection with Sp based on ® Binax NOW Sp results *

Table 4 ® Proportion by age of infection with S. pneumoniae according to Binax NOW Sp results. Age (month)

1–6 7–12 13–24 25–36 37–59 Total

Number of patients

251 155 138 52 29 625

®

Positive Binax NOW Sp results n

%

66 44 42 9 5 166

26.3 28.4 30.4 17.3 17.2 26.6

3.5 Antibody responses against pneumococcal polysaccharide according to carriage and variation of carriage according to infection with S. pneumoniae There is no significant variation (p = 0.38) in the mean IgG antibody response against pneumococcal polysaccharide according to carriage of S. pneumoniae but there is significant (p = 0.038) variation according to the presence of S. pneumoniae antigen in urine (Table 5). Table 6 shows that the presence of S. pneumoniae antigen in urine is significantly (p ≤ 0.01) higher in children with nasopharyngeal carriage of S. pneumoniae (40%) than in non-carriers (20%). 4 Discussion In this study, ELISA was used to determine levels of non-serotype specific IgG antibody against polysaccharide capsular antigen. Most of the vaccinated children included in this study (83%) were vaccinated with the PPSV-23, which is not effective in young children [7]. The type of ELISA used in this study is known to overestimate the true anti-capsular polysaccharide antibody concentration, which constitutes a limitation for this study. The primary reason was that the assay measured antibodies against pneumococcal cell wall polysaccharide (C-PS) and anti-capsular PS antibodies [8–10]. This occurred because “purified” capsular PS contains up to 5% (by weight) C-PS, which may be covalently bound to the serotypespecific PS via a peptidoglycan moiety [11]. Additionally, most people have antibodies to C-PS, perhaps in response to pneumococcal carriage or infection. This study shows a carriage rate of S. pneumoniae in children of 39%. Pneumococcal carriage is considered to be an important source of horizontal spread of this pathogen within the community. The rates of nasopharyngeal colonization of S. pneumoniae were found to be 20–40% in healthy children in Japan [12] and as high as 90% in Gambia, Africa [13]. The presence of pneumococcal antigen in urine was found in 26% of the children tested ® with the Binax Now S. pneumoniae Antigen Card. This percentage of positive tests might have been affected by the high carriage rate

Number and percentage of patients

Mean IgG level (␮g/mL)

Positive Negative

224 (38,1%) 364 (61,9%)

74,37 ± 46,61 73,20 ± 42,61

Positive Negative

155 (26.2%) 437 (73.8%)

65.7 ± 40.4 73.0 ± 41.2*

P = 0.038.

Table 6 Proportion of S. pneumoniae carriage according to presence antigen in urine based ® on Binax NOW Sp test. ®

®

Parameters

Binax NOW Sp test positive (n %)

Binax NOW Sp test negative (n %)

Presence of nasopharyngeal carriage of S. pneumoniae Absence of nasopharyngeal carriage of S. pneumoniae

80 (40%)*

118 (60%)

67 (20%)

276 (80%)

*

infection (presence of S. pneumoniae antigen in urine) according ® to Binax NOW Sp test is 26% (168/640 children). Table 4 shows that there is a decrease (p = 0.28) of infection with S. pneumoniae from children aged 1–24 months (varying from 26.3% to 30.4%) to children aged 25–59 months (varying from 17.2 to 17.3%).

3

p ≤ 0.01.

of S. pneumoniae in children, thus reducing the specificity of the test [14,15]. The results obtained in this study show that the presence of S. pneumoniae antigen in urine is significantly higher in carriers than in non-carriers (40% vs. 20%). The absence of any significant difference between the level of antibodies against C-PS according to age and vaccination status suggests a high rate of contact with S. pneumoniae in children either through recurrent infection or carriage. The mean age of vaccinated children is 9.7 ± 8.2 months (Table 1) and 96% of children vaccinated with the PPSV-23 (data not shown) were younger than 2 years old at the time of inclusion in the study, meaning that they did not reach the required age when vaccinated. The PPSV-23 vaccination is recommended in subjects older than 2 years. The safety and efficacy of the PPSV-23 vaccine has not been established in children less than 2 years of age in whom the antibody response may be low. PPSV-23 is not part of the routine vaccination program of Niger, however, vaccination can be done on request normally at the age of 2 years (1st dose) and 3 years (2nd dose) in public health services or private health services. The high level of antibodies observed was probably not related to vaccination, but may be related to contact with the organism by carriage or infection and overestimation of the values associated with the assay method used. Antibodies to capsular PS are highly protective; efforts to develop pneumococcal vaccines have focused on the use of various combinations of the most commonly identified pneumococcal capsular PS as immunogens. The PPSV-23 was introduced in 1983 and this vaccine contains 25 ␮g of each of 12 of the original 14 PS serotypes plus an additional 11 serotypes. Because these PS vaccines were not effective in young children [16], a seven-Valent Pneumococcal Conjugate Vaccine was developed [17,18]. Later, a 13-Valent Pneumococcal Conjugate Vaccine (PCV-13) was developed and approved in many countries worldwide to increase serotype coverage globally [19]. The protective concentration of IgG anti-capsular polysaccharide antibodies measured by ELISA for pneumococcal conjugate (PnC) vaccine in infants recommended by the WHO is a threshold ≥0.35 g/mL, measured one month after primary immunization [20,21]. To better manage respiratory infections in children, it is essential for developing countries that already endure a heavy burden of these infections to understand local epidemiology that will help decide initial treatments. Data from laboratory tests performed in emergencies generally lack specificity to determine the


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exact cause [22] and laboratory diagnostic capacity is not readily available in these countries. Thus, initial treatment is generally ® probabilistic. Rapid diagnostic tests such as Binax NOW Sp can also be very helpful. With the introduction of the PCV-13, a modification of the epidemiology may be expected, hence the necessity for further evaluation studies. It is also necessary for countries to control vaccinations in all health sectors for better respect of protocols. This study shows that S. pneumoniae has high circulation in children either through infection or carriage. After the introduction of the conjugate vaccine in routine vaccination programs in Niger there is need to evaluate specific immunity to circulating serotype in children and evaluate the efficacy of the vaccination and impact on the epidemiology and carriage. Funding This work was directly financed by the TOTAL Corporate Foundation through the TONIRA project. Competing interests None declared. Ethical approval Not required. Acknowledgements We warmly thank the personnel of the Bacteriology–Virology Unit and Epidemiology Unit of CERMES, the personnel of the Pediatric Department of National Reference Hospital Niamey and National Reference Hospital of Lamordé, Niamey for their contributions. References [1] Walker CL, Rudan I, Liu L, Nair H, Theodoratu E, Bhutta ZA, et al. Global burden of childhood pneumonia and diarrhoea. Lancet 2013;381:1405–16. [2] World Health Organization. Pneumonia. Fact Sheet No. 331; 2017. Updated September 2016. www.who.int/mediacentre/factsheets/fs331/en/index.html [Accessed 7 December 2016]. [3] Bogaert D, de Groot R, Hermans PWH. Streptococcus pneumonia colonization: the key to pneumococcal disease. Lancet Infect Dis 2004;4:144–54. [4] Cohen R, Levy C, Bingen E, et al. Nasopharyngeal carriage of children 6 to 60 months during the implementation of the 13-valent pneumococcal conjugate vaccine. Arch Pediatr 2012;19:1132–9.

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