Pneumococcal nasopharyngeal carriage among Bhutanese children hospitalized with clinical pneumonia: serotypes and viral co-infection

Streptococcus pneumoniae is a common cause of invasive bacterial disease (IBD), including pneumonia, meningitis and sepsis in children under five years of age. Although the prevalence of pneumococcal associated IBD and deaths has declined in the last two decades, mainly as a result of the parsimonious global introduction of pneumococcal conjugate vaccines (PCV), this pathogen still causes a significant burden. In 2015, S. pneumoniae was estimated to cause 8.9 million cases of clinical pneumonia in children aged 1 to 59 months, and 294,000 deaths among HIV-uninfected children aged 1 to 59 months. The majority of these deaths (81%) were attributed to pneumonia [1]. The vast majority of the pneumococcal burden is now concentrated in low- and middle-income countries (LMIC). In 2015, approximately 50% of all pneumococcal deaths were registered in four countries: India, Nigeria, the Democratic Republic of the Congo, and Pakistan [1].

Pneumococcal nasopharyngeal colonization (PNC) is generally considered a prerequisite for pneumococcal disease and is a source of spread between people [2, 3]. However, S. pneumoniae is part of the commensal nasopharyngeal flora and in most instances, PNC does not lead to disease [3]. While the association between PNC and development of acute otitis media is well recognised, its relationship with pneumonia is less strongly established [4, 5]. Other bacteria and viruses are common colonizers of the nasopharynx. The interplay between respiratory viruses and S. pneumoniae on the progression to the disease is still poorly understood [6, 7].

By December 2019, 145 countries had introduced PCV into their national immunization programme and 15 additional countries were planning to do so [8]. Currently, there are two WHO prequalified vaccines that are commonly used: PCV13 (Prevenar 13®, Pfizer) and PCV10 (Synflorix®, GlaxoSmithKline) that include 13 and 10 serotypes respectively [9, 10]. A third vaccine that also includes 10 serotypes (Pneumosil®, Serum Institute of India) was recently prequalified by WHO in December 2019 [9]. The introduction of PCV has substantially reduced both the burden of pneumococcal invasive disease and the rates of PNC by serotypes included in the vaccine [1, 11‐14]. The emergence of serotypes not included in the vaccines has been well documented in high-income countries but little is known in LMIC [15‐17], and surveillance data at a national level are important to identify serotype replacement and to assess most prevalent serotypes still circulating in the population.

Bhutan is a small country landlocked in the Himalayas, with an estimated population of 779,666 in 2017 [18, 19]. It is currently classified as a lower-middle income country [20]. The Constitution guarantees free essential health services for Bhutanese citizens, based on a primary health care approach [21]. Similar to other LMIC, pneumonia remains a major public health challenge in Bhutan, whereby the number of outpatient visits and hospitalizations attributed to pneumonia constitutes a considerable burden to the health system [22]. The conjugate Haemophilus influenza type b (Hib) vaccine has been routinely administered since 2011, and PCV13 was introduced in the immunization programme in January 2019 [23, 24]. Despite the burden that pneumonia represents for the country, there are scarce national data on the epidemiology and aetiology of childhood pneumonia, leading to challenges while implementing effective national preventive strategies [25]. Furthermore, while information on the pneumococcal serotypes circulating before the introduction of PCV13 is essential to monitor the impact of the vaccine, there are no data on circulating pneumococcal serotypes among the Bhutanese population [25].

We conducted this prospective study to determine the prevalence of pneumococcal carriage and to identify the pneumococcal serotypes circulating among Bhutanese children under five years of age admitted with WHO-defined pneumonia, before the introduction of PCV in the country. We also aimed to contribute to the understanding of the interplay between PNC and viral co-infections among this population.

Among the 189 children recruited in the RIBhuC study between 1st July 2017 and 30th June 2018 [26], NPW was collected in 129 children (68.3%). After microbiological screening for virus, there was insufficient sample for pneumococcal testing in seven children, and one sample showed an inhibited finding, leaving 121/189 cases (64.0%) for analysis (Fig. 1).

We found a prevalence of PNC of 62.8%, which is comparable to that of other developing countries before the introduction of PCV [6, 17, 41]. Similar studies in India found a PNC prevalence ranging between 35 and 75% [41, 42]. We found no published data on PNC in children admitted with clinical pneumonia from other neighbouring countries. We used PCR to detect PNC, which is more sensitive than culture, and thereby which could lead to a higher prevalence as compared to pneumococcal carriage studies that used culture. However, the recent multicentric study conducted in nine settings over eight developing and emerging countries also used PCR and found an overall PNC prevalence of 68.1% among children admitted with suspected pneumonia, similar to our study [41]. As expected, prior administration of antibiotics in our study appeared to reduce pneumococcal carriage detection [17, 43].

The most prevalent serotypes were 7B/7C/40 (indistinguishable by laboratory technique) identified in 43.4% of the children, followed by 6A/B (15.8%), 14 (11.8%), 23F (7.9%), 3 and 1 (6.6% each). This serotype distribution is rather different from that described in similar studies. We found a much higher proportion of children with the serotype 7B/7C/40, while the proportion of children we identified with the serotypes 19F, 6A or 6B was considerably lower than in similar studies (proportion of serotypes in children sick with respiratory symptoms) [7, 17, 41, 42, 44] or among healthy children in community-based carriage studies [43, 45‐48]. In the neighbouring context of India, 6A/B and 19F were also found to be the most prevalent serotypes in children admitted with clinical pneumonia, together with serotypes 14 and 23F. In a systematic review that included Indian studies looking at prevalence of serotype distribution among children with invasive pneumococcal disease prior to introduction of PCV, the most prevalent serotypes in decreasing order were 14, 1, 19F, 6B, 5, 6A, 9 V and 23F [49]. However, these were isolated from normally sterile sites such as blood, cerebrospinal fluid or pleural fluid. Data from other neighbouring countries are scarce, except for some data on pneumococcal serotype distribution among healthy children in Nepal [50, 51]. In our study, 19F and 6A/B were identified in 4/76 (5.3%) and 12/76 (15.8%) of the children, respectively. The proportion of the serotypes 14 and 23F, however, was similar to other studies. Serotype 1 has been identified as an important cause of highly invasive pneumococcal disease and is atypically found in carriage studies of healthy children [49, 52]. In our study, serotype 1 was the fifth most common serotype (together with serotype 3) identified, which is in line with findings of PNC in children sick with pneumonia.

PCV13, which was introduced in Bhutan after the end of the study, could potentially prevent the infection by at least one serotype in up to 57.9% of the children recruited in this study. Similar studies including two from India showed incongruences in these estimates, reporting PCV13 coverage ranging between 32 and 84% [7, 41, 42]. However, this is in line with the different serotype distribution we described above, as the most commonly identified serotypes (19F, 6A and 6B) in other studies are covered by PCV13, while the commonly identified serotypes 7B/7C/40 in our study are not.

Bacterial pneumonia secondary to viral respiratory infections such as influenza have been previously well characterized, and S. pneumoniae is the most common bacteria involved [53]. The nasopharynx is colonised by many bacteria and viruses and while the interaction between respiratory viruses and S. pneumoniae is likely to play a crucial role on the progression to the disease, this is a less well understood area [6, 7]. In the present study, respiratory viruses were detected in similar proportion of children with and without PNC. However, when looking at specific viral infections, children with PNC were less likely to be co-infected with rhinovirus (28.6% versus 47.5%; p = 0.046), and there was a trend of higher proportion of co-infection with RSV among colonized children (50.0% versus 32.5%; albeit non statistically significant, p = 0.075). Positive association of pneumococcal colonisation with influenza, RSV, adenovirus and rhinovirus have been found by previous studies, but findings have not been consistent [54‐56]. Differences between studies might be explained by different pneumococcal serotype distribution as well as different circulation of viruses depending on seasonality and local epidemiology. One of the best understood and well documented synergistic viral-bacterial interaction is that of the influenza virus, identified as a risk factor for the acquisition, colonization and development of pneumonia due to S. pneumoniae [53, 55, 57‐59]. Conversely in our study, presence of influenza virus was not related with PNC. This might be due to the small number of children with influenza and by the fact that this is a cross-sectional study with collection of NPW specimen at the time of admission, while bacterial superinfection is often sequential, occurring a few days after the viral episode.