Unlocking New Insights in Pneumococcal Carriage Detection: Nasopharyngeal vs. Saliva Samples/Exploring non-invasive sampling methods for pneumococcal carriage detection in children

Posted on November 7, 2023   by Anne Wyllie

Anne Wyllie takes us behind the scenes of their latest publication 'Saliva as an alternative sample type for detection of pneumococcal carriage in young children' published in Microbiology.

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Anne Wyllie and Annemarie Kuipers

My name is Anne Wyllie, I am a Principal Investigator and Research Scientist at the Yale School of Public Health, USA. My lab group is working to elevate saliva as a sample type for both infectious disease diagnostics and the community surveillance of respiratory pathogens. Saliva is an ideal sample type for these purposes as it is non-invasive, we have demonstrated that it can be reliably self-collected by the patient or study participant, and it holds potential for a wide range of respiratory pathogens.

Streptococcus pneumoniae, also known as pneumococcus, is a bacterium that is a common resident of the human nasopharynx. It can also cause various respiratory infections, such as otitis media and pneumonia, or even more severe conditions like meningitis and septicaemia. Children typically carry pneumococcus more frequently and for longer than adults, meaning that they are considered the main source of transmission of pneumococcus through the community. As such, understanding the prevalence of pneumococcus among children is important for informing and evaluating vaccination strategies.

The gold standard sample type for detecting pneumococcal carriage is the nasopharyngeal (NP) swabs. While detection has traditionally been through culture of NP swabs, studies are showing that the sensitivity of detection can be increased when swabs are instead tested with qPCR. NP swabs, however, are an invasive sample type which require a trained healthcare professional to collect the sample. With the discomfort they can cause and the resources they require, they are not well suited for the repeated sampling of individuals. Saliva, on the other hand, is a non-invasive sample which can be reliably self-collected - or in the instance of small children - by their parents.

During my PhD studies, my supervisor and senior author of this paper Krzysztof Trzcinski, was revisiting the early literature on Streptococcus pneumoniae (pneumococcus). He discovered that in the early 1900s, saliva was used for studying the presence of pneumococcus in healthy individuals (this is termed ‘carriage’ which contrasts to ‘infection’ or ‘disease’ in sick individuals). However, in more recent times, carriage studies have been conducted using NP swabs. Based on this finding, he wondered how these sample types differed in their ability to detect pneumococcal carriage: present day studies using NP swabs, typically found <5% of older adults carried pneumococcus compared to the 1900s studies using saliva, which typically found around 50% of adults carrying pneumococcus. We first explored the possibility of using saliva for pneumococcal carriage detection, collecting samples from local Dutch primary school students. From those promising results, we tested it more extensively for carriage detection in adults and indeed found that saliva was far more sensitive than NP swabs for detecting pneumococcal carriage in adults.

Having demonstrated that testing saliva of adults using qPCR significantly improves detection of pneumococcus, we wondered how this would compare with children, when they are already frequent carriers. Therefore, in the current study we compared the detection of pneumococcus in asymptomatic 24-month-old children when using NP swabs and saliva and testing the samples by both culture and qPCR.

Culture of NP swabs revealed pneumococcal carriage in 57% of the study participants, but detection was not possible from saliva due to abundant polymicrobial growth on culture-plates. Testing the same samples with qPCR increased the number of children pneumococci-positive to 73% when testing nasopharyngeal samples, but only 60% tested positive when their saliva was tested. Despite this difference, 11% of children were positive exclusively in their saliva samples, meaning that testing saliva contributed significantly to the overall number of carriers detected.

While in this study, testing NP swabs with qPCR proved to still be the ‘best’ or ‘most sensitive’ sample type for pneumococcal carriage detection in toddlers, it’s important to recognize that saliva still performed well. As such, there is still potential for application of this sample type in carriage detection in children. First, if collected alongside NP swabs, saliva can significantly enhance carriage detection, identifying more carriers overall. In the absence of NP swabs, the benefits of saliva collection could possibly outweigh its reduced sensitivity. First, saliva reduces participant discomfort which can overcome the increasing testing aversion that we’re seeing to swabs. This is important for longitudinal studies should one wish to study carriage and/or infection dynamics or transmission between individuals, such as in households or daycare centres. Being less resource-intensive to collect (not requiring a healthcare worker and requiring only a simple, low-cost, laboratory plastic tube to collect the sample in), the cost-saving benefits could permit more overall samples to be collected, while still at a reduced cost. More samples could facilitate more data points to overcome the reduced sensitivity. This approach could be invaluable, providing a more sustainable approach for surveillance efforts related to vaccination programmes.

This study is just one in a series of research studies and literature reviews that we have conducted where we challenge the status quo of the NP as the gold standard for respiratory commensal and pathogen detection. Exploring alternative and lower-burden sampling methods like saliva, can support greater research efforts in which research funding can go further while providing a better study experience for study participants. As such, this study not only enhances our understanding of pneumococcal carriage but also underscores the value of adaptable and less intrusive approaches in the field of microbiology and how they can be used to either complement or diversify more traditional approaches. It is important to note however, that when sampling saliva, both collection and testing methods are very important - if not done so properly this can significantly impact sensitivity. As witnessed through the pandemic, a method that works for swabs, isn’t guaranteed to work on saliva. Saliva is a more heterogeneous and complex sample type and alternative processing methods may be required.

We, and others who have been working to develop and optimize these methods for working with saliva, are generally very happy to share experiences and protocols to help the successful implementation of these approaches in your own research settings. Should you wish to explore saliva for your own research applications, do not hesitate to get in touch.

Thumbnail image: a stylised DNA helix comprised of pneumococcus, representing the molecular detection of pneumococcus. Credit: Anne Wyllie and Annemarie Kuipers