Background
Pertussis (whooping cough) is caused by the vaccine-preventable, bacterial pathogen
Bordetella pertussis [
1,
2]. Despite high vaccine coverage with the whole-cell or acellular vaccines and an initial substantial decrease following vaccine introduction, the incidence of pertussis has increased globally during the last two decades [
3‐
7]. This has been attributed to increased awareness by clinicians, more sensitive molecular techniques for diagnosis [
4,
7], serological markers for identification of infection in adolescents and adults who usually present atypically with
B.
pertussis [
3], pathogen adaptation and/or waning vaccine immunity [
6,
8].
The changing epidemiology of pertussis disease requires new control strategies but establishing the burden of pertussis disease in low-resource settings is challenging due to the absence of effective surveillance systems. As such, pertussis surveillance can potentially be linked to surveillance for other respiratory illnesses, such as influenza or pneumonia surveillance [
9]. Differences in case definitions, however, may result in reduced sensitivity for detecting pertussis cases using these platforms.
Different laboratory methods such as bacterial culture, molecular testing and serology may be used to confirm a clinical diagnosis of pertussis, however, the sensitivity of each method is dependent on the stage of disease and the age of the individual. Culture and molecular testing are recommended for diagnosis during the early (catarrhal and paroxysmal) phases of disease, whereas serology is recommended during the convalescent phase [
10,
11]. The sensitivity of both culture and PCR appear to be highest in young infants and decline in adolescents and adults [
12].
Real-time PCR detection of
B. pertussis is limited by a lack of validated and specific gene targets. The most commonly used PCR target is the multi-copy insertion sequence gene IS
481 (present in 50–250 copies per genome); however, this gene is also found in
B.
holmesii and
B.
bronchiseptica [
13,
14]. Use of a second confirmatory target such as the pertussis toxin subunit gene (
ptxS1) improves specificity of
B. pertussis detection [
7,
15,
16].
The high copy number of IS
481 enables highly sensitive detection of
B. pertussis, however, this results in uncertainty regarding the interpretation of results with high PCR cycle threshold (C
t) values that cannot always be confirmed by detection of a less sensitive, single copy
ptxS1 assay. Multiple copies of IS
481 increase the risk of laboratory contamination and detection of false positives [
17,
18]. Bacterial load in the host and resulting C
t values may be influenced by time between symptom onset and specimen collection, immune status of the host, prior use of antimicrobial therapy or quality of the sample collected [
19‐
21]. The interpretation and clinical relevance of high C
t values, in a setting where stringent laboratory control measures are available to prevent and detect IS
481 contamination, is not clearly understood.
We aimed to evaluate a multi-target real-time PCR assay and the use of Ct value cut-offs for B. pertussis detection and diagnosis in patients with mild or severe respiratory illness at two sentinel sites in South Africa.
Discussion
In the absence of pertussis-specific surveillance in South Africa, we evaluated the utility of real-time PCR detection of
B. pertussis using an existing SRI and ILI surveillance platform. In our setting, laboratory methods were standardised and
B. pertussis laboratory contamination could be excluded. As such, all individuals in whom IS
481 was detected and
B. holmesii,
B. parapertussis and
B. bronchiseptica were excluded, were considered to have
B. pertussis DNA in their respiratory tract at the time of sampling and were therefore considered positive for
B. pertussis infection.
B.
bronchiseptica has been associated with HIV-infected individuals [
26‐
29]; however, this pathogen was not detected in our study. In confirmed cases, the attributable fraction of
B. pertussis detection to respiratory disease was significant for both hospitalised (SRI) and mild (ILI) cases. However, for PCR-possible cases (both SRI and ILI) there was no significant association with
B.
pertussis disease.
In individuals with high C
t values (> 35), low bacterial loads may represent asymptomatic colonisation, or residual DNA post disease, or pertussis disease [
19,
30]. Therefore, the use of stringent C
t value cut-offs may still result in the misdiagnosis of some true pertussis cases given that adults and adolescents have been shown to harbor lower bacterial loads and may present with milder disease or atypical symptoms [
19,
30,
31]. In our study, just over one-third of
B. pertussis PCR-positive cases had low bacterial loads (C
t > 35) and were defined as possible pertussis cases. This is potentially due to the case definition that was not specific for pertussis and the enrollment of individuals of all ages. We also showed that in hospitalised patients, pertussis-possible individuals were more likely to be adults (45–64 years of age) than infants (< 1 year of age) compared with pertussis-confirmed individuals.
In our study, a subset of 17 patients that were PCR positive for
B.
pertussis (confirmed and possible cases) from 2014 were retrospectively interviewed. The majority of confirmed cases reported clinical symptoms consistent with pertussis; however, 50% (4/8) of possible cases also had clinical pertussis. Since this was only a small group of individuals, we are prospectively conducting active case follow-up to understand the association between PCR C
t values and pertussis symptoms. In Canada, Public Health Ontario Laboratories determined the association between PCR C
t with pertussis severity and symptoms, and showed that the proportion of patients with pertussis symptoms did not differ between individuals with low (C
t < 36) and high IS
481 C
t values [
19].
IS
481 is a multi-copy target with up to 250 copies per
B. pertussis genome which increases assay sensitivity, but also increases the risk of false positives due to environmental or laboratory contamination [
17,
18,
32]. As such, different C
t value cut-offs have been recommended when interpreting PCR data and these vary between studies. In a Tunisian study,
B.
pertussis cases were defined as PCR positive for
IS481 and
ptxS1 with a C
t < 45, or as
Bordetella spp. if they were positive for
IS481 only with a C
t < 45 [
7]. The assay currently used by our laboratory detects one genomic equivalent of
B. pertussis at an average C
t of 33.3 [
16]. This, together with variable PCR testing and interpretation algorithms in some settings, supports the recommendation by the authors of using a more stringent IS
481 C
t cut-off (< 35) [
16]. However, the 95% confidence intervals (28.5–38.1) imply that higher C
t values could still indicate positivity and, in a setting where testing procedures are standardised, should not be ignored. Also, C
t values in clinical specimens may vary due to specimen composition or may be affected during storage or transit, and thus may not directly emulate experimental sensitivity in a controlled laboratory environment.
Sequencing confirmed the presence of the IS481 gene from a subset of PCR-positive samples, ruling out non-specific amplification, primer dimer formation and probe degradation, however, one B. pertussis confirmed, and eight possible samples could not be confirmed by sequencing likely due to insufficient DNA concentration for Sanger sequencing. Although only 7% of possible B. pertussis cases tested positive for the ptxS1 gene due to the lower sensitivity of the assay compared to IS481, we excluded B. holmesii from all IS481 PCR-positive samples, suggesting that these were positive for B. pertussis.
Earlier studies have indicated that NP is the preferred specimen type for laboratory detection of
B. pertussis [
10,
33,
34]. The use of sputum for
B. pertussis detection has been suggested by WHO [
34], however, supporting data are lacking. In our study, testing of induced sputum in addition to NP samples increased the diagnostic yield of
B. pertussis 5-fold among hospitalised SRI patients. This is similar to another South African study where the
B.
pertussis detection rate was 3.7% when testing NP specimens and increased to 7% when induced sputum specimens were additionally included [
35].
The World Health Organization has recommended that all countries perform pertussis surveillance and have suggested that for resource-limited settings it can be linked to surveillance for other respiratory illnesses. Due to the lack of pertussis data in South Africa, our aim was to establish pertussis surveillance by linking it to an already established syndromic SRI and ILI surveillance platform. While not pertussis-specific surveillance, this enabled us to obtain pertussis data for the country at a low cost by leveraging systems and staff already in place. An additional benefit of this surveillance is that children and adults with mild and severe respiratory illness that do not necessarily present with typical pertussis symptoms were included, adding to our understanding of pertussis disease in individuals that are not clinically suspected to have pertussis. However, use of syndromic surveillance platforms for pertussis surveillance also has several drawbacks, which need to be acknowledged. As the case definition for ILI cases and SRI adult cases included fever, it is likely that some pertussis cases were missed, and the data likely represents an underestimation of disease prevalence. In addition, the interpretation and clinical relevance of PCR-positive individuals that do not present with typical pertussis symptoms is not well understood. However, despite these limitations, we detected
B. pertussis by PCR in 1.1% of patients with either mild or severe respiratory illness, which correlates with another South African study that enrolled an infant cohort from 2012 to 2014 as part of the Drakenstein Child Health Study in the Western Cape and found an overall pertussis prevalence of 2% among infants presenting with pneumonia [
36].
The study has limitations that need to be considered. The surveillance case definition was not specific for pertussis therefore the time to swabbing following disease onset could not be determined. Overall, there was a small number of pertussis cases, which limited our statistical power for analyses, and as a result, differences between the characteristics of confirmed and possible cases which may exist, may not have been detected. Pertussis vaccination history was not available for adults, and for children < 5 years of age the collected data were poor, so the impact of vaccination on pertussis disease and Ct value could not be determined.
In a controlled setting with standardised procedures and when contamination can conclusively be excluded, we suggest that IS481-positive specimens with Ct values < 40 should be considered indicative of B. pertussis infection. Low IS481 Ct values (< 35) are more likely to represent true cases of pertussis disease, whereas specimens with high Ct values may represent B. pertussis carriage (as B. pertussis was detected in controls with higher Ct values), residual B. pertussis DNA following disease, or active disease especially in older individuals who may present atypically. As such, in a respiratory surveillance setting and for public health reporting, low bacterial loads may not reflect true cases of disease. However, for individual patient management, possible cases should be interpreted in the context of other clinical and epidemiological factors including patient symptoms, age, immune status and epidemiological links with laboratory-confirmed pertussis cases. B. pertussis carriage is not well understood, and further research is required to better understand the significance of PCR detection of B. pertussis in asymptomatic individuals and individuals lacking typical pertussis symptoms.