Background
Pertussis is one of the top ten fatal infections in children, causing serious and potentially fatal complications, especially in very young infants [
1,
2]. It is a respiratory disease caused by infection with
Bordetella pertussis (B. pertussis). The well-known symptoms of pertussis include repeated paroxysmal cough, inspiratory whoop, and post-cough vomiting [
3]. However, these clinical symptoms are not limited to patients with
B. pertussis infection. Other pathogens such as adenovirus (ADV), influenza virus (IV), and
Mycoplasma pneumoniae (MP) also can cause similar clinical symptoms [
4], collectively known as pertussis-like syndrome.
Pertussis-like syndrome can occur at all ages but is more common in children. It can be very unpleasant for patients, especially young infants and their parents, as symptoms frequently interfere with daily activities and cause significant sleep disturbance. Especially in the paroxysmal stage characterized by spasmodic cough followed by post-tussive whooping and vomiting, the effect of available medications is poor leading to anxiety in parents. It is difficult to distinguish the symptoms of infection with B. pertussis from infection with viruses. In addition, there is a lack of information around the etiology of pertussis-like syndrome worldwide. As such, a greater understanding of the pathogens that cause pertussis-like syndrome is important to inform treatment decision making.
In this study, we aimed to identify the causative pathogens associated with pertussis-like syndrome and to compare clinical presentation between those with B. pertussis and pertussis-like syndrome in children admitted to the Children’s hospital of Soochow university.
Methods
Study design and population
This was a cross-sectional study designed to identify the causative pathogens associated with pertussis-like syndrome. Children with suspected pertussis who were admitted to the Children’s Hospital of Soochow University from March 2016 to September 2018 were enrolled in this study. The clinical criteria for suspected pertussis are cough lasting for > 2 weeks with one or more of the following symptoms: whoop and staccato cough, apneic paroxysm or post-tussive vomiting. The exclusion criteria were historical diagnosis of chronic lung disease, congenital heart disease, immunodeficiency or preterm birth at ≤34 weeks’ gestation. In addition, 91 children admitted to the childcare unit for health examination were enrolled in the control group, including 75 boys and 16 girls. The average age was (0.67 ± 0.58) years old. Routine blood tests and cellular immunity results were collected.
Sample collection
Nasopharyngeal aspirates were obtained from each patient within 18 h after admission using a sterile plastic catheter, which was briefly inserted into the lower pharynx via the nasal cavity. These samples were used for detection of common microorganisms, such as respiratory syncytial virus (RSV), ADV, influenza viruses A and B (IV-A and B), parainfluenza viruses 1, 2, and 3 (Pinf-1, 2, 3), human metapneumovirus (hMPV), human bocavirus (HBoV), human rhinovirus (HRV), MP, and bacteria.
Blood samples were collected immediately after admission for routine tests (Sysmex XS-500i, Hua Sin Science Co., Ltd., Guangzhou, China), liver and kidney function (ADVIA 2400, Siemens Healthineers, America), and cellular immunity.
B. Pertussis detection
B. pertussis DNA was detected in nasopharyngeal aspirates by real-time polymerase chain reaction (PCR). Pathogen detection was achieved using a TaqMan genomic assay and fluorescent real-time PCR (BIO-RAD iCycler, USA).
B. pertussis DNA was determined using two PCR assays, each specific for an independent region of the
B. pertussis genome: (i) the insertion sequence IS481 and (ii) the pertussis toxin S1 (ptxA) promoter region. IS481 is a very sensitive target for screening
B. pertussis because it is the most common insertion sequence, with multiple copies per genome [
5,
6]. PtxA is highly specific but less sensitive because it usually exists as a single or occasionally double copy [
7,
8]. Oligonucleotide primers targeting the ptxA promoter of
B. pertussis (PTp1 and PTp2, Table
1) were used to amplify a 191-bp product, while primers targeting the insertion sequence IS481 of B. pertussis (BP-1 and BP-2MOD, Table
1) were used to amplify a 145-bp product. All primers and probes were purchased from Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). Positive, negative and water blank controls were set for each PCR amplification. The judgement of positive results is generally < 34 cycles. Higher than this number, but with an amplification curve, and the curve is smooth, we would repeat the experiment. If the results of the two experiments were the same, a positive result was recorded, otherwise negative. B. pertussis infection was defined as IS481 PCR positive, whether ptxA PCR is positive or not.
Table 1Primers and probes used for duplex real-time PCR
ptxA-Pr | PTp1 | CCAACGCGCATGCGTGCAGATTCGTC |
ptxA-Pr | PTp2 | CCCTCTGCGTTTTGATGGTGCCTATTTTA |
IS481 | BP-1 | GATTCAATAGGTTGTATGCATGGTT |
IS481 | BP-2MOD | TGGACCATTTCGAGTCGACG |
Other microbiological analyses
Ten viruses and bacteria (including MP) were also tested in nasopharyngeal aspirates. Bacteria were tested by inoculating nasopharyngeal aspirates on blood plates that were read after incubating for 18–20 h. Bacterial growth was > 103 colony forming units/ml was considered significant. Viruses including RSV, ADV, IV-A and B and Pinf-1, 2, 3 were investigated by immunofluorescence tests using D3 Ultra™ Respiratory Virus Screening and LD Kit (Diagnostic Hybrids, Ohio, USA). A positive result was defined as over five inclusion bodies analyzed under a fluorescence microscope. MP and the various viruses were examined by PCR (Nucleic acid amplification fluorescent reagent kit, Ann Gene Co., Guangdong, China) according to the manufacturer’s instructions.
Cellular immunity
Venous blood was anticoagulated by heparin and antibodies were added to whole blood (anti-CD3+ and anti-CD19+ labeled with cyanobacteria-5 antibody, anti-CD4+, anti-CD23+ and anti-CD25+ labeled with fluorescein isothiocyanate, and anti-CD8+ antibody labeled with algal red protein). After mixing, the cells were incubated in a dark chamber and lysed with hemolysin, followed by further incubation in a dark chamber. Flow cytometry was performed using a BECKMAN COULTER flow cytometer and a specific kit (Immunotech, France).
Clinical data collection
Detailed demographic and clinical data such as age, gender, pertussis vaccination status, previous antibiotic consumption, family history of pertussis, and clinical symptoms were obtained from questionnaires that were answered by patients’ parents and from medical records.
Statistical analysis
Enumeration data are expressed as mean ± standard deviation (SD). Measurement data are expressed as a percentage (%). Statistical analysis was performed using SPSS 18.0 software (SPSS Inc., Chicago, IL); the rank sum test was used for comparison among groups and one-way analysis of variance was used for comparison. P < 0.05 was considered statistically significant.
Discussion
Pertussis is a series of respiratory symptoms caused by infection with
B. pertussis, including paroxysmal fits of coughing, vomiting after coughing and deep inspirium. The intermittent symptoms of coughing can last for more than 28 days and up to 3 months [
9]. However, in clinical practice, we found that other pathogens can also cause pertussis-like cough as part of pertussis-like syndrome. Stephanie Saiki-Macedo found that in Peru, there was a high prevalence of ADV,
B. pertussis, MP and IV-B infection in patients with a probable diagnosis of pertussis [
10]. In our study, detailed etiological examination was performed in 281 children who had typical pertussis-like syndrome. About half of the children were
B. pertussis positive, and about a quarter of the children had infections with other pathogens. No pathogens were detected in around a quarter of the children tested. Our study found that, in addition to
B. pertussis, the most common causative pathogen in children with pertussis-like syndrome children was MP, which was detected in more than one third of children. In addition, the rate of HRV, SP, HI and Pinf-3 infections was also high in pertussis-like syndrome.
Amal Al Maani compared pertussis and pertussis-like illness in Oman and found there was no significant difference in clinical criteria between them [
11]. In our study, no children had pertussis encephalopathy or pulmonary arterial hypertension and there was no significant difference in other clinical manifestations between the pertussis-like group and the
B. pertussis group. Spastic cough was more common in spring and summer and nearly half of these children had B.
pertussis. However, more than 75% of children had B.
pertussis infection in the winter. Therefore, the occurrence of whooping cough in winter should raise suspicion of
B. pertussis infection.
Previous studies showed that leukocytosis, especially lymphocytosis was associated with mortalities and morbidities in patients with pertussis infection [
12,
13]. In our study, the absolute lymphocyte count in the
B. pertussis group was higher than the pertussis-like group, but with no significant difference. However, the absolute neutrophil count and blood platelet count in the B.
pertussis group was significantly higher than in the pertussis-like group and control group. This may be associated with the time of onset. Joshua C. Eby studied the relationship between pertussis bacillus and neutrophils and found that the recruitment of neutrophils is suppressed by the pertussis toxin early in the course of the disease, but after the first week, the combined activities of pertussis toxin, lipooligosaccharide and adenylate cyclase toxin result in production of cytokines that generate an IL-17 response, promoting neutrophil recruitment [
14]. In our study, most children with pertussis had been ill for more than a week before being hospitalized. This could explain the significant increase of neutrophils in the children with
B. pertussis infection in our study. As for the relationship between pertussis and platelets, the study results are controversial. Pertussis toxin shows distinct early signalling events in platelet-activating factors leading to thrombocytosis and platelet activation through interaction with platelet glycoprotein Ib [
15,
16]. However, other studies showed that platelet aggregation is suppressed by the
B. pertussis adenylate cyclase toxin through increased intracellular cyclic adenosine monophosphate (cAMP), and this dysfunction resulted in hemorrhage [
17]. In our study, children infected with
B. pertussis had a significantly higher number of blood platelets, which may be related to the overactive immune inflammatory response following
B. pertussis infection.
MP is one of the most common causes of community-acquired pneumonia in children. In older children, MP infection can cause lobular pneumonia with clinical manifestations including high fever and cough. However, in young children, chest radiographs often showed interstitial inflammation which mainly manifested as cough and wheezing. Many studies showed that
MP can cause persistent cough [
18,
19]. Wang K found that MP is an important infection in children with persistent cough but the course is shorter than pertussis [
20], which is similar to our results. As both
B. pertussis and MP are mainly treated with macrolide drugs like erythromycin or azithromycin, children with persistent cough or spasmodic cough can be treated empirically with these drugs in clinical practice.
The current study has some limitations. First, the sample size is still small. In the next stage, we will continue to enroll more children with pertussis-like syndrome and follow up them to explore the long-term prognosis of these children. Second, there are still limitations in pathogen detection. Our test did not include all the pathogen that can cause pertussis-like syndrome, like
Bordetella parapertussis, which accounts for 10–14.9% of
Bordetella [
21]. We will detect more kinds of pathogens including
Bordetella parapertussis later
.
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