Introduction
Community-acquired pneumonia (CAP) remains a major cause of childhood morbidity and mortality worldwide [
1]. Its clinical microbiology has considerably changed because molecular methods allow the detection of a wide range of pathogens directly from respiratory specimens with high sensitivity [
2]. Therefore, the choice of sample type and sampling method is critical for enhancing the predictive value of these molecular methods [
3].
Owing to the convenience of specimen collection, the diagnosis of most respiratory pathogens is performed using aspirated sputum or even upper respiratory secretions such as nasal and pharyngeal swabs [
4]. Data show that these specimens are susceptible to oral colonisation, making it difficult to determine whether sputum isolates indicate infection, colonisation, or contamination [
4]. Fiberoptic bronchoscopy (FOB) with bronchoalveolar lavage (BAL) is now considered an important tool for the diagnosis and treatment of pneumonia. Although BAL fluid (BALF) is a reliable specimen for the identification of lower respiratory tract infection pathogens, identification via bronchoscopy has proven challenging in children owing to the need for anaesthesia and specialised procedural expertise [
5]. Few studies have compared the differences in bacterial detection between sputum and BALF; however, studies comparing the two specimens in detecting viruses as well as atypical bacteria are scarce [
5,
6]. According to the Infectious Diseases Society of America and the American Thoracic Society, viruses and atypical bacteria (e.g.,
Mycoplasma pneumoniae) account for a large proportion of CAP pathogenesis in children [
7]. Therefore, it is important to assess the prevalence of a wide range of pathogens in relatively accessible sputum specimens compared with alveolar lavage fluid specimens.
To the best of our knowledge, there are no reports describing the differences in the simultaneous detection of several viruses and atypical bacteria by multiplex polymerase chain reaction (PCR) between induced sputum with BALF specimens. Here, we compare the identification pf these two types of specimens for nine viruses and two atypical bacteria in children with CAP receiving BAL. This approach enables more rigorous assessment of the molecular results of different respiratory specimens in the detection of multiple pathogens.
Discussion
In this study, we used multiplex PCR to detect nine viruses and two atypical bacteria in paired sputum and BALF samples from 142 children hospitalised with CAP treated with BAL. There was slight, moderate to perfect agreement for these pathogens tested. As we know, sputum is the main specimen used in hospitalised patients in China due to its easy availability [
14]. However, sputum specimens can be easily contaminated by pathogens present in the upper respiratory tract [
15]. Alveolar lavage via fiberoptic bronchoscopy (FOB) is now a useful tool for the diagnosis and treatment of lung infections [
16]. Compare to sputum, BALF specimens are much less likely to be contaminated with oral microorganisms [
17]. As FOB is an invasive procedure, the possibility of using sputum rather than BALF is an issue that needs to be addressed, particularly in paediatric practice. To date, only a few reports have compared certain pathogenic results between BALF and other respiratory specimens in paediatric patients [
6,
18‐
21].
Adenovirus infection can cause severe CAP and is associated with acute respiratory distress syndrome (ARDS) or atelectasis, with a mortality rate of over 50% in children [
22,
23]. The persistence of adenovirus infection has been described as a possible cause of unremitting airway obstruction [
24]. In the present study, we used multiplex-PCR analysis and the false-negative rate for adenovirus detection in sputum compared with BALF was close to 70%. Wang et al. observed a false negative rate of 58.4% for adenovirus detection by NPS compared to paired BALF in children with severe CAP [
25]. These results suggest that sputum samples are occasionally not suitable for identifying the causative agent of lower respiratory tract infections, even when molecular methods are applied. Early identification of adenovirus infections from the lower respiratory tract and timely and effective treatment are important for severe CAP children in order to prevent progression of the disease.
In detecting
M. pneumoniae nucleic acid, our previous study and others reports demonstrated the superiority of sputum over nasopharyngeal swabs (NPS) or nasopharyngeal aspirate (NPA) [
20,
21,
26‐
28]. Luo et al. measured 533 paired NPA-BALF samples collected from children with pneumonia and found moderate concordance (κ = 0.407) for
M. pneumoniae [
21]. Xu et al. performed real-time PCR on 406 NPA and BALF samples from children with CAP and found a kappa value of only 0.020 to detect
M. pneumoniae [
20].To our knowledge, no article has compared the detection of
M. pneumoniae between induced sputum (IS) and BALF in pediatric patients. In recent years, there has been an increasing incidence of severe Mycoplasma pneumoniae pneumonia (SMPP) and refractory MPP (RMPP) in children, and the formation of mucus plug in SMPP or RMPP is a major indication for BAL [
29]. In our study, the positivity of
M. pneumoniae was highest in children received BAL, at approximately 40%. Comparison of BALF and sputum showed almost perfect agreement, with kappa value close to 0.9, with false negative and false positive rates being approximately 5%. These data suggested that if
M. pneumoniae has been detected in IS, repeated testing on if from BALF samples is of little significance. Similar to
M. pneumoniae, other viruses (with the exception of adenovirus) showed good concordance. Therefore, sputum can be used as an alternative to BALF to detect
M. pneumoniae, influenza virus, bocavirus and RSV if the purpose of patients undergoing FOB is diagnostic rather than therapeutic. Sputum can be used to detect these pathogens in children who exhibit contraindications to BAL or in children with CAP who are otherwise unable to obtain BALF.
We found that the inconsistency rate of sputum and BALF was significantly associated with age, which was higher in younger children. Rodrigues et al. observed that co-infection and carriage rates in children were independent of age [
30]. Verhagen et al. found that viral co-infection was more frequent in children under 4 years of age than in older children [
31]. Using a combination of clinician-ordered diagnostics and lower respiratory mNGS, Tsitsiklis et al. observed a decrease in positive detection rates with increasing age [
32]. These findings can be explained by a lack of intact immune memory, reduced innate and adaptive immunity, and physiological differences in the airway, which may increase the susceptibility of children or infants to incidental carriage of potentially pathogenic microorganisms [
33]. It is therefore important to select the appropriate specimen type for younger children to improve the detection accuracy on respiratory tract pathogens.
In the present study, we observed a total of 21 cases with bacterial pneumonia, of which 6 were sputum(+)/BALF(+), 9 were sputum(+)/BALF(-), 2 were sputum(-)/BALF(+) and 4 were sputum(-)/BALF(-). Of them, the proportion of inconsistent results is twice as high as the proportion of consistent results with adenovirus predominating. Ronda et al. observed increased bacterial (
S. aureus and GNB) colonization during viral respiratory tract infections, which may be a contributing factor to the increased risk of bacterial pneumonia [
34]. Du et al. showed that, 48.8% of the children (163/216) with severe adenovirus pneumonia had bacterial coinfection [
35]. Lai et al. found that HMPV-infected mice showed impaired recruitment of airway neutrophils, which may lead to delayed bacterial clearance and increased inflammation in the lung [
36]. Therefore, when a prior viral upper respiratory infection is suspected, it is prudent to consider whether the culprit of the pneumonia is a virus or a bacterium.
Limitations
This study has several limitations. First, although multiplex PCR requires nominal fluorescence to determine a positive result, this method cannot be used to distinguish whether the detected pathogen is a current infection or a colonised pathogen. In addition, it is important to note that viruses take longer to shed in the upper respiratory tract than in the lower respiratory tract [
37]. Future comparative studies that include the viral load measurement in a large sample size is needed. Secondly, eight patients in this study were positive for adenovirus in sputum, whereas only three patients were positive for adenovirus in BALF. Future comparative studies are needed to specifically address the differences in adenovirus detection. Third, although we kept the resorbed volume above 40% of the injected volume, the dilution of BALF may lead to missed detection of low-load pathogens. Forth, most CAP patients do not require bronchoscopy, and our comparative results were limited to patients with severe CAP, not mostly encountered CAP. Finally, although we tested most of the known pathogens causing respiratory symptoms, we cannot exclude the possibility that variants or unknown pathogens were missed.
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