Introduction

Patients with cystic fibrosis (CF) are plagued by chronic respiratory infections and inflammation leading to progressive bronchiectasis and, ultimately, respiratory failure. Pulmonary disease is the leading cause of morbidity and mortality in CF [1]. The prevalence of each microbial species varies with age. The most prevalent species are Pseudomonas aeruginosa and Staphylococcus aureus. P. aeruginosa is recognized as the main pathogen responsible for the morbidity and mortality of CF patients [2–4]. The prevalence of P. aeruginosa infection increases with age and more than 80% of patients older than 19 years are chronically infected [2, 4–6]. Chronic infections with S. aureus, both methicillin sensitive (MSSA) and methicillin resistant (MRSA) are also on the rise for the CF population as a whole [4]. Early identification of S. aureus is important, as MRSA is associated with an adverse outcome [7, 8]. Therefore, an accurate assessment of lower respiratory tract colonization is critical to inform accurate therapy.

Quarterly respiratory cultures are obtained as standard of care to inform treatment, a practice based on non-evidence based guidelines [4, 9]. Though sputum is preferred over throat culture [10], not all patients are able to spontaneously produce a sputum sample. In fact only 35–40% of pediatric patients produce sufficient sputum to be sent for culture [11, 12]. When patients are unable to produce a sputum sample, clinicians often obtain throat swabs and send them for culture as a surrogate for lower respiratory tract cultures. This practice may be useful in subjects not known to be colonized with P. aeruginosa in order to catch asymptomatic patients with first time colonization, but its clinical utility in the adult CF population is unclear [9, 13]. In fact Armstrong et al. argue that oropharyngeal cultures are poor predictors of pathogens in children with CF [14]. As it stands we don’t know how accurate sputum cultures and throat swabs are compared to the gold standard of bronchoalveolar lavage (BAL). Our aim in this study is to determine the reliability of throat cultures and sputum cultures.

Despite arguments against throat swabs as standard of care in children who cannot produce sputum, others found that throat swabs show excellent concordance with cultures obtained from a bronchoalveolar lavage [11]. In addition, in children <18 months Rosenfeld et al found that compared to BAL, oropharyngeal cultures for P. aeruginosa yield a sensitivity of 44%, specificity of 95%, positive predictive value (PPV) of 44% and negative predictive value (NPV) of 95% [15]. Ramsey et al report a PPV of 83% for P. aeruginosa and a PPV of 91% for S. aureus in non-expectorating subjects <10 years of age [11]. To complicate matters, throat swabs may underestimate bacteria associated with airway inflammation in subjects age 8–21 years [13]. While there are ample data describing the relationship between throat swab and BAL cultures in children, there are minimal data looking at the predictive value of throat swabs and sputum in adult patients with CF. In this study, we sought to determine the positive and negative predictive value of throat and sputum culture for the most common CF organisms.

Materials and Methods

Results

Patient Characteristics

A total of 20 subjects with CF were enrolled in this study. The demographic and genetic attributes of this cohort are listed in Table 1. The median age of the cohort was 27 years. All subjects possessed at least one F508del-CFTR allele.

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Table 1. Patient Characteristics.

https://doi.org/10.1371/journal.pone.0164232.t001

Microbiological Relationship between BAL Fluid Culture, Sputum Culture, and Throat Culture

BAL fluid culture and throat culture were obtained from 20 subjects with CF. Cultures were processed via standard methodology at the DHMC Microbiology Laboratory and within the guidelines of the Cystic Fibrosis Foundation [4]. In all cases, the throat swab was obtained immediately prior to the bronchoscopy. A subset of subjects (n = 12) also had a spontaneously expectorated sputum sample obtained for culture on the same day and prior to the bronchoscopy.

To assess for contamination of BAL fluid by oral flora, in a subset of subjects (n = 3) a scope was with normal saline was performed before the bronchoscope was inserted past the vocal cord. In 2 subjects, standard culture revealed only “normal oral flora” in the scope wash and did not demonstrate a predominant organism. In the third subject, no bacteria were isolated from the scope wash. These data suggest that the culture results obtained in BAL fluid do not reflect contamination of the bronchoscope by oral flora.

We calculated specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV) of throat culture results for the two most common CF pathogens as identified by Cystic Fibrosis Foundation registry data using BAL culture results as the gold standard. Comparing sputum to BAL for P. aeruginosa, we found a sensitivity of 78%, specificity of 100%, PPV of 100%, and NPV of 60%. For S. aureus found in sputum versus BAL we calculated a sensitivity of 100%, specificity of 63%, PPV of 57%, and NPV of 100%. Analyzing throat swab P. aeruginosa compared to BAL, we found a 46% sensitivity, and 100% specificity yielding a PPV of 100% and NPV of 50%. Throat swab compared to BAL for S. aureus showed a sensitivity of 100%, specificity of 23%, PPV of 41%, and NPV of 100%.

We calculated the phi coefficient to measure the degree of association between BAL and throat culture, BAL and sputum culture or throat and sputum culture for both P. aeruginosa and S. aureus (Fig 1). A phi coefficient above 0.7 signifies a strong positive association, between 0.7 and 0.3 indicates a weak positive association and below 0.3 suggests little or no association. The correlations between P. aeruginosa BAL and sputum culture and throat and sputum culture as well as S. aureus throat and sputum culture were statistically significant as determined by Fisher’s Exact Test (p < 0.05). Meanwhile, the correlations between S. aureus BAL and sputum culture and BAL and throat cultures for both pathogens failed to reach statistical significance.

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Fig 1. Comparison of Methods for Determining Bacterial Colonization.

Clinical samples were obtained from CF subjects (n = 20 for BAL and throat swab and n = 12 for sputum). The phi coefficient was used to measure the degree of association between positive and negative cultures for P. aeruginosa and S. aureus in BAL fluid, sputum and throat swabs. Throat swabs and sputum were significantly correlated for both pathogens, as were BAL fluid and sputum for P. aeruginosa (p < 0.05 as determined with Fisher’s Exact Test and indicated by an asterisk).

https://doi.org/10.1371/journal.pone.0164232.g001

Discussion

Our data demonstrate that positive sputum and throat culture findings of P. aeruginosa reflect results found in BAL fluid analysis, suggesting these are reasonable surrogates to determine lung colonization with P. aeruginosa. The diagnostic challenge lies within those subjects that have a negative throat swab or sputum culture as we cannot rely on this to rule out lung colonization as our data found a low negative predictive value for P. aeruginosa in both sputum and throat cultures. In adult subjects with CF, sputum and throat culture findings of S. aureus do not appear to reflect S. aureus colonization of the lung. However, a negative throat swab for S.aureus suggests that the organism is likely not present at all. This is very intuitive as S. aureus is known to colonize the upper airways but this does not necessarily reflect lung colonization.

Obtaining a “standard” culture may lead to identification of a particular organism, but may be subject to differences in competitive growth patterns in vitro versus in vivo. A weakness of our study is the use of conventional culture without using deep sequencing techniques. 16S ribosomal RNA (rRNA) gene sequencing allows for detection of a larger number of potential pathogens. Only a small fraction of all bacteria can be successfully cultured, while clinically significant organisms may be slow-growing, fastidious, inert, or unviable [17, 18]. Standard culture typically only shows the most abundant organism(s), whereas DNA based techniques can detect minor species, which may be just as important in influencing the pathogenicity and potential inflammatory changes [19, 20]. Consistent with prior studies [21], we recently reported that the sputum microbiome is different from the BAL microbiome in a cohort of adult patients with CF [22]. However, standard clinical practice is to perform quarterly cultures for identification of dominant pathogen(s) so our study sought to investigate how reliable throat and sputum cultures are using the standard methodology. Our future investigations will focus on differences in the microbiome using these more advanced techniques.

Our study was powered to investigate differences in identification of the two most common CF pathogens (S. aureus and P. aeruginosa). We recognize that there are many other important pathogens in CF and a potential weakness of our current study was the lack of investigation of these other important bacterial species. However, given that most eradication protocols used clinically are directed at these two most common pathogens [23, 24], early and accurate identification of colonization with S. aureus and P. aeruginosa is critical for the effective management of our patients. In addition, despite adequate power, our study is limited by its small sample size and single site. Our future studies will enroll subjects at multiple sites to increase our sample size and allow investigation of geographic differences. Although the microbiome literature suggests stability of the microbiome over time in an individual patient, it is well described that CF patients can be transiently colonized with specific bacteria. A limitation of our study is that it involves a single visit rather than evaluating serial samples.

An important aspect of our study is that it helps the clinician to identify the optimal sampling technique in a clinical context. The predictive value of throat cultures was thoroughly analyzed by Ramsey et al and they found that in non-expectorating patients less than 10 years of age a positive result for P. aeruginosa was 83% predictive (95% confidence interval 36 to 100%), while S. aureus was 91% predictive (59 to 100%). Negative throat cultures yielded 70% (48 to 86%) for P. aeruginosa and 80% (52 to 96%) for S. aureus [11]. More recently, sputum in children (both induced and spontaneously expectorated) has been compared to throat swab using quantitative PCR concluding that throat swabs may underestimate bacteria associated with airway inflammation [13]. This is consistent with our recent study demonstrating that the microbiome of sputum from CF patients is different from that found in BAL and protected brush samples [22]. Our current data clearly demonstrate that the absence of P. aeruginosa on throat culture is not adequate to determine if P. aeruginosa is present in the lung of stable adult CF patients. Further studies using deep sequencing are needed to identify the accuracy of throat and sputum culture in representing the lung microbiome. In addition, studies involving a larger and more diverse patient population will allow for more generalizable findings. If significant differences between BAL and throat swab cultures are found, then the practice of quarterly surveillance cultures using throat swabs as a surrogate for samples obtained from the lung may be challenged.