ABSTRACT
Many Legionella pneumonia patients do not produce sputum, and it is unknown whether purulent sputum is required for the identification of Legionella species. This study aimed to evaluate the identification rate of Legionella species based on sputum quality and the factors predictive of Legionella infection. This study included Legionella pneumonia patients at Kurashiki Central Hospital from November 2000 to December 2022. Sputum quality, based on gram staining, was classified as the following: Geckler 1/2, 3/6 and 4/5. Geckler 4/5 was defined as purulent sputum. The sputa of 104 of 124 Legionella pneumonia patients were cultured. Fifty-four patients (51.9%) were identified with Legionella species, most of which were Legionella pneumophila serogroup 1 (81.5%). The identification rates of Legionella species according to sputum quality were 57.1% (16/28) in Geckler 1/2 sputum, 50.0% (34/68) in Geckler 3/6 sputum, and 50.0% (4/8) in Geckler 4/5 sputum, which were not significantly different (P = 0.86). On multivariate analysis, pre-culture treatment with anti-Legionella antimicrobials (odds ratio [OR] 0.26, 95% confidence interval [CI] 0.06–0.91), Pneumonia Severity Index class ≥IV (OR 2.57 [95% CI 1.02–6.71]), and intensive care unit admission (OR 3.08, 95% CI 1.06–10.09) correlated with the ability to identify Legionella species, but sputum quality did not (OR 0.88, 95% CI 0.17–4.41). The identification rate of Legionella species in non-purulent sputum was similar to that in purulent sputum. For the diagnosis of Legionella pneumonia, sputum should be collected before administering anti-Legionella antibiotics and cultured regardless of sputum quality.
KEYWORDS: diagnosis, Legionella pneumonia, predictive factor, purulent sputum, sputum culture
INTRODUCTION
Legionella pneumonia reportedly accounts for 1% to 10% of cases of community-acquired pneumonia (CAP) (1–5). In fact, the percentage increases by 10% to 15% in severe CAP, and Legionella species are one of the important causative pathogens of CAP following Streptococcus pneumoniae (6–8). Delay of administration of anti-Legionella antimicrobials for Legionella pneumonia is reported to be a risk factor for poor prognosis (9), suggesting that early diagnosis of Legionella pneumonia and its treatment by appropriate antibiotics is essential.
Globally, the urinary antigen test (UAT) is widely used for diagnosing Legionella pneumonia (10–13), due to the simplicity of the procedure and rapid results. However, since the Legionella UAT has high specificity (99%) but only moderate sensitivity (74%) (14), the possibility of Legionella pneumonia infection should not be denied based on negative UAT results. The main reason for false-negative results with the existing Legionella UAT is the inability to identify Legionella species other than L. pneumophila serogroup (SG) 1. Therefore, culture of lower respiratory tract specimens, including sputum, is needed for detecting all SGs of L. pneumophila, including SG1 and other Legionella species in cases with both positive and negative UAT results.
Although culture of Legionella species from a respiratory specimen is the diagnostic gold standard (15), it is known to have certain drawbacks, including the fact that it needs a specific culture medium, such as buffered charcoal yeast extract-α (BCYE-α) and Wadowsky-Yee-Okuda-α (WYO-α), and takes about 3–5 days to identify the Legionella species (10). In addition, previous reports showed that 50% to 70% of Legionella pneumonia patients do not produce sputum (16–18). We have also often experienced the inability to collect purulent sputum from Legionella pneumonia patients. For identification of the causative microorganisms in sputum culture, appropriate collection and culture of purulent sputum is essential, as far as possible. However, since there is much variability in the production of sputum as a symptom and the quality of sputum, whether purulent, in Legionella pneumonia cases, the detection power of respective sputum purulence in Legionella pneumonia is unknown.
The aim of the present study was to evaluate the occurrence of sputum purulence and detection ability of Legionella species based on sputum quality, in addition to factors predictive of the identification of Legionella species in Legionella pneumonia patients.
MATERIALS AND METHODS
Study population
Legionella pneumonia patients diagnosed at Kurashiki Central Hospital, a 1,166-bed tertiary hospital, from November 2000 to December 2022 were retrospectively enrolled in this study. Patients who had abnormal shadows on radiologic examinations, along with at least one symptom, such as fever, cough, sputum, chest pain, and general malaise, and one clinical finding (abnormal auscultation findings or an increased inflammatory reaction) were diagnosed with pneumonia (19). Patients who were <15 years old and those with hospital-acquired pneumonia were excluded. This study was approved by the institutional review board of our hospital (IRB number 4168). The IRB waived the need for obtaining patient informed consent due to the retrospective nature of the study.
Study design
We investigated the clinical characteristics of Legionella pneumonia patients, including age, sex, comorbidities, symptoms, pre-culture antibiotic treatment before admission, and severity of pneumonia, including CURB-65 score [confusion, urea >7 mmol/L, respiratory rate ≥30 breaths per minute, low blood pressure (systolic <90 mmHg or diastolic ≤60 mmHg), and age ≥65 years] (20), Pneumonia Severity Index (PSI) [calculated using the total score of age, sex, resident in nursing home or not, five comorbidities, five physical examination findings, six laboratory findings, and one radiological finding] (21), A-DROP score [age ≥70 y in men and ≥75 y in women, dehydration or blood urea nitrogen ≥21 mg/dl, SpO2 ≤90%, disturbance in orientation, and systolic blood pressure ≤90 mmHg] (22), and prognosis, in addition to sputum quality and culture results. Legionella pneumonia was diagnosed in patients who satisfied at least one of the following criteria: positive results of Legionella UATs, identification of Legionella species in lower respiratory tract sputum samples, positive results of Legionella gene tests, or 4-fold increase in paired serum antibody testing. In clinical practice at our hospital, Binax NOW Legionella (Abbott Diagnostics Medical, Lake Forest, CA, USA) (Binax) was used from December 2004 to July 2016, and Immunocatch Legionella (Eiken Kagaku Corporation, Tokyo, Japan) has been used since July 2016 for the diagnosis of Legionella infection. Since June 2019, Libotest Legionella (Kyokutou Corporation, Tokyo, Japan) (Libotest) has also been used in addition to Immunocatch Legionella.
In daily clinical practice, sputum culture using WYO-α, gene testing by loop-mediated isothermal amplification (LAMP) (Eiken Kagaku Corporation, Tokyo, Japan) of sputum, and serum antibody tests were performed at the discretion of the attending physicians.
Evaluation of sputum quality and sputum culture method
Sputum quality was evaluated by experienced microbiological test engineers using Geckler’s classification (23), in which gram-stained sputum smears are rated as Geckler classes 1–6, based on the number of buccal squamous epithelial cells and leukocytes (Geckler 1, buccal squamous epithelial cells > 25 and leukocytes < 10; Geckler 2, buccal squamous epithelial cells > 25 and leukocytes 10–25; Geckler 3, buccal squamous epithelial cells > 25 and leukocytes > 25; Geckler 4, buccal squamous epithelial cells 10–25 and leukocytes > 25; Geckler 5, buccal squamous epithelial cells < 10 and leukocytes > 25; and Geckler 6, buccal squamous epithelial cells < 25 and leukocytes < 25). In the present study, we defined Geckler 4/5 as purulent sputum and the classes other than Geckler 4/5 as non-purulent sputum, as previously described (24). For sputum culture, we used WYO-α medium and acid pre-treatment of sputum to decrease bacterial contamination of the sputum from patients suspected to have Legionella pneumonia.
Study outcomes
The primary outcome was the identification rate of Legionella species in each sputum quality class according to Geckler’s classification. The secondary outcome was the identification rate of Legionella species in terms of pneumonia severity, based on CURB-65, PSI, and A-DROP scores. CURB-65 ≥3 points, PSI ≥ class IV, and A-DROP ≥3 points were defined as severe pneumonia (20–22). In addition, we evaluated factors predictive of the identification of Legionella species on sputum culture.
Statistical analysis
Continuous variables are expressed as medians and interquartile ranges, and categorical variables are expressed as numbers and percentages. Categorical variables were analyzed by Fisher’s exact test, and continuous variables by the non-parametric Mann-Whitney U-test. To evaluate the identification ability according to sputum quality, we divided sputum into the following three quality groups: Geckler 1/2, 3/6, and 4/5. Univariate analysis was performed to identify predictive factors in the identification of Legionella species on sputum culture. Multivariate analysis using stepwise logistic regression analysis was conducted for all variables that were found to have a P value of ≤ 0.05 on univariate analysis, in addition to sputum quality and pre-culture treatment using anti-Legionella antibiotics. All tests were two-tailed, and a P value of < 0.05 was considered significant. All statistical analyses were performed using EZR statistical software (version 3.0.3, Vienna, Austria) (25).
RESULTS
Patients’ baseline characteristics
A total of 124 Legionella pneumonia patients, including 119 hospitalized patients and 5 outpatients, were evaluated. Figure 1 shows the results of Legionella UAT, culture, sputum gene analyses by LAMP, and serum antibodies. Among the 124 Legionella pneumonia patients, sputum for culture was obtained from 104 patients (83.9%). The sputum culture positivity rate was 51.9% (54/104), and 14 among the 18 patients with negative UAT results were diagnosed by sputum culture (Fig. 1). The baseline clinical characteristics of Legionella pneumonia patients with and without the identification of Legionella species by sputum culture are shown in Table 1. Median patient age was 68 years, and 87.5% were male. The most common comorbidity was diabetes mellitus, followed by chronic heart disease. The most common symptom was fever (89.4%), followed by cough (39.4%) and relative bradycardia (39.4%). Forty-six patients (53.5%) without sputum as a symptom were identified with Legionella by sputum culture. LAMP was performed in 10 of the 104 patients, among whom 7 patients (70%) showed positive results. Regarding the positivity rate following LAMP in terms of sputum quality, the LAMP positive rate was 100% (1/1) in purulent sputum and 66.7% (6/9) in non-purulent sputum.
Fig 1.
Study flowchart, the number of positive results, and the results of diagnostic tests, including urinary antigen tests, sputum cultures, gene tests, and serum antibodies, among the 124 Legionella pneumonia patients are shown. LAMP, loop-mediated isothermal amplification; SG, serogroup; UAT, urinary antigen test.
TABLE 1.
Baseline clinical characteristics of Legionella pneumonia patients with positive and negative sputum culture resultsb,c
| All patients n = 104 |
Positive sputum culture n = 54 |
Negative sputum culture n = 50 |
P value | |
|---|---|---|---|---|
| Age (y) | 68 [60–76] | 66 [58–73] | 70 [65–78] | 0.043 |
| Male | 91 (87.5) | 51 (94.4) | 40 (80.0) | 0.037 |
| Smoking status | ||||
| Current +past | 83 (79.8) | 43 (79.6) | 40 (80.0) | 0.706 |
| Comorbidities | ||||
| Chronic obstructive pulmonary disease | 8 (7.7) | 3 (5.6) | 5 (10.0) | 0.477 |
| Diabetes mellitus | 30 (28.8) | 18 (33.3) | 12 (24.0) | 0.387 |
| Chronic heart disease | 19 (18.3) | 12 (22.2) | 7 (14.0) | 0.318 |
| Malignancy | 7 (6.7) | 4 (7.4) | 3 (6.0) | 1.000 |
| Chronic renal disease | 8 (7.7) | 6 (11.1) | 2 (4.0) | 0.273 |
| Chronic liver disease | 8 (7.7) | 5 (9.3) | 3 (6.0) | 0.717 |
| Cerebrovascular disease | 14 (13.5) | 5 (9.3) | 9 (18.0) | 0.254 |
| Symptoms | ||||
| Fever | 93 (89.4) | 46 (85.2) | 47 (94.0) | 0.096 |
| Cough | 41 (39.4) | 18 (33.3) | 23 (46.0) | 0.226 |
| Sputum | 18 (17.3) | 8 (14.8) | 10 (20.0) | 0.604 |
| Dyspnea | 24 (23.1) | 17 (31.5) | 7 (14.0) | 0.061 |
| Headache | 15 (14.4) | 7 (13.0) | 8 (16.0) | 0.781 |
| Abdominal pain | 0 (0) | 0 (0) | 0 (0) | NA |
| Diarrhea | 10 (9.6) | 5 (9.3) | 5 (10.0) | 1.000 |
| Arthralgia | 4 (3.8) | 3 (5.6) | 1 (2.0) | 0.619 |
| Myalgia | 2 (1.9) | 1 (1.9) | 1 (2.0) | 1.000 |
| Mental disturbance | 34 (32.7) | 17 (31.5) | 17 (34.0) | 0.834 |
| Vital signs | ||||
| Temperature (°C) | 38.9 [38.0–39.5] | 39.0 [38.0–39.5] | 38.7 [38.0–39.4] | 0.571 |
| Heart rate (beats/min) | 100 [90–114] | 105 [86–121] | 99 [91–110] | 0.307 |
| Relative bradycardia | 41 (39.4) | 19 (35.2) | 22 (44.0) | 0.424 |
| Systolic blood pressure (mmHg) | 136 [120–156] | 137 [120–156] | 133 [119–152] | 0.925 |
| Laboratory findings | ||||
| C-reactive protein (mg/L) | 250.6 [185.7–320.0] | 269.4 [201.3–337.3] | 230.0 [152.9–288.4] | 0.048 |
| Albumin (g/dL) | 2.9 [2.4–3.3] | 2.9 [2.4–3.2] | 2.9 [2.5–3.3] | 0.643 |
| Aspartate aminotransferase (U/L) | 52 [29–158] | 57 [30–181] | 51 [28–150] | 0.683 |
| Alanine aminotransferase (U/L) | 40 [21–67] | 41 [22–63] | 39 [20–71] | 0.943 |
| Lactate dehydrogenase (U/L) | 317 [227–489] | 307 [226–539] | 335 [240–443] | 0.940 |
| Blood urea nitrogen (mg/dL) | 21 [15–29] | 21 [14–34] | 21 [15–26] | 0.592 |
| Creatinine (mg/dL) | 1.02 [0.82–1.52] | 1.15 [0.86–2.04] | 0.98 [0.78–1.27] | 0.031 |
| Sodium (mmol/L) | 133 [130–138] | 133 [129–136] | 134 [131–139] | 0.271 |
| White blood cells (×103 /µL) | 11.0 [8.6–14.4] | 11.3 [9.5–14.7] | 10.5 [7.8–13.8] | 0.332 |
| Platelets (×104 /µL) | 17.2 [12.7–21.8] | 15.8 [12.0–20.0] | 18.5 [13.1–24.5] | 0.080 |
| Pre-treatment with anti-Legionella antibiotics | 15 (14.4) | 5 (9.3) | 10 (20.0) | 0.164 |
| Severity of pneumonia | ||||
| CURB-65 (points) | 0.961 | |||
| 0 | 6 (5.8) | 4 (7.4) | 2 (4.0) | |
| 1 | 35 (33.7) | 18 (33.0) | 17 (34.0) | |
| 2 | 32 (30.8) | 17 (31.5) | 15 (30.0) | |
| 3 | 23 (22.1) | 11 (20.4) | 12 (24.0) | |
| 4 | 8 (7.7) | 4 (7.4) | 4 (8.0) | |
| 5 | 0 (0) | 0 (0) | 0 (0) | |
| CURB-65 ≥ 3 points | 31 (29.8) | 15 (27.8) | 16 (32.0) | 0.673 |
| PSI (points) | 99 [86–125] | 105 [96–124] | 95 [78–128] | 0.176 |
| PSI (class) | 0.006 | |||
| I | 2 (1.9) | 1 (1.9) | 1 (2.0) | |
| II | 10 (9.6) | 1 (1.9) | 9 (18.0) | |
| III | 21 (20.2) | 9 (16.7) | 12 (24.0) | |
| IV | 49 (47.1) | 33 (61.1) | 16 (32.0) | |
| V | 22 (21.2) | 10 (18.5) | 12 (24.0) | |
| PSI class ≥IV | 71 (68.3) | 43 (79.6) | 28 (56.0) | 0.012 |
| A-DROP (points) | 0.509 | |||
| 0 | 12 (11.5) | 4 (7.4) | 8 (16.0) | |
| 1 | 35 (33.7) | 20 (37.0) | 15 (30.0) | |
| 2 | 25 (24.0) | 15 (27.8) | 10 (20.0) | |
| 3 | 23 (22.1) | 11 (20.4) | 12 (24.0) | |
| 4 | 8 (7.7) | 3 (5.6) | 5 (10.0) | |
| 5 | 1 (1.0) | 1 (1.9) | 0 (0) | |
| A-DROP ≥3 points | 32 (30.8) | 15 (27.8) | 17 (34.0) | 0.529 |
| ICU admission | 38 (36.5) | 28 (51.9) | 10 (20.0) | 0.001 |
| ICU admission within 24 hours of admission | 26 (25.0) | 19 (35.2) | 7 (14.0) | 0.014 |
| Late ICU admissiona | 12 (11.5) | 9 (16.7) | 3 (6.0) | 0.126 |
| 30-day mortality | 6 (5.8) | 3 (5.6) | 3 (6.0) | 1.000 |
| In-hospital mortality | 7 (6.7) | 3 (5.6) | 4 (8.0) | 0.708 |
Late ICU admission means ICU admission more than 24 h after admission.
A-DROP: age ≥70 years in men or ≥75 years in women, blood urea nitrogen ≥21 mg/dL or dehydration, oxyhemoglobin saturation measured by pulse oximetry ≤90% or partial pressure of oxygen in arterial blood ≤60 mmHg, confusion, and systolic blood pressure ≤90 mmHg; CURB-65: confusion, urea >7 mmol/L, respiratory rate ≥30 breaths/min, low blood pressure (systolic <90 mmHg or diastolic ≤60 mmHg), and age ≥65 y; ICU: intensive care unit; NA: not assessed; PSI: Pneumonia Severity Index.
Data are shown as numbers (%) or medians and interquartile range.
Distribution of Legionella species and Legionella pneumophila serogroups identified by sputum culture
The Legionella species identified by sputum culture is shown in Fig. 2. The most common Legionella species was L. pneumophila SG1 (81.5%), followed by L. pneumophila SG3 (11.1%). Non-L. pneumophila SG1 was identified in 18.5% of Legionella pneumonia patients.
Fig 2.
Distribution of Legionella species and Legionella pneumophila serogroups in sputum culture.
Ability to identify Legionella species by sputum culture in terms of sputum quality
Figure 3 shows the identification rate of Legionella infection in the respective sputum quality groups. Although a few patients had purulent sputum (8/104, 7.7%), most patients did not (96/104, 92.3%). Legionella species were most identified in Geckler 3/6 sputum (34/54, 63.0%), followed by Geckler 1/2 (16/54, 29.6%) and 4/5 (4/54, 7.4%) sputa. Regarding the identification rate of Legionella species, there were no significant differences in terms of sputum quality (P = 0.86). In patients with purulent sputum, Streptococcus pneumoniae was cultivated in only one patient (1/8), whereas no significant pathogens were cultivated in the others, including in four patients identified with Legionella species.
Fig 3.
Identification rate of Legionella species according to sputum quality in the 104 Legionella pneumonia patients. Almost all the patients (92.3%) had non-purulent sputum, and a few patients (7.7%) had purulent sputum. The identification rate of Legionella species was not significantly different in each sputum quality group (P = 0.86).
Ability to identify Legionella species by sputum culture according to pneumonia severity
Table 2 shows the identification rate of Legionella species in non-severe and severe pneumonia groups according to CURB-65, PSI, and A-DROP scores. There was a significant difference in the ability to identify Legionella according to the severity of pneumonia determined using PSI, but not with CURB-65 and A-DROP scores.
TABLE 2.
| All patients n = 104 | Patients identified with Legionella infection n = 54 |
Identification rate of Legionella species in each pneumonia severity group % |
P value | |
|---|---|---|---|---|
| CURB-65 (points) | 0.673 | |||
| 0–2 | 73 (70.2) | 39 (72.2) | 53.4 | |
| 3–5 | 31 (29.8) | 15 (27.8) | 48.4 | |
| PSI (class) | 0.012 | |||
| I-III | 33 (31.7) | 11 (20.4) | 33.3 | |
| IV-V | 71 (68.3) | 43 (79.6) | 60.6 | |
| A-DROP (points) | 0.529 | |||
| 0–2 | 72 (69.2) | 39 (72.2) | 54.2 | |
| 3–5 | 32 (30.8) | 15 (27.8) | 46.9 |
Data are shown as numbers (%).
A-DROP: age ≥70 years in men or ≥75 years in women, blood urea nitrogen ≥21 mg/dL or dehydration, oxyhemoglobin saturation measured by pulse oximetry ≤90% or partial pressure of oxygen in arterial blood ≤60 mmHg, confusion, and systolic blood pressure ≤90 mmHg; CURB-65: confusion, urea >7 mmol/L, respiratory rate ≥30 breaths/min, low blood pressure (systolic <90 mmHg or diastolic ≤60 mmHg), and age ≥65 y; PSI: Pneumonia Severity Index.
Predictive factors for identifying Legionella species by sputum culture
Sputum purulence was not a significant predictive factor in both univariate and multivariate analyses. In multivariate analysis, pre-culture treatment with anti-Legionella antibiotics was a negative predictive factor (odds ratio [OR] 0.26 [95% confidence interval (CI) 0.06–0.91], P = 0.044), whereas PSI class ≥IV (OR 2.57 [95% CI 1.02–6.71], P = 0.048) and intensive care unit (ICU) admission within 24 h after admission (OR 3.08 [95% CI 1.06–10.09], P = 0.048) were both positive predictive factors for the identification of Legionella species (Table 3).
TABLE 3.
Predictive factors in the identification of Legionella species by sputum culturea
| Univariate analysis | Multivariate analysis | |||
|---|---|---|---|---|
| Odds ratio (95% CI) |
P value | Odds ratio (95% CI) |
P value | |
| Age | 0.97 [0.94–1.00] | 0.099 | ||
| Sex | 4.25 [1.21–19.90] | 0.036 | ||
| PSI class ≥IV | 3.07 [1.31–7.52] | 0.011 | 2.57 [1.02–6.71] | 0.048 |
| ICU admission within 24 h | 3.33 [1.30–9.38] | 0.015 | 3.08 [1.06–10.09] | 0.048 |
| Pre-treatment with anti-Legionella antibiotics | 0.41 [0.12–1.25] | 0.127 | 0.26 [0.06–0.91] | 0.044 |
| Sputum quality | 1.22 [0.61–2.48] | 0.569 | 0.88 [0.17–4.41] | 0.877 |
CI, confidence interval; ICU, intensive care unit; NA, not assessed; PSI, Pneumonia Severity Index.
Regarding the correlation between the identification rate of Legionella species and interval between sputum collection and administration of anti-Legionella antimicrobials, the identification rate was not inferior in patients in whom sputum was sampled within 24 h after administering anti-Legionella antibiotics compared with patients who did not receive antibiotic therapy for Legionella pneumonia [75.0% (6/8) vs 54.2% (47/87)]. However, the identification rate of Legionella species decreased steeply and was significantly lower if the sputum was collected more than 24 h after administering anti-Legionella antibiotics, compared with patients without pre-treatment and with pre-treatment by anti-Legionella antibiotics within 24 h (55.8% vs 11.1%, p = 0.04) (Fig. 4).
Fig 4.
Identification rate of Legionella species by sputum culture relative to time after the administration of anti-Legionella antibiotics The identification rate of Legionella species was 54.2% (47/87) in patients without pre-culture antibiotic treatment using anti-Legionella antibiotics, 75.0% (6/8) in patients within 24 h after the administration of anti-Legionella antibiotics, 16.7% (1/6) in patients within 24 to 48 h after the administration of anti-Legionella antibiotics, and 0% (0/3) in patients more than 48 h after the administration of anti-Legionella antibiotics.
DISCUSSION
The present study showed that most Legionella pneumonia patients had non-purulent sputum, and the identification rate of Legionella species by sputum culture did not differ significantly between purulent and non-purulent sputa. Regarding the correlation between the identification rate of Legionella species and pneumonia severity, the identification rate was higher in severe pneumonia patients, defined by PSI, than in non-severe patients. In addition, antibiotic treatment for Legionella pneumonia before sputum sampling was a negative predictor, and severe pneumonia defined by PSI and ICU admission within 24 h after admission were positive predictors in the identification of Legionella species by sputum culture.
Many Legionella pneumonia patients have non-purulent sputum or sputum cannot be obtained for culture (4). Therefore, most attending physicians usually use UAT kits for the diagnosis of Legionellosis in Japan and many other countries (10–13). The main disadvantage of the existing UAT kits is their inability to diagnose Legionella pneumonia due to non-Legionella pneumophila serogroup 1 (26). In February 2019, Libotest Legionella (Kyokutou Corporation, Tokyo, Japan), which can detect all Legionella pneumophila serogroups, was launched in Japan. We previously reported that Libotest Legionella had similar diagnostic ability compared with existing UAT kits, including BinaxNOW and Q line, in addition to the ability to detect all serogroups of Legionella pneumophila (18). However, since the sensitivity of Libotest Legionella for diagnosing Legionellosis due to non-L. pneumophila SG1 is unknown and there is a substantial need for surveillance of Legionella species in Legionella pneumonia patients, performance of sputum culture for the diagnosis of Legionella pneumonia will continue to be important. In the present study, sputum culture was performed in 83.9% of Legionella pneumonia patients, and Legionella species were identified in almost half of them, although only 17.5% of the patients had sputum as a symptom. In addition, the identification rate of Legionella species was not significantly different between purulent and non-purulent sputa (50.0% vs 52.1%, p = 1.00). Cunha et al. reported that the sputum of Legionella pneumonia patients had less neutrophils and was watery (10). According to these studies, including that of our study, efforts should be made to collect sputum for the diagnosis of Legionella pneumonia even in patients without sputum as a symptom.
Two previous studies have evaluated the correlation between sputum quality and the culture results of Legionella species in Legionella pneumonia patients (27, 28). Ingram et al. investigated the correlation between sputum quality and culture results in 19 patients with Legionella pneumonia due to L. pneumophila (27). They reported that L. pneumophila was identified the most in Geckler six sputum (n = 7), followed by Geckler one sputum (n = 4), and most patients (78.9%) had non-purulent sputum (Geckler 1, 2, 3, and 6) (27). Another study by Shakeshaft et al. also showed that 46 of the 72 culture-positive Legionella pneumonia patients (63.9%) had non-purulent sputum, as defined by Murray and Washington criteria (28). The authors of these two studies concluded that all sputum samples, including non-purulent sputum, should be submitted for Legionella culture when Legionella pneumonia is suspected. The results of their studies were very significant for diagnosing Legionella pneumonia, although there were some possible limitations. First, only patients with identification of Legionella species by sputum culture were included in both studies. Therefore, the distribution of sputum quality and identification rate of Legionella species in each sputum quality group of Legionella pneumonia patients were unknown. Second, Ingram’s study included a relatively small number of patients (n = 19) (27), whereas most of the 72 culture-positive Legionella pneumonia patients included in Shakeshaft’s study were due to L. longbeachae (65.3%) (28). Therefore, our study is the first to evaluate the distribution of sputum quality and identification rate of Legionella species, including L. pneumophila and other species, according to sputum quality in over a hundred Legionella pneumonia patients, including more than 90% of L. pneumophila cases.
Regarding the correlation between pneumonia severity and the identification rate of Legionella species, our study showed that the identification rate was significantly higher in severe cases than in non-severe cases, as defined by PSI. There are several possible reasons for this observation. First, a previous report indicated that the sensitivity of Legionella UATs is higher in severe disease cases than in non-severe cases (29). According to that study, severe patients might have a greater load of Legionella species in sputum compared with urine. Second, it is possible that the attending physicians made a greater effort to collect sputum in severe pneumonia patients. However, in the present study, the collection rate of sputum was high (83.9%), and the collection rate of sputum was almost the same in PSI I-III and IV-V cases (75.0% vs 88.8%, data not shown). Therefore, sputum collection bias is not likely to have affected the results.
We also reported that administration of anti-Legionella antimicrobials before sputum evaluation was a negative predictor of the identification of Legionella species, and severe pneumonia, defined as PSI class ≥IV and ICU admission within 24 h after admission, was a positive predictive factor in the identification of Legionella species by sputum culture. A previous report showed that the sensitivity of sputum culture in diagnosing pneumococcal pneumonia decreased after antibiotic administration (93% without pre-culture antibiotics vs 74% with pre-culture antibiotics) (30). Another study by Mentasti et al. reported that the identification rate of Legionella species by sputum culture was higher in sputum collected within 2 days after admission compared with sputum collected more than 2 days after admission (79.6% vs 47.8%) (31). According to these reports, pre-culture antibiotic treatment with anti-Legionella antimicrobials might lead to a reduction in the amount of Legionella species observed in sputum. Indeed, our study showed that the identification rate of Legionella species significantly reduced from 24 h after administering anti-Legionella antibiotics. Musher et al. reported that the positive sputum culture rate for S. pneumoniae clearly decreased from 24 h after administering antibiotics compared with the rate within 24 h of antibiotic administration (28.6% in ≥24 h, 88.9% in 6–24 h, 77.8% in <6 h, and 93% without antibiotics) (30). In addition, despite a report of three cases of Legionnaires’ disease showing the correlation between positivity in polymerase chain reaction (PCR) tests and antibiotic treatment, Korosec et al. reported that Legionella amplicon intensity was highest in sputum or bronchial aspirates collected at or before the start of antibiotic therapy and decreased markedly within 3 days of antibiotic therapy (32). Therefore, sputum collection for culture should preferably be performed before or within 24 h of anti-Legionella antimicrobial administration, although treatment delay for sputum collection is not recommended. In terms of the correlation between identification rate and pneumonia severity, previous studies reported that the identification rate was higher in severe pneumonia compared with non-severe pneumonia cases, as previously mentioned (29). This is the first study to show that the identification rate was significantly higher in severe pneumonia, including cases requiring ICU admission within 24 h, than in non-severe pneumonia using multivariate analysis.
Regarding the diagnosis of Legionella pneumonia, gene tests, including PCR and LAMP, are essential for diagnosing Legionella pneumonia in patients with negative UAT results in daily clinical practice. Indeed, a previous report showed that a larger number of Legionella pneumonia patients could be diagnosed using both LAMP and UAT (33). However, PCR and LAMP could not detect the details of Legionella pneumophila SG and Legionella species. Therefore, performing sputum culture for Legionella species identification is likely to be significant for surveillance in individual areas and countries.
There are some limitations to the present study. First, this study was conducted retrospectively at a single center in Japan. Patients’ symptoms, including sputum, might have been underestimated due to its retrospective nature. Second, the attending physicians’ efforts in collecting sputum might have affected the diagnosis of Legionella pneumonia. Legionella pneumonia might have been underdiagnosed because the attending physicians did not collect sputum in pneumonia patients with negative Legionella UAT results because of the absence of sputum production as a symptom. Third, sputum samples were not obtained from 20 patients who were diagnosed with UATs. However, since the percentage of such patients was relatively small (16.1%), it is not likely to have affected the study results. Finally, since the prevalence of Legionella pneumonia and Legionella species varies between areas and countries, similar studies are needed in other countries as well. Additionally, since many Legionella pneumonia patients might be underdiagnosed due to negative UAT results, we believe that collection and culture of sputum for diagnosing Legionella pneumonia is important even in places with a low prevalence of Legionella. A strength of the present study is that it was relatively large, including 124 Legionella pneumonia patients, among whom sputum was collected from 104 patients. In addition, sputum quality was examined by experienced microbiological test engineers, and this is the first study to evaluate the identification rate of Legionella species in terms of sputum quality.
In conclusion, most Legionella pneumonia patients in this study had non-purulent sputum, and the identification rate of Legionella species was comparable between non-purulent and purulent sputum. These results suggest that regardless of sputum quality, sputum should be collected and cultured within 24 h of the administration of anti-Legionella antimicrobials in cases of suspected Legionella pneumonia.
ACKNOWLEDGMENTS
The authors would like to thank Akie Ohmori, Hiroyuki Fujii, and other microbiological test engineers for performing the sputum culture.
A.I. served as the principal author, had full access to all the study data, and takes responsibility for the integrity and accuracy of the data and data analysis. A.I., T.I., H.T., Y.N., M.K., A.Y., and Y.W. contributed to the study conception and design and to the acquisition of data. A.I., T.I., and H.T. contributed to the analysis and interpretation of data. A.I., T.I., H.T., Y.N., M.K., A.Y., and Y.W. contributed to drafting and revision of the manuscript and approval of the final version to be submitted for consideration for publication.
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. The authors report no potential conflicts of interest with any companies/organizations whose products or services might have been discussed in this article.
Contributor Information
Akihiro Ito, Email: ai12306@kchnet.or.jp.
Nathan A. Ledeboer, Medical College of Wisconsin, Milwaukee, Wisconsin, USA
DATA AVAILABILITY
The data sets used and analyzed during the current study are available from the corresponding author upon reasonable request.
REFERENCES
- 1. Ishida T, Hashimoto T, Arita M, Ito I, Osawa M. 1998. Etiology of community-acquired pneumonia in hospitalized patients: a 3-year prospective study in Japan. Chest 114:1588–1593. doi: 10.1378/chest.114.6.1588 [DOI] [PubMed] [Google Scholar]
- 2. Saito A, Kohno S, Matsushima T, Watanabe A, Oizumi K, Yamaguchi K, Oda H, Study Group . 2006. Prospective multicenter study of the causative organisms of community-acquired pneumonia in adults in Japan. J Infect Chemother 12:63–69. doi: 10.1007/s10156-005-0425-8 [DOI] [PubMed] [Google Scholar]
- 3. Lim WS, Macfarlane JT, Boswell TC, Harrison TG, Rose D, Leinonen M, Saikku P. 2001. Study of community acquired pneumonia aetiology (SCAPA) in adults admitted to hospital: implications for management guidelines. Thorax 56:296–301. doi: 10.1136/thorax.56.4.296 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. von Baum H, Ewig S, Marre R, Suttorp N, Gonschior S, Welte T, Lück C, Competence Network for Community Acquired Pneumonia Study Group . 2008. Community-acquired Legionella pneumonia: new insights from the German competence network for community acquired pneumonia. Clin Infect Dis 46:1356–1364. doi: 10.1086/586741 [DOI] [PubMed] [Google Scholar]
- 5. Cillóniz C, Ewig S, Polverino E, Marcos MA, Esquinas C, Gabarrús A, Mensa J, Torres A. 2011. Microbial aetiology of community-acquired pneumonia and its relation to severity. Thorax 66:340–346. doi: 10.1136/thx.2010.143982 [DOI] [PubMed] [Google Scholar]
- 6. Ishiguro T, Takayanagi N, Yamaguchi S, Yamakawa H, Nakamoto K, Takaku Y, Miyahara Y, Kagiyama N, Kurashima K, Yanagisawa T, Sugita Y. 2013. Etiology and factors contributing to the severity and mortality of community-acquired pneumonia. Intern Med 52:317–324. doi: 10.2169/internalmedicine.52.8830 [DOI] [PubMed] [Google Scholar]
- 7. Arancibia F, Cortes CP, Valdés M, Cerda J, Hernández A, Soto L, Torres A. 2014. Importance of Legionella pneumophila in the etiology of severe community-acquired pneumonia in Santiago, Chile. Chest 145:290–296. doi: 10.1378/chest.13-0162 [DOI] [PubMed] [Google Scholar]
- 8. Ishida T, Tachibana H, Ito A, Tanaka M, Tokioka F, Furuta K, Nishiyama A, Ikeda S, Niwa T, Yoshioka H, Arita M, Hashimoto T. 2014. Clinical characteristics of severe community-acquired pneumonia among younger patients: an analysis of 18 years at a community hospital. J Infect Chemother 20:471–476. doi: 10.1016/j.jiac.2014.04.005 [DOI] [PubMed] [Google Scholar]
- 9. Heath CH, Grove DI, Looke DF. 1996. Delay in appropriate therapy of Legionella pneumonia associated with increased mortality. Eur J Clin Microbiol Infect Dis 15:286–290. doi: 10.1007/BF01695659 [DOI] [PubMed] [Google Scholar]
- 10. Cunha BA, Burillo A, Bouza E. 2016. Legionnaires’ disease. Lancet 387:376–385. doi: 10.1016/S0140-6736(15)60078-2 [DOI] [PubMed] [Google Scholar]
- 11. Mercante JW, Winchell JM. 2015. Current and emerging Legionella diagnostics for laboratory and outbreak investigations. Clin Microbiol Rev 28:95–133. doi: 10.1128/CMR.00029-14 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Miyashita N, Aoki Y, Kikuchi T, Seki M, Tateda K, Higa F, Maki N, Uchino K, Ogasawara K, Watanabe A. 2016. Questionnaire survey results on diagnosis and treatment of Legionella. Japanese society of chemotherapy Legionella therapeutic drug evaluation committee. Jpn J Chemother 64:66–75 (in Japanese). [Google Scholar]
- 13. Kinjo T, Ito A, Ishii M, Komiya K, Yamasue M, Yamaguchi T, Imamura Y, Iwanaga N, Tateda K, Kawakami K. 2022. National survey of physicians in Japan regarding their use of diagnostic tests for legionellosis. J Infect Chemother 28:129–134. doi: 10.1016/j.jiac.2021.12.008 [DOI] [PubMed] [Google Scholar]
- 14. Shimada T, Noguchi Y, Jackson JL, Miyashita J, Hayashino Y, Kamiya T, Yamazaki S, Matsumura T, Fukuhara S. 2009. Systematic review and Metaanalysis: Urinary antigen tests for Legionellosis. Chest 136:1576–1585. doi: 10.1378/chest.08-2602 [DOI] [PubMed] [Google Scholar]
- 15. Fields BS, Benson RF, Besser RE. 2002. Legionella and Legionnaires’ disease: 25 years of investigation. Clin Microbiol Rev 15:506–526. doi: 10.1128/CMR.15.3.506-526.2002 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Fernández-Sabé N, Rosón B, Carratalà J, Dorca J, Manresa F, Gudiol F. 2003. Clinical diagnosis of Legionella pneumonia revisited: evaluation of the community-based pneumonia incidence study group scoring system. Clin Infect Dis 37:483–489. doi: 10.1086/376627 [DOI] [PubMed] [Google Scholar]
- 17. Miyashita N, Higa F, Aoki Y, Kikuchi T, Seki M, Tateda K, Maki N, Uchino K, Ogasawara K, Kiyota H, Watanabe A. 2017. Clinical presentation of Legionella pneumonia: evaluation of clinical scoring systems and therapeutic efficacy. J Infect Chemother 23:727–732. doi: 10.1016/j.jiac.2017.09.001 [DOI] [PubMed] [Google Scholar]
- 18. Ito A, Yamamoto Y, Ishii Y, Okazaki A, Ishiura Y, Kawagishi Y, Takiguchi Y, Kishi K, Taguchi Y, Shinzato T, Okochi Y, Hayashi R, Nakamori Y, Kichikawa Y, Murata K, Takeda H, Higa F, Miyara T, Saito K, Ishikawa T, Ishida T, Tateda K. 2021. Evaluation of a novel urinary antigen test kit for diagnosing Legionella pneumonia. Int J Infect Dis 103:42–47. doi: 10.1016/j.ijid.2020.10.106 [DOI] [PubMed] [Google Scholar]
- 19. Mandell LA, Wunderink RG, Anzueto A, Bartlett JG, Campbell GD, Dean NC, Dowell SF, File Jr TM, Musher DM, Niederman MS, Torres A, Whitney CG, Infectious Diseases Society of America, American Thoracic Society . 2007. Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults. Clin Infect Dis 44:S27–S72. doi: 10.1086/511159 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. Lim WS, van der Eerden MM, Laing R, Boersma WG, Karalus N, Town GI, Lewis SA, Macfarlane JT. 2003. Defining community-acquired pneumonia severity on presentation to hospital: an international derivation and validation study. Thorax 58:377–382. doi: 10.1136/thorax.58.5.377 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Fine MJ, Auble TE, Yealy DM, Hanusa BH, Weissfeld LA, Singer DE, Coley CM, Marrie TJ, Kapoor WN. 1997. A prediction rule to identify low-risk patients with community-acquired pneumonia. N Engl J Med 336:243–250. doi: 10.1056/NEJM199701233360402 [DOI] [PubMed] [Google Scholar]
- 22. Miyashita N, Matsushima T, Oka M, Japanese Respiratory Society . 2006. The JRS guidelines for the management of community-acquired pneumonia in adults: an update and new recommendations. Intern Med 45:419–428. doi: 10.2169/internalmedicine.45.1691 [DOI] [PubMed] [Google Scholar]
- 23. Geckler RW, Gremillion DH, McAllister CK, Ellenbogen C. 1977. Microscopic and bacteriological comparison of paired sputa and transtracheal aspirates. J Clin Microbiol 6:396–399. doi: 10.1128/jcm.6.4.396-399.1977 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24. Van Scoy RE. 1977. Bacterial sputum cultures. a clinician's viewpoint. Mayo Clin Proc 52:39–41. [PubMed] [Google Scholar]
- 25. Kanda Y. 2013. Investigation of the freely available easy-to-use software 'EZR' for medical statistics. Bone Marrow Transplant 48:452–458. doi: 10.1038/bmt.2012.244 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26. Phin N, Parry-Ford F, Harrison T, Stagg HR, Zhang N, Kumar K, Lortholary O, Zumla A, Abubakar I. 2014. Epidemiology and clinical management of Legionnaires’ Disease. Lancet Infect Dis 14:1011–1021. doi: 10.1016/S1473-3099(14)70713-3 [DOI] [PubMed] [Google Scholar]
- 27. Ingram JG, Plouffe JF. 1994. Danger of sputum purulence screens in culture of Legionella species. J Clin Microbiol 32:209–210. doi: 10.1128/jcm.32.1.209-210.1994 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28. Shakeshaft MK, Murdoch DR. 2020. Microscopic screening of sputum samples should not be used when testing for Legionella species. Clin Infect Dis 71:1356–1357. doi: 10.1093/cid/ciz1148 [DOI] [PubMed] [Google Scholar]
- 29. Yzerman EPF, den Boer JW, Lettinga KD, Schellekens J, Dankert J, Peeters M. 2002. Sensitivity of three urinary antigen tests associated with clinical severity in a large outbreak of Legionnaires' disease in the Netherlands. J Clin Microbiol 40:3232–3236. doi: 10.1128/JCM.40.9.3232-3236.2002 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30. Musher DM, Montoya R, Wanahita A. 2004. Diagnostic value of microscopic examination of Gram-stained sputum and sputum cultures in patients with bacteremic pneumococcal pneumonia. Clin Infect Dis 39:165–169. doi: 10.1086/421497 [DOI] [PubMed] [Google Scholar]
- 31. Mentasti M, Fry NK, Afshar B, Palepou-Foxley C, Naik FC, Harrison TG. 2012. Application of Legionella pneumophila-specific quantitative real-time PCR combined with direct amplification and sequence-based typing in the diagnosis and epidemiological investigation of Legionnaires’ disease. Eur J Clin Microbiol Infect Dis 31:2017–2028. doi: 10.1007/s10096-011-1535-0 [DOI] [PubMed] [Google Scholar]
- 32. Korosec P, Silar M, Erzen R, Kosnik M. 2006. The influence of antimicrobial therapy on the sensitivity of Legionella PCR. Scand J Infect Dis 38:925–928. doi: 10.1080/00365540600561777 [DOI] [PubMed] [Google Scholar]
- 33. Shirasaka W, Shinozaki M, Kushibiki C, Katsukawa C. 2019. Comparative study of the detection rates of the urinary antigen test and the LAMP method for Legionella species. J Jpn Soc Clin Microbiol 29:203–206. [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data sets used and analyzed during the current study are available from the corresponding author upon reasonable request.