ABSTRACT
Background and Objective:
Legionella pneumophila (Lp) has been recognized as an important cause of Hospital Acquired Pneumonia (HAP). However, the frequency distribution of Lp as etiological agents of HAP is less studied in India. The aim of this study was to determine the frequency of Lp in cases of HAP by using a combination of culture, urinary Lp sg1 antigen testing, and Lp-specific 16S RNA gene PCR.
Methods:
Admitted patients fulfilling the clinical and radiological criteria of HAP were enrolled in the study between February 2023 and June 2024. Respiratory specimens of patients were subjected to culture in BCYE agar and the Lp-specific PCR, and urine Lp sg1 antigen testing.
Results:
Of the 195 suspected HAP patients enrolled, 160 (82.05%) were microbiologically confirmed as HAP. All respiratory specimens tested negative for Lp by culture and urine antigen tests. However, 20 of the 160 microbiologically confirmed HAP cases were positive for Lp by 16S rRNA gene PCR. New-onset pulmonary infiltrates were significantly associated with Lp HAP compared to non-Legionella HAP cases (50% vs. 5%, p < 0.001). Lp-positive HAP cases had higher total protein (6.13 vs. 5.56 g/dL, p = 0.043) and globulin (3.24 vs. 2.84 g/dL, P = 0.037), and lower serum phosphate levels (2.94 vs. 4.58 mg/dL, p < 0.001) compared to negative cases. In the present study, Lp was a co-pathogen in fifteen HAP cases (Eight cases with Klebsiella pneumoniae, six with Pseudomonas aeruginosa, and one with Burkholderia cepacia complex).
Conclusion:
This study highlights the significant role of Lp non serogroup 1 as a pathogen in 12.5% of microbiologically confirmed HAP cases, using PCR, which were negative by culture and commercially available urinary antigen testing targeting Lp serogroup 1. The findings emphasize incorporating Lp molecular tests along with Ag testing in the diagnostic algorithm of HAP cases.
KEY WORDS: Hospital-acquired pneumonia, L. pneumophila, non-serogroup 1
INTRODUCTION
The Legionella genus currently comprises more than 65 species, with Legionella pneumophila (Lp) being the most frequent cause of Legionnaires’ disease (LD), primarily caused by serogroup-1 (sg-1), followed by sg-3 and sg-6, which account for 80–90% of LD cases worldwide, mostly in Europe and in the USA.[1] Non-pneumophila species, particularly L. longbeachae, are the predominant species in Australia and New Zealand. In the year 2024, multiple developed nations, including the USA, the UK, European Union nations, and Australia, reported many LD cases.[2]
Lp accounts for approximately 4.6% of cases of community-acquired pneumonia (CAP)[2] and remains a far less investigated etiological agent of hospital-acquired pneumonia (HAP). In hospital settings, admitted patients are at risk of nosocomial Lp due to inhalation of contaminated aerosols from medical humidifiers, inhalation devices, respiratory therapy equipment, etc. However, the frequency distribution of Lp as an etiological agent of HAP is less studied in many developing countries, mostly attributed to limitations of conventional laboratory diagnostic techniques.
Culture-based diagnosis of Lp is technically demanding and slow and has limited utility in clinical diagnosis and management because of suboptimal sensitivity ranging from 10% to 80%.[1] Urinary antigen testing (UAT), which primarily targets lipopolysaccharide in the Lp cell walls, is now frequently employed as a first-line screening technique. For the diagnosis of LD caused by Lp1, UATs have been shown to have a sensitivity in the range of 70–90% and a 100% specificity.[3] Sensitivity of UAT is lower (45%) for Lp HAP compared to CAP cases (80%), as HAP cases are more likely to be caused by Lp non serogroup 1, thereby limiting their utility as the sole diagnostic test in HAP cases.[4] Lp PCR, performed from respiratory tract specimens, can detect all sgs (1–16) of Lp. A systematic review demonstrated that PCR in respiratory samples showed significantly greater sensitivity than UAT, resulting in the detection of an additional 18% to 30% of Lp cases, and the sensitivity and specificity of PCR for diagnosis of LD in respiratory samples were 97.4% and 98.6%, respectively.[5] Similarly, an Italian study found that the use of real-time PCR, in addition to UAT and culture, led to an 18% increase in the detection of Lp cases.[6]
A few studies from Spain, France, Japan, and Iran have found that 0.8-19.8% of HAP cases are attributed to Lp using a combination of culture, serological, and molecular tests.[7,8,9,10,11,12,13] Few Indian studies have investigated the prevalence of Lp in patients with pneumonia and have found varying rates. In a study conducted on patients with CAP, Lp IgM positivity was reported in 15% of cases using IgM ELISA.[14] Another study on CAP patients employed culture, urinary antigen test (UAT), and ELISA (IgM and IgG) to detect Lp, there was no culture-positivity, UAT positivity was 17.69%, and overall seropositivity was 27% and one study using combination of real time PCR and serology by IgM, IgG and IgA ELISA for Lp, found that PCR positivity was reported in 6% of cases, while total serological positivity was 23%.[15,16] One study analyzed clinical, laboratory, radiological parameters, risk factors, and outcomes of hospitalized patients with or without Lp HAP.[17] One study conducted on CAP patients and water samples in South India found that 2.55% of clinical specimens and 33.3% of environmental samples were positive for Legionella spp by culture.[18] Environmental surveillance studies in India have detected Lp in various water sources. One study conducted in North India reported the presence of Lp serogroup 1 in 15.2% of samples, using both culture and real-time PCR methods.[19] Another study identified L.p serogroups 2–15 contamination in 6.66% of distal water outlets by a combination of culture and latex agglutination testing.[20] We carried out the surveillance of hospital water samples from the areas where Lp HAP cases were detected for the presence of Lp using a combination of culture and PCR. We hypothesized that a substantial proportion of HAP cases in our setting may be due to non-sg 1 Lp undetected by standard UAT.
MATERIALS AND METHODS
We conducted a prospective cross-sectional study at a tertiary hospital in Bhubaneswar, India, from February 2023 to June 2024. HAP and VAP were defined by using the modified surveillance definition adopted from the PNEU 2 and 3 algorithms in the CDC’s NHSN guidelines for enrolling suspected HAP cases [Supplementary Material].[21] For the purpose of determining the frequency of Lp in HAP cases, prevalence ranges from 8-20%, from previous published studies, were taken into account.[14,15,16,17,18] We had taken into account at least 15% prevalence (to provide a safe margin), and the sample size was calculated using the equation (n = zα^2pq/d^2). After obtaining written informed consent, 195 adult and pediatric patients with clinical and radiological suspicion of HAP were enrolled in the present study. Patients with an admission diagnosis of CAP were excluded from the study.
The attending physicians decided the type of clinical specimens (endotracheal secretion, bronchoalveolar lavage, sputum, tracheostomy secretion) to be obtained from suspected HAP cases. Collected specimens were divided into three parts: one for microscopy and semi-quantitative culture for conventional bacterial pathogens; the second part was used for culture on BCYE agar (M813, HiMedia Lab Pvt Ltd) with or without acid (KCL-HCl acid buffer) pre-treatment (in a ratio of 1 part of sample to 10 parts in low-pH KCL/HCL buffer, pH 2.2 with 4 min. of incubation at room temperature); and the third part was stored at -20°C until testing for Lp-specific polymerase chain reaction (PCR) assay. Lp ATCC 33152 [Himedia, Lot number 211-71-3, exp date 30/9/25] was used as a control for culture as well as standardization of PCR. We determined the limit of detection/analytical sensitivity by performing serial dilutions of positive control DNA (Initial concentration: 22.98 ng/μl) and found that the assay detects as low as 0.22 ng/μl of target DNA. We tested the specificity assay against negative controls, ATCC E coli 25922, to confirm that no non-specific amplification occurred, only the target sequence produced amplification, confirming assay specificity [Figure 1]. Inoculated BCYE agar was incubated at 37°C in a CO2 incubator for 5-7 days. For conventional culture, from ETA and BAL, a CFU count of 105 cfu/ml and 104 cfu/ml were considered significant, respectively.
Figure 1.
Estimation of the analytical sensitivity of PCR by diluting genomic DNA
L. pneumophila serogroup 1 (Lp1) antigen detection in urine was performed by a commercially available urine antigen test (Quick Stripe, Savyon Diagnostics Ltd) kit. 4 drops of the urine sample were added to the sample window, and reading was taken after 15 minutes, as recommended by the manufacturer.
In the present study, a total of 36 samples, including 14 biofilm swabs from ICU and ward taps, 18 bulk water samples, and four samples from various sites of cooling towers, were collected from areas where Lp HAP cases were detected. Pre-treatment with 0.1N sodium thiosulphate solution was used to neutralize residual disinfectants. Filter concentration and elution from water samples were performed by initial filtration using a 0.2 μm polycarbonate membrane filter, followed by vortexing of the filter in 5 ml of sterile water. The elute obtained from each sample was divided into two parts for culture on non-selective BCYE agar (M813I) and PCR. PCR was performed from DNA extracted from clinical and environmental samples using a commercially available PCR Kit (MBPCR006, HiMedia Lab Pvt Ltd) targeting a specific region of 16S rRNA (901 bp) of Lp. The following amounts were added to each reaction: 25 μL of master mix, 2 μL each of primer mix for Lp (DS0130), and 2 μL of the template DNA, and the final volume was adjusted to 50 μL using nuclease-free water. RNAse/DNAse-free water was used as a negative control reaction. DNA of Lp ATCC 33152 was used as a positive control in each run, along with the internal control provided in the kit for run validation. The protocol used for amplification was initial denaturation at 94°C for 10 minutes, followed by 30 cycles of denaturation at 94°C for thirty seconds, annealing at 60°C for thirty seconds, extension at 72°C for forty-five seconds, and one cycle of a final extension cycle at 72°C for 10 minutes. The amplified products were observed for visual representation of bands using an automated gel documentation system (Syngene, Germany) under UV light. The interpretation of the results was based on the presence of specific bands observed at regions corresponding to 901 bp for Lp and the internal control amplicon (285 bp) [Figure 2].
Figure 2.
PCR results of samples
Results were analyzed using the SPSS version 27 statistical software package. The chi-square test was used to detect significant differences between groups for categorical variables, and the Mann–Whitney test for continuous variables. Univariate analysis of laboratory and radiological variables influencing Lp positivity in HAP cases (P values < 0.05) was compared, and statistically significant factors were used for multivariate analysis by performing binary logistic regression. Results were expressed in the form of an odds ratio (OR) with corresponding 95% CI. A P value < 0.05 was considered statistically significant.
RESULTS
Out of the 195 suspected HAP patients enrolled during the study period, 160 (160/195, 82.05%) were microbiologically confirmed HAP cases. Most of the microbiologically confirmed HAP cases were categorized as ventilator-associated pneumonia (VAP), accounting for 151/160 (94%) of microbiologically confirmed HAP cases; non-ventilator-associated HAP (NV-HAP) accounted for 9/160 (6%) of the microbiologically confirmed HAP cases. Of the 160 microbiologically confirmed HAP cases, 129 were from the adult age group (age ≥18 years), and 31 were from the pediatric age group. Respiratory specimens of all HAP patients yielded negative results for Lp by sputum/ET/BAL culture in BCYE agar and the urine Lp sg1 antigen test. Twenty of the 160 microbiologically confirmed HAP cases were positive for Lp by specific 16S ribosomal RNA gene PCR, including eight from tracheostomy secretion samples, seven from endotracheal secretion samples, and five from bronchoalveolar lavage samples (BAL). Out of the 20 positive Lp cases diagnosed during the study period, 12 cases were from the adult age group (12/129, 9.3%), and eight cases were from the pediatric age group (8/31, 25.8%). Total percentage positivity of Lp in microbiologically confirmed HAP cases was 12.5% (20/160).
The comparison of clinical characteristics, risk factors, and laboratory and radiological parameters of the microbiologically confirmed HAP cases with or without Lp is depicted in Tables 1-3. On analysis of comorbid conditions, among microbiologically confirmed HAP cases, no pre-existing health conditions were found to be associated with Lp HAP. While comparing chest imaging findings, it was found that new-onset pulmonary infiltrates had a significant association with Lp HAP compared to HAP cases without Lp. Among biochemical parameters, total protein and serum globulin of Lp-positive cases were higher compared to Lp negative HAP cases. Serum phosphate levels were significantly lower in Lp-positive HAP patients compared to Lp-negative HAP patients. Multivariate analysis [Table 4] showed that pulmonary infiltrates and low serum phosphate levels were the independent factors influencing Lp positivity in HAP cases. Z scores were calculated to quantify the prediction of each variable, and it was shown that pulmonary infiltration has the highest influence (Z = 4.08), followed by low serum phosphate levels (Z = 3.33), whereas other parameters, such as total protein and ALP, were insignificant, as P values were >0.05.
Table 1.
Demographic details and comorbid conditions of HAP patients with or without Legionella pneumophila in the adult age group (n=129)
| HAP patient without L. pneumophila (n=117) | HAP patient with positive L. pneumophila (n=12) | P (<0.05=significant) | |
|---|---|---|---|
| Demographic details | |||
| Median age (year) (IQR) | 48 (35-63) | 47.5 (36-57.5) | 0.85 |
| Sex (M/F) | 67/50 | 9/3 | 0.234 |
| Location | |||
| ICU | 107 (91.45%) | 12 (100%) | 0.292 |
| Ward | 10 (8.55%) | nil | |
| Comorbid conditions | |||
| Diabetes mellitus | 30 (25.64%) | 3 (25%) | 0.961 |
| Hypertension | 38 (32.48%) | 1 (8.33%) | 0.083 |
| COPD | 10 (8.55%) | 2 (16.67%) | 0.356 |
| Pre - existing Cardiac disease | 5 (4.27%) | 1 (8.33%) | 0.525 |
| Pre - existing renal disease | 14 (11.97%) | 2 (16.67%) | 0.638 |
| Pre - existing neurological disease | 14 (11.97%) | 2 (16.67%) | 0.638 |
| Known malignancy | 8 (6.84%) | 1 (8.33%) | 0.846 |
Table 3.
Clinical, laboratory, and radiological parameters of HAP cases with or without Legionella pneumophila (n=160)
| HAP patient without L. pneumophila (n=140) | HAP patient with positive L. pneumophila (n=20) | P (<0.05=significant) | |
|---|---|---|---|
| Clinical findings | |||
| Signs and symptoms | |||
| Fever | 140 (100.0%) | 20 (100.0%) | NA |
| Cough | 6 (4.3%) | 0 (0.0%) | 1.000 |
| Dyspnea | 42 (30%) | 7 (35.0%) | 0.650 |
| Tachypnea (≥24/min) | 133 (95%) | 20 (100.0%) | 0.598 |
| Increased ventilatory support | 103 (73.6%) | 16 (80.0%) | 0.538 |
| Radiographic findings | |||
| Opacities | 70 (50%) | 4 (20%) | 0.012 |
| New onset and progressive Pulmonary infiltrations | 7 (5%) | 10 (50%) | <0.001 |
| New onset and progressive Lung Consolidations | 63 (45%) | 6 (30%) | 0.205 |
| Laboratory parameters | |||
| TLC (x103/mm3); mean±SD | 17.11±6.18 | 17.523±7.0269 | 0.367 |
| Total protein (g/dl) mean +/- SD | 5.5615±1.19019 | 6.129±0.93715 | 0.043 |
| Albumin (g/dl) mean +/- SD | 2.7431±0.69639 | 2.908±0.73214 | 0.310 |
| Globulin (g/dl) mean +/- SD | 2.8393±0.82407 | 3.235±0.75622 | 0.037 |
| Serum sodium (mmol/L mean +/- SD | 135.886±7.2698 | 134.9±7.31185 | 0.602 |
| Serum phosphate (mmol/L mean +/- SD | 4.5848±1.54382 | 2.9445±1.05844 | <0.001 |
| AST (IU/ml), mean +/- SD | 90.557±112.839 | 70.56±54.9894 | 0.747 |
| ALT (IU/ml), mean +/- SD | 87.2157±116.062 | 109.59±95.6613 | 0.212 |
| ALP (IU/ml), mean +/- SD | 165.743±144.722 | 180.9±113.17471 | 0.101 |
| Abnormal Hematological/biochemical findings | |||
| Leukopenia (TLC<4 x 103) | 5 | 1 | |
| Mean±S.D | (mean=1.4) | 0.137 | |
| (3.326±0.535) | |||
| Leukocytosis (TLC ≥14x 103) | 118 | 17 | |
| Mean±S.D | Mean±S.D | 0.225 | |
| (18.66±5.18) | (19.79±4.54) | ||
| Elevated AST (AST>50 IU/ml) | 62 | 11 | |
| Mean±S.D | Mean±S.D | 0.432 | |
| (164.71±137.39) | (103.67±54.63) | ||
| Elevated ALT (ALT>50 IU/ml) | 72 | 12 | |
| Mean±S.D | Mean±S.D | 0.091 | |
| (140.61±142.98) | (161.82±91.07) | ||
| Elevated ALP (ALP >120 IU/ml) | 69 | 13 | |
| Mean±S.D | Mean±S.D | 0.598 | |
| (257.26±159.83) | (225.69±117.91) | ||
| Hyponatremia (Sodium<125 mmol/L) | 3 | 1 | |
| Mean±S.D | (mean=124) | 0.564 | |
| (121.66±4.04) | |||
| Hypophosphatemia (phosphate<2.5 mmol/L) | 7 | 9 | |
| Mean±S.D | Mean±S.D | 0.915 | |
| (2.12±0.29) | (2.16±0.25) | ||
|
| |||
| Clinical outcomes of HAP patients | |||
|
| |||
| HAP patient without L. pneumophila (n=140) | HAP patient with positive L. pneumophila (n=20) | Abnormal Hematological/biochemical findings | |
|
| |||
| Length of hospital stay (Median, IQR) | 23 (15-60) | 22.5 (57.5−16.5) | P (<0.05=significant) |
| Survived | 87 (62.6%) | 9 (45%) | |
| Expired | 52 (37.4%) | 11 (55%) | 0.133 |
Table 4.
Multivariate analysis of factors influencing L. pneumophila positivity
| Variables | Odds Ratio | 95% C.I. | P |
|---|---|---|---|
| Total Protein | 0.483 | 0.187-1.247 | 0.132 |
| Globulin | 1.384 | 0.372-5.152 | 0.628 |
| Low Serum Phosphate levels | 3.207 | 1.681-6.119 | <0.001* |
| New onset of Infiltrations on Chest Xray | 18.156 | 4.065-81.085 | <0.001* |
Table 2.
Demographic details and comorbid conditions of HAP patients with or without Legionella pneumophila in the pediatric age group (n=31)
| HAP patient without L. pneumophila (n=23) | HAP patient with positive L. pneumophila (n=8) | P (<0.05=significant) | |
|---|---|---|---|
| Demographic details | |||
| Median age (year) (IQR) | 2 (1-6) | 1 (1-8) | 0.82 |
| Sex (M/F) | 20/3 | 5/3 | 0.132 |
| Location | |||
| ICU | 19 (82.61%) | 7 (87.50%) | |
| Ward | 4 (17.39%) | 1 (12.50%) | |
| Comorbid conditions | |||
| Diabetes mellitus | - | - | - |
| Hypertension | 2 (8.70%) | 1 (12.50%) | 0.754 |
| COPD | - | - | - |
| Pre-existing Cardiac disease | - | - | - |
| Pre-existing renal disease | 2 (8.70%) | - | 0.389 |
| Pre-existing neurological disease | 1 (4.35%) | 1 (12.5%) | 0..419 |
| Known malignancy | - | - | - |
In adult Lp HAP cases, the median number of days of hospital admission before Lp positivity was 14 days. There was a 50% mortality in adult HAP cases due to Lp. We observed a temporal association Lp positive HAP cases in RICU (serial nos. 7, 8, and 9) [Table 5]. Except for those three cases, all Lp positive HAP cases were from separate ICUs. Water sources collected during the study period from RICU were negative for Lp by culture and PCR. There was a 62% mortality in pediatric HAP cases due to Lp. [Table 6]. We observed three Lp positive HAP cases during a two-week interval in the PICU. Water sources collected from the PICU during that period were negative for Lp by culture and PCR.
Table 5.
Demographics, epidemiologic with risk factors, and clinical characteristics of L. pneumophila-positive adult HAP cases
| Hospital admission date | Gender, age, Location of the patient | Risk factors | Reason for hospital admission | Sample type | Time of admission to L. pneumophila positive (days) | Total duration of hospital stays | Final outcome |
|---|---|---|---|---|---|---|---|
| 04th May | 68yr, M RICU | Smoking, COPD | AE COPD | TT | 38 | 78 | Survived |
| 07th Nov | 39, M Trauma ICU | Smoking | RTA | TT | 56 | 67 | Expired |
| 19th Dec | 50, M RICU | Smoking, Alcohol intake, DM | Sepsis | ET | 12 | 19 | Expired |
| 27th dec | 33, F MICU | DM | DKA | TT | 17 | 64 | Expired |
| 17th Feb | 50, F Neurosurgery ICU | Neurological disease | glioma | TT | 25 | 47 | Survived |
| 18th Feb | 25, F MICU | nil | Unknown poisoning | TT | 21 | 51 | Survived |
| 25th Feb | 40, M RICU | DM, CKD | ILD | ET | 12 | 24 | Expired |
| 03rd Mar | 50, M RICU | nil | AKI | ET | 14 | 18 | Expired |
| 03rd Mar | 65, M RICU | Smoking, HTN, COPD, | HIE | ET | 26 | 31 | Survived |
| 24th Apr | 33, M MICU | nil | A/H/O carbamate poisoning | TT | 19 | 71 | Survived |
| 27th Apr | 75, M RICU | Smoking, CKD | ILD | BAL | 9 | 15 | Expired |
| 01st May | 45, M MICU | Alcohol, neurological disease | Quadriparesis | ET | 9 | 15 | Survived |
Table 6.
Demographics, epidemiologic with risk factors, and clinical characteristics of L. pneumophila-positive Pediatric HAP cases
| Hospital admission date | Gender, age, Location of the patient | Risk factors | Reason for hospital admission | Sample type | Time of admission to test positive (days) | Duration of stay | Outcome |
|---|---|---|---|---|---|---|---|
| 23-Oct-23 | 6-month, M PICU | nil | Neonatal cholestasis | BAL | 10 | 15 | Survived |
| 26-Dec-23 | 13, F PICU | nil | Unknown poisoning | BAL | 14 | 21 | Expired |
| 06-Feb-24 | 4-month, F PICU | nil | Paradoxical vocal cord movement | TT | 68 | 73 | Survived |
| 04-Mar-24 | 10, M pediatric ward | HTN, TB meningitis | TB meningoencephalitis | TT | 14 | 20 | Expired |
| 07-Mar-24 | 5month, M PICU | nil | BPD | BAL | 16 | 26 | Survived |
| 29-Mar-24 | 6-month, F PICU | Retinoblastoma | Bilateral retinoblastoma | ET | 4 | 10 | Expired |
| 08-May-24 | 6month, M PICU | Neurological disease | SOJIA | ET | 11 | 20 | Expired |
| 09-May-24 | 1, M PICU | nil | FTT | BAL | 4 | 13 | Expired |
In Lp-negative HAP cases, the predominant organisms isolated from respiratory samples in both the adult and pediatric groups were Klebsiella pneumoniae (60/160, 37.5%), followed by Acinetobacter baumannii complex (49/160, 30.62%) and Pseudomonas aeruginosa (13/160, 8.12%). Carbapenem susceptibility in MDR Klebsiella pneumoniae complex, Acinetobacter baumannii complex, and Pseudomonas aeruginosa was 7.9%, 4.1%, and 30.8%, respectively. Colistin susceptibility in the MDR Klebsiella pneumoniae complex, Acinetobacter baumannii complex, and Pseudomonas aeruginosa was 91.6%, 95.9%, and 92.3%, respectively.
In the present study, Lp was a co-pathogen in fifteen cases (eight cases with Klebsiella pneumoniae, six with Pseudomonas aeruginosa, and one with Burkholderia cepacia complex). Based on clinical history, laboratory, and radiological findings, it was difficult to ascertain clearly whether those cases were dual or sequential infections.
DISCUSSION
The present study enrolled 195 suspected HAP patients between February 2023 and June 2024. Of these, 82.05% (160/195, 82.05%) were microbiologically confirmed HAP cases. This study highlights the significant role of Lp as a pathogen in HAP cases, with a detection rate of 12.5% (20/160) among microbiologically confirmed HAP cases using 16S ribosomal RNA gene PCR, which were negative by culture and commercially available urinary antigen testing targeting Lp serogroup 1. In the study of Sreenath et al.,[17] Lp culture was negative in all PCR-positive cases. Delays in respiratory sample processing, the prior use of antimicrobial agents, and culture overgrowth by other oropharyngeal bacteria are factors that limit the culture yields. The urine Lp sg1 antigen test negativity in all PCR-positive cases implies that the positive cases were caused by Lp serogroups other than serogroup 1. Previous studies have also shown that hospital-acquired Legionellosis is more likely to be caused by non-Lp1 or Lp1 strains with an LPS less well-recognized by commercially available tests.[4,22] In the present study, we have used a commercially available Lp conventional PCR, which amplifies partial 16S ribosomal RNA specific to Lp. The partial 16S ribosomal RNA subunit of Lp has been used for Legionella outbreak investigation by Sánchez-Parra et al.[23] In another related study by Gadsby et al.,[24] the implementation of Legionella duplex real-time PCR targeting the partial 16S ribosomal RNA subunit of Lp and the macrophage infectivity potentiator (mip) gene detected ten additional cases due to non-sg1 L. pneumophila over a 44-month study period at a UK-based hospital. Incorporation of Lp and Legionella species molecular diagnostic assays makes a useful addition to Lp urinary antigen testing for the detection of non-sg1 Lp in HAP cases.
The finding of Lp in 12.5% of microbiologically confirmed HAP cases in this study matches the results of a previous seven-year study on severe HAP in medical and surgical ICUs from Spain, where Lp was documented in 9/67 of microbiologically confirmed HAP cases.[25] The frequency of HAP with Lp at Tehran hospitals was estimated to be 4.58% (12/262).[9] In another study from four hospitals in Tehran, Iran, Lp was documented in 24/280 (8.6%) of BAL samples from microbiologically confirmed VAP cases by real-time PCR.[13] In our study, 19/151 (12.5%) of VAP cases were attributed to Lp. Depending on geographic location, the prevalence of Lp in HAP is different, which is explained by differences in case definitions, diagnostic methods, surveillance systems, and data presentation. It has also been attributed to publication bias, e.g., in 10 small studies (<100 patients), 13.2% of patients with pneumonia had LD, but in 5 large studies (500 patients), the figure was 3.6%.[26] We could not find any Indian studies for comparison, as published Indian studies on Lp have predominantly focused on CAP or a mix of LRTI cases.[17] Out of the twenty positive Lp HAP cases diagnosed during the study period, twelve cases were from the adult age group, and eight cases were from the pediatric age group. The median age of adult HAP patients with Lp was 47.5 years, and the median age of the pediatric HAP cases with Lp was 1 year; there was a male predisposition (the male-to-female ratio was 2.3 (14 versus 6)). Fard et al.,[9] in their study of Lp HAP cases, also documented a male-to-female ratio of 1.30 (148 versus 114) and median age of 58 years (range, 39-85 years). The mean age of Lp-positive VAP cases was 69.5 ± 20.3 years, and the male-to-female ratio was 2.0 (16 vs. 8) in the study by Sakhaee et al.[13] Sakhaee et al.[13] also found age (OR, 1.164; 95% CI, 1.009–3.089; P = 0.010) as an independent factor related to Lp infectivity. There is a paucity of literature on Lp HAP in the pediatric population. In the present study, out of the 31 cases of microbiologically confirmed HAP cases in the pediatric age group, eight cases (8/31; 25.8%) were positive by specific 16S ribosomal RNA gene PCR. In the study by Mojtahedi et al.,[12] out of 96 pediatric HAP patients, the positivity rate of the Legionella urinary antigen test was 16.7%, and the positivity rate of the PCR test was 19.8%. The authors also concluded that the highest rate of atypical pathogen pneumonia, including Lp, occurs in patients under two years of age. In our study, the number of pediatric HAP cases was less (n = 31), and the finding of 25.8% Lp needs to be validated with a large population size of pediatric HAP.
In our study, 19/20 (95%) of the Lp HAP cases were from mechanically ventilated patients admitted to various ICUs. The median length of ICU stays and median days of hospital admission before Lp positivity were was -22.5 and 14 days, respectively. In the study by Sakhaee et al.,[13] the length of ICU stays (OR, 0.951; 95% CI, 0.885–0.998; P < 0.001) and duration of ventilator use (OR, 0.921; 95% CI, 0.863–0.984; P < 0.001) were independently related to Lp infectivity. In the study by Mojtahedi et al.[12] on pediatric HAP, out of 12 post-ventilation pneumonia cases, 3 cases occurred less than 5 days after ventilation (25%), while 9 other pneumonia cases appeared after 5 days of ventilation. ICU patients, particularly those on mechanical ventilation, are at risk of developing Lp pneumonia due to inhalation of contaminated aerosols from hot- and cold-water systems, humidifiers, and air conditioning cooling towers.[17] This highlights the role of mechanical ventilation as a significant risk factor for Lp-related pneumonia.
In this study, we could not find any significant association of any comorbid conditions with Lp positivity in HAP patients. Sakhaee et al.[13] also reported identical findings on the comparison of predisposing conditions between patients with and without Lp HAP in a subset of 280 VAP patients (24 Lp-positive and 256 Lp-negative). However, in their study, smoking (OR, 1.895; 95% CI, 1.001–1.995; P = 0.025) was an independent factor associated with Lp.
In our study, 63/160 HAP patients succumbed, with an overall 39.3% mortality rate. In the study by Valles et al.,[25] out of 96 episodes of severe HAP cases, 51 patients (53%) died. HAP patients generally have a poor prognosis. In our study, 11/20 of Lp HAP cases died, resulting in a 55% fatality rate compared to 52/140 (37.1%) of HAP cases without Lp (P value 0.133). A high rate of 30-day mortality (16/39, 41%) is also described in hospital-acquired Lp by Dagan et al.[27] Patients with hospital-acquired Lp infections are usually diagnosed late, and few receive empiric anti-Legionella coverage. Current guidelines for the diagnosis and management of HAP do not recommend an empiric anti-Legionella coverage. These factors partly explain the increased mortality in hospital-acquired Lp infections.[27]
In the present study, comparison of radiographic chest X-ray findings at symptom onset between HAP cases with or without Lp revealed significant differences in the presence of new-onset infiltrates (50% vs. 5%, P value < 0.001). Lp infections often present with patchy infiltrates and pleural effusion.[28] All types of infiltrates, including interstitial and nodular, can occur in Lp and are not pathognomonic.[28] In a study by Dagan et al.[27] in the cohort of Lp HAP cases (n = 36), there was a significantly higher likelihood of having bilateral findings (opacities, pleural effusions) in chest radiography; however, those differences did not reach statistical significance on CT scans.
Failure to detect any potential environmental reservoir by culture and PCR, despite detecting twenty cases over one and a half years, is a major limitation of the present study. In a tertiary care hospital in North India, Lp serogroup 1 was detected in 15.2% of the water systems of a tertiary healthcare center, including repeated isolation from a few samples.[29] Plate culture in BCYE agar after water concentration has a detection limit of 1 CFU/ml, and PCR usually has a detection limit of 480 genome units per liter.[30] The negative result in our study may be explained partly by either the absence of Lp in our hospital water system due to regular disinfection of the hospital potable storage overhead tank by chlorine-based disinfectant on a monthly basis, or by the water system harboring Lp at undetectable levels. We tested 36 water samples over 17 months of the study period; more frequent sampling or more sites could have yielded a positive result. Also, Legionella spp. are known to persist within biofilms on pipe surfaces and inside plumbing systems. Standard water sampling may not effectively capture organisms embedded in biofilms, which are often resistant to physical disruption and chemical disinfection.[31]
In the present study, the most common microorganisms implicated in HAP were MDR Klebsiella pneumoniae complex (60/160, 37.5%) and Acinetobacter baumannii complex (49/160, 30.62%), followed by Pseudomonas aeruginosa (13/160, 8.12%). MRSA was implicated in only 1/160 (0.62%) of cases. In a recent study from 34 ICUs of 13 Indian hospitals, Acinetobacter spp. was the most frequently isolated pathogen (102; 29.6%), followed by Klebsiella spp. (92; 26.7%), Pseudomonas spp. (66; 19.1%), and Escherichia coli (24; 6.9%).[32] In our study, carbapenem susceptibility in MDR Klebsiella pneumoniae complex, Acinetobacter baumannii complex, and Pseudomonas aeruginosa was 7.9%, 4.1%, and 30.8%, respectively. Colistin susceptibility in the MDR Klebsiella pneumoniae complex, Acinetobacter baumannii complex, and Pseudomonas aeruginosa was 91.6%, 95.9%, and 92.3%, respectively. Similar findings regarding colistin are reported from a network of Indian hospitals by Mathur et al.,[32] where colistin was found to be resistant in 3.2% of Enterobacterales and 6% of Acinetobacter spp. causing VAP. In the present study, Lp was a co-pathogen in fifteen cases (eight cases with Klebsiella pneumoniae, six with Pseudomonas aeruginosa, and one with Burkholderia cepacia complex). Similar findings of polymicrobial episodes of Lp with other viral, fungal, and bacterial pathogens, namely Pneumocystis, Aspergillus, Pseudomonas aeruginosa, Haemophilus influenzae, Streptococcus pneumoniae, and CMV, have been previously reported by Valles et al.[25] in a 7-year study of severe HAP requiring ICU admission. The percentages of coinfection involving Lp pneumonia cases range from 2% to 10%.[33] It is postulated that Lp infection, like certain viral infections, may predispose the respiratory tract to other bacterial infections.[33] In co-infection cases, due to limitations in diagnostic techniques, it is often difficult to ascertain if Lp infections may have preceded the bacterial infections, may have been sequential infections, or may have been concurrent infections (i.e., true coinfection).
Detection of Lp by the specific 16S ribosomal RNA gene PCR in 12.5% (20/160) of the microbiologically confirmed HAP cases serves as an important sentinel alert to investigate the possibility of undiscovered cases of Lp HAP cases in Indian settings. The findings emphasize incorporating Lp molecular tests along with Ag testing in the diagnostic algorithm of HAP cases. There is a need for a more proactive and systematic search for Lp in hospital water systems. Further studies with large sample sizes are necessary to justify the incorporation of antibiotics active against Lp in the therapeutic protocols for HAP.
This study evaluated a commercially available PCR kit for Lp, which costed approximately 412 rupees per sample. A commercially available multiplex panel like the Biofire Filmarray Pneumonia (PN) panel, which can detect 26 respiratory pathogens, including viruses and atypical bacteria, simultaneously, costs about Rs 11,000 per sample. Depending upon the patient profile of the hospitals, any of the kits can be incorporated as a first-line evaluation of HAP cases when culture doesn’t yield a result or when co-infection is suspected. Hospital water colonization by Lp can be long-lasting and associated periodically with outbreaks. Maintaining a high concentration of chlorine (hyperchlorination: >10 mg/L of free residual chlorine) at the point of delivery is advocated to reduce Lp colonization.[34]
Ethical approval and/or institutional review board (IRB) approval
Institutional ethical committee approval (IEC/AIIMS BBSR/2022-23/115, DATED 13TH Feb 2023.
Conflicts of interest
There are no conflicts of interest.
Acknowledgment(s)
The authors acknowledge ICMR for financial support in the form of a PG thesis grant, Ref Number ICMR Grant No: 3/2/Dec-2022/PG thesis –HRD. Technical support from Mr Sunil K Prusty, Mr Chandramani, Ms Subhalaxmi, Ms Biswokalyani, and staffs of Hospital Engineering section is duly acknowledged.
SUPPLEMENTARY MATERIAL
Specific Site Algorithm for Pneumonia with Common Bacterial or Filamentous Fungal Pathogens and Specific Laboratory Findings (PNU2)
| Imaging Test Evidence | Signs/Symptoms | Laboratory |
|---|---|---|
| Two or more serial chest imaging test results with at least one of the following (1,2,13): New and persistent or Progressive and persistent • Infiltrate • Consolidation • Cavitation • Pneumatoceles, in infants ≤1 year old Note: In patients without underlying pulmonary or cardiac disease (such as respiratory distress syndrome, bronchopulmonary dysplasia, pulmonary edema, or chronic obstructive pulmonary disease), at least one definitive chest imaging test result is acceptable. (1) |
At least one of the following: • Fever (> 38.0°C or > 100.4°F) • Leukopenia (≤ 4,000 WBC/mm³) or leukocytosis (≥ 12,000 WBC/mm³) • For adults > 70 years old: altered mental status with no other recognized cause And at least one of the following: • New onset of purulent sputum (3) orchange in character of sputum (4), or increased respiratory secretions, or increased suctioning requirements • Dyspnea, or tachypnea (5), or new onset or worsening cough • Rales (6) or bronchial breath sounds • Worsening gas exchange (for example, O2 desaturations [for example, PaO2/FiO2 ≤ 240] (7), increased oxygen requirements, or increased ventilator demand) |
At least one of the following: • Organism identified from blood (8,12) • Organism identified from pleural fluid (9,12) • Positive quantitative culture or corresponding semi-quantitative culture result (9) from minimally contaminated LRT specimen (specifically, BAL, protected specimen brushing, or endotracheal aspirate) • ≥ 5% BAL-obtained cells contain intracellular bacteria on direct microscopic exam (for example, Gram's stain) • Positive quantitative culture or corresponding semi-quantitative culture result (9) of lung tissue • Histopathologic exam shows at least one of the following evidences of pneumonia: ○ Abscess formation or foci of consolidation with intense PMN accumulation in bronchioles and alveoli ○ Evidence of lung parenchyma invasion by fungal hyphae or pseudohyphae |
Specific Site Algorithm for Viral, Legionella, and other Bacterial Pneumonias with Definitive Laboratory Findings (PNU2)
| Imaging Test Evidence | Signs/Symptoms | Laboratory |
|---|---|---|
| Two or more serial chest imaging test results with at least one of the following (1,2,13): New and persistent or Progressive and persistent • Infiltrate • Consolidation • Cavitation • Pneumatoceles, in infants ≤1 year old Note: In patients without underlying pulmonary or cardiac disease (such as respiratory distress syndrome, bronchopulmonary dysplasia, pulmonary edema, or chronic obstructive pulmonary disease), at least one definitive chest imaging test result is acceptable. |
At least one of the following: • Fever (> 38.0°C or > 100.4°F) • Leukopenia (≤ 4,000 WBC/mm³) or leukocytosis (≥ 12,000 WBC/mm³) • adults ≥ 70 years old, altered mental status with no other recognized cause And at least one of the following: • New onset of purulent sputum (3) or change in character of sputum (4), or increased respiratory secretions, or increased suctioning requirements Dyspnea, or tachypnea (5), or new onset or worsening cough Rales (6) or bronchial breath sounds • Worsening gas exchange (for example, O2 desaturations [for example, PaO2/FiO2 ≤ 240] (7), increased oxygen requirements, or increased ventilator demand) |
At least one of the following: • Virus, Bordetella, Legionella, Chlamydia, or Mycoplasma identified from respiratory secretions or tissue by a culture or non-culture based microbiologic testing method which is performed for purposes of clinical diagnosis or treatment (for example, not Active Surveillance Culture/Testing (ASC/AST) • Fourfold rise in paired sera (IgG) for pathogen (for example, influenza viruses, Chlamydia • Fourfold rise in Legionella pneumophila serogroup 1 antibody titer to ≥ 1:128 in paired acute and convalescent sera by indirect IFA • Detection of Legionella pneumophila serogroup 1 antigens in urine by RIA or EIA |
Specific Site Algorithm for Pneumonia in Immunocompromised Patients (PNU3)
| Imaging Test Evidence | Signs/Symptoms | Laboratory |
|---|---|---|
| Two or more serial chest imaging test results with at least one of the following (1,2,13): New and persistent or Progressive and persistent • Infiltrate • Consolidation • Cavitation • Pneumatoceles, in infants ≤1 year old Note: In patients without underlying pulmonary or cardiac disease (such as respiratory distress syndrome, bronchopulmonary dysplasia, pulmonary edema, or chronic obstructive pulmonary disease), at least one definitive chest imaging test result is acceptable. |
Patient who is immunocompromised (see definition in footnote 10) has at least one of the following: • Fever (> 38.0°C or > 100.4°F) • For adults ≥ 70 years old, altered mental status with no other recognized cause • New onset of purulent sputum (3), or change in character of sputum (4), or increased respiratory secretions, or increased suctioning requirements • Dyspnea, or tachypnea (5), or new onset or worsening cough • Rales (6) or bronchial breath sounds • Worsening gas exchange (for example, O2 desaturations [for example, PaO2/FiO2 ≤ 240] (7), increased oxygen requirements, or increased ventilator demand) • Hemoptysis • Pleuritic chest pain |
At least one of the following: • Identification of matching Candida spp. from blood and one of the following respiratory specimens: sputum, endotracheal aspirate, BAL, or protected specimen brushing (11,12); blood specimen and respiratory specimen must have collection dates that occur within the same IWP • Evidence of fungi (excluding any Candida and yeast not otherwise specified) from minimally contaminated LRT specimen (specifically BAL, protected specimen brushing or endotracheal aspirate) from one of the following: - Direct microscopic exam - Positive culture of fungi - Non-culture diagnostic laboratory test OR Any of the following from: LABORATORY CRITERIA DEFINED UNDER PNU2 |
Funding Statement
Partial financial support in the form of a PG thesis grant, Ref Number ICMR Grant No: 3/2/Dec-2022/PG thesis –HRD.
REFERENCES
- 1.Rello J, Allam C, Ruiz-Spinelli A, Jarraud S. Severe Legionnaires’ disease. Ann Intensive Care. 2024;14:51. doi: 10.1186/s13613-024-01252-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Pareek A, Singhal R, Pareek A, Chuturgoon A, Sah R, Apostolopoulos V. Global surge of Legionnaires'disease in 2024: Urgent call for heightened awareness and preparedness. Lancet Microbe. 2025;6:101031. doi: 10.1016/j.lanmic.2024.101031. [DOI] [PubMed] [Google Scholar]
- 3.Kawasaki T, Nakagawa N, Murata M, Yasuo S, Yoshida T, Ando K, et al. Diagnostic accuracy of urinary antigen tests for Legionellosis: A systematic review and meta-analysis. Respir Investig. 2022;60:205–14. doi: 10.1016/j.resinv.2021.11.011. [DOI] [PubMed] [Google Scholar]
- 4.Helbig JH, Uldum SA, Bernander S, Lück PC, Wewalka G, Abraham B, et al. Clinical utility of urinary antigen detection for diagnosis of community-acquired, travel-associated, and nosocomial legionnaires'disease. J Clin Microbiol. 2003;41:838–40. doi: 10.1128/JCM.41.2.838-840.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Avni T, Bieber A, Green H, Steinmetz T, Leibovici L, Paul M. Diagnostic accuracy of PCR alone and compared to urinary antigen testing for detection of legionella spp.: A systematic review. J Clin Microbiol. 2016;54:401–11. doi: 10.1128/JCM.02675-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Ricci ML, Grottola A, Fregni Serpini G, Bella A, Rota MC, Frascaro F, et al. Improvement of Legionnaires'disease diagnosis using real-time PCR assay: A retrospective analysis, Italy, 2010 to 2015. Euro Surveill. 2018;23:1800032. doi: 10.2807/1560-7917.ES.2018.23.50.1800032. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Sabria M, Victor LY. Hospital-acquired legionellosis: Solutions for a preventable infection. Lancet Infect Dis. 2002;2:368–73. doi: 10.1016/s1473-3099(02)00291-8. [DOI] [PubMed] [Google Scholar]
- 8.Carratalà J, Mykietiuk A, Fernández-Sabé N, Suárez C, Dorca J, Verdaguer R. Health care-associated pneumonia requiring hospital admission-epidemiology, antibiotic therapy, and clinical outcomes. Arch Intern Med. 2007;167:1393–9. doi: 10.1001/archinte.167.13.1393. [DOI] [PubMed] [Google Scholar]
- 9.Fard S, Nomanpour B, Fatolahzadeh B, Mobarez A, Darban-Sarokhalil D, Fooladi A, et al. Hospital acquired pneumonia: Comparison of culture and real-time PCR assays for detection of Legionella pneumophila from respiratory specimens at Tehran hospitals. Acta Microbiol Immunol Hung. 2012;59:355–65. doi: 10.1556/AMicr.59.2012.3.6. [DOI] [PubMed] [Google Scholar]
- 10.Miyashita N, Kawai Y, Akaike H, Ouchi K, Hayashi T, Kurihara T, et al. Clinical features and the role of atypical pathogens in nursing and healthcare-associated pneumonia (NHCAP): Differences between a teaching university hospital and a community hospital. Intern Med. 2012;51:585–94. doi: 10.2169/internalmedicine.51.6475. [DOI] [PubMed] [Google Scholar]
- 11.Botelho-Nevers E, Grattard F, Viallon A, Allegra S, Jarraud S, Verhoeven P, et al. Prospective evaluation of RT-PCR on sputum versus culture, urinary antigens and serology for Legionnaire's disease diagnosis. J Infect. 2016;73:123–8. doi: 10.1016/j.jinf.2016.04.039. [DOI] [PubMed] [Google Scholar]
- 12.Mojtahedi SY, Rahbarimanesh A, Noorbakhsh S, Shokri H, Jamali-Moghadam-Siyahkali S, Izadi A. Urinary antigen and PCR can both be used to detect Legionella pneumophila in children's hospital-acquired pneumonia. Eur J Transl Myol. 2019;29:8120. doi: 10.4081/ejtm.2019.8120. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Sakhaee F, Mafi S, Zargar M, Vaziri F, Hajiesmaeili M, Siadat SD, Fateh A. Correlation between Legionella pneumophila serogroups isolated from patients with ventilator-associated pneumonia and water resources: A study of four hospitals in Tehran, Iran. Environ Sci Pollut Res. 2022;29:41368–74. doi: 10.1007/s11356-022-18867-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Chaudhry R, Dhawan B, Dey AB. The incidence of Legionella pneumophila: A prospective study in a tertiary care hospital in India. Trop Doct. 2000;30:197–200. doi: 10.1177/004947550003000405. [DOI] [PubMed] [Google Scholar]
- 15.Javed S, Chaudhry R, Passi K, Sharma S, Padmaja K, Dhawan B, et al. Sero diagnosis of Legionella infection in community acquired pneumonia. Indian J Med Res. 2010;131:92–6. [PubMed] [Google Scholar]
- 16.Angrup A, Chaudhry R, Sharma S, Valavane A, Passi K, Padmaja K, et al. Application of real-time quantitative polymerase chain reaction assay to detect Legionella pneumophila in patients of community-acquired pneumonia in a tertiary care hospital. Indian J Med Microbiol. 2016;34:539–43. doi: 10.4103/0255-0857.195353. [DOI] [PubMed] [Google Scholar]
- 17.Sreenath K, Dey AB, Kabra SK, Thakur B, Guleria R, Chaudhry R. Legionella pneumophila in patients with pneumonia at a referral hospital, New Delhi, India, 2015–2020. Am J Trop Med Hyg. 2020;104:854. doi: 10.4269/ajtmh.20-0653. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Anbumani S, Gururajkumar A, Chaudhury A. Isolation of Legionella pneumophila from clinical and environmental sources in a tertiary care hospital. Indian J Med Res. 2010;131:761–4. [PubMed] [Google Scholar]
- 19.Chaudhry R, Sreenath K, Arvind V, Vinayaraj EV, Tanu S. Legionella pneumophila serogroup 1 in the water facilities of a tertiary healthcare center, India. Emerg Infect Dis. 2017;23:1924. doi: 10.3201/eid2311.171071. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Jinna S, Gaikwad UN. Environmental surveillance of Legionella pneumophila in distal water supplies of a hospital for early identification and prevention of hospital-acquired legionellosis. Indian J Med Res. 2018;147:611–4. doi: 10.4103/ijmr.IJMR_527_17. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Centers for Disease Control and Prevention. Pneumonia (ventilator associated [VAP] and non-ventilator associated pneumonia [PNEU] event. 2025. [[Last accessed on 2025 Aug 27]]. Available from: https://www.cdc.gov/nhsn/pdfs/pscmanual/6pscvapcurrent.pdf .
- 22.Ranc AG, Carpentier M, Beraud L, Descours G, Ginevra C, Maisonneuve E, et al. Legionella pneumophila LPS to evaluate urinary antigen tests. Diagn Microbiol Infect Dis. 2017;89:89–91. doi: 10.1016/j.diagmicrobio.2017.06.013. [DOI] [PubMed] [Google Scholar]
- 23.Sánchez-Parra B, Núñez A, Moreno DA. Preventing legionellosis outbreaks by a quick detection of airborne Legionella pneumophila . Environ Res. 2019;171:546–9. doi: 10.1016/j.envres.2019.01.032. [DOI] [PubMed] [Google Scholar]
- 24.Gadsby NJ, Helgason KO, Dickson EM, Mills JM, Lindsay DS, Edwards GF, et al. Molecular diagnosis of Legionella infections–clinical utility of front-line screening as part of a pneumonia diagnostic algorithm. J Infect. 2016;72:161–70. doi: 10.1016/j.jinf.2015.10.011. [DOI] [PubMed] [Google Scholar]
- 25.Vallés J, Mesalles E, Mariscal D, del Mar Fernández M, Peña R, Jiménez JL, et al. A7-year study of severe hospital-acquired pneumonia requiring ICU admission. Intensive Care Med. 2003;29:1981–8. doi: 10.1007/s00134-003-2008-4. [DOI] [PubMed] [Google Scholar]
- 26.Bhopal RS. Geographical variation of Legionnaires'disease: A critique and guide to future research. Int J Epidemiol. 1993;22:1127–36. doi: 10.1093/ije/22.6.1127. [DOI] [PubMed] [Google Scholar]
- 27.Dagan A, Epstein D, Mahagneh A, Nashashibi J, Geffen Y, Neuberger A, et al. Community-acquired versus nosocomial Legionella pneumonia: Factors associated with Legionella-related mortality. Eur J Clin Microbiol Infect Dis. 2021;40:1419–26. doi: 10.1007/s10096-021-04172-y. [DOI] [PubMed] [Google Scholar]
- 28.Tan MJ, Tan JS, File Jr TM, Hamor RH, Breiman RF. The radiologic manifestations of Legionnaire's disease. Chest. 2000;117:398–403. doi: 10.1378/chest.117.2.398. [DOI] [PubMed] [Google Scholar]
- 29.Eison R. Legionella pneumonia: When to suspect, diagnostic considerations, and treatment strategies for hospital-based clinicians. Curr Emerg Hosp Med Rep. 2014;2:205–13. [Google Scholar]
- 30.Walker JT, McDermott PJ. Confirming the presence of Legionella pneumophila in your water system: A review of current Legionella testing methods. J AOAC Int. 2021;104:1135–47. doi: 10.1093/jaoacint/qsab003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Filice S, Sciuto EL, Scalese S, Faro G, Libertino S, Corso D, et al. Innovative antibiofilm smart surface against Legionella for water systems. Microorganisms. 2022;10:870. doi: 10.3390/microorganisms10050870. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Mathur P, Ningombam A, Soni KD, Aggrawal R, Singh KV, Samanta P, et al. Surveillance of ventilator associated pneumonia in a network of Indian hospitals using modified definitions: A pilot study. Lancet Reg Health Southeast Asia. 2024;28:100450. doi: 10.1016/j.lansea.2024.100450. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Tan MJ, Tan JS, File TM., Jr Legionnaires disease with bacteremic coinfection. Clin Infect Dis. 2002;35:533–9. doi: 10.1086/341771. [DOI] [PubMed] [Google Scholar]
- 34.Health Protection Agency. Hospital water. Guide to Infection Control in the Hospital. London: Health Protection Agency; 2012. pp. 200–15. [Google Scholar]