|Year : 2016 | Volume
| Issue : 4 | Page : 539-543
Application of real-time quantitative polymerase chain reaction assay to detect Legionella pneumophila in patients of community-acquired pneumonia in a tertiary care hospital
A Angrup1, R Chaudhry1, S Sharma1, A Valavane1, K Passi1, K Padmaja1, S Javed1, AB Dey1, B Dhawan2, SK Kabra3
1 Department of Microbiology, All Institute of Medical Sciences, New Delhi, India
2 Department of Medicine, All Institute of Medical Sciences, New Delhi, India
3 Department of Pediatrics, All Institute of Medical Sciences, New Delhi, India
|Date of Submission||18-Apr-2015|
|Date of Acceptance||20-Sep-2016|
|Date of Web Publication||8-Dec-2016|
Department of Microbiology, All India Institute of Medical Sciences, New Delhi
Source of Support: None, Conflict of Interest: None
Legionella pneumophila is one of the important pathogen responsible for community –acquired pneumonia attributing for 1-5% of cases. Since early and accurate therapy reduces mortality, rapid and reliable diagnostic methods are needed. A total of 134 samples of blood, urine and respiratory tract fluids were collected. Blood was tested for IgG, IgM and IgA antibodies using commercially available kits. A total of 8 (6%) samples were found to be positive for L. pneumophila by quantitative reverse transcription polymerase chain reaction (qRT-PCR), compared to conventional PCR where 6 (4.4%) samples were positive. Serology was positive in a total of 32 (23%) cases though only 3 (2.2%) of the PCR-positive cases were positive by serology as well. These results suggest that real-time PCR can detect Legionella infection early in the course of the disease before serological response develops.
Keywords: Community-acquired pneumonia, Legionella pneumophila, real-time polymerase chain reaction
|How to cite this article:|
Angrup A, Chaudhry R, Sharma S, Valavane A, Passi K, Padmaja K, Javed S, Dey A B, Dhawan B, Kabra S K. 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
|How to cite this URL:|
Angrup A, Chaudhry R, Sharma S, Valavane A, Passi K, Padmaja K, Javed S, Dey A B, Dhawan B, Kabra S K. 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 [serial online] 2016 [cited 2017 Nov 22];34:539-43. Available from: http://www.ijmm.org/text.asp?2016/34/4/539/195353
| ~ Introduction|| |
Community-acquired pneumonia (CAP) is a serious life-threatening disease, in which the etiologic agent cannot be identified in more than 50% of patients despite advanced diagnostic methods. Legionella is one of the important pathogens of community-acquired pneumonia (CAP) responsible for 1–5% of cases.Legionella often presents with a rapidly progressive severe form of pneumonia. Mortality is evaluated at approximately 30% and may be affected by the timing and choice of appropriate antibiotic therapy. Diagnostic delay may also result in increased mortality because of the delay in taking therapeutic decision. Very few studies have been reported from India.
Diagnosis of Legionnaire's disease (LD) is mainly based on culture, antigen detection in urine and antibody detection in serum. Isolation of Legionella from respiratory secretions is considered the gold standard in case definition, but it is not very sensitive (10–80% sensitivity) and a positive culture is not normally available until 3 days postincubation.,
Rapid tests, such as direct fluorescent antibody stains and urinary antigen assays, have been developed., Although useful, these assays have sensitivities <100%., A diagnosis by a four-fold IgG or IgM titre increase can only be made retrospectively and rarely influence the initial treatment of the patient., Thus, there is a need for the development of a rapid test for the early diagnosis of Legionellosis. Nucleic acid amplification assays have been shown to be useful for the detection of Legionella. Real-time quantitative polymerase chain reaction (PCR) method is less time-consuming (4–5 h), it has decreased risk of false positive results, and it is able to quantify the amplified product.
The aim of this study was to diagnose Legionella infection based on real-time PCR as a rapid diagnostic method and to compare the advantages of different PCR assays with other conventional testing methods in patients with atypical pneumonia due to Legionella pneumophila. The study was approved by the Institutional Ethical Committee.
| ~ Methodology|| |
A total of 134 patients admitted between May 2005 and August 2008 and clinically diagnosed with atypical pneumonia in our hospital were included in the study after obtaining informed consent. The samples consisting of blood, urine and respiratory tract fluids (107 throat swabs, 19 nasopharyngeal aspirates, 6 endotracheal aspirates and 2 bronchoalveolar lavages) and 37 sputum samples were collected. Blood samples were also taken and the serum separated was stored at −20°C until use.
- CAP was defined as presence of at least one of the major clinical criteria (cough, sputum production and fever >37.8°C) or two of the minor criteria (pleuritic chest pain, dyspnoea, altered mental state, sign of pulmonary consolidation on examination or total leucocyte count of 12,000/µl)
- Presence of a new pulmonary infiltrate/shadow on chest X-ray suggestive of pneumonia at/within 24 h of hospitalisation
- Patient residing in community.
- Hospital-acquired pneumonia, i.e., pneumonia that developed 72 h after hospitalisation or within 7 days of discharge
- Pulmonary shadow due to a cause other than pneumonia.
Culture and genomic DNA extraction
The L. pneumophila subsp. pneumophila strain Philadelphia ATCC33152 was used as standard stain and receivedaccording to the ATCC guidelines. It was confirmed by catalase, oxidase and Hippurate hydrolysis test. DNA extraction was done by the boiling method and stored at − 20°C until used.
Polymerase chain reaction amplification
For L. pneumophila specific amplification, we used published primers. The target sequence for amplification was a 399 bp segment of the gene coding for the macrophage infectivity potentiator (MIP) protein. The final volume of the PCR mixture was 25 µl, with 1× PCR buffer, 1.5 mM MgCl2, 200 µM dNTPs, 20 pM of each primer, 1 unit of Taq polymerase (Bangalore Genei) and 3 µl of extracted DNA. The thermal cycling profile had initial denaturation for 5 min, 35 cycles of amplification, each of 94°C for 1 min, 55°C for 50 s and 72°C for 50 s and a final elongation step of 10 min at 72°C. A negative control was systematically run in parallel. This PCR reaction had been standardised for the detection of L. pneumophila in patient samples. When primers for amplification of 399 bp fragment were used to test patient sample, 19% (25 sample) of the sample were found to be positive out of a total of 134 sample. For conformation, some PCR positive samples were sent for sequencing. Sequencing results and blast results at the National Centre of bio technology information site, revealed that forward prime was crass- reading with pseudomonas spp. Thus, a new forward prime toward 3' end of pervious primer was designed [Table 1].
|Table 1: Polymerase chain reaction and real-time polymerase chain reaction primers for macrophage infectivity potentiator gene of Legionella pneumophila|
Click here to view
This new forward primer along with the reverse primer produced an amplicon of 375 bp fragment [Figure 1]. This 375 bp fragment was cloned in pGEM-T Easy Vector and used for the standardisation of real-time PCR assay.
|Figure 1: Gel photograph of polymerase chain reaction amplification product with new forward primer for Legionella pneumophila from genomic DNA. Lane 1: Marker (100 bp Plus ladder), Lane 2: Sample number 3, Lane 3: Positive control polymerase chain reaction amplification product, Lane 4: negative control|
Click here to view
Analysis of amplified samples
The PCR products were analysed on a 1% agarose gel. Electrophoresis was run in 0.5× TBE buffer followed by ethidium bromide staining.
Standardisation of real-time polymerase chain reaction
Real-time PCR assay was performed to prepare the standard curve using L. pneumophila positive control DNA template. The positive control template is the 375 bp fragment of mip gene cloned in pGEM-T Easy vector. The concentration of positive control plasmid DNA was 120 ng/µL and the copy number with the above formula was found to be 3 × 1010 copies/µL. The copy number calculation was done using the following formula:
Where, std: Standard, MW: Molecular weight.
The primers for real-time assay synthesise a 73 bp fragment internal to the 375 bp “mip gene” which is detected by Fam-dye labelled reporter sequence (Applied Biosystems, USA). The primer and reporter sequences are given in [Table 1]. Along with the positive reactions of dilutions of template DNA, a negative (no template) control was also included. The reactions were performed in a final volume of 20 µl containing 1 µl 20× assay mix (Primers and Probe), 10 µl 2× TaqMan ® Universal Master Mix (enzyme, buffer and dNTPs) and 9 µl DNA diluted in RNase-free water. Amplification and product detection were performed with the ABI Prism™ 7700 Sequencing Detection System. Once the reaction for standard curve was finalised, similar reactions were performed for DNA extracted from patient throat swab samples.
Serology was performed from blood samples for L. pneumophila. All the antibodies IgG, IgM, and IgA were detected by ELISA as reported earlier.
Legionella urinary antigen was detected in urine samples using the Legionella urinary antigen detection assay Microwell ELISA (IVD Research, USA) and BinaxNOW Legionella urinary antigenic kit (Binax, Inc. Portland, ME, USA) following the manufacturer's instructions. The results of the urine antigen test have been published in another article entitled 'Serodiagnosis of Legionella infection in community-acquired pneumonia'. Hence, we have not included those results in this article as we are focussing on the utility of real-time PCR in diagnosis.
Fisher's exact and Pearson's Chi-square tests were used to analyse clinical signs and symptoms and demographic data (age and sex).
| ~ Results|| |
Of the 134 samples collected from the people suffering from atypical pneumonia admitted in different wards of All India Institute of Medical Sciences, 92 (69%) were males and 42 (31%) were females. The study group included 37 (28%) paediatric and 97 (72%) adult patients. There were 5 (13.8%) paediatric patients and 27 (27.5%) adults positive for L. pneumophila by any test done in the study. Overall, real-time PCR assay, PCR and serology in combination could detect L. pneumophila infection in 32 patients. Detailed serological data for L. pneumophila in the same samples have been reported in an earlier paper.
Among 134 samples, none was positive when sputum/nasopharyngeal aspirate/tracheal aspirate/broncoalveolar lavage was streaked on BCYE agar.
Out of 134 patients, 32 (23%) were positive for antibodies against L. pneumophila. IgG was positive in 9.70% (13) cases, IgM was positive in 13.43% (18) cases and IgA was positive in 11.94% (16) cases. This serology data have already been reported in an earlier paper. Of the serologically positive cases, only three were positive by PCR. In these patients, IgM was seen in 3 (2.2%), IgA in 1 (0.74%) and IgG was not seen in any of the serologically positive cases.
Polymerase chain reaction
For L. pneumophila PCR was standardised and was done from genomic DNA extracted from ATCC strain no 33152 cloned in pGEM-T Easy Vector. Of total 134 samples, 4.5% (6) were positive with new forward primer, whereas with the previous forward primer, a non-specific reaction was obtained [Table 1].
Real-time polymerase chain reaction
Results obtained in real-time PCR were expressed as threshold cycle values, corresponding to the cycle at which PCR entered the exponential phase. If no increase in fluorescent signal was observed after fifty cycles, the sample was assumed to be negative.
In the real-time PCR assay for L. pneumophila, standard curves were obtained. DNA extract from patient's respiratory sample as tested with each of the standardized assays. Quantitative reverse transcription PCR (qRT-PCR) was positive in 8 of the 134 (5.9%) samples as compared to conventional PCR where 6 (4.4%) samples were positive. Serology was positive in only 3 (2.2%) of the PCR-positive cases.
Clinical signs and symptoms
Majority of the patients diagnosed as L. pneumophila based on serology, and qRT-PCR assay had cough, fever and dyspnoea. There was no significant association between the signs and symptoms in patients with serology positive results and in those with real-time PCR positive results. Three-quarters of the patients had some radiological abnormalities such as consolidation and infiltrates, which is a common feature of not only atypical pneumonia by Legionella pneumonia but also can be seen as a feature in other diseases.
| ~ Discussion|| |
Numerous observational studies of patients with CAP requiring hospitalisation have documented that the incidence of LD ranges from 2% to 9%.,,,, A recent Australian study has found that 3.4% of CAP was caused by Legionella, while another study from the UK reported the corresponding figure to be 3%. In a study of 46 patients with CAP from Malaysia, 2.1% of patients were positive by real-time PCR. Our study showed Legionella infection in 5.9% of patients with CAP by real-time PCR.
Although potentially all Legionella spp. may cause human disease, the majority (92%) of clinical cases are caused by L. pneumophila. Microbiological diagnosis is very important since LD is clinically not distinguishable from other pneumonias. Timely and appropriate treatment improves the prognosis and can be achieved by rapid diagnosis.
Many bacterial and viral infections often share clinical features and symptoms which are difficult to distinguish clinically. A sensitive and effective method of detecting these agents is required so that the correct treatment is offered and unnecessary use of antibiotics can be avoided. Although culturing remains the gold standard for the diagnosis of legionellosis, its sensitivity may be limited in clinical routine laboratories.
In the present study, a real-time PCR assay targeting the mip gene was performed along with serology and conventional PCR. qRT-PCR was positive in 8 of the 134 (5.9%) samples as compared to conventional PCR where 6 (4.4%) samples were positive. However, out of the 8 cases, positive by qRT-PCR, 5 were negative by serology. Of these 8 cases, IgM antibody was detected in 3 cases whereas IgA in one case and IgG was absent. Thus, real-time PCR was able to pick up even early cases, in which the serological response had yet not developed. During the early period of infection, organism load is usually high in the throat swab sample but detectable amount of antibodies is not generated, and hence such samples are missed by serological methods.
The majority of patients admitted were referred from other hospitals and were already on antibiotics. This could be a reason why the PCR was negative even though the serology was positive. If mycoplasmacidal antibiotic has been administered, PCR results may be negative even though the serology is positive. Furthermore, in most of the cases, the sample was sent late in the course of the disease when all other causes had been ruled out. In such cases, the organism has already cleared while the antibody response is still present.
Mycoplasma pneumoniae and L. pneumophila coinfection was seen in none of the cases by PCR and in one (0.74%) case by serology. In a study by Nomanpour et al. from Tehran, both real-time PCR and culture methods were applied on 129 respiratory specimens from patients with CAP. The results of real-time PCR were compared with those obtained by culture. Real-time PCR and culture found 11 (8.5%) and 9 (6.9%) specimens, respectively, as positive for L. pneumophila. In our study, legionellae were not isolated in any of the respiratory tract samples by culture. The reason for negative cultures could be the prior treatment of the patients with antimicrobials which decreases the chance of isolating the microorganism. Diagnosis by culture of respiratory tract secretions requires 3–5 days of incubation. Laboratories in India routinely do not attempt culture for Legionella spp. Studies from other countries reported the sensitivity of culture from respiratory tract secretions varying from 10% to 80%.
Cough (100%), fever (81.2%) and dyspnoea (59.3%) were the most common presenting features. There was no significant association between the signs and symptoms in patients with serology positive results and in those with real-time PCR positive results. In our earlier study, we reported the presence of extrapulmonary symptoms such as diarrhoea, abdominal pain and headache in patients with positive serology for L. pneumophila but no such significant findings were seen in this study. Three-quarters of the patients had some radiological abnormalities such as consolidation and infiltrates, which is a common feature of not only atypical pneumonia by Legionella pneumonia but also can be seen as a feature in other diseases, and hence for these reasons confining laboratory testing for this organism specifically to high-risk patients will overlook a notable number of cases, and our study also supports the practice of placing more emphasis on ascertaining the aetiology of pneumonia. The predictive value of clinical findings such as X-ray results, respiration rate and white blood cell count is low for diagnosing CAP and initiating empirical antibiotic therapy.
Although patchy infiltration is reported as a common radiological finding in our study, 25% of PCR positive patients had right lower lobe consolidation. PCR and urinary antigen test are able to provide a diagnosis in a time frame that are helpful in the acute stage of the disease. Because urinary antigen detection, assays detect only a limited number of serogroups of L. pneumophila, so total dependence on this diagnostic assay is not highly recommended. In areas where legionellae other than L. pneumophila serogroup I are important pathogens, urinary antigen tests are still useful but should not be used as the sole diagnostic tool.
This study is first of its kind from India, in which qRT-PCR, PCR along with three different classes of antibodies have been done in all the cases of CAP directly from clinical specimens, whereas none of the earlier studies have looked for all these battery of tests simultaneously in the same set of samples. Timely diagnosis of pathogens involved in CAP is required in early initiation of specific antibiotic therapy as the therapeutic approach to CAP due to atypical organisms is different from typical CAPs. In conclusion, the RT-PCR can help in early detection of L. pneumophila in respiratory samples when serology is still negative.
| ~ Acknowledgements|| |
Authors thank Shri Pramod Kumar, for technical support and acknowledge Department of Biotechnology, Ministry of Science and Technology, New Delhi, India, for providing financial support.
Financial support and sponsorship
The study was supported by Department of Biotechnology, Ministry of Science and Technology, New Delhi, India.
Conflicts of interest
There are no conflicts of interest.
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