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Pneumocystis jirovecii an opportunistic fungus is one of the common cause of respiratory infection in immunocompromised individuals. Pneumocystis pneumonia (PCP) is conventionally diagnosed by identifying Pneumocystis jirovecii in lower respiratory specimens using microscopy. Molecular diagnosis of PCP is an established sensitive technique with improved results. Use of hot-start touchdown PCR for the diagnosis of PCP has been evaluated in the present study.
Methods: A prospective study was conducted using rapid DNA extraction protocol followed by Hot-start touchdown polymerase chain reaction (TD-PCR) assay, targeting mitochondrial large sub-unit rRNA (mtLSU rRNA) gene to qualitatively determine P. jirovecii in bronchoalveolar lavage fluid (BALF) from both Human Immuno Deficiency Syndrome (HIV) seropositive and non-HIV immunosuppressed individuals, and to compare this assay with single round conventional PCR targeting the same gene of P. jirovecii.
Results: Based on the microscopic examination of 230 BAL fluid specimens, only 5 individuals were PCP positive. Whereas, significant differences in the positivity of PCP cases were observed using, single round conventional PCR (21/230) and Hot-start touchdown PCR (25/230).
Conclusion: The rapid DNA extraction protocol used in this study and mt LSU rRNA Hot-start touchdown PCR assay detects P. jirovecii DNA in BAL fluid with a much better yield. Thus, detection of P. jirovecii DNA by Hot-start TD-PCR assay may be a useful PCR method for diagnosis of PCP.
Over the last three decades Pneumocystis jirovecii infection has been established itself from relatively rare pathogen to one of the common causes of pneumonia in immunocompromised hosts. Pneumocystis pneumonia (PCP) is caused by Pneumocystis jirovecii, formerly known as P. carinii f.sp. hominis (1). With the emergence of HIV-1 pandemic, PCP emerged as one of the most common Acquired Immuno-Deficiency Syndrome (AIDS) defining disease in industrialized countries.
PCP being one of the initial AIDS defining manifestations, the diseases has also been associated in other non-AIDS defining hosts such as patients with malignancy, transplant recipients and patients receiving immunosuppressive therapy. Before, systematic PCP prophylaxis was introduced (2), PCP was observed as the AIDS defining event in 60% of HIV-1 infected patients and it was estimated that up to 80% of patients with CD4 counts less than 200 would eventually develop PCP. Following the introduction of primary and secondary PCP prophylaxis in the early nineties, a decline in the incidence of AIDS related PCP was observed, which further declined after the introduction of highly active antiretroviral therapy (HAART) in the mid-nineties (3). Nevertheless, PCP continues to be a common disease with substantial morbidity and mortality. In the industrialized world, PCP remains one of the most common AIDS defining events (4, 5), especially in patients unaware of being HIV-infected or for reasons that denied proper medical care. As PCP is a diagnostic challenge, the infection was previously under-diagnosed in many developing countries and exact scenario was not known. With increased awareness and recognition, PCP has been seen as one of the important cause of pneumonia in HIV-1 infected individuals (6, 7). Earlier study done in our settings revealed that special attention and treatment among HIV infected and post renal transplant recipients are needed (8).
The confirmatory diagnosis of PCP is generally established by direct demonstration of Pneumocystis organisms from respiratory specimens using the Giemsa and Grocott's Gomori methenamine silver staining (GMS). However, sensitivity of the staining techniques, is reported to be 70-92% in bronchoalveolar lavage (BAL) specimens, but suffers from low yield from aspirates and induced sputum samples 35-78 % (9). As only few organisms can be detected in sputum and the sensitivity of microscopic examination of induced sputum being variable, the ideal sample for PCP continues to be bronchoalveolar lavage (BAL) fluid.
Over a period of last two decades, use of polymerase chain reaction (PCR) assay has considerably increased the sensitivity of detection of Pneumocystis, which is reported to be 86-100 % in BALF (10, 11). The results of the various studies indicate that PCR is more sensitive than the conventional microscopic examination methods for the detection of P. jirovecii in human samples (12, 13). In spite of the increased sensitivity of PCR assays for the detection of P.Â jirovecii, these assays are not in routine use in many clinical microbiology laboratories. Lack of standardized assay and labour-intensive methods for nucleic acid extraction and detection of amplified products contribute to the difficulty in using PCR-based assays for the detection of P.Â jirovecii in the clinical laboratory.
At present, a highly sensitive technique is required for the detection of P. jirovecii in patients receiving chemoprophylaxis, because chemoprophylaxis decreases the yield of microscopic methods (14). Furthermore, empirical administration of trimethoprim-sulfamethoxazole may also result in decreased microbial load that can not be detected with microscopy.
Larsen et.al. in 1998 reported that a new single-round "touchdown" PCR method had added sensitivity and specificity for the diagnosis of PCP in persons with AIDS (15). The same had been validated by Horra et al. in 2006 by comparing single and touchdown PCR protocols for detecting Pneumocystis jirovecii DNA in paraffin-embedded lung tissue samples (16). In the present prospective study, a rapid Hot-start touchdown PCR assay was performed after a simple DNA extraction procedure with commercially available DNA extraction kit (Sigma, USA) for the detection of P.Â jirovecii in BAL specimens submitted to our laboratory from north Indian population.
MATERIALS AND METHODS
Patients and clinical specimens
The study population included individuals infected with HIV-1, transplant recipients, and immunocompromised individuals due to other illnesses. Patients with clinical manifestations such as dyspnoea more on exertion, cough, fever, and possibly accompanied by abnormal chest signs and/or abnormal chest radiographs were studied intensively. The other relevant data included CD4+ lymphocyte count, arterial blood gas analysis (ABG), microbiological investigations of the BAL specimens for bacterial cultures and anti-PCP prophylaxis. A total of 230 BALF samples were included in the study. 198Â (BALF) specimens, obtained prospectively from 190Â patients submitted to our laboratory and 32 BALF obtained from patients other than clinical diagnosis of PCP were taken as controls. Specimens were examined by microscopy using GMS staining and PCR. This study was approved by the Institutional Ethical committee.
The respiratory samples were immediately transported to the laboratory on ice and stored at 4Â°C until processed. The BAL samples obtained from patients were further divided into aliquots and were concentrated by centrifugation at 4000 rpm for 10 min. 100 Âµl of this suspended pellet was used for preparation of smears for microscopic examination using GMS staining. The remaining portions of the sediments were stored at -20Â°C and were subsequently used for PCR assays.
DNA extraction and Hot start touchdown PCR.
Extraction of DNA from clinical sample was carried out using commercially available kit and as per manufacturer's instruction (Sigma, USA). Briefly, 100 Âµl of extraction solution was pipetted out into a microcentrifuge tube supplemented with 25 Âµl of tissue preparation solution. Thereafter, 10 Âµl of the specimen sediment was added to the solution and was mixed thoroughly by vortexing. The incubation was done at room temperature for 10 min, followed by further incubation at 95Â°C for 3 min. Finally, 100 Âµl of neutralization solution was added to the sample and mixed by vortexing. Neutralized sample extract was stored at 4Â°C or tested immediately using PCR assay.
The RED Extract-N-Amp PCR Reaction Mix contained Jumpstart Taq antibody for specific hot-start amplification. PCR amplification and standardization were achieved using DNA obtained from microscopically proven positive cases of Pneumocystis pneumonia as positive control. The set of primers described by Wakefield et al. (17, 18) i.e. part of the mitochondrial gene encoding for the large subunit of rRNA were chosen as it has been shown by several studies that mtLSU rRNA gene PCR is the most specific and sensitive assay. The following pairs of primers were selected i.e. pAZ102-E (5' -GATGGCTGTTTCCAAGCCCA-3') and pAZ102-H (5'-GTG TACGTTGCAAAGTACTC-3').Primers were synthesized commercially by Bio-Link and HP Scientifics, New Delhi, India. For performing PCR assay, the reaction mixture containing 10 Âµl of REDExtract-N-Amp PCR Reaction mixture, 0.4 Âµl each of forward and reverse primer, 4 Âµl of tissue extract (DNA) and remaining double distilled PCR grade water was added to make a total volume of 20 Âµl without the loading dye. Following PCR parameters were used - initial single round PCR of the mtLSU rRNA fragment was performed by preheating for 5 min at 94 Â°C, and for 40 cycles consisting of 30 sec at 92 Â°C for denaturation, 30 sec at 55 Â°C for annealing, and 1 min at 72 Â°C for elongation. A final extension step of 5 min at 72 Â°C was done in the reaction procedure. Hot-start, TD-PCR was performed using an initial preheating step for 10 min at 95 Â°C, a touchdown for 15 sec at 94 Â°C. The annealing step was at 65 Â°C for 30 sec and temperature was decreased to 55 Â°C per cycle during the first 10 cycles, and then treated for 15 sec at 72 Â°C. The subsequent 30 cycles were performed for 15 sec at 92 Â°C, 30 sec at 55 Â°C and 15 sec at 72 Â°C.A final extension period of 5 min at 72 Â°C. The PCR assay was performed using ABI 2720, thermocycler (Applied Biosystem, USA). The product size for mt LSU rRNA gene PCR amplification reaction was 346 bp (Fig. 1). The amplified DNA was then directly loaded onto a 1.5% agarose gel after the PCR assay was completed.
To avoid contamination, each step (reagent preparation, DNA extraction and amplification) was performed in separate rooms, within the laboratory, with different sets of micropipettes and using filter tips. PCR mixtures and the extraction step were performed in a laminar-flow cabinet. To monitor for possible contamination, negative control (ultra-pure distilled water) was included in each PCR experiment. Following DNA extraction, Hot-start TD-PCR and single round conventional PCR assays were performed in a blinded manner and the extracted DNA were tested at dilutions of 1:1, 1:5, 1:25, and 1:125 (Fig. 2) in triplicates. Specificity was checked using eight microorganisms. Table 1.
DNA Extraction: The extraction of DNA for PCR amplification from BAL specimens could be completed within 15 minutes.
Hot start Touchdown PCR with BAL specimens:
Hot start Touchdown PCR results were obtained within (3hours) in comparison to single round PCR which takes 5.5hr. The results for the 230 (198+32) BAL specimens tested are given in Table 2. Out of 198 samples, only five were positive by microscopy. Hot-start touchdown PCR results were in concordance with microscopy for 173 specimens (87.8%). 20(10%) BAL samples were negative by staining and positive by Hotstart touchdown PCR and sixteen were positive by single round conventional PCR. All 32 specimens collected from individuals suspected of chest infection other than PCP were found to be negative by both microscopy and PCR.
These 25 BAL samples were obtained from 24 patients. Upon retrospective review of their medical records it was observed that all of them presented with pulmonary symptoms such as dry non-productive cough, dyspnoea on exertion and fever. On chest radiographs examinations interstitial pneumonitis was observed in nine patients and HRCT suggestive of ground glass appearance were in seven additional patients. Seventeen patients (82%) had a history of prior anti-PCP prophylaxis (trimethoprim-sulfamethoxazole). Out of these twenty five patients, two did not respond to trimethoprim-sulfamethoxazole, but responded to clindamycin. This treatment was continued during and after the episode of acute respiratory illness for a period of two weeks. Two HIV seropositive patients had co-infection with tuberculosis and three post-renal transplant patients had co-infection with cytomegalovirus (CMV) infection. Out of 24 patients, 19 had clinical improvement. Three patients died one was HIV seropositive, two were PRT patients. One post renal transplant patient previously responded to the treatment but during the second episode of PCP there was no response to the regimen. No medical record was obtained for one of the out patient department HIV positive patients having PCP on molecular examination of BAL specimen.
Sensitivity and specificity of the assay:
The sensitivity of the designed hot start touchdown PCR assay was evaluated by making serial dilutions of the DNA provided by Dr. Phillipe Hauser and was compared with the single round conventional PCR targeting the mt LSU rRNA gene. With a hot start touchdown PCR assay specific for Pneumocystis, it was possible to detect specifically Pneumocystis DNA in 2.5 hr, with lower limits of detection of 1:125 of template DNA of Pneumocystis using a gel electrophoresis without using any tracking dye.
The specificity of the assay was also assessed by using DNA of the microorganisms as listed in table 1. A positive control containing the nucleotide sequence of the mt LSU rRNA gene of P. jirovecii (GenBank accession no. AB266392) was amplified by the Hot-start touchdown PCR method. The assay using these primers for P. jirovecii did not show cross-reactivity with other related fungal and some bacterial microorganisms.
The sensitivity, specificity, for the hot start touchdown PCR test were 100 and 100%, and for conventional single round PCR were 84 and 100% respectively.
In spite, of the intensive efforts to understand the life cycle of P. jirovecii, many aspects of Pneumocystis biology has still been incomplete because of lack of culture methods. Important insights into the biology of the organism have been obtained the advancing knowledge of its genomic set up. These advances lead to the development of molecular diagnostic approaches for P. jirovecii.
In conventional PCR, the Taq DNA polymerase is active at room temperature and to a lesser degree, even on ice. In some instances, when all the reaction components are put together, nonspecific primer annealing can occur due to these low temperatures. This nonspecific primer annealing can then be extended by the Taq DNA polymerase, generating nonspecific products as well as low product yields. In case of nested PCR there are chances of crossover contamination and is tedious and time consuming. On the other hand Touchdown (TD) PCR offers a simple and rapid means to optimize PCRs, by increasing specificity, sensitivity as well as yield, without the need of lengthy optimizations and/or in redesigning the primers. TD-PCR employs an initial annealing temperature above the projected melting temperature (Tm) of the primers being used, then progressively transitions to a lower, more permissive annealing temperature over the course of successive cycles (19). Although TD-PCR offers a simple way to enhance PCR efficiency, but some times it may fail to generate product, multiple products or products of the incorrect size. To circumvent these problems, hot-start PCR was performed in conjunction with TD-PCR cycling parameters (19). Hot Start PCR significantly reduces nonspecific priming, the formation of primer-dimers and often increases the product yields (20). Classical methods of hot start PCR involves either addition of a reversible inhibitor of the polymerase or by omission of an essential reaction component before the initial denaturating step. It involves additional handling and increased risk of contamination.
To circumvent, all these issues, Jumpstart Taq polymerase has been used. In this kit Taq DNA polymerase activity has been inactivated by combining the enzyme with JumpStart Taq antibody, a neutralizing monoclonal antibody to Taq DNA polymerase. The antibody inactivation provides a simple, efficient procedure for hot start PCR. So, that polymerase activity is completely restored during the first denaturation step of thermal cycling.
In this present study, RED Extract-N-Amp PCR was used for the first time to extract P. jirovecii DNA from respiratory clinical specimens. Our results suggest that RED Extract-N-Amp PCR is as effective as proteinase K-phenol chloroform for obtaining DNA from P. jirovecii. RED Extract-N-Amp PCR assay simplified and shortened PCR template preparation by avoiding procedures such as digestion with proteinase K, purification with phenol-chloroform, and ethanol precipitation, which may likely induce contamination and some times may lead to the loss of DNA material.
The length of the hot start touchdown PCR assay was shortened to 2 hrs, instead of 3 or 3.5 hrs in conventional single round PCR without the loss of sensitivity.
The primers described by Wakefield et al. (17) were chosen because different investigators have shown that mitochondrial rRNA gene PCR is the most specific and sensitive single step PCR assay for the detection of P. jirovecii (21, 22). The increased sensitivity (up to 100 times) that we observed between single step and hot start touchdown-step PCR assay is equivalent or even more than the increased sensitivity produced by different modifications of PCR and confirmed the findings of other investigators. The results of hot start touchdown single step PCR coupled with agarose gel electrophoresis in the present study is in concordance with the other studies (23).
Additional 20 cases of P. jirovecii were detected by hot start touchdown PCR assay which were negative by staining. The high degree of sensitivity of modified PCR protocols has also shown its usefulness in detection of P. jirovecii several weeks before or after (15) an episode of microscopically proven PCP. These findings are consistent with those for experimental pneumocystosis, which have demonstrated that PCR becomes positive before the results of cytological and histological studies are positive (15) and remains positive much longer (16). Similar results were obtained in two of our patients.
In conclusion, we have evaluated a rapid DNA extraction protocol, simple, and sensitive Hot start touchdown PCR which may be useful in clinical laboratories for the routine diagnosis of PCP with BAL specimens. In addition, clinical samples containing low numbers of the P. jirovecii, might be difficult or some times even impossible to detect using conventional single round PCR. In such cases Hot start touchdown- PCR protocol appear to significantly enhance the probability of detecting P. jirovecii.