LGCmain
Indian Journal of Pathology and Microbiology
Home About us Instructions Submission Subscribe Advertise Contact e-Alerts Ahead Of Print Login 
Users Online: 1496
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size
IJPM is coming out with a Special issue on "Genitourinary & Gynecological pathology including Breast". Please submit your articles for these issues


 
  Table of Contents    
ORIGINAL ARTICLE  
Year : 2011  |  Volume : 54  |  Issue : 2  |  Page : 326-329
Role of PCR for diagnosing Pneumocystis jirovecii pneumonia in HIV-infected individuals in a tertiary care hospital in India


1 Department of Microbiology, Kasturba Medical College, Manipal, Karnataka, India
2 Department of Medicine, Kasturba Medical College, Manipal, Karnataka, India

Click here for correspondence address and email

Date of Web Publication27-May-2011
 

   Abstract 

Objectives : In developing countries like India, the diagnosis of Pneumocystis jirovecii infection is often made either by conventional staining or clinically. This study was planned to know the utility of polymerase chain reaction (PCR) in diagnosing Pneumocystis jirovecii pneumonia (PJP) in human immunodeficiency virus (HIV)-infected patients, to compare the PCR results with that of staining techniques and also to correlate the results with clinical condition of patients. Materials and Methods: A prospective study included 50 HIV-infected adult in-patients with symptoms of lower respiratory tract infection. Induced sputum, bronchoalveolar lavage or tracheal aspirate were proceeded for both staining and PCR for mitochondrial large subunit rRNA gene of P. jirovecii. Results: In our study PCR results correlated with staining findings in 14% (7/50) of cases. Another 20% (10/50) cases could be diagnosed only with PCR, where staining was negative for the presence of P. jirovecii. When compared with clinical evidence of disease, PCR showed 93.7% sensitivity and 94.1% specificity. Presence of dyspnea and CD 4 count showed statistical significance (P<0.05) in PCP-diagnosed patients. Conclusions: PCR can be used for early and accurate diagnosis of PCP in HIV-infected patients.

Keywords: HIV-infected, polymerase chain reaction; Pneumocystis jirovecii pneumonia

How to cite this article:
Chawla K, Martena S, Gurung B, Mukhopadhyay C, Varghese GK, Bairy I. Role of PCR for diagnosing Pneumocystis jirovecii pneumonia in HIV-infected individuals in a tertiary care hospital in India. Indian J Pathol Microbiol 2011;54:326-9

How to cite this URL:
Chawla K, Martena S, Gurung B, Mukhopadhyay C, Varghese GK, Bairy I. Role of PCR for diagnosing Pneumocystis jirovecii pneumonia in HIV-infected individuals in a tertiary care hospital in India. Indian J Pathol Microbiol [serial online] 2011 [cited 2019 Dec 15];54:326-9. Available from: http://www.ijpmonline.org/text.asp?2011/54/2/326/81624



   Introduction Top


Pneumocystis jirovecii, formerly known as Pneumocystis carinii f. sp. hominis, remains an important cause of serious opportunistic infection in immunocompromised patients. [1] The incidence of Pneumocystis jirovecii pneumonia (PJP) was huge after the emergence of AIDS in early 1980s, [2] but has showed a decline after the initiation of prophylactic treatment with co-trimoxazole in 1990s. [3] Though many studies done earlier have shown that incidence remains high in western countries, a recent study has demonstrated an increase in incidence in the developing countries also. [4] In most of the centers in India, the diagnosis of PJP is done by conventional microscopy which may lead to underreporting of cases. Therefore the true incidence of patients suffering from PJP is lacking. Inability to make a correct diagnosis can also lead to increase in morbidity and mortality of such cases.

Microbiological diagnosis of PJP was done, a few years back, only microscopically after visualizing the smears stained with Grocott-Gomori silver stain, Toluidine O, Giemsa or Wright Stains, for trophozoites or cysts forms of P. jirovecii. [5] But the sensitivity of staining techniques, though acceptable for broncho-alveolar lavage (BAL) samples (70-92%), [6] is very less for aspirates and induced sputum (35-78%). [7] Immunofluorescence methods developed by using monoclonal or polyclonal antibodies also face the same problem of low sensitivity. [8] Molecular diagnosis of presence of P. jirovecii was described first of all by Wakefield in 1990. [6] Since then many polymerase chain reactions (PCRs) have been developed using different target genes. These molecular techniques have shown promising results with high sensitivity and specificity. [9] From developing countries like India though few studies have been published about PJP in human immunodeficiency virus (HIV)-infected patients, [10],[11] there are only few planned studies that show the utility of PCR for detection of P. jirovecii in respiratory samples. [12] This study was done with the objective to know the utility of PCR in finding the incidence of PJP in HIV-infected patients and to compare the PCR results with that of staining techniques. An attempt was also made to correlate the results with the clinical condition of patients.


   Materials and Methods Top


The present prospective study was done in our tertiary care center from June 2009 to Dec 2009. The study was approved by the institutional ethical committee.

Selection of Cases And Sample Collection

Fifty HIV-infected adult in-patients (most of them were newly detected cases without any prophylactic treatment) with symptoms suggestive of lower respiratory tract infections were selected for the study. The selected patients were having a combination of symptoms like complaint of cough with or without expectoration, dyspnea on exertion, fever and weight loss. Induced sputum, endotracheal aspirates or BAL samples were collected from selected patients and were transported within 2 h of collection to the laboratory.

Processing of Samples And Smear Staining

BAL samples and tracheal aspirates were centrifuged at 1300 rpm for 5 min. No mucolytic agent was used for these samples to avoid non-specific inhibition. Sputum samples were first treated with equal volume of dithiothreitol, vortexed briefly, incubated at 37°C for 15 min and then centrifuged at 6000 rpm for 10 min. The resulting pellet was washed twice with phosphate buffer saline (PBS) and resuspended in 1 ml of PBS.

Staining for Pneumocystis jiroveci

A portion of resuspended pellet was used to make two smears for Grocott-Gomori methenamine silver (GMS) stain (for cysts forms of P. jirovecii) and Giemsa stain (for trophozoites of P. jirovecii), respectively. The stained smears were visualized under microscope for the presence or absence of these morphological forms. Positive result by both the methods or by any one of these was considered as positive for the presence of P. jirovecii.

DNA Extraction and PCR

DNA was extracted from other part of resuspended pellet using QIAamp DNA Mini kit (Quiagen) according to the instructions provided in kit. PCR was performed for mitochondrial large subunit rRNA gene (mtLSUrRNA) using primers as described by Wakefield et al. [6] The PCR mixture (50 μl) contained 10 pmol of each primer, pAZ102E and pAZ102H specific for mtLSUrRNA gene, 200μM dNTPs, 3 mM MgCl 2 , 5 μl 10XPCR buffer, 1.25 U Taq DNA polymerase and 10 μl purified DNA. PCR was run at 1 minute each for denaturation, annealing and extension at 95, 58 and 72°C, for 40 cycles. The resulting product of 364bp fragment was separated on 2% agarose gel and was detected under UV illumination. [Figure 1] Each run of PCR was performed with positive (BAL sample positive for P. jirovecii by both PCR and GMS staining) and negative control (autoclaved water) so as to check the performance of the test. Barrier filter tips were used to exclude the carry-over amplicon contamination.
Figure 1: Gel photograph showing the results of PCR for mtLSUrRNA gene of Pneumocystis jirovecii, 1 and 4 Positive sample, 2 and 3 Negative samples, 5 Negative control, 6 Positive control, 7 100bp DNA Ladder

Click here to view


Reproducibility of PCR

PCR was done twice for all the samples to check for the consistency of the results.

Data Collection

For all the selected patients relevant demographic, clinical, radiological and laboratory data was collected. As there is no gold standard available to compare the results of any diagnostic test (except the histopathological examination of lung tissue), the correlation of positive PCR was done with the clinical condition of patients. CD 4 count was available for 37 selected patients (13 PCR positive and 24 PCR negative patients).

Clinical Classification of Cases

Patients were classified as clinically PCP proven cases if they were showing dyspnea, non-productive cough, low-grade fever, bilateral diffuses infiltrates in chest radiology and a favorable response to co-trimoxazole.

Controls

Twenty HIV seronegative patients presenting with signs and symptoms suggestive of lower respiratory infections were selected as controls for the study. Sputum samples were collected from these controls and were subjected to staining and PCR for the presence of PJP.

Statistical Analysis

Data was summarized using proportions. The association of socio-demographic and clinical factors to PCR positivity or negativity was done using Chi-square test or Fisher's exact tests.


   Results Top


Male to female ratio of 50 HIV seropositive cases selected for study was 3.1:1, with the age range of 25-61 years. Diagnosis of PJP was made in 32% (16/50), 34% (17/50) and 14% (7/50) by clinical criteria, PCR and staining methods, respectively. Staining for P. jirovecii correlated with PCR results in 7/17 patients (41.1%). Comparison of results of PCR and staining for trophozoites and cysts of P. jirovecii are shown in [Table 1].
Table 1: Comparison of PCR and staining results for Pneumocystis jirovecii (n=50)

Click here to view


Clinically, 16 patients were classified as PJP proven cases. Out of these 16 clinically proven cases, 15 (93.7%) showed positive results for PCR. False positivity and false negativity for PCR when compared with clinical evidence of disease was noticed in two and one cases, respectively. Results of comparison of PCR with clinical evidence of disease are shown in [Table 2]. In 32 patients there was no evidence of PJP either clinically or by PCR. Most of the PJP negative (12/32) patients were diagnosed having pulmonary tuberculosis and others (20/32) were later diagnosed having bacterial pneumonia or interstitial lung disease. Three out of 16 (18.7%) clinically PJP positive cases were also diagnosed having co-infection with pulmonary tuberculosis. In six out of 16 (37.5%) clinically positive patients the clinical condition worsened on treatment. Two patients (12.5%) expired even after initiating treatment with co-trimoxazole. In control group all cases were negative for PJP either by staining or PCR.
Table 2: Comparison of PCR with clinical evidence of disease (n=50)

Click here to view


When compared with clinical evidence of disease, PCR showed sensitivity and specificity of 93.7% and 94.1%, respectively. Positive predictive value and negative predictive value of PCR was detected as 88.2% and 96.9%, respectively. Kappa agreement between clinical diagnosis and PCR results was 0.86 showing good agreement. [Table 3] shows the comparison between socio-demographic and clinical profiles of PCR positive and PCR negative patients. It is evident from this table that presence of dyspnea and CD4 count are significant (P < 0.05) variables in patients suffering from PJP as compared to other patients.
Table 3: Comparison of socio-demographic and clinical profile of PCR positive and PCR negative patients for Pneumocystis jirovecii pneumonia

Click here to view



   Discussion Top


In developing countries like India, diagnosis of PJP is mostly done clinically or at the most by staining procedures. Different types of staining methods are available for visualizing either the trophozoites or the cysts forms of the pathogen. But this technique has the disadvantage that it needs experienced person to visualize the stained slides and also there can be high background staining resulting in difficulty in visualization of various morphological forms of P. jiroveci. [13] Nowadays these staining techniques are being replaced by immunofluorescence or molecular techniques that have increased sensitivity and specificity. But only a few centers, where the facilities are available, can do PCR for microbiological diagnosis of the disease.

In our study PCR results correlated with staining results in 14% (7/50) of cases. Another 20% (10/50) cases could be diagnosed only with the PCR but staining was negative for the presence of P. jirovecii. In a study done by Takahashi et al, PJP was diagnosed both by staining and PCR in 57.7% cases, but another 26.9% cases were diagnosed only by PCR. [14] When compared with clinical evidence of disease, PCR was positive in 93.8% (15/16) of clinically proven cases but staining could make the diagnosis only in 43.7% (7/16) of PJP cases.

Primers against different genes have been used by various authors in the past. These are mtLSUrRNA, thymidylate synthetase (TS), 18S rRNA, 5S rRNA, major surface glycoprotein (MSG) and internal transcribed spacer (ITS). [15] Out of these genes, PCR for mtLSU has shown most promising results and the same was used in the present study. [12] The only shortcoming of PCR is that it cannot differentiate infection from colonization. In present study in 2/50 (4%) patients, PCR was positive (false positive) but clinically they were stable. It may represent the colonization of these patients with P. jirovecii. Unfortunately we could not follow up the cases progressively to find out the development of disease later. But study done by Turner et al has shown that if the presence of DNA of P. jirovecii is detected, these cases are prone to get the disease in a due course of time. [13] Elvin et al have also supported this assumption and found out that out of eight PCR positive but clinically normal cases, six developed the PJP in 164-352 days. [16] The study suggested that PCR may identify the asymptomatic cases with high risk of developing PJP later. So these cases should be put on prophylactic treatment before the development of disease.

In our study one case was detected where PCR showed negative results but clinically he was diagnosed having PJP. PCR can show false-negative results in cases where the cysts are empty. It is seen in those cases who are already on prophylactic c o-trimoxazole (CTX). [13] Weig et al have also suggested the limited persistence and subsequent elimination of P. jirovecii after treatment of PJP . [5]

Presently few studies have observed the emergence of resistance to CTX. It has been shown due to the mutations in one of the active sites located at amino acids 55 (Thr-Ala) and 57 (Pro-Ser) in the P. jirovecii dihydropteroate synthase enzyme, which is involved in the folate and ultimately nucleotide, biosynthetic pathway. [17],[18] The resistance may lead to treatment failure to the therapeutic drug. [19] In our study 35.3% cases showed either worsening of clinical state or were expired. It may be due to the severity of HIV infection or the presence of other clinical conditions like disseminated cytomegalovirus infection or TB or in few cases it may be due to resistance to CTX.

In our study clinically presence of dyspnea and CD4 count showed statistical significance (P<0.05) in PJP diagnosed patients. These variables have been chosen by CDC as clinical criteria to diagnose a case of PJP. [20] Earlier Delorenzo et al have described the importance of bilateral perihilar diffuse infiltrates in PJP cases. [21] In our study, chest X-ray findings have shown no significant difference between PJP positive and negative patients. It may be due to the reason that these findings are present in early stage of disease but later it may progress to interstitial alveolar butterfly pattern and may spread to apices or base. However, normal X-ray may be seen in 2-34% cases of PJP. [22],[11]

We also observed that 3/16 (18.7%) PJP cases were having associated tuberculosis (TB). In another study done from India by Udwadia et al, co-existence of TB and PJP was noticed in 10.5% cases. [10] Few more studies from developing countries have also reported about co-infection of PJP and TB in 13-66% of patients. [4],[11] This may be due to the reason that TB is endemic in India and is the most important infection seen in HIV-infected individuals.

To summarize, PCR when correlated with clinical diagnosis of PJP has shown sensitivity, specificity, positive predictive value and negative predictive value as 93.7%, 94.1%, 88.2% and 96.9%, respectively. Kappa agreement was observed as 0.86 between PCR and clinical diagnosis of PJP cases which is fairly good agreement. When compared with staining procedures, PCR has definitely helped us to diagnose many more cases that were failed to be diagnosed by staining procedures. Hence we suggest the use of PCR for early and correct diagnosis of PJP in HIV-infected patients for timely management of the disease.

 
   References Top

1.Flori P, Bellete B, Durand F, Raberin H, Cazorla C, Hafid J et al. Comparison between real-time PCR, conventional PCR and different staining techniques for diagnosing Pneumocystis jiroveci pneumonia from bronchoalveolar lavage specimens. J Med Microbiol 2004;53:603-7.  Back to cited text no. 1
    
2.Masur H, Michelis MA, Greene JB, Onorato I, Stouwe RA, Holzman RS et al. An outbreak of community-acquired Pneumocystis carinii pneumonia: Initial manifestation of cellular immune dysfunction. N Engl J Med 1981;305:1431-8.  Back to cited text no. 2
    
3.Centres for Disease Control and Prevention. Guidelines for prophylaxis against Pneumocystis carinii pneumonia for persons infected with human immunodeficiency virus. MMWR Morb Mortal Wkly Rep 1989;38:1-9.  Back to cited text no. 3
    
4.Fisk DT, Meshnick S, Kazanjian PH. Pneumocystis carinii pneumonia in patients in the developing world who have acquired immunodeficiency syndrome. Clin Infect Dis 2003;36:70-8.  Back to cited text no. 4
[PUBMED]  [FULLTEXT]  
5.Weig M, Klinker H, Bogner BH, Meier A, Gross U. Usefulness of PCR for the diagnosis of Pneumocystis carinii pneumonia in different patient groups. J Clin Microbiol 1997;35:1445-9.  Back to cited text no. 5
    
6.Wakefield AE, Pixley FJ, Banerji S, Sinclair K, Miller RF, Moxon ER et al. Detection of Pneumocystis carinii with DNA amplification. Lancet 1990;336:451-3.  Back to cited text no. 6
    
7.Cregan P, Yamamoto A, Lum A, VanDerHeide T, MacDonald M, Pulliam L. Comparison of four methods for rapid detection of Pneumocystis carinii in respiratory specimens. J Clin Microbiol 1990;28:2432-6.  Back to cited text no. 7
[PUBMED]  [FULLTEXT]  
8.Galan F, Oliver JL, Roux P, Poirot JL, Bereziat G. Detection of Pneumocystis carinii DNA by polymerase chain reaction compared to direct microscopy and immunofluorescence. J Protozool 1991;38:199S-200S.  Back to cited text no. 8
[PUBMED]    
9.Lu JJ, Chen CH, Bartlett MS, Smith JW, Lee CH. Comparison of six different PCR methods for detection of Pneumocystis carinii. J Clin Microbiol 1995;33:2785-8.  Back to cited text no. 9
[PUBMED]  [FULLTEXT]  
10.Udwadia ZF, Doshi AV, Bhaduri AS. Pneumocystis carinii pneumonia in HIV infected patients from Mumbai. J Assoc Physicians India 2005;53:437-40.  Back to cited text no. 10
[PUBMED]    
11.Rani NU, Reddy VV, Kumar AP, Kumar KV, Babu GR, Rao DB. Clinical profile of Pneumocystis carinii pneumonia in HIV infected persons. Ind J Tub 2000;47:93-6.   Back to cited text no. 11
    
12.Gupta R, Mirdha BR, Guleria R, Mohan A, Kabra SK, Kumar L et al. Use of Different Primer Directed Sequence Amplification by Polymerase Chain Reaction for Identification of Pneumocystis jirovecii in Clinical Samples. Indian J Chest Dis All Sci 2008;50:321-7.  Back to cited text no. 12
    
13.Turner D, Schwarz Y, Yust I. Induced sputum for diagnosing Pneumocystis carinii pneumonia in HIV patients: New data, new issues. Eur Respir J 2003;21:204-8.  Back to cited text no. 13
[PUBMED]  [FULLTEXT]  
14.Takahasi T, Goto M, Endo T, Nakamura T, Yusa N, Sato N et al. Pneumocystis carinii carriage in immunocompromised patients with and without human immunodeficiency virus infection. J Med Microbiol 2002;51:611-4.  Back to cited text no. 14
    
15.Thomas CF, Limper AH. Pneumocystis Pneumonia. N Engl J Med 2004;350:2487-98.  Back to cited text no. 15
    
16.Elvin K, Olsson M, Lindman C, Bjorkman A. Detection of asymptomatic Pneumocystis carinii infection by polymerase chain reaction: Predictive for subsequent pneumonia. AIDS 1996;36:1296-7.  Back to cited text no. 16
    
17.Zingale A, Carrera P, Lazzarin A, Scarpellini P. Detection of Pneumocystis carinii and characterization of mutations associated with sulfa resistance in bronchoalveolar lavage samples from Human Immunodeficiency Virus infected subjects. J Clin Microbiol 2003;41:2709-12.  Back to cited text no. 17
[PUBMED]  [FULLTEXT]  
18.Kazanjian P, Armstrong W, Hossler PA, Burgman W, Richardson J, Lee CH et al. Pneumocystis carinii mutations are associated with duration of sulfa or sulfone prophylaxis exposure in AIDS patients. J Infect Dis 2000;182:551-7.  Back to cited text no. 18
    
19.Mai Q, Gurunthan S, Masur H, Kovacs JA. Failure of co-trimoxazole in Pneumocystis carinii infection and mutations in dihydropteroate synthase gene. Lancet 1998;351:1631-2.  Back to cited text no. 19
    
20.Centres for Disease Control. Pneumocystis carinii pneumonia- Los Angeles MMWR 1980;30:250.  Back to cited text no. 20
    
21.DeLorenzo LJ, Huang CT, Maguire GP, Stone DJ. Roentgenographic patterns of PCP in 104 patients with AIDS. Chest 1987;91:323-7.  Back to cited text no. 21
[PUBMED]  [FULLTEXT]  
22.Fishman JA. Pneumocystis carinii. In: Fishman AP, Elias JA, Fishman JA, Grippi MA, Senior RM, Park AI, editors. Fishmans pulmonary disease and Disorders 4th ed, 2nd volume, section 20, chapter133. China: The Mc Graw-Hill Companies; 2008. p. 2313-32.  Back to cited text no. 22
    

Top
Correspondence Address:
Kiran Chawla
Department of Microbiology, Kasturba Medical College, Manipal. Karnataka - 576 104
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.81624

Rights and Permissions


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]

This article has been cited by
1 Evaluation of the automated Becton Dickinson MAX real-time PCR platform for detection of Pneumocystis jirovecii
Jung-Yien Chien,Chia-Jung Liu,Pei-Chien Chuang,Tai-Fen Lee,Yu-Tsung Huang,Chun-Hsing Liao,Chien-Ching Hung,Wan-Huei Sheng,Chong-Jen Yu,Po-Ren Hsueh
Future Microbiology. 2017; 12(1): 29
[Pubmed] | [DOI]
2 Pneumocystis jirovecii Infection and the Associated Dihydropteroate Synthase (DHPS) and Dihydrofolate Reductase (DHFR) Mutations in HIV-Positive Individuals from Pune, India
Arati Mane,Pankaj Gujar,Jipsi Chandra,Rahul Lokhande,Tilak Dhamgaye,Shivhari Ghorpade,Arun Risbud
Mycopathologia. 2014;
[Pubmed] | [DOI]
3 Updating Our Understanding of Pulmonary Disease Associated With HIV Infection
Miriam Estébanez-Muñoz,Clara I. Soto-Abánades,Juan J. Ríos-Blanco,Jose R. Arribas
Archivos de Bronconeumología (English Edition). 2012; 48(4): 126
[Pubmed] | [DOI]
4 Actualización en la patología pulmonar relacionada con la infección VIH
Miriam Estébanez-Muñoz,Clara I. Soto-Abánades,Juan J. Ríos-Blanco,Jose R. Arribas
Archivos de Bronconeumología. 2012; 48(4): 126
[Pubmed] | [DOI]
5 Outcome of human immunodeficiency virus–exposed and –infected children admitted to a pediatric intensive care unit for respiratory failure*
Omolemo P. Kitchin,Refiloe Masekela,Piet Becker,Teshni Moodley,Sam M. Risenga,Robin J. Green
Pediatric Critical Care Medicine. 2012; 13(5): 516
[Pubmed] | [DOI]
6 Updating Our Understanding of Pulmonary Disease Associated With HIV Infection | [Actualización en la patología pulmonar relacionada con la infección VIH]
Estébanez-Muñoz, M., Soto-Abánades, C.I., Ríos-Blanco, J.J., Arribas, J.R.
Archivos de Bronconeumologia. 2012; 48(4): 126-132
[Pubmed]
7 Cost-effectiveness analysis of diagnostic options for Pneumocystis pneumonia (PCP)
Harris, J.R., Marston, B.J., Sangrujee, N., DuPlessis, D., Park, B.
PLoS ONE. 2011; 6(8): art -e23158
[Pubmed]
8 Cost-Effectiveness Analysis of Diagnostic Options for Pneumocystis Pneumonia (PCP)
Julie R. Harris,Barbara J. Marston,Nalinee Sangrujee,Desiree DuPlessis,Benjamin Park,Vishnu Chaturvedi
PLoS ONE. 2011; 6(8): e23158
[Pubmed] | [DOI]



 

Top
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  


    Abstract
    Introduction
    Materials and Me...
    Results
    Discussion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed5233    
    Printed166    
    Emailed2    
    PDF Downloaded205    
    Comments [Add]    
    Cited by others 8    

Recommend this journal