Diagnosis of Pneumocystis Pneumonia by Bronchoalveolar Lavage Cytology: Experience at a Tertiary Care Centre in India

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1 ORIGINAL ARTICLE Diagnosis of Pneumocystis Pneumonia by Bronchoalveolar Lavage Cytology: Experience at a Tertiary Care Centre in India Sonika Dahiya, Sandeep R. Mathur, Venkateswaran K. Iyer, Kusum Kapila and Kusum Verma Cytopathology Laboratory, Department of Pathology, All India Institute of Medical Sciences, New Delhi, India ABSTRACT Background. Diagnosis of Pneumocystis pneumonia requires morphological demostration of P. carinii (now re-named as P. jiroveci). Although bronchoalveolar lavage (BAL) fluid cytology constitutes a formidable tool for detecting this infection, few studies on the utility of BAL cytology in diagnosing PCP are available from India. The present study reports the clinical spectrum, cytomorphological features and the utility of BAL cytology in diagnosing Pneumocystis infection from a tertiary care centre in India. Methods. Retrospective study of 13 patients with PCP, diagnosed on examination of BAL fluid. Results. The mean age of the patients was 41.2 years. One patient had human immunodeficiency virus (HIV) infection, while the other 10 were renal transplant receipients on immunosuppressive therapy. The immune status of two patients was unknown. Fever, cough and shortness of breath were the main presenting symptoms. Radiological diagnosis of Pneumocystis pneumonia was offered in only one case. Foamy alveolar casts were present in all cases. Silver methanamine stain enhanced the rounded, helmet or cleft forms of sporozoites. Inflammatory infiltrate was mainly polymorphonuclear. Conclusions. BAL cytology, thus, constitutes a useful diagnostic modality for morphological documentation and reliable diagnosis of Pneumocystis pneumonia in an immunocompromised host. Pneumocystis pneumonia appears to be a common opportunistic infection in renal transplant receipients in India. Key words: Pneumocystis pneumonia, Human immunodeficiency virus, Post renal transplantation, Bronchoalveolar lavage. [Indian J Chest Dis Allied Sci 2005; 47: ] INTRODUCTION Opportunistic infections of the lungs frequently occur in the immunosuppressed individuals and are a major cause of morbidity and mortality. Infections reported worldwide in such patients include Pneumocystis pneumonia, cytomegalovirus (CMV) pneumonia, tuberculosis, cryptococcosis, Aspergillosis and Candidiasis. Fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) fluid examination and is reported to be the favoured diagnostic [Received: July 26, 2004; accepted after revision: January 7, 2005] Correspondence and reprint request: Dr Kusum Verma, Professor and Head, Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi ; Tele.: ; Telefax: ; <icverma@vsnl.com.>.

2 260 Pneumocystis Pneumonia and BAL Cytology Sonika Dahiya et al procedure to aid in the rapid and accurate diagnosis of these infections 1. Although many reports have been published in the Western literature demonstrating the utility of BAL in diagnosis of Pneumocystis pneumonia, relatively fewer studies are available from India 2-9. One of the reasons suggested is that BAL fluid examination is not available in most centres in India 8,9. The present study highlights the role of BAL fluid examination in the detection of PCP, along with the clinical spectrum and morphological features of Pneumocystis pneumonia from a tertiary care centre where BAL fluid examination is routinely carried out in all patients with interstitial lung disease or suspected lung infection. MATERIAL AND METHODS Over a five-and-a-half year period from January 1998 to June 2003, 13 case records of patients in whom a diagnosis of PCP was made from BAL fluid examination at the Cytopathology Laboratory, All India Institute of Medical Sciences, New Delhi, were retrospectively studied. Cytospin preparations had been made from BAL fluid and stained with Papanicolaou (n=13) and May Grunwald Giemsa Stain (n=3). Silver methanamine (SM) stained smears and transbronchial lung biopsy (TBLB) specimens were available in 11 and 4 cases, respectively. Clinical details of these cases were obtained from the records. Cytology smears were reviewed, and diagnostic criteria propounded by Strigle et al 1 were used for diagnosing Pneumocystis pneumonia. These criteria include presence of three-dimensional configuration of exudative masses with a coarsely granular, foamy or bubbly honeycombed appearance; shadowed outline of the cyst walls within the masses with occasional minute intracystic bodies (sporozoites); and the size and shape of the exudative masses looking like distended alveolar sacs (alveolar cysts ). Numbers of such foamy alveolar casts (FAC) were graded from + to +++; and the type of inflammatory cells accompanying identified. Correlation between BAL fluid examination and TBLB findings was also studied. RESULTS Clinical Features Mean age of the patients was 41.2 years (range years); all of them were males. One patient had human immunodeficiency virus (HIV) infection, while there were 10 renal transplant receipients on immunosuppressive therapy. In remaining two cases HIV status was not known, and both were receiving antituberculosis treatment. Common presenting symptoms were fever (11/13), cough (10/13) and shortness of breath (8/13) (Table). Chest radiograph had shown parenchymal infiltrates in 11 cases and a radiological diagnosis of Pneumocystis pneumonia was rendered in only one case (Table). BAL Findings Foamy alveolar casts (FACs) were the characteristic feature and were seen in all cases although their numbers were variable. Number of FAC s were 1-2 (+) in four cases, numerous (+++) in two cases and in moderate numbers (++) in rest of the cases (Table). Papanicolaou stain showed FACs in all the cases while May - Grunwald-Giemsa (MGG) staining (n=3) showed the presence of these casts in two of the three cases. The casts had a foamy to bubbly appearance due to the lack of the staining of cyst (Figure 1). Both the sporozoites and trophozoites stained faint blue appearing as dot like structure in the centre of these foamy areas. Figure 1. Photomicrograph showing foamy alveolar casts along with alveolar macrophages in a bronchoalveolar lavage specimen (Papanicolaou 400).

3 Table. Clinical details and bronchoalveolar lavage findings in 13 patients with Pneumocystis pneumonia Case Age Sex Symptom Cough Expectoration Fever Dyspnoea Chest Radiograph/ BAL Findings No. (Years) Duration CT-scan of the (days) Chest FAC Inflammatory Density Cells 1 35 Male B/L basal haziness Male B/L lung infiltrates +++ P++ E ± 3 57* Male Right lower lobe ++ P+ non-resolving pneumonia 43* Male ?PCP ++ L+ 5 45* Male Right sided diffuse ++ P++ haziness 6 53* Male ? ++ P * Male B/L middle and lower + - zone haziness, more on the right side 8 26* Male Opacity in the upper + - segment of the lower lobe on the right side 9 36* Male B/L middle lobe infiltrates ++ P * Male B/L perihilar shadows ++ P * Male B/L lower zone haziness * Male B/L lower zone infiltrates ++ P Male Peribronchoarterial +++ L+ alveolar opacities Patients 1,2, 9 and 13 underwent transbronchial lung biopsy *= renal transplant recipient; = human immunodeficiency virus positive + = present; - = absent; B/L = bilateral; P = polymorphs; L = lymphocytes; E = eosinophils; FAC = foamy alveolar casts; BAL = bronchoalveolar lavage 2005; Vol. 47 The Indian Journal of Chest Diseases & Allied Sciences 261

4 262 Pneumocystis Pneumonia and BAL Cytology Sonika Dahiya et al These features helped to distinguish casts from mucous plugs or artifacts. Silver methanamine stain showed impregnation of the cyst wall and enhanced the rounded, helmet or cleft forms of intracystic bodies (sporozoites), and thus, confirmed the diagnosis in all 11 cases (Figure 2), where SM was available. Figure 2. Photomicrograph showing impregnation of cyst wall and the intracystic bodies (Silver methanamine 400). Polymorphs were the predominant constituent of inflammatory cell infiltrate (7/13 cases) whereas lymphocytes (2/13 cases) and eosinophils (1/13 cases) were less commonly seen (Table). Co-infection with CMV was observed in one case (Case No. 3). Bronchial Biopsy Transbronchial lung biopsies were available in four cases (Table). However, all the four cases showed non-specific features with the absence of organisms. This was due to the fact that the biopsies obtained were very tiny and it was difficult to detect the organisms on routine stains alone. Although interstitial polymorphonuclear and mononuclear infiltrates were observed, intact alveoli were lacking, in the biopsies. Special stains could not be performed due to inadequate tissue in the biopsies. DISCUSSION Though life threatening, Pneumocystis pneumonia is a treatable infection and therefore, a rapid and accurate diagnosis is mandatory. Clinically these patients present with respiratory symptoms and/or signs suggestive of pulmonary infection like cough, dyspnoea, fever or an abnormal chest radiograph. These signs and symptoms may also be observed in other opportunistic infections in patients with acquired immunodeficiency syndrome (AIDS) or other immunocompromised states Pneumocystis pneumonia was diagnosed to be the cause of respiratory symptoms in 40.5% (79/195) 10, 46.9% (420/894) 11 and 86% (36/42) 12 cases of AIDS in some of the published series. Selwyn et al 13 studied the clinical predictors of Pneumocystis pneumonia thereby distinguishing it from other opportunistic infections and observed that exertional dyspnoea and interstitial infiltrates in lung have a specificity of 92 percent. We observed the combination of these two features in only around 50% of our cases. Boiselle et al 14 had reinforced that the diagnosis of Pneumocystis pneumonia can be correctly made on the chest radiograph in 75% of cases. However, in the present study only one case could be diagnosed correctly as Pneumocystis pneumonia on the chest radiograph. Various methods including evaluation of sputum, tracheal aspirate, endobronchial brush biopsy and percutaneous needle aspiration have been employed to identify P. jiroveci. Currently, BAL fluid examination is the procedure of choice. The excellent sensitivity of BAL for diagnosis of Pneumocystis pneumonia may be related to the extensive bilateral pulmonary involvement in immunocompromised hosts 15. In the present study, majority of the cases were renal transplant recipients who were on immunosuppressive therapy. In contrast, Sternberg et al 16 observed CMV to be the commonest infectious cause of pneumonitis (8/ 48) in renal transplant recipients, whereas Pneumocystis pneumonia was found only in three patients and that too as a co-existent infection with CMV. Similarly Huerlin et al 17 found CMV as the predominant cause of

5 2005; Vol. 47 The Indian Journal of Chest Diseases & Allied Sciences 263 infectious pneumonia in post renal transplant patients. Menon et al 7 did not observe a single case of Pneumocystis pneumonia in 16 post-renal transplant cases and 14 patients on dialysis. Cytomegalovirus was diagnosed in two of the 16 post transplant cases. However, they observed Pneumocystis pneumonia in four of the eight HIV positive cases. Findings of the present study differ from reported cases as CMV was observed in only one of the 10 post transplant cases. Our laboratory had studied BAL from 15 renal transplant patients with fever and pulmonary infiltrates previously ( ) and did not observe a single case of CMV or PCP (unpublished observations). Despite a high prevalence of HIV/AIDS cases in India 18, Pneumocystis pneumonia remains less common here. This could be because BAL is not routinely done in most of the centres in India. Therefore, the published literature from India with regards to Pneumocystis pneumonia is relatively limited 2-9,18,19. Singh et al 2 reported the first few cases of Pneumocystis pneumonia in AIDS patients from India. The clinical and radiological profile of Pneumocystis pneumonia simulating tuberculosis has been reported by Arora et al 3. Mathew et al 4 reported Pneumocystis pneumonia in 5/15 (33%) AIDS patients and have stressed upon the utilisation of sensitive diagnostic tests. A case of combined tuberculosis and Pneumocystis pneumonia presenting as a cavitary lesion has been reported by Jindal et al 5 in a post-renal transplant patient. Pneumocystis pneumonia was demonstrated in 9/32 AIDS patients using induced sputum samples and indirect immunofluorescence technique by Usha et al 6. Menon et al 7 have emphasised upon the greater sensitivity of cytological examination of BAL in diagnosing Pneumocystis pneumonia over culture and biopsy. The latter found four cases of Pneumocystis pneumonia out of 38 BAL specimens from immunosuppressed hosts. Bijur et al 8 studied BAL and TBLB specimens in five HIV patients and demonstrated Pneumocystis pneumonia in three cases. They attribute the lower proportion of Pneumocystis pneumonia infection as opposed to other opportunistic infections in developing countries to the lack of use of sensitive diagnostic methods like BAL cytology. Deshmukh et al 9 have reported five cases of Pneumocystis pneumonia among 34 autopsies in patients with AIDS and have emphasised the occurrence of other co-existing opportunistic infections, like Cryptococcosis, tuberculosis, etc. Lanjewar et al 18 observed Pneumocystis pneumonia in seven out of 143 adult lung specimens from HIV-positive patients at autopsy, while Santosh et al 19 observed one case of Pneumocystis pneumonia in autopsy/biopsy series of 10 HIV-positive patients. The standard method of establishing the diagnosis of Pneumocystis pneumonia remains tinctorial staining which can be divided into two groups. The first includes cyst wall stains such as toluidine blue and SM and the other category includes MGG and Papanicolaou techniques, which stain the intracystic sporozoites as well as trophozoites 10. Both the types of stains were used in the present study for the demonstration of the organisms. Few authors 20,21 have tried to observe the clinical significance of cellular infiltrates with prognosis in immunocompromised patients with Pneumocystis pneumonia and concluded that accumulation of polymorphs in BAL is associated with more severe respiratory compromise. Unfortunately, follow up is not available in the present cases and we cannot comment upon significance of cell type associated with Pneumocystis pneumonia. Sternberg et al 16 observed that the percentage of polymorphs in the lavage fluid was significantly lower in post renal transplant receipients with Pneumocystis pneumonia as compared to other infections. Results of the present study do not support this finding. Although BAL cytology has been traditionally used for evaluation of interstitial lung diseases, it also serves as an effective and easy technique to diagnose Pneumocystis pneumonia. A careful cytological examination for foamy alveolar casts along with the use of special stains confirms the diagnosis. In this regard, BAL fluid specimen which may also be termed as a liquid biopsy, is considerably superior over a tissue biopsy as abundant material is

6 264 Pneumocystis Pneumonia and BAL Cytology Sonika Dahiya et al available for performing special stains. Furthermore, the complications associated with TBLB such as pneumothorax, bleeding, can be avoided. A cytopathologist should always be vigilant towards the presence of Pneumocystis infection besides other opportunistic infections in all immunosuppressed patients. Characteristic cytomorphological features on routine and special stains offers a sensitive tool for diagnosing this pathogen. Studies employing BAL cytology in immunosuppressed patients with a large sample size are required define the epidemiology of Pneumocystis pneumonia in India. REFERENCES 1. Strigle M, Gal AA, Koss MN. Rapid diagnosis of Pneumocystis carinii infection in AIDS by cytocentrifugation and rapid hematoxylineosin staining. Diagn Cytopathol 1990; 6: Singh YN, Singh S, Rattan A, Ray JC, Sriniwas TR, Kumar A, et al. Pneumocystis carinnii infection in patients of AIDS in India. J Assoc Physicians India 1993; 41: Arora VK, Tumbanatham A, Kumar SV, Ratnakar C. Pneumocystis carinii pneumonia simulating as pulmonary tuberculosis in AIDS. Indian J Chest Dis Allied Sci 1996; 38: Mathews MS, Mathai E. Emerging importance of Pneumocystis carinii among Indian immunosuppressed patients. Indian J Chest Dis Allied Sci 2000; 42: Jindal SK, Gupta D, Aggarwal AN, Chakrabarti A. The spectrum of respiratory mycoses in a referral hospital in north-western India. Indian J Chest Dis Allied Sci 2000; 42: Usha MM, Rajendran SP, Thyagarajan SP, Soloman S, Kumarasamy N, Yepthomi T, et al. Indentification of Pneumocystis carinii in induced sputum of AIDS patients in Chennai (Madras). Indian J Pathol Mirobiol 2000; 43: Menon LR, Divate S, Acharya VN, Mahashur AA, Natrajan G, Almeida AF. Utility of bronchoalveolar lavage in the diagnosis of pulmonary infections in immunosuppressed patients. J Assoc Physicians India 2002; 50: Bijur S, Menon L, Iyer E, Deshpande J, Sivaraman A, Vaideeswar P, et al. Pneumocystis carinii pneumonia in human immunodeficiency virus infected patients in Bombay: diagnosed by bronchoalveolar lavage cytology and transbronchial lung biopsy. Indian J Chest Dis Allied Sci 1996; 38: Deshmukh SD, Ghaisas MV, Rane SR, Bapat M. Pneumocystis carinii pneumonia and its association with other opportunistic infections in AIDS, an autopsy report of five cases. Indian J Pathol Microbiol 2003; 46: Djamin RS, Drent M, Schreurs AJ, Groen EA, Wagenaar SS. Diagnosis of Pneumocystis carinii pneumonia in HIV-positive patients: bronchoalveolar lavage vs bronchial brushing. Acta Cytol 1998; 42: Baughman RP, Dohn MN, Frame PT. The continuing utility of bronchoalveolar lavage to diagnose opportunistic infection in AIDS patients. Am J Med 1994; 97: Golden JA, Hollander H, Stulbarg MS, Gamsu G. Bronchoalveolar lavage as the exclusive diagnostic modality for Pneumocystis carinii pneumonia: A prospective study among patients with acquired immunodeficiency syndrome. Chest 1986; 90: Selwyn PA, Pumerantz AS, Durante A, Alcabes PG, Gourevitch MN, Bioselle PM, et al. Clinical predictors of Pneumocystis carinii pneumonia, bacterial pneumonia and tuberculosis in HIVinfected patients. AIDS 1998; 12: Boiselle PM, Tocino I, Hooley RJ, Pumerantz AS, Selwyn PA, Neklesa VP, et al. Chest radiograph interpretation of Pneumocystis carinii pneumonia, bacterial pneumonia and pulmonary tuberculosis in HIV-positive patients: accuracy, distinguishing features and mimics. J Thorac Imaging 1997; 12: Gal AA, Klatt EC, Koss MN, Strigle SM, Boylen CT. The effectiveness of bronchoscopy in the diagnosis of Pneumocystis carinii and cytomegalovirus pulmonary infections in acquired immunodeficiency syndrome. Arch Pathol Lab Med 1987; 111: Sternberg RI, Baughman RP, Dohn MN, First MR. Utility of bronchoalveolar lavage in assessing pneumonia in immunosuppressed renal transplant recipients. Am J Med 1993; 95:

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