The cytological diagnosis of Pneumocystis jiroveci pneumonia in bronchoalveolar lavage

INTRODUCTION

Immunosuppressed individuals are more prone for opportunistic infections and it is a chief cause of morbidity and mortality. Various organisms such as Pneumocystis jiroveci pneumonia (PJP), Cytomegalovirus (CMV), Aspergillosis, Candidiasis, and Cryptococcosis can cause lung infections in an immunocompromised individual due to lack of adequate and control immunity. PJP, previously known as Pneumocystis carinii pneumonia, is the most common opportunistic infection in affecting people living with HIV.^[1] As PJP can cause life-threatening infection to a patient, the treatment should not be delayed for these cases.^[1] Hence, a prompt and early diagnosis is always needed for such cases to reduce morbidity as well as mortality.

Fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) fluid examination is favored diagnostic procedure to help in the quick and precise diagnosis of these infections.^[2] Although many case reports have been published in the Western literature demonstrating the utility of BAL in the diagnosis of Pneumocystis pneumonia, relatively fewer studies are available from India. In this paper, we discussed the cytology along with clinic-radiological features of PJP cases in BAL diagnosed in our institution.

MATERIAL AND METHODS

This retrospective study was conducted in PGIMER, Chandigarh, India. We have reviewed 15 cases of PJP on BAL fluid cytology over a period of 8.5 years, that is, from January 2011 to June 2019. [Table 1] highlights the detailed description of all 15 cases. In this study, we have tried to correlate the cytological findings with the clinical and radiological features. The patient’s clinical data were archived from the hospital record. Cytospin preparation was made for every case and stained with May Grunwald Giemsa and Papanicolaou. Silver methanamine stained smears are available for all cases. We identified the foamy alveolar cast (FAC) in BAL fluid cytology [Figure 1] and graded the presence of FAC in BAL fluid cytology from 1+ to 3+ depending on its frequency per low power field (×10). Ten such fields were counted. The grade 1+ indicated 1-5 FACs, 2+ indicated 6–10 FACs, and 3+ indicated more than 10 FACs per 10 low power field [Figure 2]. FAC includes three-dimensional configuration of exudative masses with a coarsely granular, “foamy or bubbly honeycombed” appearance. These masses show shadowed outline of the cyst walls with rare tiny intracystic bodies (sporozoites). These exudative masses resemble bloated alveolar sacs, hence, the name alveolar “cysts.” We also observed the accompanying inflammatory cells.

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Figure 1:

Photomicrograph showing foamy alveolar casts along with alveolar macrophages in a bronchoalveolar lavage specimen (Papanicolaou ×400). Inset shows higher magnification of foamy alveolar cast highlighting cup shaped PJP organisms (Papanicolaou ×1000).

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Figure 2:

(a) Foamy alveolar cast (1+) having 1–5/10 low power field (Papanicolaou ×120). (b) Foamy alveolar cast (2+) having 6–10/10 low power field (Papanicolaou ×120). (c) Foamy alveolar cast (3+) having more than 10/10 low power field (Papanicolaou ×120).

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RESULTS

BAL findings

Both May Grunwald Giemsa and Papanicolaou stain were performed in all 15 cases. The silver methanamine was done in all cases. Presence of FACs was the distinctive feature and was noted in 14 cases (93.3%). A number of FAC were 3 + in two cases, 2 + in ten cases, and 1+ in two cases. One of the cases showed very occasional FAC. This case was diagnosed as non-specific inflammatory smear in BAL fluid cytology. The occasional cast on the cytology smear was regarded as mucous plug. The immune status of this patient was not known and computed tomography scan suggested bilateral haziness in lung suggestive of pneumonia. Transbronchial biopsy was available for this case and showed foamy intra-alveolar exudates accompanied by lymphoplasmacytic interstitial infiltrates. Silver methanamine stain was performed in histology section of the same case which showed the numerous cysts of PJP within alveoli thus confirming the diagnosis of PJP [Figure 3]. All the remaining cases showed impregnation of the cyst wall and boosted the rounded, helmet or cleft forms of intracystic bodies (sporozoites) in silver methanamine stain and thus confirmed the diagnosis of PJP. One of the cases showed the coinfection with candidiasis [Figure 4a and b]. Periodic acid Schiff stain better highlighted the candida pseudohyphae [Figure 4c]. Rest of the cases did not show any coinfection. Polymorphs were predominant cells in seven cases, lymphocytes were predominant in three cases, and histiocytes were predominant in five cases.

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Figure 3:

(a) Photomicrograph showing PJP in the histology section of lung tissue (Hematoxylin and eosin stain ×400). (b) Photomicrograph showing impregnation of cyst wall and the intracystic bodies in histology section of lung tissue (Silver methanamine × 400). (c) Photomicrograph showing immunostaining in histology section of lung tissue (Immunostaining ×400).

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Figure 4:

(a and b) Photomicrograph showing low and high power of PJP along with candidiasis in BAL fluid sample (Papanicolaou ×100 and 400 respectively). (c) Photomicrograph showing PJP along with candidial pseudohyphae in BAL fluid sample (Periodic acid Schiff ×400).

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DISCUSSION

PJP is a treatable disease and it requires quick treatment. For this purpose, an accurate and rapid diagnosis is mandatory. In our study, the most of the immune-compromised patients presented with respiratory difficulty, fever, and cough. As already described, apart from PJP infection, immunocompromised individuals are more prone for some other lung infection such as CMV and Aspergillus which can also lead to similar type of symptoms. Hence, it is vital to diagnose proper etiology for such patients.

In the present study, we reported 15 cases of PJP. Striking cytological features were the presence of polymorphs, activated macrophages, and few eosinophils apart from FACs. One case was falsely diagnosed as negative in BAL smear. Various methods such as sputum examination, tracheal aspirates, endobronchial brush biopsy, and percutaneous needle aspiration are employed to recognize P. jerovecii.^[1] BAL cytology shows excellent sensitivity in diagnosing PJP.^[3] Few histochemical special stains have been used to detect PJP in clinical specimens. These special stains include the Diff-Quik, Grocott-Gomori methenamine silver (GMS), and Calcofluor white stains.^[4]

FACs if present in enough numbers can be caught at lower magnification. Higher magnification of FACs shows PJP organisms in the form of basophilic dots within the alveolar cast. PJP is seen as aggregates of cysts and trophozoites with a granular, foamy, or honeycomb appearance. Nevertheless, the confirmation is done with the aid of GMS stain. GMS stain highlights numerous tiny cup-shaped cysts with a black dot in the center of the cyst.^[5] GMS stain helps in differentiating PJPs alveolar cast from protein alveolar proteinosis. PJP immunostaining enhances the sensitivity as well as specificity; however, the quality, as well as yield of the BAL fluid from the respiratory tract, is of vital importance for the diagnostic accuracy.^[6] The study done by Boiselle et al. diagnosed PJP pneumonia on chest radiograph in 75% cases.^[7] Unfortunately, in our case series, only five cases were correctly diagnosed in chest radiograph.

Out of 15 cases, seven are HIV positive; five patients were post-renal transplant; and one patient was known case of ALL on immunosuppression. Immune status of two patients was unknown. Sternberg et al. found CMV as the most prevalent lung infection in immunocompromised patients.^[8] Menon et al. did not identify even a single case of PJP in 16 post-renal transplant cases and 14 dialysis cases.^[9] In our study, though we have 15 cases to report, we did not find any case of CMV infection in BAL fluid of immunocompromised patients which differ from the previous study done by Sternberg et al. and Menon et al.^[8,9]

[Table 2] shows a comparison of different studies of PJP in BAL cytology. Dahiya et al.^[1] described 13 cases of PJP in the year 2005, among which ten cases were post-renal transplant, and only one case was HIV-positive. These results were in discordance with our results, as we found predominant population HIV-positive (46.7%). FACs were characteristic features in their study too. Coinfection was seen in one case with CMV. Young et al.^[10] described 15 cases of PJP in the year 1986, among which 12 cases were post-renal transplant, two cases were of SLE, and one case of leukemia. None of the cases was HIV-positive. These results were too in discordance with our results suggesting increase in PJP infection in people living with HIV. FAC was seen in all the cases, though background inflammatory cells were less frequent compared to the other studies. Coinfection was seen in five cases, two with CMV and three with candidiasis. In the early stage of PJP, the organism attaches mainly to Type 1 alveoli and in the early stage of the disease lymphomononuclear infiltrate are the main cellular component associated with PJP and polymorphs are less in number.^[11] Our study indicates that the histiocytes are the predominant cellular inflammatory infiltrate along with polymorphs and lymphocytes in BAL fluid of PJP patients. Robert et al. concluded in their study that the presence of polymorph in BAL fluid in PJP patients is associated with more severe respiratory distress; however, in our study, we did not grade the respiratory difficulty due to lack of follow-up of these patients.^[12]

Djamin et al., while evaluating PJP infection in immunocompromised patients, found that bronchial brushing has limited value and maximum cases were detected in BAL fluid preparation only.^[13] Menon et al. also found in their study that BAL cytology technique is a more helpful, non-invasive, and rapid diagnostic tool than microbiological culture in diagnosing PJP infection in immunocompromised patients.^[9] In our study, out of 15 cases, 14 cases were accurately diagnosed in BAL. Only one case was missed in BAL fluid sample which was further detected in biopsy specimen of the patient. BAL fluid specimen is termed as a liquid biopsy and it is a rapid technique to detect the PJP infection where early treatment to a patient can be administered.^[1] Furthermore, the risk of pneumothorax and bleeding is less during sample collection by BAL technique than transbronchial biopsy technique. Although BAL offers the quickest diagnosis, polymerase chain reaction performed in conjunction with BAL gives more accurate diagnosis.^[14] In recent times, molecular detection has also become part of PJP diagnosis.^[15]

SUMMARY

Over time, PJP infection is on an increasing trend in people living with HIV as compared to the previous studies. Moreover, the rate of coinfection has decreased over time, with one coinfection seen in our study. Cytopathologists should be aware of the existence of PJP infection besides other opportunistic infection in an immunocompromised patient. A careful and thorough screening can minimize the false-negative rate in the detection of PJP infection. Distinctive cytomorphological features on routine, as well as special stain, offer a sensitive tool for diagnosing this pathogen.