Diagnosis of Pneumocystosis pneumonia with two staining methods using various specimens collected from animal model
M Hajia, A Mahmoodzadeh, M Rezaiemanesh, H Morovati
pcp, specimens, staining method
M Hajia, A Mahmoodzadeh, M Rezaiemanesh, H Morovati. Diagnosis of Pneumocystosis pneumonia with two staining methods using various specimens collected from animal model. The Internet Journal of Microbiology. 2007 Volume 5 Number 2.
Correct diagnosis of pneumocystosis pneumonia depends on suitable sampling procedure and staining method. The aim of this study was comparison two staining methods “Giemsa and GMS” in three collected samples from studied animal model “oral swab, BAL and lung homogenate”.
Twenty two female rats of two month-ages (Sprague-Dawley) with 150-200 grams of weight were used. Methyl prednizolone acetate was used to stimulate pneumocystosis. Oral swab (OS), broncho-alveolar lavage (BAL) and lung homogenate (LH) were collected during studied weeks from control and test groups. All samples were stained with Giemsa and GMS (Gomori's Methenamine Silver). These specimens were classified in four stages based on 35 microscopic fields (100X) for counting of cyst numbers, tested by two staining methods for common specificity such as number of cysts, required time for observations, cost, and possibility of improper diagnosis.
All controls were negative in all specimens by both staining methods. Oral swabs were negative by both staining methods. The Lavage specimens were negative from weeks zero to five by Giemsa method but were positive from week two to the end by GMS method. Lung homogenate specimens were negative in weeks 0 and 2 by Giemsa but negative in week 0 by GMS. These samples were positive in later weeks in two staining methods.
Based on the analyzed results Giemsa and GMS had no enough sensitivity in oral swab specimens. The best sensitivity was obtained by GMS use of LH specimens.
Direct observation of
GMS is a silver staining method that is usually used for the observation of fungi in tissue specimens. This method is the specific procedure for the cyst wall and is used as a gold standard method for diagnosis of the pneumocystosis (8,9,10,11).
Proper diagnosis depends on applied staining method, kind of specimens and properly sampling from infected area. Specimens are provided by either non-invasive methods (such as sputum or stimulation procedure) or even invasive methods like broncho-alveolar lavage, lung homogenate and lung biopsy. Invasive methods have higher sensitivity (12). Those samples collected from HIV positive patients have also higher sensitivity because of high load of the organisms in these samples (13).
With looking through advantages and disadvantages these two methods, we want to evaluate the efficiency of the Giemsa and GMS in diagnosis of the PCP in animal model in different samples and applied conditions.
Materials and Methods
Procedure for collecting specimens
Eight circular bodies (4-8 µM)
Purple banana shape (sporozoite) along each other
Observation of trophozoite (2-4 µM) with blue nucleolus and pink cytoplasm that are usually in mass shape near each other or separated
Observed cysts by Giemsa staining method
Observed cysts by GMS Staining method
Scoring results of staining methods
71.8% of tested specimens were negative and the rest 28.2% had just 1+ and 2+ grade by Giemsa. Negative rate of GMS was less than Giemsa. 51.3% of GMS had negative results while 15.4% and 5.1% had 3+ and 4+ grades (Table No. 2).
Three specimens (oral swab, lung homogenate and BAL) have been compared with each other in this study. Analyzed results revealed none of oral swab samples were positive even in the presence of the infection. These samples had the highest positive rate in lung homogenate and then in BAL specimens just in the early stage of the infection, while both had the same sensitivity in the last weeks.
It is frequently reported that microscopic examination of stained smears on specimens is standard method for diagnosis of PCP. Staining method is usually based on diagnosis of the cysts, because trophozoites are normally mistaken with counter-stain materials (13, 17). Giemsa method is the easiest, cheapest and the most rapid procedure, enable to differentiate those empty cysts from those containing organisms or even with yeast cells (5,6). Despite these advantages, diagnosis of the PCP is rather difficult and requires skilled microscopist because differentiation of the organisms from smears materials is not easy (17). Sensitivity of this method is also expected to be lower than GMS because of incapability of those empty cysts.
On the other hand, GMS method is usually used to observed fungi in tissue specimens. This method is specific for cyst wall and is applied as a gold standard method for diagnosis of pneumocystosis. Having long steps of staining and requirement of more than one hour time to perform are of its limitations. This method also can not diagnose trophozoite. There is possibility infection can not be identified in those mild infections or those cases with high ratio of trophozoite to cyst (6,7). Misdiagnosing is other limitation (1). Additionally, effect of anti-pneumocystosis drugs can not be determined because those empty cysts are not differentiated from the cysts containing trophozoites.
It is frequently observed HIV positive patients had pneumocystis cysts in their lung after treatment. Sporozoites of these cysts that have been destroyed by the drugs are responsible for these situations (18). We can differentiate pneumocystis cysts from other organisms because of two dark spots attached to each other inside the cysts, if GMS staining method to be performed in best conditions. We could decrease require necessary time for staining steps up to 30 min in this study.
It is reported Giemsa method has low sensitivity among studied staining procedures (8, 17,19). Obtaining results in this study is in agreement with the other reports. Analyzed Results revealed GMS has higher sensitivity than Giemsa. Gurpreet has reported that routine staining method has enough sensitivity in those specimens containing high organisms such as biopsy, BAL specimens and taken specimens of those HIV positive patients (11, 20). Obviously, this routine method has high false positive results in HIV negative patients even in BAL specimens (15, 21,22).
The standard staining method is microscopic observation of Pneumocystis in specimens such as BAL, Stimulated sputum, or biopsy (23,24,25). Oral swab sampling is non-invasive method that has been applied in animal model (26,27) with no positive results. Diagnosis of PCP is usually made by stimulated sputum sampling in HIV positive patients that is rarely positive in HIV negative patients with immunodeficiency problem. Correct diagnosis requires proper sampling such as BAL or lung biopsy. However, stimulated sputum sampling is more applicable in HIV positive patients because of higher load of organisms in this patients group (27,28,29). The sensitivity will be increase in those invasive samples. The quality of the result will be better in those invasive specimens that sampling is near to the colonized area (11, 30). BAL sample is an invasive method that is occasionally facing with danger, although is a selective sample (31). Its sensitivity by staining methods is about 55-78 percent (11, 32). False negative is even high in BAL sample in HIV negative patients (21,22). Besides false negative rate is high in non-invasive sampling methods and those patients that are in early stage of the disease (14). Open lung biopsy is the most invasive method that require for operating room. Use of this technique enables us to have the best specimens, although patients are facing with side effects (12).
Staining is necessary for diagnosis of pneumocystis if no other alternative method is available. Therefore, it is underlining that Giemsa procedure to be considered as the last alternative method. However, it is recommended to use other new diagnostic method such as PCR to decrease the risk of danger invasive sampling.