Page 87 - AIDSBK23C

Page 87

Mature cyst forms of P jiroveci (carinii) contain up to eight sporozoites. When the cysts
rupture, the released sporozoites mature into trophozoites and repeat the cycle. In tissue
sections, the cysts are identified by cell wall stains such as Gomori methenamine silver (GMS),
cresyl violet, or toluidine blue. Gomori methenamine silver staining gives the best contrast for
screening of a tissue section or cytologic smear because the red cell-sized P jiroveci (carinii)
organisms have a dark brown-black color. The 5 to 7 micron cysts usually occur in cohesive
clusters. They are round to elliptical in shape with sharp but sometimes slightly folded edges
resembling crushed ping-pong balls. Folding or rupture produces crescentic (parenthesis-
shaped) or cup-shaped forms. The lightly stained folds of the P jiroveci (carinii) cell membranes
may appear as a central dark dot. Endothelial cell nuclei, in contrast, have a granular to stippled
appearance. Red blood cells may be concave, folded, or crescentic, but they are smaller, do not
typically appear clustered in alveoli, and have no central dot. Precipitated stains yield artefacts
that are variably sized, have angular borders and are distributed haphazardly throughout the slide
with no regard for tissue structures. [401]
The P jiroveci (carinii) organisms are typically found within a foamy to granular pink
exudate within alveoli. This foamy exudate is seen by electron microscopy to be composed of
cysts and trophozoites with little or no fibrin. The organisms appear to be held together by
slender membranotubular extensions growing from their surfaces. The uneven contours of the
organisms lead to the formation of voids that contribute to the characteristic light microscopic
appearance of the foamy exudate. P jiroveci (carinii) cysts have a characteristic folded or
crescentic appearance.[401]
The GMS stain is most frequently used for diagnosis of Pneumocystis jiroveci (carinii).
There are a variety of methods published for the performance of this stain, and some of them
employ a microwave oven or pre-treatment with oxidizers to reduce the time needed for
completion to under 20 minutes and improve recognition of organisms.[403,404] Regardless of
which method is chosen, it is crucial that this stain be performed as consistently as possible to
avoid both false positive and false negative diagnoses.
False negative methenamine silver stains result from under stained preparations in which
the P jiroveci (carinii) cysts are too faint to be recognized. Over-staining results in false
negative results if there is so much black staining precipitate on the slide that it obscures the
organisms. False positive preparations come from over staining so that red blood cells,
endothelial cell nuclei, or precipitated stain that appear the same overall size and shape as
Pneumocystis organisms are misinterpreted.[405]
Several methods are available in addition to GMS staining for identification of P jiroveci
(carinii) in smears. Giemsa or Diff-Quik staining identifies the small, delicate intracystic bodies,
or sporozoites (up to 8), under oil immersion (1000X) arranged in a clock face to haphazard
distribution within the cyst and appearing as 1 to 2 micron dark blue dots. Giemsa or Diff-Quik
stains cannot demonstrate the organism's cell wall, so contrast is poor, limiting this technique
primarily to cytologic material obtained from bronchoalveolar lavage or sputum specimens. This
method is preferred by some pathologists because the appearance is quite characteristic, giving a
high specificity, and the method is simple and quick.[406]
The calcofluor white stain can also be utilized to detect cysts of P jiroveci (carinii) in
pulmonary specimens.[408] With calcofluor white, a chemofluorescent stain, cysts of P jiroveci
(carinii) will appear under fluorescence microscopy with light peripheral staining along with a
double-parenthesis-like structure near the center of the cysts.[407] The sensitivity of this stain is
as good as GMS for detection of P jiroveci (carinii) cysts.[407]