The Cytological Diagnosis of Pneumocystis Carinii By Fluorescence Microscopy of Papanicolaou Stained Bronchoalveolar Lavage Specimens

In a retrospective and prospective analysis fluorescence microscopy of Papanicolaou stained bronchoalveolar lavage specimens has been applied to the diagnosis of Pneumocystis carinii (PC) in routine cytology. The pneumocysts presented as circular structures of 5 microns in diameter and of brilliant green-yellow fluorescence surrounding two mirror image reniform structures. Fluorescent inclusions of 1-3 microns diameter within the alveolar macrophages could be identified as remnants of pneumocysts by a follow-up of all steps of degradation ending in very small irregular granules. By applying both criteria, i.e. pneumocysts with reniform bodies and degradation inclusions within macrophages, Pneumocystis carinii pneumonitis (PCP) could be detected in 100% of cases. Transbronchial biopsy permitted the correct diagnosis in only 65.2% of cases. Retrospective analysis of slides is possible after a long period as no significant loss of fluorescence occurs after 4 years. Thus fluorescence microscopy permits the diagnosis of Pneumocystis carinii without any additional staining or loss of time.     

Multi-Gene Family of Major Surface Glycoproteins of Pneumocystis carinii: Full-Size cDNA Cloning and Expression

The major surface glycoprotein (MSG) of Pneumocystis cariniiplays a crucial role in the fatal pneumonia caused by this organismin AIDS patients. A cDNA encoding a full-length MSG polypeptidewas isolated from a phage library of rat-derived P. cariniicDNAs. The deduced MSG, referred to as the MSG5 subtype, isa 120,765-Da protein composed of 1,076 amino acids and containsan anchoring hydrophobic sequence at the C-terminus of the protein.Sequence analyses of cloned MSG-cDNAs revealed an MSG-gene familywith 70% protein sequence identity between subtypes. P. cariniikaryotype hybridization analyses indicated that the MSG genefamily members are scattered throughout most of the P. cariniichromosomes. These recombinant MSG proteins reacted with theantiserum from P. carinii-infected rats, as expected, and antiserumgenerated against P. carinii-infected mice, indicating the existenceof common determinants in MSG polypeptides. The family of MSGproteins is rich in cysteine residues and these cysteine arehighly conserved in all MSG subtypes regardless of species specificity,suggesting the structural and/or functional importance of thesecysteine. The pathobiological significance of the MSG gene familyand its sequence diversity in P. carinii is discussed.     

Improved Intracellular Morphology of Pneumocystis Carinii From Rat Lung by Postfixation with a Mixture of Potassium Ferrocyanide and Osmium Tetroxide

Pneumocystis carinii infected rat lungs were postfixed with a mixture of OsO₄ and K₄Fe(CN)₆. A marked improvement in staining of cell membranes, endoplasmic reticulum, nuclear membranes and glycogen was observed. These improvements were seen in both the trophic and cystic forms of the organisms. The addition of K₄Fe(CN)₆ did not improve the staining of cell walls, microtubules or ribosomes. Trophozoites were seen attached to both type 1 and type 2 pneumocytes by filopodia and/or intercalation of the cell body of P. carinii with the host lung cells. It is expected that the improvement in ultrastructural detail will allow better understanding of the ultrastructure of P. carinii and provide insights into the modes of action of various antimicrobial compounds on this organism.     

Pneumocystis carinii: Genetic Diversity and Cell Biology

As an important opportunistic pulmonary pathogen, Pneumocystis carinii has been the focus of extensive research over the decades. The use of laboratory animal models has permitted a detailed understanding of the host–parasite interaction but an understanding of the basic biology of P. carinii has lagged due in large part to the inability of the organism to grow well in culture and to the lack of a tractable genetic system. Molecular techniques have demonstrated extensive heterogeneity among P. carinii organisms isolated from different host species. Characterization of the genes and genomes of the Pneumocystis family has supported the notion that the family comprises different species rather than strains within the genus Pneumocystis and contributed to the understanding of the pathophysiology of infection. Many of the technical obstacles in the study of the organisms have been overcome in the past decade and the pace of research into the basic biology of the organism has accelerated. Biochemical pathways have been inferred from the presence of key enzyme activities or gene sequences, and attempts to dissect cellular pathways have been initiated. The Pneumocystis genome project promises to be a rich source of information with regard to the functional activity of the organism and the presence of specific biochemical pathways. These advances in our understanding of the biology of this organism should provide for future studies leading to the control of this opportunistic pathogen.     

Comparative morphology and ecology of two species of Litomosoides (Nematoda: Filarioidea) of rodents in Florida, with a key to the species of Litomosoides Chandler, 1931

From April 1970 to August 1971, 235 cotton rats (Sigmodon hispidus) and 281 rice rats (Oryzomys palustris) were trapped in salt and fresh water marshes in north-central Florida and examined for natural infections of filarial worms, Litomosoides spp. Cotton rats from both types of marshes were infected (28–44 per cent prevalence), whereas only rice rats from the salt marsh were infected (56 per cent prevalence). Older animals were more commonly infected than younger ones. In cotton rats the worms were located in the pleural cavity, whereas in rice rats the worms were located primarily in the abdominal cavity. Filarial worms from rice rats were transmitted experimentally to laboratory-reared rice rats and cotton rats, but worms from cotton rats were transmitted only to cotton rats. Morphological studies on adult forms and microfilariae indicated that the worms in rice rats were distinct from those in cotton rats and are therefore described as Litomosoides scotti sp. n. The cotton rat filariids were referable to Litomosoides carinii (Travassos, 1919) Vaz, 1934. L. scotti differs from L. carinii in the ratio of the spicules, in the shape of the distal end of the right spicule and in having shorter microfilariae.     

The value of direct fluorescent antibody (DFA) testing for the detection of Pneumocystis Carinii In cytological specimens

A direct fluorescent antibody (DFA) method was compared with methenamine silver staining (MSS) for the detection of Pneumocystis carinii in 384 cytological specimens. DFA testing was more sensitive than the MSS, with P. carinii detected in 31 specimens with DFA and 24 with the MSS. Results of the two methods disagreed in 17 specimens, all of which were sputa. Twelve sputum specimens were DFA positive/MSS negative and five were MSS positive/DFA negative. It is concluded that the DFA technique, although relatively expensive, is simple to perform and offers superior sensitivity to the MSS. However, in sputum specimens the combined use of DFA and MSS leads to optimal sensitivity for the detection of P. carinii.