- Volume 45, Issue 2, 1996

Detection and quantification of different Pneumocystis carinii (PC) life cycle forms were performed by polymerase chain reaction (PCR) and by morphological stains on bronchoalveolar lavage fluids (BALF) from HIV-infected patients with P. carinii pneumonia (PCP). The number of PC trophozoites was higher in patients with PCP who were receiving prophylaxis than in those not receiving prophylaxis. Also the cyst: trophozoite ratio was lower in the first group. No difference was observed between patients receiving different prophylactic medications. The imbalance between PC forms in BALF from patients with PCP receiving anti-PC prophylaxis may hamper the sensitivity of cyst stains. Multiple stains or PCR examination should be performed on BALF from patients with clinically suspected PCP who are receiving prophylaxis.

Vaginal swabs for microbiological culture were taken from 174 pregnant women whose vaginal flora had been evaluated by Gram’s stain; 50 had grade III flora (bacterial vaginosis, BV), 50 grade II (intermediate), 41 had vaginal flora graded as abnormal which then reverted to grade I (revertants) and 33 had normal flora (controls). The aim was to determine whether bacterial species isolated from women with grade II flora differed from those with grade III flora. Isolation of 120Lactobacillus spp. decreased from grade I to grade III and that of other aerobic and anaerobic bacterial species increased. There was little difference in the species isolated from women with grade II and grade III flora, but there was a distinct order in which organisms in different species increased in numbers. The vaginal flora of revertants was intermediate between that of healthy controls and those with grade II flora. Coagulase-negative Staphylococcus spp. were isolated from a greater number of revertants than grade I controls but the incidence did not increase in grade II or grade III. Bifidobacterium spp. were isolated from a greater number of revertants than grade I controls and increased further in grade II and grade III. However, Gardnerella vaginalis and Mycoplasma hominis were isolated from a much larger number of women with grade III flora than the other groups. The conclusion is that grade II is a transitional phase between grade I and grade III and that some organisms such as G. vaginalis and M. hominis only reach large numbers in the late stage. The sequence of appearance of the various bacterial species may be a result of the pathophysiological alteration of the vaginal ecosystem associated with BV.

A convenient dipstrip method (Bacteruritest; Mast Diagnostics) for bacterial quantification was evaluated with 42 endotracheal aspirates. For 31 specimens, the dipstrip method yielded counts within a 10-fold range of surface plate counts. Two specimens yielded counts by the dipstrip within a 100-fold range of plate counts. Six specimens yielded confluent growth at the greatest dilution tested by the dipstrip method, and counts > 1010 cfu/ml in the surface plate method. Three specimens yielded no detectable growth by the dipstrip and surface plate counts <102 cfu/ml. Dipstrips provide a cheap, convenient method for the routine quantification of the bacterial load in endotracheal aspirates.

A large study of the distribution of Clostridium difficile in the environment of the Cardiff area of South Wales was performed with a methodology designed to maximise recovery. A total of 2580 samples was taken, with 184 (7.1%) yielding isolates. The highest yield for C. difficile was obtained from river waters, with 14 (87.5%) of 16 samples from four rivers positive, and from sea water samples with 7 (44%) of 15 positive from six beaches on the Bristol Channel. In addition, 7 (46.7%) of 15 samples of lake water were also positive. Twenty-two (21%) of 104 soil samples, taken from random sites in Cardiff, were positive, as were 20% of environmental samples from four Cardiff hospitals. C. difficile was also isolated from 50% of eight swimming pool waters examined and 1 (5.5%) of 18 of mains tap water. Carriage of C. difficile in 524 faecal samples of assorted farm animals was c. 1%, and was 10% in dogs and 2% in cats. In private residences, the organism was present in 12 (2.2%) of 550 samples. While 2.4% of 300 raw vegetable samples were positive, none of 107 assorted fish gut contents was. These findings indicate that C. difficile may be more widely distributed in the general environment, particularly water, than was previously thought.

Cytotoxic strains of Helicobacter pylori cause an intense vacuolar degeneration of cells, due to the enlargement of late endosomes in the presence of membrane permeant weak bases. Bafilomycins, specific inhibitors of the vacuolar-type (V-) ATPase proton pump, prevent vacuole formation. The presence of the V-ATPase on vacuolar membranes was demonstrated by immunofluorescence with a monoclonal antibody (MAb) specific for the human 116-kDa regulatory subunit. The V-ATPase co-localised with the late endosomal marker rab7 on vacuolar membranes. In contrast, the early recycling endosomal compartment was not altered by the VacA cytotoxin, although it was endowed with the V-ATPase. Endocytosis of a MAb against the 116-kDa regulatory subunit of V-ATPase blocked endosomal acidification in HeLa cells and prevented VacA action. These results indicate that selective swelling of late endosomes, due to accumulation of osmotically active weak bases driven by the V-ATPase, is essential for vacuole formation.

After the introduction of the dengue-2 (16681) virus by intradermal (i.d.) injection into the footpads of mice, Langerhans cells (LCs) increased in numbers within 24 h at the site of injection and neutralising antibody developed. On comparing the i.d. and intramuscular (i.m.) routes, antibody was produced more rapidly and at higher levels when the virus was injected by the i.d. route. Subsequent re-challenge by the i.d. route produced an even more rapid serological response with all mice producing significant neutralising titres within 12 h. Numbers of ATPase-positive LCs varied with time. A significant sharp drop in LC densities in the early post-injection phase directly correlated with the increased numbers of dendritic cells in the superficial dermis and interfollicular sinuses of draining lymph nodes (LN). Immunofluorescence showed the presence of viral antigen in the footpad epidermis and draining LN within minutes or within 2 h of challenge, respectively.