A phenotype-genotype approach to predicting CYP450 and P-glycoprotein drug interactions with the mixed inhibitor/inducer tipranavir/ritonavir.

The effects of tipranavir/ritonavir (TPV/r) on hepatic and intestinal P-glycoprotein (P-gp) and cytochrome P450 (CYP) enzyme activity were evaluated in 23 volunteers. The subjects received oral (p.o.) caffeine, warfarin + vitamin K, omeprazole, dextromethorphan, and midazolam and digoxin (p.o. and intravenous (i.v.)) at baseline, during the first three doses of TPV/r (500 mg/200 mg b.i.d.), and at steady state. Plasma area under the curve (AUC)(0-infinity) and urinary metabolite ratios were used for quantification of protein activities. A single dose of TPV/r had no effect on the activity of CYP1A2 and CYP2C9; it weakly inhibited CYP2C19 and P-gp; and it potently inhibited CYP2D6 and CYP3A. Multiple dosing produced weak induction of CYP1A2, moderate induction of CYP2C19, potent induction of intestinal P-gp, and potent inhibition of CYP2D6 and CYP3A, with no significant effects on CYP2C9 and hepatic P-gp. Several P450/transporter single-nucleotide polymorphisms correlated with the baseline phenotype but not with the extent of inhibition or induction. Although mixed induction and inhibition are present, this approach offers an understanding of drug interaction mechanisms and ultimately assists in optimizing the clinical use of TPV/r.

Vaccination of seropositive subjects with CHIRON CMV gB subunit vaccine combined with MF59 adjuvant for production of CMV immune globulin.

The safety and immunogenicity of four different regimens of CHIRON cytomegalovirus (CMV) gB subunit vaccine combined with MF59 adjuvant and administered to seropositive plasma donors were evaluated to ascertain whether vaccination of seropositive subjects would significantly increase antibody titer to gB glycoprotein. This was done to select the best vaccination regimen for generating high-titered plasma for manufacture of CMV immune globulin. No serious adverse events were attributed to this vaccine, and the vaccine was well tolerated. Only the first dose of vaccine in each regimen stimulated a four-fold or greater antibody response to gB glycoprotein and each regimen induced similar antibody titers. However, initial vaccination followed by a 1 week rest from plasmapheresis and two booster vaccinations at 8 and 24 weeks, each followed with another 1 week rest from plasmapheresis, maintained the highest geometric mean gB ELISA titer of the four regimens over the 34-week post-vaccination period. CMVIG manufactured from a pool of high titered plasma units from two of four subject groups had gB ELISA and neutralizing antibody titers nine and six times higher, respectively, compared to Cytogam, indicating that vaccination of seropositive subjects with CHIRON gB vaccine combined with MF59 adjuvant prior to harvesting plasma can enhance functional antibody in a CMVIG product.

Evaluation of the modified Visuwell Strep-A enzyme immunoassay for detection of group-A Streptococcus from throat swabs.

The modified Visuwell Strep-A enzyme immunoassay (EIA) was compared with culture for detection of group-A Streptococcus from throat swabs. Throat swabs in modified Stuarts medium obtained after culture at two institutions were tested in Visuwell. Cumulative results were n = 417, sensitivity 87.8%, specificity 89.9% predictive value positive (PVP) 67.9%, predictive value negative (PVN) 96.8%, and accuracy 89.5%. At another site, swabs were delivered to the laboratory without transport medium, cultured, and subsequently tested by Visuwell (n = 202, sensitivity 79.6%, specificity 84.5%, PVP 65.2%, PVN 91.9%, accuracy 83.2%). When 1+ culture-positive specimens were considered negative, the sensitivity and PVN increased from 79.6% to 90.2% and 91.9% to 97.1% respectively. Overall performance of the modified Visuwell was comparable with that of the initial assay for throat swabs transported with or without modified Stuarts medium. Cross reaction with organisms other than group-A Streptococcus normally found in the oropharynx was negligible in Visuwell and the limit of detection of group-A Streptococcus was 5 x 10(4) colony-forming units.

Comparison of Visuwell enzyme immunoassay to culture for detection of group A Streptococcus in throat swab specimens.

A microwell enzyme immunoassay (Visuwell) for direct detection of Group A streptococcal antigen from throat swab specimens has been developed. It incorporates urease conjugated antibody as the detector and is easily interpreted by a yellow to purple color change. Throat specimens obtained on rayon-tipped swabs were transported moist in modified Stuarts medium and cultured before being tested in Visuwell (n = 585, prevalence 17.1%, sensitivity 88%, specificity 92.4%, predictive value positive 70.4%, predictive value negative 97.4%, and accuracy 91.6%). In instances of discrepancy between culture and Visuwell, throat swab extracts were tested in a latex agglutination test. In 21 of 37 instances of Visuwell-positive, culture-negative specimens, latex agglutination was positive. Throat specimens obtained using double rayon swabs and transported to the laboratory dry had one swab cultured and the other tested in Visuwell (n = 280, prevalence 20.4%, sensitivity 75.4%, specificity 88.3%, predictive value positive 62.3%, predictive value negative 93.4%, and accuracy 85.7%). When 1+ culture positive specimens were considered negative, a sensitivity of 97.6% was obtained. In 14 of 26 instances of Visuwell-positive, culture-negative specimens, latex agglutination was positive. Cross-reaction with organisms other than Group A Streptococcus found in the oropharynx was negligible in Visuwell. Limit of detection of Group A streptococcal antigen was equivalent for Visuwell and latex agglutination.

The effectiveness of six disinfectants in inactivation of reovirus 3.

Reovirus 3 in the presence of foetal bovine serum was exposed to six disinfectants for times of 10, 20 and 30 sec. At the end of such exposure times the addition of skim milk terminated disinfectant activity, and residual virus was assayed using the plaque technique. The six disinfectants considered were Javex (a sodium hypochlorite disinfectant), sodium hydroxide, ethanol, Wescodyne, One Stroke Ves-Phene, and Sonacide. Ethanol (95% v/v) and 1/75 Javex (800 ppm chlorine) were the most effective virucides. Both of these agents inactivated 10(5) plaque forming units (PFU) in 30 sec. Undiluted Sonacide, 0.25% (w/v) sodium hydroxide and 1/200 Wescodyne each inactivated between 10(2) and 10(3) PFU in 30 sec. Javex at a dilution of 1/100 (600 ppm chlorine) was next in effectiveness, inactivating 10(1.5) PFU in 30 sec and was more effective than 1/50 Ves-Phene which inactivated 10(1) PFU in 30 sec. Ethanol in 70% (v/v) solution was totally ineffective in inactivating reovirus 3. Ethanol (95% v/v) after dilution in the test system was 76% (v/v) and ethanol (70% v/v) was really 56% (v/v).

Comparative in vitro activity of ceftizoxime, cefoperazone, and cefoxitin against anaerobic bacteria.

Against 482 obligate anaerobes studied by the agar dilution technique, ceftizoxime was significantly more active than both cefoxitin and cefoperazone (P less than 0.001); the latter two agents were comparable in activity. The enhanced activity of ceftizoxime, as compared with the activity of cefoxitin, was against both gram-positive and gram-negative anaerobes (especially Lactobacillus and bacteroides spp). Cefoperazone, however, was more active than cefoxitin against gram-positive anaerobes (particularly Lactobacillus spp.) but was less active than cefoxitin against gram-negative anaerobes (particularly Bacteroides fragilis and Veillonella spp.).

The relative resistance of f2 bacteriophage to inactivation by disinfectants.

f2 bacteriophage in the presence of fetal calf serum (at a final concentration of 10%) was exposed to six commonly used disinfectants for times of 10, 20 and 30 sec. At the end of exposure times skim milk neutralized the disinfectant activity and residual virus was assayed using the plaque technique. The 6 disinfectants considered were Javex, sodium hydroxide, ethanol, Wescodyne, One Stroke Ves-Phene and Sonacide. A 0.25% (w/v) solution of sodium hydroxide and 1/50 Javex (1200 parts/10(6) chlorine) were the most effective of the six disinfectants considered since 10(5) f2 bacteriophage were inactivated in 30 seconds in each instance. Since a 0.25% (w/v) solution of sodium hydroxide had a pH of 12.5 this made it too caustic to use as a disinfectant in many practical situations. It was concluded therefore that Javex at some dilution less than 1/50 (greater than 1200 parts/10(6) chlorine) was the most practical of the six disinfectants to use. Ethanol (95%, v/v) inactivated 10(3) f2 bacteriophage in 30 seconds while 1/20 Wescodyne and undiluted Sonacide inactivated 10(1)-virus particles. Ves-Phene at a dilution of 1/50 was a completely ineffective virucide during the 30 sec exposure. The resistance of f2 bacteriophage to inactivation by these six disinfectants was compared with that of echovirus 11 and coxsackievirus B5. In all instances except exposure to undiluted Sonacide, f2 was comparable in resistance to inactivation and in many cases had greater resistance.

The relative effectiveness of commonly used disinfectants in inactivation of coxsackievirus B5.

Coxsackievirus B5 in the presence of fetal calf serum was exposed to six commonly used disinfectants for times of 10, 20 and 30 s. At the end of exposure times skim milk neutralized the disinfectant activity, with residual virus assayed by the plaque technique. The six disinfectants considered were Javex, sodium hydroxide, ethanol, Wescodyne, One Stroke Ves-Phene and Sonacide. Although 95% (v/v) ethanol was significantly more virucidal than dilutions of the other five disinfectants tested causing a 10(6) reduction in 20 s, it may not be practical to use in many instances. Next to 95% (v/v) ethanol, 1/75 (800 parts/10(6) Javex, 0.25% (w/v) sodium hydroxide and 1/200 Wescodyne were the most effective virucides. These disinfectants were equal in effectiveness causing a 10(5) reduction of coxsackievirus B5 in 30 s. Of these three disinfectants Javex is the most practical to use since sodium hydoroxide is caustic and Wescodyne is selective in its virucidal action. Undiluted Sonacide was a less effective virucide causing a less than 10-fold reduction of coxsackievirus B5 in 30 s. A 1/50 dilution of One Stroke Ves-Phene was the least effective virucide tested since it did not significantly inactivate coxsackievirus B5 in 30 s.

The relative effectiveness of commonly used disinfectants in inactivation of echovirus 11.

Echovirus 11 in the presence of fetal calf serum was exposed to six commonly used disinfectants for times of 10, 20 and 30 s. At the end of such exposure times, skim milk neutralized disinfectant activity and residual virus was assayed using the plaque technique. The six disinfectants studied were Javex, sodium hydroxide, ethanol, Wescodyne, One Stroke Ves-Phene, and Sonacide. Although 0.25% (w/v) sodium hydroxide and 95% (v/v) ethanol were equally virucidal and significantly more so than the other four disinfectants, causing 10(6) reduction in 20 s, they may not be practical to use in many instances. Javex at a dilution of 1/50 (1200 parts/10(6) chlorine) proved to be virucidal causing 10(3.5) reduction of echovirus 11 in 30 s. Wescodyne (1/50) and undiluted Sonacide were relatively ineffective causing 10 reduction or less of echovirus 11 in 30 s. One Stroke Ves-Phene (1/50) was ineffective causing no significant inactivation in 30 s.

Wescodyne: lack of activity against poliovirus in the presence of organic matter.

The National Institute of Health Guidelines for Recombinant DNA Research recommends 2% aqueous Wescodyne, an iodophore that is used in many hospitals and laboratories as a disinfectant, as a decontaminant for biological safety cabinets and 5% for a spill outside a cabinet. A contact time of 10 to 15 minutes was given for the 2% solution and 20 minutes was considered adequate for the 5% concentration. The results indicate: 1. Aqueous Wescodyne (5%) is ineffective when used for 80 minutes against poliovirus in a test mixture containing 8.5% bovine serum albumin (a mixture equivalent in protein concentration to the higher range in serum). 2. Wescodyne (10%) employed under the same conditions for 40 minutes is also ineffective. 3. Wescodyne (10% v/v) in 50% ethanol (w/w) was effective and this mixture, originally recommended for hand washing, should be considered for use in biohazard situations, particularly for decontamination of work surfaces and biological safety cabinets. These results are of significance for if a virucide cannot inactivate poliovirus one would be concerned about using the virucide against hepatitis B or SV40 viruses.

Anti-immunoglobulin analysis by diffusion patterns of inhibition and facilitation of complementary lysis in agar. II. Diffusion-lysis as a method for recognizing in vivo immunosuppressive activity of anti-immunoglobulins.

This paper provides evidence that it is possible to prepare facilitating anti-mouse immunoglobulin (that is, anti-mouse immunoglobulin which facilitates complementary lysis of red cells sensitized with mouse-produced haemolysin) which, when injected into mice 24 hours before an injection of sheep red cells, very markedly reduced the number of haemolysin-producing cells detectable in spleen four days later. The diffusion-lysis method was used to recognize this and other anti-Ig's in heterologous antiserum and fractions thereof. The effective antibody was in the gamma2 fraction of antiserum produced in guinea pigs by injecting them with guinea pig red cells sensitized with mouse-produced haemolysin. This method of immunizing was used in order to stimulate the production of antibody against immunoglobulin which had undergone the configurational change characteristically occurring when antibody unites with antigen. The 19S fraction of the antiserum contained inhibiting anti-mouse immunoglobulin (anti-mouse immunoglobulin which inhibits complementary lysis of red cells sensitized with mouse-produced haemolysin) and interfered with immune depression by the gamma2 fraction. It is postulated that the gamma2 fraction induces complementary lysis only of lymphocytes whose surface immunoglobulin receptors have bound antigen and undergone configurational change. It is suggested that facilitating anti-immunoglobulin of the type described is responsible for immune suppression by anti-lymphocyte serum (ALS). Facilitating anti-mouse immunoglobulin was demonstrated in two samples of ALS (anti-mouse) which were active in suppressing graft rejection, but inhibiting anti-mouse immunoglobulin only was found in a sample which was ineffective in suppressing graft rejection.