Emergence and Spread of Neisseria Gonorrhoeae Antimicrobial Resistance in California

The development of antimicrobial resistance in Neisseria gonorrhoeae has severely limited the treatment options available for uncomplicated urogenital infection. As of August 2012, dual therapy with an injectable third generation cephalosporin and a macrolide is the only Centers for Disease Control and Prevention recommended treatment regimen for empirical therapy in the United States. Verified treatment failures with oral cephalosporins used for urethral infections and with injectable cephalosporins used for pharyngeal infections have been reported in Asia, Europe and Australia. At this time, there have been no verified treatment failures with injectable cephalosporins in a urethral infection. Isolates with reduced susceptibility to the both oral and injectable cephalosporins have been described in the U.S., but no treatment failures have been described. This dissertation is formatted as three self-contained research papers. The first paper compares Etest to agar dilution for the determination of N. gonorrhoeae minimum inhibitory concentrations. The second paper describes the genotypic and phenotypic surveillance of N. gonorrhoeae resistance to third generation cephalosporins and macrolides in California in 2011. The third paper details the development of a new molecular assay for the detection of genes associated with reduced susceptibility to cephalosporins

Neisseria gonorrhoeae Strain with Reduced Susceptibilities to Extended-Spectrum Cephalosporins

The spread of Neisseria gonorrhoeae strains with reduced susceptibility to extended-spectrum cephalosporins is an increasing public health threat. Using Etest and multiantigen sequence typing, we detected sequence type 1407, which is associated with reduced susceptibilities to extended-spectrum cephalosporins, in 4 major populated regions in California, USA, in 2012

Standardization and validation of a cytometric bead assay to assess antibodies to multiple Plasmodium falciparum recombinant antigens

Abstract Background Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple Plasmodium falciparum antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to P. falciparum antigens, and to compare results of multiplex CBA to ELISA. Methods Antibodies to ten recombinant P. falciparum antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex vs multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared. Results Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (r = 0.88-0.99, P values from <0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA. Conclusion With optimization, CBA may be the preferred method of testing for antibodies to P. falciparum antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in positive samples and lower background readings for blank samples than ELISA

Evidence for globally shared, cross-reacting polymorphic epitopes in the pregnancy-associated malaria vaccine candidate VAR2CSA

Pregnancy-associated malaria (PAM) is characterized by the placental sequestration of Plasmodium falciparum-infected erythrocytes (IEs) with the ability to bind to chondroitin sulfate A (CSA). VAR2CSA is a leading candidate for a pregnancy malaria vaccine, but its large size (∼350 kDa) and extensive polymorphism may pose a challenge to vaccine development. In this study, rabbits were immunized with individual VAR2CSA Duffy binding-like (DBL) domains expressed in Pichia pastoris or var2csa plasmid DNA and sera were screened on different CSA-binding parasite lines. Rabbit antibodies to three recombinant proteins (DBL1, DBL3, and DBL6) and four plasmid DNAs (DBL1, DBL3, DBL5, and DBL6) reacted with homologous FCR3-CSA IEs. By comparison, antibodies to the DBL4 domain were unable to react with native VAR2CSA protein unless it was first partially proteolyzed with trypsin or chymotrypsin. To investigate the antigenic relationship of geographically diverse CSA-binding isolates, rabbit immune sera were screened on four heterologous CSA-binding lines from different continental origins. Antibodies did not target conserved epitopes exposed in all VAR2CSA alleles; however, antisera to several DBL domains cross-reacted on parasite isolates that had polymorphic loops in common with the homologous immunogen. This study demonstrates that VAR2CSA contains common polymorphic epitopes that are shared between geographically diverse CSA-binding lines

Human Factor H Domains 6 and 7 Fused to IgG1 Fc Are Immunotherapeutic against Neisseria gonorrhoeae

Novel therapeutics against multidrug-resistant Neisseria gonorrhoeae are urgently needed. Gonococcal lipooligosaccharide often expresses lacto-N-neotetraose (LNnT), which becomes sialylated in vivo, enhancing factor H (FH) binding and contributing to the organism\u27s ability to resist killing by complement. We previously showed that FH domains 18-20 (with a D-to-G mutation at position 1119 in domain 19) fused to Fc (FHD1119G/Fc) displayed complement-dependent bactericidal activity in vitro and attenuated gonococcal vaginal colonization of mice. Gonococcal lipooligosaccharide phase variation can result in loss of LNnT expression. Loss of sialylated LNnT, although associated with a considerable fitness cost, could decrease efficacy of FHD1119G/Fc. Similar to N. meningitidis, gonococci also bind FH domains 6 and 7 through Neisserial surface protein A (NspA). In this study, we show that a fusion protein comprising FH domains 6 and 7 fused to human IgG1 Fc (FH6,7/Fc) bound to 15 wild-type antimicrobial resistant isolates of N. gonorrhoeae and to each of six lgtA gonococcal deletion mutants. FH6,7/Fc mediated complement-dependent killing of 8 of the 15 wild-type gonococcal isolates and effectively reduced the duration and burden of vaginal colonization of three gonococcal strains tested in wild-type mice, including two strains that resisted complement-dependent killing but on which FH6,7/Fc enhanced C3 deposition. FH/Fc lost efficacy when Fc was mutated to abrogate C1q binding and in C1q-/- mice, highlighting the requirement of the classical pathway for its activity. Targeting gonococci with FH6,7/Fc provides an additional immunotherapeutic approach against multidrug-resistant gonorrhea

Standardization and validation of a cytometric bead assay to assess antibodies to multiple <it>Plasmodium falciparum</it> recombinant antigens

<p>Abstract</p> <p>Background</p> <p>Multiplex cytometric bead assay (CBA) have a number of advantages over ELISA for antibody testing, but little information is available on standardization and validation of antibody CBA to multiple <it>Plasmodium falciparum</it> antigens. The present study was set to determine optimal parameters for multiplex testing of antibodies to <it>P. falciparum</it> antigens, and to compare results of multiplex CBA to ELISA.</p> <p>Methods</p> <p>Antibodies to ten recombinant <it>P. falciparum</it> antigens were measured by CBA and ELISA in samples from 30 individuals from a malaria endemic area of Kenya and compared to known positive and negative control plasma samples. Optimal antigen amounts, monoplex <it>vs</it> multiplex testing, plasma dilution, optimal buffer, number of beads required were assessed for CBA testing, and results from CBA vs. ELISA testing were compared.</p> <p>Results</p> <p>Optimal amounts for CBA antibody testing differed according to antigen. Results for monoplex CBA testing correlated strongly with multiplex testing for all antigens (<it>r</it> = 0.88-0.99, <it>P</it> values from <0.0001 - 0.004), and antibodies to variants of the same antigen were accurately distinguished within a multiplex reaction. Plasma dilutions of 1:100 or 1:200 were optimal for all antigens for CBA testing. Plasma diluted in a buffer containing 0.05% sodium azide, 0.5% polyvinylalcohol, and 0.8% polyvinylpyrrolidone had the lowest background activity. CBA median fluorescence intensity (MFI) values with 1,000 antigen-conjugated beads/well did not differ significantly from MFI with 5,000 beads/well. CBA and ELISA results correlated well for all antigens except apical membrane antigen-1 (AMA-1). CBA testing produced a greater range of values in samples from malaria endemic areas and less background reactivity for blank samples than ELISA.</p> <p>Conclusion</p> <p>With optimization, CBA may be the preferred method of testing for antibodies to <it>P. falciparum</it> antigens, as CBA can test for antibodies to multiple recombinant antigens from a single plasma sample and produces a greater range of values in positive samples and lower background readings for blank samples than ELISA.</p