ISOLATION OF PENICILLIN-RESISTANT STRAINS OF NEISSERIA GONORRHOEAE AND HAEMOPHILUS INFLUENZAE HAS INCREASED SUBSTANTIALLY IN THE U.

ISOLATION OF PENICILLIN-RESISTANT STRAINS OF NEISSERIA GONORRHOEAE AND HAEMOPHILUS INFLUENZAE HAS INCREASED SUBSTANTIALLY IN THE U.S. OVER THE LAST FEW YEARS. IN SOME REGIONS THIS RAPID INCREASE IN THE NUMBER OF RESISTANT STRAINS HAS HEIGHTENED THE CONCERN OF PUBLIC HEALTH AGENCIES AND HAS RESULTED IN REVISED METHODS OF CONTROL. IF THE SPREAD OF RESISTANT STRAINS IS NOT ADEQUATELY CONTROLLED, THIS FAILURE WILL BE REFLECTED IN HIGHER INCIDENCES OF DISEASE AND INCREASED COSTS OF TREATMENT WITH ALTERNATIVE ANTIBIOTICS. ONE IMPROVEMENT IN THE METHODS FOR CONTROL WOULD BE TO PROVIDE A MEANS OF MORE RAPID BACTERIAL DETECTION AND IDENTIFICATION OF RESISTANCE FACTORS. THIS METHOD MUST BE FASTER THAN THE 2-4 DAYS NECESSARY FOR CULTURE-DEPENDENT TECHNIQUES BUT POSSESS A SIMILAR DEGREE OF ACCURACY AND SENSITIVITY. WE INTEND TO DEVELOP A DIRECT, CULTURE-INDEPENDENT ASSAY UTILIZING MONOCLONAL ANTIBODIES. SINCE RESISTANCE IS CONFERRED BY UNIQUE, PLASMID-ENCODED PROTEINS (I.E., BETA-LACTAMASE) TARGET MOLECULES CAN BE IDENTIFIED BY A SENSITIVE IMMUNOFLUORESCENCE ASSAY TO STAIN INDIVIDUAL BACTERIA IN A DIRECT PATIENT SMEAR. FOR THIS PURPOSE, WE WILL UTILIZE MONOCLONAL ANTIBODIES DIRECTED AGAINST TWO "FUNCTIONAL" TARGETS. THE FIRST WILL PROVIDE THE PHYLOGENETIC IDENTIFICATION OF THE BACTERIA (I.E., ANTIBODIES TO MEMBRANE PROTEINS OR LPS), AND THE SECOND WILL IDENTIFY THE SPECIFIC PLASMID-ENCODED RESISTANCE FACTOR (I.E., ANTIBODY TO BETA-LACTAMASE). PATIENT SPECIMEN WILL BE SMEARED ONTO MICROSCOPE SLIDES, FIXED, AND STAINED WITH THE ANTIBODIES. THE SLIDES WILL THEN BE SCANNED UNDER A FLUORESCENCE MICROSCOPE FOR THE PRESENCE OF STAINED ORGANISMS. SINCE BETA-LACTAMASE ACCUMULATE IN THE PERIPLASMIC SPACES BETWEEN THE CELL WALL AND OUTER MEMBRANE, IT MAY BE POSSIBLE TO IDENTIFY THE BACTERIA POSSESSING THE RESISTANCE FACTOR BY A CHARACTERISTIC STAINING MORPHOLOGY. WE SHOULD BE ABLE TO PERFORM THE TEST IN LESS THAN AN HOUR WITH A DEGREE OF SENSITIVITY AND ACCURACY COMPARABLE TO CULTURE.