Clostridium difficile infections are a major cause of antibiotic-associated diarrhea in hospital and care facility patients. In spite of the availability of effective antibiotic treatments, C. difficile infection (CDI) is still a major cause of patient suffering, death, and substantial health care costs. Clostridium difficile exerts its major pathological effects through the actions of two protein exotoxins, TcdA and TcdB, which bind to and disrupt gut tissue. Antibiotics target the infecting bacteria but not the exotoxins. Administering neutralizing antibodies against TcdA and TcdB to patients receiving antibiotic treatment might modulate the effects of the exotoxins directly. We have developed a mixture of three humanized IgG1 monoclonal antibodies (MAbs) which neutralize TcdA and TcdB to address three clinical needs: reduction of the severity and duration of diarrhea, reduction of death rates, and reduction of the rate of recurrence. The UCB MAb mixture showed higher potency in a variety of in vitro binding and neutralization assays (ϳ10-fold improvements), higher levels of protection in a hamster model of CDI (82% versus 18% at 28 days), and higher valencies of toxin binding (12 versus 2 for TcdA and 3 versus 2 for TcdB) than other agents in clinical development. Comparisons of the MAb properties also offered some insight into the potential relative importance of TcdA and TcdB in the disease process.

A total of 100 herpes simplex viruses isolated from lesions not responding to acyclovir (ACV) therapy were recovered from 51 patients infected with human immunodeficiency virus. In vitro analysis of these isolates included testing their susceptibility to ACV and determining their thymidine kinase (TK) phenotypes. Of the 100 isolates evaluated, 23 were ACV sensitive and 77 were ACV resistant. Seventy-four of these ACV-resistant isolates were of the TK-deficient or low-TK-producer phenotype and three were of the TK-altered phenotype. The TKs of isolates that represented each of the different autoradiographic phenotypes were further characterized by enzyme kinetics. The ability of selected isolates to cause disease in vivo was evaluated-by using several mouse virulence models. Cutaneous virulence in normal and immunocompromised mice was evaluated, and neurovirulence in normal mice was determined. Latent infections were assayed by the cocultivation of trigeminal ganglia recovered from mice that had survived acute infection. These reactivated viruses were evaluated in vitro and compared with the original infecting isolate. The mechanisms of resistance and pathogenicity of these herpes simplex virus isolates recovered from patients positive for human immunodeficiency virus are similar to those reported for isolates recovered from normal and immunocompromised patients without AIDS.

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In vitro and in vivo studies were done on a berpes simplex virus type 2 strain recovered from a patient on acyclovir (ACV) which was ACV resistant but expressed thymidine (dThd) kinase (EC 2.7.1.21) activity. Plaque-purified clones derived from the original clinical sample were heterogeneous with respect to plaque size and drug susceptibility. The heterogeneity of this viral mixture was also evident from varied I251-labeled 5-iodo-2'-deoxycytidine autoradiographic patterns and from varied expression of dThd kinase-associated phosphorylating activities. Four clones from this mixture were 1-0i-D-arabinofuranosylthymine (ara-T) susceptible and ACV resistant. Extracts of cells infected with these 0lones catalyzed the phosphorylation of ara-T but little of ACV. The virus-coded dThd kinase was purified from one of these clones to determine whether its substrate specificity was altered. The amount of virus-coded dThd phosphorylating activity with the cell extracts was estimated to be sevenfold lower with the resistant clone than with the MS strain of herpes simplex virus type 2. The dThd kinase eluted from a dThd-agarose affinity column under the same conditions with extracts from both sources and substrate saturations of both enzymes by acyclic nucleoside analog phosphate acceptors were classical hyperbolic functions. However, there were significant differences in the kinetic parameters of substrates between the two enzymes. Apparent Km (Kin') values for dThd, deoxycytidine, ara-T, ACV, and the acyclic guanosine analog 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine (BW B759U) were 2-to 60-fold higher with the variant enzyme than with the enzyme from laboratory strain MS. Comparing these two enzymes, relative maximal phosphorylation rates (Vm) were eightfold lower for ACV but unchanged for BW B759U. In contrast, the relative rates for deoxycytidine and ara-T were eight-and twofold higher, respectively. The surprisingly good substrate activity with BW B759U compared with that of ACV (Vm/Km' = 0.39 versus 0.01) coincided with susceptibility of the ACV-resistant virus to BW B759U. This clinical variant retained its pathogenicity for mice and was only moderately less neurovirulent than wild-type virus. Although such mutants have the potential to induce illness less responsive to therapy, the recurrence from which the isolate was obtained was typical for this patient in severity and duration. Since this episode, the patient has been treated successfully with ACV.

Zovirax (acyclovir, ACV) is now widely accepted as a safe and effective treatment for the management of herpes simplex virus (HSV) infections in normal and immunocompromised patients. However, a common concern with regard to the widespread use of any antimicrobial agent is resistance. The virus specific mechanism of action of ACV involves two virus encoded enzymes, thymidine kinase (TK) and DNA polymerase. Any alteration in the genes coding for these two enzymes would therefore be expected to confer resistance. The findings from two extensive resistance monitoring programs have shown that in immunocompetent patients receiving ACV for the management of acute HSV disease, the incidence of resistance is extremely rare. The situation in the immunocompromised is different. In this patient group HSV disease is severe and protracted often requiring prolonged therapy thus increasing the exposure of the virus to drug. As a result HSV isolates resistant to ACV have occasionally been recovered. Biochemical and genetic analysis of the resistant clinical isolates has shown that resistance in the most part is due to an inability of the virus to produce TK which mirrors the findings with cell culture derived resistant virus. Laboratory studies would indicate that TK-deficient virus would have little clinical impact. Significantly, resistance has rarely been attributed to alterations in the substrate specificity of TK or DNA polymerase. The biological significance of these mutants is unclear but to date there has been no evidence of transmission of resistant virus.