The Journal of Infectious Diseases

Resiquimod, an investigational immune response modifier and Toll-like receptor (TLR) 7 and 8 agonist, stimulates production of cytokines that promote an antigen-specific T helper type 1 (Th1)--acquired immune response. In animal models, induction of Th1-specific responses modifies experimental herpes simplex virus (HSV) infection. We conducted a randomized, double-blind, vehicle-controlled trial to assess the efficacy of resiquimod 0.01% gel for reducing human anogenital HSV-2 mucosal reactivation. Adults with genital HSV-2 applied resiquimod or vehicle topically to herpes lesions 2 times weekly for 3 weeks and then collected daily anogenital swabs for 60 days for HSV DNA polymerase chain reaction. Recurrences during the subsequent 7 months were treated with study gel. During the final treatment-free 60 days, participants again collected daily swabs to assess shedding. The median lesion and shedding rates were lower for resiquimod compared with vehicle recipients during the initial sampling period (10% vs. 16% [P=.03] and 10% vs. 17% [P=.08], respectively) and during the final sampling period (3% vs. 22% [P<.001] and 10% vs. 26% [P=.009], respectively). Resiquimod did not influence recurrence length. These findings suggest that the immunological control of HSV-2 reactivation and lesion clearance may differ and that TLR7 and TLR8 agonists can reduce the frequency of mucosal HSV-2 reactivation.

We conducted a large national cohort study to examine the risk of cancer among men and women with genital warts (GW). By use of the Danish National Patient Register, we identified 16,155 men and 32,933 women who received a diagnosis of GW during 1978-2008. Standardized incidence ratios (SIRs) were computed as estimates of the relative risk of specific cancers or sites. A diagnosis of GW was strongly related to anal (SIR for men, 21.5; SIR for women, 7.8), vulvar (SIR, 14.8), vaginal (SIR, 5.9), cervical (SIR, 1.5), penile (SIR, 8.2), and head and neck cancer (SIR, 2.8), including subsites of head and neck cancer with confirmed HPV association (SIR for men, 3.5; SIR for women, 4.8). The risks remained elevated for >10 years following GW diagnosis. In addition, we found moderately increased SIR estimates for nonmelanoma skin cancer, smoking-related cancers, and Hodgkin and non-Hodgkin lymphoma. Individuals with GW have a long-term increased risk of anogenital cancers and head and neck cancers. The elevated risks of nonmelanoma skin cancers might indicate an association with HPV, while excess risks of other cancers could point to differences in other risk factors between individuals with GW and the general population.

Molluscum contagiosum virus (MCV) is a poxvirus that causes localized papules in healthy persons. We evaluated a woman with severe immunodeficiency and disseminated MCV. During treatment with CMX-001, an antiviral with activity against other poxviruses, MCV DNA was detected in 20% of plasma samples. When the patient was not receiving CMX-001, MCV DNA was detected in 50% of samples. We also noted improvement in warts on her fingers during CMX-001 therapy. Although MCV is caused by direct inoculation of virus into skin in healthy persons, in a severely immunocompromised person MCV DNA was present in blood and may spread by viremia.

Background. We report the first-in-human safety and immunogenicity assessment of a prototype Ad26 vector-based human immunodeficiency virus (HIV) vaccine in humans. Methods. Sixty Ad26-seronegative, healthy, HIV-uninfected subjects were enrolled in a randomized, double-blinded, placebo-controlled, dose-escalation phase 1 study. Five groups of 12 subjects received 10⁹–10¹¹ vp of the Ad26-EnvA vaccine (N = 10/group) or placebo (N = 2/group) at weeks 0 and 24 or weeks 0, 4, and 24. Safety and immunogenicity were assessed. Results. Self-limited reactogenicity was observed after the initial immunization at the highest (10¹¹ vp) dose. No product-related SAEs were observed. All subjects who received the Ad26-EnvA vaccine developed Ad26 NAb titers, EnvA-specific enzyme-linked immunosorbent assays (ELISA) titers, and EnvA-specific enzyme-linked immunospot assays (ELISPOT) responses. These responses persisted at week 52. At week 28 in the 10⁹, 10¹⁰, 10¹¹ vp 3-dose and the 10¹⁰ and 5 × 10¹⁰ vp 2-dose groups, geometric mean EnvA ELISA titers were 6113, 12 470, 8545, 3470, and 9655 and mean EnvA ELISPOT responses were 397, 178, 736, 196, and 1311 SFC/10⁶ peripheral blood mononuclear cells, respectively. Conclusion. This Ad26 vectored vaccine was generally safe and immunogenic at all doses tested. Reactogenicity was minimal with doses of 5 × 10¹⁰ vp or less. Ad26 is a promising new vaccine vector for HIV-1. Clinical Trials Registration. NCT00618605.

Background. Adenovirus serotype 26 (Ad26) has been developed as a novel candidate vaccine vector for human immunodeficiency virus type 1 (HIV-1) and other pathogens. The primary safety and immunogenicity data from the Integrated Preclinical/Clinical AIDS Vaccine Development Program (IPCAVD) 001 trial, the first-in-human evaluation of a prototype Ad26 vector-based vaccine expressing clade A HIV-1 Env (Ad26.ENVA.01), are reported concurrently with this article. Here, we characterize in greater detail the humoral and cellular immune responses elicited by Ad26.ENVA.01 in humans. Methods. Samples from the IPCAVD 001 trial were used for humoral and cellular immunogenicity assays. Results. We observed a dose-dependent expansion of the magnitude, breadth, and epitopic diversity of Env-specific binding antibody responses elicited by this vaccine. Antibody-dependent cell-mediated phagocytosis, virus inhibition, and degranulation functional activity were also observed. Env-specific cellular immune responses induced by the vaccine included multiple CD8⁺ and CD4⁺ T-lymphocyte memory subpopulations and cytokine secretion phenotypes, although cellular immune breadth was limited. Baseline vector-specific T-lymphocyte responses were common but did not impair Env-specific immune responses in this study. Conclusion. Ad26.ENVA.01 elicited a broad diversity of humoral and cellular immune responses in humans. These data support the further clinical development of Ad26 as a candidate vaccine vector. Clinical Trials Registration. NCT00618605.

Background: We report the first-in-human safety and immunogenicity assessment of a prototype hexon chimeric adenovirus (Ad) serotype 5 (Ad5) vector containing the hexon hypervariable regions of Ad serotype 48 (Ad48) and expressing human immunodeficiency virus (HIV) type 1 EnvA. Methods: Forty-eight Ad5 and Ad48 seronegative, HIV-uninfected subjects were enrolled in a randomized, double-blind, placebo-controlled, dose escalation phase 1 study. Four groups of 12 subjects received 10(9) to 10(11) viral particles (vp) of the Ad5HVR48.EnvA.01 vaccine (n = 10 per group) or placebo (n = 2 per group) at week 0 or weeks 0, 4, and 24. Safety and immunogenicity were assessed. Results: Self-limited reactogenicity was observed after the initial immunization in the highest (10(11) vp) dose group. Responses in vaccinees included Ad48 neutralizing antibody (nAb) titers higher than Ad5 nAb titers, EnvA-specific enzyme-linked immunosorbent assay titers, and EnvA-specific enzyme-linked immunospot assay responses, and these responses generally persisted at week 52. At week 28 in the 10(9), 10(10), and 10(11) vp 3-dose groups, geometric mean EnvA enzyme-linked immunosorbent assay titers were 5721, 10 929, and 3420, respectively, and Ad48 nAb titers were a median of 1.7-fold higher than for Ad5. Conclusions: Ad5HVR48.ENVA.01 was safe, well tolerated, and immunogenic at all doses tested. Vector-elicited nAb responses were greater for Ad48 than Ad5, confirming that Ad-specific nAbs in humans are primarily, but not exclusively, directed against the hexon hypervariable regions. Clinical Trials Registration. NCT00695877.

Genotypes that confer drug resistance were evaluated in human immunodeficiency virus (HIV) proviral DNA obtained from peripheral blood mononuclear cells (PBMC) and lymphoid tissue at baseline and after 8 weeks of therapy with zidovudine alone or in combination with didanosine from 22 patients (8 zidovudine-naive and 14 zidovudine-experienced). There was evidence of zidovudine resistance at codon 215 in 27.3% (6/22) of patients. All 20 patients evaluable for codon 74 (site of didanosine resistance) had virus that remained wild type during the 8-week study period. When HIV proviral DNA from PBMC was compared with that from lymphoid tissue, 94.7% (18/19) of evaluable samples were concordant at codon 215 at baseline, while 85.7% (12/14) were concordant at week 8. Resistance in PBMC (but not in lymphoid tissue) developed in 1 of 8 zidovudine-naive patients; an increased proportion of resistant strains in PBMC (but not in lymphoid tissue) was observed in 2 of 14 zidovudine-experienced patients. These results suggest high concordance for drug resistance mutations in HIV proviral DNA from blood and lymph node tissue.

Alterations of the genital mucosal barrier may influence the number of viruses transmitted from a human immunodeficiency virus–infected source host to the newly infected individual. We used heteroduplex tracking assay and single-genome sequencing to investigate the effect of a tenofovir-based microbicide gel on the transmission bottleneck in women who seroconverted during the CAPRISA 004 microbicide trial. Seventy-seven percent (17 of 22; 95% confidence interval [CI], 56%–90%) of women in the tenofovir gel arm were infected with a single virus compared with 92% (13 of 14; 95% CI, 67%–>99%) in the placebo arm (P = .37). Tenofovir gel had no discernable impact on the transmission bottleneck.

The Centre for the AIDS Programme of Research in South Africa 004 (CAPRISA 004) study demonstrated that vaginally applied tenofovir gel is a promising intervention for protecting women from sexually acquiring human immunodeficiency virus (HIV). However, the potential for emergence of tenofovir resistance remains a concern in women who seroconvert while using the gel despite the lack of plasma virus resistance as assessed by population sequencing during the trial. We applied highly sensitive polymerase chain reaction–based assays to screen for tenofovir resistance in plasma and vaginal swab specimens. The absence of mutation detection suggested little immediate risk of tenofovir-resistant HIV-1 emergence and forward transmission in settings in which gel users are closely monitored for HIV seroconversion.

Pyrimethamine (50 mg) with folinic acid (15 mg) given three times weekly was assessed as primary prophylaxis for toxoplasmic encephalitis (TE) in 554 human immunodeficiency virus-infected patients seropositive for Toxoplasma gondii and with <200 CD4 cells/mm3. At 1 year, the incidence of TE was similar in pyrimethamine, 12%, and placebo, 13%, groups (relative risk [RR], 0.9; 95% confidence interval [CI], 0.6–1.4), and the survival rate was also similar, 85% and 80%, respectively (RR, 0.9; 95% CI, 0.7–1.2). Rash was the only adverse event that appeared significantly more frequently in the pyrimethamine arm (7% vs. 1%). In the on-treatment analysis, the incidence of TE was lower in the pyrimethamine arm, 4%, than in the placebo arm, 12% (P < .006). Thus, pyrimethamine cannot be recommended as a first-line regimen for primary prophylaxis of TE if the patient can take cotrimoxazole. However, it should be considered for patients who are intolerant to cotrimoxazole, especially in high-risk patients with <100 CD4 cells/mm3.

In the first preventative human immunodeficiency virus (HIV) vaccine study to be carried out in Africa, 40 HIV-seronegative Ugandan volunteers were randomly assigned to receive a canarypox vector containing HIV-1 clade B (env and gag-pro) antigens (ALVAC-HIV; n=20), control vector containing the rabies virus glycoprotein G gene (n=10), or saline placebo (n=10). Cytotoxic T lymphocyte activity against target cells expressing clade A, B, and D antigens was assessed using standard chromium-release and confirmatory interferon-γ enzyme-linked immunospot (ELISPOT) assays. Neutralizing antibody responses to cell line–adapted strains and primary isolates in all 3 clades were also tested. Twenty percent of vaccine recipients generated detectable cytolytic responses to either Gag or Env, and 45% had vaccine-induced HIV-specific CD8+ T cell responses, as measured by the ELISPOT assay. In contrast, only 5% of the control group had vaccine-specific responses. Neutralizing antibodies against primary and laboratory-adapted HIV-1 clade B strains were seen in 10% and 15% of vaccine recipients, respectively, but responses against clades A and D were not detected. Although the immunogenicity of this clade B–based vaccine was low, ALVAC-HIV elicited CD8+ T cell responses with detectable cross-activity against clade A and D antigens in a significant proportion of vaccine recipients

Nonenterotoxigenic strains no. 1196-78 and no. 1074-78 of Vibrio cholerae serogroup O1 (biotype El Tor, serotype Ogawa) were isolated from sewage water in Brazil and fed to 20 volunteers. Neither strain caused diarrhea. None of the seven volunteers who ingested Ogawa strain no. 1074-78 (10(6) organisms) excreted the organism whereas eight of the 13 volunteers who ingested Ogawa strain no. 1196-78 (10(6) or 10(8) organisms) did excrete the organism in their stools. None of 114 stool-culture isolates yielded cholera enterotoxin, and none of the 20 volunteers had significant increases in serum titers of antitoxin as measured by enzyme-linked immunosorbent assay although six of the volunteers had slightly elevated vibriocidal antibody levels. Six volunteers used as controls and four volunteers who had stool cultures positive for strain no. 1196-78 of V. cholerae were challenged with pathogenic El Tor Ogawa strain no. E7946 (10(6) organisms) to determine if previous ingestion of the Brazilian strain would induce protective immunity. All 10 of the volunteers developed diarrhea, and the severity of the illness was similar in both groups.

The discovery of 11 persons infected with Vibrio cholerae serogroup O1 (biotype EI Tor, serotype Inaba) in southwestern Louisiana in 1978 provided an opportunity to evaluate the serologic response to this agent in North Americans with naturally acquired infection. One antibacterial assay (vibriocidal assay) and two antitoxin assays (enzymelinked immunosorbent assays [ELISA] and rabbit skin permeability factor assay) were used. Antitoxin levels were elevated longer than vibriocidal antibody levels, and asymptomatic infected persons had levels of antitoxin and vibriocidal antibody as high as those of persons with clinical cholera. With use of serologic criteria derived from these studies, one additional person infected with V. cholerae O1 was discovered, and a relatively low (4%–7%) prevalence rate of elevated levels of vibriocidal antibody and antitoxin was found in a serum survey of a community with several known cases. Comparison of the results from the ELISA and the rabbit skin permeability factor assay demonstrated similar rates of elevated levels of antitoxin, but the ELISA required less time and less serum per sample analyzed.

A new Vibrio cholerae serogroup 0139 strain of unknown origin recently emerged in India and Bangladesh, causing a major outbreak of cholera. The genetic relationship between this epidemic strain and the 01 strain responsible for the 7th pandemic ofcholera was studied by analyzing the DNA polymorphism of V. cholerae by pulsed-field gel electrophoresis and arbitrarily primed polymerase chain reaction. The restriction patterns of the reference strain 0139 Bengal and 10 wild 0139 strains isolated early in the Indian outbreak strikingly resemble that of the pandemic 01 strain of V. cholerae El Tor, thus suggesting a close genetic relationship among these strains. This similarity contrasts with the genetic heterogeneity of sporadic non-O1 strains isolated in various parts of the world. Study results strongly suggest that the new epidemic 0139 strain is closely related to and might be derived from the pandemic 01 strain of V. cholerae .

Vibrio cholerae can shift to a “rugose” colonial morphology associated with expression of an amorphous exopolysaccharide that promotes cell aggregation. Flow cytometric studies indicated that up to 3% of particles in rugose cultures represented aggregates of >5 bacterial cells. Rugose variants of our test strains displayed resistance to killing by chlorine, with viable cells persisting for >30 min in 2 mg/L free chlorine; strains also showed resistance to killing by complement-mediated serum bactericidal activity. Six volunteers fed 106 cfu of a rugose variant of V. cholerae 01 EI Tor Inaba N16961 developed symptoms typical of cholera, with a mean diarrheal stool volume of 2.2 L (range, 1.4-4.3). Isolates recovered from the stool of infected volunteers retained the rugose phenotype. The data suggest that rugose strains cause human disease. The role of these strains in the epidemiology of cholera remains to be determined.

Uveitis occurred in a substantial proportion of AIDS patients receiving rifabutin, 600 mg daily, together with clarithromycin and ethambutol for treatment of Mycobacterium avium complex bacteremia. A case-control study was undertaken to examine potential risk factors for developing uveitis. Of eight parameters examined, only baseline body weight predicted the development of uveitis by both univariate and multivariate analyses (P = .001). The incidence of uveitis was 14% in patients weighing >65 kg, 45% in patients between 55 and 65 kg, and 64% in patients <55 kg. Concomitant therapy with fluconazole, a drug known to raise serum rifabutin concentrations, was not associated with an increased incidence of uveitis. The risk of uveitis was markedly reduced when rifabutin was given at 300 mg daily in combination with clarithromycin and ethambutol.

The potential immunoprotective role of antiserum to an Escherichia coli 15 mutant derived from E. coli 0111:B4 was demonstrated in an experimental mouse model. Overwhelming bacterial inocula masked the effects of cross-reactive immunoprotection due to antiserum to strain 15. Enhanced bacterial clearance was observed in mice receivingantiserum to strain 15 in sublethal infections but not from lethal doses. Incorporation of hemoglobin with the bacterial inocula decreased the 50% lethal dose of challenge organisms, allowing the demonstration of protective activity of antiserum to strain 15 in lethal infection. Pretreatment of mice with antiserum to strain 15 did not protect against lethal doses of endotoxin. The protective factor was demonstrated by exhaustive adsorption experiments to be an antibody specific for strain 15 lipopolysaccharide. The protective activity of antiserum to strain 15 was abolished only after adsorption with strain 15 lipopolysaccharide but not with Salmonella typhimurium mutants with or without enterobacterial common antigen.

Eighty-eight Escherichia coli strains of the enteropathogenic (EPEC) group 0114 that were isolated from humans and animals in geographically different places and over more than 30 years were examined for virulence markers, O:H serotypes, and for electrophoretic types by multilocus enzyme electrophoresis. Four major genetically tightly related clusters of strains showed close correlation between electrophoretic types and other phenotypic characters. Cluster I contained 35 EPEe class II strains of serotypes 0114:H9 and 0114:H− and 5 enterotoxigenic E. coli belonging to 0114:H21 and 0114:H49. Clusters II and III comprised 36 0114:H4, 0114:H32, and 0114:H− strains; most were of doubtful pathogenicity except one Verotoxin-positive 0114:H4 strain isolated from a human with diarrhea. Cluster IV contained 9 classic EPEC strains of serogroup 0114:H2 that were characterized by localized adherence to HEp-2 cells and by the EPEC adherence factor.

Enterotoxigenic Escherichia coli (ETEC) of serotype O114:H21, which produced only heat-labile enterotoxin (LT), gave mannose-resistant hemagglutination (MRHA) with bovine erythrocytes. One strain, m0738A, wasshown to possess fimbriae of ∼7.5 nm diameter. On SOS-PAGE two possible fimbrial polypeptides of molecular masses 17.5and 15.5kDa were seen; the 17.5-kDa band was the most prominent. Loss of LT and MRHA together from strain E20738A was associated with loss of a l00-MDa plasmid. An absorbed anti-strain E20738A serum reacted specifically with the 17.5-and 15.5-kDa polypeptides and bound to the intact fimbriae. This antiserum reacted positively in an ELISA with LT-positive E. coli strains of serogroups O8, O15, O48, O114, and O146. The antiserum did not react with ETEC carrying known colonization factors. The term coli-surface-associated antigen (CS) 17 has been used to describe the fimbriae.

In Uganda, the HIV Network for Prevention Trials (HIVNET) 012 study recently demonstrated that single-dose nevirapine (Nvp) prophylaxis is effective for preventing mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1). This exploratory study examines the relationship between HIV-1 subtype, MTCT, and the development of Nvp resistance (NvpR) in women enrolled in HIVNET 012. For 102 women (32 whose infants were HIV-1 infected by age 6–8 weeks and 70 whose infants were uninfected), HIV-1 subtypes included 50 (49%) subtype A, 35 (34%) subtype D, 4 (4%) subtype C, 12 (12%) recombinant subtype, and 1 unclassified. There was no apparent difference in the rate of MTCT among women with subtype A versus D (adjusted odds ratio [OR], 1.24; 95% confidence interval [CI], 0.45–3.43). NvpR mutations were detected more frequently at 6–8 weeks postpartum in women with subtype D than in women with subtype A (adjusted OR, 4.94; 95% CI, 1.21–20.22). Additional studies are needed to further define the relationship between HIV-1 subtype and NvpR among women receiving Nvp prophylaxis

The availability of enfuvirtide enables assessment of whether human immunodeficiency virus (HIV) decay can be enhanced by targeting reverse transcriptase, protease, and fusion. We performed a 12-week study of 22 patients randomized to receive ritonavir-boosted saquinavir and efavirenz with (the 3-target arm) or without (the 2-target arm) enfuvirtide. We observed no difference in the mean±SD elimination-rate constant for overall decay (0.142±0.040 per day and 0.128 ± 0.033 per day in the 2- and 3-target arms, respectively; P>.1) or for modeled first-phase decay rate (-0.62±0.34 per day and -0.51±0.16 per day; P>.1). Antiretroviral therapy that inhibits HIV reverse transcriptase and protease exerts potent antiviral effects that might not be augmented by the addition of an HIV fusion inhibitor

The HIV Network for Prevention Trials (HIVNET) 012 trial showed that NVP resistance (NVPR) emerged in some women and children after the administration of single-dose nevirapine (SD-NVP). We tested whether K103N-containing human immunodeficiency virus (HIV)-1 variants persisted in women and infants 1 year or more after the administration of SD-NVP. We analyzed samples from 9 women and 5 infants in HIVNET 012 who had NVPR 6-8 weeks after the administration of SD-NVP. Samples were analyzed with the ViroSeq system and with 2 sensitive resistance assays, LigAmp and TyHRT. ViroSeq detected the K103N mutation in 8 of 9 women and in 2 of 5 infants. LigAmp detected the K103N mutation at low levels in 8 of 9 women and in 4 of 5 infants. K103N was not detected by ViroSeq 12-24 months after the administration of SD-NVP but was detected by LigAmp in 3 of 9 women and in 1 of 5 infants. K103N was also detected in those samples by use of the TyHRT assay. K103N-containing variants persist in some women and infants for 1 year or more after the administration of SD-NVP. Sensitive resistance assays may provide new insight into the impact of antiretroviral drug exposure on HIV-1 evolution.

Previous studies have identified two bacterial factors involved in enteropathogenic Escherichia coli (EPEC) infection. A plasmid-mediated EPEe adherence factor (EAF) is responsible for initial and localized adherence. A chromosomally encoded E. coli attachment and effacement factor (eae) is involved in effacement of the eukaryotic cell surface and characteristic “pedestal” formation. By using isogenic strains deficient in either EAF, eae, or both, the process of EPEC adherence and entry in vitro was examined. While EAF proved necessary and sufficient for efficient bacterial association with HEp-2 cells, both EAF and eae were required for efficient effacement of and entry into these cells and other cultured cell lines. Invasion mediated by eae was markedly inhibited by cytochalasin D and colchicine. Afimbrial adhesin or type I pili from uropathogenic strains of E. coli substituted for EAF in EAF−Eae+ strains to provide initial adherence to HEp-2 cells and to facilitate actin condensation.

In October 1992, a non-Ol strain of Vibrio cholerae emerged as a cause of epidemic cholera in India and Bangladesh. This antigenically novel clone has been designated serogroup 0139 synonym Bengal. Since its emergence, V. cholerae 0139 has caused a massive cholera epidemic throughout and beyond the Indian subcontinent. Molecular analysis of virulence determinants in clinical isolates suggests that 0139 strains are highly related to El Tor 01 strains. Unlike other non-O 1 strains, 0139 strains carry multiple copies of the cholera toxin genetic element and also genes for the toxin-coregulated pilus. These results guided construction of a live V. cholerae 0139 vaccine prototype through deletion of genes for at least four specific virulence determinants (ctxA, ace, zot, and cep) as well as other factors involved in site-specific and homologous recombination (RS1, attRS1, and recA). It is hoped that this attenuated live vaccine will help control the pandemic spread of V. cholerae 0139.

To determine the source and extent of an outbreak of Vibrio cholerae 0139 Bengal infections among 630 cruise ship passengers to Southeast Asia, a retrospective cohort study was done. Questionnaires were sent to all passengers from the United States, Canada, and the United Kingdom, and serum samples were requested from all passengers reporting diarrhea. A case was defined as diarrheal illness with onset between 8 and 28 February 1994 and a cholera antitoxic antibody titer ⩾800. Six passengers, including 1 with bacteremia, met the case definition. Illness was associated with eating yellow rice at a buffet restaurant in Bangkok on 10 February (relative risk undefined, P = .005). This international outbreak demonstrates foodborne transmission of Vibrio cholerae 0139 Bengal, an emerging cause of epidemic cholera in Asia, to tourists from Western countries. Physicians should suspect infection with either V. cholerae 01 or 0139 in any patient with severe watery diarrhea after travel to the developing world.

Samples of blood and urine were collected at baseline, week 4, and week 8 and then every 8 weeks from 310 patients entering a controlled trial of prophylaxis with valaciclovir versus acyclovir. Samples were tested under code by polymerase chain reaction (PCR) in one laboratory. The median number of samples collected from each patient was 5 for blood (range, 0-15) and 5 for urine (range, 0-15). Both baseline PCR viremia and PCR viruria were significantly associated with future cytomegalovirus (CMV) disease (P = .002 and P = .02, respectively). The greatest effect of valaciclovir on CMV disease was seen in patients who were PCR-positive in blood at baseline (P = .002), although a significant effect was also seen in those who were PCR-negative in urine (P = .02). Thus, PCR viremia provides prognostic information about CMV disease in AIDS patients, and valaciclovir showed activity as both a preemptive and prophylactic agent.

Cytomegalovirus (CMV) disease is a common complication of advanced human immunodeficiency virus (HIV) infection. Administration of oral valaciclovir, a valine ester of acyclovir, achieves sufficient plasma acyclovir levels to inhibit many clinical isolates. Acyclovir has been associated with enhanced survival in AIDS but not with CMV disease prevention. CMV-seropositive patients (1227) with CD4 cell counts <100/mm3 were enrolled in a randomized, double-blind trial. Valaciclovir, 8 g/day, was compared with acyclovir, 3.2 or 0.8 g/day, for CMV prevention; all three arms were compared for survival. The confirmed CMV disease rate was 11.7% among valaciclovir recipients and 17.5% in the pooled acyclovir arms, a 33% reduction in risk. Time to confirmed CMV disease was significantly longer for the valaciclovir group (P = .03). A trend toward earlier mortality for valaciclovir recipients was seen (P = .06). Toxicity and earlier medication discontinuation were more common in this group. Valaciclovir significantly reduces the risk of CMV disease. Further exploration of a better-tolerated dose is warranted.

Escherichia coli serotype O157:H7 has been epidemiologically linked to outbreaks of hemorrhagic colitis associated with fast-food restaurants and nursing homes. Sporadic cases now exceed those associated with outbreaks. The incidence of the organism in patients with common diarrhea syndromes and in asymptomatic persons is unknown. Routine serotyping of E. coli isolates is impractical for most clinical microbiology laboratories. We developed a screening plate by utilizing sorbitol fermentation as a biochemical marker to identify organisms for serotyping. A total of 2,552 stool samples were screened. In 106 (4.1%), sorbitol-negative E. coli were identified. Of these, two were serotype O157:H7, and both produced a Vero cell toxin. One patient had hemorrhagic colitis and the other a mild, febrile, self-limited diarrhea with no other bacterial pathogen identified. This plate provides an easy, effective method of screening for sorbitol-negative E. coli, a process facilitating the selection of organisms for serotyping and one that may help clarify this organism's role in human disease.

An outbreak of Escherichia coli 0157:H7 hemorrhagic colitis at a Minnesota junior high school in October 1988 comprised 32 cases among 1562 students (attack rate, 2.0%). Four children were hospitalized; none developed hemolytic-uremic syndrome. Case children were more likely than controls to have eaten heat-processed meat patties (odds ratio, 6.2; 95% confidence interval, 2.0–20.1; P < .001) in the school cafeteria on a specific day. The minimum estimated attack rate among students who ate these patties was 8%. The patties should have been sufficientlycooked by the manufacturer to destroy enteric pathogens before they were frozen and distributed. E. coli were cultured from frozen patties that were manufactured at the same plant on the same dates as the implicated patties, but serotype 0157:H7 was not isolated. Heat-processed meat patties may serve as vehicles for E. coli 0157:H7 infection, and currently there are no federal or state regulatory standards to ensure the safety of these products.

Enterohemorrhagic Escherichia coli (EHEC) cause hemorrhagic colitis and hemolytic uremic syndrome (HUS), make potent cytotoxins (Verotoxins [VT] or Shiga-like toxins), and possess a plasmid (∼60 megadaltons) that encodes a new fimbrial antigen and promotes attachment to epithelial cells.Weevaluated the use of a DNA probe, prepared from a 3.4-kilobase segment of the EHEC plasmid, to detect EHEC. The probe hybridized with 106 (99%) of 107 O157:H7 and 34 (77%) of 44 O26:H11, VT-positive strains from patients with colitis, HUS, and diarrheal disease and hybridized with 21 (81%) of 26 VT-positive E. coli of serotypes other than O157:H7 or O26:H11 from patients with hemorrhagic colitis and HUS. Weexamined 601 other strains, including 18 serotype O26 isolates of H types other than H11, 306 enteropathogenic E. coli, 60 enteroinvasive E. coli, 119 enterotoxigenic E. coli, and 20 isolates from the urinary tract and 77 isolates from the normal intestinal flora; only one (O127:H-) was positive (specificity, 99.8%). Serotype O26:H11, previously considered a classic enteropathogenic E. coli serotype, is now shown to be EHEC.

Fifty-two patients were studied prospectively to determine the etiology of postdiarrheal hemolytic uremic syndrome (DUS). Escherichia coli 0157:H7 was isolated from 33 patients (63.4%). If stool obtained within 2 days of the onset of diarrhea was cultured for E. coli 0157:H7, the recovery rate was 100%. This rate decreased to 91.7% and 33.3% if stool was cultured for this pathogen 3–6 or ⩾7 days, respectively, after diarrhea began. The culture-positive group was more likely to have had bloody diarrhea and fecal leukocytes and to have received transfusions than the culture-negative group but was otherwise similar in clinical characteristics. E. coli 0157:H7 is the predominant pathogen associated with HUS in western Washington. Recovery ofthis pathogen is highly dependent on obtaining stool cultures within 6 days of onset of diarrhea.

Infection with Escherichia coli O157:H7 can lead to hemolytic uremic syndrome (HUS) in some children. Epidemiologic data suggest that Shiga toxin (Stx) 2—producing strains are more frequently associated with HUS than are Stx1-producing strains. Less clear is whether strains that express Stx2 alone are more frequently associated with HUS than strains that express Stx1 and Stx2. Isogenic mutants 933stx1− and 933stx2− were produced from strain 933 (Stx1 and Stx2 producer), and 86-24stx2− was produced from strain 86-24 (Stx2 producer). Neurologic lesions or symptoms developed in 18 (90%) of 20 gnotobiotic piglets orally infected with strain 86-24, in 15 (85%) of 18 infected with mutant 933stx1−, in 9 (31%) of 29 infected with strain 933, in 0 of 5 infected with mutant 86-24stx2−, and in 0 of 6 infected with mutant 933stx2−. It was concluded that strains expressing Stx2 alone are more neurotropic for piglets when fed orally than are those strains expressing Stx1 and 2, whereas Stx1-producing strains induce only diarrhea. It is also conceivable that strains that produce Stx2 may constitute a significant predictive risk factor for HUS in humans.

To better define the bovine reservoir of Escherichia coli 0157:H7, cattle were tested monthly by bacteriologic culture for fecal excretion of E. coli. E. coli 0157:H7 was isolated from feces of 56 cattle sampled an average of 6.98 times (2–12 samples). By broth enrichment culture, immunomagnetic separation, or both, 35 cattle had 1 positive sample, 12 had 2, 7 had 3, and 1 each had 4 and 5. Five cattle with ⩾2 positive samples were in a herd in which 5 pulsed-field gel electrophoretic (PFGE) types were simultaneously present; in 3 of these cattle, different PFGE types were detected in different samples. The duration of detected excretion E. coli 0157:H7 by individual cattle in this study was <1 month in 35 (63%) of 56 cattle. Both serial and concurrent excretion of different E. coli O157:H7 strains by individual cattle was observed.

Between 23 June and 15 July 1994, 21 cases (19 primary and 2 secondary) of Escherichia coli 0157:H7 infection were identified in the Bethel, Connecticut, area. Three pulsed-field gel electrophoresis (PFGE) patterns from 15 isolates (I, n = 13; II, n = 2; and III, n = 1) were observed. A casecontrol study that excluded secondary cases and patients with PFGE II and III patterns (n = 16) demonstrated that consumption of food from one supermarket was associated with illness (15/16 cases vs. 31/47 geographically matched controls, odds ratio [OR] undefined, lower 95% confidence interval OR = 1.45,P = .018). No one food was associated with illness. Inspection of the supermarket revealed deficiencies in hygiene and meat handling practices. The 2 cases with PFGE II ate raw beef and raw lamb from a second supermarket. These outbreaks demonstrate the value of PFGE in supporting epidemiologic investigations and the potential for outbreaks arising from retail outlets.

O-specific polysaccharide (O-PS) isolated from serotype 18 Escherichia coli lipopolysaccharide (LPS) was covalently coupled to either Pseudomonas aeruginosa toxin A (TA) or cholera toxin (CT). The conjugates were nontoxic and nonpyrogenic. The conjugates were well tolerated on parenteral administration to human volunteers, with only mild, transient local reactions reported. Immunizatio nengendered an IgG antibody response to both the O-PS and carrier protein. Anti-LPS antibody promoted the uptake and killing of an E. coli O18 strain bearing the K1 capsule by human polymorphonuclear leukocytes, whichwascomplementdependent. Antibodyto carrier protein neutralized the activity of native TA or CT in cell culture assays. Passively transferred IgG isolated from the serum of immunized donors provided a significant (P < .01) degree of protection against fatal experimental E. coli O18 sepsisin mice. This study illustrates the potential use of such conjugates as vaccines against E. coli extraintestinal infections.

We identified an unusual case of human immunodeficiency virus type 1 (HIV-1) infection in a patient (GM43) who exhibited a persistently low antibody response and undetectable viral load during a 5-year follow-up period. GM43 harbored HIV-1 circulating recombinant form 01_AE with gross deletions in the nef/long terminal repeat (LTR) region. The sizes of the deletions increased progressively from 84 to >400 bp during the 5-year period. GM43 appeared to have acquired defective variants from her husband. The genetic alterations in the nef/LTR region were remarkably similar to those that have been reported in slow progressors (such as the slow progressors in the Sydney Blood Bank Cohort). The present study is the first report of slow disease progression due to gross genetic alterations in the nef/LTR region in a person infected with an HIV-1 non–subtype B strain

Enteroviruses have been associated with type 1 diabetes mellitus (T1DM), but it is unclear whether there is a distinct disease subtype. Plasma and stool samples from 206 consecutively diagnosed children and 160 healthy control children were analyzed by reverse-transcription polymerase chain reaction for the RNA of 60 enteroviruses. More children with diabetes tested positive for enterovirus RNA (30% vs. 4%; odds ratio, 11.1; 95% confidence interval, 4.7–25.7; P<.001). Enterovirus 71 was detected in 25% of children; these were temporally associated with outbreaks in Southeast Asia and Australia. Fewer children with the diabetes-associated human leukocyte antigen DRB1*03-DQB1*02 genotype tested positive for enterovirus RNA (P=.02). More children presenting with severe diabetic ketoacidosis (pH, <7.1) tested positive for enterovirus RNA (P=.04). These results suggest that there is a subgroup of patients with T1DM, who are at low genetic risk, in whom enteroviruses contribute to diabetes onset

Stimulation of peripheral blood mononuclear cells (PBMC) with allogeneic PBMC (ALLO) can result in activity that inhibits the replication of human immunodeficiency virus (HIV). The present study demonstrates that strong anti-HIV activity is dependent on expression of HLA-A*02 by the responding PBMC. Anti-HIV activity was equally effective against 2 primary isolates that use different coreceptors. Neither ALLO-stimulated cell proliferation nor cytokine and β-chemokine production was associated with the expression of HLA-A*02. ALLO-stimulated production of strong anti-HIV activity required intact PBMC and was not inhibited by monoclonal antibodies directed against nonpolymorphic regions of human leukocyte antigens (HLAs). Anti-HIV activity was generated by ALLO-stimulated CD4+ cells, CD8+ T lymphocytes, and monocytes from HLA-A*02—positive patients. These findings provide the first evidence that the production of an HIV inhibitory factor or factors is associated with certain HLA genes and raise new possibilities concerning the role of the major histocompatibility complex in controlling viral infections via alloantigen stimulation.

How the cellular immune response copes with diverse antigenic competition is poorly understood. Responses of virus-specific cytotoxic T lymphocytes (CTL) were examined longitudinally in an individual coinfected with human immunodeficiency virus type 1 (HIV-1), Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CTL responses to all 3 viruses were quantified by limiting dilution analysis and staining with HLA-A*0201 tetrameric complexes folded with HIV-1, EBV, and CMV peptides. A predominance of CMV-pp65-speciflc CTL was found, with a much lower frequency of CTL to HIV-1 Gag and Pol and to EBV-BMLF1 and LMP2. The high frequency of CMV-speciflc CTL, compared with HIV-1- and EBV-specific CTL, was confirmed in an additional 16 HLA-A*0201-positive virus-coinfected subjects. Therefore, the human immune system can mount CTL responses to multiple viral antigens simultaneously, albeit with different strengths.

Major histocompatibility class II alleles of 351 persons living in an area endemic for Schistosoma mansoni in northeastern Brazil were characterized at three loci (DRBl, DQAl, and DQBl). Contingency analyses were used to compare allele frequencieswitfihigh egg excretion, proliferative response to schistosome soluble egg antigens (SEA), and occurrence of severe, biopsy-confirmed hepatosplenic disease. There were no associations of HLA-DR or DQ with egg excretion. Patients positive for DRBl*01, DQAl*0101, or DQBl*0501 were less likely to respond to SEA than was the overall study population. However, using stringent Bonferroni correction (multiplying P values by the number of alleles tested; P × 35), none of these associations with SEA responsiveness remained significant. Hepatosplenic disease was less likely in patients positive for DRBI*11 and was more likely in patients positive for DRBl*07 or DQBl*0201. However, only the DQBl*0201 association remained significant (odds ratio = 3.72; P < .005) following Bonferroni correction.

CD8+ T cells could make an important contribution to protection against tuberculosis (TB), but the antigenic determinants recognized in the context of major histocompatibility complex class I molecules remain ill defined. Our aim was to identify nonamer peptides derived from the acr/16-kDa antigen. Two immunogenic peptides (p21–29 and p120–128) were identified by their ability to elicit cytotoxic CD8+ T cells from juvenile patients recovering from TB. Epitope-specific recognition was demonstrated by the lysis of both Mycobacterium tuberculosis–infected and peptide-pulsed macrophages, the release of cytotoxic granules, and interferon-γ and tumor necrosis factor–α production. CD8+ T cell responses to p21–29 and p120–128 were detected ex vivo in freshly isolated peripheral blood mononuclear cells from patients with TB but not in those from healthy control subjects. Our data suggest that these antigenic peptides can play a critical role in effective immunity against mycobacterial infection and TB

Trimetrexate is a powerful inhibitor of the dihydrofolate reductase of Pneumocystis carinii. AIDS patients (n = 215) with moderate to severe P. carinii pneumonia were enrolled in a doubleblind study of trimetrexate plus leucovorin versus trimethoprim-sulfamethoxazole (TMP-SMZ) for 21 days. By study day 10, study therapy failed because of lack of efficacy in 16% of patients assigned to TMP-SMZ and 27% assigned to trimetrexate (P = .064), and the Pao2 − Pao2 improved significantly faster with TMP-SMZ. By study day 21, failure rates were 20% with TMP-SMZ and 38% with trimetrexate (P = .008), with respective mortality rates of 12% and 20% (P = .088). By study day 49, the difference in mortality (16% vs. 31%) was significant (P = .028). The cumulative incidence of serious and treatment-terminating adverse events including hematologic toxicities was less with trimetrexate (P < .001). Thus, trimetrexate plus leucovorin was effective, albeit inferior to TMP-SMZ,Jor moderately severe P. carinii pneumonia but was better tolerated than TMP-SMZ.

To compare human immunodeficiency virus (HIV) type 1 disease progression in patients infected by the predominant strain circulating recombinant form (CRF) 02_AG in western and west-central Africa and in patients infected by other strains, a prospective multicenter cohort study was conducted in Cameroon and Senegal. Among the 335 patients, a broad HIV-1 group M subtype diversity was observed in the envelope V3–V5 region, but strain CRF02_AG predominated in both Cameroon and Senegal (61.2% and 62.9%, respectively; P<.8). Multivariate analyses showed no difference between patients infected by CRF02 strains and those infected by other strains in terms of survival (adjusted hazards ratio [HR], 1.16; 95% confidence interval [CI], 0.76–1.78; P=.5), clinical disease progression (HR, 0.79; 95% CI, 0.50–1.25; P=.3), or square root CD4 cell decline (regression coefficient, –0.01; 95% CI, −0.82 to 0.81; P=.9). This study suggests that the predominance of HIV-1 CRF02_AG strain in western and west-central Africa should have no major clinical consequences

Previous genomewide association studies (GWASs) of AIDS have targeted end points based on the control of viral load and disease nonprogression. The discovery of genetic factors that predispose individuals to rapid progression to AIDS should also reveal new insights into the molecular etiology of the pathology. We undertook a case-control GWAS of a unique cohort of 85 human immunodeficiency virus type 1 (HIV-1)-infected patients who experienced rapid disease progression, using Illumina HumanHap300 BeadChips. The case group was compared with a control group of 1352 individuals for the 291,119 autosomal single-nucleotide polymorphisms (SNPs) passing the quality control tests, using the false-discovery rate (FDR) statistical method for multitest correction. Novel associations with rapid progression (FDR, < or = 25%) were identified for PRMT6 (P = 6.1 x 10(-7); odds ratio [OR], 0.24), SOX5 (P = 1.8 x 10(-6); OR, 0.45), RXRG (P = 3.9 x 10(-6); OR, 3.29), and TGFBRAP1 (P = 7 x 10(-6); OR, 0.34). The haplotype analysis identified exonic and promoter SNPs potentially important for PRMT6 and TGFBRAP1 function. The statistical and biological relevance of these associations and their high ORs underscore the power of extreme phenotypes for GWASs, even with a modest sample size. These genetic results emphasize the role of the transforming growth factor beta pathway in the pathogenesis of HIV-1 disease. Finally, the wealth of information provided by this study should help unravel new diagnostic and therapeutic targets.