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Intranuclear inclusions typical of cytomegalovirus infections were first noticed in 1881 by German scientists who thought they represented protozoa. After viruses were grown in cell cultures, Weller, Smith and Rowe independently isolated and grew CMV from man and mice in 1956-1957. Antibodies in 30-100% of normal adults indicate not only a past infection, but the presence of a present latent infection. The presence of CMV DNA in tissues and most organs surveyed indicates the ubiquity of latent infection. CMV disease requires the virus and some deficiency of immunity such as occurs in the immature fetus, in AIDS, and in transplant patients on immunosuppressive drugs. Antiviral agents can inhibit CMV replication but they cannot prevent or cure latent infections. A pharmacological approach using the many leads in understanding latency is needed.

Summary Avoparcin, a vancomycin analogue, was banned as a feed additive in Taiwan in 2000. A nationwide surveillance was conducted to study the prevalence of vancomycin-resistant enterococci (VRE) on chicken farms between 2000 and 2003. Among the 1021 E. faecalis and 967 E. faecium isolates studied, resistance to tetracycline, erythromycin, high-level aminoglycosides, ciprofloxacin and chloramphenicol either increased or remained high except vancomycin. The proportion of VRE decreased, between 2000 and 2003, from 13.7% (22/161) to 3.7% (11/299) for E. faecalis, and 3.4% (4/119) to 0% (0/300) for E. faecium. Only 8.8% (7/80) of the chicken farms surveyed harboured VRE in 2003 compared with 25% (15/60) in 2000. All VRE were resistant to tetracycline and erythromycin. All VRE possess the vanA gene but nearly all (79 of 83 isolates) were susceptible to teicoplanin, indicating VanB phenotype. Some clones were detected from different farms in various regions over the years. We conclude that the frequency of VRE in chicken farms decreased in association with a ban on avoparcin; and the continued presence of VRE may be due to the ability of some strains to persist in the farms, transfer of vancomycin resistance determinants or co-selection by the continued use of other antibiotics.

In a nationwide surveillance of antimicrobial resistance (Taiwan Surveillance of Antimicrobial Resistance, TSAR), isolates were collected from 21 medical centers and regional hospitals throughout Taiwan over a three-month period in 2000 (TSAR II). This report summarizes susceptibility data of 7 common Gram-negative bacilli (Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Proteus mirabilis, Enterobacter cloacae, Pseudomonas aeruginosa, and Acinetobacter baumannii) in the TSAR II collection and compared selected key forms of resistance by epidemiologic factors and with isolates collected in 1998 (TSAR I) as well as with data from international surveillance studies. Resistance of the 5 Enterobacteriaceae species to most of the commonly prescribed "first-line" antimicrobials in Taiwan, such as ampicillin (78% in E. coli, 68% in P. mirabilis), gentamicin (19% in K. pneumonia to 66% in S. marcescens), and trimethoprim/sulfamethoxazole (29% in K. pneumoniae to 70% in P. mirabilis), was high, several of which are higher than other countries. Resistance to certain broad-spectrum antimicrobials is also more acute in Taiwan than most Western countries, such as ceftazidime resistant A. baumannii (73%) and ciprofloxacin resistant E. coli (12%). Differences in geographic regions and specimen types were associated with certain forms of resistance in TSAR II; however, the resistance problem is prevalent among both inpatients and outpatients of not only medical centers but also regional hospitals throughout Taiwan.

Objective:To determine the susceptibilities ofCandidaspecies isolated from Taiwan to amphotericin B and fluconazole.Design:Prospective surveillance study.Methods:Each hospital was asked to submit up to 10C. albicansand 40non-albicans Candidaspecies during the collection period, from April 15 to June 15, 1999. One isolate was accepted from each episode of infection. The broth microdilution method was used to determine susceptibilities to amphotericin B and fluconazole.Results:Only 3 of 632 isolates, one each ofC. famata, C. krusei,andC. tropicalis,were resistant to amphotericin B. A total of 53 (8.4%) of 632 clinical yeast isolates, consisting of 4%C. albicans,8%C. glabrata,15%C. tropicalis,and 70%C. krusei,were resistant to fluconazole. In contrast, noC. parapsilosisisolate was resistant to fluconazole. Isolates from tertiary-care medical centers had higher rates of resistance to fluconazole than did those from regional and local hospitals (11.4% vs 6.6%). Isolates from different sources showed different levels of susceptibility to fluconazole. All of the isolates with the exception ofC. tropicalisandC. kruseiisolated from blood were susceptible to fluconazole. A pattern of co-resistance to both amphotericin B and fluconazole was observed.Conclusions:Non-albicans Candidaspecies had higher rates of resistance to fluconazole than didC. albicans(44 of 395 [11.2%] vs 9 of 237 [3.8%];P= .002). The increasing rate of fluconazole resistance inC. tropicalis(15%) is important becauseC. tropicalisis one of the most commonly isolatednon-albicans Candidaspecies.

Susceptibilities to amphotericin B and fluconazole of 383 Candida species isolated from blood were determined. Candida albicans was the most common species (55.6%), followed by Candida parapsilosis (17.5%), Candida tropicalis (16.5%), Candida glabrata (5.2%), Candida guilliermondii (2.3%), and others (2.9%). All but three isolates, Candida ciferrii, C. tropicalis, and C. glabrata, one each, were susceptible to amphotericin B. A total of 367 (95.8%) and 15 (4.2%) isolates were susceptible and susceptible-dose dependent to fluconazole, respectively. Only one isolate, a C. glabrata, was resistant to fluconazole. Few patients (13%) having prior fluconazole treatments may explain the low rate of resistance to fluconazole in this study.

In a survey of 541 Klebsiella pneumoniae isolates from 44 hospitals in Taiwan, three distinct populations were identified by the disk diffusion method according to the disribution of zone diameters of ciprofloxacin. Isolates with resistant, reduced-susceptible, and susceptible to fluoroquinolone were defined as CIP zone diameters ofor = 15 mm, 16-26 mm, andor = 27 mm, respectively. Thus, in addition to 38 (7%) resistant isolates, there were 30 (5.5%) reduced-susceptible isolates and 473 (87.5%) susceptible isolates. A total of 34 isolates consisting of nine resistant, 13 reduced-susceptible, and 12 susceptible isolates were assessed for point mutations in gyrA and parC and the outer membrane profiles. The susceptibility to fluoroquinolone of 13 reduced-susceptible isolates was not altered in the presence of carbonyl cyanide m-chlorophenylhydrazone, an efflux inhibitor, showing that efflux is not a major contributor to reduced susceptibility. In addition to single mutation in gyrA, OmpK35 porin loss can also be the first step for developing fluoroquinolone resistance. No strain possesses a parC mutation without the simultaneous presence of a gyrA mutation, suggesting that mutations in parC play a complementary role for higher-level of fluoroquinolone resistance and fluoroquinolone resistance is a multistep process.

From July, 1998, to June, 1999, pneumococcal isolates from 288 patients with invasive disease in Taiwan were serogrouped and tested for their susceptibility to various antibiotics. Automated ribotyping was used to study their molecular epidemiology. The mortality rate among thoseor = 65 years was higher than those 18 or 19-64 years (p0.001). The total incidence of infection was significantly higher during the cooler season than the warmer season (p = 0.017). Among strains isolated from children agedor = 18 years, 76% were not susceptible to penicillin, a rate that was significantly higher (p0.001) than that for adults (45%), as was the susceptibility to azithromycin, erythromycin, and trimethoprim-sulfamethoxazole (p0.005). The most prevalent serogroup encountered in the invasive isolates was 23, followed by 6, 14, 19, and 3. Isolation of Streptococcus pneumoniae in cerebrospinal fluid was at high rate in children under 5 years (p = 0.00012). Molecular typing revealed a high degree of polymorphism among the isolates. Among serogroup 23 and 19 isolates, a high proportion had the same ribotypes, the Taiwan23F-15 and Taiwan19F-14 isolates, suggesting the circulation of a Taiwanese epidemic strain. In Taiwan, S. pneumoniae isolates should be tested for their resistance profile for childrenor = 18 years old, as these are more likely to harbor high-level resistance. Control of pneumococcal infection with the 7-valent-conjugated vaccine should also be considered because it is estimated that it would cover nearly 90% of the serotypes among pediatric invasive disease.

We determined the clinical and microbiologic characteristics of community-acquired Acinetobacter baumannii bacteremia in 19 adult patients. We found that malignancy was the most frequent underlying disease. The overall mortality rate was 58%. All 14 available isolates were identified as genomic species 2 ( A. baumannii ) by 16S ribosomal DNA sequencing and were found to be genetically distinct by pulsed-field gel electrophoresis.

This chapter provides an overview of the 68 types of enterovirus serotypes including the 3 polioviruses, 29 coxsackieviruses, and 32 echoviruses. Enteroviruses are no longer differentiated into these three different groups, and beginning with enterovirus 68, they were named numerically in sequence. Aseptic meningitis, along with less specific respiratory, gastrointestinal, or other skin manifestations, may accompany enterovirus infections. One of the characteristics of Enterovirus 71 (EV 71) outbreaks is that other enteroviruses frequently cocirculate with EV 71. This outbreak was characterized by cocirculation of a number of other enteroviruses, the most prominent one being coxsackievirus A16. There were 226 coxsackievirus A16 isolates, of which 28% were from inpatients and 37% were from outpatients. Significantly more EV 71 isolates were from inpatients and significantly more coxsackievirus A16 isolates were from outpatients. EV 71 can cause large epidemics, and most recently these have occurred in the Far East. These outbreaks are frequently mixtures of EV 71 and other viruses such as coxsackievirus A16, but serious disease and fatalities are caused only by EV 71. Hallmark clinical manifestations are hand-foot-and-mouth disease (HFMD), flaccid paralysis, brain stem encephalitis, and pulmonary edema. EV 71 must be diagnosed by specific virologic procedures.

A survey of 1,203 Escherichia coli isolates from 44 hospitals in Taiwan revealed that 136 (11.3%) isolates were resistant to fluoroquinolones and that another 261 (21.7%) isolates had reduced susceptibility. Resistance was more common in isolates responsible for hospital-acquired (mostly in intensive care units) infections (17.5%) than in other adult inpatient (11.4%; P = 0.08) and outpatient isolates (11.9%; P > 0.1). Similarly, reduced susceptibility was more common in isolates responsible for hospital-acquired infections (30.9%) than in other adult inpatient (21.0%; P = 0.04) and outpatient (21.4%; P = 0.06) isolates. Isolates from pediatric patients were less likely to be resistant (1.3 versus 12.0%; P < 0.01) but were nearly as likely to have reduced susceptibility (17.7 versus 21.9%; P > 0.1) as nonpediatric isolates. There was an inverse relationship in the proportion of isolates that were resistant versus the proportion that had reduced susceptibility among isolates from individual hospitals ( R = 0.031; P < 0.05). In an analysis of isolates from two hospitals, all 9 resistant strains possessed double point mutations in gyrA and all 19 strains with reduced susceptibility strains had single point mutations; no mutations were found among fully susceptible strains. Risk factors for resistance included underlying cancer (odds ratio [OR], 83; 95% confidence interval [CI 95 ], 7.3 to 2,241; P < 0.001), exposure to a quinolone (OR, undefined; P = 0.02), and exposure to a nonquinolone antibiotic (OR, 20; CI 95 , 2.2 to 482; P < 0.001); underlying cancer was the only independent risk factor (OR, 83; CI 95 , 8.6 to 807; P < 0.001). There were no significant associations between any of these factors and reduced susceptibility. Whereas acute and chronic quinolone use in cancer patients is a major selective pressure for resistance, other undetermined but distinct selective pressures appear to be more responsible for reduced susceptibility to fluoroquinolones in E. coli .

A total of 113 blood culture isolates of Klebsiella pneumoniae from 10 hospitals in northern Taiwan were studied for SHV and TEM b-lactamase production. blaSHV was amplified from all isolates by PCR. TEM-type resistance, was found in 32 of the isolates and was of the TEM-1 type in all isolates. SHV-1, -2, -5, -11, and -12 and two novel enzymes were identified. These novel enzymes were designated SHV-25 and SHV-26 and had pIs of 7.5 and 7.6, respectively. Amino acid differences in comparison to the amino acid sequence of blaSHV-1 were found at positions T18A (ThrACC3AlaGCC), L35Q (LeuCTA3GluCAA), and M129V (MetATG3ValGTG) for SHV-25 and at position A187T (AlaGCC3ThrACC) for SHV-26. The results of substrate profiles and MIC determinations showed that the novel enzymes did not hydrolyze extended-spectrum cephalosporins, rendering the isolates susceptible to these agents. Inhibition profiles revealed that the 50% inhibitory concentration for SHV-26 was higher than those for SHV-1 and SHV-25, resulting in an intermediate resistance to amoxicillinclavulanic acid. Forty-nine ribotypes were identified, suggesting that major clonal spread had not occurred in any of the hospitals. According to the amino acid sequence, SHV b-lactamases in Taiwan may basically be derived through stepwise mutation from SHV-1 or SHV-11 and further subdivided by four routes. The stepwise mutations initiated from SHV-1 or SHV-11 to SHV-2, SHV-5, and SHV-12 comprise the evolutionary change responsible for extended-spectrum b-lactamase (ESBL) production in Taiwan. The stepwise mutations that lead to a non-ESBL (SHV-25) and the b-lactamase (SHV-26) with reduced susceptibility to clavulanic acid are possibly derived from SHV-11 and SHV-1, respectively. The results suggest a stepwise evolution of SHV b-lactamases in Taiwan. Extended-spectrum b-lactamases (ESBLs) are predominantly derived from plasmid-mediated TEM or SHV b-lactamases through a mutation or mutations that lead to one or more amino acid changes and that result in the alteration of the binding to and hydrolysis of specific substrates at the active site (6; G. A. Jacoby and K. Bush, http://www.lahey.org/studies /webt.htm). These enzymes, especially TEM and SHV, are most commonly found in Klebsiella pneumoniae and Escherichia coli and have been reported worldwide (6, 17, 19; Jacoby and Bush, http://www.lahey.org/studies/webt.htm). ESBLs have also recently been found in other members of the enteric bacteria (2, 8, 16). Most of these ESBLs are associated with nosocomial infections and treatment with antibiotics prior to the infection. Selective pressure has been suggested to enhance stepwise mutations in the nucleotides of classic b-lactamases (9).

Sixty-five ciprofloxacin-resistant clinical Escherichia coli isolates were collected from a Taiwan Medical Center from December 1998 to February 1999. All 65 clinical isolates were resistant (MICsor = 4 microg/mL) to the following fluoroquinolones: ofloxacin, levofloxacin, sparfloxacin, and trovafloxacin. These isolates were cross-resistant to chloramphenicol (65 isolates, 100%), tetracycline (65 isolates, 100%), cefuroxime (64 isolates, 98.5%), ampicillin (57 isolates, 87.7%), gentamicin (53 isolates, 81.5%), and cephalothin (24 isolates, 36.9%). Pulsed-field gel electrophoresis (PFGE) revealed a high diversity among the genomes of these isolates and indicated that clonal spread was not responsible for the prevalence of ciprofloxacin resistance in the hospital. Sequencing of the polymerase chain reaction (PCR) amplified products of the quinolone resistance determining regions (QRDRs) of gyrA and parC showed that all isolates carrying double mutations in gyrA at codon 83 and 87 and at least one parC mutation at codon 80 and/or 84. The mutation at codon 83 of GyrA from serine to leucine (S83L) was present in all the clinical isolates. The most prevalent pattern was the S83L mutation and the mutation at codon 87 from an aspartate to an asparagine (D87N) of GyrA plus a mutation from a serine to an isoleucine (S80I) at codon 80 of ParC (63.2%). This indicated that the presence of high-level resistance to quinolones in clinical E. coli isolates were associated with mutations at hot spots, codon 83 and 87 in GyrA and followed by subsequent mutation in either codon 80 and/or 84 in ParC.

Thirteen patients who had 16 episodes of bacteremia were observed between 1993 and 1997 in a pediatric oncology ward with a high background isolation rate of cefotaxime- or aztreonam-resistant gram-negative bacteria. Four blood isolates were Escherichia coli and 12 were Klebsiella pneumoniae , and these isolates harbored extended-spectrum β-lactamases (ESBLs). All episodes of bacteremia were nosocomial, all except one of the episodes occurred in neutropenic patients, and all patients were treated with piperacillin or ceftazidime with amikacin and cefazolin prior to the onset of bacteremia. Nine of 13 patients were receiving extended-spectrum β-lactam treatment when the bacteremias caused by ESBL producers occurred. Molecular studies revealed that four K. pneumoniae SHV-2-producing isolates from 1994 were of the same clone. Other ESBL producers, including six that carried both TEM-1 and SHV-5, five that carried SHV-5, and one that carried SHV-2 alone, were unrelated. In conclusion, SHV-5 was present in 11 of the 16 isolates and coexisted with TEM-1 in 6 isolates. Acquisition of resistance genes probably occurred under antibiotic selection pressure. This study highlights the importance of routine checks for and detection of ESBL producers. Effective therapy against ESBL producers should be considered early for children who have malignancies and neutropenia and who are septic, despite treatment with a regimen that includes an extended-spectrum β-lactam, in a clinical setting of an increased incidence of ESBL-producing bacteria.

Background Enteroviruses can cause outbreaks of hand-foot-and-mouth disease (characterized by vesicular lesions on the hands, feet, and oral mucosa) or herpangina, usually without life-threatening manifestations. In 1998 an epidemic of enterovirus 71 infection caused hand-foot-and-mouth disease and herpangina in thousands of people in Taiwan, some of whom died. Methods We assessed the epidemiologic aspects of this outbreak. Cases of hand-foot-and-mouth disease or herpangina in ambulatory patients were reported to the Taiwan Department of Health by a mean of 818 sentinel physicians. Severe cases in hospitalized patients were reported by 40 medical centers and regional hospitals. Viruses were isolated by 10 hospital laboratories and the department of health. Results The sentinel physicians reported 129,106 cases of hand-foot-and-mouth disease or herpangina in two waves of the epidemic, which probably represents less than 10 percent of the estimated total number of cases. There were 405 patients with severe ...

Epstein-Barr virus (EBV) plays a major role in the pathogenesis of posttransplant lymphoproliferative disease (PTLD). Patients who undergo primary EBV infection after transplantation are at greater risk of developing PTLD. In this retrospective study, the incidence of EBV infection and associated PTLD in 40 consecutive adult recipients who were seronegative for EBV at the time of liver transplantation were investigated, and risk factors for PTLD were analyzed. Of 37 patients with available timely posttransplant serum samples, 35 (95%) developed primary EBV infection. Of the 40 patients, 13 (33%) developed PTLD a median of 126 days (range, 48-776) after liver transplantation. The factor significantly associated with the development of PTLD was cytomegalovirus disease (relative risk, 7.3; 95% confidence interval, 2.36-22.6; P = .0006). Cytomegalovirus disease is a predictor for the development of PTLD in primary EBV infection after liver transplantation, and it may be a target for prophylactic intervention.

To determine if use of antiherpes drugs protects against the development of AIDS-associated Kaposi's sarcoma (KS), data from 935 homosexual men with AIDS from the Multicenter AIDS Cohort Study were analyzed. In nested case-control analysis, neither acyclovir use for human immunodeficiency virus infection (odds ratio [OR], 0.84 ; 95% confidence interval [CI], 0.56-1.26 ; P =.39) nor acyclovir use for any indication (OR, 1.02 ; 95% CI, 0.76-1.38 ; P =.89) was associated with a reduced risk of KS as initial AIDS diagnosis. In longitudinal analysis, acyclovir was also not protective against developing KS as a late manifestation of AIDS (after initial non-KS AIDS diagnosis). Among men with cytomegalovirus disease, ganciclovir use (relative risk [RR], 0.56 ; 95% CI, 0.22-1.44 ; P =.23) and foscarnet use (RR, 0.40 ; 95% CI, 0.051-3.10 ; P =.38) were associated (although not significantly) with a reduced risk of KS. Thus, acyclovir use does not appear to reduce the risk of KS, but further study of other antiherpes drugs such as ganciclovir and foscarnet is warranted.

While the combination of zidovudine and didanosine is used in HIV-infected patients with more advanced disease, the possibility of a pharmacokinetic interaction between these two drugs remains controversial. Zidovudine doses of 50, 100, and 200 mg, combined with 67, 167, and 250 mg of didanosine were evaluated in 11 asymptomatic HIV-infected patients after receiving 24 weeks of combination therapy in AIDS Clinical Trials Group protocol 143. The pharmacokinetic parameters of zidovudine and didanosine were similar to those obtained with each drug given as monotherapy in other previously published studies. The renal clearance and urinary recovery of glucuronidated zidovudine was reduced when zidovudine was given in combination with didanosine, possibly due to competition for renal tubular secretion. These data suggest that no clinically important pharmacokinetic interaction occurs when zidovudine and didanosine are given together.

To evaluate the safety and efficacy of didanosine (ddl) monotherapy and three different combinations of zidovudine (ZDV) and ddl in asymptomatic human immunodeficiency virus-1 (HIV-1) infection, we conducted an open-label, phase I/II study in 126 asymptomatic HIV-1-infected hemophilic and nonhemophilic subjects with a CD4 count of 200 to 500/mm3 stratified for prior zidovudine treatment and baseline CD4 count. Study arms included arm A, low-dose combination (ZDV 150 mg and ddl 134 mg, daily); arm B, moderate-dose combination (ZDV 300 mg and ddI 334 mg, daily); arm C, high-dose combination (ZDV 600 mg and ddl 500 mg, daily), and arm D, ddl monotherapy (ddl 500 mg, daily). Earlier, more frequent hepatotoxicity was experienced by hemophilic subjects (P = .008), but there were no differences in toxicity between treatment arms (P = .51), nor were there any differences in the rate of development of clinical endpoints by treatment (P = .41). Smaller median CD4 increases occurred over the first 12 weeks for arms A and D, 44/mm3 and 42/mm3, than arms B and C, 105/mm3 and 114/mm3, respectively, (P = .015). Hemophilia status (P = .0004) and prior ZDV experience (P = .044) independently predicted weaker CD4 responses during the first 12 weeks of treatment. Using a regression model and adjusting for hemophilia status, prior ZDV treatment, and baseline CD4, there was a significant reduction in quantitative viral load from baseline by week 12 for all treatment arms combined (P = .0001), with significantly lower median percent reduction for arm A (56.3%) than arms B, C, and D (94.6%, 98.5%, and 91.9%, respectively, P = .015). Although greater hepatoxicity and weaker CD4 responses occur in hemophilic subjects, didanosine monotherapy and combination therapy with zidovudine are safe and effective in asymptomatic HIV-1-infected patients.

Thirty randomly selected patients with advanced chronic liver disease, which had been evaluated for possible liver transplantation, were sampled endoscopically at 7 alimentary tract locations to assess the frequency and amount of Candida carriage. Eightyone percent (127/156) of the samples obtained contained Candida and 53% (82/156) yielded high counts (> 300 CFU/ml). The most predominant Candida species isolated at each site was Candida albicana, which accounted for 103 (64%) of the 160 fungal isolates. The other Candida species isolated included C tropicalis 30 (19%), C krusei 16 (10%), and C glabrata 11 (7%), Although the number of sites at which yeast was present and the quantities of yeast at each site varied widely among the patients studied, 100% of the patients had Candida in at least one site of the gastrointestinal tract. Eighty-six percent (24/28) of the duodenal aspirates contained Candida and 50% (14/28) of the duodenal samples contained greater than 300 CFU/ml. A positive culture from the stomach was a reliable predictor of the presence of Candida in the duodenum (P=0.0001), but a positive culture at no other site readily predicted the presence of Candida at yet another site. Importantly, there was no correlation between the presence or absence of Candida in either oral or rectal swabs and colonization at other anatomic sites within the gastrointestinal tract, These findings are important in liver transplantation, particularly in those cases in which the bowel has been opened to create a choledochojejunostomy anastomosis. The operative attempts to reduce gastrointestinal fungal carriage using oral antifungal agents may be justified before liver transplantation in an effort to lower the risk of posttransplantation fungal infections, particularly in those patients expected to have a Roux-en-Y choledochojejunostomy biliary reconstruction. © 1994 by Williams and Wilkins.

Cytolytic T lymphocytes play an important role in host defense against viral infections, including human immunodeficiency virus (HIV). In a phase I clinical trial (protocol 080 of the AIDS Clinical Trials Group), generation of CD8+ effector cells from peripheral blood of patients with acquired immunodeficiency syndrome (AIDS)-related complex (ARC) or AIDS and safety of autologous adoptive transfer of these cells were evaluated. For therapeutic infusions, CD8+ T cells were purified by positive selection on anti-CD8 monoclonal antibody-coated flasks from leukapheresed peripheral blood of seven patients. These CD8+ T cells were cultured in the presence of interleukin-2 and phytohemagglutinin for up to 3 weeks to obtain cells sufficient for therapeutic infusions (10(8) to 10(10)). All 31 cell cultures established from the seven patients and used for therapy were highly enriched in CD8+ (mean, 97%), CD8+HLA-DR+ (50%), cytotoxic CD8+CD11b- (82%), and memory CD29+ (78%) T lymphocytes. In vitro expanded CD8+ cells had excellent cytotoxic function at the time they were used for therapy, including HIV-specific activity against autologous targets infected with vaccinia vectors expressing HIV-IIIb antigens, gag, pol, and env. Anti-HIV activity of cultured CD8+ cells was significantly higher than that of autologous fresh peripheral blood lymphocytes. Our results show that CD8+ T lymphocytes obtained from peripheral blood of symptomatic HIV-infected patients can be purified, cultured to obtain large numbers of cells with enhanced anti-HIV activity, and safely infused into patients with AIDS as a form of immunotherapy.

Based on preclinical studies showing that CD8+ T lymphocytes of human immunodeficiency syndrome (HIV)-infected subjects have anti-HIV activities, a phase 1 study was undertaken to determine the safety and feasibility of infusing in vitro purified, activated, and expanded CD8+ cells as a therapeutic measure in seven patients with acquired immunodeficiency syndrome (AIDS)-related complex (ARC) or AIDS. Autologous CD8+ cells were first selectively isolated in monoclonal antibody-coated flasks from peripheral blood mononuclear cells recovered by leukapheresis. They were then cultured and expanded with phytohemagglutinin and recombinant interleukin-2 (rIL-2) before infusion. Five cycles of isolations and infusions of increasing numbers of CD8+ T cells were achieved in five of seven subjects. Five cycles could not be completed in two subjects with AIDS whose CD4+ cell counts were < or = 48/microliters. Infusions of CD8+ cells alone were well tolerated. Four patients received rIL-2 by continuous infusion for 5 days with their final cycle of CD8+ cells. All developed reversible adverse effects attributable to rIL-2. After infusion, 111In-labeled CD8+ cells quickly accumulated in the lungs, with less than 10% of the labeled cells remaining in the circulation. After 24 hours, labeled CD8+ cells were reduced in the lungs, but increased and persisted in liver, spleen, and bone marrow. Four of five patients who were treated with multiple infusions of CD8+ cells have improved or remained clinically stable, and the fifth developed Pneumocystis carinii pneumonia but recovered. This study demonstrated that infusion of autologous, in vitro expanded and activated CD8+ cells was feasible and clinically well tolerated in five of seven subjects with advanced HIV infections.

Epstein-Barr virus (EBV) is associated with the development of two human B-cell malignancies, Burkitt's lymphoma and lymphomas that occur in the immunosuppressed host. The latter category of disease has become important recently as it is seen primarily in organ transplant recipients and individuals with acquired immunodeficiency syndrome. One possible mechanism for lymphoma development involves a reduction in or lack of EBV-specific cytotoxic T-cell recognition. In support of this model are previous observations that the expression of EBV nuclear antigen 2 (EBNA2) and latent membrane protein, two viral antigens associated with major histocompatibility complex class I-restricted T- cell killing, are downregulated in Burkitt's lymphoma and in early passage lymphoblastoid cell lines (LCL) derived from the malignant lesions. To determine whether a similar mechanism could occur in the development of posttransplant lymphoproliferative disorders (PTLD), we compared EBV gene expression among 23 PTLD tumor lesions obtained from 11 solid organ transplant recipients and among LCL derived from 3 of these lesions. In this report, we demonstrate, by Southern blot, Western blot, and immunofluorescence analysis, that (1) the tumor lesions exhibit varying patterns of restricted viral gene expression; (2) LCL derived from these lesions may represent the in vitro selection of cell subpopulations; and (3) immunosuppressed individuals have a markedly reduced antibody response to the latent cycle antigens, EBNA1, EBNA2, and EBNA-LP, but not to the lytic cycle viral capsid antigen when compared with normal immunocompetent controls.

Objective To determine the frequency of Epstein-Barr virus (EBV) oropharyngeal shedding during HIV infection in homosexual men in the Multicenter AIDS Cohort Study. Design The cohort consisted of 210 men who were HIV-seronegative at their baseline study visit, 39 of whom seroconverted to HIV at a later date, and 73 asymptomatic and mildly symptomatic men with HIV infection of indeterminate duration. Methods EBV in throat washings was detected by transformation of newborn cord blood lymphocytes. Results EBV was isolated from 49% (35 out of 71) of the HIV-seropositive and 16% (33 out of 204) of the HIV-seronegative homosexual men tested at their baseline visit. Elevated EBV shedding frequency was noted 6 months before, as well as during the first HIV-seropositive clinic visit, in the men who seroconverted to HIV. Seronegative men who shed EBV at their baseline visit seroconverted to HIV within a shorter period than did non-shedders during 5 years of follow-up. Shedding of EBV was not significantly associated with either abnormal T-cell numbers, clinical symptoms or risk for development of AIDS. Conclusions There is an increased rate of EBV shedding in HIV-seropositive homosexual men that occurs very early in the course of HIV infection.

We studied the effect of the different types of interferons on the production of cytotoxin by human peripheral blood mononuclear cells (PBMCs) stimulated with the mitogen phytohemagglutinin (PHA). Maximum secreted levels of cytotoxin were observed at day 3 in culture and consisted of both tumor necrosis factor alpha (TNF-alpha) and lymphotoxin as determined by specific antibodies. Type I interferons (IFN-alpha and IFN-beta) consistently suppressed cytotoxin production. Both TNF-alpha and lymphotoxin were significantly suppressed. Mean suppression by IFN-alpha and IFN-beta (1000 U/ml) was 56 and 66%, respectively, in PBMCs from 18 different donors. The suppressive effects of IFN-alpha and IFN-beta on cytotoxin production were dose responsive over a range of 10 to 1000 U/ml. Type II interferon (IFN-gamma) did not have consistent significant effects. Pretreatment with IFN-alpha or IFN-beta for 24 or 48 h prior to PHA stimulation also resulted in significant suppression. Supplementation with interleukin-2 (10 U/ml) or IFN-gamma (1000 U/ml) did not overcome cytotoxin suppression by IFN-alpha or IFN-beta. Cytotoxin suppression by IFN-alpha and IFN-beta together appeared to be noninteractive. Suppression appeared not to be due to blockade of the cytotoxin release, since both cell-associated cytotoxin and secreted cytotoxin were suppressed to the same level. These results demonstrated that cytotoxin and lymphotoxin production by PHA-stimulated PBMCs could be down-regulated by type I interferons and that there is a substantial difference between the action of type I interferons and type II interferons (IFN-gamma) in modulating the biosynthesis of cytotoxins.

A mathematical model based on the median effect principle was used to demonstrate its usefulness in studying cytokine interactions. The inhibition of cytopathological effects due to encephalomyocarditis virus (EMCV) by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF) in WISH cells using a microtiter assay was employed. The model was used to analyze the characteristics of the dose-effect relationships of IFN-gamma, TNF, and their combination. The method also was used to define synergism as opposed to additive interaction. This application of the median-effect model proved to be easy and accurate and could be useful in the study of interactions in cytokine research.

The combinations of ampligen and zidovudine at ratios of 100:1, 25:1, 10:1, and 1:50 acted synergistically to reduce cytopathology caused by HIV in MT-2 cell cultures. Combination indices were less than 1 at all of these ratios representing different combinations of concentrations and at 3 effective doses (ED30, ED50, ED70). Combination of drugs which show synergism at a wide range of ratios of combinations suggest that they may be useful clinically, and that the antiviral efficacy of ZDV may be increased in combination with ampligen. Synergism was also found between ampligen and zidovudine by reduction of HIV-produced plaques in a HeLa cell line expressing CD-4 receptors. However the combination of ampligen and dideoxyinosine against HIV in MT-2 cells was only additive and not synergistic.

OBJECTIVE To assess the presence of covert HIV-1 infection. SETTING High-risk seronegative homosexual men from the Pittsburgh portion of the Multicenter AIDS Cohort Study were examined for the presence of HIV-1 infection. PATIENTS, PARTICIPANTS Ten men (group 1) were examined prospectively for the presence of HIV-1 in their freshly-obtained peripheral blood mononuclear cells (PBMC). Furthermore, cryopreserved PBMC from 26 men (group 2) at their first visit (1984-1985) were examined retrospectively for the presence of HIV-1. MAIN OUTCOME MEASURES PBMC samples from groups 1 and 2 were examined for HIV-1 by polymerase chain reaction (PCR) using gag, env and strong-stop (long terminal repeat) specific primers. In addition, fresh PBMC samples from group 1 were examined for HIV-1 by virus culture. RESULTS None of the 10 PBMC samples from group 1 were positive for virus culture and PCR. Only one of the 26 men from group 2 was positive for gag and strong-stop DNA sequences. This PCR-positive, seronegative subject was found to be negative for HIV-1 by PCR at follow-up visits up to 48 months later. None of 15 seronegative, low-risk homosexual men and 12 seronegative heterosexual men were found to be PCR-positive for HIV-1. However, six HIV-1-seropositive men were positive by PCR for gag, env, and strong-stop HIV-1 DNA sequences. CONCLUSIONS These results suggest a low prevalence of covert HIV-1 infection in high-risk seronegative homosexual men in our geographic area.

Twenty-five patients with AIDS in AIDS Clinical Trials Group Protocol 002 were treated with either low or high dosages of zidovudine. This resulted in moderate, transient increases by 10 and 20 weeks in lymphocyte blastogenesis and interferon-gamma (IFN-gamma) production in vitro in response to phytohemagglutinin with and without recombinant interleukin-2. Immune responses to cytomegalovirus and herpes simplex virus type 1 antigens were augmented less frequently during therapy. Natural killer (NK) cell lysis of uninfected and human immunodeficiency virus-infected cells was also transiently increased by 10 and 20 weeks. IFN-gamma production, the only immune parameter directly associated with increases in numbers of CD4+ T cells, peaked at 10 weeks of treatment. The limited efficacy of zidovudine treatment in AIDS patients is associated with moderate, temporary increases in nonspecific and herpesvirus-specific T lymphocyte responses and NK cell function.

Although used widely, the effectiveness of zidovudine therapy and primary prophylaxis for Pneumocystis carinii pneumonia (PCP) in HIV-1-infected individuals, has not been assessed in a large cohort. We have done an observational study between October, 1986, and October, 1990, of a cohort of 2145 HIV-1-seropositive men and 371 who seroconverted during the study. A Markov chain transitional analysis was used to examine the effect of zidovudine and PCP prophylaxis on the probability of progression of H IV-1 infection to AIDS (after 6, 12, 18, and 24 months) after follow-up visits categorised into one of six disease states. The six starting states were based on CD4 + lymphocyte counts and the presence of HIV-related symptoms. Use of pre-AIDS zidovudine and PCP prophylaxis was associated with significant reductions in rates of progression to AIDS at 6, 12, 18, and 24 months for participants starting with less than 350 CD4 + lymphocytes/μl. For those starting with 350 or more CD4 + lymphocytes/μl, non-significant protective trends were seen during 12, 18, and 24 month intervals. In multivariate log-linear models virtually all the treatment effect was due to zidovudine. However, after adjusting for the effects of zidovudine, PCP prophylaxis reduced significantly the probability of progression to a first episode of PCP during 6, 12, 18, and 24 month intervals. This study suggests that early primary PCP prophylaxis is effective in preventing first episodes of PCP, and that the efficacy of zidovudine demonstrated in clinical trials can be translated to the population level.

Zidovudine therapy of AIDS patients has been shown to cause only transient improvements in the numbers of circulating CD4+ cells and the in vitro functional activities of cultured lymphocytes. The present studies were undertaken to determine whether prolonged zidovudine therapy enhanced reactivity in two sensitive assays of T-cell function: the ability of phytohemagglutinin (PHA)-stimulated cells to form T-cell colonies and their capacity to express receptors for the growth factor interleukin-2 (IL-2). Treated patients, studied over periods of 20-60 weeks, showed no improvement in colony formation at any time interval, even in plates supplemented with exogenous IL-2. However, mitogen-stimulated T lymphocytes showed a significant increase in the capacity to express IL-2 receptors (CD25). This enhanced expression resulted primarily from activation of the CD8+ cell subset.

Background. The initially tested dose of zidovudine for the treatment of patients with advanced disease caused by the human immunodeficiency virus type 1 (HIV) was 1500 mg. Although this dose is effective, it is associated with substantial toxicity. Methods. To evaluate the efficacy and safety of a reduced dose, we conducted a randomized controlled trial in 524 subjects who had had a first episode of Pneumocystis carinii pneumonia. The subjects were assigned to receive zidovudine in either a dose of 250 mg taken orally every four hours (the standard-treatment group, n = 262) or a dose of 200 mg taken orally every four hours for four weeks and thereafter 100 mg taken every four hours (the low-dose group, n = 262). Results. The median length of follow-up was 25.6 months. At 18 months the estimated survival rates were 52 percent for the standard-treatment group and 63 percent for the low-dose group (P = 0.012 by the log-rank test). At 24 months the estimated survival rates were 27 percent for the st...

The determinants of cytomegalovirus (CMV) infection and disease can now be understood from studies of newborns, recipients of organ or bone marrow transplants, subjects infected with human immunodeficiency virus, and recipients of blood transfusions. CMV is transmitted to the neonate transplacentally, by passage through a contaminated birth canal, or by ingestion of infected breast milk; to the adult by heterosexual and homosexual sex with an infected partner; and to the transplant recipient by infected organs. A major unsolved problem in the study of CMV is the nature of viral latency. Knowledge regarding the requirements for activation of latent infection at the molecular, cellular, or host level is incomplete. Both viral and host factors may contribute to the successful transmission of CMV by latently infected cells in transplanted organs and transfused blood.

Women attending family planning clinics in western Pennsylvania were enrolled into a prospective epidemiologic study of herpes simplex virus (HSV) infection. Detection of antibodies to HSV-1 and HSV-2 was based on an immunodot assay using type-specific glycoproteins gG-1 and gG-2. Serologic and historical data at enrollment were analyzed for 4527 subjects; the seroprevalence of HSV-2 was 21.6%. By multivariate analysis, HSV-2 infection as determined by seroprevalence was significantly and independently associated with age, years of sexual activity, race, one or more episodes of other genital infections, lower annual family income, and multiple sexual partners. The presence of antibody to HSV-1 was associated with a lower frequency of antibody to HSV-2, suggesting that developed immunity to HSV-1 protected against HSV-2 infection. Of 926 participants who had antibody to HSV-2, only 117 (12.6%) reported a history of genital herpes. Hence the vast majority of HSV-2 infections were inapparent. Although seroprevalence of HSV-2 was higher among black (35.4%) than white (18.5%) women, black women appeared to have significantly less symptomatic genital herpes than white women (7.7% vs. 14.7%, P less than .01).