Quantitative image analysis of HIV-1 infection in lymphoid tissue.

Tracking human immunodeficiency virus-type 1 (HIV-1) infection at the cellular level in tissue reservoirs provides opportunities to better understand the pathogenesis of infection and to rationally design and monitor therapy. A quantitative technique was developed to determine viral burden in two important cellular compartments in lymphoid tissues. Image analysis and in situ hybridization were combined to show that in the presymptomatic stages of infection there is a large, relatively stable pool of virions on the surfaces of follicular dendritic cells and a smaller pool of productively infected cells. Despite evidence of constraints on HIV-1 replication in the infected cell population in lymphoid tissues, estimates of the numbers of these cells and the virus they could produce are consistent with the quantities of virus that have been detected in the bloodstream. The cellular sources of virus production and storage in lymphoid tissues can now be studied with this approach over the course of infection and treatment.

Enterobacter bacteremia. An analysis of 50 episodes.

During a six-year period, 147 patients had Enterobacter bacteremia (3.8% of the episodes of bacteremia), with an incidence of 1.25 per 1,000 admitted patients. We chose a random group of 50 cases for analysis. The disease was community acquired in 24% of the cases and nosocomially acquired in the remaining 76%. The bacteremia was unimicrobial in 70% and part of a polymicrobial bacteremia in 30%. The species most commonly causing bacteremia was Enterobacter cloacae. Portals of entry, in decreasing order of frequency, were unknown, surgical wound, respiratory tract, and urinary tract. The most common clinical finding was fever (92%). Shock occurred in 30% of the patients, and only two patients had evidence of disseminated intravascular coagulation. Of the Enterobacter isolates, 12% were resistant to gentamicin. Overall mortality was 42%; factors associated with a poor prognosis were inadequacy of antimicrobial chemotherapy, septic shock, type of underlying disease, clinical condition, and requirement of intensive care.

Analysis of HIV-1 reverse transcriptase and protease sequences in paired plasma and lymphoid tissue specimens from HIV-1 infected individuals.

OBJECTIVES: To determine how representative the genotype of HIV-1 circulating in plasma is of the genotype of the virus present in lymphoid tissue. METHODS: Paired plasma and tonsillar tissue samples were prospectively obtained from patients with various levels of plasma HIV-1 RNA who were receiving combination antiretroviral therapy. HIV-1 reverse transcriptase and protease sequences were amplified from plasma and lymphoid tissue specimens by nested polymerase chain reaction and analyzed using an automated sequencing system. Results were compared with consensus HIV-1 sequences to determine whether drug-resistance mutations were present in the regions analyzed. RESULTS: HIV-1 protease sequences were compared in 11 plasma/tissue pairs obtained from eight patients; HIV reverse transcriptase sequences were compared in 12 plasma/tissue pairs obtained from nine patients. Sequence homology between plasma and tissue RNA, tissue RNA and DNA, and plasma and tissue DNA ranged from 97% to 100%. Few discrepancies were found when the percentage of mutant sequences at resistance codons was compared among paired samples. In most instances, tissue RNA or plasma contained a higher percentage of mutant sequences than did tissue DNA. CONCLUSION: The genotype of plasma HIV-1 is similar to the genotype of the virus in lymphoid tissue. Resistance studies using plasma samples should provide accurate information regarding the genotype of HIV-1 in lymphoid tissues.

Neurological outcomes in late HIV infection: adverse impact of neurological impairment on survival and protective effect of antiviral therapy. AIDS Clinical Trial Group and Neurological AIDS Research Consortium study team.

OBJECTIVE: In a large multi-center clinical trial of combination reverse transcriptase inhibitors (RTIs), we assessed the impact of antiretroviral therapy on neurological function, the relationship between neurological and systemic benefit, and the prognostic value of neurological performance in late HIV-1 infection. DESIGN: Neurological evaluations incorporated in a randomized, multi-center trial of combination antiretroviral therapy. SETTING: Forty-two AIDS Clinical Trials Group sites and seven National Hemophilia Foundation sites. PATIENTS: Adult HIV-infected patients (n = 1313) with CD4 counts < 50 x 10(6) cells/l. INTERVENTIONS: Four combinations of reverse transcriptase inhibitors consisting of zidovudine (ZDV), alternating monthly with didanosine (ddl), or in combination with zalcitabine (ddC), ddl or ddl and nevirapine. MAIN OUTCOME MEASURES: Mean change from baseline of a four-item quantitative neurological performance battery score, the QNPZ-4, administered to 1031 subjects. RESULTS: Triple therapy and ZDV/ddl combination preserved or improved neurological performance over time compared with the alternating ZDV/ddl and ZDV/ddC regimens (P < 0.001), paralleling their impact on survival in the same trial as previously reported. QNPZ-4 scores were predictive of survival (P < 0.001), after adjusting for CD4 counts and HIV-1 plasma RNA concentrations. CONCLUSIONS: Combination antiretroviral therapy can have a salutary effect on preserving or improving neurological function. Superior systemic treatments may likewise better preserve neurological function. The significant association of poor neurological performance with mortality, independent of CD4 counts and HIV-1 RNA levels indicates that neurological dysfunction is an important cause or a strong marker of poor prognosis in late HIV-1 infection. This study demonstrates the value of adjunctive neurological measures in large therapeutic trials of late HIV-1 infection.

Virologic responses to a ritonavir--saquinavir-containing regimen in patients who had previously failed nelfinavir.

OBJECTIVES: The effectiveness of a second protease inhibitor in patients who failed an initial protease inhibitor is unclear but believed to be low. It has been postulated, however, that patients who fail nelfinavir may respond differently. We therefore assessed the virologic response to a ritonavir-saquinavir-containing regimen in patients who had previously failed nelfinavir. METHODS: A total of 26 patients enrolled in the nelfinavir clinical trials AG506 and AG511 at our two sites who failed (two consecutive HIV viral loads > 5000 copies/ml; branched DNA assay) were switched to a combination of stavudine 40 mg twice daily, lamivudine 150 mg twice daily, ritonavir 400 mg twice daily and saquinavir 400 mg twice daily. RESULTS: The mean viral load at enrollment in this study was 46 674 copies/ml (range, 1075-146400 copies/ml). The median CD4 cell count was 222 x 10(6)/l (range, 82-448 x 10(6)/l). The median duration of nelfinavir use with a detectable viral load before the switch occurred was 48 weeks. Two patients discontinued the study at 3 weeks. All of the remaining patients (n = 24) reached undetectable viral loads (< 500 copies/ml) that were sustained at week 24 in 17 (71%) out of 24 subjects. The most frequent baseline mutations in the protease gene prior to switching were D30N (13 out of 18), N88D (eight out of 18) and M36I (eight out of 18). The presence or absence of these mutations was not predictive of a short-term virologic response. CONCLUSIONS: Most patients who failed a nelfinavir-containing regimen responded to a switch to a combination regimen with saquinavir-ritonavir.

Cytomegalovirus (CMV) and human immunodeficiency virus (HIV) burden, CMV end-organ disease, and survival in subjects with advanced HIV infection (AIDS Clinical Trials Group Protocol 360).

We undertook a prospective study to analyze cytomegalovirus (CMV) end-organ disease (EOD) in subjects with advanced human immunodeficiency virus (HIV) infection. Of 403 individuals without prior CMV EOD who were followed up for a median of 151 weeks, 56 died and 21 developed CMV EOD. Twenty of the subjects with CMV EOD had CD4 cell counts of < or =50 cells/mm3 and HIV RNA level of >10,000 copies/mL of plasma at baseline; in these 20 subjects, an increase of CMV DNA level to greater than the quantification limits was associated with CMV EOD. A CD4 cell count of < or =100 cells/mm3 and an HIV RNA level of >10,000 copies/mL of plasma at baseline, a CMV DNA level of >200 copies/mL of blood during follow-up, or development of CMV EOD were all associated with decreased survival. HIV-infected subjects with CD4 cell counts of < or =50 cells/mm3 and HIV RNA levels of >10,000 copies/mL of plasma should have blood fractions screened for CMV DNA; if CMV DNA is detected, CMV prophylaxis might be considered.

Concentration-controlled zidovudine therapy.

BACKGROUND: Heterogeneity in the response to antiretroviral therapy has been attributed to pharmacologic, immunologic, and virologic differences between patients. Currently available antiretroviral agents used for the treatment of human immunodeficiency virus (HIV) infection in adults are administered in standard fixed doses. The active moiety of nucleoside anti-HIV drugs is the intracellular anabolite. Therefore the heterogeneity in response to nucleoside agents may arise as a result of pharmacologic variability at both the systemic and cellular level. OBJECTIVES: To determine whether a novel concentration-controlled zidovudine regimen could improve anti-HIV response compared with the standard fixed-dose approach. DESIGN: At the Outpatient Clinic of the General Clinical Research Center at the University of Minnesota, 20 persons with HIV infection received an oral regimen of zidovudine designed to achieve a target concentration in plasma of 0.7 mumol/L and the 500 mg/day standard dose in a randomized, crossover 24-week study. RESULTS: The concentration-controlled regimen achieved overall higher systemic concentrations with reduced interpatient variability: steady-state average zidovudine plasma concentrations were 0.76 mumol/L (coefficient of variation, 12%) versus 0.62 mumol/L (coefficient of variation, 32%) for the standard regimen. There was no difference in safety and tolerance between regimens. Intracellular zidovudine triphosphate concentrations averaged 160 fmol/10(6) peripheral blood mononuclear cells (PBMCs) with concentration-controlled versus 92 fmol/10(6) PBMCs for standard therapy. The percentage change from baseline in CD4 cells was a 22% increase for the concentration-controlled regimen versus a 7% decrease with standard therapy. CONCLUSIONS: These data indicate that pharmacologic variability affects antiretroviral response. Furthermore, these findings provide a framework to characterize the pharmacologic determinants of effect and quantitate their contribution to the heterogeneity in clinical response to optimize therapeutic benefit.

Management of acyclovir-resistant herpes simplex and varicella-zoster virus infections.

Persons with AIDS who have CD4+ counts < or = 100 and transplant patients, especially bone marrow allograft recipients, may experience clinically significant infections with acyclovir-resistant herpes simplex virus (HSV) or varicella-zoster virus (VZV). Patients who have received prior repeated acyclovir treatment appear to be at the highest risk of harboring acyclovir-resistant strains. Algorithms for the management of these infections were developed at a recent roundtable symposium. The consensus of the panelists was that treatment with foscarnet should be initiated within 7-10 days in patients suspected to have acyclovir-resistant HSV or VZV infections. Foscarnet therapy should be continued for at least 10 days or until lesions are completely healed.

Treatment of invasive cytomegalovirus disease in solid organ transplant patients with ganciclovir.

The occurrence of cytomegalovirus infection after solid organ transplantation has been correlated with decrease patient and allograft survival. The disease has not been conquered for two majors reasons: the length of time to establish the diagnosis of CMV has been excessive, and suitable, nontoxic antiviral agents have not been available for use. The purpose of this study was to examine the current incidence and impact of tissue-invasive cytomegalovirus (TI-CMV) disease that developed in 93 patients who underwent solid organ transplantation at University of Minnesota Hospitals (3/1/87 and 6/30/89) and who were treated with antiviral agent ganciclovir ( [9-(1,3-dihydroxy-2-2-propoxymethyl)-guanine [DHPG]). During this same period of time 323 patients received kidney transplants and 71 received kidney-pancreas transplants. Three patient groups were defined: (1) no CMV; (2) CMV infection (cultural or serologic evidence of noninvasive CMV infection); and (3) evidence of TI-CMV disease based upon initial complaints of fever, malaise, dyspnea, or abdominal pain, leukopenia (WBC less than 3000/ml), and evidence of a positive CMV rapid antigen test, CMV culture, or the presence of characteristic CMV inclusion bodies upon examination of material obtained by means of bronchoscopy, upper-gastrointestinal endoscopy, colonoscopy, or liver or renal biopsy. Patients with solely fever, leukopenia, but without a rising CMV serum titer, or positive CMV urine or blood cultures were excluded from the study. A multivariate analysis revealed that rejection therapy, age greater than 50 years, and receiving an organ from a seropositive donor were all significant variables that predisposed to TI-CMV. Analysis of patient and kidney allograft survival indicated that asymptomatic CMV infection had little current impact upon patient or allograft survival, while patients who developed TI-CMV exhibited higher rates of allograft loss and mortality, despite DHPG therapy. Comparison with historical group of patients indicated that TI-CMV DHPG-treated patients exhibited a trend toward improved allograft survival that may be relevant because the historical group of patients included patients with mild CMV infection. DHPG therapy was well tolerated and produced minimal toxicity, and excellent 30-day cure rates (89.2%), although 21.2% of patients required retreatment subsequently. We are currently conducting a trial to compare the ability of DHPG administered plus an anti-CMV immune globulin preparation with acyclovir to prevent posttransplant TI-CMV disease.

A prospective randomized study of acyclovir versus ganciclovir plus human immune globulin prophylaxis of cytomegalovirus infection after solid organ transplantation.

Cytomegalovirus disease occurs frequently after solid organ transplantation and has been associated with decreased patient and allograft survival. We hypothesized that CMV transmission or reactivation begins immediately or soon after transplantation, and that a short-duration ganciclovir (GCV)-based regimen would obviate the need for long-term antiviral agent administration, perhaps serving to interdict CMV infection and disease as well as, or perhaps even more effectively than, a more prolonged, oral acyclovir (ACV)-based form of prophylaxis. A total of 311 patients were stratified according to allograft type, age, and presence or absence or diabetes mellitus, and were then randomized to receive either long-duration ACV prophylaxis (800 mg orally or 400 mg i.v. q.i.d. for 12 weeks after transplantation or 6 weeks after any antirejection therapy) versus short-duration GCV (5 mg/kg/12 hr i.v. for 7 days after transplant or after any antirejection therapy) plus human immune globulin (HIg; Sandoglobulin or Minnesota CMV immune globulin) 100 mg/kg i.v. administered on days 1, 4, and 7 after transplant or after any antirejection therapy. A total of 266 patients (ACV, n = 133; GCV+HIg, n = 133) completed the protocol and were available for follow-up. CMV disease occurred in fewer patients (n = 28, 21.0%) in the ACV group, while significantly more patients (n = 42, 31.6%) in the GCV + HIg group developed group developed CMV disease slightly later (2.83 +/- 0.70 months) than those who received GCV/HIg (2.15 +/- 0.21 months, P > 0.01). Multivariate analysis demonstrated (2.15 +/- 0.21 months, P > 0.1). Multivariate analysis demonstrated that receiving antirejection therapy, a liver transplant, or a donor organ from a CMV-seropositive individual if the recipient was CMV seronegative were major risk factors for the development of CMV disease (P < 0.001), while the difference between ACV versus GCV + HIg prophylaxis was also significant (P = 0.054). No differences in actuarial patient or allograft survival, however, were noted between the 2 prophylaxis groups. Overall, ACV prophylaxis appeared to be more effective in reducing the incidence of posttransplant CMV disease, although this effect was diminished in high-risk groups of patients. Our findings indicate that CMV transmission or reactivation may best be prevented by long-term antiviral agent administration, and that the primary morbidity of CMV disease is the need for rehospitalization when either prolonged ACV or short-duration GCV + HIg prophylaxis is used in this patient population.

Adenovirus in the gastrointestinal tracts of immunosuppressed patients.

Adenovirus was cultured from various gastrointestinal sites and one lung specimen from seven immunosuppressed patients, four of whom were bone marrow transplant recipients. Histologic examination and in situ hybridization studies of the lung demonstrated adenovirus in the bronchial epithelium and alveolar lining cells. In contrast, none of the 34 gastrointestinal biopsies performed within 30 days of the positive adenovirus culture showed histologic or molecular evidence of invasive adenovirus in the gastrointestinal mucosa. These results suggest that isolation of adenovirus from gastrointestinal biopsy specimens taken from immunosuppressed patients probably does not indicate an invasive adenoviral infection.

Evaluation of centrifugation cultures of bronchoalveolar lavage fluid for the diagnosis of cytomegalovirus pneumonitis.

Cytomegalovirus (CMV) pneumonitis is one of the most severe manifestations of CMV disease among immunocompromised patients. The diagnosis of CMV pneumonitis traditionally has required the use of invasive procedures such as lung biopsy. In this retrospective study, we evaluated a centrifugation culture method in samples of bronchoalveolar fluid for the noninvasive diagnosis of CMV pneumonitis. During a 9-mo period, 75 bronchoalveolar lavage samples were collected from 58 patients with pneumonitis. We analyzed the data from 21 patients in whom lung tissue samples were obtained within 14 days of the bronchoalveolar lavage. Centrifugation cultures of bronchoalveolar fluid were positive for CMV in 12 cases. CMV pneumonitis was confirmed in samples of lung tissue from five (42%) of the 12 patients, whereas no evidence of CMV pneumonitis was found in the remaining seven (58%) cases. Of nine patients with negative centrifugation cultures, CMV pneumonitis was confirmed in two (22%). When compared with conventional cultures, we found bronchoalveolar lavage fluid centrifugation cultures to be highly sensitive (100%) and specific (92%) for the detection of CMV infection. However, detection of CMV by centrifugation culture proved to be only moderately sensitive (71%) and nonspecific (50%) for the diagnosis of CMV pneumonitis.

Serratia bacteremia.

During a 6-yr period, 146 patients at our institution had Serratia bacteremia (3.8% of the total number of episodes of bacteremia), with an incidence of 1.24/1000 admitted patients. We chose a random group of 50 cases for clinical analysis in the present study. The disease was community-acquired in 8% of the cases and nosocomially-acquired in the remaining 92%. The bacteremia was unimicrobial in 84% and part of a polymicrobial bacteremia in 16% of the episodes. The most frequently isolated species of the Serratia genus was S. marcescens. Portals of entry, in decreasing order of frequency, were: urinary, unknown, respiratory, and surgical wound infections. Clinically, the most frequent finding was fever (100%). Shock occurred in 28% of the patients, and none of our cases showed evidence of disseminated intravascular coagulation. We found 62% of Serratia isolates resistant to gentamicin. Overall mortality was 38% and factors associated with a poor prognosis were: severity of the underlying disease, critical clinical situation at onset of bacteremia, presence in the intensive care unit (I.C.U.), occurrence of shock or polymicrobial bacteremia, portal of entry in the respiratory tract, and inadequate treatment.

Interlaboratory concordance of DNA sequence analysis to detect reverse transcriptase mutations in HIV-1 proviral DNA. ACTG Sequencing Working Group. AIDS Clinical Trials Group.

Thirteen laboratories evaluated the reproducibility of sequencing methods to detect drug resistance mutations in HIV-1 reverse transcriptase (RT). Blinded, cultured peripheral blood mononuclear cell pellets were distributed to each laboratory. Each laboratory used its preferred method for sequencing proviral DNA. Differences in protocols included: DNA purification; number of PCR amplifications; PCR product purification; sequence/location of PCR/sequencing primers; sequencing template; sequencing reaction label; sequencing polymerase; and use of manual versus automated methods to resolve sequencing reaction products. Five unknowns were evaluated. Thirteen laboratories submitted 39043 nucleotide assignments spanning codons 10-256 of HIV-1 RT. A consensus nucleotide assignment (defined as agreement among > or = 75% of laboratories) could be made in over 99% of nucleotide positions, and was more frequent in the three laboratory isolates. The overall rate of discrepant nucleotide assignments was 0.29%. A consensus nucleotide assignment could not be made at RT codon 41 in the clinical isolate tested. Clonal analysis revealed that this was due to the presence of a mixture of wild-type and mutant genotypes. These observations suggest that sequencing methodologies currently in use in ACTG laboratories to sequence HIV-1 RT yield highly concordant results for laboratory strains; however, more discrepancies among laboratories may occur when clinical isolates are tested.

Pharmacokinetics and safety of concentration-controlled oral zidovudine therapy.

STUDY OBJECTIVE: To evaluate the pharmacokinetics, safety, and feasibility of concentration-controlled oral zidovudine therapy. DESIGN: Randomized, crossover, open-label study. SETTING: University-affiliated general clinical research center. PATIENTS: Eight individuals infected with the human immunodeficiency virus with CD4+ lymphocyte counts of 100 cells/microliter or greater. INTERVENTION: During the 24-week study, patients received oral zidovudine regimens that consisted of a standard fixed dose of 500 mg/day and a concentration-controlled regimen designed to maintain a steady-state plasma concentration (Css) of 0.187 +/- 0.04 mg/L (0.7 +/- 0.14 microM). MEASUREMENTS AND MAIN RESULTS: The mean Css during standard therapy was 0.170 +/- 0.024 mg/L versus 0.205 +/- 0.021 mg/L with the concentration-controlled regimen (p = 0.025). Respective mean changes in hemoglobin were -0.02 g/dl (range -0.9-0.9 g/dl) and -0.30 g/dl (range -1.5-0.4 g/dl, p = 0.67). The absolute neutrophil count decreased 0.90 x 10(9)/L during standard therapy and increased 0.40 x 10(9)/L during concentration-controlled therapy (p = 0.07). The regimens did not differ in toxicity. CONCLUSION: Concentration-controlled oral antiretroviral therapy with zidovudine is feasible and safe, and provides pharmacologic data to determine the regimen's virologic and immunologic benefits.

Visceral leishmaniasis in patients infected with human immunodeficiency virus. Co-operative Group for the Study of Leishmaniasis in AIDS.

We describe 40 HIV-seropositive patients who developed visceral leishmaniasis. All the patients lived in areas endemic for visceral leishmaniasis and belonged to groups at risk for AIDS. Twenty-three patients (57.2%) had definitive AIDS before or after diagnosis of leishmaniasis and 77.5% were classified as belonging to CDC group IV. Fever was present in 95% patients and enlargement of the liver and/or spleen in 92.5%. Lymphopenia was found in 78.3%, depression of the absolute number of CD4 lymphocytes in 90% and depression of the CD4 to CD8 ratio in all evaluated cases but leishmania antibodies were found in only 35.2%. Parasites were demonstrated in the bone marrow or liver in every case. Thirty patients (75%) showed an initial good response to antimonial drugs, although the leishmaniasis followed a chronic or relapsing course in 17 (42.5%). HIV-related mortality was 40%. A significant correlation was found only between the relapsing course of the disease and mortality. In a multivariate linear regression model, the relapsing course was the only variable that influenced mortality. Visceral leishmaniasis is an opportunistic disease that should be suspected in HIV-infected patients. We suggest that it should be included in the CDC group IV C-1 and considered as a disease indicative of AIDS.

Outpatient biopsies of the palatine tonsil: access to lymphoid tissue for assessment of human immunodeficiency virus RNA titers.

OBJECTIVES: Our objective was to assess the feasibility of using tonsillar lymphoid biopsy specimens obtained on an outpatient basis to quantitate a patient's lymphoid human immunodeficiency virus (HIV) RNA titers. DESIGN: A pilot cohort study was performed. PATIENTS: We evaluated ten HIV-seropositive patients who ranged in age from 26 to 48 years and had CD4+ cell counts ranging from 110 to 833 at enrollment. MAIN OUTCOME MEASURES: The main outcome measures were tolerance and safety of outpatient tonsil biopsies and quantitation of HIV RNA titers in tonsillar lymphoid biopsy specimens, plasma, and peripheral blood mononuclear cells determined by a new method of HIV RNA signal amplification with branched DNA probes. RESULTS: Outpatient tonsil biopsies were well tolerated and were performed without complications. Nine of 10 tonsil biopsies from the HIV-seropositive patients examined were positive for significant concentrations of HIV RNA, ranging from 106 to 101 HIV RNA equivalents per gram of tissue. All of the HIV RNA-positive tonsillar lymphoid specimens had HIV RNA titers that were 101 to 104 times greater than those recovered from plasma (per milliliter) of the same patient obtained at the time of biopsy. CONCLUSIONS: Sufficient tonsillar tissue can be obtained in an outpatient clinic setting to quantitate lymphoid HIV titers by the new branched-DNA signal amplification method with relative ease and without complication. The biopsy method described here affords ready access to the lymphoreticular system, which may help to advance our understanding of the pathogenesis of myriad immune diseases without the need for excisional node biopsies.

[Lymphoma of the rectum associated with lymphoid nodular hyperplasia in a common variable immunodeficiency]. / Linfoma rectal asociado a hiperplasia nodular linfoide en el seno de una inmunodeficiencia variable común.

A patient with common variable immunodeficiency developed lymphoid nodular hyperplasia and, subsequently, a follicular non-Hodgkin lymphoma with excellent response to chemotherapy. The patient remained in remission after 4 years. The very unusual type of this lymphoma and its localization are discussed, and the possible relations between these different conditions as a single spectrum of B lymphocyte are analyzed.