Tuberculosis-related mortality in people living with HIV in Europe and Latin America: an international cohort study

BACKGROUND Management of tuberculosis in patients with HIV in eastern Europe is complicated by the high prevalence of multidrug-resistant tuberculosis, low rates of drug susceptibility testing, and poor access to antiretroviral therapy (ART). We report 1 year mortality estimates from a multiregional (eastern Europe, western Europe, and Latin America) prospective cohort study: the TB:HIV study. METHODS Consecutive HIV-positive patients aged 16 years or older with a diagnosis of tuberculosis between Jan 1, 2011, and Dec 31, 2013, were enrolled from 62 HIV and tuberculosis clinics in 19 countries in eastern Europe, western Europe, and Latin America. The primary endpoint was death within 12 months after starting tuberculosis treatment; all deaths were classified according to whether or not they were tuberculosis related. Follow-up was either until death, the final visit, or 12 months after baseline, whichever occurred first. Risk factors for all-cause and tuberculosis-related deaths were assessed using Kaplan-Meier estimates and Cox models. FINDINGS Of 1406 patients (834 in eastern Europe, 317 in western Europe, and 255 in Latin America), 264 (19%) died within 12 months. 188 (71%) of these deaths were tuberculosis related. The probability of all-cause death was 29% (95% CI 26-32) in eastern Europe, 4% (3-7) in western Europe, and 11% (8-16) in Latin America (p<0·0001) and the corresponding probabilities of tuberculosis-related death were 23% (20-26), 1% (0-3), and 4% (2-8), respectively (p<0·0001). Patients receiving care outside eastern Europe had a 77% decreased risk of death: adjusted hazard ratio (aHR) 0·23 (95% CI 0·16-0·31). In eastern Europe, compared with patients who started a regimen with at least three active antituberculosis drugs, those who started fewer than three active antituberculosis drugs were at a higher risk of tuberculosis-related death (aHR 3·17; 95% CI 1·83-5·49) as were those who did not have baseline drug-susceptibility tests (2·24; 1·31-3·83). Other prognostic factors for increased tuberculosis-related mortality were disseminated tuberculosis and a low CD4 cell count. 18% of patients were receiving ART at tuberculosis diagnosis in eastern Europe compared with 44% in western Europe and 39% in Latin America (p<0·0001); 12 months later the proportions were 67% in eastern Europe, 92% in western Europe, and 85% in Latin America (p<0·0001). INTERPRETATION Patients with HIV and tuberculosis in eastern Europe have a risk of death nearly four-times higher than that in patients from western Europe and Latin America. This increased mortality rate is associated with modifiable risk factors such as lack of drug susceptibility testing and suboptimal initial antituberculosis treatment in settings with a high prevalence of drug resistance. Urgent action is needed to improve tuberculosis care for patients living with HIV in eastern Europe. FUNDING EU Seventh Framework Programme.

Developing a point-of-care electronic medical record system for TB/HIV co-infected patients: experiences from Lighthouse Trust, Lilongwe, Malawi.

BACKGROUND Implementation of user-friendly, real-time, electronic medical records for patient management may lead to improved adherence to clinical guidelines and improved quality of patient care. We detail the systematic, iterative process that implementation partners, Lighthouse clinic and Baobab Health Trust, employed to develop and implement a point-of-care electronic medical records system in an integrated, public clinic in Malawi that serves HIV-infected and tuberculosis (TB) patients. METHODS Baobab Health Trust, the system developers, conducted a series of technical and clinical meetings with Lighthouse and Ministry of Health to determine specifications. Multiple pre-testing sessions assessed patient flow, question clarity, information sequencing, and verified compliance to national guidelines. Final components of the TB/HIV electronic medical records system include: patient demographics; anthropometric measurements; laboratory samples and results; HIV testing; WHO clinical staging; TB diagnosis; family planning; clinical review; and drug dispensing. RESULTS Our experience suggests that an electronic medical records system can improve patient management, enhance integration of TB/HIV services, and improve provider decision-making. However, despite sufficient funding and motivation, several challenges delayed system launch including: expansion of system components to include of HIV testing and counseling services; changes in the national antiretroviral treatment guidelines that required system revision; and low confidence to use the system among new healthcare workers. To ensure a more robust and agile system that met all stakeholder and user needs, our electronic medical records launch was delayed more than a year. Open communication with stakeholders, careful consideration of ongoing provider input, and a well-functioning, backup, paper-based TB registry helped ensure successful implementation and sustainability of the system. Additional, on-site, technical support provided reassurance and swift problem-solving during the extended launch period. CONCLUSION Even when system users are closely involved in the design and development of an electronic medical record system, it is critical to allow sufficient time for software development, solicitation of detailed feedback from both users and stakeholders, and iterative system revisions to successfully transition from paper to point-of-care electronic medical records. For those in low-resource settings, electronic medical records for integrated care is a possible and positive innovation.

In Utero Exposure to Antiretroviral Drugs: Effect on Birth Weight and Growth Among HIV-exposed Uninfected Children in Brazil.

BACKGROUND There are concerns about the effects of in utero exposure to antiretroviral drugs (ARVs) on the development of HIV-exposed but uninfected (HEU) children. The aim of this study was to evaluate whether in utero exposure to ARVs is associated with lower birth weight/height and reduced growth during the first 2 years of life. METHODS This cohort study was conducted among HEU infants born between 1996 and 2010 in Tertiary children's hospital in Rio de Janeiro, Brazil. Weight was measured by mechanical scale, and height was measured by measuring board. Z-scores for weight-for-age (WAZ), length-for-age (LAZ) and weight-for-length were calculated. We modeled trajectories by mixed-effects models and adjusted for mother's age, CD4 cell count, viral load, year of birth and family income. RESULTS A total of 588 HEU infants were included of whom 155 (26%) were not exposed to ARVs, 114 (19%) were exposed early (first trimester) and 319 (54%) later. WAZ were lower among infants exposed early compared with infants exposed later: adjusted differences were -0.52 (95% confidence interval [CI]: -0.99 to -0.04, P = 0.02) at birth and -0.22 (95% CI: -0.47 to 0.04, P = 0.10) during follow-up. LAZ were lower during follow-up: -0.35 (95% CI: -0.63 to -0.08, P = 0.01). There were no differences in weight-for-length scores. Z-scores of infants exposed late during pregnancy were similar to unexposed infants. CONCLUSIONS In HEU children, early exposure to ARVs was associated with lower WAZ at birth and lower LAZ up to 2 years of life. Growth of HEU children needs to be monitored closely.

Pitfalls of practicing cancer epidemiology in resource-limited settings: the case of survival and loss to follow-up after a diagnosis of Kaposi's sarcoma in five countries across sub-Saharan Africa.

BACKGROUND Survival after diagnosis is a fundamental concern in cancer epidemiology. In resource-rich settings, ambient clinical databases, municipal data and cancer registries make survival estimation in real-world populations relatively straightforward. In resource-poor settings, given the deficiencies in a variety of health-related data systems, it is less clear how well we can determine cancer survival from ambient data. METHODS We addressed this issue in sub-Saharan Africa for Kaposi's sarcoma (KS), a cancer for which incidence has exploded with the HIV epidemic but for which survival in the region may be changing with the recent advent of antiretroviral therapy (ART). From 33 primary care HIV Clinics in Kenya, Uganda, Malawi, Nigeria and Cameroon participating in the International Epidemiologic Databases to Evaluate AIDS (IeDEA) Consortia in 2009-2012, we identified 1328 adults with newly diagnosed KS. Patients were evaluated from KS diagnosis until death, transfer to another facility or database closure. RESULTS Nominally, 22% of patients were estimated to be dead by 2 years, but this estimate was clouded by 45% cumulative lost to follow-up with unknown vital status by 2 years. After adjustment for site and CD4 count, age <30 years and male sex were independently associated with becoming lost. CONCLUSIONS In this community-based sample of patients diagnosed with KS in sub-Saharan Africa, almost half became lost to follow-up by 2 years. This precluded accurate estimation of survival. Until we either generally strengthen data systems or implement cancer-specific enhancements (e.g., tracking of the lost) in the region, insights from cancer epidemiology will be limited.

Diagnosis of sexually transmitted infections in developing nations using syndromic management: Is it working?

Sexually transmitted infections (STIs) are a major public health problem, and controlling their spread is a priority. According to the World Health Organization (WHO), there are 340 million new cases of treatable STIs among 15–49 year olds that occur yearly around the world (1). Infection with STIs can lead to several complications such as pelvic inflammatory disorder (PID), cervical cancer, infertility, ectopic pregnancy, and even death (1). Additionally, STIs and associated complications are among the top disease types for which healthcare is sought in developing nations (1), and according to the UNAIDS report, there is a strong connection between STIs and the sexual spread of HIV infection (2). In fact, it is estimated that the presence of an untreated STI can increase the likelihood of contracting and spreading HIV by a factor up to 10 (2). In addition, developing countries are poorer in resources and lack inexpensive and precise diagnostic laboratory tests for STIs, thereby exacerbating the problem. Thus, the WHO recommends syndromic management of STIs for delivering care where lab testing is scarce or unattainable (1). This approach utilizes the use of an easy to use algorithm to help healthcare workers recognize symptoms/signs so as to provide treatment for the likely cause of the syndrome. Furthermore, according to the WHO, syndromic management offers instant and legitimate treatment compared to clinical diagnosis, and that it is also more cost-effective for some syndromes over the use of laboratory testing (1). In addition, even though it has been shown that the vaginal discharge syndrome has low specificity for gonorrhea and Chlamydia and can lead to over treatment (1), this is the recommended way to manage STIs in developing nations. Thus, the purpose of this paper is to specifically address the following questions: is syndromic management working to lower the STI burden in developing nations? How effective is it, and should it still be recommended? To answer these questions, a systematic literature review was conducted to evaluate the current effectiveness of syndromic management in developing nations. This review examined published articles over the past 5 years that compared syndromic management to laboratory testing and had published sensitivity, specificity, and positive predicative value data. Focusing mainly on vaginal discharge, urethral discharge, and genital ulcer algorithms, it was seen that though syndromic management is more effective in diagnosing and treating urethral and genial ulcer syndromes in men, there still remains an urgent need to revise the WHO recommendations for managing STIs in developing nations. Current studies have continued to show decreased specificity, sensitivity and positive predicative values for the vaginal discharge syndrome, and high rates of asymptomatic infections and healthcare workers neglecting to follow guidelines limit the usefulness of syndromic management. Furthermore, though advocate d as cost-effective by the WHO, there is a cost incurred from treating uninfected people. Instead of improving this system, it is recommended that better and less expensive point of care and the development of rapid test diagnosis kits be the focus and method of diagnosis and treatment in developing nations for STI management. ^

An analysis of the effects of a peer mentor program and other factors on HIV patients' retention in care and clinical outcomes at Thomas Street Health Clinic, Houston Texas

Research examining programs designed to retain patients in health care focus on repeated interactions between outreach workers and patients (Bradford et al. 2007; Cheever 2007). The purpose of this study was to determine if patients who are peer-mentored at their intake exam remain in care longer and attend more physicians' visits than those who were not mentored. Using patients' medical records and a previously created mentor database, the study determined how many patients attended their intake visit but subsequently failed to establish regular care. The cohort study examined risk factors for establishing care, determined if patients lacking a peer mentor failed to establish care more than peer mentor assisted patients, and subsequently if peer mentored patients had better health outcomes. The sample consists of 1639 patients who were entered into the Thomas Street Patient Mentor Database between May 2005 and June 2007. The assignment to the mentored group was haphazardly conducted based on mentor availability. The data from the Mentor Database was then analyzed using descriptive statistical software (SPSS version 15; SPSS Inc., Chicago, Illinois, USA). Results indicated that patients who had a mentor at intake were more likely to return for primary care HIV visits at 90 and 180 days. Mentored patients also were more likely to be prescribed ART within 180 days from intake. Other risk factors that impacted remaining in care included gender, previous care status, time from diagnosis to intake visit, and intravenous drug use. Clinical health outcomes did not differ significantly between groups. This supports that mentoring did improve outcomes. Continuing to use peer-mentoring programs for HIV care may help in increasing retention of patients in care and improving patients' health in a cost effective manner. Future research on the effects of peer mentoring on mentors, and effects of concordance of mentor and patient demographics may help to further improve peer-mentoring programs. ^

Outcomes of tuberculosis positive non-injection drug users in Harris County from October 1995 through September 2004

Background. The population-based Houston Tuberculosis Initiative (HTI) study has enrolled and gathered demographic, social, behavioral, and disease related data on more than 80% of all reported Mycobacterium Tuberculosis (MTB) cases and 90% of all culture positive patients in Houston/Harris County over a 9 year period (from October 1995-September 2004). During this time period 33% (n=1210) of HTI MTB cases have reported a history of drug use. Of those MTB cases reporting a history of drug use, a majority of them (73.6%), are non-injection drug users (NIDUs). ^ Other than HIV, drug use is the single most important risk factor for progression from latent to infectious tuberculosis (TB). In addition, drug use is associated with increased transmission of active TB, as seen by the increased number of clonally related strains or clusters (see definition on page 30) found in this population. The deregulatory effects of drug use on immune function are well documented. Associations between drug use and increased morbidity have been reported since the late 1970's. However, limited research focused on the immunological consequence of non-injection drug use and its relation to tuberculosis infection among TB patients is available. ^ Methods. TB transmission patterns, symptoms, and prevalence of co-morbidities were a focus of this project. Smoking is known to suppress Nitric Oxide (NO) production and interfere with immune function. In order to limit any possible confounding due to smoking two separate analyses were done. Non-injection drug user smokers (NIDU-S) were compared to non-drug user smokers (NDU-S) and non-injection drug user non-smokers (NIDU-NS) were compared to non-drug user non-smokers (NDU-NS) individually. Specifically proportions, chi-square p-values, and (where appropriate) odds ratios with 95% confidence intervals were calculated to assess characteristics and potential associations of co-morbidities and symptoms of TB among NIDUs HTI TB cases. ^ Results. Significant differences in demographic characteristics and risk factors were found. In addition drug users were found to have a decreased risk for cancer, diabetes mellitus, and chronic pulmonary disease. They were at increased risk of having HIV/AIDS diagnosis, liver disease, and trauma related morbidities. Drug users were more likely to have pulmonary TB disease, and a significantly increased amount of clonally related strains of TB or "clusters" were seen in both smokers and non-smoker drug users when compared to their non-drug user counterparts. Drug users are more likely to belong to print groups (clonally related TB strains with matching spoligotypes) including print one and print three and the Beijing family group, s1. Drug users were found to be no more likely to experience drug resistance to TB therapy and were likely to be cured of disease upon completion of therapy. ^ Conclusion. Drug users demographic and behavioral risk factors put them at an increased risk contracting and spreading TB disease throughout the community. Their increased levels of clustering are evidence of recent transmission and the significance of certain print groups among this population indicate the transmission is from within the social family. For these reasons a focus on this "at risk population" is critical to the success of future public health interventions. Successful completion of directly observed therapy (DOT), the tracking of TB outbreaks and incidence through molecular characterization, and increased diagnostic strategies have led to the stabilization of TB incidence in Houston, Harris County over the past 9 years and proven that the Houston Tuberculosis Initiative has played a critical role in the control and prevention of TB transmission. ^

Psychosocial predictors of HIV testing behaviors among high risk heterosexuals

Though a lot of progress has been made in the treatment, prevention, and in increasing the knowledge and awareness of HIV/AIDS, the CDC reports that over 21% of the people infected with HIV are unaware of their HIV serostatus. Thirty-one percent of people infected with HIV are diagnosed late in the disease progression, often too late to prevent the transmission or the progression of HIV to AIDS. CDC has set a goal to increase by the year 2010, the number of people aware of the HIV serostatus by 5%. ^ This study examined the association between decision-making and risk-taking (assessed using the decision-making confidence and risk-taking scales of the Texas Christian University Self Rating Form) and HIV testing behaviors within a population of heterosexuals at risk for HIV infections living in Harris County, Texas (N=923). Data used in the study was obtained during the first cycle of the National HIV Behavioral Surveillance among heterosexuals at risk for HIV infection (NHBS-HET1), conducted from October, 2006 to June, 2007. Eighty percent of the study population reported testing for HIV at some point in their lives. The results showed that individuals who scored high (>3.3) on the decision-making confidence scale of the TCU/SRF were more likely to be tested for HIV when compared to those who scored low on the scale (OR= 2.02, 95% CI= 1.44–2.84), and that individuals who score low on the risk-taking scale of the TCU/SRF were more likely to have been tested for HIV when compared to those who scored high on the scale (OR= 1.65, 95% CI= 1.2–2.31). Several demographic factors were also assessed for their association with HIV testing behaviors. Only sex was found to be associated with HIV testing. ^ The findings suggest that risk-taking and decision-making are predictors of HIV testing behaviors such as prior HIV testing within heterosexuals living in high-risk areas of Houston, Texas, and that intervention designed to improve the risk-taking and decision-making attributes of this population might improve HIV testing within this population.^

HIV-associated malignancies among children attending the Baylor Clinic in Kampala, Uganda

Introduction: HIV-associated malignancies such as Kaposi’s sarcoma and Non-Hodgkin’s lymphoma occur in children and usually lead to significant morbidity and mortality. No studies have been done to establish prevalence and outcome of these malignancies in children in a hospital setting in Uganda. ^ Research question: What proportion of children attending the Baylor-Uganda COE present with HIV-associated malignancies and what are the characteristics and outcome of these malignancies? The objective was to determine the prevalence, associated factors and outcome of HIV-associated malignancies among children attending the Baylor-Uganda Clinic in Kampala, Uganda. Study Design: This was a retrospective case series involving records review of patients who presented to the Baylor-Clinic between January 2004 and December 2008. Study Setting: The Baylor-Uganda Clinic, where I worked as a physician before coming to Houston, is a well funded, well staffed; Pediatric HIV clinic located in Mulago Hospital, Kampala, Uganda and is affiliated to Makerere University Medical School. Study Participants: Medical charts of patients aged 6 weeks to 18 years who enrolled for care at the clinic during the years 2004 to 2008 were retrieved for data abstraction. Selection Criteria: Study participants had to be patients of Baylor-Uganda seen during the study period; they had to be aged 6 weeks to 18 years; and had to be HIV positive. Patients with incomplete data or whose malignancies were not confirmed by histology were excluded. Study Variables: Data on patient’s age, sex, diagnosis, type of malignancy, anatomic location of the malignancy; pathology report, baseline laboratory results and outcome of treatment, were abstracted. Data Analysis: Cross tabulation to determine associations between variables using Pearson’s chi square at 95% level of significance was done. Proportions of malignancies among different groups were determined. In addition, Kaplan Meier survival analysis and comparison of survival distributions using the log-rank test was done. Change in CD4 percentages from baseline was assessed with the Wilcoxon signed rank test. Results: The proportion of children with malignancies during the study period was found to be 1.65%. Only 2 malignancies: Kaposi’s sarcoma and Non-Hodgkin’s lymphoma were found. 90% of the malignancies were Kaposi’s sarcoma. Lymph node involvement in children with Kaposi’s sarcoma was common, but the worst prognosis was seen with visceral involvement. Deaths during follow-up were seen in the first few weeks to months. Upon starting treatment the CD4 cell percentage increased significantly from a baseline median of 6% to 14% at 6 months and 15.8% at 12 months of follow-up.^

Factors governing late-recurrence of disease in a cohort of early-stage breast cancer survivors

Purpose. To determine the risk of late breast cancer recurrence (5 years after treatment) in a population of women diagnosed with early-stage breast cancer at The University of Texas M.D. Anderson Cancer Center (MDACC) between 1985-2000 and to examine the effect of this population’s BMI, smoking history, reproductive history, hormone use, and alcohol intake at the time of diagnosis on risk of late recurrence.^ Methods. Patients included 1,913 members of the Early Stage Breast Cancer Repository recruited at MDACC who had survived without a recurrence for at least five years after their initial diagnosis of early stage breast cancer. Clinical and epidemiological information was ascertained twice on participants during the study—first by medical record abstraction then by patient interview at least five years after receipt of adjuvant treatment. A total of 223 late breast cancer recurrences were captured, with an average follow-up of 10.6 years. Cox proportional hazards models were used to calculate hazard ratios (HR) and 95% confidence intervals (CI). ^

Tuberculosis in HIV-infected individuals in Ho Chi Minh City, Vietnam

Early and accurate detection of TB disease in HIV-infected individuals is a critical step for a successful TB program. In Vietnam, the diagnosis of TB disease, which is based predominantly on the clinical examination, chest radiography (CXR) and acid fast bacilli (AFB) sputum smear, has shown to be of low sensitivity in immunocompromised patients. The sputum culture is not routinely performed for patients with AFB negative smears, even in HIV-infected individuals.^ In that background, we conducted this cross-sectional study to estimate the prevalence of sputum culture-confirmed pulmonary tuberculosis (PTB), smear-negative PTB, and multidrug-resistant TB (MDR-TB) in the HIV-infected population in Ho Chi Minh City (HCMC), the largest city in Vietnam where both TB and HIV are highly prevalent. We also evaluated the diagnostic performance of various algorithms based on routine available tools in Vietnam such as symptoms screening, CXR, and AFB smear. Nearly 400 subjects were consecutively recruited from HIV-infected patients seeking care at the An Hoa Clinic in District 6 of Ho Chi Minh City from August 2009 through June 2010. Participants’ demographic data, clinical status, CXR, and laboratory results were collected. A multiple logistic regression model was developed to assess the association of covariates and PTB. ^ The prevalence of smear-positive TB, smear-negative TB, resistant TB, and MDR-TB were 7%, 2%, 5%, 2.5%, and 0.3%, respectively. Adjusted odds ratios for low CD4+ cell count, positive sputum smear, and CXR to positive sputum culture were 3.17, 32.04, and 4.28, respectively. Clinical findings alone had poor sensitivity, but the combination of CD4+ cell count, sputum smear, and CXR proved to perform a more accurate diagnosis.^ This study results support the routine use of sputum culture to improve the detection of TB disease in HIV-infected individuals in Vietnam. When routine sputum culture is not available, an algorithm combining CD4+ cell count, sputum smear, and CXR is recommended for diagnosing PTB. Future studies on more affordable, rapid, and accurate tests for TB infection would also be necessary to timely provide specific treatments for patients in need, reduce mortality, and minimize TB transmission to the general population.^

Posttranscriptional modification of retroviral primers is required for late stages of DNA replication

During reverse transcription of retroviral RNA, synthesis of (−) strand DNA is primed by a cellular tRNA that anneals to an 18-nt primer binding site within the 5′ long terminal repeat. For (+) strand synthesis using a (−) strand DNA template linked to the tRNA primer, only the first 18 nt of tRNA are replicated to regenerate the primer binding site, creating the (+) strand strong stop DNA intermediate and providing a 3′ terminus capable of strand transfer and further elongation. On model HIV templates that approximate the (−) strand linked to natural modified or synthetic unmodified tRNA3Lys, we find that a (+) strand strong stop intermediate of the proper length is generated only on templates containing the natural, modified tRNA3Lys, suggesting that a posttranscriptional modification provides the termination signal. In the presence of a recipient template, synthesis after strand transfer occurs only from intermediates generated from templates containing modified tRNA3Lys. Reverse transcriptase from Moloney murine leukemia virus and avian myoblastosis virus shows the same requirement for a modified tRNA3Lys template. Because all retroviral tRNA primers contain the same 1-methyl-A58 modification, our results suggest that 1-methyl-A58 is generally required for termination of replication 18 nt into the tRNA sequence, generating the (+) strand intermediate, strand transfer, and subsequent synthesis of the entire (+) strand. The possibility that the host methyl transferase responsible for methylating A58 may provide a target for HIV chemotherapy is discussed.

Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy

Nucleic acid sequence-based amplification (NASBA) has proved to be an ultrasensitive method for HIV-1 diagnosis in plasma even in the primary HIV infection stage. This technique was combined with fluorescence correlation spectroscopy (FCS) which enables online detection of the HIV-1 RNA molecules amplified by NASBA. A fluorescently labeled DNA probe at nanomolar concentration was introduced into the NASBA reaction mixture and hybridizing to a distinct sequence of the amplified RNA molecule. The specific hybridization and extension of this probe during amplification reaction, resulting in an increase of its diffusion time, was monitored online by FCS. As a consequence, after having reached a critical concentration of 0.1–1 nM (threshold for unaided FCS detection), the number of amplified RNA molecules in the further course of reaction could be determined. Evaluation of the hybridization/extension kinetics allowed an estimation of the initial HIV-1 RNA concentration that was present at the beginning of amplification. The value of initial HIV-1 RNA number enables discrimination between positive and false-positive samples (caused for instance by carryover contamination)—this possibility of discrimination is an essential necessity for all diagnostic methods using amplification systems (PCR as well as NASBA). Quantitation of HIV-1 RNA in plasma by combination of NASBA with FCS may also be useful in assessing the efficacy of anti-HIV agents, especially in the early infection stage when standard ELISA antibody tests often display negative results.

Proteasome inhibition interferes with Gag polyprotein processing, release, and maturation of HIV-1 and HIV-2

Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6gag, a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6gag. Consistent with this, viruses with mutations in PR or p6gag were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.

Identification of inflections in T-cell counts among HIV-1-infected individuals and relationship with progression to clinical AIDS

Studies of circulating T (CD3+) lymphocytes have shown that on a population basis T-cell numbers remain stable for many years after HIV-1 infection (blind T-cell homeostasis), but decline rapidly beginning approximately 1.5–2.5 years before the onset of clinical AIDS. We derived a general method for defining the loss of homeostasis on the individual level and for determining the prevalence of homeostasis loss according to HIV status and the occurrence of AIDS in more than 5,000 men enrolled in the Multicenter AIDS Cohort Study. We used a segmented regression model for log10 CD3+ cell counts that included separate T-cell trajectories before and after a time (the T-cell inflection point) where the loss of T-cell homeostasis was most likely to have occurred. The average slope of CD3+ lymphocyte counts before the inflection point was close to zero for HIV− and HIV+ men, consistent with blind T-cell homeostasis. After the inflection point, the HIV+ individuals who developed AIDS generally showed a dramatic decline in CD3+ cell counts relative to HIV− men and HIV+ men not developing AIDS. A CD3+ cell decline of greater than 10 percent per year was present in 77% of HIV+ men developing AIDS but in only 23% of HIV+ men with no onset of AIDS. Our findings at the individual level support the blind T-cell homeostasis hypothesis and provide strong evidence that the loss of homeostasis is an important mechanism in the pathogenesis of the severe immunodeficiency that characterizes the late stages of HIV infection.