Optimising DTwP-containing vaccine infant immunisation schedules (OptImms) - a protocol for two parallel, open-label, randomised controlled trials.

BACKGROUND: Universal immunisation is the cornerstone of preventive medicine for children, The World Health Organisation (WHO) recommends diphtheria-tetanus-pertussis (DTP) vaccine administered at 6, 10 and 14 weeks of age as part of routine immunisation. However, globally, more than 17 unique DTP-containing vaccine schedules are in use. New vaccines for other diseases continue to be introduced into the infant immunisation schedule, resulting in an increasingly crowded schedule. The OptImms trial will assess whether antibody titres against pertussis and other antigens in childhood can be maintained whilst adjusting the current Expanded Programme on Immunisation (EPI) schedule to provide space for the introduction of new vaccines. METHODS: The OptImms studies are two randomised, five-arm, non-inferiority clinical trials in Nepal and Uganda. Infants aged 6 weeks will be randomised to one of five primary vaccination schedules based on age at first DTwP-vaccination (6 versus 8 weeks of age), number of doses in the DTwP priming series (two versus three), and spacing of priming series vaccinations (4 versus 8 weeks). Additionally, participants will be randomised to receive their DTwP booster at 9 or 12 months of age. A further sub-study will compare the co-administration of typhoid vaccine with other routine vaccines at one year of age. The primary outcome is anti-pertussis toxin IgG antibodies measured at the time of the booster dose. Secondary outcomes include antibodies against other vaccine antigens in the primary schedule and their safety. DISCUSSION: These data will provide key data to inform policy decisions on streamlining vaccination schedules in childhood. TRIAL REGISTRATIONS: ISRCTN12240140 (Nepa1, 7th January 2021) and ISRCTN6036654 (Uganda, 17th February 2021).

Priority Activities in Child and Adolescent Tuberculosis to Close the Policy-Practice Gap in Low- and Middle-Income Countries.

Over the past 15 years, and despite many difficulties, significant progress has been made to advance child and adolescent tuberculosis (TB) care. Despite increasing availability of safe and effective treatment and prevention options, TB remains a global health priority as a major cause of child and adolescent morbidity and mortality-over one and a half million children and adolescents develop TB each year. A history of the global public health perspective on child and adolescent TB is followed by 12 narratives detailing challenges and progress in 19 TB endemic low and middle-income countries. Overarching challenges include: under-detection and under-reporting of child and adolescent TB; poor implementation and reporting of contact investigation and TB preventive treatment services; the need for health systems strengthening to deliver effective, decentralized services; and lack of integration between TB programs and child health services. The COVID-19 pandemic has had a significant negative impact on case detection and treatment outcomes. Child and adolescent TB working groups can address country-specific challenges to close the policy-practice gaps by developing and supporting decentral ized models of care, strengthening clinical and laboratory diagnosis, including of multidrug-resistant TB, providing recommended options for treatment of disease and infection, and forging strong collaborations across relevant health sectors.

A qualitative study of the development and utilization of health facility-based immunization microplans in Uganda.

BACKGROUND: In 2006, Uganda adopted the Reaching Every District strategy with the goal of attaining at least 80% coverage for routine immunizations in every district. The development and utilization of health facility/district immunization microplans is the key to the strategy. A number of reports have shown suboptimal development and use of microplans in Uganda. This study explores factors associated with suboptimal development and use of microplans in two districts in Uganda to pinpoint challenges encountered during the microplanning process. METHODS: A qualitative study was conducted comparing two districts: Kapchorwa, with low immunization coverage, and Luwero with high immunization coverage. Data were collected through multilevel observation of health facilities, planning sessions and planning meetings; records review of microplans, micromaps and meeting minutes; 57 interviews with health workers at the ministry level and lower-level health facility workers. Data were analysed using NVivo 8 qualitative text analysis software. Transcripts were coded, and memos and display matrices were developed to examine the process of developing and utilizing microplans, including experiences of health workers (implementers). RESULTS: Three key findings emerged from this study. First, there are significant knowledge gaps with regard to the microplanning process among health workers at all levels (community and district health facility and nationally). Limited knowledge about communities and programme catchment areas greatly hinders the planning process by limiting the ability to identify hard-to-reach areas and to prioritize areas according to need. Secondly, the microplanning tool is bulky and complex. Finally, microplanning is being implemented in the context of already overtasked health personnel who have to conduct several other activities as part of their daily routines. CONCLUSIONS: In order to achieve quality improvement as outlined in the Reaching Every District campaign, the microplanning process should be revised. Health workers' misunderstanding and limited knowledge about the microplanning process, especially at peripheral health facilities, coupled with the complex, bulky nature of the microplanning tool, reduces the effectiveness of microplanning in improving routine immunization in Uganda. This study reveals the need to reduce the complexity of the tool and to identify ways to train and support workers in the use of the revised tool, including support in incorporating the microplanning process into their busy schedules.

Evaluating a Mobile Phone-Delivered Text Message Reminder Intervention to Reduce Infant Vaccination Dropout in Arua, Uganda: Protocol for a Randomized Controlled Trial.

BACKGROUND: Globally, suboptimal vaccine coverage is a public health concern. According to Uganda's 2016 Demographic and Health Survey, only 49% of 12- to 23-month-old children received all recommended vaccinations by 12 months of age. Innovative ways are needed to increase coverage, reduce dropout, and increase awareness among caregivers to bring children for timely vaccination. OBJECTIVE: This study evaluates a personalized, automated caregiver mobile phone-delivered text message reminder intervention to reduce the proportion of children who start but do not complete the vaccination series for children aged 12 months and younger in select health facilities in Arua district. METHODS: A two-arm, multicenter, parallel group randomized controlled trial was conducted in four health facilities providing vaccination services in and around the town of Arua. Caregivers of children between 6 weeks and 6 months of age at the time of their first dose of pentavalent vaccine (Penta1; containing diphtheria, tetanus, pertussis, hepatitis B, and Haemophilus influenzae type b antigens) were recruited and interviewed. All participants received the standard of care, defined as the health worker providing child vaccination home-based records to caregivers as available and providing verbal instruction of when to return for the next visit. At the end of each day, caregivers and their children were randomized by computer either to receive or not receive personalized, automated text message reminders for their subsequent vaccination visits according to the national schedule. Text message reminders for Penta2 were sent 2 days before, on the day of, and 2 days after the scheduled vaccination visit. Reminders for Penta3 and the measles-containing vaccine were sent on the scheduled day of vaccination and 5 and 7 days after the scheduled day. Study personnel conducted postintervention follow-up interviews with participants at the health facilities during the children's measles-containing vaccine visit. In addition, focus group discussions were conducted to assess caregiver acceptability of the intervention, economic data were collected to evaluate the incremental costs and cost-effectiveness of the intervention, and health facility record review forms were completed to capture service delivery process indicators. RESULTS: Of the 3485 screened participants, 1961 were enrolled from a sample size of 1962. Enrollment concluded in August 2016. Follow-up interviews of study participants, including data extraction from the children's vaccination cards, data extraction from the health facility immunization registers, completion of the health facility record review forms, and focus group discussions were completed by December 2017. The results are expected to be released in 2021. CONCLUSIONS: Prompting health-seeking behavior with reminders has been shown to improve health intervention uptake. Mobile phone ownership continues to grow in Uganda, so their use in vaccination interventions such as this study is logical and should be evaluated with scientifically rigorous study designs. TRIAL REGISTRATION: ClinicalTrials.gov NCT04177485; https://clinicaltrials.gov/ct2/show/NCT04177485. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/17262.

Population differences in vaccine responses (POPVAC): scientific rationale and cross-cutting analyses for three linked, randomised controlled trials assessing the role, reversibility and mediators of immunomodulation by chronic infections in the tropics.

INTRODUCTION: Vaccine-specific immune responses vary between populations and are often impaired in low income, rural settings. Drivers of these differences are not fully elucidated, hampering identification of strategies for optimising vaccine effectiveness. We hypothesise that urban-rural (and regional and international) differences in vaccine responses are mediated to an important extent by differential exposure to chronic infections, particularly parasitic infections. METHODS AND ANALYSIS: Three related trials sharing core elements of study design and procedures (allowing comparison of outcomes across the trials) will test the effects of (1) individually randomised intervention against schistosomiasis (trial A) and malaria (trial B), and (2) Bacillus Calmette-Guérin (BCG) revaccination (trial C), on a common set of vaccine responses. We will enrol adolescents from Ugandan schools in rural high-schistosomiasis (trial A) and rural high-malaria (trial B) settings and from an established urban birth cohort (trial C). All participants will receive BCG on day '0'; yellow fever, oral typhoid and human papilloma virus (HPV) vaccines at week 4; and HPV and tetanus/diphtheria booster vaccine at week 28. Primary outcomes are BCG-specific IFN-γ responses (8 weeks after BCG) and for other vaccines, antibody responses to key vaccine antigens at 4 weeks after immunisation. Secondary analyses will determine effects of interventions on correlates of protective immunity, vaccine response waning, priming versus boosting immunisations, and parasite infection status and intensity. Overarching analyses will compare outcomes between the three trial settings. Sample archives will offer opportunities for exploratory evaluation of the role of immunological and 'trans-kingdom' mediators in parasite modulation of vaccine-specific responses. ETHICS AND DISSEMINATION: Ethics approval has been obtained from relevant Ugandan and UK ethics committees. Results will be shared with Uganda Ministry of Health, relevant district councils, community leaders and study participants. Further dissemination will be done through conference proceedings and publications. TRIAL REGISTRATION NUMBERS: ISRCTN60517191, ISRCTN62041885, ISRCTN10482904.

Financial cost analysis of a strategy to improve the quality of administrative vaccination data in Uganda.

BACKGROUND: High-quality vaccination data are critical to planning, implementation and evaluation of immunization programs. However, sub-optimal administrative vaccination data quality in low- and middle-income countries persist for heterogeneous reasons, though most relate to organizational factors and human behavior. The nationwide Data Improvement Team (DIT) strategy in Uganda aimed to strengthen human resource capacity to generate quality administrative vaccination data at the health facility. METHODS: A financial cost analysis of the Uganda DIT strategy (2014-2016) was conducted from the program funder perspective. Activity-based micro-costing from funder financial and program monitoring records was used to estimate total and unit costs by program area (in 2016 US dollars). Hypothetical scenarios were developed to illustrate potential approaches to reducing costs. RESULTS: Over 25 months the DIT strategy was implemented in all 116 operational districts and 3443 (89%) health facilities in Uganda at a total financial cost of US $575 275. Training and deployment of DITs accounted for the highest proportion of expenditure across program areas (69%). Transport, per diems, lodging, and honoraria for DIT members and national supervisors were the main cost drivers of the strategy. Deployment of 557 DIT members cost US $839 per DIT member, US $4 030 per district, and US $136 per health facility. The estimated opportunity cost of government staff time wasn't a major cost driver (2.5%) of total cost. CONCLUSION: The results provide the first estimates of the magnitude and drivers of cost to implement a national workforce capacity building strategy to improve administrative vaccination data quality in a low- or middle-income country. Financial costs are a critical input to combine with future outcome data to describe the cost of strategies relative to performance outcomes. The operational costs of the strategy were modest (0.5-1.6%) relative to the estimated operational costs of Uganda's national immunization program.

Comparing static and outreach immunization strategies and associated factors in Uganda, Nov-Dec 2016.

INTRODUCTION: the government of Uganda aims at reducing childhood morbidity through provision of immunization services. We compared the proportion of children 12-33 months reached using either static or outreach immunization strategies and factors affecting utilization of routine vaccination services in order to inform policy updates. METHODS: we adopted the 2015 vaccination coverage cluster survey technique. The sample selection was based on a stratified three-stage sample design. Using the Fleiss formula, a sample of 50 enumeration areas was sufficient to generate immunization coverages at each region. A total of 200 enumeration areas were selected for the survey. Thirty households were selected per enumeration area. Epi-Info software was used to calculate weighted coverage estimates. facility. RESULTS: among the 2231 vaccinated children aged 12-23 months who participated in the survey, 68.1% received immunization services from a health unit and 10.6% from outreaches. The factors that affected utilization of routine vaccination services were; accessibility, where 78.2% resided within 5km from a health. 29.7% missed vaccination due to lack of vaccines at the health facility. Other reasons were lack of supplies at 39.2% and because the caretaker had other things to do, 26.4%. The survey showed 1.8% (40/2271) respondents had not vaccinated their children. Among these, 70% said they had not vaccinated their child because they were busy doing other things and 27.5% had not done so because of lack of motivation. CONCLUSION: almost 7 in 10 children aged 12-23 months access vaccination at health facilities. There is evidence of parental apathy as well as misconceptions about vaccination.

Building health workforce capacity for planning and monitoring through the Strengthening Technical Assistance for routine immunization training (START) approach in Uganda.

INTRODUCTION: The Global Vaccine Action Plan identifies workforce capacity building as a key strategy to achieve strong immunization programs. The Strengthening Technical Assistance for Routine Immunization Training (START) approach aimed to utilize practical training methods to build capacity of district and health center staff to implement routine immunization (RI) planning and monitoring activities, as well as build supportive supervision skills of district staff. METHODS: First implemented in Uganda, the START approach was executed by trained external consultants who used existing tools, resources, and experiences to mentor district-level counterparts and, with them, conducted on-the-job training and mentorship of health center staff over several site visits. Implementation was routinely monitored using daily activity reports, pre and post surveys of resources and systems at districts and health centers and interviews with START consultants. RESULTS: From July 2013 through December 2014 three START teams of four consultants per team, worked 6 months each across 50 districts in Uganda including the five divisions of Kampala district (45% of all districts). They conducted on-the-job training in 444 selected under-performing health centers, with a median of two visits to each (range 1-7, IQR: 1-3). More than half of these visits were conducted in collaboration with the district immunization officer, providing the opportunity for mentorship of district immunization officers. Changes in staff motivation and awareness of challenges; availability and completion of RI planning and monitoring tools and systems were observed. However, the START consultants felt that potential durability of these changes may be limited by contextual factors, including external accountability, availability of resources, and individual staff attitude. CONCLUSIONS: Mentoring and on-the-job training offer promising alternatives to traditional classroom training and audit-focused supervision for building health workforce capacity. Further evidence regarding comparative effectiveness of these strategies and durability of observed positive change is needed.

Factors that affect immunization data quality in Kabarole District, Uganda.

INTRODUCTION: Reliable and timely immunization data is vital at all levels of health care to inform decisions and improve program performance. Inadequate data quality may impair our understanding of the true vaccination coverage and also hinder our capability to meet the program objectives. It's therefore important to regularly assess immunization data quality to ensure good performance, sound decision making and efficient use of resources. METHODS: We conducted an immunization data quality audit between July and August 2016. The verification factor was estimated by dividing the recounted diphtheria, pertussis and tetanus third dose vaccination for children under 1 year (DPT3<1 year) by reported DPT3<1 year. The quality of data collection processes was measured using quality indices for the 3 different components: recording practices, storage/reporting, monitoring and evaluation. These indices were applied to the different levels of the health care service delivery system. Quality index score was estimated by dividing the total question or observation correctly answered by the total number of answers/ observations for a particular component. RESULTS: The mean health center verification factor was 87%. Sixty five percent (32/49) of the health centers had consistent data, 27% (13/49) over reported and 4% (2/49) under-reported. Health center 11s and 111s contributed to over-reporting and under-reporting. All the health centers' reports were complete and timely between January and June and from November to December. The mean quality indices for the 3 different componets assessed were; recording practices 66%, storing/reporting 75%, monitoring and evaluation 43%. There was a weak positive correlation between the health center verifaction factor and quality index though this was not statistically significant (r = 0.014; p = 0.92). CONCLUSION: Lower level health centers contributed significantly to the inconsistencies in immunization data; there were wide variation between the quality indices of recording practices, storage/reporting, monitoring and evaluation. We recommended that District Local Governments and Ministry of Health focus on improving data quality at lower levels of health service delivery.

Enhancing Workforce Capacity to Improve Vaccination Data Quality, Uganda.

In Uganda, vaccine dose administration data are often not available or are of insufficient quality to optimally plan, monitor, and evaluate program performance. A collaboration of partners aimed to address these key issues by deploying data improvement teams (DITs) to improve data collection, management, analysis, and use in district health offices and health facilities. During November 2014-September 2016, DITs visited all districts and 89% of health facilities in Uganda. DITs identified gaps in awareness and processes, assessed accuracy of data, and provided on-the-job training to strengthen systems and improve healthcare workers' knowledge and skills in data quality. Inaccurate data were observed primarily at the health facility level. Improvements in data management and collection practices were observed, although routine follow-up and accountability will be needed to sustain change. The DIT strategy offers a useful approach to enhancing the quality of health data.

Positive predictive value and effectiveness of measles case-based surveillance in Uganda, 2012-2015.

INTRODUCTION: Disease surveillance is a critical component in the control and elimination of vaccine preventable diseases. The Uganda National Expanded Program on Immunization strives to have a sensitive surveillance system within the Integrated Disease Surveillance and Response (IDSR) framework. We analyzed measles surveillance data to determine the effectiveness of the measles case-based surveillance system and estimate its positive predictive value in order to inform policy and practice. METHODS: An IDSR alert was defined as ≥1 suspected measles case reported by a district in a week, through the electronic Health Management Information System. We defined an alert in the measles case-based surveillance system (CBS) as ≥1 suspected measles case with a blood sample collected for confirmation during the corresponding week in a particular district. Effectiveness of CBS was defined as having ≥80% of IDSR alerts with a blood sample collected for laboratory confirmation. Positive predictive value was defined as the proportion of measles case-patients who also had a positive measles serological result (IgM +). We reviewed case-based surveillance data with laboratory confirmation and measles surveillance data from the electronic Health Management Information System from 2012-2015. RESULTS: A total of 6,974 suspected measles case-persons were investigated by the measles case-based surveillance between 2012 and 2015. Of these, 943 (14%) were measles specific IgM positive. The median age of measles case-persons between 2013 and 2015 was 4.0 years. Between 2013 and 2015, 72% of the IDSR alerts reported in the electronic Health Management Information System, had blood samples collected for laboratory confirmation. This was however less than the WHO recommended standard of ≥80%. The PPV of CBS between 2013 and 2015 was 8.6%. CONCLUSION: In conclusion, the effectiveness of measles case-based surveillance was sub-optimal, while the PPV showed that true measles cases have significantly reduced in Uganda. We recommended strengthening of case-based surveillance to ensure that all suspected measles cases have blood samples collected for laboratory confirmation to improve detection and ensure elimination by 2020.

Understanding the policy environment for immunization supply chains: Lessons learned from landscape analyses in Uganda and Senegal.

As immunization programs around the world undergo rapid change and expansion, supply chain and logistics systems have become strained, making it increasingly challenging for national public health systems to provide reliable, safe, and efficient access to vaccines. Governments and immunization partners have been aware of this problem for several years, and in 2010, the World Health Organization (WHO) launched the Effective Vaccine Management (EVM) process to help countries identify shortcomings in their immunization supply chains and develop plans for systematic improvement. EVM improvement plans now exist in all Gavi-eligible countries plus many middle- and upper-income countries; however, implementation has been slow and in many cases fraught with financial, managerial, structural, and political roadblocks. Recognizing that significant change of any kind requires a supportive policy environment and strong leadership, PATH began working in Uganda and Senegal to landscape the policy environment around immunization and identify relevant policies, administrative and technical roles and responsibilities, and other issues that may be affecting the supply chain for immunization. The policy landscape assessments included a desk review and a series of structured, in-depth interviews with key international, national, and local stakeholders. The findings highlighted a number of critical issues and challenges in both countries that may be preventing supply chains from functioning optimally. These challenges include a need for better coordination and planning between immunization programs and supply chain managers; the need for sufficient, timely and reliable financing for all aspects of immunization programs; the need for high-level managers trained in immunization supply chain management; and an urgent need for better, more timely data for decision-making. Overcoming these challenges will require the involvement of high-level political actors-including ministers of health and finance, parliamentarians, and other officials who have the ability to approve and influence policy, personnel, and structural changes; ensure work plans are backed with adequate resources for implementation; and hold program managers accountable for achieving agreed indicators.

Influence of efavirenz pharmacokinetics and pharmacogenetics on neuropsychological disorders in Ugandan HIV-positive patients with or without tuberculosis: a prospective cohort study.

BACKGROUND: HIV infection, anti-tuberculosis and efavirenz therapy are associated with neuropsychological effects. We evaluated the influence of rifampicin cotreatment, efavirenz pharmacokinetics and pharmacogenetics on neuropsychiatric disorders in Ugandan HIV patients with or without tuberculosis coinfection. METHODS: 197 treatment naïve Ugandan HIV patients, of whom 138 were TB co-infected, enrolled prospectively and received efavirenz based HAART. TB-HIV confected patients received concomitant rifampicin based anti-TB therapy. Genotypes for CYP2B6 (*6, *11), CYP3A5 (*3, *6, *7), ABCB1 (c.3435C>T and c.4036 A/G rs3842), CYP2A6 (*9, *17) and NR1I3 rs3003596 T/C were determined. Efavirenz plasma concentrations were serially quantified at 3rd day, 1st, 2nd, 4th, 6th, 8th and 12th weeks during therapy. Efavirenz neuropsychiatric symptoms were evaluated in terms of sleep disorders, hallucinations and cognitive effects at baseline, at two and twelve weeks of efavirenz treatment using a modified Mini Mental State Examination (MMSE) score. RESULTS: During the first twelve weeks of ART, 73.6% of the patients experienced at least one efavirenz related neuropsychiatric symptom. Commonest symptoms experienced were sleep disorders 60.5% (n=124) and hallucination 30.7% (n=63). Neuropsychiatric symptoms during HAART were significantly predicted by efavirenz plasma concentrations consistently. Rifampicin cotreatment reduced plasma efavirenz concentrations significantly only during the first week but not afterwards. There was no significant difference in the incidence of neuropsychiatric symptoms between patients receiving efavirenz with or without rifampicin cotreatment. CYP2B6*6 and ABCB1 c.4036 A/G genotype significantly predicted efavirenz concentrations. The tendency of CYP2B6*6 genotype association with higher incidence of having vivid dream (p=0.05), insomnia (p=0.19) and tactile hallucination (p=0.09) was observed mainly at week-2. CONCLUSIONS: Efavirenz related neuropsychiatric symptoms are common among Ugandan HIV patients receiving ART and is mainly predicted by higher efavirenz plasma concentrations and CYP2B6 genotype but not by rifampicin based anti-TB co-treatment.

A randomized, double-blind, placebo-controlled trial of the use of prednisolone as an adjunct to treatment in HIV-1-associated pleural tuberculosis.

BACKGROUND: Active tuberculosis may accelerate progression of human immunodeficiency virus (HIV) infection by promoting viral replication in activated lymphocytes. Glucocorticoids are used in pleural tuberculosis to reduce inflammation-induced pathology, and their use also might reduce progression of HIV by suppressing immune activation. We examined the effect that prednisolone has on survival in HIV-1-associated pleural tuberculosis. METHODS: We conducted a randomized, double-blind, placebo-controlled trial of prednisolone as an adjunct to tuberculosis treatment, in adults with HIV-1-associated pleural tuberculosis. The primary outcome was death. Analysis was by intention to treat. RESULTS: Of 197 participants, 99 were assigned to the prednisolone group and 98 to the placebo group. The mortality rate was 21 deaths/100 person-years (pyr) in the prednisolone group and 25 deaths/100 pyr in the placebo group (age-, sex-, and initial CD4+ T cell count-adjusted mortality rate ratio, 0.99 [95% confidence interval, 0.62-1.56] [P =.95]). Resolution of tuberculosis was faster in the prednisolone group, but recurrence rates were slightly (though not significantly) higher, and use of prednisolone was associated with a significantly higher incidence of Kaposi sarcoma (4.2 cases/100 pyr, compared with 0 cases/100 pyr [P =.02]). CONCLUSIONS: In view of the lack of survival benefit and the increased risk of Kaposi sarcoma, the use of prednisolone in HIV-associated tuberculous pleurisy is not recommended.

Human immunodeficiency virus type 1 (HIV-1) quasispecies at the sites of Mycobacterium tuberculosis infection contribute to systemic HIV-1 heterogeneity.

We have recently reported an increased heterogeneity in the human immunodeficiency virus type 1 (HIV-1) envelope gene (env) in HIV-1-infected patients with pulmonary tuberculosis (TB) compared to patients with HIV-1 alone. This increase may be a result of dissemination of lung-derived HIV-1 isolates from sites of Mycobacterium tuberculosis infection and/or the systemic activation of the immune system in response to TB. To distinguish between these two mechanisms, blood and pleural fluid samples were obtained from HIV-1-infected patients with active pleural TB in Kampala, Uganda (CD4 cell counts of 34 to 705 cells/microl, HIV-1 plasma loads of 2,400 to 280,000 RNA copies/ml, and HIV-1 pleural loads of 7,600 to 4,500,000 RNA copies/ml). The C2-C3 coding region of HIV-1 env was PCR amplified from lysed peripheral blood mononuclear cells and pleural fluid mononuclear cells and reverse transcriptase-PCR amplified from plasma and pleural fluid HIV-1 virions of eight HIV-1 patients with pleural TB. Phylogenetic and phenetic analyses revealed a compartmentalization of HIV-1 quasispecies between blood and pleural space in four of eight patients, with migration events between the compartments. There was a trend for a greater genetic heterogeneity in the pleural space, which may be the result of an M. tuberculosis-mediated increase in HIV-1 replication and/or selection pressure at the site of infection. Collectively, these findings suggest that HIV-1 quasispecies in the M. tuberculosis-infected pleural space may leak into the systemic circulation and lead to increased systemic HIV-1 heterogeneity during TB.