Quantitative Assessment of Systematic Bias: A Guide for Researchers.

Observational research provides valuable opportunities to advance oral health science but is limited by vulnerabilities to systematic bias, including unmeasured confounding, errors in variable measurement, or bias in the creation of study populations and/or analytic samples. The potential influence of systematic biases on observed results is often only briefly mentioned among the discussion of limitations of a given study, despite existing methods that support detailed assessments of their potential effects. Quantitative bias analysis is a set of methodological techniques that, when applied to observational data, can provide important context to aid in the interpretation and integration of observational research findings into the broader body of oral health research. Specifically, these methods were developed to provide quantitative estimates of the potential magnitude and direction of the influence of systematic biases on observed results. We aim to encourage and facilitate the broad adoption of quantitative bias analyses into observational oral health research. To this end, we provide an overview of quantitative bias analysis techniques, including a step-by-step implementation guide. We also provide a detailed appendix that guides readers through an applied example using real data obtained from a prospective observational cohort study of preconception periodontitis in relation to time to pregnancy. Quantitative bias analysis methods are available to all investigators. When appropriately applied to observational studies, findings from such studies can have a greater impact in the broader research context.

Leveraging routine data in impact evaluation: Understanding data systems in primary healthcare prior to a matched cluster-randomised evaluation of adherence guidelines in South Africa.

BACKGROUND: An essential part of providing high-quality patient care and a means of efficiently conducting research studies relies upon high-quality routinely collected medical information. OBJECTIVES: To describe the registers, paper records and databases used in a sample of primary healthcare clinics in South Africa (SA) with the view to conduct an impact evaluation using routine data. METHODS: Between October 2015 and December 2015, we collected information on the presence, quality and completeness of registers, clinical stationery and databases at 24 public health facilities in SA. We describe each register and type of clinical stationery we encountered, their primary uses, and the quality of completion. We also mapped the ideal flow of data through a site to better understand how its data collection works. RESULTS: We identified 13 registers (9 standard, 4 non-standard), 5 types of stationery and 4 databases as sources of medical information within a site. Not all clinics used all the standardised registers, and in those that did, registers were kept in various degrees of completeness: a common problem was inconsistent recording of folder numbers. The quality of patient stationery was generally high, with only the chronic patient record being considered of varied quality. The TIER.Net database had high-quality information on key variables, but national identification (ID) number was incompletely captured (42% complete). Very few evaluation sites used electronic data collection systems for conditions other than HIV/AIDS. CONCLUSION: Registers, databases and clinical stationery were not implemented or completed consistently across the 24 evaluation sites. For those considering using routinely collected data for research and evaluation purposes, we would recommend a thorough review of clinic data collection systems for both quality and completeness before considering them to be a reliable data source.

Incidence and predictors of sexually transmitted infections among adult HIV-positive patients receiving antiretroviral therapy at Themba Lethu HIV clinic in Johannesburg, South Africa.

BACKGROUND: Sexually transmitted infections (STIs) among people living with HIV/AIDS can facilitate the spread of HIV. OBJECTIVES: To estimate STI incidence and identify predictors of STI acquisition among HIV-positive patients during their first 24 months of antiretroviral therapy (ART) in Johannesburg, South Africa. METHODS: We conducted a cohort study using prospectively collected routine data on patients who initiated ART between January 2004 and January 2015 at the Themba Lethu HIV clinic in Johannesburg. Kaplan-Meier analysis was used to estimate STI incidence rates (based on evidence of laboratory diagnosis and STI syndromic treatment prescription records). STI predictors were identified using Cox regression analysis. RESULTS: Among 26 762 adult patients on ART, there were 1 906 (7.1%) cases of STI (incidence 4.8/100 person-years; 95% confidence interval (CI) 4.7 - 5.1). Non-pregnant women were 60% more likely than men to be diagnosed with an STI (adjusted hazard ratio (aHR) 1.6; 95% CI 1.4 - 1.8). The risk of STI decreased with increasing baseline CD4 count (aHR 0.8, 0.5 and 0.4 for CD4 counts 101 - 200, 201 - 350 and >350 cells/µL, respectively, compared with CD4 count <100 cells/µL). Patients with advanced baseline World Health Organization (WHO) clinical stages had a higher risk (aHR 1.6 for WHO stage 4; 95% CI 1.3 - 1.9) compared with those with WHO stage 1. However, there was a 20% increase in the risk of STI among obese patients compared with underweight patients (aHR 1.3; 95% CI 1.0 - 1.7). Over 80% of obese patients diagnosed with an STI had a CD4 count <200 cells/µL. CONCLUSIONS: STIs are common in HIV-infected patients who are receiving ART. While both ART and the syndromic management of STIs are high-impact interventions for controlling the spread of HIV, closer monitoring of STI occurrences is warranted, particularly among immunologically vulnerable patients.

Treatment outcomes among HIV-positive orphaned and non-orphaned children on antiretroviral therapy in Johannesburg, South Africa.

BACKGROUND: Limited research investigating treatment outcomes for HIV-positive orphans compared with non-orphans has shown mixed results, with several studies indicating that HIV-positive orphans are at greater risk of delayed access to HIV care and poor antiretroviral therapy (ART) adherence, while other data suggest that ART outcomes of orphans can be similar to those of non-orphans. Understanding the impact of orphan status on short-term ART outcomes could improve targeted intervention strategies, and subsequent long-term treatment and developmental outcomes, for HIV-positive infants, children and adolescents. OBJECTIVES: To evaluate the relationship between orphan status and ART outcomes among HIV-positive infants, children and adolescents initiating ART at two large public sector HIV clinics in Johannesburg, South Africa. METHODS: This was a retrospective cohort study of HIV-positive children aged <18 years initiating standard first-line ART between June 2004 and May 2013. Using propensity scores, orphans and non-orphans were matched for age, sex, World Health Organization stage and ART regimen. The effect of orphanhood on attrition from care (all-cause mortality and loss to follow-up) was evaluated using Cox proportional hazards regression analysis, and its effect on having a detectable viral load (≥400 copies/mL) at 12 months on ART using binomial regression analysis with modified Poisson distribution. RESULTS: A total of 251 (29.4%) orphans (maternal, paternal or both) and 603 (70.6%) non-orphans were included at ART initiation. Following multiple imputation for missing data and propensity score matching, 222 orphans and 222 non-orphans were included. Orphans had a median age of 8.0 years (interquartile range (IQR) 4.9 - 10.7) and non-orphans 7.4 years (IQR 4.2 - 10.2). A total of 12 (5.4%) orphans and 33 (14.9%) non-orphans experienced attrition from care during the first 12 months on ART (adjusted hazard ratio 0.32, 95% confidence interval (CI) 0.17 - 0.63). Among those alive and in care, with a viral load at 12 months on ART, 18.0% of orphans (33/183) and 14.8% of non-orphans (24/162) had a detectable viral load (adjusted risk ratio 1.15, 95% CI 1.04 - 1.28). CONCLUSIONS: Orphans were less likely than non-orphans to experience attrition, but among those in care at 12 months, orphans were more likely to have detectable viral loads. Lower attrition among orphans may be due to their being in institutional or foster care, ensuring that they make their visits; however, their higher rates of non-suppression may result from lack of psychosocial support or stigma resulting in struggles to adhere. Additional research investigating age-specific outcomes will be important to elucidate these effects further.

"Even if you're HIV-positive there's life after if you take your medication": experiences of people on long-term ART in South Africa: a short report.

South Africa's national antiretroviral treatment (ART) programme, initiated in 2004, is the largest HIV treatment programme in the world with an estimated 4.2 million people on ART. Today, an HIV diagnosis is no longer associated with certain death, but is rather a manageable chronic disease, with all HIV-positive patients now eligible to receive treatment. In this study, we explore patient experiences at the onset of the ART programme, including facilitators and barriers around decision-making along the HIV care cascade (HIV testing, ART initiation, retention, and adherence). We conducted twenty-four in-depth interviews among adults (≥18 years old) who initiated ART between April 2004 and March 2005 and were alive, on treatment at enrolment (October 2015-March 2016) at a large public-sector clinic in Johannesburg, South Africa. Data were analysed using a thematic analysis approach. Patients cited physical wellbeing, responsibility for raising children, supportive clinic staff and noticeable improvements in health on ART as key facilitators to continued care. In contrast, changing clinic conditions, fear of side-effects and stigma were mentioned as barriers. This study provides a unique lens through which to evaluate factors associated with long-term retention and adherence to ART at a crucial time in ART programming when more people will be initiating life-long treatment. We must continue to focus on supportive and empathetic clinic environments, more convenient ways to access medication for patients, and developing tools or interventions that continue to address the issues of stigma and discrimination and build the support networks for all those on treatment.

Treatment outcomes among HIV-positive orphaned and non-orphaned children on antiretroviral therapy in Johannesburg, South Africa

Background. Limited research investigating treatment outcomes for HIV-positive orphans compared with non-orphans has shown mixed results, with several studies indicating that HIV-positive orphans are at greater risk of delayed access to HIV care and poor antiretroviral therapy (ART) adherence, while other data suggest that ART outcomes of orphans can be similar to those of non-orphans. Understanding the impact of orphan status on short-term ART outcomes could improve targeted intervention strategies, and subsequent long-term treatment and developmental outcomes, for HIV-positive infants, children and adolescents.Objectives. To evaluate the relationship between orphan status and ART outcomes among HIV-positive infants, children and adolescents initiating ART at two large public sector HIV clinics in Johannesburg, South Africa.Methods. This was a retrospective cohort study of HIV-positive children aged <18 years initiating standard first-line ART between June 2004 and May 2013. Using propensity scores, orphans and non-orphans were matched for age, sex, World Health Organization stage and ART regimen. The effect of orphanhood on attrition from care (all-cause mortality and loss to follow-up) was evaluated using Cox proportional hazards regression analysis, and its effect on having a detectable viral load (≥400 copies/mL) at 12 months on ART using binomial regression analysis with modified Poisson distribution.Results. A total of 251 (29.4%) orphans (maternal, paternal or both) and 603 (70.6%) non-orphans were included at ART initiation. Following multiple imputation for missing data and propensity score matching, 222 orphans and 222 non-orphans were included. Orphans had a median age of 8.0 years (interquartile range (IQR) 4.9 - 10.7) and non-orphans 7.4 years (IQR 4.2 - 10.2). A total of 12 (5.4%) orphans and 33 (14.9%) non-orphans experienced attrition from care during the first 12 months on ART (adjusted hazard ratio 0.32, 95% confidence interval (CI) 0.17 - 0.63). Among those alive and in care, with a viral load at 12 months on ART, 18.0% of orphans (33/183) and 14.8% of non-orphans (24/162) had a detectable viral load (adjusted risk ratio 1.15, 95% CI 1.04 - 1.28).Conclusions. Orphans were less likely than non-orphans to experience attrition, but among those in care at 12 months, orphans were more likely to have detectable viral loads. Lower attrition among orphans may be due to their being in institutional or foster care, ensuring that they make their visits; however, their higher rates of non-suppression may result from lack of psychosocial support or stigma resulting in struggles to adhere. Additional research investigating age-specific outcomes will be important to elucidate these effects further

Can routine inpatient mortality data improve HIV mortality estimates? Inpatient mortality at an urban hospital in South Africa.

BACKGROUND: South Africa (SA) has one of the world's largest HIV treatment programmes, to which a dramatic increase in life expectancy has been attributed. However, there continue to be concerns regarding the reporting of HIV-related mortality in SA, which varies by source. As accurate HIV mortality estimates are key to measuring the success of the national programme as well as identifying areas for improvement, we propose a complementary approach to monitoring changes in HIV-related mortality using routine inpatient records to examine trends in causes of death and HIV status over time. OBJECTIVES: To investigate the feasibility of this approach by calculating mortality due to natural causes in the medical ward of a hospital during 2010 by HIV status. METHODS: We conducted a cross-sectional study of inpatient mortality at a regional hospital in Johannesburg, SA, analysing all deaths due to natural causes among adult medical ward inpatients. Cause of death was recorded from the mortuary register. HIV status was ascertained directly from the mortuary register or from laboratory tests specific for HIV diagnosis or monitoring. RESULTS: Of 1 167 inpatients who died, the majority were HIV-positive (58%). HIV positivity among males (55%) was slightly lower than that among females (61%), and HIV-positive patients were younger (median 40 years) than those who were HIV-negative (56 years) and of unknown HIV status (68 years). 'Infections and parasites' was the most common cause of natural death (29%). On average, HIV-positive patients were admitted for slightly longer (mean 10.5 days) than HIV-negative patients (9.6 days) and those of unknown HIV status (8.9 days), yet HIV-positive inpatient deaths accounted for the majority (62%) of the total bed days. CONCLUSIONS: Even with widespread access to antiretroviral therapy, the majority of inpatient natural deaths at a large public sector hospital in 2010 were of HIV-positive patients and were probably related to HIV. In view of the importance of accurate data on causes of death, both for the HIV programme and to track other diseases, large-scale expansion of this approach over a longer period should be considered.

Improved simplified clinical algorithm for identifying patients eligible for immediate initiation of antiretroviral therapy for HIV (SLATE II): protocol for a randomized evaluation.

BACKGROUND: The World Health Organization recommends rapid (≤ 7 days) or same-day initiation of antiretroviral treatment (ART) for HIV-positive patients. South Africa adopted this recommendation in 2017, but multiple clinic visits, long waiting times, and delays for laboratory tests remain common. Streamlined approaches to same-day initiation that allow the majority of patients to start ART immediately, while ensuring that patients who do require additional services receive them, are needed to achieve national and international treatment program goals. METHODS/DESIGN: The SLATE II (Simplified Algorithm for Treatment Eligibility) study is an individually randomized evaluation of a clinical algorithm to reliably determine a patient's eligibility for immediate ART initiation without waiting for laboratory results or additional clinic visits. It differs from the earlier SLATE I study in management of patients with symptoms of tuberculosis (under SLATE II these patients may be started on ART immediately) and other criteria for immediate initiation. SLATE II will randomize (1:1) 600 adult, HIV-positive patients who present for HIV testing or care and are not yet on ART in South Africa. Patients randomized to the standard arm will receive standard-of-care ART initiation from clinic staff. Patients randomized to the intervention arm will be administered a symptom report, medical history, brief physical exam, and readiness assessment. Symptomatic patients will also have a tuberculosis (TB) module with lipoarabinomannan antigen of mycobacteria test. Patients who have satisfactory results for all four components will be dispensed antiretrovirals (ARVs) immediately, at the same clinic visit. Patients who have any negative results will be referred for further investigation, care, counseling, tests, or other services prior to being dispensed ARVs. Follow-up will be by passive medical record review. The primary outcomes will be ART initiation in  ≤ 7 days and retention in care 8 months after study enrollment. DISCUSSION: SLATE II improves upon the SLATE I study by reducing the number of reasons for delaying ART initiation and allowing more patients with TB symptoms to start ART on the day of diagnosis. If successful, SLATE II will provide a simple and streamlined approach that can readily be adopted in other settings without investment in additional technology. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03315013 . Registered on 19 October 2017.

Can routine inpatient mortality data improve HIV mortality estimates? Inpatient mortality at an urban hospital in South Africa

Background. South Africa (SA) has one of the world's largest HIV treatment programmes, to which a dramatic increase in life expectancy has been attributed. However, there continue to be concerns regarding the reporting of HIV-related mortality in SA, which varies by source. As accurate HIV mortality estimates are key to measuring the success of the national programme as well as identifying areas for improvement, we propose a complementary approach to monitoring changes in HIV-related mortality using routine inpatient records to examine trends in causes of death and HIV status over time.Objectives. To investigate the feasibility of this approach by calculating mortality due to natural causes in the medical ward of a hospital during 2010 by HIV status.Methods. We conducted a cross-sectional study of inpatient mortality at a regional hospital in Johannesburg, SA, analysing all deaths due to natural causes among adult medical ward inpatients. Cause of death was recorded from the mortuary register. HIV status was ascertained directly from the mortuary register or from laboratory tests specific for HIV diagnosis or monitoring.Results. Of 1 167 inpatients who died, the majority were HIV-positive (58%). HIV positivity among males (55%) was slightly lower than that among females (61%), and HIV-positive patients were younger (median 40 years) than those who were HIV-negative (56 years) and of unknown HIV status (68 years). 'Infections and parasites' was the most common cause of natural death (29%). On average, HIV-positive patients were admitted for slightly longer (mean 10.5 days) than HIV-negative patients (9.6 days) and those of unknown HIV status (8.9 days), yet HIV-positive inpatient deaths accounted for the majority (62%) of the total bed days.Conclusions. Even with widespread access to antiretroviral therapy, the majority of inpatient natural deaths at a large public sector hospital in 2010 were of HIV-positive patients and were probably related to HIV. In view of the importance of accurate data on causes of death, both for the HIV programme and to track other diseases, large-scale expansion of this approach over a longer period should be considered

Changes in elevated cholesterol in the era of tenofovir in South Africa: risk factors, clinical management and outcomes.

OBJECTIVES: Antiretroviral therapy (ART) has been associated with unfavourable lipid profile changes and increased risk of cardiovascular disease (CVD). With a growing population on ART in South Africa, there has been concern about the increase in noncommunicable diseases such as CVD. We determined risk factors associated with increased total cholesterol (TC) in a large cohort on ART and describe the clinical management thereof. METHODS: We conducted an observational cohort study of ART-naïve adults initiating standard first-line ART in a large urban clinic in Johannesburg, South Africa. TC was measured annually for most patients. A proportional hazards regression model was used to determine risk factors associated with incident high TC (≥ 6 mmol/L). RESULTS: Significant risk factors included initial regimen non-tenofovir vs. tenofovir [hazard ratio (HR) 1.54; 95% confidence interval (CI) 1.14-2.08], age ≥40 vs. <30 years (HR 3.22; 95% CI 2.07-4.99), body mass index (BMI) ≥ 30 kg/m2 (HR 1.65; 95% CI 1.18-2.31) and BMI 25-29.9 kg/m2 (HR 1.70; 95% CI 1.30-2.23) vs. 18-24.9 kg/m2 , and baseline CD4 count < 50 cells/µL (HR 1.55; 95% CI 1.10-2.20) and 50-99 cells/µL (HR 1.40; 95% CI 1.00-1.97) vs. > 200 cells/µL. Two-thirds of patients with high TC were given cholesterol-lowering drugs, after repeat TC measurements about 12 months apart, while 31.8% were likely to have received dietary counselling only. CONCLUSIONS: Older age, higher BMI, lower CD4 count and a non-tenofovir regimen were risk factors for incident elevated TC. Current guidelines do not indicate regular cholesterol testing at ART clinic visits, which are the main exposure to regular clinical monitoring for most HIV-positive individuals. If regular cholesterol monitoring is conducted, improvements can be made to identify and treat patients sooner.

Incident tuberculosis in HIV-positive children, adolescents and adults on antiretroviral therapy in South Africa.

OBJECTIVE: To evaluate the association between age and incident tuberculosis (TB) among human immunodeficiency virus (HIV) infected patients receiving antiretroviral treatment (ART) in South Africa. DESIGN: Prospective cohort analysis among HIV-infected patients initiating ART between April 2004 and April 2012. Generalized estimating equations (GEE) were used with modified Poisson regression clustered by treatment site as a function of sex, age, nucleoside reverse transcriptase inhibitor, CD4 count, hemoglobin levels and year of ART initiation. Cumulative incidence functions stratified by age and controlling for death as a competing risk were used to graphically display incident TB. RESULTS: Although non-significant, GEE models showed that patients aged <1 year had a 40% increase in risk of TB compared to those aged 30-39.9 years. Results also showed that male patients, those with low CD4, those with low hemoglobin and those who initiated ART before 2010 were at increased risk of TB. CONCLUSIONS: Our results show that patients aged <1 year, males, patients with low CD4 and those with low hemoglobin at ART initiation are at increased risk of incident TB in the first 24 months of ART. Given the known transmission risk factors for children living in households with a TB contact, reducing TB incidence in HIV-positive adults could substantially impact the risk of TB in young children.

Clinician Compliance with Laboratory Monitoring and Prescribing Guidelines in HIV-1-Infected Patients Receiving Tenofovir

BACKGROUND:Tenofovir is part of the preferred first-line regimen for HIV-infected patients in South Africa (SA); but is associated with kidney toxicity. SA antiretroviral therapy (ART) guidelines recommend creatinine monitoring at baseline (ART start) and at 3; 6 and 12 months; and substituting tenofovir with zidovudine; stavudine or abacavir should creatinine clearance (CrCl) decrease to etlt;50 mL/min. OBJECTIVE:To assess clinician compliance with tenofovir monitoring and prescribing guidelines.METHODS:We described the proportion of adult patients on tenofovir-based first-line ART who were screened for baseline renal impairment; were monitored according to the SA antiretroviral treatment guidelines; and were switched from tenofovir if renal function declined.RESULTS:We included 13 168 patients who started ART from 2010 to 2012. Creatinine concentrations were recorded in 11 712 (88.9%) patients on tenofovir at baseline; 9 135/11 657 (78.4%) at 3 months; 5 426/10 554 (51.4%) at 6 months; and 5 949/ 8 421 (70.6%) at 12 months. At baseline; 227 (1.9%) started tenofovir despite a CrCl etlt;50 mL/min. While on tenofovir; 525 patients had at least one CrCl of etlt;50 mL/min. Of 382 patients with =3 months' follow-up after a CrCl etlt;50 mL/min; 114 (29.8%) stopped tenofovir within 3 months. Clinicians were more likely to stop tenofovir in patients with lower CrCl and CD4 count. Of 226 patients who continued to receive tenofovir and had further CrCls available; 156 (69.0%) had a CrCl =50 mL/min at their next visit.CONCLUSIONS:Creatinine monitoring is feasible where access to laboratory services is good. Kidney function recovered in most patients who continued to receive tenofovir despite a CrCl etlt;50 mL/min. Further research is needed to determine how best to monitor renal function with tenofovir in resource-limited settings

The early effects of stavudine compared with tenofovir on adipocyte gene expression, mitochondrial DNA copy number and metabolic parameters in South African HIV-infected patients: a randomized trial.

OBJECTIVES: Stavudine is being phased out because of its mitochondrial toxicity and tenofovir (TDF) is recommended as part of first-line highly active antiretroviral therapy (HAART) in South Africa. A prospective, open-label, randomized controlled trial comparing standard- and low-dose stavudine with TDF was performed to assess early differences in adipocyte mtDNA copy number, gene expression and metabolic parameters in Black South African HIV-infected patients. METHODS: Sixty patients were randomized 1:1:1 to either standard-dose (30-40 mg) or low-dose (20-30 mg) stavudine or TDF (300 mg) each combined with lamivudine and efavirenz. Subcutaneous fat biopsies were obtained at weeks 0 and 4. Adipocyte mtDNA copies/cell and gene expression were measured using quantitative polymerase chain reaction (qPCR). Markers of inflammation and lipid and glucose metabolism were also assessed. RESULTS: A 29% and 32% decrease in the mean mtDNA copies/cell was noted in the standard-dose (P < 0.05) and low-dose stavudine (P < 0.005) arms, respectively, when compared with TDF at 4 weeks. Nuclear respiratory factor-1 (NRF1) and mitochondrial cytochrome B (MTCYB) gene expression levels were affected by stavudine, with a significantly (P < 0.05) greater fall in expression observed with the standard, but not the low dose compared with TDF. No significant differences were observed in markers of inflammation and lipid and glucose metabolism. CONCLUSIONS: These results demonstrate early mitochondrial depletion among Black South African patients receiving low and standard doses of stavudine, with preservation of gene expression levels, except for NRF1 and MTCYB, when compared with patients on TDF.

Tuberculosis in HIV programmes in lower-income countries: practices and risk factors.

BACKGROUND: Tuberculosis (TB) is a common diagnosis in human immunodeficiency virus (HIV) infected patients on antiretroviral treatment (ART). OBJECTIVE: To describe TB-related practices in ART programmes in lower-income countries and identify risk factors for TB in the first year of ART. METHODS: Programme characteristics were assessed using standardised electronic questionnaire. Patient data from 2003 to 2008 were analysed and incidence rate ratios (IRRs) calculated using Poisson regression models. RESULTS: Fifteen ART programmes in 12 countries in Africa, South America and Asia were included. Chest X-ray, sputum microscopy and culture were available free of charge in respectively 13 (86.7%), 14 (93.3%) and eight (53.3%) programmes. Eight sites (53.3%) used directly observed treatment and five (33.3%) routinely administered isoniazid preventive treatment (IPT). A total of 19 413 patients aged ≥ 16 years contributed 13,227 person-years of follow-up; 1081 new TB events were diagnosed. Risk factors included CD4 cell count (>350 cells/µl vs. <25 cells/µl, adjusted IRR 0.46, 95%CI 0.33-0.64, P < 0.0001), sex (women vs. men, adjusted IRR 0.77, 95%CI 0.68-0.88, P = 0.0001) and use of IPT (IRR 0.24, 95%CI 0.19-0.31, P < 0.0001). CONCLUSIONS: Diagnostic capacity and practices vary widely across ART programmes. IPT prevented TB, but was used in few programmes. More efforts are needed to reduce the burden of TB in HIV co-infected patients in lower income countries.

Impact of human immunodeficiency virus infection on Streptococcus pneumoniae colonization and seroepidemiology among Zambian women.

Nasopharyngeal colonization with Streptococcus pneumoniae precedes invasive pneumococcal disease. Human immunodeficiency virus (HIV) infection increases rates of invasive pneumococcal disease, and its effect on colonization is unknown. In a longitudinal cohort of Zambian mothers with or without HIV infection, HIV infection increased the risk of colonization (risk ratio [RR], 1.9; 95% confidence interval [CI], 1.3-2.8) and repeat colonization (RR, 2.4; 95% CI, 1.1-5.3) and reduced the time to new colonization (P = .01). Repeat colonization with homologous sero/factor types occurred only among HIV-positive mothers. Pediatric serotypes 6, 19, and 23 accounted for excess colonization among HIV-positive mothers. HIV infection significantly increases the risk of pneumococcal colonization. Increased rates of colonization by pediatric serotypes suggest a potential role for the 7-valent pneumococcal vaccine in HIV-infected adults.

Effect of presumptive co-trimoxazole prophylaxis on pneumococcal colonization rates, seroepidemiology and antibiotic resistance in Zambian infants: a longitudinal cohort study.

OBJECTIVE: To ascertain the microbiological consequences of WHO's recommendation for presumptive co-trimoxazole prophylaxis for infants with perinatal HIV exposure. METHODS: Using a longitudinal cohort design, we followed HIV-exposed and HIV-unexposed infants trimonthly for up to 18 months per infant. HIV-exposed infants received daily co-trimoxazole prophylaxis from 6 weeks to > or = 12 months of age. Using Streptococcus pneumoniae as our sentinel pathogen, we measured how co-trimoxazole altered nasopharyngeal colonization, pneumococcal resistance to antibiotics and serotype distribution as a function of co-trimoxazole exposure. FINDINGS: From 260 infants followed for 3096 patient-months, we detected pneumococci in 360/1394 (25.8%) samples. HIV-exposed infants were colonized more frequently than HIV-unexposed infants (risk ratio, RR: 1.4; 95% confidence interval, CI: 1.0-1.9, P = 0.04). Co-trimoxazole prophylaxis reduced colonization by ca 7% but increased the risk of colonization with co-trimoxazole-resistant pneumococci within 6 weeks of starting prophylaxis (RR: 3.2; 95% CI: 1.3-7.8, P = 0.04). Prophylaxis with co-trimoxazole led to a small but statistically significant increase of nasopharyngeal colonization with pneumococci not susceptible to clindamycin (RR: 1.6; 95% CI: 1.0-2.6, P = 0.04) but did not increase the risk of non-susceptibility to penicillin (RR: 1.1; 95% CI: 0.7-1.7), erythromycin (RR: 1.0; 95% CI: 0.6-1.7), tetracycline (RR: 0.9; 95% CI: 0.6-1.5) or chloramphenicol (RR: 0.8; 95% CI: 0.3-2.3). Co-trimoxazole prophylaxis did not cause the prevailing pneumococcal serotypes to differ from those that are targeted by the 7-valent conjugate pneumococcal vaccine (RR: 1.0; 95% CI: 0.7-1.6). CONCLUSION: Co-trimoxazole prophylaxis modestly suppresses pneumococcal colonization but accelerates infant acquisition of co-trimoxazole- and clindamycin-resistant pneumococci. Co-trimoxazole prophylaxis appears unlikely to compromise the future efficacy of conjugate vaccines.

Identification of candidate genes in ulcerative colitis and Crohn's disease using cDNA array technology.

Inflammatory bowel disease (IBD) follows a multigenic mode of inheritance, encompassing the clinically discrete phenotypes of ulcerative colitis (UC) and Crohn's disease (CD). The risk of malignant transformation of the colon increases with the duration and extent of IBD and is particularly high for patients with a longstanding history of UC. We wished to identify candidate genes that might be involved in disease pathogenesis based on functional plausibility and their putative role in IBD carcinogenesis. Polyadenylated mRNA (PolyA+ mRNA) preparation from inflamed intestinal mucosa of patients with a longstanding history of UC and CD was performed with subsequent hybridization of alpha phosphorus [alpha-32P]-deoxyadenotriphosphate-labeled complementary deoxyribonucleic acid (DNA) populations to nucleic acid arrays. Of 588 different human gene transcripts arrayed, secreted apoptosis-related protein 1 (Sarp1), frizzled (fz) homologues, and disheveled (dvl) were differentially expressed, being elevated in UC as compared to CD. These genes encode proteins involved in the Wingless-type (Wnt)/beta-catenin signaling pathway. The autonomous expression of Sarp1 and Sarp1-compatible fz receptor genes suggests that the Wnt pathway may be involved in UC carcinogenesis.

Effect of the ratio of free to total prostate-specific antigen on interassay variability in proficiency test samples.

BACKGROUND: Up to sevenfold differences were observed between total prostate-specific antigen (PSA) methods for New York State Proficiency Test samples prepared with seminal fluid PSA in human female serum. Because the PSA was mainly in its free form under these conditions, we wanted to determine whether a defined mixture of free and complexed PSA would reduce the interassay differences. METHODS: We prepared a series of five solutions of 60 g/L bovine serum albumin with 10 microgram/L total PSA consisting of varied proportions of free, noncomplexible PSA, and alpha(1)-antichymotrypsin (ACT)-complexed PSA from 0% to 100%. Two hundred seventy laboratories measured the total PSA in these samples, and 16 laboratories also analyzed the samples for free PSA. The results were used to calculate free/total PSA ratios. RESULTS: Interassay CVs for total PSA measurements were approximately 7% at 10-15% free PSA but became gradually larger as the free/total PSA ratio increased. Measured free-PSA concentrations were similar within each sample (mean CV, 12%), and the results were relatively independent of the proportion of free PSA in the samples. Twofold discrepancies between actual and expected ratios were observed with some methods at 100% free PSA and to a lesser degree at 30% free PSA. At 100% free PSA, the relatively higher total-PSA values measured by nonequimolar methods yielded low free/total PSA ratios of 50-60%. In contrast, the lower total PSA values obtained by equimolar methods yielded ratios close to the expected 100%. CONCLUSIONS: Preparing proficiency test samples with a 10:90 mixture of free, noncomplexible PSA:PSA-ACT is a viable alternative to the use of seminal fluid PSA. Furthermore, the method used to measure total PSA may have a substantial impact on the calculated proportion of free PSA and hence may have clinical relevance.

Cyclin E, a redundant cyclin in breast cancer.

Cyclin E is an important regulator of cell cycle progression that together with cyclin-dependent kinase (cdk) 2 is crucial for the G1/S transition during the mammalian cell cycle. Previously, we showed that severe overexpression of cyclin E protein in tumor cells and tissues results in the appearance of lower molecular weight isoforms of cyclin E, which together with cdk2 can form a kinase complex active throughout the cell cycle. In this study, we report that one of the substrates of this constitutively active cyclin E/cdk2 complex is retinoblastoma susceptibility gene product (pRb) in populations of breast cancer cells and tissues that also overexpress p16. In these tumor cells and tissues, we show that the expression of p16 and pRb is not mutually exclusive. Overexpression of p16 in these cells results in sequestering of cdk4 and cdk6, rendering cyclin D1/cdk complexes inactive. However, pRb appears to be phosphorylated throughout the cell cycle following an initial lag, revealing a time course similar to phosphorylation of glutathione S-transferase retinoblastoma by cyclin E immunoprecipitates prepared from these synchronized cells. Hence, cyclin E kinase complexes can function redundantly and replace the loss of cyclin D-dependent kinase complexes that functionally inactivate pRb. In addition, the constitutively overexpressed cyclin E is also the predominant cyclin found in p107/E2F complexes throughout the tumor, but not the normal, cell cycle. These observations suggest that overexpression of cyclin E in tumor cells, which also overexpress p16, can bypass the cyclin D/cdk4-cdk6/p16/pRb feedback loop, providing yet another mechanism by which tumors can gain a growth advantage.

Deregulation of cyclin E in breast cancer.

Cyclin E, a regulatory subunit of cyclin dependent kinase-2, is thought to be rate limiting for the G1/S transition during the mammalian cell cycle. Previously, we showed severe alterations in cyclin E protein expression in human mammary epithelial cell lines and in surgical material obtained from patients with various malignancies. To understand the functional basis of these alterations we analyse here the regulation of cyclin E in breast cancer cells. We find that while cyclin E protein and its associated kinase activity in normal cells are cell cycle regulated, in tumor cells it remains in an active complex throughout the cell cycle. We also analysed cyclin E for possible deletions which could result in its constitutive function and found two novel truncated variants in its coding region. These variant forms of cyclin E were detected in several normal and tumor cell lines and tissue specimens. However, Western blot analysis indicated that only the multiple isoforms of cyclin E protein were expressed in tumor but not the normal tissue specimen, suggesting post transcriptional regulation of cyclin E. Lastly, in vitro analyses indicated that these truncated variant forms of cyclin E are biochemically active in their ability to phosphorylate histone H1. Collectively these observations suggest the presence of more than one form of cyclin E mRNA in all cells, normal and tumor. Once translated in tumor cells, the protein products of these truncated forms could give rise to a constitutively active form of cyclin E containing complexes.