Test For Recent Infection Research Articles

Recent HIV Infection Surveillance Using a Rapid Test for Recent Infection among Persons Newly Diagnosed with HIV in Thailand: Establishment, Implementation, Initial outcomes, and Lessons Learned (Preprint)

Recent HIV Infection Surveillance Using a Rapid Test for Recent Infection among Persons Newly Diagnosed with HIV in Thailand: Establishment, Implementation, Initial outcomes, and Lessons Learned (Preprint)

Establishing Quality Assurance for HIV-1 Rapid Test for Recent Infection in Thailand through the Utilization of Dried Tube Specimens.

The present study focuses on establishing the quality assurance of laboratories for recent infections (RTRI) in Thailand. We developed a cold-chain independent method, using fully characterized plasma obtained from the Thai Red Cross Society, and prepared as dried tube specimens (DTS). Twenty microliters of HIV-seronegative, recent, and long-term infected samples were aliquoted into individual tubes and dried at room temperature, 20-30 degrees Celsius, in a biosafety cabinet overnight to ensure optimal preservation. The DTS external quality control and external quality assessment were tested for homogeneity and stability following the ISO/Guide 35 guidelines. The DTS panels were distributed to 48 sites (FY 2022) and 27 sites (FY 2023) across 14 and 9 provinces, respectively, in Thailand. The results from participating laboratories were collected and evaluated for performance. The results were scored, and acceptable performance criteria were defined as the proportion of panels correctly tested, which was set at 100%. The satisfactory performance ranged from 96% to 100% and was not significantly different among the 13 health regions. The developed and implemented DTS panels can be used to monitor the quality of RTRI testing in Thailand.

Continuous quality evaluation of the Asanté rapid test for recent infection for robust kit lot quality verification.

The Sedia Biosciences Asanté rapid test for recent infection (RTRI) can identify HIV infections and characterize HIV-1 as recent or long-term infection via the positive verification (V) line and long-term line (LT) line, respectively. Tracking with Recency Assays to Control the Epidemic (TRACE) program uses RTRI assays. Successful implementation of TRACE requires high-quality test performance. The goal of this study is to evaluate the additional quality practices established for new kit lots prior to field use. Asanté lot quality control data from the manufacturer is reviewed by the Centers for Disease Control and Prevention International Laboratory Branch (CDC-ILB) in the Division of Global HIV and TB using. If a lot passes manufacturer quality control and CDC-ILB review, test kits are sent to CDC-ILB for further evaluation. Evaluation by CDC includes inter-rater reliability and linear regressions comparing the V and LT lines against reference data as well as V and LT line data between testers. A Bland-Altman analysis was conducted to assess bias and systematic error. Overall, CDC-ILB passed 29 (91%) out of 32 Sedia Biosciences Asanté kit lots that initially passed manufacturing quality control from July 2017 to May 2020. Regression analyses demonstrate that test kits are performing as expected with consistent R2≥0.92 for both V and LT lines. On average, inter-rater reliability kappa was 0.9, indicating a strong level of agreement. Bland-Altman analyses demonstrate high agreement with little to no systematic error and bias. Ongoing evaluation of new RTRI kit lots is important to ensure high quality test performance. Rejecting 9% of kit lots highlight the importance of continuing to work with manufacturers to ensure consistent kit production and quality assurance (QA) activities. Investing in effective QA measures, conducting both pre- and post-market performance data reviews, could help improve RTRI accuracy and outcomes in similar testing programs.

HIV-1 Incidence in the Era of Rapid Tests for Recent Infection in Livingstone District, Zambia

HIV-1 Incidence in the Era of Rapid Tests for Recent Infection in Livingstone District, Zambia

Classification of recent and long-term infection among high-risk individuals using HIV-1 rapid recency testing

Background: HIV prevention with a significant reduction of new infections is important for epidemic control strategies. Antibody-based tests for recent infection (TRI) that can help distinguish recent from long-term HIV infection have been used since the mid-1990s to estimate population-level HIV incidence. Because the tests are done centrally, test results are not available to providers immediately. Point of care rapid testing for recent HIV infection (RTRI) was used for distinguishing recent from long-term status and estimated HIV incidence for preventing HIV infection. The usefulness of RTRI for recent HIV infection detection can be accelerated prevention of HIV infection transmission. Objectives: To demonstrate RTRI assay utility to distinguish recent from long-term HIV infection among high-risk individuals. Materials and methods: Between September and October 2022, HIV seropositive plasma samples of a high-risk group (N=90) and HIV-negative group (N=90) were collected from the outpatient department of Khon Kaen Hospital. All Specimens were tested by AsanteTM Rapid Recency Assay (ARRA) and classified as negative, recent and long-term infection based on the presence or absence of specific lines (control line [CL], Positive verification line [PVL] and Long-term line ([TL]) by visual reading. Results: Among 90 HIV seropositive plasma samples, 14.4% (13/90) were recent HIV infection and 84.4% (76/90) were long-term HIV infection. There were no significant differences between recent and long-term infections among men who have sex with men (MSM), transgender women (TG), heterosexuals and sex workers. Most of recently infected HIV patients were the MSM/TG group (53.9%, 7/13) and patients aged between 18 and 25 years old (76.9%, 10/13). CD4 count among recent cases was 396.7±278.4 cells/mm3 . ARRA performance has resulted in a sensitivity of 98.89% (95% CI: 93.96-99.97) and specificity of 100.00% (95% CI: 95.98-100.00). Conclusion: ARRA performance showed excellent agreement with high sensitivity and specificity to comparing with a standard algorithm for HIV diagnosis. Its ability to classify new infections is crucial in HIV intervention and prevention strategies.

A Stable Dried Tube Specimen for Quality Assurance and Training Programs for HIV Rapid Test for Recent Infection.

The HIV epidemic is still one of the world's most serious public health challenges, affecting about 38 million people worldwide, especially in sub-Saharan African and Southeast Asian countries. In recent years, tests have been developed to discriminate recent from long-term infection in HIV-infected populations, and these tools can help identify new outbreaks and networks of transmission and target prevention and treatment plans. New rapid tests for recent infection are being deployed in point-of-care settings; however, quality assurance programs need to be implemented to ensure consistency and reliability of the results. We have developed a dried tube specimen (DTS) stabilized with disaccharide trehalose as a quality control reagent for rapid recency testing that can be stored unrefrigerated prior to reconstitution at temperatures up to 37°C for up to 12 weeks. Analysis of 10 trehalose-stabilized DTSs showed that they maintained the same recency classification in all of the samples stored at 4°C and 37°C up to 12 weeks and at 56°C for 2 weeks, while the DTSs prepared without trehalose changed their classification from long-term to recent or recent to negative after storage at 37°C for 12 weeks. Development of DTS quality control reagents will facilitate proficiency and training programs, particularly in settings without cold chain capability in field environments. IMPORTANCE Implementation of stabilized dried tube specimens (DTSs) for quality control and training would facilitate HIV recency programs, especially in point-of-care settings without cold chain availability. This study shows that addition of the disaccharide trehalose to DTSs prior to drying the samples increased stability of the samples across a range of temperatures. This finding provides an affordable way to increase the availability of these key reagents for quality control in resource-constrained settings.

Characterising persons diagnosed with HIV as either recent or long-term using a cross-sectional analysis of recent infection surveillance data collected in Malawi from September 2019 to March 2020

ObjectivesIn Malawi, a recent infection testing algorithm (RITA) is used to characterise infections of persons newly diagnosed with HIV as recent or long term. This paper shares results from recent...

Performance evaluation of the Asante Rapid Recency Assay for verification of HIV diagnosis and detection of recent HIV-1 infections: Implications for epidemic control.

We previously described development of a rapid test for recent infection (RTRI) that can diagnose HIV infection and detect HIV-1 recent infections in a single device. This technology was transferred to a commercial partner as Asante Rapid Recency Assay (ARRA). We evaluated performance of the ARRA kits in the laboratory using a well-characterized panel of specimens. The plasma specimen panel (N = 1500) included HIV-1 (N = 570), HIV-2 (N = 10), and HIV-negatives (N = 920) representing multiple subtypes and geographic locations. Reference diagnostic data were generated using the Bio-Rad HIV-1-2-O EIA/Western blot algorithm with further serotyping performed using the Multispot HIV-1/2 assay. The LAg-Avidity EIA was used to generate reference data on recent and long-term infection for HIV-1 positive specimens at a normalized optical density (ODn) cutoff of 2.0 corresponding to a mean duration of about 6 months. All specimens were tested with ARRA according to the manufacturer's recommendations. Test strips were also read for line intensities using a reader and results were correlated with visual interpretation. ARRA's positive verification line (PVL) correctly classified 575 of 580 HIV-positive and 910 of 920 negative specimens resulting in a sensitivity of 99.1% (95% CI: 98.0-99.6) and specificity of 98.9% (95% CI: 98.1-99.4), respectively. The reader-based classification was similar for PVL with sensitivity of 99.3% (576/580) and specificity of 98.8% (909/920). ARRA's long-term line (LTL) classified 109 of 565 HIV-1 specimens as recent and 456 as long-term compared to 98 as recent and 467 as long-term (LT) by LAg-Avidity EIA (cutoff ODn = 2.0), suggesting a mean duration of recent infection (MDRI) close to 6 months. Agreement of ARRA with LAg recent cases was 81.6% (80/98) and LT cases was 93.8% (438/467), with an overall agreement of 91.7% (kappa = 0.72). The reader (cutoff 2.9) classified 109/566 specimens as recent infections compared to 99 by the LAg-Avidity EIA for recency agreement of 81.8% (81/99), LT agreement of 9% (439/467) with overall agreement of 91.9% (kappa = 0.72). The agreement between visual interpretation and strip reader was 99.9% (95% CI: 99.6-99.9) for the PVL and 98.1% (95% CI: 96.6-98.9) for the LTL. ARRA performed well with HIV diagnostic sensitivity >99% and specificity >98%. Its ability to identify recent infections is comparable to the LA-Avidity EIA corresponding to an MDRI of about 6 months. This point-of-care assay has implications for real-time surveillance of new infections among newly diagnosed individuals for targeted prevention and interrupting ongoing transmission thus accelerating epidemic control.

Acceptability and feasibility of HIV recent infection surveillance by healthcare workers using a rapid test for recent infection at HIV testing sites — Malawi, 2019

BackgroundThe Malawi Ministry of Health implemented a new surveillance activity in April 2019 to detect recent HIV infections using a rapid test for recent infection (RTRI) to identify areas of ongoing transmission and guide response activities.SettingAt 23 health facilities in Blantyre District, healthcare workers (HCWs) were trained to conduct recent infection testing. In September 2019, we conducted a cross-sectional survey at these sites to explore the acceptability and feasibility of integrating this activity into routine HIV testing services (HTS).MethodsResearch assistants interviewed HCWs using a semi-structured survey. Descriptive statistics were used to summarize quantitative responses and thematic analysis was used to group open-ended text.ResultsWe interviewed 119 HCWs. Eighty-two percent of participants reported the RTRI was easy-to-use. HCWs perceived high client acceptability; 100% reported clients as ‘somewhat’ or ‘very accepting’. Challenges included 68% of HCWs estimating they spend ≥20 min beyond routine HTS per client for this activity and 51% performing at least two additional finger pricks to complete the testing algorithm. HCWs differed in their perceptions of whether results should be returned to clients.ConclusionThis study assessed HCW experiences using point-of-care RTRIs for HIV recent infection surveillance. Overall, HCWs perceived RTRIs to be acceptable, easy-to-use, and valuable. Though only clients with new HIV diagnoses are tested for recent infection, additional time may be substantial at high-volume health service delivery points. Providing response plans or aggregated recent infection results to HCWs and/or clients may support motivation and sustainability of this novel surveillance activity.

Geospatial Transmission Hotspots of Recent HIV Infection - Malawi, October 2019-March 2020.

Persons infected with HIV are more likely to transmit the virus during the early stages (acute and recent) of infection, when viral load is elevated and opportunities to implement risk reduction are limited because persons are typically unaware of their status (1,2). Identifying recent HIV infections (acquired within the preceding 12 months)* is critical to understanding the factors and geographic areas associated with transmission to strengthen program intervention, including treatment and prevention (2). During June 2019, a novel recent infection surveillance initiative was integrated into routine HIV testing services in Malawi, a landlocked country in southeastern Africa with one of the world's highest prevalences of HIV infection.† The objectives of this initiative were to collect data on new HIV diagnoses, characterize the epidemic, and guide public health response (2). New HIV diagnoses were classified as recent infections based on a testing algorithm that included results from the rapid test for recent infection (RTRI)§ and HIV viral load testing (3,4). Among 9,168 persons aged ≥15 years with a new HIV diagnosis who received testing across 103 facilities during October 2019-March 2020, a total of 304 (3.3%) were classified as having a recent infection. Higher proportions of recent infections were detected among females, persons aged <30 years, and clients at maternal and child health and youth clinics. Using a software application that analyzes clustering in spatially referenced data, transmission hotspots were identified with rates of recent infection that were significantly higher than expected. These near real-time HIV surveillance data highlighted locations across Malawi, allowing HIV program stakeholders to assess program gaps and improve access to HIV testing, prevention, and treatment services. Hotspot investigation information could be used to tailor HIV testing, prevention, and treatment to ultimately interrupt transmission.

Performance of a novel rapid test for recent HIV infection among newly-diagnosed pregnant adolescent girls and young women in four high-HIV-prevalence districts-Malawi, 2017-2018.

Tests for recent HIV infection (TRI) distinguish recent from long-term HIV infections using markers of antibody maturation. The limiting antigen avidity enzyme immunoassay (LAg EIA) is widely used with HIV viral load (VL) in a recent infection testing algorithm (RITA) to improve classification of recent infection status, estimate population-level HIV incidence, and monitor trends in HIV transmission. A novel rapid test for recent HIV infection (RTRI), Asanté™, can determine HIV serostatus and HIV recency within minutes on a lateral flow device through visual assessment of test strip or reader device. We conducted a field-based laboratory evaluation of the RTRI among pregnant adolescent girls and young women (AGYW) attending antenatal clinics (ANC) in Malawi.We enrolled pregnant AGYW aged <25 years testing HIV-positive for the first time at their first ANC visit from 121 ANCs in four high-HIV burden districts. Consenting participants provided blood for recency testing using LAg EIA and RTRI, which were tested in central laboratories. Specimens with LAg EIA normalized optical density values ≤2.0 were classified as probable recent infections. RTRI results were based on: (1) visual assessment: presence of a long-term line (LT) indicating non-recent infection and absence of the line indicating recent infection; or (2) a reader; specimens with LT line intensity units <3.0 were classified as probable recent infections. VL was measured for specimens classified as a probable recent infections by either assay; those with HIV-1 RNA ≥1,000 copies/mL were classified as confirmed recent infections. We evaluated the performance of the RTRI by calculating correlation between RTRI and LAg EIA results, and percent agreement and kappa between RTRI and LAg EIA RITA results.Between November 2017 to June 2018, 380 specimens were available for RTRI evaluation; 376 (98.9%) were confirmed HIV-positive on RTRI. Spearman's rho between RTRI and LAg EIA was 0.72 indicating strong correlation. Percent agreement and kappa between RTRI- and LAg EIA-based RITAs were >90% and >0.65 respectively indicating substantial agreement between the RITAs.This was the first field evaluation of an RTRI in sub-Saharan Africa, which demonstrated good performance of the assay and feasibility of integrating RTRI into routine HIV testing services for real-time surveillance of recent HIV infection.

Short Communication: Assessing Estimates of HIV Incidence with a Recent Infection Testing Algorithm That Includes Viral Load Testing and Exposure to Antiretroviral Therapy.

A recent infection testing algorithm (RITA) that includes a test for recent HIV infection and a viral load (VL) test is the recommended strategy to estimate national HIV incidence, reducing false-recent misclassification to <1%. The inclusion of information on exposure to antiretroviral therapy (ART), as a supplement to VL testing, could improve RITA performance by further lowering false-recent misclassification of true long-term infection. In 2012, Kenya and South Africa conducted national population-based surveys that collected information on HIV recency (i.e., HIV antibody seroconversion, on average, in the past 130 days) using the Limiting Antigen avidity (LAg-Avidity) enzyme immunoassay, HIV RNA levels, and ART exposure among HIV-infected respondents aged 15-49 years. In Kenya, ART exposure was defined as testing positive for one or more antiretroviral (ARV) drugs using high-performance liquid chromatography coupled with tandem mass spectrometry, and, if not ARV-positive, self-reporting a history of ART exposure. In South Africa, ART exposure was defined as testing ARV-positive. Two RITA strategies were compared: RITA #1 defined recent infection as testing LAg-Avidity-recent with unsuppressed VL (HIV RNA ≥1,000 copies/ml), and RITA #2 defined recent infection as testing LAg-Avidity-recent with unsuppressed VL and, if unsuppressed, having no ART exposure. RITA-derived incidence among persons aged 15-49 years in Kenya was 0.9% on RITA #1 and 0.8% on RITA #2. In South Africa, RITA-derived incidence was 2.2% on RITA #1 and 1.7% on RITA #2. Among specimens testing recent with unsuppressed VL in Kenya and South Africa, 16.0% and 19.7% had evidence of ART exposure, respectively. Although the performance of a VL- and ART-based RITA was encouraging, additional research is needed across HIV-1 subtypes and subpopulations to calibrate and validate this algorithm.

Short Communication: Defining Optimality of a Test for Recent Infection for HIV Incidence Surveillance

The estimation of HIV incidence from cross-sectional surveys using tests for recent infection has attracted much interest. It is increasingly recognized that the lack of high performance recent infection tests is hindering the implementation of this surveillance approach. With growing funding opportunities, test developers are currently trying to fill this gap. However, there is a lack of consensus and clear guidance for developers on the evaluation and optimization of candidate tests. A fundamental shift from conventional thinking about test performance is needed: away from metrics relevant in typical public health settings where the detection of a condition in individuals is of primary interest (sensitivity, specificity, and predictive values) and toward metrics that are appropriate when estimating a population-level parameter such as incidence (accuracy and precision). The inappropriate use of individual-level diagnostics performance measures could lead to spurious assessments and suboptimal designs of tests for incidence estimation. In some contexts, such as population-level application to HIV incidence, bias of estimates is essentially negligible, and all that remains is the maximization of precision. The maximization of the precision of incidence estimates provides a completely general criterion for test developers to assess and optimize test designs. Summarizing the test dynamics into the properties relevant for incidence estimation, high precision estimates are obtained when (1) the mean duration of recent infection is large, and (2) the false-recent rate is small. The optimal trade-off between these two test properties will produce the highest precision, and therefore the most epidemiologically useful incidence estimates.

Short Communication: HIV Incidence Among Vulnerable Populations in Honduras: Results from an Integrated Behavioral and Biological Survey Among Female Sex Workers, Men Who Have Sex with Men, and Garifuna in Honduras, 2006

Honduras has one of the highest HIV prevalence rates in Central America. Data on HIV incidence are needed to identify groups at greatest need of prevention interventions to inform the national HIV response. We applied a test for recent infection to HIV-positive specimens from a biological and behavioral survey to estimate assay-derived incidence among men who have sex with men (MSM), female sex workers (FSW), and the Garifuna population in Honduras. Assay-derived estimates were compared to the mathematically modeled estimates in the same populations to assess plausibility of the assay-based estimates. Assay-derived incidence was 1.1% (95% CI 0.2-2.0) among MSM, 0.4% (95% CI 0.1-0.8) among the Garifuna, and 0% (95% CI 0-0.01) among FSWs. The modeled incidence estimates were similar at 1.03% among MSM, 0.30% among the Garifuna, and 0.23% among FSWs. HIV incidence based on the assay was highest among MSM in Honduras, lowest among FSWs, and similar to modeled incidence in these groups. Targeted programs on HIV prevention, care, and treatment are urgently needed for the MSM population. Continued support for existing prevention programs for FSWs and Garifuna are recommended.

P8 Tests of recent HIV infection in clinical practice: the patient perspective

BackgroundA test for recent infection (avidity test) is offered for all patients newly diagnosed with HIV in England and Wales as part of an HIV incidence surveillance programme. The UK...

Identification of recent HIV-1 infection among newly diagnosed cases in Catalonia, Spain (2006-08)

Quantification and description of patients recently infected by HIV can provide an accurate estimate of the dynamics of HIV transmission. Between 2006 and 2008 in Catalonia, we estimated the prevalence of recent HIV infection among newly diagnosed cases, described the epidemiological characteristics of the infection according to whether it was recent, long-standing or advanced, and identified factors associated with recent infection. A Test for Recent Infection (TRI) was performed in serum samples from patients newly diagnosed with HIV. Two different TRI were used: the Vironostika-LS assay (January 2006-May 2007) and the BED-CEIA CEIA (June 2007 onwards). Samples were obtained within the first 6 months of diagnosis. Patients whose samples tested positive in the TRI were considered recently infected. Of 1125 newly diagnosed patients, 79.9% were men (median age, 35.4 years), 38.7% were born outside Spain, 48.9% were men who have sex with men (MSM) and 10.6% presented other sexually transmitted infections. The overall percentage of recent infection was 23.0%, which increased significantly, from 18.1% in 2006 to 26.2% in 2008. This percentage was higher for patients from South America (27.6%). Factors associated with recent infection were acquiring infection through sexual contact between MSM [odds ratio (OR) 2.0; 95% confidence interval (95% CI) 1.1-3.9], compared with acquiring infection through heterosexual relations and being under 30 years of age (OR 5.9; 95% CI 1.9-17.4), compared with being over 50 years of age. The highest percentage of recent infection was identified in MSM, suggesting either a higher incidence or a greater frequency of HIV testing. Information regarding testing patterns is necessary to correctly interpret data from recently infected individuals. Systems to monitor the HIV epidemic should include both parameters.

Beyond detuning: 10 years of progress and new challenges in the development and application of assays for HIV incidence estimation

Beyond detuning: 10 years of progress and new challenges in the development and application of assays for HIV incidence estimation

HIV Incidence Estimation Using the BED Capture Enzyme Immunoassay

HIV incidence estimates are essential for understanding the evolution of the HIV epidemic and the impact of interventions. Tests for recent HIV infection allow incidence estimation based on a single cross-sectional survey. The BED IgG-Capture Enzyme Immunoassay (BED assay) is a commercially available and widely used test for recent HIV infection. In a systematic literature search for BED assay studies, we identified 1181 unique studies, 1138 of which were excluded based on titles or abstracts. We conducted reviews of the 43 remaining publications and a further 23 studies identified on conference Web sites or by colleagues. Thirty-nine articles were included in the final review. We investigated the sensitivity of incidence values to various estimation methods and parameter choices. BED assay surveys have been conducted on 5 continents in general populations and high-risk groups, using 1 or more of 10 distinct incidence formulae. Most studies used estimators that do not account for assay imperfection. Those studies that correct for assay imperfection commonly do not use locally valid assay parameters. Incidence estimates were very sensitive to methodological and parameter choices. Most confidence intervals provided good assessment of uncertainty due to counting error, but only a few incorporated parameter uncertainty. BED assay surveys can produce valid HIV incidence estimates, but many studies have not sufficiently accounted for assay imperfection. Future studies should (1) report all information necessary for incidence point and uncertainty estimation, (2) use an unbiased estimator with locally valid assay calibration parameters, and (3) compute confidence intervals that take into account parameter uncertainty.

Using tests for recent infection to estimate incidence: problems and prospects for HIV

Tests for recent infection (TRIs), such as the BED assay, provide a convenient way to estimate HIV incidence rates from cross-sectional survey data. Controversy has arisen over how the imperfect performance of a TRI should be characterised and taken into account. Recent theoretical work is providing a unified framework within which to work with a variety of TRI- and epidemic-specific assumptions in order to estimate incidence using imperfect TRIs, but suggests that larger survey sample sizes will be required than previously thought. This paper reviews the framework qualitatively and provides examples of estimator performance, identifying the characteristics required by a TRI to estimate incidence reliably that should guide the future development of TRIs.

Rethinking the role of the local community in HIV epidemic spread in sub-Saharan Africa: a proximate-determinants approach.

The spread of HIV in sub-Saharan Africa continues largely unabated. To improve prevention interventions, a better understanding of the determinants of HIV infection is required. Conceptual frameworks can guide epidemiological investigation and prevent a misguided focus on single risk factors in isolation. Existing frameworks of HIV infection focus on transmission. However, the transmitting individual is rarely known. By contrast, data on individual HIV acquisition are available from longitudinal studies and tests for recent HIV infection. From the perspective of individuals susceptible to HIV, it is important to distinguish between factors determining the individual's biological disposition and sexual behavior and community-level factors, which can affect both HIV acquisition and the likelihood that a sex partner chosen from a community will be infected with HIV and transmit the infection. We propose a framework that takes the susceptible individual as a starting point and links distal, proximate and biological determinants of HIV infection at both the individual and the community level. We describe three necessary ingredients for the use of the framework (identification of the relevant community, multilevel analysis and methods for causal inference).