Central American countries were among the first in the world to implement HIV rapid recency testing and recognize its potential for improved surveillance and public health response.  While HIV testing and counseling (HTC) is offered to all contacts of people recently diagnosed with HIV, recency assays allow healthcare workers to identify and prioritize for testing the sexual partners of HIV-diagnosed patients who were infected in the past 12 months, when the likelihood of transmission is high. PEPFAR, CDC, and implementing partner Universidad del Valle de Guatemala (UVG) organized a regional workshop in October 2017 to introduce recency testing to stakeholders whose understanding and adoption of this new strategy was needed for successful regional implementation, including National AIDS Programs, clinicians, laboratorians, and community-based organizations (CBOs).  In light of Central America’s concentrated epidemic, engaging with the men who have sex with men (MSM) community was of crucial importance.  We approached this component of our recency testing introduction strategy using a two-pronged framework: 1) community consultations before implementation, and 2) client surveys during early implementation.  

Community Consultations

During the first phase of planning and stakeholder engagement, we recruited champions from MSM CBOs who understood the public health benefits of recency testing in their communities and could facilitate the process of implementation. CDC and UVG convened meetings with MSM CBOs to introduce recency testing as an additional and innovative component of the tailored HIV combination prevention package available for key populations. During these consultations, CDC and UVG provided background information and explained the significance of recency testing. Community partners raised their concerns, especially regarding confidentiality.  The team shared the process for recency testing, consent, and results delivery; the opportunity for prioritized index testing (i.e., the testing of exposed contacts) among people with recent infection; and procedures for HIV rapid recency data collection, analysis, and expected actions.  

These initial meetings led to increased interest from CBOs in recency testing, the establishment of partnerships for recency testing service promotion and data use, and the development of joint recency testing/index testing promotional materials and FAQs.  In Guatemala, leading MSM CBO Colectivo Amigos contra el Sida (CAS) led an initiative to develop a demand creation strategy for recency testing across its social media outlets (see Figure 1).   In El Salvador, PEPFAR/CDC and the National AIDS Program organized a follow-up half-day workshop with MSM organizations to address community concerns about the legal framework around recency testing and index testing, including confidentiality, and potential intimate partner violence.  PEPFAR and MoH representatives addressed these concerns by sharing client-centered guidance from the recency standard operating procedures, confidentiality measures taken at the site level, and highlighting that a local ethics committee reviewed and cleared the national recency protocol. As an outcome of this workshop, MSM CBOs embraced recency testing and became enthusiastic advocates for its use in El Salvador.

Having established an ongoing collaboration between Ministries of Health, PEPFAR/CDC, and MSM CBOs enabled productive and efficient community consultations regarding this new initiative.  It would have been much more difficult to build trust between the CBOs and the MoH if rapid recency testing was the first attempt of partnership.  It was also helpful for CDC and UVG to clearly link rapid recency testing to programmatic action that could improve health outcomes in the MSM community, i.e., prioritization of partners of people with recent infection for index testing. 

Client surveys

Secondly, the team conducted surveys among health care workers and MSM community members to gain insights on acceptability and preferences related to recency testing, index testing, and assisted partner notification.  A total of 393 MSM seeking HIV testing completed the survey at facilities serving key populations in Guatemala, Honduras, and Nicaragua.  Over 90% of them agreed that, in case of a positive HIV diagnosis, it would be important for them to know if their infection was recent and to refer their sexual partners for testing.  Key motivators for recency testing and index testing acceptance were the perception of rigorous service confidentiality, the opportunity to provide early HIV treatment for their partners, and to prevent new HIV infections in the MSM community.  A participant shared, “rapid recency testing enhances the service package at the clinic and its results help improve awareness of prevention options for me and my partners.” Clinicians and laboratory workers stated that recency did not significantly increase their workload.  They also overwhelmingly supported the return of recency results to users.

Many participants also expressed they would accept recency testing in order to identify which sexual partner transmitted the infection to them.  This finding was critical as it allowed the team to update recency counseling scripts to clarify that recency results, by themselves, do not indicate direction of HIV transmission.  Users expressed that they would not accept recency if they felt that their results would not be shared in a confidential manner. 

With these significant milestones and successes, next steps for HIV rapid recency testing in Central America include:

1. Incorporating this strategy into national testing algorithms;
2. Adding it to the regional list of approved tests in integrated regional purchase mechanisms;
3. Scaling up partner testing of recent index cases; and
4. Developing a protocol to identify and respond to clusters of recent infection.  

Community engagement, and the ownership and sustainability it brings along, will be a critical component in successfully achieving each of these steps. 

University of California, San Francisco (UCSF)

Recency testing using the limiting antigen (LAg) avidity assay was one of several new breakthroughs in HIV control discussed at the 10th International AIDS Society Conference on HIV Science, which was held in Mexico City from Sunday, July 21, 2019 to Wednesday, July 24, 2019. The conference kicked off with a satellite session sponsored by TRACE partners UCSF and ICAP.

The satellite session included opening remarks by Ambassador Deborah Birx where she shared her vision for expanding recency testing in routine settings to all PEPFAR countries. She emphasized the urgency to understanding the epidemic and responding in real-time in order to achieve our goals of epidemic control. Check out the video of her opening remarks on the TRACE eLearning hub here. The session continued with presentations sharing experiences from Rwanda, Ethiopia, Malawi, and Vietnam, all countries currently implementing recency testing. The last hour of the session was a panel discussion on using recency data to benefit PLHIV, ethical considerations, and program implications.

The session was moderated by Andrea Kim, one of the TRACE co-leads from CDC. The panel was comprised of Amalia Girón from Universidad de la Valle, Guatemala where they have been doing recency testing and returning results for over a year; Rachel Baggaley, who is working on WHO guidance regarding the use of and ethical implications of recency testing; Gregorio Millett from AmfAR, who represented the views of the community; Wame Mosime from ITPC, who brought perspectives from grass roots community organizations; and Jerome Singh, an ethicist from University of KwaZulu-Natal who brought the perspective of ethical and legal implications. The panel discussed the use cases of recency results at the individual, surveillance, and program levels and additional scientific evidence that is needed to understand the benefits/risks of returning individual results. Finally, the panel discussed how to engage the PLHIV in rolling out recency testing. The panel discussion will soon be posted on the TRACE eLearning hub as well.

In addition to the panel discussion, there were multiple abstracts, symposia, and oral presentations which dealt with incident infection, partner notification, investigation of HIV clusters, and genotypic linkage of newly diagnosed cases. There was a relatively modest number that focused specifically on point-of-care testing for recent HIV infection using LAg avidity testing.

Point-of-care LAg avidity testing was featured in two presentations, one a poster presentation from the MeSH Consortium at the London School of Hygiene and Tropical Medicine[1] and another oral presentation from the Vietnam AIDS Authority Council (VAAC)[2] in an oral abstract session entitled “Interrupting transmission using new tools.”

In the first of these, Risher and colleagues reported on data from a study conducted among pregnant women attending antenatal clinics (ANC) in western Kenya.¹ The investigators compared incidence estimates in adolescent girls and women ≥13 years old attending one of 14 ANC derived from LAg avidity testing with incidence estimates from women who could be matched to serial seroprevalence surveys from the Health and Demographic Surveillance Site (HDSS) that covers the Gem district. HDSS data are often viewed as the gold standard for population-based estimates. Seropositive ANC patients were tested recent infection using the Maxim HIV-1 LAg Avidity EIA® and the recent infection testing algorithm (RITA) method (both viral load testing and matching clinic records for antiretroviral therapy); for DSS participants, incidence was based on known seroconversions. Using LAg avidity testing and RITA, of 426 women with newly diagnosed HIV infection, 10 (2.3%) were found to have recent infection (Figure 1). This equated to an incidence in this specific population of 1.1 per 100 person years. Among ANC patients who  participated in the HDSS, there were 25 seroconversions in 1,877 person years of follow up and an estimated incidence of 1.3 per 100 person years. Thus, the results obtained from LAg avidity testing and directly observed seroconversions in the same population were very similar. Additionally, the investigators reported that women in their first trimester of pregnancy has 9.6 times the adjusted odds of testing positive for recent infection compared to women in their second and third trimesters. The investigators concluded that (1) the acceptance rate for recency testing was high, (2) HIV incidence estimates using LAg and RITA were similar to those obtained from linked seroconversions at the local HDSS site, and (3) LAg testing with RITA is a promising avenue for ANC surveillance, where the population at risk can be clearly defined.

In the Vietnamese study, ² Vu and colleagues reported results from a cohort study of men who have sex with men (MSM) living in Hanoi and recruited through time-location sampling. Inclusion criteria were being a Vietnamese national, male at birth, ≥16 years old, having lived in Hanoi ≥3 months, and having had oral or anal sex with another man or transgendered women in the past 12 months. Baseline specimens were tested for HIV infection using three third- and fourth-generation tests serially, then specimens testing positive for HIV on both were tested for viral load. Then those with viral load <1,000 copies/mL were tested for recent infection using the Sedia Asanté HIV-1 Rapid Recency® assay. Note, that this is the reverse of the usual algorithm where viral load testing is done only for those with positive recency assays. They derived incidence estimates using SACEMA R inctools package (http://www.incidence-estimation.org/) using a mean duration of recent infection of 161 days and a false positive rate of 0%. Of the 800 men in the cohort, 80 (10.9% when weighted for study design) tested positive for HIV infection, and 75 continued with further testing. Of these, 27 (36%) had confirmed recent infection (Figure 2). This corresponded to an annual incidence of 5.8 per 100 person years (0.8-10.6 per 100 person years). In multivariate analysis, men who had been recently infected had higher adjusted odds than men who had been infected more than one year previously to be low income, to not have been exposed to chemsex, and to not have syphilis. The prevalence and incidence found in this study are substantially higher than results based on the Asian Epidemic Model (4.8% and 0.36 per 100 person years). The investigators will extend recency screening to the other two arms of this cohort (MSM recruited by respondent-driven sampling and MSM recruited by Internet-based respondent-driven sampling), and Vietnam will be scaling up recency testing more broadly in the coming year.

In addition to these two studies that utilized point-of-care LAg avidity assays, three other studies used conventional LAg avidity testing to estimated incidence. These studies came from cohorts of MSM and transgender women in Tijuana, Mexico,[3] adolescent girls and young women in five southern African countries,[4] and injection drug users in Athens, Greece.[5] Both the Mexican and the Greek studies used recency testing with RITA to identify recently infected cohort members and then to use them to construct network analyses.

In the Mexican study, Proyecto Enclaces, investigators recruited a new cohort of MSM and transgender women using venue-based sampling, partners of infected cohort members identified through contact tracing and respondent-driven sampling.³ Inclusion criteria were age ≥18 years, anal sex with a man or a transgender woman in the past 4 months, and either a self-reported HIV infection or a laboratory-confirmed infection. In addition to testing participants using the LAg avidity assay, samples were also used to generate pol-1 sequences by PCR and Sanger sequencing and then constructed a network map using TRAnsmission Cluster Engine (TRACE), a tool for large-scale molecular epidemiology. Of the 233 participants enrolled, 195 were tested for recent infection. Of these, 22 (11%) had positive LAg assays. Participants who had lived in Tijuana longer, those who had used cocaine in the past month and had ever experienced sexual abuse had higher adjusted odds of recent infection compared to older infection. Overall, 9 (60%) of 15 participants who had both LAg and pol-1 sequences clustered compared to 40 (31%) of 130 with more long-standing infection. The investigators concluded that because the recent infections were not linked to each other, there was most likely steady, ongoing transmission of HIV in this community in Tijuana.

In the Greek study,⁵ (9.8%) of highly connected core cohort members (k score=4 or 5) were recently infected compared to 3 (2.0%) of 147 participants with fewer connection (k score ≤3) (p=0.038).⁵ These investigators concluded that being in a more connected part of the risk network in this extensive outbreak was associated with recent infection as the outbreak slowed and transmission waned and suggested the utility of contact tracing from those with new infections to control outbreaks at least in the early stages.

The other study that utilized laboratory-based LAg avidity testing was from the Population-based HIV Impact Assessment (PHIA) program.⁴ This paper combined data on HIV-infected 15-to-24-year-old adolescent girls and young women (AGYW) and their cohabiting male partners from five southern African countries where PHIA surveys had been conducted: Eswatini, Lesotho, Malawi, Zambia, and Zimbabwe. Its focus was primarily on characterizing male partners of AGYW with prevalent infection. Recent infection was defined as LAg avidity assay positive, viral load >1,000 copies/mL, and no detectable antiretrovirals. HIV prevalence among both AGYW and their cohabiting male partners was 6%, and HIV incidence in AGYW ranged from 0 per 100 person years among those with 15-to-24-year-old male partners to 3.0 per 100 person years among those with 35-to-44-year-old male partners. The odds of recent AGYW infection increased 8% with each additional year of partner age difference (OR=1.08, 95%CI 1.05-1.11) and was associated with increasing viremia among older male partners but not age per se. The investigators concluded that improving ART coverage among key male age groups (25-44 years old) may advance HIV prevention among AGYW.

What are the take-home lessons? First, recency testing is largely acceptable to individuals seeking routine care and participating in studies. Secondly, in the two studies in which point-of-care testing was used, the test performed well. Third, and this was brought out in the question-and-answer session following the oral abstract session, incidence is only meaningful when there is a defined population from which the sample has been drawn. So, estimates of incidence from clinic samples, unless those samples are representative of a population (like all pregnant women in a specific area), should be taken with a grain of salt. In studies where a representative baseline sample is used, as in the Greek, Mexican, and 5-county PHIA study, incidence calculations are meaningful. Because of the likely representativeness of the Kenyan and Vietnamese studies, these calculations are also likely meaningful, especially in Kenya where they were compared against a known standard. Finally, examining cohorts for differences between those recently infection and those infected in the past is similarly tricky. People with long-term infection had recent infection a few years before, so the best way to interpret these data are as secular trends. Finally, recent infection can be used to construct network maps and improve our understanding of transmission dynamics. As more data come in from incident infection surveillance systems and index partner testing programs, the utility of recency testing for disease control purposes will become clearer.

1.     Risher K, Ambia J, Calvert C, et al. Incidence in pregnant women attending antenatal clinics in western Kenya [MOPEC371]. 10^th International AIDS Society Conference on HIV Science, Mexico City, Mexico, July 22, 2019.

2.     Vu D, Le G, An L, et al. High burden of recent and prevalent infection among men who have sex with men (MSM) in Hanoi, Vietnam [WEAC0103]. 10^th International AIDS Society Conference on HIV Science, Mexico City, Mexico, July 24, 2019.

3.     Skaathun B, Pines HA, Patterson TL, et al. HIV incidence among men who have sex with men (MSM) and transgender women (TW) in Tijuana, Mexico [WEAC0106]. 10^th International AIDS Society Conference on HIV Science, Mexico City, Mexico, July 24, 2019.

4.     Ayton S, Schwitters A, Mantell J, et al. Male partner age and HIV infection among young women cohabiting partnerships in five countries in southern Africa [MOPEC344]. 10^th International AIDS Society Conference on HIV Science, Mexico City, Mexico, July 24, 2019.

5.     Friedman SR, Williams LD, Paraskevis D, et al. A highly-connected risk network position was associated with recent HIV infection approximately three years after the large HIV outbreak began among people who inject drugs (PWID) in Athens. {MOPEC334]. 10^th International AIDS Society Conference on HIV Science, Mexico City, Mexico, July 24, 2019.

Between 2012 and 2016 Ireland experienced at 50% increase in the number of HIV diagnosis. In order to understand whether there was an upsurge in the epidemic or if the increase was a result of detecting previously undiagnosed cases they integrated recent infection testing into their diagnosis-based HIV surveillance system. The goal was to identify recent infections in order to better monitor the HIV epidemic and target prevention programs. They integrated Lag Avidity testing into their routine testing and used demographic, clinical and epidemiological information routinely collected through their Computerised Infectious Disease Reporting (CIDR) system to complete the recent infection testing algorithm. The National Virus Reference Laboratory (NVRL) tested sera from all new HIV cases with the Sedia HIV-1 limiting antigen-avidity EIA assay. They also tested for the p24 antigen which is an indicator of infection in the last 3 weeks.

They applied RITA to diagnoses notified between 1 January 2016 and 31 December 2016 with an avidity index (AI) result. Surveillance data were extracted from CIDR in August 2017, after the annual data validation process was completed. The RITA used classified cases with an AI of less than 1.5 as likely to be a recent infection unless epidemiological criteria (CD4 count <200 cells/mm3; viral load <400 copies/ml; the presence of AIDS-defining illness; prior antiretroviral therapy use; history of PrEP or PEP use in previous 6 months) indicated a potential false-recent result. They calculated a second estimate of the proportion of recent infections that included cases who were p24 antigen positive, but did not have avidity testing carried out. The denominator for this estimate included all HIV diagnoses notified in 2016 with an available AI result, or with a positive p24 antigen test.

RITA results were returned to clinicians with interpretation guidance (drafted in partnership with patient organizations). There were anecdotal reports that results are valuable at both individual and population level.

Of 508 diagnoses in 2016, they linked 448 (88.1%) to an avidity test result. RITA classified 12.5% of diagnoses as recent, with the highest proportion (26.3%) amongst people who inject drugs. In multivariable logistic regression, recent infection was more likely with a concurrent sexually transmitted infection (aOR 2.59; 95% CI 1.04–6.45). Data were incomplete for at least one RITA criterion in 48% of cases.

The study highlights the need to monitor trends in HIV testing patterns by sub-group as a critical step to interpreting recent infection testing data. They identified recent infections among persons previously diagnosed outside of Ireland; inclusion criteria of previous diagnosis <1 year should be considered.

The study demonstrated the feasibility of integrating RITA into routine surveillance and showed ongoing HIV transmission and a high proportion of recent infection among PWID. To improve the interpretation of RITA, further efforts are required to improve completeness of the required epidemiological data.

Author(s): 

Eduard Grebe1, Gary Murphy2, Sheila M. Keating3, Dylan Hampton3, Michael P. Busch3, Shelley Facente4, Kara Marson4, Christopher D. Pilcher4, Andrew Longosz5, Susan H. Eshleman5, Thomas C. Quinn5, Alex Welte1, Neil Parkin6, Oliver Laeyendecker5

1 South African Centre for Epidemiological Modelling and Analysis, Stellenbosch, South Africa; 2 Public Health England, London, UK; 3 Blood Systems Research Institute, San Francisco, CA, USA; 4 University of California San Francisco, San Francisco, CA, USA; 5 Johns Hopkins University School of Medicine, Baltimore, MD; 6 Data First Consulting, Belmont, CA, USA

Abstract Body: 

We evaluated Sedia Limiting Antigen Avidity EIA (LAg)-based recent infection testing algorithms (RITAs) for incidence surveillance, using data from seroconverter cohorts, consisting of 10,322 specimens from 2,297 subjects. We investigated the impact of HIV-1 subtype and sex on mean duration of recent infection (MDRI) and false-recent rate (FRR).

We estimated MDRI and FRR for a range of RITAs consisting of LAg ODn thresholds, alone, and in combination with viral load. To estimate MDRI, we harmonised time-since-infection estimates for all subjects based on diagnostic testing histories. We fitted regression models for the probability of exhibiting the recent biomarker as a function of time since infection, for each threshold combination and separately for each HIV-1 subtype, and integrated the function from zero to the time cut-off T (2 years) to obtain MDRI. To evaluate surveillance performance, we defined epidemiological scenarios (subtype distribution, incidence, prevalence, treatment coverage) based on (I) the South African, (II) the Kenyan, and (III) a concentrated MSM epidemic. We estimated context-adapted MDRI (weighted average of subtype-specific MDRIs, adjusted for screening test) and context-specific FRR (weighted according to density of the times-since-infection and treatment coverage in the population). Uncertainty in epidemiological parameters was incorporated. Performance was defined as precision of incidence estimates.

Using all subtypes, MDRI for a RITA with ODn≤1.5 and VL>1000 was 143 days (95% CI:132,155) and FRR in untreated subjects 1.88% (1.26,2.68). Subtype MDRIs (in days) were as follows: A: 165 (128,210), B: 161 (136,190), C: 130 (116,143) and D: 184 (130,248). Differences between subtype-specific MDRIs were statistically significant for B&D (at 90% confidence level, p=0.071) and C&D (pMDRI and FRR vary by substantially HIV-1 subtype and sex. Optimal performance was achieved at ODn thresholds from 1.0-2.5. RITA properties depend strongly on population-level subtype and sex distributions.

PERFORMANCE COMPARISON OF THE MAXIM AND SEDIA LIMITING ANTIGEN AVIDITY ASSAYS

Author(s): 

Joseph B. Sempa1, Gary Murphy2, Jake Hall2, Sheila M. Keating3, Dylan Hampton3, Shelley Facente4, Kara Marson4, Christopher D. Pilcher4, Neil Parkin5, Michael P. Busch3, Alex Welte1, Eduard Grebe1

1 South African Centre for Epidemiological Modelling and Analysis, Stellenbosch, South Africa; 2 Public Health England, London, UK; 3 Blood Systems Research Institute, San Francisco, CA, USA; 4 University of California San Francisco, San Francisco, CA, USA; 5 Data First Consulting, Belmont, CA, USA

Abstract Body: 

The HIV-1 Limiting Antigen Avidity EIA (LAg assay) used for detecting ‘recent’ HIV infection is mainly from two manufacturers: Maxim Biomedical and Sedia Biosciences. We assessed and compared the performance, for incidence estimation, of the Maxim and Sedia LAg assays.

We ran both assays on a panel of 2,500 well-characterised HIV-1-infected specimens, most with estimable duration of HIV infection. We analysed concordance of assay results, assessed reproducibility using repeat testing, and estimated the critical performance characteristics of a test for recent infection – mean duration of recent infection (MDRI) and false-recent rate (FRR) – for a range of normalised optical density (ODn) recency discrimination thresholds, in combination with viral load thresholds. We further specified three surveillance scenarios defined by incidence, prevalence, treatment coverage, and subtype and infection-time distributions based on A) South African B) Kenyan, and C) concentrated MSM epidemics. Overall performance was measured as precision of incidence estimates.

ODn measurements produced by the two assays on the same specimens were highly correlated (R2=0.91). The Maxim assay produced systematically lower ODn values (mean ODn of 0.643 vs 0.749), largely as a result of higher calibrator readings. Correlation was greater for non-normalised OD readings (R2=0.94) and the slope was closer to 1 (1.054 for OD vs 0.899 for ODn). Reproducibility of repeat testing (25 replicates of 3 blinded control specimens) was slightly greater for the Maxim assay (CoV 8.9% to 14.8% vs 13.2% to 15.0%). At the ‘standard’ recency discrimination threshold of ODn≤1.5, in combination with a viral load threshold (>1000), the Maxim assay had a longer MDRI of 201 days (95% CI: 180,223) vs 171 days (152,191) for Sedia, and a higher FRR in treatment-naive subjects (1.7% vs 1.1%). Under surveillance scenario A, the minimal relative standard errors achieved, in combination with viral load, were 22.8% (at ODn≤3.25 & VL>1000) for Maxim, and 23.4% (at ODn≤3.00 & VL>1000) for Sedia.

Maxim LAg ODn values can be approximately inferred from Sedia values with a conversion factor of 1.172, arising from differences in the reactivity of calibrators supplied in the assay kits. Performance for surveillance purposes was indistinguishable, although different thresholds were nominally optimal, and, crucially, different values of MDRI and FRR must be used in survey planning and incidence estimation.

FIRST USE OF POINT-OF-CARE HIV RECENCY TESTS AS A SURVEILLANCE TOOL IN NICARAGUA

Author(s):

Sanny Northbrook1, Carlos Vargas2, Ricardo Mendizabal-Burastero2, Jose Rodas1, Andrea Kim3, Nasim Farach1, Aleyda Solorzano2, Luz Maria Romero2, Bharat Parekh

1 CDC-DGHT Central America Regional Office; 2 Universidad del Valle de Guatemala; 3 CDC Headquarters, Atlanta, GA, USA

Abstract Body:

Background

Routine assessment of transmission dynamics facilitates the universal test-and-treat approach for persons living with HIV (PLHIV) and ensures that interventions target those with highest risk. Rapid recency tests can distinguish recent (on average, in the past 6 months) from non-recent infection, enabling healthcare workers to detect, monitor, and respond to recent infections. We describe the first use of point-of-care recency tests as a surveillance tool in Nicaragua.

Methods

From January to July 2018, all persons diagnosed with HIV per the national HIV testing algorithm at 205 public testing sites had blood samples sent to the National Center for Diagnostic and Reference (CNDR) for confirmation. The rapid recency assay for HIV-1 (SEDIA Biosciences) was performed onsite for PLHIV diagnosed at key population clinics and at the CNDR for all other samples. Viral load testing was performed using COBAS Amplicor/Ampliprep HIV 2.0 (Roche Diagnostics) on all samples testing recent to confirm recency of infection (≥1,000 cps/mL). Surveillance variables (age, sex, sexual orientation, and place of residence) were recorded in a database. We conducted univariate analysis to describe characteristics of PLHIV with recent infection (Stata 13.0) and identified areas with statistically significant recent HIV infection (p values <0.05) by comparing observed versus expected number of recently infected individuals (SatScan).

Results

Of the 452 PLHIV with a new diagnosis and recency test performed during the assessment period, 84 (19%) tested recent; of these, 58 (69%) had a viral load ≥1,000 copies/mL. The median age (33 years; range, 14–64 years) among PLHIV with recent infections was significantly lower than the median age  (49 years; range, 14–73) among PLHIV with long-term infections (p=0.03). Of those with a confirmed recent infection, 83% were men, 31% were men who have sex with men, and 59% lived in Managua. Of 17 clusters identified, 2 had high rates of HIV recency with 2.9–6.1 times more recent infections than expected, with Managua having the highest relative risk.

Conclusions

Most male and Managua PLHIV residents tested recent. A visual dashboard and maps identifying potential clusters of recent transmission were developed and are updated weekly to facilitate real-time analysis and response.

From March 15th to 24th, 2019, colleagues from the Centers for Disease Control and Prevention (CDC), Malawian Ministry of Health (MOH) and National HIV Reference Laboratory (NHRL), I-TECH Malawi, and University of California, San Francisco (UCSF) conducted three step-down training sessions with more than 200 participants in total. Those attending the trainings to support the first phase of national roll-out included HIV testing service (HTS) providers such as health diagnostic assistants (HDAs) and other key stakeholders such as site coordinators and district supervisors from across Blantyre.

Nearly all participants achieved a perfect score in their proficiency testing with the Maxim Swift Recent Infection Assay as well as with the Asante HIV-1 Rapid Test for Recent Infection (98% and 89%, respectively). In addition to the practical test, participants scored 83% on average on the complementary written test, meaning that the vast majority of participants understood the information presented. 

General principles around HIV as well as recency-specific principles were reinforced during the trainings such as: 

1. Incidence is the rate of new HIV infections in a population, helping to target surveillance  and prevention programs, and helping to track the leading edge of the epidemic; 
2. HIV-1 RTRIs are unable to detect HIV-1 infection before seroconversion – just as the majority of HIV rapid tests that cannot work before seroconversion because they are antibody-based;
3. Antibodies made during the early (recent) phase of infection have very low avidity, or binding strength of antibodies to antigen, and very low titers, or quantities of antibodies; and
4. Individuals who were recently infected and not on ART have weak immune systems and high viral loads.

Feedback from the trainings was well-received by the coordinators with participants commenting that they enjoyed many of the sessions and material covered. Highlights from participants’ surveys included enjoying a refresher of the basics of HIV, learning about recent HIV infections, and practicing the RTRI testing and dried blood spot preparation methods. Participants remarked that the most useful information that they learned was how to conduct recent infection testing alongside the national HIV testing algorithm, how to conduct the rapid tests themselves, and how to complete the accompanying Recent Infection HIV Surveillance Register.

Following the step-down trainings, the CDC, I-TECH Malawi, and UCSF’s in-country partner, Global AIDS Interfaith Alliance (GAIA), have been actively supporting site activations across the district of Blantyre. During site activation, officers visited sites as they prepared for the roll-out of recent HIV testing in order to assess sites’ readiness for implementation. This included ensuring that health officials had been adequately trained on RTRI methods, that sites were equipped with all necessary testing supplies, and that systems were in place for ensuring quality of care and service delivery at all HTS entry points at each site.

University of California, San Francisco (UCSF)

Soon after the first-generation HIV test was licensed in 1985, there was interest in understanding what went on in infection before measurable HIV antibody appeared. This was largely driven by blood banks’ need to be able to identify donors with HIV infection before they became antibody positive (approximately three weeks from exposure in the original tests). The advent of the ability to test for an HIV antigen, the p24 nucleocapsid protein, which is produced in excess by the replicating virus and enters the circulation, marked the beginning of our technical ability to detect recent HIV infection from laboratory tests. As shown in Figure 1, p24 appears in the circulation at approximately 14 days post-infection. If a patient tests positive for p24 antigen but not for HIV antibody, that person is likely between 14- and 21-days post-infection. This is a very narrow window, and large samples are required to find people in this window. Blood tests have continued to evolve, and blood banks now screen with third-generation tests, which detect antibody by around 21 days post-infection, and antigen testing. HIV RNA testing is another earlier marker of infection that appears even earlier than p24 antigen; it is, however, used primarily for testing newborns and infants for HIV infection (who can have transplacentally transfused maternal antibody).

Figure 1. HIV antibody, p24 and RNA levels as a function of time since exposure.

What properties should an ideal test for incident HIV infection have? It should be universally positive in recent infection and universally negative later in infection. It should be unaffected by HIV subtype, mode of transmission, antiretroviral therapy, presence of opportunistic infections and demographic factors such as age, sex, race and pregnancy. Finally, for the purposes of calculating incidence, it should have a relatively long and uniform window period. There are two broad categories of ways to calculate incidence: direct and indirect methods (Table 1). Below we examine one approach to direct measurement, estimation using laboratory tests for recent infection.

Table 1. Direct and indirect methods for estimating incidence.

Antigen and nucleic acid testing in the window period. Early interest in acute HIV infection was two-fold. First, clinicians were interested in the diagnosis of acute HIV infection, a clinical syndrome that included symptoms such as fever, rash, myalgias and arthralgias [^[1]]. The second was to use laboratory markers of acute infection to estimate HIV incidence in cross-sectional samples. In 1995, Brookmeyer and colleagues used p24 antigen testing (without regard to HIV antibody) to calculate HIV incidence in a sample of HIV-seronegative patients attending a sexually transmitted disease clinic in Pune, India [^[2],^[3]]. They found 15 of 1,241 patients to be antigen-positive, antibody-negative and, using a window period of 22.5 days, calculated an incidence rate of 19.6 per 100 person years in this sample. This general method, but using HIV RNA testing instead of p24 testing, was used by Pilcher and colleagues to estimate HIV incidence in North Carolina among individuals testing HIV-negative in state testing facilities [^[4]]. They found 23 nucleic acid-positive specimens among 108,644 HIV-uninfected individuals and, combining these results with results from the sensitive/less-sensitive EIA, described below, calculated an HIV incidence of 2.2 infections per 1,000 per year. Similar techniques were proposed for estimating incidence among blood donors [^[5]]. However, whether using p24 antigen testing or HIV RNA testing, these methods have not been subsequently used on large samples to estimate incidence largely due to the sample sizes required and the uncertainty as to what the sample represents [^[6]].

Serological Testing Algorithm for Recent HIV Seroconversion. In 1998 a new method for identifying patients with recent infection appeared, the Serological Testing Algorithm for Recent HIV Seroconversion (STAHRS) [^[7]]. This method tested serum twice, once with a relatively more poorly performing first-generation antibody test (the less-sensitive or detuned test) and the second with a state-of-the-art third generation test (the sensitive test). The less sensitive test was calibrated to detect HIV antibodies approximately 150 days after infection while the sensitive test could detect it within 21 days. Patients who were positive by the new test but negative by the older test were categorized as recently infected, that is, they were in a window period of 21 to 150 days post-infection (Figure 2).

Estimating incidence using STAHRS, however, turned out to be problematic because of variable window periods among different HIV-1 clades. The original window period of 129 days had been estimated using North American type B clades, and the poor performance with non-type B clades [^[8]] and problems with false-positive results (that is, false recent results) among patients on antiretroviral therapy (ART) [^[9]] led to its eventual discontinuation as a way to estimate incidence. Interestingly, though, the authors saw that the test might be used for something more than incidence estimates and wrote, “It is useful … at the clinical level for patient care … and at the public health level for focusing and evaluating HIV prevention efforts.”

Figure 2. More-sensitive/less-sensitive (detuned) EIA for recent infection

Because of the clade-to-clade variability and other issues that limited the utility of STAHRS, attention turned to tests that would have similar performance across clades. The next series of assays assessed particular antibody characteristics – concentration [^[10],^[11]], proportion [^[12]], isotype [^[13]] or avidity [^[14]]. All these characteristics appear between the earliest serological response and established infection, that is, the window period. Of these, two, the BED-capture EIA (or BED-CEIA) incidence assay and the avidity assay, have been widely used to estimate incidence in cross-sectional samples.

            BED-CEIA. The BED-CEIA uses a quantitative IgG-capture EIA to determine the relative ratio of HIV-1-specific IgG to total IgG [12]. This ratio is generally lower in earlier infection and higher in longer-term infection. The attraction of the BED-CEIA was that a synthesized peptide, representing a conserved region in the B, E (now CRF-01AE) and D clades of HIV-1, allowed for detection of early HIV infection in several clades [^[15]]. The problem, however, remained misclassification of results as recent infections, particularly among those with late-stage infection, low CD4 cell counts and low viral loads and among those on ART [^[16],^[17],^[18],^[19]]. Moreover, clade-to-clade differences persisted, with variable window periods for various clades [^[20]]. Because of these ongoing limitations, testing algorithms were introduced that combined to compensate for misclassification of long-term infections, such as testing for low CD4 counts, low viral loads or the presence of antiretroviral drug residua or even combining incidence assays, the so-called Recent Infection Testing Algorithm (RITA) [^[21]]. Despite these attempts at improved performance, however, the UNAIDS Reference Group on Estimates, Modelling and Projections compared results from BED-CEIA testing in several African countries and Thailand and found that BED-CEIA overestimated incidence derived from a variety of methods. As a result, the Group recommended that BED-CEIA not be used for routine surveillance applications [^[22]]. However, investigators continued to work with BED-CEIA and the newer limiting avidity (LAg) assay and using RITAs to improve performance [^[23],^[24],^[25]] largely by minimizing false recent results.

            Limiting antigen avidity testing. LAg testing has avoided several of the pitfalls of BED-CEIA testing and is now the basis for the point-of-care recency tests now being widely used in PEPFAR-funded projects. Avidity assays are based on the strength of antibody-antigen binding and the observation that early antibodies bind less strongly (less avidly) to HIV antigens than antibodies produced by a more mature immune reaction [^[26],^[27]]. Beyond a certain cut-off point, the antibody response is considered more mature, and the infection is classified as long-term. Unless ART is begun during primary infection, there should be minimal false positives due to viral suppression, whether therapeutically with ART or naturally among elite suppressors [^[28]]. Moreover, the maturing avidity pattern has been shown to be similar in patients with clade B and non-clade B infections [^[29]]. However, there is some evidence that avidity declines in patients with opportunistic infections, which may lead to misclassification [^[30]].

In a 2014 meeting in Barcelona, WHO’s Incidence Assay Working Group reviewed recent data directly comparing the LAg, BED-CEIA, less-sensitive/detuned Vitros, Vitros avidity and BioRad avidity tests, which found that LAg had the lowest false recency rate (Table 2) [^[31]]. Moreover, they endorsed the use of RITAs to reduce false recency rates [^[32]], which at the time of the meeting had been used successfully to validate incidence measurements from Kenya [^[33]], South Africa [^[34]] and Eswatini [^[35]].

Table 2. Estimated test properties (and 95% confidence intervals) for each assay, for various specimen sets [31].

            Since the 2014 statement, numerous investigators have employed LAg assays with various permutations of RITAs to estimate incidence from representative samples. For instance, Kim and Rehle used the LAg assay plus two different algorithms to estimate incidence in representative national samples from Kenya and South Africa [^[36]]. One algorithm used the LAg assay plus viral load testing; the other used the LAg assay, viral load testing and serum testing for antiviral drugs. The investigators tested HIV-positive specimens qualitatively for the presence of antiretroviral drugs using high-performance liquid chromatography with tandem mass spectrometry. In Kenya drugs tested for were lamivudine (3TC), nevirapine (NVP), efavirenz (EFV) and lopinavir (LPV). In South Africa they were 3TC, NVP, EFV, LPV, zidovudine, atazanavir and darunavir, reflecting drugs recommended in the national guidelines. They found that 4 participants in Kenya and 18 in South Africa who had tested positive by the LAg assay and had high viral loads, also tested positive for antiretrovirals, which, when removed by the RITA, led to an almost 20% reduction in incidence estimates (Table 3).

Table 3. Annualized HIV incidence among persons aged 15-49 years by recent infection testing algorithm, Kenya and South Africa, 2012.

            Other countries, notably Botswana [^[37]], South Africa [^[38]] and several of the Population-Based HIV Impact Assessments (PHIA) [^[39],^[40]] have used LAg avidity-based RITAs to estimate incidence (Table 4). PHIA investigators estimated incidences ranging from 0.06 per 100 person years in Ethiopia [^[41]] to 1.36 per 100 person years in Eswatini [^[42]]. They generally used RITAs that included viral load, but they also were able to add testing for antiretroviral drugs and were able to compare results (Table 4) [^[43],^[44],^[45]].

Table 4. PHIA incidence results by country, 2016-2019.

            Rapid point-of-care LAg testing. Rapid point-of-care (RT-POC) LAg avidity tests are the newest wrinkle and are being deployed in a number of PEPFAR-supported countries. Two kits are commercially available from Sedia Biosciences (Asanté) [^[51]] and Maxim Biomedical (Swift) [^[52]]. The tests can be done with capillary blood and, as they are rapid tests, can be applied in routine HIV testing settings. Some preliminary data are available from Central America, Kenya and Malawi. In Central America, they are used to identify patients presenting to HIV testing sites in Guatemala, Nicaragua and Honduras. In Guatemala, 69% of patients testing positive by RT-POC identified sexual partners who were HIV-infected compared to 39% of those with long-term HIV infection while in Nicaragua the yield increased from 55% to 75% [^[53]]. In Malawi, RT-POC LAg avidity tests were used to characterize recent infection among 15-to-24-year-old women [^[54]]. Of 589 women who were newly diagnosed with HIV infection, 68 (11.7%) had recent infection, which translated to an estimated incidence of 0.59 per 100 person years. Older women and those residing in Blantyre had significantly higher incidence rates. Another report from Grebe and colleagues [^[55]] found that in a pooled seroconverter group using Sedia® LAg-avidity assay and RITAs the mean duration of recent infection (MDRI) was approximately 40 days longer in women than in men; there was no difference between pregnant and non-pregnant women. Other field trials have recently been completed, and more data will be presented at the International AIDS Society’s Conference on HIV Science in Mexico City in July 2019.

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