Key Factors Influencing the Emergence of Human Immunodeficiency Virus Drug Resistance in Low- and Middle-Income Countries.

The emergence and spread of human immunodeficiency virus (HIV) drug resistance from antiretroviral roll-out programs remain a threat to long-term control of the HIV-AIDS epidemic in low- and middle-income countries (LMICs). The patterns of drug resistance and factors driving emergence of resistance are complex and multifactorial. The key drivers of drug resistance in LMICs are reviewed here, and recommendations are made to limit their influence on antiretroviral therapy efficacy.

Implementation of HIV and Tuberculosis Diagnostics: The Importance of Context.

BACKGROUND: Novel diagnostics have been widely applied across human immunodeficiency virus (HIV) and tuberculosis prevention and treatment programs. To achieve the greatest impact, HIV and tuberculosis diagnostic programs must carefully plan and implement within the context of a specific healthcare system and the laboratory capacity. METHODS: A workshop was convened in Cape Town in September 2014. Participants included experts from laboratory and clinical practices, officials from ministries of health, and representatives from industry. RESULTS: The article summarizes best practices, challenges, and lessons learned from implementation experiences across sub-Saharan Africa for (1) building laboratory programs within the context of a healthcare system; (2) utilizing experience of clinicians and healthcare partners in planning and implementing the right diagnostic; and (3) evaluating the effects of new diagnostics on the healthcare system and on patient health outcomes. CONCLUSIONS: The successful implementation of HIV and tuberculosis diagnostics in resource-limited settings relies on careful consideration of each specific context.

Eliminating perinatal HIV in the United States: mission possible?

: In 2015, only 53 infants born in the United States acquired HIV - the lowest recorded number of perinatal HIV infections. Recognizing this significant achievement, we must acknowledge that the United States has not yet reached the goal of eliminating perinatal HIV transmission. This analysis describes different approaches to perinatal HIV preventive services among five states and the District of Columbia as case studies. Continuous focus on improving identification, surveillance and prevention of HIV infection in pregnant women and their infants is necessary to reach the goal of eliminating perinatal HIV transmission in the United States.

World Health Organization/HIVResNet Drug Resistance Laboratory Strategy.

With rapidly increasing access to antiretroviral drugs globally, HIV drug resistance (HIVDR) has become a significant public health issue. This requires a coordinated and collaborative response from country level to international level to assess the extent of HIVDR and the establishment of efficient and evidence-based strategies to minimize its appearance and onward transmission. In parallel with the rollout of universal access to HIV treatment, countries are developing protocols based on the recommendations of the World Health Organization (WHO) to measure, at a population level, both transmitted HIVDR and HIVDR emerging during treatment. The WHO in collaboration with international experts (HIVResNet Laboratory Working Group), has developed a laboratory strategy, which has the overall goal of delivering quality-assured HIV genotypic results on specimens derived from the HIVDR surveys. The results will be used to help control the emergence and spread of drug resistance and to guide decision makers on antiretroviral therapy policy at national, regional and global level. The HIVDR Laboratory Strategy developed by the WHO includes several key aspects: the formation of a global network of national, regional and specialized laboratories accredited to perform HIVDR testing using a common set of WHO standard and performance indicators; recommendations of acceptable methods for collection, handling, shipment and storage of specimens in field conditions; and the provision of laboratory technical support, capacity building and quality assurance for network laboratories. The WHO/HIVResNet HIVDR Laboratory Network has been developed along the lines of other successful laboratory networks coordinated by the WHO. As of August 2007, assessment for accreditation has been conducted in 30 laboratories, covering the WHO's African, South-East Asia, Western Pacific, and the Caribbean Regions.

Diminished human immunodeficiency virus type 1 DNA yield from dried blood spots after storage in a humid incubator at 37 degrees C compared to -20 degrees C.

Collecting whole blood on filter paper simplifies the processing, transport, and storage of specimens used for the diagnosis of human immunodeficiency virus type 1 (HIV-1) and other tests. Specimens may be collected in tropical or rural areas with minimal facilities for handling specimens. To compare simulated tropical conditions with freezer storage, we examined the stability of HIV-1 DNA in dried blood spots (DBS) stored in humid heat and at -20 degrees C. DBS were created by spotting 50-microl aliquots of whole blood on 903 filter paper. DNA was extracted from DBS at baseline and after 2, 6, or 12 months of storage at -20 degrees C or at 37 degrees C with approximately 85% humidity. The DNA was tested undiluted or diluted using the Amplicor HIV-1 DNA PCR (Roche), version 1.5. Each reaction was scored positive, negative, or indeterminate based on optical density. Results were compared between storage conditions and over time. A total of 1,832 reactions from 916 DBS were analyzed, including 100 DBS at baseline, 418 stored at -20 degrees C, and 398 stored at 37 degrees C. A chi-square test showed fewer positive reactions for DBS stored at 37 degrees C (55%) than for those stored at -20 degrees C (78%) (P < 0.0001). Samples stored at -20 degrees C showed little change in the probability of detection of HIV-1 DNA over time; the odds ratio (OR) was 0.93 after storage for 1 year. Samples stored at 37 degrees C demonstrated a significant change in detection at 1 year (OR, 0.29). We conclude that exposure of DBS to 37 degrees C and high humidity impaired the recovery of HIV-1 DNA from DBS, whereas DNA recovery was preserved when DBS were stored frozen.

HIV birth testing and linkage to care for HIV-infected infants.

: On 5-6 May 2016, the division of AIDS of the National Institute of Allergy and Infectious Diseases convened a workshop on 'HIV Birth Testing and Linkage to Care for HIV Infected Infants.' The goal of the workshop was to evaluate birth testing for early infant diagnosis (EID) of HIV, delineate technological resources for advancing a point-of-care (POC) HIV test implementable at birth and chart out the implementation hurdles for initiating early antiretroviral therapy to HIV-infected infants diagnosed at birth. The workshop addressed research and regulatory needs involved in the optimization of POC EID testing and challenges associated with implementation of EID, focusing on testing at birth. Scientific gaps and areas of intervention to accelerate and scale-up EID initiatives and birth testing were identified. These include discussion of the evidence supporting an early mortality peak among HIV-infected infant and justifying a role for birth HIV testing, including POC testing; evaluation of the current POC EID technology pipeline and test performance characteristics required for effective programmatic uptake; mathematical modeling of different testing scenarios and solutions with inclusion of birth testing; the adoption of setting-specific EID testing algorithms to achieve efficient linkage to care including early antiretroviral therapy initiation; the development of appropriate quality assurance programs to ensure accuracy of test results and enable sustainability of the testing program. Addressing these gaps and answering these challenges will be important in helping improve outcomes for HIV-infected infants and accelerate achieving the Joint United Nations Program for HIV and AIDS 90-90-90 targets in children.

Simultaneous identification of orthopoxviruses and alphaviruses by oligonucleotide macroarray with special emphasis on detection of variola and Venezuelan equine encephalitis viruses.

The development of a method in macroarray format for the identification of alphaviruses and orthopoxviruses in samples of concern in biodefense is reported. Capture oligonucleotides designed to bind generic members of the orthopox- or alphavirus families and a collection of additional oligonucleotides to bind specifically nucleic acids from five individual alphaviruses, including Venezuelan equine encephalitis, or DNA from each of four orthopoxviruses, including variola virus (VAR) were deposited onto nylon membranes. Hybridization of digoxigenin labeled PCR products to the macroarray produced results easily observable to the naked eye. Multiplex RT-PCR utilizing both orthopox- and alphavirus-generic primers yielded amplification of DNA corresponding to the expected sizes of the orthopoxvirus and alphavirus fragments, respectively. Hybridization of samples to capture oligonucleotides in the macroarray membranes identified correctly generic orthopox- or alphaviral sequences. The hybridizations correctly identified each of the three alphaviruses and two orthopoxviruses tested. We observed cross-hybridization only once (between two alphaviruses) that was less intense than the spots formed by correct hybridization. The macroarray test described below is easy to perform, inexpensive, relatively fast, uncomplicated to interpret, and its end point is read visually without the need of additional equipment. This nucleic acid hybridization assay onto nylon membranes in macroarray format can help in detecting or excluding the presence of threat viruses in environmental samples and appears promising for a variety of biodefense applications.

HIV-1 Drug Resistance Mutations: Potential Applications for Point-of-Care Genotypic Resistance Testing.

The increasing prevalence of acquired and transmitted HIV-1 drug resistance is an obstacle to successful antiretroviral therapy (ART) in the low- and middle-income countries (LMICs) hardest hit by the HIV-1 pandemic. Genotypic drug resistance testing could facilitate the choice of initial ART in areas with rising transmitted drug resistance (TDR) and enable care-providers to determine which individuals with virological failure (VF) on a first- or second-line ART regimen require a change in treatment. An inexpensive near point-of-care (POC) genotypic resistance test would be useful in settings where the resources, capacity, and infrastructure to perform standard genotypic drug resistance testing are limited. Such a test would be particularly useful in conjunction with the POC HIV-1 viral load tests that are currently being introduced in LMICs. A POC genotypic resistance test is likely to involve the use of allele-specific point mutation assays for detecting drug-resistance mutations (DRMs). This study proposes that two major nucleoside reverse transcriptase inhibitor (NRTI)-associated DRMs (M184V and K65R) and four major NNRTI-associated DRMs (K103N, Y181C, G190A, and V106M) would be the most useful for POC genotypic resistance testing in LMIC settings. One or more of these six DRMs was present in 61.2% of analyzed virus sequences from ART-naïve individuals with intermediate or high-level TDR and 98.8% of analyzed virus sequences from individuals on a first-line NRTI/NNRTI-containing regimen with intermediate or high-level acquired drug resistance. The detection of one or more of these DRMs in an ART-naïve individual or in a individual with VF on a first-line NRTI/NNRTI-containing regimen may be considered an indication for a protease inhibitor (PI)-containing regimen or closer virological monitoring based on cost-effectiveness or country policy.

Laboratory challenges conducting international clinical research in resource-limited settings.

There are many challenges to performing clinical research in resource-limited settings. Here, we discuss several of the most common laboratory issues that must be addressed. These include issues relating to organization and personnel, laboratory facilities and equipment, standard operating procedures, external quality assurance, shipping, laboratory capacity, and data management. Although much progress has been made, innovative ways of addressing some of these issues are still very much needed.

Workshop summary: Novel biomarkers for HIV incidence assay development.

Reliable methods for measuring human immunodeficiency virus (HIV) incidence are a high priority for HIV prevention. They are particularly important to assess the population-level effectiveness of new prevention strategies, to evaluate the community-wide impact of ongoing prevention programs, and to assess whether a proposed prevention trial can be performed in a timely and cost-efficient manner in a particular population and setting. New incidence assays and algorithms that are accurate, rapid, cost-efficient, and can be performed on easily-obtained specimens are urgently needed. On May 4, 2011, the Division of AIDS (DAIDS), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), sponsored a 1-day workshop to examine strategies for developing new assays to distinguish recent from chronic HIV infections. Participants included leading investigators, clinicians, public health experts, industry, regulatory specialists, and other stakeholders. Immune-based parameters, markers of viral sequence diversity, and other biomarkers such as telomere length were evaluated. Emerging nanotechnology and chip-based diagnostics, including algorithms for performing diverse assays on a single platform, were also reviewed. This report summarizes the presentations, panel discussions, and the consensus reached for pursuing the development of a new generation of HIV incidence assays.

Evaluation of dried blood spots for human immunodeficiency virus type 1 drug resistance testing.

Dried blood spots (DBS) are simpler to prepare, store, and transport than plasma or serum and may represent a good alternative for drug resistance genotyping, particularly in resource-limited settings. However, the utility of DBS for drug resistance testing is unknown. We investigated the efficiency of amplification of large human immunodeficiency virus type 1 (HIV-1) pol fragments (1,023 bp) from DBS stored at different temperatures, the type of amplified product(s) (RNA and/or DNA), and the similarity between plasma and DBS sequences. We evaluated two matched plasma/DBS panels stored for 5 to 6 years at several temperatures and 40 plasma/DBS specimens collected from untreated persons in Cameroon and stored for 2 to 3 years at -20 degrees C. The amplification of HIV-1 pol was done using an in-house reverse transcriptase-nested PCR assay. Reactions were done with and without reverse transcription to evaluate the contribution of HIV DNA to pol sequences from DBS. Amplification was successful for the DBS samples stored for 5 to 6 years at -20 degrees C or at -70 degrees C but not for those stored at room temperature. Thirty-seven of the 40 (92.5%) DBS from Cameroon were amplifiable, including 8/11 (72.7%) with plasma virus loads of <10,000 RNA copies/ml and all 29 with plasma virus loads of >10,000. Proviral DNA contributed significantly to DBS sequences in 24 of the 37 (65%) specimens from Cameroon. The overall similarity between plasma and DBS sequences was 98.1%. Our results demonstrate the feasibility of DBS for drug resistance testing and indicate that -20 degrees C is a suitable temperature for long-term storage of DBS. The amplification of proviral DNA from DBS highlights the need for a wider evaluation of the concordance of resistance genotypes between plasma and DBS.