National HIV Reference Laboratory Research Articles

HIV-1 infection among women in Israel, 2010\u20132018

IntroductionAlthough women comprise 33% of the HIV-1-carriers in Israel, they have not previously been considered a risk group requiring special attention. Immigration waves from countries in Africa and in East Europe may have changed the local landscape of women diagnosed with HIV-1. Here, we aimed to assess viral and demographic characteristics of HIV-1-positive women identified in Israel between 2010 and 2018.MethodsAll > 16 year-old, HIV-1-infected women, diagnosed in Israel in 2010–2018, (n = 763) registered in the National HIV reference laboratory were included in this cross-sectional study. Demographic and clinical characteristics were extracted from the database. Viral subtypes and transmitted drug resistance mutations (TDRM) were determined in 337 (44.2%) randomly selected samples collected from treatment-naive women.ResultsMedian age at diagnosis was 38 years. Most (73.3%) women were immigrants from the former Soviet Union (FSU) (41.2%, 314) or sub-Saharan Africa (SSA) (32.2%, 246) and carried subtype A (79.7%) or C (90.3%), respectively. Only 11.4% (87) were Israeli-born women. Over the years, the prevalence of women from SSA decreased while that of women from FSU increased significantly (p < 0.001). The median CD4+ cell count was 263 cells/mm3, and higher (391 cells/mm3) in Israeli-born women. TDRM were identified in 10.4% of the tested samples; 1.8, 3 and 7.1% had protease inhibitors (PI), nucleotide reverse transcriptase inhibitors (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) TDRM, respectively. The prevalence of women with NNRTI TDRM significantly increased from 4.9% in 2010–2012 to 13.3% in 2016–2018. Israeli-born women had the highest prevalence (16.3%) of NNRTI TDRM (p = 0.014). NRTI A62 (5.6%), NNRTI E138 and K103 (5.6 and 4.2%, respectively) were the most prominent mutated sites.ConclusionsMost HIV-1-positive women diagnosed in Israel in 2010–2018 were immigrants, with the relative ratio of FSU immigrants increasing in recent years. The high proportion of women diagnosed with resistance mutations, particularly, the yearly increase in the frequency of NNRTI mutations, support the national policy of resistance testing at baseline.

Prevalence of cryptococcal antigen (CrAg) among HIV-positive patients in Eswatini, 2014-2015.

BackgroundCryptococcal meningitis is a leading cause of death amongst people living with HIV. However, routine cryptococcal antigen (CrAg) screening was not in the national guidelines in Eswatini.ObjectivesA cross-sectional study was conducted between August 2014 and March 2015 to examine CrAg prevalence at Mbabane Government Hospital in Eswatini.MethodsWe collected urine and whole blood from antiretroviral-therapy-naïve patients with HIV and a cluster of differentiation 4 (CD4) counts < 200 cells/mm3 for plasma and urine CrAg lateral flow assay (LFA) screening at the national HIV reference laboratory. Two CD4 cut-off points were used to estimate CrAg prevalence: CD4 < 100 and < 200 cells/mm3. Sensitivity and specificity of urine CrAg LFA was compared to plasma CrAg LFA.ResultsPlasma CrAg prevalence was 4% (8/182, 95% confidence interval [CI]: 2–8) amongst patients with CD4 counts of < 200 cells/mm3, and 8% (8/102, 95% CI: 3–15) amongst patients with CD4 counts of < 100 cells/mm3. Urine CrAg LFA had a sensitivity of 100% (95% CI: 59–100) and a specificity of 80% (95% CI: 72–86) compared with plasma CrAg LFA tests for patients with CD4 < 200 cells/mm3. Forty-three per cent of 99 patients with CD4 < 100 were at World Health Organization clinical stages I or II.ConclusionThe prevalence of CrAg in Eswatini was higher than the current global estimate of 6% amongst HIV-positive people with CD4 < 100 cell/mm3, indicating the importance of initiating a national screening programme. Mechanisms for CrAg testing, training, reporting, and drug and commodity supply issues are important considerations before national implementation.

From Sequence Data to Patient Result: A Solution for HIV Drug Resistance Genotyping With Exatype, End to End Software for Pol-HIV-1 Sanger Based Sequence Analysis and Patient HIV Drug Resistance Result Generation.

Introduction:With the rapid scale-up of antiretroviral therapy (ART) to treat HIV infection, there are ongoing concerns regarding probable emergence and transmission of HIV drug resistance (HIVDR) mutations. This scale-up has to lead to an increased need for routine HIVDR testing to inform the clinical decision on a regimen switch. Although the majority of wet laboratory processes are standardized, slow, labor-intensive data transfer and subjective manual sequence interpretation steps are still required to finalize and release patient results. We thus set out to validate the applicability of a software package to generate HIVDR patient results from raw sequence data independently.Methods:We assessed the performance characteristics of Hyrax Bioscience’s Exatype (a sequence data to patient result, fully automated sequence analysis software, which consolidates RECall, MEGA X and the Stanford HIV database) against the standard method (RECall and Stanford database). Exatype is a web-based HIV Drug resistance bioinformatic pipeline available at sanger.exatype.com. To validate the exatype, we used a test set of 135 remnant HIV viral load samples at the National HIV Reference Laboratory (NHRL).Result:We analyzed, and successfully generated results of 126 sequences out of 135 specimens by both Standard and Exatype software. Result production using Exatype required minimal hands-on time in comparison to the Standard (6 computation-hours using the standard method versus 1.5 Exatype computation-hours). Concordance between the 2 systems was 99.8% for 311,227 bases compared. 99.7% of the 0.2% discordant bases, were attributed to nucleotide mixtures as a result of the sequence editing in Recall. Both methods identified similar (99.1%) critical antiretroviral resistance-associated mutations resulting in a 99.2% concordance of resistance susceptibility interpretations. The Base-calling comparison between the 2 methods had Cohen’s kappa (0.97 to 0.99), implying an almost perfect agreement with minimal base calling variation. On a predefined dataset, RECall editing displayed the highest probability to score mixtures accurately 1 vs. 0.71 and the lowest chance to inaccurately assign mixtures to pure nucleotides (0.002–0.0008). This advantage is attributable to the manual sequence editing in RECall.Conclusion:The reduction in hands-on time needed is a benefit when using the Exatype HIV DR sequence analysis platform and result generation tool. There is a minimal difference in base calling between Exatype and standard methods. Although the discrepancy has minimal impact on drug resistance interpretation, allowance of sequence editing in Exatype as RECall can significantly improve its performance.

The performance of BD FACSPresto™ for CD4 T-cell count, CD4% and hemoglobin concentration test in Ethiopia.

IntroductionIn Ethiopia, CD4+ T-cell counting is still required for all patients at baseline before antiretroviral therapy (ART) and to determine eligibility and follow-up of opportunistic infection prophylaxis. However, access to CD4+ T cell count in rural health facilities remains a major challenge in Ethiopia like other resource-limited settings.MethodologyBoth capillary and venous blood was drawn from each of 325 study participant recruited in Addis Ababa and surroundings. The CD4+ T-cell count, CD4%, and hemoglobin (Hgb) were tested at one of the four study health facilities using capillary blood and BD FACSPresto™ device. These tests were also done at the national HIV reference laboratory, using venous blood with BD FACSCalibur™, Sysmex XT-1800i™, and BD FACSPresto™.ResultsBD FACSPresto™ had an absolute mean bias of -13.3 cells/ul (-2.99%) and 28.3 cells/μl (6.4%) using venous and capillary blood, respectively, compared with BD FACSCalibur™. The absolute CD4 assay on the BD FACSPresto™ had a regression coefficient (R2) of 0.87 and 0.92 using capillary blood and venous blood samples, respectively, compared with BD FACSCalibur™. The percentage similarity of the BD FACSPresto™ using capillary and venous blood was 105.2% and 99.3%, respectively. The sensitivity of the FACSPresto™ using threshold of 500 cells/μl for ART eligibility using capillary and venous blood was 87.9 and 94.3%, while the specificity was 91.4 and 83.8%, respectively. Furthermore, the BD FACSPresto™ had an absolute mean bias of -0.2 dl/μl (0.0%) (95% LOA: -1.7, 1.3) and -0.59 dl/μl (0.1%) (95% LOA: -1.49, 0.31) for Hgb using capillary and venous blood compared with the Sysmex XT-1800i™, respectively.ConclusionOur results showed acceptable agreement between the BD FACSPresto™ and BD FACSCalibur™ for CD4+ T-cell counting and CD4%; and between the BD FACSPresto™ and Sysmex XT-1800i™for measuring Hgb concentration.

Attaining ISO 15189 accreditation through SLMTA: A journey by Kenya's National HIV Reference Laboratory.

BackgroundThe National HIV Reference Laboratory (NHRL) serves as Kenya’s referral HIV laboratory, offering specialised testing and external quality assessment, as well as operating the national HIV serology proficiency scheme. In 2010, the Kenya Ministry of Health established a goal for NHRL to achieve international accreditation.ObjectivesThis study chronicles the journey that NHRL took in pursuit of accreditation, along with the challenges and lessons learned.MethodsNHRL participated in the Strengthening Laboratory Management Toward Accreditation (SLMTA) programme from 2010–2011. Improvement projects were undertaken to address gaps in the 12 quality system essentials through development of work plans, team formation, training and mentorship of personnel. Audits were conducted and the scores used to track progress along a five-star grading scale. Standard quality indicators (turn-around time, specimen rejection rates and service interruptions) were measured. Costs of improvement projects and accreditation were estimated based on expenditures.ResultsNHRL scored 45% (zero stars) at baseline in March 2010 and 95% (five stars) after programme completion in October 2011; in 2013 it became the first public health laboratory in Kenya to attain ISO 15189 accreditation. From 2010–2013, turn-around times decreased by 50% – 95%, specimen rejections decreased by 93% and service interruptions dropped from 15 to zero days. Laboratory expenditures associated with achieving accreditation were approximately US $36 500.ConclusionInternational accreditation is achievable through SLMTA, even for a laboratory with limited initial quality management systems. Key success factors were dedication to a shared goal, leadership commitment, team formation and effective mentorship. Countries wishing to achieve accreditation must ensure adequate funding and support.

Laboratory Informatics and AIDS Indicator Surveys

Background and Purpose: National AIDS indicator surveys generate large volumes of specimens and associated data, necessitating the preparedness of systems and processes in the reference laboratory. Specimen management is often more routine while management of data becomes secondary. However, complete and accurate laboratory results are essential to ensure data quality. Robust laboratory informatics solutions for managing data are needed to increase data accuracy and reduce turnaround time. Methods: During the 2012 Kenya AIDS Indicator Survey (KAIS), 62,435 samples were collected from respondent households and transferred to the National HIV Reference Laboratory (NHRL) for testing. The Association of Public Health Laboratories (APHL) collaborated with the Kenya Ministry of Health in laboratory process improvement, laboratory information system design and governance focusing on preparing the laboratory to manage the large volume of samples and associated data. This task included developing detailed work flow and data flow diagrams for the entire process, from field specimen and data collection, to receipt, testing and storage. These diagrams were used for training and for reference during the survey process. They were also used to define KAIS 2012 requirements for the existing laboratory information management system (LIMS). Laboratory processes were documented and matched up with the configurations developed in the LIMS. The team also designed specimen tracking and receiving forms based on KAIS 2012 protocol and LIMS requirements. They matched these forms with data elements in the LIMS for data harmonization. The KAIS survey team developed contingency plans for every KAIS 2012 laboratory activity to ensure NHRL implemented uniform procedures when normal operations did not occur. These plans focused on the management and standardization of human resources as appropriate personnel and associated procedures are integral to optimal information system performance. The team defined staff roles and responsibilities and developed procedures for managing forms, specimens and electronic data, including quality and use aligned with survey protocols. During the project, the KAIS team provided knowledge transfer to NHRL to ensure the development of local capacity and long-term sustainability. Results: Due to the informatics processes in place, NHRL was able to share laboratory test results from KAIS with other stakeholders within a day of being received. NHRL was also able to immediately access data on storage locations of all samples that were previously managed using paper systems – a significant challenge before the project. Visualization, mapping and contingency planning for each step of the process ensured that laboratory support to KAIS 2012 was timely, and of high quality, regardless of digressions from standard operating procedures caused by unexpected circumstances. While contingency plans are not routine, they can be critical to the success of the survey. Conclusions: Preparedness is key. Finalization of the survey protocol three months before the pilot, training and piloting all aspects of laboratory services a month prior to the survey ensured informatics solutions are appropriate and robust. Engaging all stakeholders -- particularly survey, laboratory and LIMS staff -- in laboratory informatics solutions planning, implementation, monitoring is critical to developing the most efficient systems and processes for a large national sero survey. During execution, constant and ongoing communication is essential to adapt to the evolving needs of the survey and its participants. Keywords: LIMS/LIS, Informatics, survey

Men Who Have Sex With Men, Risk Behavior, and HIV Infection: Integrative Analysis of Clinical, Epidemiological, and Laboratory Databases

Centralized data collection and analytic tools facilitate tracing HIV transmission trends at the patient-population level with increasing resolution, complementing behavioral studies while avoiding sampling biases. By several measures, the rate of HIV infection among men who have sex with men (MSM) in Israel increased in the past several years more rapidly than was expected. We describe features of the data that connect this increase to behavioral changes. We retrospectively analyzed data from the national HIV reference laboratory and the national HIV and sexually transmitted infections registries. We examined changes in selected epidemiologic and clinical parameters and in the pattern of drug-resistant virus transmission among MSM in Israel. In particular, virus isolates from 296 MSM (23.8% of all MSM who received a diagnosis) were genotyped, drug-resistance conferring mutations were characterized, and phylogenetic trees were constructed. Compared with earlier years, during 2007-2009 MSM were more often infected with drug-resistant virus before treatment initiation, were coinfected with syphilis, and received a diagnosis during acute retroviral syndrome. Phylogenetic analysis suggested frequent transmission of drug-resistant HIV by drug-treated individuals to >1 partner. Secondary transmission of resistant virus by drug-naive patients is also consistent with the phylogenetic patterns. In addition, non-B HIV subtypes began to appear among MSM. Together, our findings suggest that the sexual behavior of MSM, both HIV-infected and uninfected, has become riskier, contributing to the number of those seeking early clarification of status, to syphilis comorbidity, and to the spread of drug resistance. These findings call for action by public health planners and community-based organizations aimed at increasing awareness of the risks, bringing a change in attitude and establishing safe sex norms.

HIV-1 antibody testing strategy: evaluation of ELISA screening and Western blot profiles in a mixed low risk/high risk patient population

The Westmead HIV-1 antibody testing strategy showed that, regardless of ELISA screening kit manufacturer, sera which were repeatedly positive by two ELISA screening assays (one indirect and the other competitive format) had a 97–98% chance of being confirmed positive by Western blot for HIV-1 antibody or a less than 3% chance of either being identified as a seroconverter (1%) or a late stage AIDS patient (1.2%). Sera which were discordant by two ELISA screening assays had a less than 4% chance of either being confirmed positive by Western blot (2.5%) or identified as a seroconverter (1.3%). The incidence of non-specific indeterminate Western blot profiles were shown to be inversely proportional to the specificity of the ELISA screening kits used. The use of a recombinant envelope ELISA was able to confirm the viral specificity of HIV-1 envelope bands (gp160, 120 or 41) on Western blot. Guidelines suggested by the Australian National HIV Reference Laboratory, Fairfield Hospital, Melbourne, which categorized indeterminate or atypical Western blot profiles into four reaction groups were found to be useful for the interpretation of Western blot patterns. A Western blot profile which is reactive for HIV-1 viral glycoproteins (gp160, 120 and 41) alone or in combination with not more than two other viral proteins (Indeterminate Group 4) and which is confirmed viral envelope specific by a recombinant envelope ELISA can be used as a predictor of seroconversion.

Evaluation of enzyme-linked immunosorbent assays: a method of data analysis

Diagnostic tests are usually evaluated in terms of simple qualitative measures of sensitivity and specificity. When comparing different quantitative assays such as ELISAs, it is often more useful to deal with actual values (sample optical density/cut-off optical density ratio (OD ratio)) rather than the qualitative relationship to the cut-off, i.e. positive or negative. This allows for a statistical approach to the questions of sensitivity and specificity. The National HIV Reference Laboratory of Australia has developed an approach for determining statistical estimates of sensitivity and specificity in terms of delta (δ). Delta is defined as the distance of the mean OD ratio of the sample population from the cut-off measured in standard deviation units. This paper discusses the derivation of this measurement and its usefulness when evaluating ELISA tests.