Point-of-Care Detection of Nonadherence to Antiretroviral Treatment for HIV-1 in Resource-Limited Settings Using Drug Level Testing for Efavirenz, Lopinavir, and Dolutegravir: A Validation and Pharmacokinetic Simulation Study.

BACKGROUND: Virological failure during antiretroviral treatment (ART) may indicate the presence of drug resistance, but may also originate from nonadherence. Qualitative detection of ART components using drug level testing may be used to differentiate between these scenarios. We aimed to validate and implement qualitative point-of-care drug level tests for efavirenz (EFV), lopinavir (LPV), and dolutegravir (DTG) in rural South Africa. METHODS: Qualitative performance of immunoassays for EFV, LPV, and DTG was assessed by calculating limit of detection (LoD), region of uncertainty, and qualitative agreement with a reference test. Minimum duration of nonadherence resulting in a negative drug level test was assessed by simulation of treatment cessation using validated population pharmacokinetic models. RESULTS: LoD was 0.05 mg/L for EFV, 0.06 mg/L for LPV, and 0.02 mg/L for DTG. Region of uncertainty was 0.01-0.06 mg/L for EFV, 0.01-0.07 mg/L for LPV, and 0.01-0.02 mg/L for DTG. Qualitative agreement with reference testing at the LoD in patient samples was 95.2% (79/83) for EFV, 99.3% (140/141) for LPV, and 100% (118/118) for DTG. After simulated treatment cessation, median time to undetectability below LoD was 7 days [interquartile range (IQR) 4-13] for EFV, 30 hours (IQR 24-36) for LPV, and 6 days (IQR 4-7) for DTG. CONCLUSIONS: We demonstrate that qualitative ART drug level testing using immunoassays is feasible in a rural resource-limited setting. Implementation of this technology enables reliable detection of recent nonadherence and may allow for rapid and cost-effective differentiation between patients in need for adherence counseling and patients who require drug resistance testing or alternative treatment.

Acute Gastroenteritis Disease Burden in Infants With Medical Risk Conditions in the Netherlands.

BACKGROUND: Infants with medical risk conditions are vulnerable to childhood infections including acute gastroenteritis (AGE). To guide prevention programs, we quantified AGE incidence, severity and virus prevalence among medical risk infants in the Netherlands. METHODS: This prospective cohort-study was part of the RIVAR-project recruiting infants with prematurity, low birth weight or severe congenital conditions in 13 hospitals. Follow-up included 18 monthly health questionnaires detailing AGE symptoms and healthcare usage. Parents were also instructed to notify when an infant developed AGE, to collect a stool sample and complete a daily severity score (Modified Vesikari Severity). Stool samples were analyzed by real-time polymerase chain reaction for rotavirus, norovirus, adenovirus and astrovirus. RESULTS: Between November 2014 and October 2017, 631 infants participated during 9125 person-months of observation. In total, 559 episodes were identified. The mean AGE incidence rate was 73.5 per 100 person-years (PY) (95% confidence interval: 67.6-79.9) and increased with age [incidence rate: 48.3 (39.8-58.3) vs. 80.2 (73.0-88.1)/100 PY for ages 1-5 vs. 6-18 months, respectively]. Healthcare was attended for 38.1% (213/559) and 26.8% (68/254) were classified as severe based on the Modified Vesikari Severity. Stool samples were obtained from 254 AGE episodes. Norovirus was identified in 65 (25.6%) and rotavirus in 44 (17.7%). Adenovirus and astrovirus together accounted for 8.3% (N = 21). Severe AGE occurred most frequently in rotavirus positive episodes. CONCLUSION: The observed AGE incidence, severity and healthcare usage among medical risk infants confirms substantial disease burden. Norovirus and rotavirus are the dominant pathogens and severe episodes occurred most frequently in children with rotavirus infection. AGE prevention in medical risk infants should be prioritized.

The Evolving Landscape of HIV Drug Resistance Diagnostics for Expanding Testing in Resource-Limited Settings.

Global scale-up of antiretroviral treatment has dramatically changed the prospects of HIV/AIDS disease, rendering life-long chronic care and treatment a reality for millions of HIV-infected patients. Affordable technologies to monitor antiretroviral treatment are needed to ensure long-term durability of limited available drug regimens. HIV drug resistance tests can complement existing strategies in optimizing clinical decision-making for patients with treatment failure, in addition to facilitating population-based surveillance of HIV drug resistance. This review assesses the current landscape of HIV drug resistance technologies and discusses the strengths and limitations of existing assays available for expanding testing in resource-limited settings. These include sequencing-based assays (Sanger sequencing assays and nextgeneration sequencing), point mutation assays, and genotype-free data-based prediction systems. Sanger assays are currently considered the gold standard genotyping technology, though only available at a limited number of resource-limited setting reference and regional laboratories, but high capital and test costs have limited their wide expansion. Point mutation assays present opportunities for simplified laboratory assays, but HIV genetic variability, extensive codon redundancy at or near the mutation target sites with limited multiplexing capability have restricted their utility. Next-generation sequencing, despite high costs, may have potential to reduce the testing cost significantly through multiplexing in high-throughput facilities, although the level of bioinformatics expertise required for data analysis is currently still complex and expensive and lacks standardization. Web-based genotype-free prediction systems may provide enhanced antiretroviral treatment decision-making without the need for laboratory testing, but require further clinical field evaluation and implementation scientific research in resource-limited settings.

Design of the PROUD study: PCR faeces testing in outpatients with diarrhoea.

BACKGROUND: Infectious intestinal disease (IID) is an important cause of morbidity in developed countries and a frequent reason for general practitioner (GP) consultation. In recent years polymerase chain reaction (PCR) based techniques have gradually replaced conventional enteropathogen detection techniques like microscopy and culture in primary care patients suspected of IID. PCR features testing of multiple enteropathogens in a single faecal sample with shorter turnaround times and greater sensitivity compared to conventional techniques. However, the associated costs and benefits have not been quantified. Furthermore, primary care incidence and prevalence estimates of enteropathogens associated with IID are sparsely available and predominantly based on conventional techniques. The PROUD-study (PCR diagnostics in Outpatients with Diarrhoea) determines: 1) health (care) effects and 2) cost-effectiveness of PCR introduction in primary care patients suspected of IID; 3) occurrence of major enteropathogens in primary care patients suspected of IID. METHODS: A before-after cohort study will be performed of patients with suspected IID consulting a GP in the Utrecht General Practitioner Network (UGPN), covering the before period (2010-2011) with conventional testing and the after period (2013-2014) with PCR testing. Prospective study data on patient characteristics and primary outcome measures (i.e. healthcare use and disease outcome) will be collected from electronic patient and laboratory records in 2015 and 2016. The effect of PCR introduction is investigated by comparing the primary outcome measures and their associated healthcare costs between the conventional period and the PCR period, and is followed by a cost-effectiveness analysis. To determine the occurrence of enteropathogens associated with IID in primary care, routine care faeces samples from the year 2014 will be screened using PCR. DISCUSSION: The PROUD-study will quantify the costs and effects of the introduction of PCR techniques for enteropathogens in primary care patients suspected of IID and generate up-to-date and sensitive estimates of enteropathogen occurrence among primary care patients.

Clinical Evaluation of an Affordable Qualitative Viral Failure Assay for HIV Using Dried Blood Spots in Uganda.

BACKGROUND: WHO recommends regular viral load (VL) monitoring of patients on antiretroviral therapy (ART) for timely detection of virological failure, prevention of acquired HIV drug resistance (HIVDR) and avoiding unnecessary switching to second-line ART. However, the cost and complexity of routine VL testing remains prohibitive in most resource limited settings (RLS). We evaluated a simple, low-cost, qualitative viral-failure assay (VFA) on dried blood spots (DBS) in three clinical settings in Uganda. METHODS: We conducted a cross-sectional diagnostic accuracy study in three HIV/AIDS treatment centres at the Joint Clinical Research Centre in Uganda. The VFA employs semi-quantitative detection of HIV-1 RNA amplified from the LTR gene. We used paired dry blood spot (DBS) and plasma with the COBASAmpliPrep/COBASTaqMan, Roche version 2 (VLref) as the reference assay. We used the VFA at two thresholds of viral load, (>5,000 or >1,000 copies/ml). RESULTS: 496 paired VFA and VLref results were available for comparative analysis. Overall, VFA demonstrated 78.4% sensitivity, (95% CI: 69.7%-87.1%), 93% specificity (95% CI: 89.7%-96.4%), 89.3% accuracy (95% CI: 85%-92%) and an agreement kappa = 0.72 as compared to the VLref. The predictive values of positivity and negativity among patients on ART for >12 months were 72.7% and 99.3%, respectively. CONCLUSIONS: VFA allowed 89% of correct classification of VF. Only 11% of the patients were misclassified with the potential of unnecessary or late switch to second-line ART. Our findings present an opportunity to roll out simple and affordable VL monitoring for HIV-1 treatment in RLS.

Impact of rapid detection of viral and atypical bacterial pathogens by real-time polymerase chain reaction for patients with lower respiratory tract infection.

BACKGROUND: Rapid diagnostic tests with a high sensitivity for lower respiratory tract infection (LRTI) could lead to improved patient care and reduce unnecessary antibiotic use and associated costs. Diagnostic yields, feasibility, and costs of real-time polymerase chain reaction (PCR) of nasopharyngeal and oropharyngeal swab specimens in the routine diagnostic work-up for LRTI were determined. METHODS: In a randomized controlled trial, nasopharyngeal and oropharyngeal swab specimens from patients admitted for antibiotic treatment of LRTI were evaluated by means of real-time PCR for respiratory viruses and atypical pathogens, as well as by conventional diagnostic procedures. Real-time PCR results for patients in the intervention group were reported to the treating physician; results for patients in the control group were not made available. RESULTS: A total of 107 patients (mean age [+/- standard deviation], 63.6+/-16.3 years) were included, of whom 55 were allocated to the intervention group. The pathogens detected most frequently were influenza virus (14 patients), Streptococcus pneumoniae (8), coronavirus (6), Staphylococcus aureus (5), and rhinoviruses (5). Real-time PCR increased the diagnostic yield from 23 cases (21% of patients) to 47 cases (43% of patients), compared with conventional diagnostic tests. The detection of viral pathogens by PCR was associated with the winter season, less infiltrates on chest radiographs, lower C-reactive protein levels, and shorter duration of symptoms. Use of real-time PCR results resulted in partial or total cessation of antibiotic treatment for 6 patients (11%; 95% confidence interval, 2-19), but overall antibiotic use was comparable in the intervention group and the control group (median duration of treatment, 10.0 vs. 9.0 days; P=not significant). Use of real-time PCR increased treatment and diagnostic costs with 318.17 per patient. CONCLUSIONS: Implementation of real-time PCR for the etiological diagnosis of LRTI increased the diagnostic yield considerably, but it did not reduce antibiotic use or costs.

Prevalence of drug-resistant HIV-1 variants in untreated individuals in Europe: implications for clinical management.

BACKGROUND: Infection with drug-resistant human immunodeficiency virus type 1 (HIV-1) can impair the response to combination therapy. Widespread transmission of drug-resistant variants has the disturbing potential of limiting future therapy options and affecting the efficacy of postexposure prophylaxis. METHODS: We determined the baseline rate of drug resistance in 2208 therapy-naive patients recently and chronically infected with HIV-1 from 19 European countries during 1996-2002. RESULTS: In Europe, 1 of 10 antiretroviral-naive patients carried viruses with > or = 1 drug-resistance mutation. Recently infected patients harbored resistant variants more often than did chronically infected patients (13.5% vs. 8.7%; P=.006). Non-B viruses (30%) less frequently carried resistance mutations than did subtype B viruses (4.8% vs. 12.9%; P<.01). Baseline resistance increased over time in newly diagnosed cases of non-B infection: from 2.0% (1/49) in 1996-1998 to 8.2% (16/194) in 2000-2001. CONCLUSIONS: Drug-resistant variants are frequently present in both recently and chronically infected therapy-naive patients. Drug-resistant variants are most commonly seen in patients infected with subtype B virus, probably because of longer exposure of these viruses to drugs. However, an increase in baseline resistance in non-B viruses is observed. These data argue for testing all drug-naive patients and are of relevance when guidelines for management of postexposure prophylaxis and first-line therapy are updated.

Evaluation of antiretroviral therapy results in a resource-poor setting in Blantyre, Malawi.

OBJECTIVE: To evaluate treatment results of the paying antiretroviral therapy (ART) clinic of Queen Elizabeth Central Hospital, a large public and teaching hospital in Blantyre, Malawi. The only ART was a fixed drug combination of stavudine, lamivudine and nevirapine. METHODS: Cross sectional study with interviews, laboratory tests (CD4 count, viral load, nevirapine plasma levels, transaminases) and data extraction from files. RESULTS: A total of 422 (59%) of the patients who started ART since 2000 were lost to follow-up. The 176 patients enrolled in the study had good virological and excellent clinical treatment results. The most common side effect was peripheral neuropathy. Nevirapine plasma levels were remarkably high and associated with successful virological treatment results. Two simple adherence questions pertaining to the use of medication in the previous 8 days corresponded well with nevirapine levels. The most important reasons for non-adherence were shortage of drugs in the hospital pharmacy and personal financial constraints. CONCLUSIONS: (1) Many patients were lost to follow-up. (2) High nevirapine levels contributed to good therapy results in those studied. (3) Simple adherence questions predicted subtherapeutic nevirapine levels. (4) Antiretroviral drug supply needs to be uninterrupted and free of charge, to prevent avoidable non-adherence.

Assessment of adherence to HIV protease inhibitors: comparison and combination of various methods, including MEMS (electronic monitoring), patient and nurse report, and therapeutic drug monitoring.

BACKGROUND: Adherence to protease inhibitor-containing antiretroviral therapy is crucial, but difficult to measure. OBJECTIVE: To compare and combine various methods of measuring adherence to the strict protease inhibitor-containing regimens. METHODS: The following methods were used: medication event monitoring system (MEMS) caps (electronic monitoring), therapeutic drug monitoring, pill count, pharmacy refill data, questionnaires, diaries (for registration of food patterns and special events related to the use of MEMS), adherence assessment by the physician and clinical nurse specialist, and in-depth interviews. In addition, ultrasensitive viral load and resistance testing was performed. RESULTS: Twenty-eight patients were included; data could be evaluated in 26. According to MEMS data, 25% of the patients took fewer than 95% of all doses, and two thirds of the patients took fewer than 95% of the doses on time. Only 43% of the patients showed good adherence with food restrictions. Methods that showed significant correlations with MEMS results were patients' self-reported adherence; therapeutic drug monitoring, indicating plasma levels outside predefined ranges; and estimation of adherence by a clinical nurse specialist, especially by in-depth interview. CONCLUSION: Diary-corrected MEMS data gave a detailed insight into patients' adherence patterns. Patients' self-report and therapeutic drug monitoring were significantly correlated with the MEMS data, and the clinical nurse specialist may also play a role in identifying patients who are imperfectly adherent.