Point and interval estimation in two-stage adaptive designs with time to event data and biomarker-driven subpopulation selection

In personalized medicine, it is often desired to determine if all patients or only a subset of them benefit from a treatment. We consider estimation in two‐stage adaptive designs that in stage 1 recruit patients from the full population. In stage 2, patient recruitment is restricted to the part of the population, which, based on stage 1 data, benefits from the experimental treatment. Existing estimators, which adjust for using stage 1 data for selecting the part of the population from which stage 2 patients are recruited, as well as for the confirmatory analysis after stage 2, do not consider time to event patient outcomes. In this work, for time to event data, we have derived a new asymptotically unbiased estimator for the log hazard ratio and a new interval estimator with good coverage probabilities and probabilities that the upper bounds are below the true values. The estimators are appropriate for several selection rules that are based on a single or multiple biomarkers, which can be categorical or continuous

Adjusting for treatment selection in phase II/III clinical trials with time to event data

Phase II/III clinical trials are efficient two‐stage designs that test multiple experimental treatments. In stage 1, patients are allocated to the control and all experimental treatments, with the data collected from them used to select experimental treatments to continue to stage 2. Patients recruited in stage 2 are allocated to the selected treatments and the control. Combined data of stage 1 and stage 2 are used for a confirmatory phase III analysis. Appropriate analysis needs to adjust for selection bias of the stage 1 data. Point estimators exist for normally distributed outcome data. Extending these estimators to time to event data is not straightforward because treatment selection is based on correlated treatment effects and stage 1 patients who do not get events in stage 1 are followed‐up in stage 2. We have derived an approximately uniformly minimum variance conditional unbiased estimator (UMVCUE) and compared its biases and mean squared errors to existing bias adjusted estimators. In simulations, one existing bias adjusted estimator has similar properties as the practically unbiased UMVCUE while the others can have noticeable biases but they are less variable than the UMVCUE. For confirmatory phase II/III clinical trials where unbiased estimators are desired, we recommend the UMVCUE or the existing estimator with which it has similar properties

Performance of methylcellulose and Avicel overlays in plaque and focus assays of Chikungunya virus

Background: Chikungunya virus is a re-emerging pathogen that is responsible for Chikungunya fever periodic outbreaks along the Kenyan coast and in other African countries.  Epidemiological data from the World Health Organization show that in 2014-2015, there was a major outbreak of Chikungunya fever in the Americas and Pacific Islands.  Surveillance and correct diagnosis are therefore key in controlling the spread and management of the disease. Plaque and focus assays are key techniques in viral characterization or quantification, and both assays typically require overlay with gelling polymers to limit the spread of viruses in cell culture.  There are anecdotal reports that Avicel may be superior to methylcellulose in assay of Influenza virus. However, it is unclear whether this would apply to other viruses. Objective: The objective of this study was to determine the performance of methylcellulose and Avicel overlays in plaque and focus assays of Chikungunya virus. Methods: Confluent Vero cells were seeded in 6- or 96-well plates for plaque and focus assays respectively. Cells were inoculated with serially diluted Chikungunya virus, and incubated to allow adherence of the virus to the cells. The inoculum was removed; replaced with Avicel or methylcellulose overlay at various concentrations and stained with crystal violet or immunostained.  Statistical significance was computed using the Holm-Sidak test. Results: The size of plaques formed by Chikungunya virus was dependent on the concentration of both Avicel and methylcellulose gels used as overlays, with Avicel overlays giving consistently larger plaques than methylcellulose.  Chikungunya virus formed plaques nearly 2.5 times larger in diameter (2 vs 0.8 mm) with 1.2 % Avicel than with 1.25 % methylcellulose after 60 hr growth.  Plaques formed with Avicel were better defined and easier to count after 48 hr growth period compared to a 60 hr period. However, methycellulose overlays provided smaller, more distinct and better defined foci in focus assays. Conclusion: Both methylcellulose and Avicel are good overlay media for viral assays. Avicel is marginally better for plaque assays while methylcellulose provides more distinct and easier to count foci in focus assays. Key words: Chikungunya virus, plaque assay, focus assay, methylcellulose, Avice

Development of Dromedary Antibody-based Enzyme Linked Immunosorbent Assay for Detecting Chikungunya virus Infections

Background: Chikungunya virus (CHIKV) is an arthropod-borne Togavirus belonging to the genus Alphavirus that is responsible for sporadic worldwide outbreaks of Chikungunya fever, an acute febrile illness often associated with severe polyarthralgia. In Kenya, Chikungunya virus is of great epidemiological concern, with the last major outbreak occurring in 2016 in North Eastern Kenya. Reliable detection of CHIKV infections is key to controlling this re-emerging pathogen, for which no cure currently exists.  Current diagnostic methods for CHIKV employ a combination of tests, particularly immunologic, serologic or virologic techniques.  However, the independent scientific reviews on the validity and sensitivity of currently available commercial assays have been conflicting. Objective: This study aimed to develop and validate a dromedary antibody-based enzyme linked immunosorbent assay for detecting Chikungunya virus infections. Methods: To produce sufficient antigen for camel immunization, Chikungunya virus (strain Lamu 33) was propagated in confluent C6-36 E2 cells using Cytodex microcarrier system. Purified and inactivated CHIKV immunogen was used to inoculate two camels reared at the University of Nairobi farm in Kibwezi, Kenya. Camel serum samples collected over the entire immunization period were assayed for the presence of anti-Chikungunya IgG by indirect ELISA. Purification of camel Heavy Chain IgG antibodies was performed by lectin affinity chromatography on protein A and protein G-Sepharose columns; then conjugated with horse radish peroxidase (HRP). The HRP-conjugated camel Heavy Chain IgG2 and IgG3 were optimized for ELISA, with optical density measured using a microplate reader set at 492nm.   A total of 188 human sera samples were assayed using the developed dromedary-based enzyme linked immunosorbent assay to determine Chikungunya virus infections. Results: The sensitivity of the dromedary HCAb IgG2 assay was 91.3% (95% CI: 0.831 - 0.994); while that for HCAb IgG3 assay was 95.7% (95% CI: 0.898 - 1.01).  The specificity of HCAb IgG2 assay was 92.3% (95% CI: 0.879 - 0.967); while the specificity of HCAb IgG3 method was 90% (95% CI: 0.851 - 0.949). For HCAb IgG2 and IgG3 based assays, the positive predictive values were 79.2% and 75.8 % respectively; while the negative predictive values were 97% and 98.4% for HCAb IgG2 and IgG3 based assays respectively. Conclusion: The camel antibody based assay was found to be reliable assay with very good sensitivity and specificity, and can be deployed for detection of Chikungunya virus infections. Key words: Chikungunya, ELISA, camel antibodies, diagnosi

Evaluation of malaria rapid diagnostic tests among children in a malaria endemic region in coastal Kenya

Background: In Kenya, malaria case management is based on clinical suspicion and detection of parasite in blood by parasitological or confirmatory diagnosis. Confirmatory diagnosis can be achieved with either microscopy or Rapid diagnostic tests (RDTs). RDTs are relatively new technologies, and their performance in actual conditions of use needs to be evaluated to provide information for appropriate use and to support decision making in procurement. Objectives: To evaluate performance and operational characteristics of three malaria RDTs: CareStart™, First Response®, and SD Bioline™ in the field for diagnosis of infection by Plasmodium falciparum monospecies as well as mixed infections with P. ovale and P. malariae. Methodology: A prospective study with blind comparisons to a gold standard was carried out at Pingilikani dispensary in Kilifi County, Kenya. Blood samples were obtained from 500 febrile children. Three RDTs: CareStart™, First Response® and SD Bioline™ were evaluated against microscopy of Giemsa stained blood films for detection of Plasmodium falciparum and non-falciparum malarial parasites. RDTs specific for P. falciparum only (HRP2 RDTs) and non-falciparum malarial parasites (HRP2/pLDH (Pf/pan) RDTs) were evaluated. Results: Plasmodium sp were detected by microscopy in 242 (48.40%) study participants. Plasmodium falciparum species were the most prevalent (93.3%) in comparison with other Plasmodium species: P. ovale and P. malariae whose prevalence were 2.89% and 3.71% respectively. Compared to microscopy the sensitivities of CareStart™, SD Bioline™, and First Response® RDTs for Plasmodium falciparum using Pf (mono species) kits were: 95.04% (95% CI: 92.34 - 97.73), 95.04% (95% CI: 92.34 - 97.73) and 94.21% (95% CI: 91.3 - 97.11) respectively while the specificities were 78.12% (95% CI: 72.98 - 83.25), 81.10% (95% CI: 76.23 - 85.96) and 78.74%  (95% CI: 73.65 - 83.82) respectively. Sensitivities of CareStart™, SD Bioline™ and First Response® RDTs for Plasmodium falciparum using Pf/Pan kits were: 99.02% (95% CI: 98.92 - 99.15), 99.04% (95% CI: 98.92 – 99.15) and 97.56% (95% CI: 97.78 – 97.99), respectively while the specificities were 78.46% (95% CI: 77.61 - 79.30), 78.46% (95% CI: 75.78 - 81.13) and 80.28% (95% CI: 76.73 - 83.82) respectively. CareStart™, SD Bioline™, and First Response® RDTs for non-falciparum sp using Pf/Pan kits both had 100% sensitivity and specificity. Conclusion: Data from this study demonstrate that CareStart™, SD Bioline™ and First Response® RDTs have good operational characteristics and are reliable alternatives to microscopy for diagnosing malaria in children. Key words: malaria, rapid diagnostic tests, microscopy, Plasmodiu

Global, regional, and national disability-adjusted life-years (DALYs) for 333 diseases and injuries and healthy life expectancy (HALE) for 195 countries and territories, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016

BACKGROUND: Measurement of changes in health across locations is useful to compare and contrast changing epidemiological patterns against health system performance and identify specific needs for resource allocation in research, policy development, and programme decision making. Using the Global Burden of Diseases, Injuries, and Risk Factors Study 2016, we drew from two widely used summary measures to monitor such changes in population health: disability-adjusted life-years (DALYs) and healthy life expectancy (HALE). We used these measures to track trends and benchmark progress compared with expected trends on the basis of the Socio-demographic Index (SDI). METHODS: We used results from the Global Burden of Diseases, Injuries, and Risk Factors Study 2016 for all-cause mortality, cause-specific mortality, and non-fatal disease burden to derive HALE and DALYs by sex for 195 countries and territories from 1990 to 2016. We calculated DALYs by summing years of life lost and years of life lived with disability for each location, age group, sex, and year. We estimated HALE using age-specific death rates and years of life lived with disability per capita. We explored how DALYs and HALE differed from expected trends when compared with the SDI: the geometric mean of income per person, educational attainment in the population older than age 15 years, and total fertility rate. FINDINGS: The highest globally observed HALE at birth for both women and men was in Singapore, at 75·2 years (95% uncertainty interval 71·9-78·6) for females and 72·0 years (68·8-75·1) for males. The lowest for females was in the Central African Republic (45·6 years [42·0-49·5]) and for males was in Lesotho (41·5 years [39·0-44·0]). From 1990 to 2016, global HALE increased by an average of 6·24 years (5·97-6·48) for both sexes combined. Global HALE increased by 6·04 years (5·74-6·27) for males and 6·49 years (6·08-6·77) for females, whereas HALE at age 65 years increased by 1·78 years (1·61-1·93) for males and 1·96 years (1·69-2·13) for females. Total global DALYs remained largely unchanged from 1990 to 2016 (-2·3% [-5·9 to 0·9]), with decreases in communicable, maternal, neonatal, and nutritional (CMNN) disease DALYs offset by increased DALYs due to non-communicable diseases (NCDs). The exemplars, calculated as the five lowest ratios of observed to expected age-standardised DALY rates in 2016, were Nicaragua, Costa Rica, the Maldives, Peru, and Israel. The leading three causes of DALYs globally were ischaemic heart disease, cerebrovascular disease, and lower respiratory infections, comprising 16·1% of all DALYs. Total DALYs and age-standardised DALY rates due to most CMNN causes decreased from 1990 to 2016. Conversely, the total DALY burden rose for most NCDs; however, age-standardised DALY rates due to NCDs declined globally. INTERPRETATION: At a global level, DALYs and HALE continue to show improvements. At the same time, we observe that many populations are facing growing functional health loss. Rising SDI was associated with increases in cumulative years of life lived with disability and decreases in CMNN DALYs offset by increased NCD DALYs. Relative compression of morbidity highlights the importance of continued health interventions, which has changed in most locations in pace with the gross domestic product per person, education, and family planning. The analysis of DALYs and HALE and their relationship to SDI represents a robust framework with which to benchmark location-specific health performance. Country-specific drivers of disease burden, particularly for causes with higher-than-expected DALYs, should inform health policies, health system improvement initiatives, targeted prevention efforts, and development assistance for health, including financial and research investments for all countries, regardless of their level of sociodemographic development. The presence of countries that substantially outperform others suggests the need for increased scrutiny for proven examples of best practices, which can help to extend gains, whereas the presence of underperforming countries suggests the need for devotion of extra attention to health systems that need more robust support. FUNDING: Bill & Melinda Gates Foundation

Preliminary In Vivo Evidence of Oral Selenium Supplementation as a Potentiating Agent on a Vector-Based COVID-19 Vaccine in BALB/c Mice

Evidence of efficacy and toxicity of oral selenium supplementation in vaccine administration against severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) in mice models is scarce. In this study, 4 × 109 virus particles (40 µL) dose of Janssen COVID-19 intramuscular injection vaccine was supplemented with a commercial selenium supplement and sodium selenite orally in BALB/c mice (N = 18). Qualitative determination of anti-spike IgG antibody response using indirect Enzyme-Linked Immunosorbent Assay (ELISA) showed significant (p ≤ 0.001) increase in anti-spike IgG antibody response for mice groups immunized with vaccine and supplemented selenium. Furthermore, cytokine profiling using real-time quantitative polymerase chain reaction also showed an increase in IL-6 and IL-10 mRNA levels normalized using hypoxanthine phosphoribosyl transferase 1 (Hprt1) and glyceraldehyde 3-phosphate dehydrogenase (Gadph) housekeeping genes. There was no statistical significance (p < 0.465) among treated and untreated groups for alanine transaminase (ALT), aspartate transaminase (AST), urea, and creatinine parameters. The study presents preliminary findings and suggests that supplementing Janssen COVID-19 vaccines with selenium can generate more robust immune responses

Increase in the Immune Response in Balb/c Mice after the Co-Administration of a Vector-Based COVID-19 Vaccine with Cytosine Phosphoguanine Oligodeoxynucleotide

The effects of cytosine phosphoguanine oligodeoxynucleotides (CPG ODNs) on immune response have been demonstrated for different vaccines; however, such information is limited for the vector-based Coronavirus disease 2019 (COVID-19). This paper aims to demonstrate the potential effect of CPG ODNs on immunological response against the vector-based COVID-19 vaccine on Balb/c mice using a JNJ-78436735 Ad26.COV2-S recombinant as a model vaccine. A total of 18 BALB/c mice clustered into six groups were used. All groups were observed for 14- and 28-days post immunization. Qualitative determination of IgG was performed using indirect Enzyme-Linked Immunosorbent Assay (ELISA) and qPCR for cytokine profiling. A significant (p ≤ 0.001) rise in antibody response was observed for groups 3 and 4, who also showed increased expression levels of Tumor Necrosis Factor (TNF) and Interferon Gamma (IFN-γ). Immunological parameters for toxicity were normal in all treatment groups. We conclude that supplementing vector-based COVID-19 vaccines with CpG ODNs has the potential to boost the body’s immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection