Waranya Wongwit

Lead levels of Culex mosquito larvae inhabiting lead utilizing factory

OBJECTIVE To determine lead level primarily in Culex quinquefasciatus (Cx. quinquefasciatus), and Culex gelidus (Cx. gelidus) larvae inhabiting lead consuming factories, and to putatively estimate eco-toxicological impact of effluents from the firms. METHODS Third instars larvae were sampled by standard dipping method and lead concentrations in the larvae and their respective surrounding factory aquatic environments were determined through standard atomic absorption spectrophotometry (AAS). RESULTS Cx. quinquefasciatus was the most abundant species followed by Cx. gelidus. The levels of lead were higher in the Cx. quinquefasciatus (1.08-47.47 µg/g), than in the wastewaters surface (0.01-0.78 µg/mL) from the factories or closer areas around factories. Other species were not reaching the criteria for lead determination. CONCLUSIONS The Cx. quinquefasciatus larvae can bio-accumulate the metal and can potentially serve as a biomarker of lead contamination, to complemente conventional techniques.

Genetic Variations of Glutathione S-Transferase Influence on Blood Cadmium Concentration

The glutathione S-transferases (GSTs) are involved in biotransformation and detoxification of cadmium (Cd). Genetic polymorphisms in these genes may lead to interindividual variation in Cd susceptibility. The objective of this study was to assess the association of GSTs (GSTT1, GSTM1, and GSTP1 Val105Ile) polymorphisms with blood Cd concentrations in a nonoccupationally exposed population. The 370 blood samples were analyzed for Cd concentration and polymorphisms in GSTs genes. Geometric mean of blood Cd among this population was 0.46 ± 0.02 μg/L (with 95% CI; 0.43–0.49 μg/L). Blood Cd concentrations in subjects carrying GSTP1 Val/Val genotype were significantly higher than those with Ile/Ile and Ile/Val genotypes. No significant differences in blood Cd concentrations among individual with gene deletions of GSTT1 and GSTM1 were observed. GSTP1/GSTT1 and GSTP1/GSTM1 combinations showed significantly associated with increase in blood Cd levels. This study indicated that polymorphisms of GSTP1 combined with GSTT1 and/or GSTM1 deletion are likely to influence on individual susceptibility to cadmium toxicity.

Effectiveness of Using Mobile Phone Image Capture for Collecting Secondary Data: A Case Study on Immunization History Data Among Children in Remote Areas of Thailand

Background Entering data onto paper-based forms, then digitizing them, is a traditional data-management method that might result in poor data quality, especially when the secondary data are incomplete, illegible, or missing. Transcription errors from source documents to case report forms (CRFs) are common, and subsequently the errors pass from the CRFs to the electronic database. Objective This study aimed to demonstrate the usefulness and to evaluate the effectiveness of mobile phone camera applications in capturing health-related data, aiming for data quality and completeness as compared to current routine practices exercised by government officials. Methods In this study, the concept of “data entry via phone image capture” (DEPIC) was introduced and developed to capture data directly from source documents. This case study was based on immunization history data recorded in a mother and child health (MCH) logbook. The MCH logbooks (kept by parents) were updated whenever parents brought their children to health care facilities for immunization. Traditionally, health providers are supposed to key in duplicate information of the immunization history of each child; both on the MCH logbook, which is returned to the parents, and on the individual immunization history card, which is kept at the health care unit to be subsequently entered into the electronic health care information system (HCIS). In this study, DEPIC utilized the photographic functionality of mobile phones to capture images of all immunization-history records on logbook pages and to transcribe these records directly into the database using a data-entry screen corresponding to logbook data records. DEPIC data were then compared with HCIS data-points for quality, completeness, and consistency. Results As a proof-of-concept, DEPIC captured immunization history records of 363 ethnic children living in remote areas from their MCH logbooks. Comparison of the 2 databases, DEPIC versus HCIS, revealed differences in the percentage of completeness and consistency of immunization history records. Comparing the records of each logbook in the DEPIC and HCIS databases, 17.3% (63/363) of children had complete immunization history records in the DEPIC database, but no complete records were reported in the HCIS database. Regarding the individual’s actual vaccination dates, comparison of records taken from MCH logbook and those in the HCIS found that 24.2% (88/363) of the children’s records were absolutely inconsistent. In addition, statistics derived from the DEPIC records showed a higher immunization coverage and much more compliance to immunization schedule by age group when compared to records derived from the HCIS database. Conclusions DEPIC, or the concept of collecting data via image capture directly from their primary sources, has proven to be a useful data collection method in terms of completeness and consistency. In this study, DEPIC was implemented in data collection of a single survey. The DEPIC concept, however, can be easily applied in other types of survey research, for example, collecting data on changes or trends based on image evidence over time. With its image evidence and audit trail features, DEPIC has the potential for being used even in clinical studies since it could generate improved data integrity and more reliable statistics for use in both health care and research settings.

Ethical issues in research involving minority populations: the process and outcomes of protocol review by the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University, Thailand

BackgroundRecruiting minorities into research studies requires special attention, particularly when studies involve “extra-vulnerable” participants with multiple vulnerabilities, e.g., pregnant women, the fetuses/neonates of ethnic minorities, children in refugee camps, or cross-border migrants. This study retrospectively analyzed submissions to the Ethics Committee of the Faculty of Tropical Medicine (FTM-EC) in Thailand. Issues related to the process and outcomes of proposal review, and the main issues for which clarification/revision were requested on studies, are discussed extensively.MethodsThe study data were extracted from proposals and amendments submitted to the FTM-EC during the period October 2009 – September 2012, and then analyzed qualitatively and quantitatively. The main issues for clarification/revision were analyzed by thematic content analysis.Results373 proposals were submitted; 44 studies involved minority groups with 21 extra-vulnerable minorities. All clinical and 2/3 of non-clinical studies submitted for initial review underwent full-board review. For combined clinical and non-clinical study submissions, 92.1% were referred back to the investigators and approved after clarification/revision, while 2.7% were deferred due to major/critical changes, and 2.1% not approved due to substantial violations of ethical principles. The main issues needing clarification/revision differed between all studies and those involving minorities: participant information sheet (62.2% vs. 86.4%), informed consent/assent form (51.2% vs. 86.4%), and research methodology (80.7% vs. 84.1%), respectively. The main ethical issues arising during the meetings, regarding studies involving minorities, included ensuring no exploitation, coercion, or pressure on the minority to participate; methodology not affecting their legal status; considering ethnicity and cultural structure; and providing appropriate compensation.ConclusionDelays in the approval or non-approval of studies involving minorities were mainly due to major or minor deviations from acceptable ethical standards and/or unclear research methodology. The FTM-EC has employed several mechanisms in its operations, including transparency in the review process, building good relationships via open communication with investigators, requesting investigators to consider closely the necessity to enroll minority groups and the risk-benefits for individuals and their communities, and the inclusion of minority-community engagement when developing the proposal. Other effective activities include annual study-site inspections, and offering refresher courses to raise awareness of minority and vulnerability issues among researchers.

Antibody germline characterization of cross-neutralizing human IgGs against 4 serotypes of dengue virus

Abstract Dengue virus (DENV), a re-emerging virus, constitutes the largest vector-borne disease virus, with 50–100 million cases reported every year. Although DENV infection induces lifelong immunity against viruses of the same serotypes, the subsequent infection with the heterologous serotypes can cause more severe form of the disease, such as Dengue Haemorrhagic Fever (DHF) or Dengue Shock Syndrome (DSS). However, there is neither approved vaccine nor specific drugs available to treat this disease. In this study, previously developed 19 human monoclonal antibodies (HuMAbs) showing strong to moderate cross neutralizing activity were selected. Most of them (13/19) were targeted to domain II of envelop glycoprotein. To understand and clarify the recognition properties, the maturation mechanisms comprising Variable/Diversity/Joining (VDJ) recombination, Variable Heavy (VH)/Variable Light (VL) chain pairing, variability at junctional site, and somatic hypermutation (SHM) of those antibodies were studied and compared with their predecessor germline sequences. IMGT/V-QUEST database was applied to analyze the isolated VH and VL sequences. To confirm the correction of isolated VH/VL, 3 HuMAbs (1A10H7, 1B3B9, 1G7C2) was transiently expressed in HEK293T cell. All three clones of the expressed recombinant IgG (rIgG) showed the same binding and neutralizing activity as same as those from hybridomas. The data obtained in this study will elucidate the properties of those HuMAbs for further genetic modification, and its binding epitopes.

Human single-chain variable fragment antibody expressed in E. coli with optimal in vitro cross-neutralizing and no enhancing activity

Single chain fragment variable (scFv) is a small molecule antibody comprising of only the variable region of heavy and light chain responsible for antigen binding. For dengue disease, the Fc region of antibody molecule was reported to be involved with dengue complication caused by Antibody-dependent enhancement (ADE). We attempted to produce small molecule scFv human monoclonal antibody (HuMAb), which lacking the Fc portion to eliminate the ADE effect of the IgG. This scFv antibody was produced in Escherichia coli. The biologically active form of scFv antibody was successfully generated. 23-1C2D2-scFv showed neutralizing activity similar to the IgG obtained from parental hybridoma, but lacked enhancing activity in all studied concentrations. This antibody was targeted to the 101WXN103 motif of dengue envelop protein domain II, studied by western blot analysis with truncated E protein and random peptide phage display. This scFv is verified as a candidate for further development as therapeutic candidate for DENV infection.

Generation and characterization of cross neutralizing human monoclonal antibody against 4 serotypes of dengue virus without enhancing activity

Background Dengue disease is a leading cause of illness and death in the tropics and subtropics. Most severe cases occur among patients secondarily infected with a different dengue virus (DENV) serotype compared with that from the first infection, resulting in antibody-dependent enhancement activity (ADE). Our previous study generated the neutralizing human monoclonal antibody, D23-1B3B9 (B3B9), targeting the first domain II of E protein, which showed strong neutralizing activity (NT) against all four DENV serotypes. However, at sub-neutralizing concentrations, it showed ADE activity in vitro. Methods In this study, we constructed a new expression plasmid using the existing IgG heavy chain plasmid as a template for Fc modification at position N297Q by site-directed mutagenesis. The resulting plasmid was then co-transfected with a light chain plasmid to produce full recombinant IgG (rIgG) in mammalian cells (N297Q-B3B9). This rIgG was characterized for neutralizing and enhancing activity by using different FcγR bearing cells. To produce sufficient quantities of B3B9 rIgG for further characterization, CHO-K1 cells stably secreting N297Q-B3B9 rIgG were then established. Results The generated N297Q-B3B9 rIgG which targets the conserved N-terminal fusion loop of DENV envelope protein showed the same cross-neutralizing activity to all four DENV serotypes as those of wild type rIgG. In both FcγRI- and RII-bearing THP-1 cells and FcγRII-bearing K562 cells, N297Q-B3B9 rIgG lacked ADE activity against all DENV serotypes at sub-neutralizing concentrations. Fortunately, the N297Q-B3B9 rIgG secreted from stable cells showed the same patterns of NT and ADE activities as those of the N297Q-B3B9 rIgG obtained from transient expression against DENV2. Thus, the CHO-K1 stably expressing N297Q-B3B9 HuMAb can be developed as high producer stable cells and used to produce sufficient amounts of antibody for further characterization as a promising dengue therapeutic candidate. Discussion Human monoclonal antibody, targeted to fusion loop of envelope domainII (EDII), was generated and showed cross-neutralizing activity to 4 serotypes of DENV, but did not cause any viral enhancement activity in vitro. This HuMAb could be further developed as therapeutic candidates.