Performance evaluation of alternate ESR measurement method using BC-780 automated hematology analyzer: a comparison study with the Westergren reference method.

OBJECTIVES: Implementation of alternate erythrocyte sedimentation rate (ESR) measurement method is increasing worldwide due to its various advantages. In this study, we aim to evaluate the analytical performance of the BC-780 automated hematology analyzer in measurement of ESR value. METHODS: Analyzer performance including precision study, carryover, sample stability and potential interferences are examined. Samples with ESR values spanning the whole analytical ESR range are included for method comparison study. Samples with different hematocrit (Hct) and mean corpuscular volume (MCV) values are also analyzed and compared with the results obtained from the Westergren reference method. RESULTS: Precisions and carryover results are consistent with the manufacturers' claim. ESR values do not change significantly in the samples stored at 2-8 °C for 24 h (h) or at room temperature (RT) for 8 h, but significantly decreased (p<0.001) when stored at RT for 24 h. Significant increase in ESR value is documented in samples that are hemolyzed (hemoglobin concentration ranged from 1.28-6.01 g/L) (p=0.010) or lipemic (triglyceride above 4.75 mmol/L) (p=0.001). Method comparison study yields a proportional difference with a regression equation=3.08+ 0.98x. Bland-Altman analysis shows a mean absolute bias of 3.12 mm. The obtained absolute mean biases are below 5 mm in all analytical categories except for the group where MCV>100 fL. CONCLUSIONS: Most tested parameters met the manufacturer's specifications and were comparable to the reference method. Despite the presence of positive bias, it falls within acceptable criteria. Extensive validation against potential interferences such as hemolysis/lipemia is still necessary in future.

Randomised controlled trial of glucose-6-phosphate dehydrogenase deficient versus non-deficient red blood cell transfusion in patients with hypoproliferative anaemia.

BACKGROUND: Recent studies revealed the glucose-6-phosphate dehydrogenase (G-6-PD) deficiency prevalence of 7.7-10% among Thai blood donors. Transfusion of red blood cells (RBCs) from these subjects potentially causes haemolysis in recipients. METHODS: RBC units from the National Blood Centre were sampled to assess G-6-PD levels using spectrophotometry. Patients with pure underproduction anaemia requiring blood transfusion were randomised to receive G-6-PD-deficient versus normal ABO-matched RBCs. Pre- and 48-h post-transfusion indirect bilirubin, haemoglobin, haematocrit, lactate dehydrogenase (LDH) and haptoglobin were measured. RESULTS: From April 2020 to March 2021, 374 RBC units were tested for G-6-PD, and that 25 were found to be G-6-PD deficient. Twelve units of G-6-PD-deficient RBCs and 14 units of normal RBCs were given to patients who met the inclusion criteria. The median (interquartile range) increases of indirect bilirubin in G-6-PD-deficient (N = 11) versus normal RBCs (N = 13) were + 0.12 (0.27) versus + 0.01 (1.3) mg/dl, p = 0.030), respectively. The median increases of haemoglobin were 1.00 (0.50) versus + 0.80 (0.95), p = 0.910, respectively. The increases in haematocrit were 2.59 (1.9) versus 2.29 (2.1), p = 0.733, respectively. There were no significant differences in changes of LDH and haptoglobin levels and no transfusion reactions. DISCUSSION: G-6-PD-deficient packed red cells were associated with mildly elevated indirect bilirubin after transfusion, but there was no observed clinical symptoms.

Immunogenicity and protective efficacy of SARS-CoV-2 mRNA vaccine encoding secreted non-stabilized spike in female mice.

Establishment of an mRNA vaccine platform in low- and middle-income countries (LMICs) is important to enhance vaccine accessibility and ensure future pandemic preparedness. Here, we describe the preclinical studies of "ChulaCov19", a SARS-CoV-2 mRNA encoding prefusion-unstabilized ectodomain spike protein encapsulated in lipid nanoparticles (LNP). In female BALB/c mice, ChulaCov19 at 0.2, 1, 10, and 30 µg elicits robust neutralizing antibody (NAb) and T cell responses in a dose-dependent relationship. The geometric mean titers (GMTs) of NAb against wild-type (WT, Wuhan-Hu1) virus are 1,280, 11,762, 54,047, and 62,084, respectively. Higher doses induce better cross-NAb against Delta (B.1.617.2) and Omicron (BA.1 and BA.4/5) variants. This elicited immunogenicity is significantly higher than those induced by homologous CoronaVac or AZD1222 vaccination. In a heterologous prime-boost study, ChulaCov19 booster dose generates a 7-fold increase of NAb against Wuhan-Hu1 WT virus and also significantly increases NAb response against Omicron (BA.1 and BA.4/5) when compared to homologous CoronaVac or AZD1222 vaccination. Challenge studies show that ChulaCov19 protects human-ACE-2-expressing female mice from COVID-19 symptoms, prevents viremia and significantly reduces tissue viral load. Moreover, anamnestic NAb response is undetectable in challenge animals. ChulaCov19 is therefore a promising mRNA vaccine candidate either as a primary or boost vaccination and has entered clinical development.

Minority HIV-1 resistant variants in recent infection and in patients who failed first-line antiretroviral therapy with no detectable resistance-associated mutations in Thailand.

Through the Thai National AIDS Program, approximately 200,000 patients infected with HIV are on antiretroviral (ARV) therapy. Although studies have shown low prevalence of primary HIV-1 resistance transmission in Thailand and in Southeast Asia where subtype CRF01_AE is predominant, minority HIV-1 drug resistance has not been studied. Two groups of patients, whose conventional genotyping results showed no drug resistance-associated mutations, were investigated: 104 homosexual men recently infected with HIV-1, naïve to ARV treatment and 22 first-line non-nucleoside reverse transcriptase inhibitor (NNRTI)-based failure patients. Pyrosequencing (PSQ) assay was developed to detect and quantify minority Y181C and M184V variants from the patients' plasma samples. The sensitivity of PSQ to detect minority Y181C and M184V variants was approximately 1%. 1/104 (0.5%) and 3/101 (3%) samples were found harboring Y181C and M184V in the group of homosexual men recently infected with HIV-1. In patients with first-line treatment failure, one had a minority M184V mutation (4.5%). The prevalence of Y181C and M184V minority variants in homosexual men recently infected and naïve to treatment was low in Thailand. Systematic monitoring of primary resistance transmission in Thailand and this region is essential to guide whether genotypic resistance test is required prior to commencing the first-line highly active antiretroviral therapy (HAART).

DNA vaccine candidate encoding SARS-CoV-2 spike proteins elicited potent humoral and Th1 cell-mediated immune responses in mice.

More than 65 million people have been confirmed infection with SARS-CoV-2 and more than 1 million have died from COVID-19 and this pandemic remains critical worldwide. Effective vaccines are one of the most important strategies to limit the pandemic. Here, we report a construction strategy of DNA vaccine candidates expressing full length wild type SARS-CoV-2 spike (S) protein, S1 or S2 region and their immunogenicity in mice. All DNA vaccine constructs of pCMVkan-S, -S1 and -S2 induced high levels of specific binding IgG that showed a balance of IgG1/IgG2a response. However, only the sera from mice vaccinated with pCMKkan-S or -S1 DNA vaccines could inhibit viral RBD and ACE2 interaction. The highest neutralizing antibody (NAb) titer was found in pCMVkan-S group, followed by -S1, while -S2 showed the lowest PRNT50 titers. The geometric mean titers (GMTs) were 2,551, 1,005 and 291 for pCMVkan-S, -S1 and -S2, respectively. pCMVkan-S construct vaccine also induced the highest magnitude and breadth of T cells response. Analysis of IFN-γ positive cells after stimulation with SARS-CoV-2 spike peptide pools were 2,991, 1,376 and 1,885 SFC/106 splenocytes for pCMVkan-S, -S1 and -S2, respectively. Our findings highlighted that full-length S antigen is more potent than the truncated spike (S1 or S2) in inducing of neutralizing antibody and robust T cell responses.

Optimization of a Der p 2-based prophylactic DNA vaccine against house dust mite allergy.

DNA vaccines encoding allergens are promising immunotherapeutics to prevent or to treat allergy through induction of allergen-specific Th1 responses. Despite anti-allergy effects observed in small rodents, DNA-based vaccines are weak immunogens in primates and humans and particularly when administered by conventional injection. The goal of the present study was to improve the immunogenicity of a prophylactic vaccine encoding the major house dust mite allergen Der p 2. In this context, we evaluated the influence of different DNA backbones including notably intron and CpG enriched sequence, the DNA dose, the in vivo delivery by electroporation as well as the heterologous prime boost regimen on the vaccine efficiency. We found that a minimal allergen expression level threshold must be reached to induce the production of specific antibodies but beyond this limit, the intensity of the immune response was independent on the DNA dose and allergen expression. The in vivo DNA delivery by electroporation drastically enhanced the production of specific antibodies but not the IFNg secretion. Vaccination of naïve mice with DNA encoding Der p 2 delivered by electroporation even at very low dose (2µg) prevented the development of house dust mite allergy through Th1-skewed immune response characterized by the drastic reduction of allergen-specific IgE, IL-5 and lung inflammation together with the induction of strong specific IgG2a titers and IFNg secretion. CpG cassette in the DNA backbone does not play a critical role in the efficient prophylaxis. Finally, comparable protective immune responses were observed when using heterologous DNA prime/protein boost or homologous DNA prime/boost. Taken together, these data suggest that the potent Th1 response induced by DNA-based vaccine encoding allergens through electroporation provides the rationale for the evaluation of DNA encoding Der p 2 into HDM allergy clinical trials.

A novel immunodominant CD8+ T cell response restricted by a common HLA-C allele targets a conserved region of Gag HIV-1 clade CRF01_AE infected Thais.

BACKGROUND: CD8+ T cell responses play an important role in the control of HIV-1. The extensive sequence diversity of HIV-1 represents a critical hurdle to developing an effective HIV-1 vaccine, and it is likely that regional-specific vaccine strains will be required to overcome the diversity of the different HIV-1 clades distributed world-wide. Unfortunately, little is known about the CD8+ T cell responses against CRF01_AE, which is responsible for the majority of infections in Southeast Asia. METHODOLOGY/PRINCIPAL FINDINGS: To identify dominant CD8+ T cell responses recognized in HIV-1 clade CRF01_AE infected subjects we drew upon data from an immunological screen of 100 HIV-1 clade CRF01_AE infected subjects using IFN-gamma ELISpot to characterize a novel immunodominant CD8+ T cell response in HIV-1 Gag restricted by HLA-Cw*0102 (p24, (277)YSPVSILDI(285), YI9). Over 75% of Cw*0102+ve subjects targeted this epitope, representing the strongest response in more than a third of these individuals. This novel CD8 epitope was located in a highly conserved region of HIV-1 Gag known to contain immunodominant CD8 epitopes, which are restricted by HLA-B*57 and -B*27 in clade B infection. Nonetheless, viral escape in this epitope was frequently observed in Cw*0102+ve subjects, suggestive of strong selection pressure being exerted by this common CD8+ T cell response. CONCLUSIONS/SIGNIFICANCE: As HLA-Cw*0102 is frequently expressed in the Thai population (allelic frequency of 16.8%), this immunodominant Cw*0102-restricted Gag epitope may represent an attractive candidate for vaccines specific to CRF01_AE and may help facilitate further studies of immunopathogenesis in this understudied HIV-1 clade.