Anti‐SARS‐CoV‐2 IgG antibody levels among Thai healthcare providers receiving homologous and heterologous COVID‐19 vaccination regimens

BackgroundWe examined SARS‐CoV‐2 anti‐spike 1 IgG antibody levels following COVID‐19 vaccination (AstraZeneca [AZ], Sinovac [SV], Pfizer‐BioNTech [PZ]) among Thai healthcare providers.MethodsBlood specimens were tested using enzyme‐linked immunosorbent assay. We analyzed seven vaccination regimens: (1) one dose of AZ or SV, (2) two doses of homologous (2AZ, 2SV) or heterologous (1AZ + 1PZ) vaccines, and (3) three doses of heterologous vaccines (2SV + 1AZ, 2SV + 1PZ). Differences in antibody levels were assessed using Kruskal–Wallis statistic, Mann–Whitney test, or Wilcoxon matched‐pairs signed‐rank test. Antibody kinetics were predicted using fractional polynomial regression.ResultsThe 563 participants had median age of 39 years; 92% were female; 74% reported no underlying medical condition. Antibody levels peaked at 22–23 days in both 1AZ and 2SV vaccinees and dropped below assay''s cutoff for positive (35.2 binding antibody units/ml [BAU/ml]) in 55 days among 1AZ vaccinees compared with 117 days among 2SV vaccinees. 1AZ + 1PZ vaccination regimen was highly immunogenic (median 2279 BAU/ml) 1–4 weeks post vaccination. 2SV + 1PZ vaccinees had significantly higher antibody levels than 2SV + 1AZ vaccinees 4 weeks post vaccination (3423 vs. 2105 BAU/ml; p‐value < 0.01), and during weeks 5–8 (3656 vs. 1072 BAU/ml; p‐value < 0.01). Antibodies peaked at 12–15 days in both 2SV + 1PZ and 2SV + 1AZ vaccinees, but those of 2SV + 1AZ declined more rapidly and dropped below assay''s cutoff in 228 days while those of 2SV + 1PZ remained detectable.Conclusions1AZ + 1PZ, 2SV + 1AZ, and 2SV + 1PZ vaccinees had substantial IgG levels, suggesting that these individuals likely mounted sufficient anti‐S1 IgG antibodies for possible protection against SARS‐CoV‐2 infection.     

A transgenic rat expressing green fluorescent protein (GFP) in peripheral nerves provides a new hindlimb model for the study of nerve injury and regeneration

The norepinephrine transporter (NET) is an important target for a wide variety of antidepressants and psychostimulants. Despite its prominence as a drug target, there is only one radioligand in use for NET competitive binding assays, [³H]nisoxetine. However, traditional [³H]nisoxetine binding protocols often give an underestimation for the affinity of certain classes of NET ligands, particularly cocaine and other tropanes. Here, we explore the feasibility of using the phenyltropane [³H]CFT for labeling human NET (hNET) in heterologous cell-based binding studies. Assays were optimized for time and protein content and specific, one-site binding was observed. Potencies of tested NET ligands for inhibition of [³H]CFT binding to whole cells (at physiological [Na⁺] and 25 °C) were similar to potencies observed in the [³H]NE uptake assay. Inhibition constants (K_i) for binding assays were highly correlated with uptake inhibition constants for all compounds tested (R² = 0.99, p < 0.0001). Cell-free membrane preparations did not display the same pharmacological profile. Under conditions routinely used for measuring [³H]nisoxetine binding to membrane preparations (4 °C for 3 h, [Na⁺] at 295 mM), the potency of nisoxetine and desipramine in inhibiting [³H]CFT binding became greater than that measured in a functional assay of [³H]NE uptake at physiological [Na⁺]. However, the opposite was true for CFT and cocaine. Interestingly, while investigating [³H]CFT as a potential NET radioligand, we uncovered evidence suggesting that CFT and nisoxetine are not mutually exclusive in binding to the NET. Dixon plots of the interaction between nisoxetine and CFT in inhibition of [³H]dopamine uptake by the NET indicate that the two compounds can simultaneously bind to the transporter.     

Drinking water: a possible source of Blastocystis spp. subtype 1 infection in schoolchildren of a rural community in central Thailand

In January 2005, a survey of intestinal parasitic infections was performed in a primary school, central Thailand. Of 675 stool samples, Blastocystis was identified with a prevalence of 18.9%. Genetic characterization of Blastocystis showed subtype 1 (77.9%) and subtype 2 (22.1%). Study of the water supply in this school was performed to find the possible sources of Blastocystis. Blastocystis from one water sample was identified as subtype 1, which had a nucleotide sequence of small subunit (SSU) ribosomal RNA (rRNA) gene that was 100% identical to that of Blastocystis infected in schoolchildren. Our information supports the evidence of water-borne transmission in this population.     

Nerve endoneurial microstructure facilitates uniform distribution of regenerative fibers: a post hoc comparison of midgraft nerve fiber densities

Despite their inferiority to nerve autograft, clinical alternatives are commonly used for reconstruction of peripheral nerve injuries because of their convenient off-the-shelf availability. Previously, our group compared isografts with NeuraGen(?) (Integra, Plainsboro, NJ) nerve guides, which are a commercially available type I collagen conduit and processed rat allografts comparable to Avance(?) (AxoGen, Alachua, FL) human decellularized allograft product. From this study, qualitative observations were made of distinct differences in the pattern of regenerating fibers within conduits, acellular allografts, and isografts. In the current post hoc analysis, these observations were quantified. Using nerve density, we statistically compared the differential pattern of regenerating axon fibers within grafts and conduit. The conduits exhibited a consistent decrease in midgraft density when compared with the isograft and acellularized allografts at two gap lengths (14 mm and 28 mm) and time points (12 and 22 weeks). The decrease in density was accompanied by clustered distribution of nerve fibers in conduits, which contrasted the evenly distributed regeneration seen in processed allografts and isografts. We hypothesize that the lack of endoneurial microstructure of conduits results in the clustering regenerating fibers, and that the presence of microstructure in the acellularized allograft and isografts facilitates even distribution of regenerating fibers.     

A systematic evaluation of Schwann cell injection into acellular cold-preserved nerve grafts

Peripheral nerve regeneration after injury depends on environmental cues and trophic support. Schwann cells (SCs) secrete trophic factors that promote neuronal survival and help guide axons during regeneration. The addition of SCs to acellular nerve grafts is a promising strategy for enhancing peripheral nerve regeneration; however, inconsistencies in seeding parameters have led to varying results. The current work sought to establish a systematic approach to seeding SCs in cold-preserved acellular nerve grafts. Studies were undertaken to (1) determine the needle gauge for optimal cell survival and minimal epineurial disruption during injection, (2) track the seeded SCs using a commercially available dye, and (3) evaluate the seeding efficiency of SCs in nerve grafts. It was determined that seeding with a 27-gauge needle resulted in the highest viability of SCs with the least damage to the epineurium. In addition, Qtracker(?) dye, a commercially available quantum dot nanocrystal, was used to label SCs prior to transplantation, which allowed visualization of the seeded SCs in nerve grafts. Finally, stereological methods were used to evaluate the seeding efficiency of SCs in nerve grafts immediately after injection and following a 1- or 3-day in vitro incubation in SC growth media. Using a systematic approach, the best needle gauge and a suitable dye for SC visualization in acellular nerve grafts were identified. Seeding efficiency in these grafts was also determined. The findings will lead to improvements ability to assess injection of cells (including SCs) for use with acellular nerve grafts to promote nerve regeneration.     

Comparison of acellular nerve allograft modification with Schwann cells or VEGF

Background

Individual contributions of exogenous Schwann cells (SCs) and vascular endothelial growth factor (VEGF) were evaluated in acellular nerve allografts (ANAs). ANA processing removes SCs and vasculature, likely contributing to reduced regeneration compared to autografts. Exogenous SCs may improve the regenerative microenvironment, and VEGF has been shown to stimulate angiogenesis. Replacing these components in ANAs may improve regeneration.

Methods

A rat sciatic nerve transection model was used to study 20-mm grafts. Four graft types were studied: (1) isograft, (2) ANA, (3) ANA-SCs, and (4) ANA-VEGF. After 10 weeks in vivo, the midgraft and distal nerve to the grafts were analyzed for axonal regeneration using histomorphometry to assess total myelinated axon counts, density, width, and percent neural tissue.

Results

The most axons in the distal nerve were regenerated in the isograft followed by the ANA- SC group, with 9171 ± 1822 and 7103 ± 1576 regenerated axons respectively. Both the ANA and ANA-VEGF groups had significantly fewer regenerated axons compared to the isograft (p < 0.05) with 5225 ± 2994 and 5709 ± 2657 regenerated axons, respectively. The ANA and ANA-VEGF groups also had significantly reduced fiber density and percent nerve compared to the isograft; the isograft and ANA-SC groups were not significantly different (p < 0.05).

Conclusions

These results show that SCs improve axonal regeneration in a 20 mm ANA to a greater extent than VEGF. VEGF treatment showed a trend toward increased axonal regeneration but was not significantly different compared to the untreated ANA. The role of VEGF may be clearer in longer grafts where ischemia is a greater factor.     

Multiple modes of transmission of giardiasis in primary schoolchildren of a rural community, Thailand

In February 2005, we conducted a cross-sectional study to determine the prevalence and the risk factors of giardiasis in 531 primary schoolchildren of a rural community, Chacheongsao province, Thailand. Using both sedimentation and flotation techniques to detect Giardia duodenalis, the prevalence of giardiasis was 6.2%. Assemblage A, subgenotype II and assemblage B, subgenotype IV were identified by PCR-RFLP of glutamate dehydrogenase gene. Our data might indicate that, in this population, only assemblage A, subgenotype II of G. duodenalis was transmitted via water. Using multivariate analysis, significant risk factors for giardiasis were children of age 5-9 years, households with > or = 3 children under the age of 12 years, low parental educational level, drinking bottled water, and living in close contact with dogs. Washing hands before meals had a protective effect. From these significant risk factors, multiple modes of transmission of G. duodenalis were suggested in this population.     

Genotypic Characterization of Enterocytozoon bieneusi in Specimens from Pigs and Humans in a Pig Farm Community in Central Thailand

We determined that 15.7% of pigs and 1.4% of humans in a pig farm community in central Thailand harbored Enterocytozoon bieneusi. Genotyping of E. bieneusi from pigs showed genotypes O, E, and H. However, only genotype A was found in human subjects. This indicates nonzoonotic transmission of E. bieneusi in this community.Enterocytozoon bieneusi is an opportunistic organism causing diarrhea in human immunodeficiency virus (HIV)-positive patients, in whom it has a prevalence of 2 to 50% (5). The infection not only has been reported to occur in immunocompromised hosts but also has been found in healthy individuals (14, 20). This organism can infect a broad range of animals (4, 12, 16, 18, 22, 23). Genotypes of E. bieneusi in humans and animals are differentiated using the polymorphisms of the internal transcribed spacer (ITS) sequence of the rRNA gene (4, 11). To date, at least 70 ITS genotypes have been reported to infect humans and animals (2, 6). The zoonotic nature of E. bieneusi was confirmed because ITS genotypes found in domestic and wild animals had been reported to occur in immunosuppressed hosts (22). In Thailand, we reported genotypes E, O, and PigEBITS 7, which have previously been identified in pigs (3, 4) and in Thai HIV-infected patients (9). This study aimed to identify the ITS genotypes of E. bieneusi in pigs and humans who worked in or lived near pig farms to investigate the transmission of E. bieneusi among these host species.A cross-sectional study of E. bieneusi infection was conducted in a community in Nakorn Pathom Province, Central Thailand, January 2005. This community is composed primarily of four pig farms, a residential area, and a school. The residential area, but not the school, was near the pig farms. Fecal specimens were collected from school children and those who were living in this community, including pig farmers. Fecal specimens were also collected from pigs of four farms and examined for E. bieneusi. The study was approved by the Ethics Committee of the Royal Thai Army, Medical Department. Informed consent was obtained from each adult individual and from parents of school children before enrollment in the study.Fecal specimens from pigs and humans were examined for microsporidian spores using gram-chromotrope staining under light microscopy (13). DNA was prepared from water-ethyl acetate-concentrated stool specimens using FTA filter paper (Whatman Bioscience, United Kingdom) as previously described (21). Amplification of the ITS region of the small-subunit rRNA gene was performed using primers under conditions described by Katzwinkel-Wladarsch et al. (8). For specimens with PCR-negative results, PCR amplification was repeated at least twice. DNA sequencing was conducted by Macrogen Inc., Seoul, Republic of Korea. Nucleotide sequences were determined using the Sequencher program (Gene Codes Corporation, Inc.), and multiple alignment was performed using Clustal X 1.83 for Windows (24). The genotype of each specimen was confirmed by determining the homology of the sequenced PCR product with the published sequence.A total of 268 pig fecal samples were collected. Pigs aged between 21 days and 22 months were examined for E. bieneusi infection. Microsporidial spores were found in 0.7% of pig fecal samples using gram-chromotrope staining, while a greater prevalence of E. bieneusi infection, 15.7%, was detected by PCR. The prevalences of E. bieneusi infection among the four farms and different age groups are presented in Table ​Table1.1. A significantly higher prevalence of E. bieneusi was found in pigs aged 2 to 3.9 months than in pigs of other age groups (chi-square test, P < 0.001). Multivariate analysis confirmed that pigs aged 2 to 3.9 months had a 5.3-times-greater risk of infection than pigs in other age groups (95% confidence interval, 2.6 to 10.6; P < 0.001). Of these 42 E. bieneusi-positive samples, 21 (50%) were successfully characterized by sequencing analysis, and the organism was identified as being of genotypes E (12 samples [57.1%]), O (8 samples [38.1%]), and H (1 sample [4.8%]).

TABLE 1.

Prevalence of E. bieneusi positivity in pig specimens as determined by PCR

Incidence and risk factors of hookworm infection in a rural community of central Thailand

A cohort study to identify incidence and risk factors of hookworm infection was conducted in a rural community, central Thailand from November 2005 to February 2007. Stool specimens were examined for hookworm eggs using wet preparation, Kato thick smear, and water-ethyl acetate sedimentation technique. The incidence rate of hookworm infection was 7.5/100 person-years. The independent risk factors for acquiring hookworm infection were barefoot walking (incidence rate ratio [IRR] = 4.2, 95% confidence interval [CI] = 1.2-14.5) and raising buffaloes around the house (IRR = 4.8, 95% CI = 1.9-11.8). Sequencing of internal transcribed spacer 1 (ITS1)-5.8S-ITS2 region of the ribosomal RNA gene were performed for identifying species of hookworm. Necator americanus was the most common hookworm identified in this population. Ancylostoma duodenale and A. ceylanicum were also detected. Our data suggest transmission of both human and animal hookworms in this community. Thus, prevention and control strategies of hookworm infection should cover both human and animal infection.