Comparative Genomics Reveals Insights into the Divergent Evolution of Astigmatic Mites and Household Pest Adaptations.

Highly diversified astigmatic mites comprise many medically important human household pests such as house dust mites causing ∼1-2% of all allergic diseases globally; however, their evolutionary origin and diverse lifestyles including reversible parasitism have not been illustrated at the genomic level, which hampers allergy prevention and our exploration of these household pests. Using six high-quality assembled and annotated genomes, this study not only refuted the monophyly of mites and ticks, but also thoroughly explored the divergence of Acariformes and the diversification of astigmatic mites. In monophyletic Acariformes, Prostigmata known as notorious plant pests first evolved, and then rapidly evolving Astigmata diverged from soil oribatid mites. Within astigmatic mites, a wide range of gene families rapidly expanded via tandem gene duplications, including ionotropic glutamate receptors, triacylglycerol lipases, serine proteases and UDP glucuronosyltransferases. Gene diversification after tandem duplications provides many genetic resources for adaptation to sensing environmental signals, digestion, and detoxification in rapidly changing household environments. Many gene decay events only occurred in the skin-burrowing parasitic mite Sarcoptes scabiei. Throughout the evolution of Acariformes, massive horizontal gene transfer events occurred in gene families such as UDP glucuronosyltransferases and several important fungal cell wall lytic enzymes, which enable detoxification and digestive functions and provide perfect drug targets for pest control. This comparative study sheds light on the divergent evolution and quick adaptation to human household environments of astigmatic mites and provides insights into the genetic adaptations and even control of human household pests.

Genome assembly and annotation of Periplaneta americana reveal a comprehensive cockroach allergen profile.

BACKGROUND: One of the most common cockroach types in urban areas, the American cockroach (Periplaneta americana), has been reported to impose an increased risk of allergies and asthma. Limited groups of allergens (Per a 1-13) have been identified in this species due to the lack of genome-related information. METHODS: To expand the allergen profile of P. americana, genomic, transcriptomic, and proteomic approaches were applied. With the support of a high-quality genome assembled using nanopore, Illumina, and Hi-C sequencing techniques, potential allergens were identified based on protein homology. Then, using enzyme-linked immunosorbent assay, selected allergens were tested in Thai patients allergic to P. americana. RESULTS: A chromosomal-level genome of P. americana (3.06 Gb) has been assembled with 94.6% BUSCO completeness, and its contiguity has been significantly improved (N50 = 151 Mb). A comprehensive allergen profile has been characterized, with seven novel groups of allergens, including enolase (Per a 14), cytochrome C (Per a 15), cofilin (Per a 16), alpha-tubulin (Per a 17), cyclophilin (Per a 18), porin3 (Per a 19), and peroxiredoxin-6 (Per a 20), showing IgE sensitivity in enzyme-linked immunosorbent assay. A new isoallergen of tropomyosin (Per a 7.02) and multiple potential isoallergens of Per a 5 were revealed using bioinformatics and proteomic approaches. Additionally, comparative analysis of P. americana with the closely related Blattodea species revealed the possibility of cross-reaction. CONCLUSION: The high-quality genome and proteome of P. americana are beneficial in studying cockroach allergens at the molecular level. Seven novel allergen groups and one isoallergen in Per a 7 were identified.

Sandwich dot-immunogold filtration assay (DIGFA) for specific immunodiagnosis of active neuroangiostrongyliasis.

Serological tests may yield false-negative results for specific antibodies detection before or at the early seroconversion phase. Tests that detect circulating antigens of Angiostrongylus cantonensis would therefore be of value in diagnosis to distinguish current or past infection. Here, a quick, easy to perform, portable and inexpensive diagnostic device for detection of 31-kDa A. cantonensis specific antigens had been developed. This sandwich dot-immunogold filtration assay (AcDIGFAAg), for detecting active angiostrongyliasis was produced using anti-A. cantonensis polyclonal antibody dotted on the nitrocellulose membrane as a capture agent and colloidal gold-labelled anti-31 kDa A. cantonensis antibody as a detection agent. A well-defined pink dot, indicating positivity, was seen readily by naked eye within 10-15 min. The AcDIGFAAg detected A. cantonensis-specific antigens in cerebrospinal fluid samples from 4 out of 10 serologically confirmed angiostrongyliasis cases and 2 out of 5 suspected cases with negative anti-A. cantonensis antibodies. Among the 19 patient sera with A. cantonensis infection, 2 showed positive reaction by AcDIGFAAg. No positive AcDIGFAAg reaction was observed in all the serum samples with other parasitic diseases, and the healthy controls. The present 'AcDIGFAAg' enables rapid qualitative detection of the specific 31-kDa antigens of A. cantonensis in clinical samples with potential for application even under resource-limited settings.

A simple monochromatic flow cytometric assay for assessment of intraerythrocytic development of Plasmodium falciparum.

BACKGROUND: Gold standard microscopic examination of Plasmodium falciparum intraerythrocytic stage remains an important process for staging and enumerating parasitized erythrocytes in culture; however, microscopy is laborious and its accuracy is dependent upon the skill of the examiner. METHODS: In this study, ViSafe Green (VSG), which is a nucleic acid-binding fluorescent dye, was used for assessing in vitro development of P. falciparum using flow cytometry. RESULTS: Fluorescence intensity of VSG was found to depend on the developmental stage of parasites. Specifically, multiple-nuclei-containing schizonts were observed in the VSGhigh population, and growing trophozoites and ring-shaped forms were observed in the VSGintermediate and VSGlow populations. The efficacy of VSG-based assay was found to be comparable to the microscopic examination method, and it demonstrated an ability to detect as low as 0.001% of the parasitaemia estimated by Giemsa staining. Moreover, when applying VSG for anti-malarial drug test, it was able to observe the growth inhibitory effect of dihydroartemisinin, the front-line drug for malaria therapy. CONCLUSIONS: Taken together, the results of this study suggest the VSG-based flow cytometric assay to be a simple and reliable assay for assessing P. falciparum malaria development in vitro.

Cloning, Expression, and Immunological Characterization of Formosan Subterranean Termite (Blattodea: Rhinotermitidae) Arginine Kinase.

Several parts of the world regularly consume termites. Arthropod arginine kinase proteins often cross-react with human immunoblobulin E (IgE) antibodies and they are considered pan-allergens. The Formosan subterranean termite Coptotermes formosanus (C. formosanus (Shiraki) [Isoptera: Rhinotermitidae]), along with cockroaches, belong to the order Blattodea and they are common household pests in tropical and subtropical parts of the world. An sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) band migrating at approximately 37 kDa in C. formosanus termite extracts cross-reacted with IgE from five cockroach allergic patient samples by immunoblot. Liquid chromatography-mass spectrometry analysis of gel slices from the corresponding region of a gel indicated several peptides from the excised region were identical to the American cockroach arginine kinase allergen, Per a 9. The sequence of the full-length C. formosanus arginine kinase gene indicates the protein it encodes is 96% identical to American cockroach Per a 9, 94% identical to German cockroach Bla g 9, and 82-84% identical to shrimp arginine kinase proteins Pen m 2, Lit v 2, and Cra c 2. Full-length C. formosanus arginine kinase was fused to a glutathione S-transferase tag and recombinantly expressed and purified from Escherichia coli by affinity chromatography. The recombinant protein was recognized by IgE from 11 of 12 cockroach or shrimp allergic samples, but did not cross-react with dust mite allergic or peanut/tree nut allergic samples. The results of this study indicate the C. formosanus arginine kinase cross-reacts with cockroach and shrimp allergic IgE, and if consumed would likely act as an allergen.

Concordance of skin prick test and serum-specific IgE to locally produced component-resolved diagnostics for cockroach allergy.

BACKGROUND: Diagnosis of Periplaneta americana (American cockroach, ACR) allergy is commonly performed based on clinical history and skin prick test (SPT) or specific serum IgE (sIgE) measurement. The concordance of the findings with the SPT and sIgE results has never been investigated. OBJECTIVE: To compare the results of SPT with commercial ACR-extract (C-ACE) and sIgE measurement, using commercial kit and in-house enzyme-linked immunosorbent assay (ELISA) to the locally produced ACR extract (L-ACE) and native Per a 1, Per a 5, Per a 7, and Per a 9. METHODS: Sera from 66 individuals clinically diagnosed with chronic allergic rhinitis were included; 46 were positive SPT to C-ACE, and 20 were negative. Specific serum IgE levels were established by using a commercial test kit (ImmunoCap) and an in-house IgE-ELISA RESULTS: The percentage the C-ACE SPT-positive cases that were positive by the ImmunoCap-sIgE was 32.6%, indicating low concordance of the 2 assays. With the in-house ELISA, Per a 9 gave the highest sensitivity (98.00%), positive predictive value (PPV; 95.74%), and negative predictive value (NPV; 94.74%) of the sIgE quantification. The correlation coefficients (R) of the L-ACE-SPT and sIgE to L-ACE, Per a 1, Per a 5, Per a 7, and Per a 9 and ImmunoCap sIgE were 0.133, 0.278, 0.419, 0.280, and 0.432, and 0.256, respectively. CONCLUSION: Skin prick test and sIgE measurement using commercial reagents have low concordance. Data of this study showed that sIgE to the native Per a 9 should be considered as an adjunct to the clinical history in diagnosis of ACR sensitization/allergy, particularly when the SPT and the nasal challenge, which is the gold standard method, cannot be performed.

Quantification of Fel d 1 in house dust samples of cat allergic patients by using monoclonal antibody specific to a novel IgE-binding epitope.

BACKGROUND: Avoidance of allergen exposure is an effective measure for preventing naÏve and allergic individuals from sensitization (primary intervention) and disease aggravation (secondary intervention), respectively. Regular monitoring of the allergens in the environment is required for the effective intervention. Thus, there is a need for cost-effective test kits for environmental allergen quantifications. OBJECTIVE: To invent a test kit for quantification of cat major allergen, Fel d 1. METHODS: A mouse monoclonal antibody (MAb) specific to the newly identified IgE-binding conformational epitope of the cat major allergen (Fel d 1) and rabbit polyclonal IgG to recombinant Fel d 1 were used as allergen capture and detection reagents, respectively. Native Fel d 1 was used in constructing a standard curve. RESULTS AND CONCLUSION: Sixteen of 36 dust samples collected from houses of cat allergic subjects in Bangkok contained Fel d 1 above 0.29 µg/gram of dust which is considered as a novel threshold level for causing cat allergy sensitization or symptoms. Among them, 7 samples contained the allergen exceeding 2.35 µg/gram of dust which is the level that would aggravate asthma. Results of the allergen quantification using the locally made test kit showed strong correlation (r = 0.923) with the allergen quantification using commercialized reagents. The assay using MAb to Fel d 1 IgE-binding epitope of this study has potential application as an economic and practical tool for cat allergy intervention measure especially in localities where health resources are relatively limited.

Proteome, Allergenome, and Novel Allergens of House Dust Mite, Dermatophagoides farinae.

Dermatophagoides farinae mite is a predominant source of indoor allergens causing high incidence of allergy worldwide. People with different genetic background respond differently to the mite components, and thus the component-resolved diagnosis (CRD) is preferred to the conventional allergy test based on crude mite extract. In this study, proteome and culprit components in the D. farinae whole body extract that sensitized the allergic patients were studied by using SDS-PAGE (1DE) and 2DE-IgE immunoblotting followed by LC-MS/MS and database search for protein identification. From the 1DE, the mite extract revealed 105 proteins that could be classified into seven functionally different groups: allergens, structural components, enzymes, enzyme inhibitor, receptor proteins, transporters, and binding/regulatory/cell signaling proteins. From the 2DE, the mite extract produced 94 spots; 63 were bound by IgE in sera of 20 D. farinae allergic patients. One more protein that was not revealed by the 2DE and protein staining reacted with IgE in 2 allergic patients. Proteins in 40 spots could be identified as 35 different types. Three of them reacted to IgE of >50% of the allergic patients, and hence they are major allergens: tropomyosin or Der f 10 (75%), aconitate hydratase (70%), and one uncharacterized protein (55%). Aconitate hydratase is a novel D. farinae major allergen unraveled in this study. Several mite minor allergens that have never been previously reported are also identified. The data have clinical applications in the component-resolved diagnosis for tailor-designed allergen-specific immunotherapy.

A novel IgE-binding epitope of cat major allergen, Fel d 1.

Information on the antigenic repertoire, especially the IgE-binding epitopes of an allergen is important for understanding the allergen induced immune response and cross-reactivity, as well as for generating the hypoallergenic variants for specific component resolved immunotherapy/diagnosis (CRIT and CRD). Data on the IgE-binding epitopes of cat allergens are scarce. In this study, a novel IgE-binding epitope of the cat major allergen, Fel d 1, was identified. Mouse monoclonal antibody (MAb) specific to the Fel d 1 was produced. Computerized intermolecular docking was used for determining the residues of the Fel d 1 bound by the specific MAb. The presumptive surface exposed residues of the Fel d 1 intrigued by the MAb are located on the chain 1. They are: L34 and T37 (helix 1); T39 (between helices 1 and 2); P40, E42 and E45 (helix 2); R61, K64, N65 and D68 (helix 3); and E73 and K76 (helix 4). The MAb competed efficiently with the cat allergic patients' serum IgE for Fel d 1 binding in the competitive IgE binding assay, indicating allergenicity of the MAb epitope. The newly identified allergenic epitope of the Fel d 1 is useful in a design of the CRIT and CRD for cat allergy.

ADIPOQ POLYMORPHISMS AMONG THAIS WITH PRE-DIABETES.

Studies have shown that polymorphisms of adiponectin gene (ADIPOQ) are associated with risk of developing type 2 diabetes mellitus (T2DM). However, no studies have investigated the association between genetic variants of ADIPOQ and pre-diabetes, a group at higher risk for developing T2DM. A total of 75 pre-diabetes and 130 normal subjects were recruited from volunteers in Bangkok, Thailand. Individuals with pre-diabetes were selected based on American Diabetes Association diagnostic criteria. Six ADIPOQ polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism technique. ADIPOQ polymorphism rs266729 C>G is significantly associated with pre-diabetes (p = 0.006). CG/GG genotypes were found among 60% and 40% of pre-diabetes and normal subjects, respectively. SNP rs266729 C>G was associated with increased pre-diabetes risk (OR = 2.64; 95% CI: 1.18-5.89, p = 0.018). No significant differences were found between pre-diabetes and normal subjects for other ADIPOQ polymorphisms. However, haplotype analysis revealed that haplotype GGTAAT is significantly associated with pre-diabetes when compared with GCGAAC reference haplotype (OR = 22.31; 95% CI: 1.37-361.93, p = 0.03). Our data indicate that ADIPOQ rs266729 C>G polymorphism may contribute to the genetic risk of pre-diabetes and provide preliminary data useful in genetic screening for pre-diabetes among Thais.

Allergenicity of native and recombinant major allergen groups 1 and 2 of Dermatophagoides mites in mite sensitive Thai patients.

BACKGROUND AND OBJECTIVE: Natural allergenic extracts using for diagnosis and immunotherapy may have batch-to-batch variations and contaminations with unrefined allergens or non-allergenic components. Thus, recombinant allergen is believed to overcome these shortcomings. In this study, native and recombinant allergens of group 1 and 2 of Dermatophagoides mites were produced and their allergenicities were compared. METHODS: Native allergens were prepared by MAb affinity chromatography. All recombinant allergens were produced in E. coli expression system. IgE reactivities of these allergens were determined by IgE-ELISA. RESULTS: The native and recombinant Der p 1, Der p 2, Der f 1, Der f 2 had molecular weights of approximately 25, 15, 25 and 15 kDa, respectively. IgE reactivities of nDer p 1, nDer f 1, rDer p 1 and rDer f 1 were 96.67%, 90%, 43.33% and 46.67%, respectively. Allergenicities of nDer p 2, nDer f 2, rDer p 2 and rDer f 2 were 86.67%, 96.43%, 76.67% and 89.29%, respectively. The findings indicated that recombinant group-1 products were minor allergens which revealed no correlation with their native forms. In contrast, recombinant group-2 allergens were major allergens and showed a significant correlation to their native allergens. CONCLUSION: We successfully produced native and recombinant group-1 and group-2 allergens. According to their allergenicities, recombinant Der p 2 and rDer f 2 have potential to replace native allergen in diagnostic and therapeutic extracts. Moreover, they can employ as a standard reagent to measure the amount of group 2 allergen in the environment by sandwich-ELISA and utilise this as an immunogen for MAb production.

Dust mite infestation in cooking flour: experimental observations and practical recommendations.

BACKGROUND: The first documented case of oral mite anaphylaxis has recently been reported in Thailand, with mites possibly originating from cooking flour. OBJECTIVE: Our study was designed to assess the effects of cooking flours enhancement and storage conditions on mite proliferation and to provide practical recommendations to prevent mite anaphylaxis. METHODS: In a factorial experiment, six commercial brands of cooking flours were selected and either inoculated or set free of mites and stored in one of the four containers chosen for the study: original package, plastic bag, plastic box and glass bottle. The resulting experimental units where then stored at either room temperature or in a refrigerator (+4C). In order to determine levels of Der f 1 allergen, 0.1 gram of flour was sampled from each experimental unit and tested by ELISA. Sampling was carried out immediately after inoculation and subsequently at week 2, 4, 6, 8, 10, 12, 16 and 20. RESULTS: Levels of Der f 1 allergen in the inoculated samples increased significantly in all conditions 6 weeks after inoculation (p <0.001) and reached the highest levels at week 8. While experimental units left at room temperature showed higher levels of mite growth (p <0.001), no statistical differences were found among types of containers. The highest amount of Der f 1 was observed for Gogi, followed by Gold Label, tempura flour, corn flour, wheat flour and tapioca starch, respectively (p <0.01). CONCLUSIONS: In the context of our experiment, mites preferably grew in cooking flours containing high amounts of wheat at room temperature, particularly after 8 week of storage. According to our results, we thus advise to keep household cooking flour refrigerated and while the type of container does not matter, storage should not exceed 20 weeks.

Proteome and allergenome of Asian wasp, Vespa affinis, venom and IgE reactivity of the venom components.

Vespa affinis (Asian wasp, Thai banded tiger wasp, or local name: Tor Hua Seua) causes the most frequent incidence of medically important Hymenoptera sting in South and Southeast Asia. However, data on the venom components attributable to the sting derived-clinical manifestations (local reactions, IgE mediated-anaphylaxis, or systemic envenomation) are lacking. This study provides the first set information on V. affinis venom proteome, allergenome, and IgE reactivity of individual venom components. From 2DE-gel based-proteomics, the venom revealed 93 protein spots, of which proteins in 51 spots could be identified and classified into three groups: typical venom components and structural and housekeeping proteins. Venom proteins in 32 spots reacted with serum IgE of wasp allergic patients. Major allergenic proteins that reacted to IgE of >50% of the wasp allergic patients included PLA1 (100%), arginine kinase (73%), heat shock 70 kDa protein (73.3%), venom allergen-5 (66.7%), enolase (66.7%), PLA1 magnifin (60%), glyceraldehyde-3-phosphate dehydrogenase (60%), hyaluronidase (53.3%), and fructose-bisphosphate aldolase (53.3%). The venom minor allergens were GB17876 transcript (40%), GB17291 transcript (20%), malic enzyme (13.3%), aconitate hydratase (6.7%), and phosphoglucomutase (6.7%). The information has diagnostic and clinical implications for future improvement of case diagnostic sensitivity and specificity, component-resolve diagnosis, and design of specific Hymenoptera venom immunotherapy.

Homocysteine but not serum amyloid A, vitamin A and E related to increased risk of metabolic syndrome in post-menopausal Thai women.

This study aims to investigate serum amyloid A, homocysteine, and biochemical-anthropometric measurements in post-menopausal women with and without metabolic syndrome (MS), and determine whether serum amyloid A and homocysteine are linked to MS among this group. This study was performed with 405 post-menopausal Thai volunteers with a mean age of 57.95±5.90 years (135 subjects with MS and 270 subjects without MS). The levels of serum amyloid A, homocysteine, vitamins, glucose, and lipids were measured. Homocysteine levels were significantly higher in the group with MS than in that without MS (p<0.001), whereas for serum amyloid A, vitamin A, vitamin E and vitamin B12, there were no significant differences. There were significant differences between the groups in folate, HDL-C, and anthropometric measurements (p<0.001). Thirty seven percent of the group with MS and 14.1% of the group without MS were classified as having hyperhomocysteinemia (p<0.001). Furthermore, logistic regression analysis revealed that hyperhomocysteinemia (odds ratio (OR): 2.67, 95% confidence interval (95%CI): 1.57-4.58), low folate (OR: 1.79, 95%CI: 1.11-2.89), and BMI (OR: 1.25, 95%CI: 1.16-1.33) were significantly related to MS. These findings suggest that increased homocysteine levels and decreased folate concentrations may influence susceptibility to MS and this effect may be an early event in the development of cardiovascular diseases among post-menopausal women. Therefore, there is a need to evaluate homocysteine levels, especially among post-menopausal Thai women.

Effects of PCSK1 genetic variants on obesity among Thai children and their family members: in relation to health risk, and biochemical and anthropometric parameters.

Single nucleotide polymorphisms (SNPs) in PCSK1, namely, rs6234, rs6235, and rs271939 have been linked to obesity in European population; and rs3811951 has also been connected to type 2 diabetes and obesity parameters in Chinese population. In this family-based case-control study, we analyzed links between PCSK1 genetic variants and obesity in Thai children and their families. Eleven obese children with a percent weight for height > or = 140 who had family history of obesity and 69 family members were recruited. SNPs rs6234, rs6235, rs3811951, and rs271939 of PCSK1 were analyzed using PCR and gene sequencing methods. DNA of 200 normal weight subjects was used as control. Participants with variant genotypes in the rs6234-6235 pair are at significantly more risk of being obese [OR = 2.44 (1.35-4.43), p = 0.003], and also at increased risk of being severely obese (obese class III) [OR = 3.03 (1.20-7.66), p = 0.015]. Variant rs3811951 showed no association with being obese, but is significantly linked to an increased risk of being severely obese [OR = 3.59 (1.42-9.08) p = 0.005]. Moreover, high density lipoprotein (HDL)-C levels between normal and variant rs3811951 group differed considerably, with patients with variant genotype having a lower HDL-C level (p = 0.037). Thus, Thais carrying SNPs rs6234-5 are at increased risk of being obese, and the risk of severe obesity increases when carrying both rs6234-5 and rs3811951, but not with rs271939. Furthermore, patients with genetic variations at rs3811951 are at risk of having low HDL-C levels.

Intranasal, liposome-adjuvanted cockroach allergy vaccines made of refined major allergen and whole-body extract of Periplaneta americana.

BACKGROUND: Cockroach (CR) allergens frequently cause severe asthma in CR-sensitized subjects. Allergen-specific immunotherapy causes a shift of allergic Th2 responses towards Th1 and/or regulatory T cell (Treg) responses which reduce airway inflammation and prevent disease progression. Data are relatively limited on immunotherapy via CR allergy vaccine. METHODS: The therapeutic efficacy of an intranasal liposome-adjuvant vaccine made of a refined Periplaneta americana arginine kinase (AK) was compared to the liposome-entrapped P. americana crude extract (CRE) vaccine. Adult BALB/c mice were rendered allergic to CRE. Three allergic mouse groups were immunized intranasally on alternate days with 8 doses of liposome-entrapped CRE (L-CRE), liposome-entrapped AK and placebo, respectively. One week later, all mice received a nebulized CRE provocation. Evaluation of vaccine efficacy was performed 1 day after provocation. RESULTS: Liposome-entrapped native AK attenuated airway inflammation after the CRE provocation and caused a shift of allergic Th2 to Th1 and Treg responses. The L-CRE also induced a shift from the Th2 to the Th1 response but did not induce a Treg response and could not attenuate the airway inflammation upon allergen reexposure. CONCLUSIONS: Intranasal liposome-adjuvant CR allergy vaccine containing native AK (Per a 9) is better than L-CRE in attenuating allergic airway inflammation. The findings of this study not only document a more comprehensive and beneficial immune response induced by the refined allergen vaccine but also raise the point that the shift from the Th2 to the Th1 response alone might not correlate with improved airway histopathology, clinical outcome and quality of life.

Association of MTHFR C677T polymorphism with bone mineral density of osteoporosis in postmenopausal Thai women.

BACKGROUND: Osteoporosis and osteopenia is rising with the increase in numbers of postmenopausal women. Methylenetetrahydrofolate reductase (MTHFR), a homocysteine catabolizing enzyme, is involved in the regulation of bone mineral density (BMD). The association between MTHFR C677T polymorphism with osteoporosis in postmenopausal Thai women is hitherto unclear. OBJECTIVE: To investigate the association between MTHFR C677T and BMD in postmenopausal Thai women. MATERIAL AND METHOD: The study subjects consisted of 346 postmenopausal Thai women volunteers. Standard dual-energy X-ray absorptiometry (DXA) was used for measurement of BMD T-score. Restriction fragment length polymorphism (RFLP) analysis was used for measurement of MTHFR C677T polymorphism. RESULTS: In the evaluation of 346 postmenopausal Thai women heterozygous (CT) genotype had a risk of osteopenia than normal control (odds ratio (OR) = 5.66, p < 0.001). BMD T-scores at each bone position revealed that heterozygous (CT) genotype had increased risk of osteopenic bones than normal controls at lumbar spines 1, 2, and 4 (OR = 2.48, p < 0.001, OR = 1.98, p = 0.008 and OR = 1.83, p = 0.016 respectively), ward's triangle (OR = 2.08, p = 0.008), and head of radius (OR = 2.95, p = 0.008). CONCLUSION: These results indicate the possibility of using MTHFR C677T polymorphism to identify postmenopausal Thai women at high risk of osteopenia.

Rapid Single-Step Immunochromatographic Assay for Angiostrongylus cantonensis Specific Antigen Detection.

Angiostrongylus cantonensis is the major etiological nematode parasite causing eosinophilic meningitis and/or eosinophilic meningoencephalitis in humans. The rapid global spread of Angiostrongylus cantonensis and the emerging occurrence of the infection have exposed the shortcomings of traditional/conventional diagnostics. This has spurred efforts to develop faster, simpler and more scalable platforms that can be decentralized for point-of-need laboratory testing. By far, the point-of-care immunoassays such as the lateral flow assay (LFA) are the best-placed. In this work, a LFA in the form of an immunochromatographic test device (designated AcAgQuickDx), based on the detection of a circulating Angiostrongylus cantonensis-derived antigen, was established using anti-31 kDa Angiostrongylus cantonensis antibody as the capture reagent and anti-Angiostrongylus cantonensis polyclonal antibody as the indicator reagent. The AcAgQuickDx was evaluated for its diagnostic potential with a total of 20 cerebrospinal fluids (CSF) and 105 serum samples from patients with angiostrongyliasis and other clinically related parasitic diseases, as well as serum samples from normal healthy subjects. Three of the ten CSF samples from serologically confirmed angiostrongyliasis cases and two of the five suspected cases with negative anti-Angiostrongylus cantonensis antibodies showed a positive AcAgQuickDx reaction. Likewise, the AcAgQuickDx was able to detect Angiostrongylus cantonensis specific antigens in four serum samples of the 27 serologically confirmed angiostrongyliasis cases. No positive reaction by AcAgQuickDx was observed in any of the CSF (n = 5) and serum (n = 43) samples with other parasitic infections, or the normal healthy controls (n = 35). The AcAgQuickDx enabled the rapid detection of active/acute Angiostrongylus cantonensis infection. It is easy to use, can be transported at room temperature and does not require refrigeration for long-term stability over a wide range of climate. It can supplement existing diagnostic tests for neuroangiostrongyliasis under clinical or field environments, particularly in remote and resource-poor areas.

Formalin Inactivation of Virus for Safe Downstream Processing of Routine Stool Parasite Examination during the COVID-19 Pandemic.

During the COVID-19 pandemic, the parasitology laboratories dealing with fecal samples for the diagnosis of gastrointestinal parasitic infections are confronting the unsaved virus-containing samples. To allow for safe downstream processing of the fecal samples, a protocol for preparing a fecal smear is urgently needed. Formalin was tested with or without isotonic forms for virus inactivation using porcine epidemic diarrhea virus (PEDV) as a representative, as it belongs to the Coronaviridae family. The results revealed complete inactivation activity of 10% formalin and 10% isotonic formalin on coronavirus after 5 min of treatment at room temperature. Both also inhibited Naegleria fowleri growth after 5 min of treatment at 37 °C without disruption of the structure. In addition to these key findings, it was also found that isotonic formalin could stabilize both red and white blood cells when used as a solution to prepare fecal smears comparable to the standard method, highlighting its value for use instead of 0.9% normal saline solution for the quantification of blood cells without active virus. The 10% isotonic formalin is useful to safely prepare a fecal smear for the diagnosis of parasites and other infections of the gastrointestinal tract during the COVID-19 pandemic.