Orapan Sripichai

Background: Fungal infections pose a significant global health threat, particularly in Asia, with Thailand experiencinghigh prevalence due to its tropical climate. Increased capacity for fungal diagnosis leads to the appropriate diagnosis and treatment, and most importantly, a reduction in illness and death. Objectives: To assess and update the current status of fungal diagnosis capacity in Thailand. Materials and methods: This study utilized an online platform to conduct a survey. The questionnaires collected the relevant information of the laboratory capacity including the capability of the on-site fungal identification, methods for fungal identification, the most commonly identified fungal pathogen and the availability of antifungal susceptibility testing (AFST). Results: There were 206 healthcare facilities across country participated in this survey, including both government and private hospitals, as well as one reference laboratory. Nearly all high-tier hospitals utilized advanced automated systems for yeast identification; however, mold identification remained limited, primarily relying on traditional culture and microscopy methods. According to the survey results, the most commonly identified yeast was Candida albicans, while the most commonly identified mold was Aspergillus spp. Although many laboratories could identify fungal pathogens, they often struggled to specify the species, which is essential for effective treatment and managing antifungal resistance. AFST was accessible in high-tier hospitals, but low-tier hospitals did not have this capability. Conclusion: These findings underscore the importance of enhancing the capacity for fungal diagnosis, which will help elucidate the national burden of fungal diseases and improve resource allocation in Thailand.

Background: Vancomycin-resistant enterococci (VRE), particularly Enterococcus faecium and E. faecalis , are responsible for various human infections and represent a significant global public health concern in both nosocomial and community settings. To facilitate early detection of these pathogens, we developed a rapid method for detecting E. faecium , E. faecalis , and vancomycin resistance genes ( vanA and vanB ) in a single reaction using multiplex recombinase polymerase amplification (M-RPA) combined with a chromatographic printed-array strip (C-PAS). Methods: A total of 106 samples, including 46 E. faecium (35 vanA -type and one vanB -type strains), 39 E. faecalis (one vanA -type and two vanB -type strains), four Enterococcus spp., and 17 non- Enterococcus spp. isolates were tested using the M-RPA-C-PAS method and compared with the conventional polymerase chain reaction (PCR) method. Specific tagged and biotinylated M-RPA primers were designed for detecting ddl ( E. faecium ), sodA ( E. faecalis ), vanA , and vanB . The M-RPA reaction was performed under isothermal conditions at 37 °C for 20 min, and visual detection using the C-PAS for DNA signal detection within another 20 min. The performance of the M-RPA-C-PAS was initially evaluated in 12 spiked rectal swabs and 12 positive blood culture samples from patients compared with the PCR method. Results: Compared with the PCR-based method, the M-RPA-C-PAS method demonstrated sensitivities of 95.7%, 94.9%, and 100.0% and specificities of 100.0%, 92.5%, and 100.0% for ddl , sodA , and vanA , respectively, and 93.2% specificity for vanB . The lower detection limits of the M-RPA-C-PAS method for detecting the ddl and sodA genes were 10⁴ and 10³ CFU/mL, respectively, whereas those of the PCR method were 10⁵ and 10⁴ CFU/mL, respectively. The lower limit of detection of both methods for vanA and vanB was 10⁴ CFU/mL. The M-RPA-C-PAS assay showed 100% agreement with PCR for identifying the four genes in the spiked and clinical samples. Conclusion: The M-RPA-C-PAS method is an alternative rapid approach with high sensitivity and specificity, demonstrating comparable performance to conventional PCR. Therefore, the M-RPA-C-PAS method represents a promising point-of-care diagnostic tool for the rapid detection of VRE, supporting timely clinical management and infection control strategies.

Defective hemoglobin production and ineffective erythropoiesis contribute to the pathophysiology of thalassemia syndromes. Previous studies in the field of erythropoiesis mainly focused on the severe forms of thalassemia, such as β-thalassemia major, while mechanisms underlying the pathogenesis of other thalassemia syndromes remain largely unexplored. The current study aimed to investigate the intrinsic pathophysiological properties of erythroid cells derived from the most common forms of thalassemia diseases, including α-thalassemia (hemoglobin H and hemoglobin H-Constant Spring diseases) and β-thalassemia (homozygous β⁰-thalassemia and β⁰-thalassemia/hemoglobin E diseases), under an identical in vitro erythroid culture system. Cell proliferation capacity, differentiation velocity, cell death, as well as globin synthesis and the expression levels of erythropoiesis modifying factors were determined. Accelerated expansion was found in erythroblast cells derived from all types of thalassemia, with the highest degree in β⁰-thalassemia/hemoglobin E. Likewise, all types of thalassemia showed limited erythroid cell differentiation, but each of them manifested varying degrees of erythroid maturation arrest corresponding with the clinical severity. Robust induction of HSP70 transcripts, an erythroid maturation-related factor, was found in both α- and β-thalassemia erythroid cells. Increased cell death was distinctly present only in homozygous β⁰-thalassemia erythroblasts and associated with the up-regulation of pro-apoptotic (Caspase 9, BAD, and MTCH1) genes and down-regulation of the anti-apoptotic BCL-XL gene.

Anemia in β-thalassemia is associated with ineffective erythropoiesis and a shortened lifespan of erythroid cells. The limited differentiation of β-thalassemic erythroblasts has been documented, but the characteristic feature of terminal erythroid maturation and its physiological relevance are not clearly described in β-thalassemias. Here, the red blood cell and reticulocyte cellular characteristics were determined in patients with β⁰-thalassemia/HbE in comparison to patients with iron deficiency anemia and healthy normal subjects. Severely affected β⁰-thalassemia/HbE patients showed the highest increase in immature reticulocytes, but the number of total erythrocytes was the lowest. Despite similar ranges of hemoglobin levels, β⁰-thalassemia/HbE patients had a higher number of reticulocytes and a greater proportion of immature fraction than patients with iron deficiency anemia did. In vitro CD34⁺ hematopoietic progenitor cells’ culture and flow cytometry analysis were conducted to investigate the erythroid maturation and mitochondrial clearance in β⁰-thalassemia/HbE erythroid cells as compared to normal cells. The delayed erythroid maturation and evidence of impaired mitochondria clearance were observed in β⁰-thalassemia/HbE cells at the terminal stage of differentiation. Additionally, increased transcript levels of genes related to erythroid mitophagy, BNIP3L and PINK1, were revealed in β⁰-thalassemia/HbE erythroblasts. The findings indicate that the erythroid maturation is physiologically relevant, and that the restoration of terminal maturation represents a potential therapeutic target for β-thalassemias.

β-Thalassemia (β-thal) is highly prevalent in Myanmar, but limited data are available on the molecular basis and the clinical manifestations in Myanmar patients. In this study, we investigated the clinical features and β-globin gene abnormalities in 15 homozygous β-thal and 60 Hb E (HBB: c.79G>A)/β-thal pediatric patients who attended Yangon Children Hospital, the biggest thalassemia day care unit center in Myanmar. Eight different β⁰-thal mutations were identified, with four accounting for 88.9% of alleles studied (excluding the Hb E variant). A genotype-phenotype correlation was found; all homozygous β⁰-thalassemias had severe clinical courses, whereas the highly variable disease severity was demonstrated among Hb E/β⁰-thal patients. Interactions of IVS-I-1 (G>T) (HBB: c0.92+1G>T) β⁰-thal with Hb E are associated with milder clinical symptoms. The number of mildly affected Hb E/β-thal patients was lower than expected, suggesting that there may be a considerable number of patients in the population who have either not been admitted to hospital or diagnosed with carrying the disease. Although the clinical severity in the Myanmar β-thal patients seems to be similar to that in other populations, the levels of hemoglobin (Hb) appears to be very low. These findings indicate the need for the improvement of patient management and the development of prevention and control programs for β-thal in Myanmar.

Introduction: β-Thalassemia/hemoglobin E represents one-half of all the clinically severe β-thalassemias worldwide. Despite similar genetic backgrounds, patients show clinical heterogeneity ranging from nearly asymptomatic to transfusion-dependent thalassemia. The underlying disease modifying factors remain largely obscure. Methods: To elucidate the correlation between ineffective erythropoiesis and β⁰-thalassemia/hemoglobin E (HbE) disease severity, in vitro culture of erythroid cells derived from patients with different clinical symptoms was established. Cell proliferation, viability, and differentiation were investigated. To identify potential molecular mechanisms leading to the arrested erythroid maturation, the expression levels of erythropoiesis modifying factors were measured. Results: The β⁰-thalassemia/HbE cells exhibited enhanced proliferation, limited differentiation, and impaired erythroid terminal maturation but did not show accelerated erythroblast differentiation and increased cell death. Erythroblasts derived from mild patients showed the highest proliferation rate with a faster cell division time, while erythroblasts derived from severe patients displayed extremely delayed erythroid maturation. Downregulation of growth differentiation factor 11 and FOXO3a was observed in mild β⁰-thalassemia/HbE erythroblasts, while upregulation of heat shock protein 70 and activin receptor 2A was revealed in severe erythroblasts. Discussion/Conclusion: The degree of erythroid expansion and maturation arrest contributes to the severity of β⁰-thalassemia/HbE patients, accounting for the disease heterogeneity. The findings suggest a restoration of erythroid maturation as a promising targeted therapy for severe patients.