Mongkol Kunakorn

Development and Evaluation of an In-House Enzyme-Linked Immunosorbent Assay for Early Diagnosis and Monitoring of Human Pythiosis

ABSTRACT Human pythiosis is an emerging, fatal, infectious disease caused by Pythium insidiosum and occurs in both tropical and subtropical countries. Thalassemic patients, farmers, and aquatic-habitat residents are predisposed to this disease. Delayed treatment due to the long time required for isolation and identification of the causative organism, as well as the difficulty in obtaining internal organ specimens, results in high morbidity and mortality. To facilitate rapid diagnosis, an in-house enzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin G antibodies against P. insidiosum was developed and evaluated for the diagnosis and monitoring of human pythiosis. Sixteen sera were collected from seven culture-proven human pythiosis cases. A total of 142 sera from thalassemic patients, from patients with other infectious diseases, and from healthy blood donors served as controls. All sera were tested in duplicate. By choosing a suitable cutoff point to maximize sensitivity and specificity, sera from pythiosis cases were all determined to be positive, whereas sera from control groups were all determined to be negative. ELISA signals from serial samples of sera taken from treated patients showed gradually declining levels of antibodies to P. insidiosum. The ELISA test was highly sensitive (100%) and specific (100%) and was useful for early diagnosis and for monitoring the treatment for pythiosis.

Identification of a Novel 74-Kilodalton Immunodominant Antigen of Pythium insidiosum Recognized by Sera from Human Patients with Pythiosis

ABSTRACT The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the etiologic agent of pythiosis, a life-threatening infectious disease of humans and animals that has been increasingly reported from tropical, subtropical, and temperate countries. Human pythiosis is endemic in Thailand, and most patients present with arteritis, leading to limb amputation and/or death, or cornea ulcer, leading to enucleation. Diagnosis of pythiosis is time-consuming and difficult. Radical surgery is the main treatment for pythiosis because conventional antifungal drugs are ineffective. The aims of this study were to evaluate the use of Western blotting for diagnosis of human pythiosis, to identify specific immunodominant antigens of P. insidiosum, and to increase understanding of humoral immune responses against the pathogen. We performed Western blot analysis on 16 P. insidiosum isolates using 12 pythiosis serum samples. These specimens were derived from human patients with pythiosis who had different forms of infection and lived in different geographic areas throughout Thailand. We have identified a 74-kDa immunodominant antigen in all P. insidiosum isolates tested. The 74-kDa antigen was also recognized by sera from all patients with pythiosis but not by control sera from healthy individuals, patients with thalassemia, and patients with various infectious diseases, indicating that Western blot analysis could facilitate diagnosis of pythiosis. Therefore, the 74-kDa antigen is a potential target for developing rapid serodiagnostic tests as well as a therapeutic vaccine for pythiosis. These advances could lead to early diagnosis and effective treatment, crucial factors for better prognosis for patients with pythiosis.

Role of the Stationary Growth Phase Sigma Factor RpoS of Burkholderia pseudomallei in Response to Physiological Stress Conditions

The Burkholderia pseudomallei rpoS gene was identified, and an rpoS null mutant was constructed. The mutant was shown to have an increased sensitivity to carbon starvation and oxidative stress. By using rpoS-lacZ fusions, transcription of rpoS was shown to be growth phase regulated, reaching a peak upon entry into stationary phase.

Ocular pythiosis: is it under-diagnosed?

PURPOSE To increase awareness of ocular pythiosis by presenting a typical case and summarizing clinical data of 11 ocular pythiosis cases in Ramathibodi Hospital. DESIGN Interventional case report. METHODS A 48-year-old healthy woman with a history of 3-week painful corneal ulcer of left eye was treated with enucleation. RESULTS The histopathology of enucleated eye revealed endophthalmitis and ulcerative keratitis with numerous hyphae in full-thickness of corneal stroma. The culture identification of the causative organism was Pythium insidiosum. The final diagnosis was ocular pythiosis. CONCLUSIONS Pythium insidiosum is a causative agent of pythiosis and is distributed worldwide. Ocular pythiosis may not be uncommon, as it may be underdiagnosed due to unfamiliarity among clinicians and microbiologists. Diagnosis of pythiosis is difficult. The disease has high morbidity, as evidenced by nearly evisceration or enucleation among all patients at Ramathibodi Hospital. Early detection and effective treatment are needed for possible cure.

ABO blood-typing using an antibody array technique based on surface plasmon resonance imaging.

In this study, readily available antibodies that are used in standard agglutination tests were evaluated for their use in ABO blood typing by a surface plasmon resonance imaging (SPR imaging) technique. Five groups of antibodies, including mixed clones of anti-A, anti-B, and anti-AB, and single clones of anti-A and anti-B, were used to construct the five-line detection arrays using a multichannel flow cell in the SPR imager. The red blood cell (RBC) samples were applied to a multichannel flow cell that was orthogonal to the detection line arrays for blood group typing. We found that the blood samples were correctly grouped in less than 12 min by the SPR imaging technique, and the results were consistent with those of the standard agglutination technique for all 60 samples. We found that mixed clones of antibodies provided 33%–68% greater change in the SPR signal than the single-clone antibodies. Applying the SPR imaging technique using readily available antibodies may reduce the costs of the antibodies, shorten the measurement time, and increase the throughput.

HemoglobinA1c level in healthy Thai adults: Reference interval and fasting plasma glucose

AIM To establish reference interval of HbA1c IFCC in Thai. MATERIALS AND METHODS 699 whole blood samples were used. Samples had fasting plasma glucose >or=126 mg/dl (7.00 mmol/L), renal problem or hemoglobinopathy was excluded. RESULTS Reference interval of HbA1c IFCC was 2.90-4.90%. CONCLUSION Effect of age should be determined.

Miltenberger blood group typing by real-time polymerase chain reaction (qPCR) melting curve analysis in Thai population.

To develop reliable and convenient methods for Miltenberger (Mia) blood group typing.

Rh blood phenotyping (D, E, e, C, c) microarrays using multichannel surface plasmon resonance imaging

The application of Surface Plasmon Resonance Imaging (SPRi) for the detection of transmembrane antigen of the Rhesus (Rh) blood group system is demonstrated. Clinically significant Rh blood group system antigens, including D, C, E, c, and e, can be simultaneously identified via solid phase immobilization assay, which offers significant time savings and assay simplification. Red blood cells (RBCs) flowed through the micro-channel, where a suitable condition for Rh blood group detection was an RBC dilution of 1:10 with a stop-flow condition. Stop flow showed an improvement in specific binding compared to continuous flow. Rh antigens required a longer incubation time to react with the immobilized antibody than A and B antigens due to the difference in antigen type and their location on the RBC. The interaction between the immobilized antibodies and their specific antigenic counterpart on the RBC showed a significant difference in RBC removal behavior using shear flow, measured from the decay of the SPR signal. The strength of the interaction between the immobilized antibody and RBC antigen was determined from the minimum wall shear stress required to start the decay process in the SPR signal. For a given range of immobilized antibody surface densities, the Rh antigen possesses a stronger interaction than A, B, and AB antigens. Identification of 82 samples of ABO and Rh blood groups using SPRi showed good agreement with the standard micro-column agglutination technique. A wider coverage of antigenic recognition for RBC when using the solid phase immobilization assay was demonstrated for the RBC with the antigenic site located on the transmembrane protein of the clinically significant Rh antigen. Given the level of accuracy and precision, the technique showed potential for the detection of the Rh minor blood group system.

Microfluidic PMMA-based microarray sensor chip with imaging analysis for ABO and RhD blood group typing

Solid phase microarrays have been described for use in blood typing; red blood cells (RBCs) captured on immobilized antibodies were detected using surface plasmon resonance or fluorescence. We present antibody microarray on Poly (methylmethacrylate) (PMMA) surface coupled with microfluidic system for ABO and RhD blood typing. After immobilized by antigen–antibody interaction, the RBCs were detected by image recognition.

Colorimetric Detection by Gold Nanoparticle DNA Probes for Miltenberger Series (GP.Mur, GP.Hop, and GP.Bun) Identification

Miltenberger (Mi) series are the collective glycophorin hybrids in the MNS blood group system. Mi series are composed of several subtypes, for examples, GP.Mur, GP.Hop, and GP.Bun. The incompatibility of Mi series blood transfusion poses the risk of hemolysis. Due to the lack of standard antibodies for Mi series blood typing, colorimetric gold nanoparticle (AuNP) DNA probes were therefore explored for Mi series identification.