Development of flow cytometry for detection and quantitation of red cell bound immunoglobulin G in autoimmune hemolytic anemia with negative direct Coombs test

About 2-10% of patients with warm-antibody autoimmune hemolytic anemia (WAIHA) exhibit a negative direct Coombs test (DAT), requiring more sensitive tests, including detection of RBC-bound immunoglobulins by flow cytometry, for diagnosis. In this study, the optimal conditions for detection and quantitation of RBC-bound IgG by flow cytometry were studied using blood samples from six patients with AIHA and two healthy individuals. Quantitation of RBC-bound IgG was performed using quantum simply cellular (QSC) beads coated with goat anti-mouse IgG antibodies. For detection of RBC bound IgG, a 60-minute incubation of all blood samples with 40 microl of 1:10 dilution of FITC-conjugated mouse anti-human IgG gave mean fluorescent intensity (MFI) values comparable to experiments using larger amounts or higher concentrations of the anti-human IgG. The acquired antibody binding capacity (ABC) values (or IgG molecules) for each QSC bead level, at 40 microl of 1:5 and 1:10 dilution of anti-human IgG for 60 minutes were close to the manufacturer-assigned ABC values. The IgG molecules per RBC in all six patients with positive DAT of 4+, 3+, 2+, 1+, trace and negative DAT were 31,725, 3,823, 1,753, 524, 260 and 88 respectively and in two healthy individuals with negative DAT they were 104 and 78.     

Domain inhibitory and bacteriostatic activities of the five-domain Kazal-type serine proteinase inhibitor from black tiger shrimp Penaeus monodon

Serine proteinase inhibitors (SPIs) in multi-cellular organisms are important modulators of proteinase activities in various biological processes. A five-domain Kazal-type SPI SPIPm2 from the black tiger shrimp Penaeus monodon is presumably involved in innate immune response. The SPIPm2 with the domain P1 residues T, A, E, K and E was isolated from the hemocyte cDNA libraries and found to strongly inhibit subtilisin and elastase, and weakly inhibit trypsin. To unravel further the inhibitory activity of each domain, we subcloned, over-expressed and purified each individual SPI domain. Their inhibitory specificities against trypsin, subtilisin and elastase were determined. Domain 1 was found to be inactive. Domains 2, 3 and 5 inhibited subtilisin. Domain 2 inhibited also elastase. Domain 4 weakly inhibited subtilisin and trypsin. The intact SPIPm2 inhibitor was found to possess bacteriostatic activity against the Bacillus subtilis but not the Bacillus megaterium, Staphylococcus aureus, Vibrio harveyi 639 and Escherichia coli JM109. Domains 2, 4 and 5 contributed to this bacteriostatic activity.     

In vitro intestinal glucose uptake is inhibited by galactomannan from Canadian fenugreek seed (Trigonella foenum graecum L) in genetically lean and obese rats

Galactomannan, a soluble fiber, has been reported to reduce postprandial blood glucose response. Using this fiber, extracted from Canadian-grown fenugreek seeds (Trigonella foenum graecum L), we conducted an in vitro study to determine if galactomannan affects intestinal glucose uptake in genetically determined lean and obese rats. The segments of jejunum and ileum from these animals were incubated with labeled glucose (2 or 32 mmol/L) in the presence of different concentrations of galactomannan ranging from 0.1% to 0.5% (wt/wt). The uptake of low or high concentration of glucose was significantly and progressively reduced by increasing concentrations of galactomannan in both lean and obese rats. No significant difference was observed in the uptake of glucose between the 2 groups. The viscosity of various concentrations of galactomannan solutions was determined after stirring for 60 minutes at a temperature-controlled (37°C) fixed sheer rate of 1.29 (1/s). The inhibitory effect of galactomannan on glucose uptake was found to be in parallel with the degree of viscosity of the fiber solutions. These results suggest that the galactomannan, because of its viscous property, has the potential to reduce intestinal absorption of low or high concentrations of glucose and hence for the benefit of blood glucose management.     

The potential usefulness of the modified Kato thick smear technique in the detection of intestinal sarcocystosis during field surveys

A total of 479 stool specimens were collected from rural communities of Ubon Ratchathani Province, Thailand and examined by two techniques: the modified Kato thick smear and the direct smear. The prevalence of Opisthorchis viverrini (14.8%), hookworm (10.2%), Sarcocystis spp (4.6%), Taenia spp (2.9%), Strongyloides stercoralis (2.1%), Giardia lamblia (1.2%), Echinostoma spp (0.6%), Ascaris lumbricoides (0.4%), Entamoeba histolytica (0.2%), Chilomastix mesnili (0.2%) and Endolimax nana (0.2%) were determined. The morphology of the Sarcocystis spp sporocysts examined by both procedures looked similar and was found to be easily recognizable. Among these specimens, 22 cases (4.6%) were positive for Sarcocystis infection detected by the modified Kato technique, whereas only one case (0.2%) was detected by both techniques. These differences were found to be statistically significant (p < 0.05), indicating that the modified Kato technique was decidedly more sensitive than the direct smear procedure in identifying Sarcocystis infection. An epidemiological survey was conducted in Khon Kaen Province involving 1124 stool samples using the modified Kato technique. The greatest frequency was Opisthorchis viverrini at 32.0% while the second highest was Sarcocystis spp at 8.0%. The prevalences of hookworm, Echinostoma spp, Taenia spp, Trichuris trichiura and Enterobius vermicularis were 2.7, 2.1, 1.0, 0.2 and 0.2%, respectively. Other than opisthorchiasis, northeastern Thailand may be an endemic area for sarcocystosis. This is the first report of the applicability and potential usefulness of the Kato thick smear technique for the diagnosis of Sarcocystis infection in a field survey.     

Incidence of urinary tract infections in patients with short-term indwelling urethral catheters: a comparison between a 3-day urinary drainage bag change and no change regimens

BACKGROUND: The current practice of caring for hospitalized patients with indwelling urethral catheters in Siriraj Hospital is to change the drainage bag every 3 days. In an extensive medical literature search, no evidence was noted to support this practice. OBJECTIVE: The purpose of this study was to compare the incidence of catheter-associated urinary tract infections (UTI) in hospitalized patients with indwelling catheters who receive a drainage bag change every 3 days with the incidence of UTI in patients who receive no bag change. DESIGN: This study was a randomized controlled trial.Participants and study procedures: Of the patients at Siriraj Hospital, 153 with an indwelling urinary catheter for at least 3 days were randomized to a 3-day drainage bag change or a no change regimen. A urine sample was obtained from each patient for culture every 7 days, on the day the catheter was removed, or the day the patient was suspected of having a UTI. RESULTS: Of the 153 study patients, 79 were randomized to the 3-day bag change regimen, and 74 patients were in the no-change group. Both groups were comparable for all baseline characteristics. The incidence of symptomatic UTI was 13.9% in the 3-day drainage bag change group and 10.8% in the no change group (P =.7). The incidence of asymptomatic UTI was 36.7% in the 3-day bag change group and 36.5% in the no change group (P =.9). CONCLUSION: There is no evidence for the necessity of a bag change every 3 days at Siriraj Hospital; the urine bag can be left longer than 3 days. However, the appropriate frequency of urinary drainage bag change needs additional study because the sample size in this study does not rule out a false-negative result.     

Defective hepatitis B virus DNA is not associated with disease status but is reduced by polymerase mutations associated with drug resistance

Defective hepatitis B virus DNA (dDNA) is reverse-transcribed from spliced hepatitis B virus (HBV) pregenomic messenger RNA (pgRNA) and has been identified in patients with chronic HBV (CH-B). The major 2.2-kb spliced pgRNA encodes a novel HBV gene product, the hepatitis B splice protein (HBSP) via a deletion and frame shift within the polymerase. Although spliced RNA and HBSP expression have been associated with increased HBV DNA levels and liver fibrosis, the role of dDNA in HBV-associated disease is largely undefined. Our aims were to (1) compare the relative proportions of dDNA (% dDNA) in a range of HBV-infected serum samples, including patients with human immunodeficiency virus (HIV)/HBV coinfection and HBV-monoinfected persons with differing severities of liver disease, and (2) determine the effect of mutations associated with drug resistance on defective DNA production. Defective DNA was detected in 90% of persons with CH-B. There was no significant difference in the relative abundance of dDNA between the monoinfected and HIV/HBV-coinfected groups. We also found no association between the % dDNA and alanine aminotransferase, hepatitis B e antigen status, HBV DNA levels, fibrosis levels, compensated or decompensated liver cirrhosis, genotype, or drug treatment. However, the % dDNA was significantly lower in individuals infected with lamivudine-resistant (LMV-R) HBV compared with wild-type HBV (P < 0.0001), indicating that antiviral drug resistance alters the balance between defective and genomic length DNA in circulation. Experiments in vitro using HBV encoding LMV-R mutations confirmed these results. CONCLUSION: Our results identified no association between dDNA and parameters associated with disease status and suggested that the relative abundance of dDNA is largely dependent on the integrity of the HBV polymerase and is unrelated to the severity of liver disease.     

HIV Prevalence,Risk Behavior,Hormone Use and Surgical History Among Transgender Persons in Thailand

While Male-to-female transgender persons (TG) are believed to often engage in sex work and have high HIV infection risk, little is known about demographics, surgical and hormone use history, risk behaviors and HIV prevalence. Between March and October 2005, 474 TG from Bangkok, Chiangmai, and Phuket were surveyed using venue-day-time sampling. Of 474 participants, overall HIV prevalence was 13.5%. Most participants had completed at least secondary or vocational education (79.2%), gender self-identified as female (89.0%), had received money, gifts or valuables for sex (60.8%), and reported hormone use (88.6%). Surgical history was taken from 325 participants. Of these, 68.6% reported some form of surgery and 11.1% had undergone penile-vaginal reconstructive surgery. In multivariate analysis, being recruited from a park/street; older age, anal sex role identification as “versatile” and anal sex debut before age 13 were independently associated with HIV prevalence. The development, implementation and evaluation of culturally appropriate sexual health interventions for Thai TG is urgently needed.     

A case of successful treatment of severe COVID‐19 pneumonia with favipiravir and tocilizumab in post–kidney transplant recipient

We report a case of COVID‐19 in kidney transplant patient in Thailand. A 58‐year‐old 2 years post–kidney transplant recipient, with maintenance immunosuppression of tacrolimus, mycophenolate mofetil (MMF), and prednisolone, presented with acute diarrhea which followed by fever on day 12. Symptoms of pneumonia together with lymphopenia from complete blood count were developed on day 7 after onset of fever with the x‐ray finding of bilateral multifocal patchy infiltration. COVID‐19 infection has been confirmed by reverse real‐time polymerase chain reaction (PCR) in nasal swab as well as found in stool. Darunavir together with ritonavir, hydroxychloroquine, azithromycin, and favipiravir was initiated on the first day of admission at primary hospital. Patient has been transferred to our hospital on day 2 of admission in which tacrolimus together with MMF was discontinued. High‐flow nasal cannula oxygen therapy was required on days 4‐5 of hospitalization. Tocilizumab was administered after rising of serum IL‐6 level. Symptoms of pneumonia were improved in which no oxygen treatment required from day 10 of hospitalization. Drug interaction between tacrolimus and anti‐viral treatment leads to severely high level of tacrolimus which caused reversible acute kidney injury (AKI) after supportive treatment.