Adaptation of the plaque assay methodology for dengue virus infected HepG2 cells

The HepG2 cell line is a useful tool for studying dengue virus-cell interactions but as it grows in clumps rather than monolayers, it does not readily adapt itself to the standard plaque assay technique. We therefore sought to develop an indirect plaque assay methodology. Initially HepG2 cells were infected with dengue virus serotype 2 and post-infection incubated for between 0 and 16 h before being treated with trypsin to separate the cells, followed by dilution and plating onto pre-grown monolayers of Vero cells in six well plates. After 7 days incubation and crystal violet staining, plaques were observed at all time points, although there was a relationship between number of plaques and post-infection incubation time, with the longest post-infection incubation time giving the highest number of plaques. To validate the assay with respect to virus input, the experiment was repeated at both the 0 and 16 h post-infection incubation times with different virus: cell levels. At both post-infection incubation times the response of input virus to plaque number was linear. This is a useful adaptation of the plaque assay methodology and one that may be applicable to other virus/cell line combinations.     

Moringa oleifera Pod Inhibits Inflammatory Mediator Production by Lipopolysaccharide-Stimulated RAW 264.7 Murine Macrophage Cell Lines

Pro-inflammatory mediators produced during inflammatory response have been demonstrated to initiate and aggravate pathological development of several chronic diseases. Plant bioactive constituents have been reported to exert anti-inflammatory activities. Various parts of Moringa oleifera have long been used as habitual diets and traditional remedy along the tropical region. Anti-inflammatory activity of boiled M. oleifera pod extract was assessed by measuring pro-inflammatory mediator expression in the lipopolysaccharide-induced murine RAW264.7 macrophage cells. Prior treatment with 31–250 μg/mL M. oleifera extract for 1 h inhibited elevation of mRNA and protein level of interleukine-6, tumor necrosis factor-alpha, inducible nitric oxide synthase, and cyclooxygenease-2, induced by lipopolysaccharide for 24 h in a dose-dependent manner. The suppressive effect was mediated partly by inhibiting phosphorylation of inhibitor kappa B protein and mitogen-activated protein kinases. These results indicate that the anti-inflammatory activity from bioactive compounds present in the M. oleifera pod constituents may contribute to ameliorate the pathogenesis of inflammatory-associated chronic diseases.     

Survey of leptospirosis of small mammals in Thailand

During 1999-2000, kidney tissues of approximately 15% of 1310 rodents trapped from northeastern provinces of Thailand were tested for the presence of leptospires. Our direct immunofluorescent assay (DFA) for detection of leptospires showed 100% sensitivity and 94% specificity with the culture data. Both methods identified R. norvegicus as the highest source of infection. Among isolated Leptospira, 137 were serotyped by cross agglutinin absorption and/or a microscopic agglutination, and gave some variations and similarities at the serovar level to the DFA results. DFA data demonstrated over half of the positive animals were infected with several serovars of Leptospira interrogans. A subsequent DFA study in Bangkok in 2002 revealed leptospiral infection in 33% of 42 rats and shrews. The most common infecting serovars were Autumnalis and Canicola identified in rural and urban animals, respectively. This finding suggests that wild small mammals may act as important sources of pathogenic leptospires and warrant active surveillance to understand the epidemiology of transmission and control of carrier animals.     

Atypical avian influenza (H5N1)

We report the first case of avian influenza in a patient with fever and diarrhea but no respiratory symptoms. Avian influenza should be included in the differential diagnosis for patients with predominantly gastrointestinal symptoms, particularly if they have a history of exposure to poultry.     

Diagnosis of human leptospirosis by monoclonal antibody-based antigen detection in urine

Hybridomas secreting specific monoclonal antibodies (MAb) to all members of the genus Leptospira (clone LF9) and those that are specific only to the pathogenic species (clones LD5 and LE1) were produced. MAb LF9, which was immunoglobulin G1 (IgG1), reacted to a 38-kDa component of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell lysates of all Leptospira spp., while MAb LD5 and MAb LE1, which were IgG1 and IgG2a, respectively, reacted to the 35- to 36-kDa components of all serogroups of the pathogenic species of LEPTOSPIRA: The MAb LD5 was used in a dot blot-enzyme-linked immunosorbent assay (dot-ELISA) for detecting Leptospira antigen in urine samples serially collected from two groups of patients diagnosed with leptospirosis, i.e., 36 clinically diagnosed patients and 25 Leptospira culture confirmed patients. Their serum samples were tested serologically by IgM Dipstick assay, indirect immunofluorescence assay (IFA), and/or microscopic agglutination test (MAT). Urine samples of 26 patients diagnosed with other illnesses and 120 healthy individuals served as controls. For the first group of patients, who had been ill for an average of 3.4 days before hospitalization, the IgM Dipstick test, IFA, and MAT were positive for 69.4, 70.0, and 85.7% of patients, while the Leptospira antigenuria tested by the MAb-based dot-ELISA was positive for 75.0, 88.9, 97.2, 97.2, and 100% of patients on days 1, 2, 3, 7, and 14 of hospitalization, respectively. All but 1 of 11 patients whose serum samples collected on the first day of hospitalization were IgM seronegative, were positive by urine antigen test on day 1. This is strong evidence that detection of antigen in urine can provide diagnostic information that could be useful in directing early therapeutic intervention. The MAT was positive in 10 of 12 patients (83.3%) of the 25 culture-positive Leptospira patients who had been ill for an average of 5.04 days before hospitalization, and the Leptospira antigen was found in 64.0, 84.0, 96.0, 100, 100, 100, and 100% of the patients' urine samples collected on days 1, 2, 3, 4, 5, 6, and 7 of hospitalization, respectively. Leptospira antigenuria was found in 3 of the 26 patients diagnosed with other illnesses and 1 of the 120 healthy controls. The reasons for this positivity are discussed. The detection of antigen in urine by the monoclonal antibody-based dot-ELISA has high potential for rapid, sensitive, and specific diagnosis of leptospirosis at a low cost.     

Successful treatment of acquired amegakaryocytic thrombocytopenic purpura refractory to corticosteroids and intravenous immunoglobulin with antithymocyte globulin and cyclosporin

Four patients with acquired amegakaryocytic thrombocytopenic purpura, who had failed corticosteroids, intravenous immunoglobulin and cyclophosphamide therapy, were treated with antithymocyte globulin, followed by cyclosporin. Three patients achieved complete remission in 28-178 days and the response duration was 16-60 months from the beginning of treatment. One patient achieved a partial response for 2 months followed by myelodysplastic syndrome 5 months later. He died in 9 months due to intracerebral bleeding. Marrow cytogenetics showed 47, XY, +21.     

Types,Clinical Features,and Survival Outcomes of Patients with Acute Myeloid Leukemia in Thailand: A 3-Year Prospective Multicenter Study from the Thai Acute Leukemia Study Group (TALSG)

BackgroundAcute myeloid leukemia (AML) is a common, challenging hematologic malignancy worldwide. Thai data on its characteristics and outcomes have never been systematically reported, to our knowledge. The objective of this study was to determine the clinical features and outcomes of Thai patients with AML.Patients and MethodsThis was a prospective observational study of nine academic hospitals. Patients with newly diagnosed AML were invited to register online.ResultsA total of 679 patients with AML were included. The presence of circulating peripheral blood blasts was correlated with a high white blood cell count. Acute promyelocytic leukemia (APL) had predominantly lower white blood cell counts and higher proportions without peripheral blood blasts compared with non-APL AML. Disseminated intravascular coagulation was commonly presented in APL (37.7%). Splenomegaly and normal platelet count were more frequently seen in patients with Philadelphia chromosome–positive AML. The median follow-up time for those who survived more than 1 year was 28.0 months. One-year overall survival rates for non-APL AML and APL were 31.9% and 88.2%, respectively; 2-year overall survival rates were 29.6% and 88.2%, respectively. Hematopoietic stem cell transplantation could improve survival in non-APL AML.ConclusionAPL should be considered despite absence of peripheral blood blast. This study demonstrates poor outcome of Thai AML and more research to improve outcomes are underway. Expanding access to hematopoietic stem cell transplantation should be considered in Thailand.     

Post-Loop Electrosurgical Excision Procedure Complications in Srinagarind Hospital

The purpose of this study was to evaluate the prevalence and predictors of post-Loop Electrosurgical Excision Procedure (LEEP) complications in Srinagarind Hospital, Khon Kaen, Thailand. Retrospective chart review was performed for 200 patients undergoing LEEP during January 2012 to February 2013. Their mean age was 45 years-old. Fifty-three (26.5%) were menopausal. The three most common preceding abnormal cervical cytology were high-grade squamous intraepithelial lesion (HSIL; 50%), atypical squamous cell cannot exclude HSIL (ASC-H; 10.5%), and low-grade squamous intraepithelial lesion (LSIL; 10%). The overall complications prevalence rate was 16.5% (95%CI, 11.4-21.6). Complications included bleeding (11%; 95%CI, 6.66-15.3), offensive discharge (4%; 95%CI, 1.28-6.72), and pelvic inflammatory disease (1.5%; 95%CI, 0.18-3.18). Only mode of delivery was an independent predictor of post-LEEP complications. Women with previous caesarean sections carried an increased risk of complications by 3.9 times (95%CI, 1.21-12.56) compared with vaginal delivery. In conclusion, LEEP is generally safe with an acceptable complication rate. Previous caesarean section was the only independent predictor for post-LEEP complications. However, this predictor still needs prudent evaluation as no clear cause-effect relationship was identified.     

The risk of kidney stones following bariatric surgery: a systematic review and meta-analysis

Background With rising prevalence of morbid obesity, the number of bariatric surgeries performed each year has been increasing worldwide. The objective of this meta-analysis was to assess the risk of kidney stones following bariatric surgery. Methods A literature search was performed using MEDLINE, EMBASE, and Cochrane Database of Systematic Reviews from inception through July 2015. Only studies reporting relative risks, odd ratios or hazard ratios (HRs) to compare risk of kidney stones in patients who underwent bariatric surgery versus no surgery were included. Pooled risk ratios (RR) and 95% confidence interval (CI) were calculated using a random-effect, generic inverse variance method. Results Four studies (One randomized controlled trial and three cohort studies) with 11,348 patients were included in analysis to assess the risk of kidney stones following bariatric surgery. The pooled RR of kidney stones in patients undergoing bariatric surgery was 1.22 (95% CI, 0.63–2.35). The type of bariatric surgery subgroup analysis demonstrated an increased risk of kidney stones in patients following Roux-en-Y gastric bypass (RYGB) with the pooled RR of 1.73 (95% CI, 1.30–2.30) and a decreased risk of kidney stones in patients following restrictive procedures including laparoscopic banding or sleeve gastrectomy with the pooled RR of 0.37 (95% CI, 0.16–0.85). Conclusions Our meta-analysis demonstrates an association between RYGB and increased risk of kidney stones. Restrictive bariatric surgery, on the other hand, may decrease kidney stone risk. Future study with long-term follow-up data is needed to confirm this potential benefit of restrictive bariatric surgery.