Rapid detection and identification of dengue viruses by polymerase chain reaction (PCR).

A polymerase chain reaction (PCR) method using sets of newly designed primers for rapid detection and simultaneous identification of dengue virus serotypes was developed and tested. The test is based on two sets of primers specific within the envelope (E) and non-structural (NS1) regions of the dengue-virus genome. Two sets of universal primers that bind to two target sequences which are shared by all the four serotypes of the virus within the E and NS1 regions are used. The resulting products are further amplified by another pair of inner or nested universal primers, which also bind to another set of shared sequences within the E and NS1 regions, respectively. The nested PCR of both the E and NS1 regions can detect dengue virus of all the four serotypes at a sensitivity of 1 plaque forming unit (pfu) or less. For the identification of serotypes, a mixture of four pairs of serotype-specific primers, specific to the E region, was used. The primers have been designed to bind to serotype specific sequences within the regions flanked by the outer universal primers, and giving the amplified products of different sizes, each corresponds to one particular serotype (405 bp for Den1, 346 bp for Den2, 196 bp for Den3, and 143 bp for Den4). A protocol has been developed and successfully applied to detect dengue virus in cell-culture supernatants and patients sera. The technique is simple and rapid, capable of not only detecting the dengue virus but also identifying the serotypes of the virus in clinical specimens.

Anti-dengue IgG detection by an indirect ELISA.

Protein-free culture media were originally developed for hybridomas to simplify downstream processing and purification. For the same reasons, we have used these protein-free media for passaging dengue 2 virus in C6/36 cells. This provided us with an infected supernatant (DenPF) which could then be used as coating antigens for an indirect enzyme-linked immunosorbent assay (ELISA) to determine dengue IgG levels. Using this preparation, the main immunogenic band as seen by immunoblot appeared to be viral envelope protein (E). Without the high concentrations of "competing" proteins from fetal calf serum (FCS), the Den2PF could be directly coated onto 96-well ELISA plates. The amount of viral proteins in Den2PF appeared to be sufficient so that there was no need for further purification steps, eg polyethylene glycol (PEG) precipitation, which made this preparation cost effective. It compared favorably with the dengue dot enzyme immunoassay (DEIA; sensitivity of 95.7% and specificity of 95.2%).

Glomerulonephritis in beta-thalassemia Hb-E disease: clinical manifestations, histopathologic studies and outcome.

We presented 8 patients with beta-thal/Hb E with glomerular diseases. Diverse glomerular lesions were seen, but diffuse endocapillary glomerulonephritis was the most common. The clinical manifestations of acute glomerulonephritis in beta-thal/Hb E differed from typical cases in the older age group, female preponderance, longer duration of edema, less hypertension, marked proteinuria, hypoalbuminemia and hypertriglyceridemia and also a longer period of recovery but their outcome was still favorable despite many risk factors of renal injury. Renal biopsy was necessary in doubtful cases to detect the correct diagnosis and give proper management. The association and mechanism of glomerulonephritis in these patients require further prospective study.

Lupus nephritis in males: 8-year experience at Siriraj Hospital.

During 1984 to 1991, 54 out of 569 lupus nephritis patients at Siriraj Hospital were male (F:M sex ratio = 10:1). Mean age of the males was 29.8 +/- 14.6 years, range 12 to 69. The three most common extrarenal manifestations were anemia, cutaneous, and musculoskeletal involvement (74.5, 51.1, and 43.9%, respectively). The major renal manifestations were edema (75.9%) with heavy proteinuria over 3.5 g/day in 62.2% and nephrotic/nephritic findings in 51.9% of cases. Hypertension was found in 35.2%. Mean serum creatinine was 2.0 +/- 1.4 mg/dl while 60.5% of cases had creatinine clearance below 50 ml/minute. Mean serum albumin was 2.6 +/- 0.8 g/dl, cholesterol 262.8 +/- 129.5 and triglycerides 343.2 +/- 244.6 mg/dl. Interestingly, hypercholesterolemia (> 250 mg/dl) was found only in 44.8% of cases with nephrotic syndrome. Antinuclear antibody was demonstrated in 91.5%, anti-dDNA antibody in 64.4% and LE cells in 40.4% of cases. Renal biopsy was done in 45 patients and 30 cases (66.7%) were classified as diffuse proliferative nephritis (WHO type IV), 15.6% of type II, 6.7% each of type III and V, with the rest of type V plus IV (4.4%). Tubulointerstitial inflammation was found in 77.3% of cases. During the follow-up period (42 +/- 35.8 months), 6 patients died. The cause of death were uremia in 3, infection in 2, and cardiac failure in 1. By life-table analysis, the probabilities of survival for 1 and 5 years were 89.5 and 80.6%, respectively. In comparison between sexes, except for a higher amount of urinary protein excretion (4.5 +/- 3.1 vs 3.5 +/- 3.0 g/day, p < 0.05), there were no statistically significant differences in clinical and pathological parameters, and probability of survival.

Analysis of serum urinary proteins in normal and renal diseases by cellulose acetate electrophoresis.

A method using dried polyacrylamide gel to concentrate urine samples has been described, tested and used for the purpose of urine protein analysis. Concentrated urine samples from 10 normals and 100 patients with IgM nephropathy and systemic lupus erythematosus (SLE) were analysed by cellulose acetate electrophoresis (CAE). The results demonstrated that the patterns of proteins in the electrophoresis could be used to discriminate the two diseases. The best discriminating power was found in the logarithm of gamma globulin to albumin ratio. In IgM nephropathy the ratio of gamma globulin to albumin is much smaller than the ratio in SLE, indicating that relatively larger gamma globulins were excreted in SLE. In addition, the ratio can be used to discriminate subgroups of patients with IgM nephropathy. Urine from patients with IgM nephropathy with focal and segmental changes showed a significantly higher ratio. The study indicated the usefulness of the technique in discriminating the two common glomerular diseases.

Urinary constituents in an endemic area of stones and renal tubular acidosis in northeastern Thailand.

Nephrolithiasis and endemic renal distal tubular acidosis are common in northeastern Thailand. The etiology is still unknown. It is generally accepted that urine electrolytes influence the capacity of urine to inhibit or promote renal and also bladder stones. The purpose of this study was to analyse the composition of the urine in the indigenous population in the northeast area and compare their values with data obtained from a group of age matched adults, living in Bangkok. Twenty-four hour urine samples from 23 normal adult villagers from six villages within the province of Khon Kaen and 34 normal adults living in Bangkok were collected, and the daily excretion of creatinine, uric acid, calcium and inorganic phosphate, sodium, potassium, chloride, magnesium and oxalate were assayed. Daily urinary sodium, potassium, chloride and phosphate of the villagers were significantly lower than those of Bangkokians. No difference in the urinary excretion of calcium, magnesium, uric acid, oxalate and creatinine was found. The Na/Ca, and Ca/PO4 ratios of villagers were significantly lower than those of the Bangkok subjects. The villagers excreted significantly lower amounts of Na in the face of relatively higher urinary Ca. The above data, combined with our previous study showing the low values of urinary citrate in the villagers in the same areas, strongly indicate that the indigeneous population is at high risk in developing urolithiasis. The causes for these electrolyte abnormalities are still unknown. Low contents of the major electrolytes in their diets might play an important role. Low phosphate output indicates low protein diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Severe cases of green pit viper snake venom poisoning.

Two severe cases of green pit viper bites were reported. The clinical manifestations were severe bleeding and diarrhea. The first patient had acute renal failure complicated from severe diarrhea and hypovolemia. Hematological findings showed hypofibrinogenemia and thrombocytopenia. Renal biopsy performed in the first case showed a picture compatible with that of the recovery phase of acute tubular necrosis. Green pit viper antivenine serum together with symptomatic and supportive treatment was given. Both patients recovered uneventfully.

Complement and dengue haemorrhagic fever/shock syndrome.

The complement system is activated in DHF/DSS. The peak of activation and the presence of C3a and C5a anaphylatoxins coincided with the onset of shock and leakage. The levels of C3a correlated well with disease severity. This indicated an important role of the complement system in the pathogenesis of shock. Circulating immune complexes as assayed by two standard techniques were not detected in the majority of patients, and if detected were found in small amount. The role of circulating immune complexes in the activation of complement in DHF/DSS needs to be reinvestigated, and other possible mechanisms leading to complement activation should be sought.