Comparison of PCR-based genotyping methods for hepatitis B virus.

Hepatitis B virus (HBV) is classified into eight genotypes (A to H). In this study, three genotyping methods were compared for their sensitivity and accuracy, namely PCR-RFLP on the S region, PCR-RFLP on the pre-S region and nested PCR with type specific primers. Sixty HBV samples from infected sera were genotyped. The nested PCR with type specific primers was found to be the most sensitive and produced substantial numbers of co-infections by genotypes B and C. The sensitivities for both PCR-RFLP methods were lower and did not reveal co-infections. Generally, the three methods produced consistent genotyping results in samples infected by single genotypes but for co-infections by genotypes B and C, the two PCR-RFLP methods yielded only single genotypic results. For the cases of single genotypic infections, genotypes ascertained by sequencing were in concordance across all three methods. However, when co-infections occurred, PCR analysis on clones revealed only single genotypic infections.

Influence of race, age and sex on the lymphocyte subsets in peripheral blood of healthy Malaysian adults.

The lymphocyte subsets in the peripheral blood of healthy Malaysian adults (212 subjects, age 18-71 years) were analysed using a flow cytometer FACScan in an effort to establish a reference range for the lymphocyte subsets. The lymphocyte subsets studied were T cells (CD3), B cells (CD19), natural killer (NK) cells (CD3- CD16+/CD56+), helper/inducer cells (CD4), cytotoxic/suppressor cells (CD8) and the helper/suppressor ratio (CD4/CD8). The distributions of T cells, CD4 cells and CD8 cells were symmetric about their means while B cells, NK cells and CD4/CD8 ratio followed a skewed distribution. Differences in race were observed for T cells, NK cells, CD4 cells and CD4/CD8 ratio where the Indians were significantly different from the Malays and the Chinese (higher T cells, CD4 cells and CD4/CD8 ratio and lower NK cells). The B cells were significantly lower in the Chinese than the Malays and the Indians. Age differences were seen only in the Chinese where increased CD4 cells and CD4/CD8 ratio, and decreased CD8 cells were observed. A sex difference was observed only in the Chinese where the CD4/CD8 ratio was significantly higher in females than males.

Serum ferritin concentrations in male Malaysian blood donors.

Serum ferritin and haemoglobin estimates were carried out on 78 first time blood donors with a view to determining iron store status. Of these 30 were Malays, 20 were Chinese and 28 were Indians. The ferritin level in Malay donors ranged from 16-160 mg/ml (mean 83 +/- 49.4 mg/ml in chinese donors is ranged from 36-500 mg/ml (mean 242.8 +/- 132 mg/ml), and in the Indian donors it ranged from 5 - 270 mg/ml (mean 94.6 +/- 67.9 mg/ml). The haemoglobin concentration for the whole group was 14.9 +/- 1.49 g/dl. There was no correlation of haemoglobin concentration with serum ferritin levels.

Some factors affecting the transformation of the yeast-like to the mycelial-like forms of Candida albicans.

Various carbohydrates tried as inducing agents for mycelia formation in Candida albicans in liquid medium have revealed that mycelia formation was very pronounced in the presence of any of the sugars like xylose, galactose, lactose and trehalose. Glucose and fructose favoured lavish yeast growth. Arabinose supported neither yeast nor mycelia growth to any appreciable extent. In a growth medium containing a mixture of glucose and galactose, a glucose concentration of 0.7% (w/v) and above was required to maintain the fungus completely in the yeast form. The possible reason as to why sufficient glucose maintained the yeast form while galactose favoured the mycelial form was discussed.

Prevalence of anti-HBc in Malaysian male blood donors and its correlation with DNA polymerase activity.

A study of Kuala Lumpur blood donors for HBsAG, anti-HBc and DNA polymeraes showed that 5.5% in the sample population was positive for HBsAG, 50.1% for anti-HBc and 10.1% for DNA polymerase activity. There was no significant difference of the HBsAG among the Malay, Chinese and Indian groups. However, a significant difference was observed for the anti-HBc and DNA polymerase activity between the Indian and the Malay/Chinese groups. Both analysis were significantly lower in the Indians but there was no significant difference between the Chinese and the Malays.

HBeAG and anti-HBe in the Malaysian population and in Malaysian prisoners.

The incidence of HBsAg in random blood donors was found to be twice that of the prisoner population. The anti-HBe however, was about twice that in the prisoners when compared with the random blood donors. Both the random blood donors and the prisoners had similar incidence of HBeAg. The percentage frequency of HBsAg positivity with anti-HBe positivity was also similar in both groups. The 18 normal non-blood donors did not have HBsAg, HBeAg or anti-HBe.

The cellular immune status of HBsAg-positive carriers in Malaysia.

The percentage of lymphocyte subsets from the peripheral blood of healthy adults and hepatitis B surface antigen (HBsAg) carriers were analyzed by flow cytometry. The five lymphocyte subsets studied were:- T (CD3) cells, B (CD19) cells, CD4 cells, CD8 cells, Natural Killer (CD3- CD16+/CD56+) cells (NK cells) and the CD4/CD8 ratio. The percentage (mean +/- SD) for the five lymphocyte subsets from the healthy adults were (67.5 +/- 8.5)%, (12.4 +/- 4.5)%, (35.5 +/- 7.8)%, (36.8 +/- 8.5)%, (17.9 +/- 8.1)% and 1.1 +/- 0.6, respectively. HBsAg carriers positive for HBV-DNA had a lower CD4/CD8 ratio than the healthy population (P = 0.030). The percentage of CD8 cells in HBsAg carriers increased significantly (r = 0.28; P = 0.019) with an increase in ALT levels but the values remained within normal range. The percentage of NK cells and CD4/CD8 ratio in HBsAg carriers positive for anti-HBe were higher than HBsAg carriers negative for anti-HBe (92% of which are HBeAg positive) (P = 0.045 and P = 0.035, respectively). The CD4/CD8 ratio in HBsAg carriers negative for anti-HBe (92% positive for HBeAg) was also lower than in the healthy population (P = 0.042). HBsAg carriers positive for HBV-DNA, HBeAg and raised ALT levels had a lower CD4/ CD8 ratio than did the healthy population. The lower ratio was due to an increase in the percentage of CD8 cells. This suggests an activated immune response triggered by the infection in an attempt to clear the virus. HBsAg carriers with normal ALT levels and who are negative for HBV-DNA may be in a state of tolerance.

A comparative study of two methods for the isolation of human leucocytes for DNA extraction.

The 'Dextran' and the 'Buffy-coat' methods for isolation of human leucocytes for DNA extraction were compared on the basis of DNA yield from the same amounts (10 ml) of blood. Human leucocytes from a total of 11 samples were isolated using both methods for each sample after which DNA was extracted. Extracted DNA samples were treated with ribonucleases and proteinase K after which the yields were quantitated by measuring absorbance at 260 nm. The 'Buffy-coat' method yielded a mean concentration of DNA of 476.7 micrograms/ml (range: 212 to 700 micrograms/ml) while the 'Dextran' method yielded 188.4 micrograms/ml (range: 64 to 340 micrograms/ml). The difference was confirmed by subjecting the extracted DNA samples to agarose gel electrophoresis.

An in-house assay for serum deoxythymidine kinase.

An in-house method which utilizes 14C-thymidine as a substrate was used to assay deoxythymidine kinase in serum. The method is sensitive enough to detect normal levels of serum deoxythymidine kinase and the assay procedure also enables rapid handling of multiple samples. With a total reaction volume of 60 ul, the enzyme reaction was found to be linear with concentrations for up to 650 U/L of TK activity. On studying serum deoxythymidine kinase (s-TK) activity with incubation time, there was a proportional increase in activity with the length of incubation time. "Within-batch" precision showed a coefficient of variation (CV) of 4.7% for serum with extremely high s-TK levels and a CV of 8.8% for serum with normal s-TK levels. S-TK showed a CV of less than 16.0% in its activity when stored at -8 degrees C and at -20 degrees C. The normal reference range obtained for s-TK activity was 8.6 +/- 7.5 U/L.

The distribution of immunoregulatory cells in the peripheral blood of normal Malaysian adults.

The distribution of immunoregulatory cells in the peripheral blood of an individual has now been established as an important tool in helping the management of several diseases. It is necessary to set the normal ranges of these cells for the laboratory. We have undertaken in this study to establish the reference ranges for normal Malaysian adults. We found that the mean percentages of T cells, B cells, T Helper cells (CD4), T suppressor cells (CD8), NK cells and the ratio of CD4/CD8 were 70.91%, 11.38%, 38.15%, 37.76%, 17.45%, and 1.00 respectively. There was no significant difference between the sexes. In certain parameters, there was significant differences between Malay, Chinese and Indians. The Chinese and Indians were significantly different in the distribution of B cells and in the CD4/CD8 ratio. In the case of CD4 and NK cells, the Indians were different from the other two groups.

Isolation of human surfactant protein A from amniotic fluid.

Surfactant protein A (SP-A) is one of the four known surfactant-associated proteins found in human lungs. It plays a major role in determining regulation of surfactant uptake and resecretion. Qualitative and quantitative deficiencies of SP-A may contribute to neonatal respiratory distress syndrome. The measurement of its level in amniotic fluid or neonatal tracheal aspirate may be useful in the assessment of replacement therapy using natural or synthetic surfactants. In order to develop an in-house immunoassay to detect the level of SP-A, we used a discontinuous sucrose density gradient to isolate SP-A from amniotic fluid. Polyacrylamide gel electrophoresis was carried out on the isolates with low molecular weight markers. We successfully isolated SP-A from 12 out of 31 samples of amniotic fluid. The isolates were found to be relatively pure and have a molecular weight of about 35 kD. The isolated SP-A were used as immunogens to raise antibodies in rabbits for the immunoassay.

An in-house ELISA method for measuring surfactant protein A.

An in-house enzyme-linked immunoabsorbant assay (ELISA) for SP-A was successfully developed using in-house polyclonal anti SP-A and a commercial polyclonal anti-rabbit immunoglobulin horseradish peroxidase conjugate system. The standard curve, generated by using 50 ng of SP-A to coat the plate and 1:500 dilution of polyclonal anti SP-A as a primary antibody, was linear for concentrations of SP-A ranging from 4 micrograms/l to 4000 micrograms/l and reproducible. Results of recovery study of SP-A from a known sample of tracheal aspirate ranged from 94%-114%. Intra- and inter-assay coefficients of variations were 2.7% and 5.6% respectively for a known sample of tracheal aspirate. Interference study showed that tracheal aspirate did not interfere with the assay. The assay developed was intended to be used for SP-A measurement in tracheal aspirates obtained from neonates with and without respiratory distress syndrome.

Effects of glycyrrhizic acid on 11 ß-hydroxysteroid dehydrogenase (11 ßHSD1 and 2) activities and HOMA-IR in rats at different treatment periods.

Glycyrrhizic acid (GA) has been reported to inhibit postprandial blood glucose rise and 11 ß-hydroxysteroid dehydrogenase 1 (11 ßHSD1) activity. As not much work has been done on GA effects on 11 ßHSD1 and 2 and HOMA-IR at different treatment periods, this work was conducted. 60 male Sprague Dawley rats fed AD LIBITUM were assigned into six groups of control and treated that were given GA at different duration namely 12, 24 and 48 h. Treated and control groups were intraperitoneally administered with GA (50 mgkg (-1)) and saline respectively. Blood and subcutaneous (ATS) and visceral adipose tissue (ATV), abdominal (MA) and quadriceps femoris muscle (MT), liver (L) and kidney (K) were examined. HOMA-IR in GA-treated rats decreased in all groups (P<0.05). In the 12-h and 24-h treated rats, 11 ßHSD1 activities decreased in all tissues (P<0.05) except MA and MT (P>0.05) in the former and ATV (P>0.05) in the latter. However, 11 ßHSD1 activities decreased significantly in all tissues ( P<0.05) in the 48-h treated rats. Significant decrease in 11 ßHSD2 (P>0.05) activities were observed in the L of all treatment groups and K in the 24-h and 48-h treated rats (P<0.05). Histological analysis on ATS showed increase in the number of small-size adipocytes while ATV adipocytes showed shrinkage after GA administration. Increased glycogen deposition in the L was observed in the GA-administered rats in all the treatment periods. In conclusion, GA treatment showed a decrease in the HOMA-IR and both 11 ßHSD1 and 2 activities in all tissues, with more profound decrease in the 48-h treated rats.