Molecular and serological detection of occult hepatitis B virus among healthy hepatitis B surface antigen-negative blood donors in Malaysia.

BACKGROUND: Occult hepatitis B infections are becoming a major global threat, but the available data on its prevalence in various parts of the world are often divergent. OBJECTIVE: This study aimed to detect occult hepatitis B virus in hepatitis B surface antigen-negative serum using anti-HBc as a marker of previous infection. PATIENT AND METHODS: A total of 1000 randomly selected hepatitis B surface antigen-negative sera from blood donors were tested for hepatitis B core antibody and hepatitis B surface antibody using an ELISA and nested polymerase chain reaction was done using primers specific to the surface gene (S-gene). RESULTS: Of the 1000 samples 55 (5.5%) were found to be reactive, of which 87.3% (48/55) were positive for hepatitis B surface antibody, indicating immunity as a result of previous infection however, that does not exclude active infection with escaped mutant HBV. Nested PCR results showed the presence of hepatitis B viral DNA in all the 55 samples that were positive for core protein, which is in agreement with the hepatitis B surface antibody result. CONCLUSION: This study reveals the 5.5% prevalence of occult hepatitis B among Malaysian blood donors as well as the reliability of using hepatitis B core antibody in screening for occult hepatitis B infection in low endemic, low socioeconomic settings.

Design and validation of small interfering RNA on respiratory syncytial virus M2-2 gene: A potential approach in RNA interference on viral replication.

Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in infants and young children globally and is a significant pathogen of the elderly and immunocompromised. The M2-2 protein of respiratory syncytial virus (RSV) is particularly important in regulation of viral RNA transcription and replication that could be a potential anti-viral candidate against RSV infection. In this study, we designed and validated siRNAs that specifically target the RSV M2-2 gene. Four siRNAs targeting different regions of the M2-2 gene were designed using web tool. In-vitro evaluation of silencing effect was performed by using RSV infected Vero cell line. Viral M2-2 linked GFP recombinant plasmid was co-transfected with non-targeted siRNA, Pooled siRNA, siRNA 1, siRNA 2, siRNA 3 and siRNA 4 using synthetic cationic polymer. The silencing effect of M2-2 gene at the protein level was measured both qualitatively and quantitatively by using fluorescence microscopy and flow cytometry. Meanwhile, the silencing effect at the mRNA level was assessed by using RT-qPCR. This study showed that all four designed siRNAs can effectively and efficiently silence M2-2 gene. siRNA 2 showed the highest (98%) silencing effect on protein level and siRNA 4 with 83.1% at the mRNA level. The viral assay showed no significant cytopathic effects observed after 6days post-infection with siRNAs. In conclusion, this study showed the effectiveness of siRNA in silencing M2-2 gene both at the protein and mRNA level which could potentially be used as a novel therapeutic agent in the treatment of RSV infection. However, further study is warranted to investigate the silencing effect of M2-2 protein and inhibition of RSV infection.

An Overview of Hepatitis B Virus Surface Antigen Mutant in the Asia Pacific.

Hepatitis B virus infection is a serious health problem worldwide, and more than 350 million people are chronic carriers, constituting a major global threat. Southeast Asia and the Western Pacific have the highest levels of endemicity in the world, with an estimated seroprevalence ranging between 2% and 31%. Mutations in the hepatitis B surface antigen (HBsAg) have been reported in many parts of the world but are most common in Asian infants; such mutants have several clinical effects, such as the development of hepatocellular carcinoma. Diagnostic failures by commercial assays have reduced the diagnostic effectiveness of HBsAg detection. For example the substitution of an amino acid in the major hydrophilic region of the S gene reduces the binding of hepatitis B surface antibodies leading to immune escape. The safety of blood transfusion may be compromised by current screening tests due to escape from being neutralised by antibodies induced by HBsAg mutants, and undetectable levels of viral surface protein. Data on the epidemiology of HBsAg mutation in Asia Pacific are scant; however, this manuscript has reviewed the available information on the epidemiology of HBsAg mutation in Asia Pacific.

Antibody and immune memory persistence post infant hepatitis B vaccination.

OBJECTIVES: This study aimed to evaluate the level of hepatitis B immunity among undergraduate students 23 years after commencement of the nationwide hepatitis B childhood immunization program in Malaysia. METHODS: A total of 402 serum samples obtained from volunteer undergraduate students were screened for the presence of hepatitis B surface antibodies using qualitative ELISA. RESULTS: Results showed that 62.7% of volunteers had protective anti-hepatitis B surface antigens (≥10 IU/L), of whom 67.9% received three doses of the vaccine. The estimated post-vaccination immunity was found to be at least 20 years, indicating persistent immunity against hepatitis B and a significant association (P < 0.05) with duration of vaccination. Anamnestic response 1 month post-hepatitis B booster was 94.0% and highly significant (P < 0.01). Isolated antihepatitis B core antigen (anti-HBc) prevalence was found to be 5.0%, all having had a positive anamnestic response. CONCLUSION: Immunity after primary vaccination with hepatitis B recombinant vaccine persists for at least 20 years post-vaccination, with significant association with the number of vaccinations. Furthermore, the presence of anamnestic response to booster vaccine indicates long-lasting immunity despite decreasing antibody levels; therefore, the need for hepatitis B vaccine boosters may not be of significant benefit after complete infant vaccination.

Free radical scavenging, antimicrobial and immunomodulatory activities of Orthosiphon stamineus.

Orthosiphon stamineus is considered an important traditional folk medicine. In this study ethanol and aqueous extracts of O. stamineus were evaluated in vitro for their antioxidant, antimicrobial as well as for their immunomodulatory properties on human peripheral blood mononuclear cells (PBMCs). The DPPH radical scavenging method was used for the determination of antioxidant activity, while the antibacterial efficacy was investigated by both disc diffusion method and Minimum Inhibitory Concentration (MIC) against four bacterial strains (Gram-positive and Gram-negative). Furthermore, the immunomodulatory potential of the extracts was investigated through the MTT assay. Aqueous extract of O. stamineus exhibited significant free radical scavenging activity with IC50 50 9.6 µg/mL, whereas the IC50 for the ethanol extract was 21.4 µg/mL. The best antimicrobial activity was shown by the aqueous extract of O. stamineus against Staphylococcus aureus, with inhibition zone of 10.5 mm and MIC value 1.56 mg/mL. Moreover, the results observed from the MTT assay showed that both plant extracts stimulated the PBMCs proliferation in vitro in a concentration-dependent manner, but the aqueous extract has remarkable activity against PBMCs. These findings indicate that O. stamineus showed high antioxidant activity and may be considered as an immunomodulatory agent.

Comparison between allicin and fluconazole in Candida albicans biofilm inhibition and in suppression of HWP1 gene expression.

Candida albicans is an opportunistic human pathogen with the ability to differentiate and grow in filamentous forms and exist as biofilms. The biofilms are a barrier to treatment as they are often resistant to the antifungal drugs. In this study, we investigated the antifungal activity of allicin, an active compound of garlic on various isolates of C. albicans. The effect of allicin on biofilm production in C. albicans as compared to fluconazole, an antifungal drug, was investigated using the tetrazolium (XTT) reduction-dependent growth and crystal violet assays as well as scanning electron microscopy (SEM). Allicin-treated cells exhibited significant reduction in biofilm growth (p<0.05) compared to fluconazole-treated and also growth control cells. Moreover, observation by SEM of allicin and fluconazole-treated cells confirmed a dose-dependent membrane disruption and decreased production of organisms. Finally, the expression of selected genes involved in biofilm formation such as HWP1 was evaluated by semi-quantitative RT-PCR and relative real time RT-PCR. Allicin was shown to down-regulate the expression of HWP1.

Comparison between efficacy of allicin and fluconazole against Candida albicans in vitro and in a systemic candidiasis mouse model.

The efficacy of allicin compared with fluconazole in alleviating systemic Candida albicans infections was evaluated both in vitro and in vivo through a systemic candidiasis mouse model. Determination of in vitro minimum inhibitory concentrations (MICs) for different C. albicans isolates revealed that both allicin and fluconazole showed different MICs that ranged from 0.05 to 12.5 µg mL(-1) and 0.25 to 16 µg mL(-1) , respectively. A time-kill study showed a significant effect of allicin (P<0.01) against C. albicans, comparable to that of fluconazole. Scanning electron microscopy observation revealed that, similar to fluconazole, allicin produced structural destruction of C. albicans cell surface at low MIC and lysis or puncture at high MIC concentrations. Treatment of BALB/c mice systemically infected with C. albicans showed that although the allicin treatment (at 5 mg kg(-1) day(-1) ) was slightly less efficacious than fluconazole treatment in terms of the fungal load reduction and host survival time, it was still effective against C. albicans in terms of mean survival time, which increased from 8.4 to 15.8 days. These results demonstrate the efficacy of anticandidal effects of allicin both in vitro and in an animal model of candidiasis and affirm the potential of allicin as an adjuvant therapy to fluconazole.

Expression analysis of SIR2 and SAPs1-4 gene expression in Candida albicans treated with allicin compared to fluconazole.

One of the main factors for virulence of fungus such as Candida albicans is the ability to change its morphology from yeast to hyphae. Allicin, one of the volatile sulfur-oil compounds from freshly crushed garlic, has a variety of antifungal activities. In this study, the effect of allicin on growth and hyphae production in C. albicans as compared to fluconazole, an antifungal drug was investigated using survival time in vitro and microscopic image at different time intervals. Additionally, the expression of selected genes involved in hyphae formation and development such as SIR2 and SAP1-4 was evaluated by semi-quantitative RT-PCR and relative real time RT-PCR. Allicin was shown to down-regulate the expression of SIR2 (5.54 fold), similar to fluconazole (3.48 fold) at 2x MIC concentrations. Interestingly, allicin had no effect on SAPs1-4 expression, whereas fluconazole was able to suppress SAP4 expression. Our findings showed that allicin was effective in suppressing hyphae development of C. albicans to an extent that is sometimes equal or more than fluconazole. Moreover, allicin and fluconazole seemed to share a common anti-Candida mechanism through inhibition of SIR2 gene, while fluconazole appeared to also exert its fungistatic effect through another pathway that involved SAP4 suppression.

The therapeutic efficacy of sulfadoxine/pyrimethamine against Plasmodium falciparum in Yemen.

OBJECTIVE: The aim of this study was to determine the sensitivities of Plasmodium falciparum clinical isolates to sulfadoxine/pyrimethamine (SP) using in vivo and in vitro methods. SUBJECTS AND METHODS: In vivo and Mark III in-vitro test techniques according to World Health Organization protocols of antimalarial drug tests were used to determine the SP susceptibility of the P. falciparum isolates from 100 malaria patients of both sexes between the ages of 3.5 and 45 years and living in Tihamah, Yemen. The study was conducted between 19 March and 12 May 2005. RESULTS: In vivo: no therapeutic failure occurred; the clinical outcome matched the parasitological response and all patients were parasite free by day 3 and remained so on days 7, 14 and 28. In vitro: all the P. falciparum isolates developed to schizonts in zero-drug-concentration wells, but were inhibited in 40 nmol/l of SP; the mean effective concentration (EC(99)) was 67.17 nmol/l. CONCLUSION: Our findings showed that the SP combination is still effective for the treatment of uncomplicated P. falciparum malaria in Yemen. It is recommended that further studies be carried out to address the importance of dihydropteroate synthetase/dihydrofolate reductase mutations as predictive markers of sulfadoxine/pyrimethamine resistance in Yemen.

The prevalence and degree of resistance of Plasmodium falciparum to first-line antimalarial drugs: an in vitro study from a malaria endemic region in Yemen.

BACKGROUND: Unpublished studies on antimalarial drug efficacy have found low levels of chloroquine resistance in Yemen. This study was carried out to determine the current prevalence of drug resistance in Plasmodium falciparum in Yemen to the main anti-malarial drugs and to determine the effective concentration(EC) values. METHODS: The WHO standard protocol was used for the selection of subjects, collection of blood samples, culture techniques, examination of post-culture blood slides and interpretation of results. The in vitro micro-test Mark III was used for assessing susceptibility of P. falciparum isolates. RESULTS: The criteria for blood parasite density was met by 219 P. falciparum malaria patients. Chloroquine resistance was found in 47% of isolated P. falciparum schizonts. Mefloquine resistanfce was found in 5.2%. In addition, the EC50 and EC95 values in blood that inhibited schizont maturation in resistant isolates were higher than the normal therapeutic level of mefloquine. No resistance occurred against quinine or artemisinin, with no growth at hte cut-off level of quinine and inhibition of low concentrations of artemisinin. CONCLUSION: Our study confirmed the occurrence of chloroquine-resistant P. falciparum and a slow increase in the rate of this resistance; it is likely that resistance will increase further and spread over all the foci of malaria in Yemen. The low rate of mefloquine-resistant P. falciparum, was lower than that reported in Africa or Southeast Asia, but it is the first report of mefloquine resistance in Yemen. Finally, the isolates were sensitive to low-concentrations of quinine and artemisinin.

Increasing single and multi-antibiotic resistance in Shigella species isolated from shigellosis patients in Sana'a, Yemen.

OBJECTIVE: The epidemiology and antibiotic susceptibility of Shigella species changes over time. Updated susceptibility knowledge is necessary for appropriate empirical antibiotic treatment. Thus, this research aimed to study these changes in 2 time periods with an interval of 10 years. METHODS: Two hundreds and three Shigella strains, isolated from stool samples of diarrheic patients at the Central Health Laboratory in Sana'a, Yemen in 2 time periods (1993 and 2003) with a 10-year interval, were examined for serotyping and drug resistance pattern. Resistance patterns of the strains to 12 commonly used antimicrobial agents and minimum inhibitory concentrations of the antibiotics were tested. RESULTS: Shigella flexneri (60%) was found to be the most common isolate of the total Shigella species, followed by Shigella dysenteriae (28.6%) and Shigella boydii (11.3%). In Shigella flexneri strains, Shigella flexneri 3 (30.5%) was the most prevalent serotype, followed by Shigella flexneri 6 (17.2%), and Shigella flexneri 1 (12.3%). All strains were found equally susceptible to cefotaxime, ceftriaxone, ciprofloxacin, and gentamicin, but more than 80% of the strains of 2003 were resistant to tetracycline, co-trimoxazole, and 52% of the same strains were resistant to ampicillin. Resistance to chloramphenicol was found in 61%, cefuroxime in 56.2%, and cephradine, 52% of the strains. Overall, Shigella species showed statistically significant increase in resistance against tetracycline, cephradine, trimethoprim/sulfamethoxazole, nalidixic acid, and aztreonam (p<0.05) over the 10 years period. This indicates decreased efficacy of co-trimoxazole and nalidixic acid for the empirical treatment of shigellosis in Sana'a, Yemen. Almost 55.2% of the strains were resistant to 4 drugs. CONCLUSION: This is one of the first studies reporting epidemiological pattern of Shigella species in Sana'a, Yemen with regard to serotypes and antibiotic resistance patterns. Based on these antibiotic resistance pattern findings, it is suggested that the commonly in use antibiotics including ampicillin, trimethoprim/sulfamethoxazole, tetracycline, and chloramphenicol should not be used for empirical treatment of shigellosis in Yemen.