H5N1 Oseltamivir-resistance detection by real-time PCR using two high sensitivity labeled TaqMan probes

A single amino acid substitution, from histidine to tyrosine at position 274 of the neuraminidase gene has converted Oseltamivir sensitive H5N1 influenza A virus into a resistant strain. Currently, Oseltamivir is being stockpiled in many countries potentially affected by the influenza A virus subtype H5N1 epidemic. To identify this change in Oseltamivir-treated patients, a method based on real-time PCR using two labeled TaqMan probes was developed for its rapid detection. In order to validate the method, Oseltamivir specimen from treated (Oseltamivir-resistant strain from a Vietnamese patient, two Oseltamivir-treated tigers) and untreated subjects have been used for this study. The results thus obtained as well as those derived from clone selection and sequencing showed that TaqMan probes could clearly discriminate wild type H274 from the mutant 274Y variant. The sensitivity of this assay was as low as 10 copies/microl and allowed the detection of the mutation in a mixture of wild type and mutant. Overall, the assay based on real-time PCR with two labeled TaqMan probes described here should be useful for detecting Oseltamivir-resistant H274Y H5N1 influenza A virus in many species and various sources of specimens with high sensitivity and specificity. Such studies can address potential differences in the diagnostic outcomes between patients who develop detectable Oseltamivir resistance and those who retain only the wild type strain of H5N1.     

Thailand intensive external quality assessment schemes as dynamic tools for improving laboratory quality and standard

The term 'external quality assessment (EQA)' has been recommended by WHO consultative meeting in 1980. It has been using to run the retrospective and com-parative statistic tests' reliability among the EQA participants of their organizers. EQA schemes in all fields of laboratory pathology should provide objective information as tools to assist laboratory analysts ensuring the test result quality and on the contrary deciding error-eradicated actions, or more or less improving national test performance quality and standard. Scoring assessment has been designed in the EQA schemes to assist monitoring, and encouraging quality and standard of laboratory performance in the long run of consecutive trials. The Faculty of Medical Technology of Mahidol University now as a collaborating WHO:SEARO EQA organizers, has provided wider EQA schemes, servicing participants all around Thailand, including 400 labs in clinical chemistry of EQAC scheme, 150 labs in clinical hormone of EQAH scheme, 200 labs in clinical microscopy of EQAM scheme, and 190 labs in clinical immunology and serology of EQAI schemes. Regular EQA newsletters, and yearly summer-time seminar and training prove to be the essential education tools promoting both laboratory quality and standard intensively.     

Posterior leukoencephalopathy following cisplatin,bleomycin and vinblastine therapy for germ cell tumor of the ovary

A 31-year-old female developed multiple episodes of grand mal seizures after combination chemotherapy with cisplatin, vinblastine and bleomycin for germ cell ovarian cancer stage Ic. The clinicoradiologic features in this patient were consistent with posterior leukoencephalopathy, which is a rare complication of chemotherapy. Seizures were controlled by the anticonvulsive agent Dilantin (Pfizer, Khet Klongtoey, Bangkok) and she returned home without any permanent neurologic deficits. Follow-up magnetic resonance imaging 2 weeks later showed complete resolution of the abnormalities. This syndrome has been previously reported following cisplatin-based chemotherapy. Physicians should remain alert to the potential hazards of chemotherapy to the central nervous system. Risks and benefits should be seriously considered before starting treatment.     

Efficacy of pulsed dyed laser (585 nm) in the treatment of molluscum contagiosum subtype 1

Molluscum contagiosum is a common cutaneous disease that may be difficult to treat when there are multiple lesions; especially in children. This study was conducted to determine the efficacy of pulsed dye laser (585 nm) in the treatment of molluscum contagiosum in 20 children. In the treated group, 70.5% of lesions healed after the first treatment; the remaining 10.6% after the second treatment (2 weeks later). The overall cure rate was significantly different from the control group (p< 0.01). The therapy was also well tolerated. Only mild transient hypopigmentation and erythema were observed. None encountered infectious events. In conclusion, pulsed dye laser is a good alternative treatment for molluscum contagiosum due to high efficacy and mild transient side effects.     

Mineralization of 3D Osteogenic Model Based on Gelatin-Dextran Hybrid Hydrogel Scaffold Bioengineered with Mesenchymal Stromal Cells: A Multiparametric Evaluation

Gelatin–dextran hydrogel scaffolds (G-PEG-Dx) were evaluated for their ability to activate the bone marrow human mesenchymal stromal cells (BM-hMSCs) towards mineralization. G-PEG-Dx1 and G-PEG-Dx2, with identical composition but different architecture, were seeded with BM-hMSCs in presence of fetal bovine serum or human platelet lysate (hPL) with or without osteogenic medium. G-PEG-Dx1, characterized by a lower degree of crosslinking and larger pores, was able to induce a better cell colonization than G-PEG-Dx2. At day 28, G-PEG-Dx2, with hPL and osteogenic factors, was more efficient than G-PEG-Dx1 in inducing mineralization. Scanning electron microscopy (SEM) and Raman spectroscopy showed that extracellular matrix produced by BM-hMSCs and calcium-positive mineralization were present along the backbone of the G-PEG-Dx2, even though it was colonized to a lesser degree by hMSCs than G-PEG-Dx1. These findings were confirmed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), detecting distinct lipidomic signatures that were associated with the different degree of scaffold mineralization. Our data show that the architecture and morphology of G-PEG-Dx2 is determinant and better than that of G-PEG-Dx1 in promoting a faster mineralization, suggesting a more favorable and active role for improving bone repair.     

Typing (A/B) and subtyping (H1/H3/H5) of influenza A viruses by multiplex real-time RT-PCR assays

In this study, a specific and sensitive one-step multiplex real-time RT-PCR was developed in two assays by using primers and a number of specific locked nucleic acid (LNA)-mediated TaqMan probes which increase the thermal stability of oligonucleotides. The first assay consisted of primers and probes specific to the matrix (M1) gene of influenza A virus, matrix (M1) gene of influenza B virus and GAPDH gene of host cells for typing of influenza virus and verification by an internal control, respectively. The other assay employed primers and probes specific to the hemagglutinin gene of H1, H3 and H5 subtypes in order to identify the three most prominent subtypes of influenza A capable of infecting humans. The specificity results did not produce any cross reactivity with other respiratory viruses or other subtypes of influenza A viruses (H2, H4 and H6-H15), indicating the high specificity of the primers and probes used. The sensitivity of the assays which depend on the type or subtype being detected was approximately 10 to 10(3)copies/microl that depended on the types or subtypes being detected. Furthermore, the assays demonstrated 100% concordance with 35 specimens infected with influenza A viruses and 34 specimens infected with other respiratory viruses, which were identified by direct nucleotide sequencing. In conclusion, the multiplex real-time RT-PCR assays have proven advantageous in terms of rapidity, specificity and sensitivity for human specimens and thus present a feasible and attractive method for large-scale detection aimed at controlling influenza outbreaks.     

Knowledge,Attitudes and Behavior of Bangkok Metropolitan Women Regarding Cervical Cancer Screening

Purpose: To assess knowledge, attitudes and cervical cancer screening behavior of Bangkok Metropolitan women. Materials and Methods: Thai women, aged 25-to-65 years old, having lived in Bangkok for 5 years or more were invited to participate in the study. After signing informed consent, all women were asked to complete a self-questionnaire (Thai language) with literate assistance if needed. The questionnaire was divided into 3 parts: (I) demographic data; (II) knowledge about cervical cancer screening; and (III) behavior and attitudes, towards cervical cancer screening. Adequate screening was defined as women who had two cervical cancer screening tests except women aged 25-30 years who may have only one screening, and the last screen was within 5 year or had had regular screening. Results: Of 4,339 women, there were 1,857 (42.8%) with adequate screening and 2,482 (57.2%) with inadequate screening. Significant factors associated with inadequate screening included age < 45 years, pre-menopausal status, family monthly income no knowledge, lack of awareness and poor attitudes. Three major reasons provided by women for inadequate screening were no symptoms (54.4%), fear of pain (33.2%), and embarrassment (34.6%). Conclusions: Personal features, knowledge, and attitudes influence screening behavior of Bangkok Metropolitan women. The three most common reasons of women for not undergoinging screening are no symptoms, fear of pain, and embarrassment. These factors should be the focus of attention to improve coverage of cervical cancer screening in Bangkok.     

Influenza virus (H5N1) in live bird markets and food markets, Thailand

A surveillance program for influenza A viruses (H5N1) was conducted in live bird and food markets in central Thailand during July 2006-August 2007. Twelve subtype H5N1 viruses were isolated. The subtype H5N1 viruses circulating in the markets were genetically related to those that circulated in Thailand during 2004-2005.     

Lupinifolin from <Emphasis Type="Italic">Derris reticulata</Emphasis> possesses bactericidal activity on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> by disrupting bacterial cell membrane

In this study, lupinifolin, a prenylated flavonoid, was isolated from Derris reticulata stem, identified by NMR spectra and confirmed with mass spectrometry. Lupinifolin was freshly prepared by solubilizing in 0.1 N NaOH and immediately diluted in Müller–Hinton broth for antibacterial testing. The data showed that Gram-positive bacteria were more susceptible to lupinifolin than Gram-negative bacteria. Of four strains of Gram-positive bacteria tested, Staphylococcus aureus was the most susceptible. Using the two-fold microdilution method, it was found that lupinifolin possessed antimicrobial activity against S. aureus with minimum inhibitory concentration and minimum bactericidal concentration of 8 and 16 µg/ml, respectively, which is less potent than ampicillin. However, from the time–effect relationship, it was shown that lupinifolin had faster onset than ampicillin. The faster onset of lupinifolin was confirmed by scanning electron microscopy. To investigate the mechanism of action of lupinifolin, transmission electron microscopy (TEM) was performed to observe the ultrastructure of S. aureus. The TEM images showed that lupinifolin ruptured the bacterial cell membrane and cell wall. Due to its fast onset, it is suggested that the action of lupinifolin is likely to be the direct disruption of the cell membrane. This hypothesis was substantiated by the data from flow cytometry using DiOC₂ as an indicator. The result showed that the red/green ratio which indicated bacterial membrane integrity was significantly decreased, similar to the known protonophore carbonyl cyanide 3-chlorophenylhydrazone. It is concluded that lupinifolin inhibits the growth of S. aureus by damaging the bacterial cytoplasmic membrane.