Displaying publications 1 - 20 of 29 in total

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  1. Salleh FM, Anuar TS, Yasin AM, Moktar N
    J Microbiol Methods, 2012 Oct;91(1):174-8.
    PMID: 22986100 DOI: 10.1016/j.mimet.2012.08.004
    Permanent staining of faecal smears by Wheatley's trichrome technique has been used by many scientists for the detection of parasites in the past and it was found to be highly sensitive. This study was conducted to evaluate the use of Wintergreen oil in comparison with xylene in Wheatley's trichrome staining technique, as the reference technique. In a blind comparison study, 500 collected faecal samples from aboriginal communities were examined. Wintergreen oil was found to be more superior than xylene as a clearing agent in the Wheatley's trichrome staining of polyvinyl alcohol-fixed faecal smears for the identification of intestinal protozoa. Elimination of toxic, carcinogenic, and fire hazards makes Wintergreen oil the preferred choice in routine parasitology examinations.
    Matched MeSH terms: Staining and Labeling/methods*
  2. Tan GM, Lim HJ, Yeow TC, Movahed E, Looi CY, Gupta R, et al.
    Proteomics, 2016 05;16(9):1347-60.
    PMID: 27134121 DOI: 10.1002/pmic.201500219
    Chlamydia trachomatis is the leading causative agent of bacterial sexually transmitted infections worldwide which can lead to female pelvic inflammatory disease and infertility. A greater understanding of host response during chlamydial infection is essential to design intervention technique to reduce the increasing incidence rate of genital chlamydial infection. In this study, we investigated proteome changes in epithelial cells during C. trachomatis infection by using an isobaric tags for relative and absolute quantitation (iTRAQ) labeling technique coupled with a liquid chromatography-tandem mass spectrometry (LC-MS(3) ) analysis. C. trachomatis (serovar D, MOI 1)-infected HeLa-229 human cervical carcinoma epithelial cells (at 2, 4 and 8 h) showed profound modifications of proteome profile which involved 606 host proteins. MGST1, SUGP2 and ATXN10 were among the top in the list of the differentially upregulated protein. Through pathway analysis, we suggested the involvement of eukaryotic initiation factor 2 (eIF2) and mammalian target of rapamycin (mTOR) in host cells upon C. trachomatis infection. Network analysis underscored the participation of DNA repair mechanism during C. trachomatis infection. In summary, intense modifications of proteome profile in C. trachomatis-infected HeLa-229 cells indicate complex host-pathogen interactions at early phase of chlamydial infection.
    Matched MeSH terms: Staining and Labeling/methods
  3. Al-Mekhlafi MA, Fatmah MS, Anisah N, Azlin M, Al-Mekhlafi HM, Norhayati M
    PMID: 21323160
    Abstract. The species identification of Enterocytozoon bieneusi and Encephalitozoon intestinalis is only possible using transmission electron microscopy (TEM), mo lecular techniques and immunofluorescence antibody assays (IFA). In this study, 50 positive and 50 negative fecal specimens for microsporidial spores using the Weber modified trichrome (WMT) staining technique were examined using IFA-MAbs. Of the 100 specimens examined, the microsporidial spores identified by IFA-MAbs were Enterocytozoon Bieneusi 42 (75%) Encephalitozoon intestinalis 7 (12.5%) and mixed infections 7 (12.5%). The sensitivity and specificity of IFA-MAbs in detecting microsporidial spores were 98% and 86%, respectively. The agreement between the WMT staining technique and IFA-MAbs was statistically significant by Kappa statistics (K = 0.840; p < 0.001). E. bieneusi was the commonest Microsporidia species isolated from the studied population; the presence of microsporidial spores detected by IFA-MAbs should be confirmed by other methods.
    Matched MeSH terms: Staining and Labeling/methods
  4. Leow SN, Luu CD, Hairul Nizam MH, Mok PL, Ruhaslizan R, Wong HS, et al.
    PLoS One, 2015;10(6):e0128973.
    PMID: 26107378 DOI: 10.1371/journal.pone.0128973
    To investigate the safety and efficacy of subretinal injection of human Wharton's Jelly-derived mesenchymal stem cells (hWJ-MSCs) on retinal structure and function in Royal College of Surgeons (RCS) rats.
    Matched MeSH terms: Staining and Labeling/methods
  5. Kaur G, Madhavan M, Basri AH, Sain AH, Hussain MS, Yatiban MK, et al.
    PMID: 15689086
    The objective of this study was to determine the sensitivity, specificity, positive (PPV), and negative predictive values (NPV) of Diff-Quik-stained gastric imprint cytology smears in the detection of H. pylori compared with histology. Air-dried imprint smears of gastric biopsies from 150 patients were stained by the Diff-Quik method in the endoscopy suite and examined for H. pylori, providing results within minutes. The presence of inflammation and intestinal metaplasia were documented. The same biopsy was processed and stained with H&E and Warthin-Starry stains, and reviewed by a different pathologist blind to the imprint cytology results. Ninety-four of the 150 patients were male with a mean age of 50 years. Based on histology, the H. pylori prevalence was very low at 8%. The sensitivity and specificity of imprint cytology in the detection of H. pylori were 83.3% and 100%, respectively. The PPV and NPV were 100% and 98.6%, respectively. There were two false negatives and no false positives. A combination of imprint cytology and histology achieved 100% sensitivity. Imprint smears did not provide added value over histology with regards to inflammation and metaplasia. Gastric imprint smears stained with Diff-Quik method is a rapid, cheap, and reliable method for the detection of H. pylori and have their best results when complemented with histology.
    Matched MeSH terms: Staining and Labeling/methods*
  6. Afzan MY, Suresh K
    Parasitol Res, 2012 Jul;111(1):371-81.
    PMID: 22398830 DOI: 10.1007/s00436-012-2848-3
    Trichomonas vaginalis, a flagellated protozoan parasite causes a variety of adverse health consequences in both men and women. The parasite exists in the trophozoite and the pseudocystic stage. The study reports for the first time that pseudocyst forms of T. vaginalis isolated from cervical neoplasia (CN) patients demonstrated distinct, different and significant in vitro growth profiles when grown in vitro cultures from day 1 up to day 5 (p<0.05, Mann-Whitney test) when compared with the same life cycle stages isolated from non-cervical neoplasia but symptomatic patients (NCN). Pseudocysts from CN and NCN isolates remained viable in distilled water until 3 h 10 min and 2 h 10 min, respectively. The nucleus of pseudocysts in CN isolates using acridine orange and DAPI showed more intense staining revealing higher nuclear content. The FITC-labeled Concanavalin A stained stronger green fluorescence with surface of pseudocysts in CN isolates showing more rough and creased surface with higher numbers of deep micropores with larger numbers of chromatin masses, vacuoles, and hydrogenosomes. The study confirms that pseudocystic stage from CN, despite the uniformity in appearance of being rounded and showing no motility without a true cyst wall under light microscopy, demonstrated different biochemical, surface, and ultrastructural properties. The study provides evidence that phenotypic variant forms of pseudocysts does exist and possibly does play a role in exacerbating cervical cancer.
    Matched MeSH terms: Staining and Labeling/methods
  7. Lono A, Kumar GS, Chye TT
    Trans R Soc Trop Med Hyg, 2010 Mar;104(3):214-8.
    PMID: 19716577 DOI: 10.1016/j.trstmh.2009.07.006
    Microsporidia are ubiquitous parasites thought to be closely related to fungi. Their presence in the environment means that humans are frequently exposed to infection. Stool samples were collected from 151 indigenous villagers from the eastern state of Pahang in 2005. The samples were concentrated with water-ether sedimentation, stained with modified trichrome stain and examined under oil-immersion microscopy. Thirty-two specimens (21.2%) were positive for microsporidia. Microsporidia were observed as ovoid or rounded ovoid shapes measuring approximately 1mum, with a bright pink outline containing a central or posterior vacuole. PCR amplification with specific primers on microscopy-positive specimens amplified Encephalitozoon intestinalis DNA from five of the ten specimens used.
    Matched MeSH terms: Staining and Labeling/methods
  8. Ithoi I, Ahmad AF, Mak JW, Nissapatorn V, Lau YL, Mahmud R
    PMID: 22299400
    Seven stains were studied to determine the best color and contrast for staining the developmental stages of free living pathogenic Acanthamoeba and Naegleria species. The acid-fast bacilli stain (AFB) produced a blue color without contrast; trichrome-eosin and modified Field's showed various color contrasts; Giemsa, iron-hematoxylin, modified AFB and Gram produced only one color which distinguished the nucleus, nucleolus, cytoplasm, food- and water-vacuoles. The motile organs (acanthopodia, pseudopodia, lobopodia and flagella) were also clearly differentiated but produced a similar color as the cytoplasm. These motile organelles were first induced by incubating at 37 degrees C for at least 15 minutes and then fixing with methanol in order to preserve the protruding morphology prior to staining. The trichrome-eosin and iron-hematoxylin stains showed good color contrast for detecting all three stages, the trophozoite, cyst and flagellate; Giemsa and Gram stained the trophozoite and flagellate stages; the modified Field's and modified AFB stains stained only the trophozoite stage. Depending on the purpose, all these stains (except the AFB stain) can be used to identify the developmental stages of Acanthamoeba and Naegleria for clinical, epidemiological or public health use.
    Matched MeSH terms: Staining and Labeling/methods*
  9. Ragavan AD, Govind SK
    Parasitol Res, 2015 Mar;114(3):1163-6.
    PMID: 25614298 DOI: 10.1007/s00436-014-4296-8
    Dientamoeba fragilis, a trichomonad parasite is usually found in the gastrointestinal tract of human, and it is known to be the cause for gastrointestinal disease. The parasite is globally distributed and mostly found in rural and urban areas. The parasite is found in humans and nonhuman primates such as the macaques, baboons, and gorillas. Often, the parasite is confused with another largely found organism in stools called Blastocystis sp. especially when seen directly under light microscopy on culture samples containing both parasites. Both sometimes are seen with two nuclei with sizes tending to be similar which complicates identification. Stools were collected fresh from nine previously diagnosed persons infected with D. fragilis who also were found to be positive for Blastocystis sp. Samples were then cultured in Loeffler's medium and were stained with Giemsa, iron hematoxylin, and modified Fields' (MF) stain, respectively. D. fragilis was differentiated from Blastocystis sp. when stained with MF stain by the presence of a thinner outer membrane with clearly demarcated nuclei in the center of the cell whilst Blastocystis sp. had a darker and thicker stained outer membrane with the presence of two nuclei. The staining contrast was more evident with modified Fields' stain when compared with the other two. The simplicity in preparing the stain as well as the speed of the staining procedure make MF stain an ideal alternate. The modified Fields' stain is faster and easier to prepare when compared to the other two stains. MF stain provides a better contrast differentiating the two organisms and therefore provides a more reliable diagnostic method to precisely identify one from the other especially when cultures show mixed infections.
    Matched MeSH terms: Staining and Labeling/methods*
  10. Afzan MY, Sivanandam S, Kumar GS
    Diagn Microbiol Infect Dis, 2010 Oct;68(2):159-62.
    PMID: 20846588 DOI: 10.1016/j.diagmicrobio.2010.06.005
    Trichomonas vaginalis, a flagellate protozoan parasite commonly found in the human genitourinary tract, is transmitted primarily by sexual intercourse. Diagnosis is usually by in vitro culture method and staining with Giemsa stain. There are laboratories that use Gram stain as well. We compared the use of modified Field's (MF), Giemsa, and Gram stains on 2 axenic and xenic isolates of T. vaginalis, respectively. Three smears from every sediment of spun cultures of all 4 isolates were stained, respectively, with each of the stains. We showed that MF staining, apart from being a rapid stain (20 s), confers sharper staining contrast, which differentiates the nucleus and the cytoplasm of the organism when compared to Giemsa and Gram staining especially on parasites from spiked urine samples. The alternative staining procedure offers in a diagnostic setting a rapid stain that can easily visualize the parasite with sharp contrasting characteristics between organelles especially the nucleus and cytoplasm. Vacuoles are more clearly visible in parasites stained with MF than when stained with Giemsa.
    Matched MeSH terms: Staining and Labeling/methods*
  11. Ren X, Evangelista-Leite D, Wu T, Rajab TK, Moser PT, Kitano K, et al.
    Biomaterials, 2018 11;182:127-134.
    PMID: 30118980 DOI: 10.1016/j.biomaterials.2018.08.012
    Decellularized native extracellular matrix (ECM) biomaterials are widely used in tissue engineering and have reached clinical application as biomesh implants. To enhance their regenerative properties and postimplantation performance, ECM biomaterials could be functionalized via immobilization of bioactive molecules. To facilitate ECM functionalization, we developed a metabolic glycan labeling approach using physiologic pathways to covalently incorporate click-reactive azide ligands into the native ECM of a wide variety of rodent tissues and organs in vivo, and into the ECM of isolated rodent and porcine lungs cultured ex vivo. The incorporated azides within the ECM were preserved after decellularization and served as chemoselective ligands for subsequent bioconjugation via click chemistry. As proof of principle, we generated alkyne-modified heparin, immobilized it onto azide-incorporated acellular lungs, and demonstrated its bioactivity by Antithrombin III immobilization and Factor Xa inhibition. The herein reported metabolic glycan labeling approach represents a novel platform technology for manufacturing click-reactive native ECM biomaterials, thereby enabling efficient and chemoselective functionalization of these materials to facilitate tissue regeneration and repair.
    Matched MeSH terms: Staining and Labeling/methods
  12. Abu Bakar N
    Trop Biomed, 2015 Sep;32(3):485-93.
    PMID: 26695209 MyJurnal
    Studies show that the pH of the malaria parasite's digestive vacuole (DV) plays a key role in the physiological functions of this organelle and antimalarial drug accumulation, and yet is technically difficult to measure. In this study, a flow cytometry-based technique was developed to measure the DV pH using a ratiometric pH indicator, FITC-dextran loaded into the DV of saponin-permeabilized parasites. To calculate the DV pH, a standard pH calibration curve was generated by incubating the saponin-permeabilized cells in buffers with different pH in the presence of an ionophore, CCCP. The measured average pH of the DV was 5.27 ± 0.03 that is approximately the same in the parasites observed microscopically by Hayward et al. (2006) (5.50 ± 0.14) using the same probe. The removal of glucose from the medium, causing a rapid depletion of parasite ATP, resulted in an alkalization of the DV. The DV was reacidified upon restoration of glucose to the medium. This technique provides a rapid, simple and quantitative measurement of the DV pH on a large number of cells. It will also be useful in future attempts to evaluate the effect of antimalarial drugs (i.e. chloroquine and artemisinin-based drugs) in pH changes of the DV.
    Matched MeSH terms: Staining and Labeling/methods
  13. Kimura M, Teramoto I, Chan CW, Idris ZM, Kongere J, Kagaya W, et al.
    Malar J, 2018 Feb 07;17(1):72.
    PMID: 29415724 DOI: 10.1186/s12936-018-2214-8
    BACKGROUND: Rapid diagnosis of malaria using acridine orange (AO) staining and a light microscope with a halogen lamp and interference filter was deployed in some malaria-endemic countries. However, it has not been widely adopted because: (1) the lamp was weak as an excitation light and the set-up did not work well under unstable power supply; and, (2) the staining of samples was frequently inconsistent.

    METHODS: The halogen lamp was replaced by a low-cost, blue light-emitting diode (LED) lamp. Using a reformulated AO solution, the staining protocol was revised to make use of a concentration gradient instead of uniform staining. To evaluate this new AO diagnostic system, a pilot field study was conducted in the Lake Victoria basin in Kenya.

    RESULTS: Without staining failure, malaria infection status of about 100 samples was determined on-site per one microscopist per day, using the improved AO diagnostic system. The improved AO diagnosis had both higher overall sensitivity (46.1 vs 38.9%: p = 0.08) and specificity (99.0 vs 96.3%) than the Giemsa method (N = 1018), using PCR diagnosis as the standard.

    CONCLUSIONS: Consistent AO staining of thin blood films and rapid evaluation of malaria parasitaemia with the revised protocol produced superior results relative to the Giemsa method. This AO diagnostic system can be set up easily at low cost using an ordinary light microscope. It may supplement rapid diagnostic tests currently used in clinical settings in malaria-endemic countries, and may be considered as an inexpensive tool for case surveillance in malaria-eliminating countries.

    Matched MeSH terms: Staining and Labeling/methods*
  14. Tan ZN, Wong WK, Nik Zairi Z, Abdullah B, Rahmah N, Zeehaida M, et al.
    Trop Biomed, 2010 Apr;27(1):79-88.
    PMID: 20562817 MyJurnal
    Entamoeba histolytica causes about 50 million infections worldwide with a death rate of over 100,000 annually. In endemic developing countries where resources are limited, microscopic examinations based on Wheatley trichrome staining is commonly used for diagnosis of intestinal amoebiasis. Other than being a time-consuming method, it must be performed promptly after stool collection as trophozoites disintegrate rapidly in faeces. The aim of this study was to compare the efficacies of Eosin-Y, Wheatley trichrome and Iodine stains in delineating the diagnostic features of the parasite, and subsequently to determine the suitable microscopy observation period for detection of erythrophagocytic and non-erythrophagocytic trophozoites spiked in semi-solid stool sample. Wheatley trichrome staining technique was performed using the standard method while the other two techniques were performed on the slides by mixing the respective staining solution with the spiked stool sample. One million of axenically cultured non-erythrophagocytic E. histolytica and erythrophagocytic E. histolytica were separately spiked into 2 g of fresh semisolid faeces. Percentage viability of the trophozoites in the spiked stool sample was determined at 30 minute intervals for eight hours using the 0.4% Trypan blue exclusion method. The results showed that Eosin-Y and Wheatley trichrome stained the karyosome and chromatin granules better as compared to Iodine stain. The percentage viability of non-erythrophagocytic trophozoites decreased faster than the erythrophagocytic form in the first 5 hours and both dropped to ~10% in the 6th hour spiked sample. In conclusion, Eosin-Y staining technique was found to be the easiest to perform, most rapid and as accurate as the commonly used Wheatley trichrome technique; Eosin-Y stained slide sealed with DPX could also be kept as a permanent record. A period not exceeding 6 hours after stool collection was found to be the most suitable in order to obtain good microscopy results of viable trophozoites.
    Matched MeSH terms: Staining and Labeling/methods*
  15. Pirehma M, Suresh K, Sivanandam S, Anuar AK, Ramakrishnan K, Kumar GS
    Parasitol Res, 1999 Oct;85(10):791-3.
    PMID: 10494803
    Acanthamoeba sp. is a free-living amoeba known to cause chronic central nervous system infection or eye infection in humans. Many cases remain undetected for want of a good detection system. We report for the first time a rapid staining method to facilitate the identification of Acanthamoeba sp. using the modified Field's staining technique. A. castellanii, which was used in the present experiment, is maintained in our laboratory in mycological peptone medium (Gibco). The cultures were pooled together and smears were made on glass slides for staining purposes. Different types of stains such as Field's stain, modified Field's stain, Wright's stain, Giemsa stain, Ziehl-Neelsen stain, and trichrome stain were used to determine the best stain for the identification of this amoeba. The concentration of various stains and the duration of staining were varied to provide the best color and contrast for each stain. Acanthamoeba was also obtained from the brain of experimentally infected mice and was stained with various stains as mentioned above to determine the best stain for use in identifying the presence of this parasite in experimentally infected animals. The modified Field's stain gives a very good color contrast as compared with other stains. Furthermore, it takes only 20 s to be carried out using the least number of reagents, making it suitable for both laboratory and field use.
    Matched MeSH terms: Staining and Labeling/methods*
  16. Fazlina N, Maha A, Jamal R, Zarina AL, Cheong SK, Hamidah H, et al.
    Hematology, 2007 Feb;12(1):33-7.
    PMID: 17364990
    The expression of the multidrug resistance (MDR) proteins may influence the outcome of treatment in patients with acute leukemia. The aim of this study was to determine the IC50 of cytotoxic drugs (cytosine arabinoside, ara-C and daunorubicin, dnr) using the in vitro 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt (MTS) assay method. A total of 82 newly diagnosed acute leukemia cases (43 adult myeloid leukaemia, AML cases and 39 acute lymphoblastic leukaemia, ALL cases) and 16 relapsed cases (8 AML cases and 8 ALL cases) were studied. The MTS assay was performed using two cytotoxic drugs, dnr and ara-C. Cells were incubated with different concentrations of drugs for 4 days and the IC50 was extrapolated from the viability curve. In newly diagnosed cases, we found that childhood ALL samples showed higher IC50 values of dnr (0.040 +/- 2.320) compared to adult AML samples (0.021 +/- 0.158). In contrast, newly diagnosed adult AML samples showed higher IC50 values of ara-C (0.157 +/- 0.529) compared to childhood ALL samples (0.100 +/- 2.350). In relapsed cases, two samples of childhood ALL showed IC50 values of dnr (0.910 +/- 1.760) and ara-C (1.310 +/- 2.390), which was higher compared to childhood AML samples (0.129 +/- 0.214 and 0.210 +/- 0.003, respectively). However, there was no correlation between IC50 values of these drugs tested with clinical outcome. In conclusion, we found that MTS assay is an easy, rapid and non laborious method to study in vitro drug resistance in acute leukaemia cases.
    Matched MeSH terms: Staining and Labeling/methods
  17. Salleh FM, Al-Mekhlafi AM, Nordin A, Yasin 'M, Al-Mekhlafi HM, Moktar N
    Diagn Microbiol Infect Dis, 2011 Jan;69(1):82-5.
    PMID: 21146718 DOI: 10.1016/j.diagmicrobio.2010.08.028
    This study was conducted to evaluate the modification of the usual Gram-chromotrope staining technique developed in-house known as Gram-chromotrope Kinyoun (GCK) in comparison with the Weber Modified Trichrome (WMT) staining technique; as the reference technique. Two hundred and ninety fecal specimens received by the Microbiology Diagnostic Laboratory of Hospital Universiti Kebangsaan Malaysia were examined for the presence of microsporidial spores. The sensitivity and specificity of GCK compared to the reference technique were 98% and 98.3%, respectively. The positive and negative predictive values were 92.5% and 99.6%, respectively. The agreement between the reference technique and the GCK staining technique was statistically significant by Kappa statistics (K = 0.941, P < 0.001). It is concluded that the GCK staining technique has high sensitivity and specificity in the detection of microsporidial spores in fecal specimens. Hence, it is recommended to be used in the diagnosis of intestinal microsporidiosis.
    Matched MeSH terms: Staining and Labeling/methods*
  18. Anuar TS, Al-Mekhlafi HM, Abdul Ghani MK, Abu Bakar E, Azreen SN, Salleh FM, et al.
    J Microbiol Methods, 2013 Mar;92(3):344-8.
    PMID: 23361047 DOI: 10.1016/j.mimet.2013.01.010
    This study was conducted to evaluate two routinely microscopic diagnostic methods in comparison with single-round PCR assay as the reference technique to detect Entamoeba histolytica/dispar/moshkovskii. Examination was performed on 500 stool samples obtained from Orang Asli communities in different states of Malaysia using formalin-ether sedimentation, trichrome staining and single-round PCR techniques. Ninety-three stool samples were detected E. histolytica/dispar/moshkovskii positive by routine microscopy, while single-round PCR detected 106 positive samples. Additional positives detected by PCR assay were eventually confirmed to be negative by both microscopic techniques. Detection rate of E. histolytica/dispar/moshkovskii was highest in combination techniques (18.6%), followed by trichrome staining (13.4%) and formalin-ether sedimentation (11.2%) techniques. Single-round PCR detected 21.2% of the stool samples. The sensitivity and specificity of formalin-ether sedimentation and trichrome staining techniques compared to the reference technique were 31.1% (95% CI: 29.0-36.0) and 94.2% (95% CI: 89.8-98.9), and 53.8% (95% CI: 46.0-76.2) and 97.5% (95% CI: 92.8-99.1), respectively. However, the sensitivity [59.4% (95% CI: 48.9-78.5)] of the method increased when both techniques were performed together, but the specificity decreased to 92.4% (95% CI: 81.0-98.0). The agreement between the reference technique, trichrome staining and combination techniques were statistically significant by Kappa statistics (trichrome staining: K = 0.592, p < 0.05; combination techniques: K = 0.543, p < 0.05). Hence, the combination technique is recommended to be used as a screening method in the diagnosis of E. histolytica/dispar/moshkovskii infections either for clinical or epidemiological study.
    Matched MeSH terms: Staining and Labeling/methods
  19. Ummul Haninah A, Vasan SS, Ravindran T, Chandru A, Lee HL, Shamala Devi S
    Trop Biomed, 2010 Dec;27(3):611-23.
    PMID: 21399603 MyJurnal
    This paper reports the development of a one-step SYBR-Green I-based realtime RT-PCR assay for the detection and quantification of Chikungunya virus (CHIKV) in human, monkey and mosquito samples by targeting the E1 structural gene. A preliminary evaluation of this assay has been successfully completed using 71 samples, consisting of a panel of negative control sera, sera from healthy individuals, sera from patients with acute disease from which CHIKV had been isolated, as well as monkey sera and adult mosquito samples obtained during the chikungunya fever outbreak in Malaysia in 2008. The assay was found to be 100-fold more sensitive than the conventional RT-PCR with a detection limit of 4.12x10(0) RNA copies/μl. The specificity of the assay was tested against other related viruses such as Dengue (serotypes 1-4), Japanese encephalitis, Herpes Simplex, Parainfluenza, Sindbis, Ross River, Yellow fever and West Nile viruses. The sensitivity, specificity and efficiency of this assay were 100%, 100% and 96.8% respectively. This study on early diagnostics is of importance to all endemic countries, especially Malaysia, which has been facing increasingly frequent and bigger outbreaks due to this virus since 1999.
    Matched MeSH terms: Staining and Labeling/methods
  20. Sirskyj D, Weltzin R, Golshani A, Anderson D, Bozic J, Diaz-Mitoma F, et al.
    J Virol Methods, 2010 Feb;163(2):459-64.
    PMID: 19913054 DOI: 10.1016/j.jviromet.2009.11.014
    Several critical factors of an influenza microneutralization assay, utilizing a rapid biotin-streptavidin conjugated system for detecting influenza virus subtypes A and B, are addressed within this manuscript. Factors such as incubation times, amount of virus, cell seeding, sonication, and TPCK trypsin were evaluated for their ability to affect influenza virus neutralization in a microplate-based neutralization assay using Madin-Darby canine kidney (MDCK) cells. It is apparent that the amount of virus used in the assay is the most critical factor to be optimized in an influenza microneutralization assay. Results indicate that 100xTCID(50) of influenza A/Solomon Islands/03/2006 (H1N1) virus overloads the assay and results in no, to low, neutralization, in both ferret and macaque sera, respectively, whereas using 6xTCID(50) resulted in significantly improved neutralization. Conversely, strong neutralization was observed against 100xTCID(50) of B/Malaysia/2506/04 virus. In this manuscript the critical factors described above were optimized and the results indicate that the described biotin-streptavidin conjugated influenza microneutralization assay is a rapid and robust method for detecting the presence of functional, influenza virus-neutralizing antibodies.
    Matched MeSH terms: Staining and Labeling/methods
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