Evelyn Siew-Chuan Koay

Real-Time PCR for Detection and Identification of Plasmodium spp.

ABSTRACT Rapid and accurate detection of malaria parasites in blood is needed to institute proper therapy. We developed and used a real-time PCR assay to detect and distinguish four Plasmodium spp. that cause human disease by using a single amplification reaction and melting curve analysis. Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and SYBR Green was used for detection in a LightCycler instrument. Patient specimens infected at 0.01 to 0.02% parasitemia densities were detected, and analytical sensitivity was estimated to be 0.2 genome equivalent per reaction. Melting curve analysis based on nucleotide variations within the amplicons provided a basis for accurate differentiation of Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. For assay validation, 358 patient blood samples from the National University Hospital in Singapore and Evanston Northwestern Healthcare in Illinois were analyzed. Of 76 blinded patient samples with a microscopic diagnosis of P. falciparum, P. vivax, or P. ovale infection, 74 (97.4%) were detected by real-time PCR, including three specimens containing mixed P. falciparum-P. vivax infections. No Plasmodium DNA was amplified in any of the 82 specimens sent for malaria testing but that were microscopically negative for Plasmodium infection. In addition, 200 blood samples from patients whose blood was collected for reasons other than malaria testing were also determined to be negative by real-time PCR. Real-time PCR with melting curve analysis could be a rapid and objective supplement to the examination of Giemsa-stained blood smears and may replace microscopy following further validation.

Reliability of Tissue Microarrays in Detecting Protein Expression and Gene Amplification in Breast Cancer

Tissue microarrays allow high throughput molecular profiling of diagnostic or predictive markers in cancer specimens and rapid validation of novel potential candidates identified from genomic and proteomic analyses in a large number of tumor samples. To validate the use of tissue microarray technology for all the main biomarkers routinely used to decide breast cancer prognostication and postsurgical adjuvant therapy, we constructed a tissue microarray from 97 breast tumors, with a single 0.6 mm core per specimen. Immunostaining of tissue microarray sections and conventional full sections of each tumor were performed using well-characterized prognostic markers (estrogen receptor ER, progesterone receptor PR and c-erbB2). The full section versus tissue microarray concordance for these stains was 97% for ER, 98% for PR, and 97% for c-erbB2, respectively, with a strong statistical association (kappa value more than 0.90). Fluorescence in situ hybridization analysis for HER-2/neu gene amplification from the single-core tissue microarray was technically successful in about 90% (87/97) of the cases, with a concordance of 95% compared with parallel analyses with the full sections. The correlation with other pathological parameters was not significantly different between full-section and array-based results. It is concluded that the constructed tissue microarray with a single core per specimen ensures full biological representativeness to identify the associations between biomarkers and clinicopathological parameters, with no significant associated sampling bias.

A diagnostic polymerase chain reaction assay for Zika virus.

Zika virus (ZIKV) is a mosquito‐borne flavivirus. Infection results in a dengue‐like illness with fever, headache, malaise, and a maculopapular rash. Nearly all cases are mild and self‐limiting but in 2007, a large outbreak of ZIKV was reported from the island of Yap (in Micronesia, northwest of Indonesia). Singapore is already endemic for dengue, and its impact on public health and economic burden is significant. Other dengue‐like infections (e.g., Chikungunya virus) are present. Yet only 10% of reported dengue cases have laboratory confirmation. The identification and control of other dengue‐like, mosquito‐transmitted infections is thus important for the health of Singapores population, as well as its economy. Given that ZIKV shares the same Aedes mosquito vector with both dengue and Chikungunya, it is possible that this virus is present in Singapore and causing some of the mild dengue‐like illness. A specific and sensitive one‐step, reverse transcription polymerase chain reaction (RT‐PCR) with an internal control (IC) was designed and tested on 88 archived samples of dengue‐negative, Chikungunya‐negative sera from patients presenting to our hospital with a dengue‐like illness, to determine the presence of ZIKV in Singapore. The assay was specific for detection of ZIKV and displayed a lower limit of detection (LoD) of 140 copies viral RNA/reaction when tested on synthetic RNA standards prepared using pooled negative patient plasma. Of the 88 samples tested, none were positive for ZIKV RNA, however, the vast majority of these were from patients admitted to hospital and further study may be warranted in community‐based environments. J. Med. Virol. 84:1501–1505, 2012.

Proteasome inhibition by lactacystin in primary neuronal cells induces both potentially neuroprotective and pro-apoptotic transcriptional responses: A microarray analysis

Although inhibition of the ubiquitin proteasome system has been postulated to play a key role in the pathogenesis of neurodegenerative diseases, studies have also shown that proteasome inhibition can induce increased expression of neuroprotective heat‐shock proteins (HSPs). The global gene expression of primary neurons in response to treatment with the proteasome inhibitor lactacystin was studied to identify the widest range of possible pathways affected. Our results showed changes in mRNA abundance, both at different time points after lactacystin treatment and at different lactacystin concentrations. Genes that were differentially up‐regulated at the early time point but not when most cells were undergoing apoptosis might be involved in an attempt to reverse proteasome inhibitor‐mediated apoptosis and include HSP70, HSP22 and cell cycle inhibitors. The up‐regulation of HSP70 and HSP22 appeared specific towards proteasome inhibitor‐mediated cell death. Overexpression of HSP22 was found to protect against proteasome inhibitor‐mediated loss of viability by up to 25%. Genes involved in oxidative stress and the inflammatory response were also up‐regulated. These data suggest an initial neuroprotective pathway involving HSPs, antioxidants and cell cycle inhibitors, followed by a pro‐apoptotic response possibly mediated by inflammation, oxidative stress and aberrant activation of cell cycle proteins.

An international standardization programme towards the application of gene expression profiling in routine leukaemia diagnostics: the Microarray Innovations in LEukemia study prephase

Gene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies. Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study. Each laboratory prepared two cell line samples, together with three replicate leukaemia patient lysates in two distinct stages: (i) a 5‐d course of protocol training, and (ii) independent proficiency testing. Unsupervised, supervised, and r2 correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra‐laboratory reproducibility and with comparable quality and reliability.

Prophylactic lamivudine prevents hepatitis B reactivation in chemotherapy patients

Background : Chronic hepatitis B virus carriers receiving chemotherapy develop a high hepatitis B virus reactivation rate (38–53%) with a high mortality (37–60%). Few studies have characterized the efficacy of lamivudine in the treatment of chemotherapy‐induced hepatitis B virus reactivation.

Plumbagin inhibits invasion and migration of breast and gastric cancer cells by downregulating the expression of chemokine receptor CXCR4

BackgroundIncreasing evidence indicates that the interaction between the CXC chemokine receptor-4 (CXCR4) and its ligand CXCL12 is critical in the process of metastasis that accounts for more than 90% of cancer-related deaths. Thus, novel agents that can downregulate the CXCR4/CXCL12 axis have therapeutic potential in inhibiting cancer metastasis.MethodsIn this report, we investigated the potential of an agent, plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone), for its ability to modulate CXCR4 expression and function in various tumor cells using Western blot analysis, DNA binding assay, transient transfection, real time PCR analysis, chromatin immunoprecipitation, and cellular migration and invasion assays.ResultsWe found that plumbagin downregulated the expression of CXCR4 in breast cancer cells irrespective of their HER2 status. The decrease in CXCR4 expression induced by plumbagin was not cell type-specific as the inhibition also occurred in gastric, lung, renal, oral, and hepatocellular tumor cell lines. Neither proteasome inhibition nor lysosomal stabilization had any effect on plumbagin-induced decrease in CXCR4 expression. Detailed study of the underlying molecular mechanism(s) revealed that the regulation of the downregulation of CXCR4 was at the transcriptional level, as indicated by downregulation of mRNA expression, inhibition of NF-κB activation, and suppression of chromatin immunoprecipitation activity. In addition, using a virtual, predictive, functional proteomics-based tumor pathway platform, we tested the hypothesis that NF-κB inhibition by plumbagin causes the decrease in CXCR4 and other metastatic genes. Suppression of CXCR4 expression by plumbagin was found to correlate with the inhibition of CXCL12-induced migration and invasion of both breast and gastric cancer cells.ConclusionsOverall, our results indicate, for the first time, that plumbagin is a novel blocker of CXCR4 expression and thus has the potential to suppress metastasis of cancer.

Evaluation of Advanced Reverse Transcription-PCR Assays and an Alternative PCR Target Region for Detection of Severe Acute Respiratory Syndrome-Associated Coronavirus

ABSTRACT First-generation reverse transcription-PCR (RT-PCR) assays for severe acute respiratory syndrome-associated coronavirus (SARS-CoV) gave false-negative results in a considerable fraction of patients. In the present study, we evaluated two second-generation, replicase (R) gene-based, real-time RT-PCR test kits—the RealArt HPA coronavirus LC kit (Artus, Hamburg, Germany) and the LightCycler SARS-CoV quantification kit (Roche, Penzberg, Germany)—and a real-time RT-PCR assay for the nucleocapsid (N) gene. Detecting the N-gene RNA might be advantageous due to its high abundance in cells. The kits achieved sensitivities of 70.8% (Artus) and 67.1% (Roche) in 66 specimens from patients with confirmed SARS (samples primarily from the upper and lower respiratory tract and stool). The sensitivity of the N-gene assay was 74.2%. The differences in all of the sensitivities were not statistically significant (P = 0.680 [analysis of variance]). Culture cells initially contained five times more N- than R-gene RNA, but the respective levels converged during 4 days of virus replication. In clinical samples the median concentrations of R- and N-gene RNA, respectively, were 1.2 × 106 and 2.8 × 106 copies/ml (sputum and endotracheal aspirates), 4.3 × 104 and 5.5 × 104 copies/ml (stool), and 5.5 × 102 and 5.2 × 102 copies/sample (throat swabs and saliva). Differences between the samples types were significant but not between the types of target RNA. All (n = 12) samples from the lower respiratory tract tested positive in all tests. In conclusion, the novel assays are more sensitive than the first-generation tests, but they still do not allow a comprehensive ruling out of SARS. Methods for the routine sampling of sputum without infection risk are needed to improve SARS RT-PCR.

Differing Symptom Patterns in Early Pandemic vs Seasonal Influenza Infections

BACKGROUND Singapore is a tropical country with a temperature range of 23 degrees C to 35 degrees C and relative humidity of 48% to 100% throughout the year. Influenza incidence peaks in June through July and November through January, though influenza cases can be detected throughout the year. METHODS Between May 1 and July 28, 2009, a novel dual-gene diagnostic polymerase chain reaction assay targeting the hemagglutinin (HA) and nucleoprotein (NP) genes of the new influenza A(H1N1/2009) virus was specifically designed for enhanced influenza surveillance using nasopharyngeal swabs collected from symptomatic patients (including their close contacts) and returning travelers returning from influenza A(H1N1/2009)-affected areas, presenting to affiliated primary care clinics as well as the main hospital emergency department. RESULTS From the week of June 16 to June 23, 2009, this pandemic influenza A(H1N1/2009) displaced and then replaced the seasonal influenzas (H3N2, H1N1, and B). Of 2683 samples tested during this 12-week surveillance period, 742 (27.6%) were positive for any influenza virus using this assay, with 547 cases of A(H1N1/2009) (20.4%), 167 cases of A(H3N2) (6.2%), 14 cases of A(H1N1) (0.5%), and 12 cases of influenza B (0.4%). Results of multivariate analysis showed that age (P < .001), fever (P < .001), cough (P < .001), sore throat (P = .002), rhinorrhea (P = .001), and dyspnea (P < .001) were significantly different among these groups. CONCLUSIONS From this large prospective study, there was a lower incidence of fever and dyspnea in patients with pandemic influenza A(H1N1/2009) infection. Similar to reports from elsewhere, it was also found that this pandemic virus tends to infect younger people, though with fewer symptoms, on average, than seasonal influenza. Early pandemic influenza A(H1N1/2009) infections appeared to be slightly milder than seasonal influenza as indicated by different symptom patterns in the presentation of more than 500 cases of influenza A(H1N1/2009) during April through July to a large teaching hospital in Singapore.

A Novel Diagnostic Target in the Hepatitis C Virus Genome

Background Detection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5′-noncoding region (5′-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays. Methods and Findings In this study we determined by de novo sequencing that the 3′-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1–6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3–24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10−9 IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1–6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5′-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US