We report the results of a laboratory evaluation of the BioRad Urinary Metanephrines Reagent Kit. The test was designed for the quantitative measurement of normetanephrine and metanephrine in urine by high performance liquid chromatography. The kit was evaluated in view of improving assay reliability and specificity as compared to the manual method based on cation exchange chromatography and spectrophotometry.
DNA extraction was carried out on 32 medicinal plant samples available in Malaysia using the TriOmic(TM) extraction kit. Amounts of 0.1 g flowers or young leaves were ground with liquid nitrogen, lysed at 65°C in RY1(plus) buffer and followed by RNAse treatment. Then, RY2 buffer was added to the samples and mixed completely by vortexing before removal of cell debris by centrifugation. Supernatants were transferred to fresh microcentrifuge tubes and 0.1 volume RY3 buffer was added to each of the transferred supernatant. The mixtures were applied to spin columns followed by a centrifugation step to remove buffers and other residues. Washing step was carried out twice by applying 70% ethanol to the spin columns. Genomic DNA of the samples was recovered by applying 50 μL TE buffer to the membrane of each spin column, followed by a centrifugation step at room temperature. A modification of the TriOmic(TM) extraction procedure was carried out by adding chloroform:isoamyl alcohol (24:1) steps in the extraction procedure. The genomic DNA extracted from most of the 32 samples showed an increase of total yield when chloroform:isoamyl alcohol (24:1) steps were applied in the TriOmicTM extraction procedure. This preliminary study is very important for molecular studies of medicinal plants available in Malaysia since the DNA extraction can be completed in a shorter period of time (within 1 h) compared to manual extraction, which entails applying phenol, chloroform and ethanol precipitation, and requires 1-2 days to complete.
MicroRNAs regulate gene expression at the post-transcriptional level. Differential expression of miRNAs can potentially be used as biomarkers for early diagnosis and prediction for outcomes. Failure in validation of miRNA profiles is often caused by variations in experimental parameters. In this study, the performance of five extraction kits and three RT-qPCR systems were evaluated using BioMark high-throughput platform and the effects of different experimental parameters on circulating miRNA levels were determined. Differences in the performance of extraction kits as well as varying accuracy, sensitivity and reproducibility in qPCR systems were observed. Normalisation of RT-qPCR data to spike-in controls can reduce extraction bias. However, the extent of correlation for different qPCR systems varies in different assays. At different time points, there was no significant fold change in eight of the plasma miRNAs that we evaluated. Higher level of miRNAs was detected in plasma as compared to serum of the same cohort. In summary, we demonstrated that high-throughput RT-qPCR with pre-amplification step had increased sensitivity and can be achieved with accuracy and high reproducibility through stringent experimental controls. The information provided here is useful for planning biomarker validation studies involving circulating miRNAs.
A simple and sensitive double-antibody radioimmunoassay for human growth hormone (HGH) was developed, optimised and validated. The anti-hGH sera raised in 2 rabbits were highly specific with low cross-reactions of 0.19% and 0.3% with human placental lactogen and 0.21% and 0.13% with human prolactin. The mean sensitivity of the assay determined from 28 assays was found to be 0.4 +/- 0.2 mIU/L. Mean recovery of added exogenous hGH was 98.8 +/- 6.8%. Linearity studies of samples diluted at 1:2, 1:4 and 1:8 gave values of 101.3 +/- 5.3%, 109.6 +/- 13.4% and 97.3 +/- 13% respectively of those expected. The reproducibility of the assay was good; within assay coefficient of variation for serum samples with GH concentrations of 2.7, 13.6 and 28.2 mU/l ranged from 5.1 to 8.3% while the inter-assay precision varied from 4.9 to 10.3%. The in-house assay showed good correlation (r = 0.96, p less than 0.001) with a commercial HGH RIA kit (Dainabot, Japan). A reference normal adult fasting GH level of less than 7 mIU/l was established from 95 samples assayed by this method.
Commercially available diagnostic test kits for detection of dengue virus (DENV) non-structural protein 1 (NS1) and anti-DENV IgM were evaluated for their sensitivity and specificity and other performance characteristics by a diagnostic laboratory network developed by World Health Organization (WHO), the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Pediatric Dengue Vaccine Initiative (PDVI). Each network laboratory contributed characterized serum specimens for the panels used in the evaluation. Microplate enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT formats) were represented by the kits. Each ELISA was evaluated by 2 laboratories and RDTs were evaluated by at least 3 laboratories. The reference tests for IgM anti-DENV were laboratory developed assays produced by the Armed Forces Research Institute for Medical Science (AFRIMS) and the Centers for Disease Control and Prevention (CDC), and the NS1 reference test was reverse transcriptase polymerase chain reaction (RT-PCR). Results were analyzed to determine sensitivity, specificity, inter-laboratory and inter-reader agreement, lot-to-lot variation and ease-of-use. NS1 ELISA sensitivity was 60-75% and specificity 71-80%; NS1 RDT sensitivity was 38-71% and specificity 76-80%; the IgM anti-DENV RDTs sensitivity was 30-96%, with a specificity of 86-92%, and IgM anti-DENV ELISA sensitivity was 96-98% and specificity 78-91%. NS1 tests were generally more sensitive in specimens from the acute phase of dengue and in primary DENV infection, whereas IgM anti-DENV tests were less sensitive in secondary DENV infections. The reproducibility of the NS1 RDTs ranged from 92-99% and the IgM anti-DENV RDTs from 88-94%.
Objective. To determine the diagnostic accuracy of placental alpha microglobulin-1 assay and standard diagnostic methods for detecting rupture of membrane. Study Design. Prospective diagnostic study, between June 2011 to November 2011 at a tertiary centre. Initial evaluation included both the standard diagnostic methods for rupture of membranes and placental alpha microglobulin-1 immunoassay. The actual rupture of membranes was diagnosed on review of the medical records after delivery (absence of membrane or a positive pad chart). Main Outcome Measures. Placental alpha microglobulin-1 immunoassay and standard diagnostic methods for diagnosis of rupture of membrane. Results. A total of 211 patients were recruited. At initial presentation, 187 patients (88.6%) had ruptured membranes, while 24 patients (11.4%) had intact membranes. Placental alpha microglobulin-1 immunoassay confirmed rupture of membranes at initial presentation with a sensitivity of 95.7% (179 of 187), specificity of 100% (24 of 24), positive predictive value of 100% (179 of 179), and negative predictive value of 75.0% (24 of 32). By comparison, the conventional standard diagnostic methods had a sensitivity of 78.1% (146 of 187), specificity of 100% (24 of 24), positive predictive value of 100% (146 of 146), and negative predictive value of 36.9% (24 of 65) in diagnosing rupture of membrane. Conclusion. Placental alpha-microglobulin-1 immunoassay is a rapid and accurate method for confirming the diagnosis of rupture of membrane. It was superior to conventional standard diagnostic methods (pooling, nitrazine, and ferning), the nitrazine test alone or fern test alone.
The aim of this study is to determine the sensitivity, specificity and the predictive value of the BV(®) Blue Test Kit in the diagnosis of bacterial vaginosis and to observe the risk factors associated with bacterial vaginosis (BV) in the study population. A prospective, cross-sectional study on 151 non-pregnant women who presented or referred to HUKM with presence of vaginal discharge, abnormal vaginal odour, pruritus vulvae of lower genital tract or incidental finding of abnormal PV discharge on pelvic examination. Samples of vaginal discharge were tested for bacterial vaginosis infection using Amsel's criteria, BV(®) Blue test and Gram stain (Nugent's score). Gram stain interpretation was made blinded without knowledge of other test result. Using Gram stain's criteria as a gold standard, the sensitivity, specificity, positive and negative predictive value of BV(®) Blue test and each of Amsel's criteria were estimated. The use of vaginal douches increased the risk of BV. The risk of BV with vaginal douching was 2.8 (95% CI 1.0-7.8) compared to never users. BV(®) Blue test showed a sensitivity of 100.0%, specificity of 98.3%, positive predictive value (PPV) of 94.4% and negative predictive value (NPV) of 100.0% compared to Gram stain (Nugent's method). BV(®) Blue test had excellent agreement with Gram stain which was 98.7%. BV(®) Blue test is a simple, rapid and reliable test allowing immediate diagnosis and prompt treatment of BV in the absence of microscopy which would greatly benefit majority of women at the greatest risk of sequel of bacterial vaginosis.
A commercial Dengue Duo rapid test kit was evaluated for early dengue diagnosis by detection of dengue virus NS1 antigen and immunoglobulin M (IgM)/IgG antibodies. A total of 420 patient serum samples were subjected to real-time reverse transcription-polymerase chain reaction (RT-PCR), in-house IgM capture enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition assay, and the SD Dengue Duo rapid test. Of the 320 dengue acute and convalescent sera, dengue infection was detected by either serology or RT-PCR in 300 samples (93.75%), as compared with 289 samples (90.31%) in the combined SD Duo NS1/IgM. The NS1 detection rate is inversely proportional, whereas the IgM detection rate is directly proportional to the presence of IgG antibodies. The sensitivity and specificity in diagnosing acute dengue infection in the SD Duo NS1/IgM were 88.65% and 98.75%, respectively. The assay is sensitive and highly specific. Detection of both NS1 and IgM by SD Duo gave comparable detection rate by either serology or RT-PCR.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the commonest causes of neonatal jaundice in Malaysia. Screening of cord blood for G6PD deficiency by the semiquantitative fluorescent spot test (FST) is performed in Malaysia but this test can miss cases of partial G6PD deficiency. The OSMMR-D kit assay measures G6PD activity and hemoglobin (Hb) concentration, allowing direct expression of results in U/gHb. We evaluated this method and established the normal range for G6PD activity in normal term neonates and adults. EDTA blood from 94 neonates and 295 adults (age 15-59 years old) with normal Hb and FST were selected. The normal means for G6PD activity for neonates and adults were 12.43 +/- 2.28 U/gHb and 9.21 +/- 2.6 U/gHb, respectively; the reference ranges for normal G6PD activity in neonates and adults were 10.15-14.71 U/gHb and 6.61-11.81 U/gHb respectively. There were no significant differences in mean normal G6PD activity between the Malays and Chinese racial groups or between genders. The upper and lower limit cut-off points for partial deficiency in neonates were 7.4 U/gHb (60% of the normal mean) and 2.5 U/gHb (20% of the normal mean), respectively. For adults, the upper and lower limit cut-off points for partial deficiency in adults were 5.52 U/gHb (60% of the normal mean) and 1.84 U/gHb (20% of the normal mean), respectively. The quantitation of G6PD enzymes using this OSMMR-D kit with Hb normalization was simple since the Hb was analyzed simultaneously and the results were reproducible with a CV of less than 5%.
The performance of a commercial rapid immunochromatographic dengue IgG/IgM assay device was evaluated against an in-place dengue IgM-capture ELISA in the National Public Health laboratory. Of the 239 serum samples from patients with clinical diagnosis of acute dengue illness, 140 and 99 samples were tested positive and negative respectively for anti-dengue IgM by the in-placed ELISA. Comparatively, 72 and 76 samples were tested positive and negative respectively, and 91 samples gave equivocal results by the rapid dengue test device. The rapid immunochromatographic assay device gave a relative sensitivity of 49.3% and a relative specificity of 62.6%. Though the rapid immunochromatographic assay device has the advantages of rapid testing which simultaneously detects both IgG and IgM and can also be performed with whole blood, serum or plasma, the user has to exercise extreme caution with the interpretation of the test result.
Evaluation of binding between analytes and its relevant ligands on surface plasmon resonance (SPR) biosensor is of considerable importance for accurate determination and screening of an interference in immunosensors. Dengue virus serotype 2 was used as a case study in this investigation. This research work compares and interprets the results obtained from analytical analysis with the experimental ones. Both the theoretical calculations and experimental results are verified with one sample from each category of dengue serotypes 2 (low, mid, and high positive), which have been examined in the database of established laboratorial diagnosis. In order to perform this investigation, the SPR angle variations are calculated, analyzed, and then validated via experimental SPR angle variations. Accordingly, the error ratios of 5.35, 6.54, and 3.72% were obtained for the low-, mid-, and high-positive-specific immune globulins of patient serums, respectively. In addition, the magnetic fields of the biosensor are numerically simulated to show the effect of different binding mediums.
The effect of HbE, a hemoglobin variant, on the determination of HbA1/HbA1c using 4 commercial kits based on cation-exchange resin, cation-exchange column chromatography and specific antibody techniques was studied. Fifty-eight normal and 63 HbE heterozygous subjects were tested for HbA1 and HbA1c using 4 commercial kits i.e. Eagles Diagnostics, Boehringer Mannehim (BM), Diastat and Ames DCA 2000. Analyses of the samples by the 4 kits were done within one week and samples were stored at 4 degrees C before analysis. The results showed that HbE affects the determination of glycosylated hemoglobin using cation-exchange based and not kits based on specific antibody techniques.
Forty-four serum samples of various reactivities to rickettsial antigens demonstrated by the indirect immunoperoxidase technique were tested with INDX Dip-S-Ticks (INDX Integrated Diagnostics Inc., USA) Kit for the detection of tick borne diseases. The kit utilised Rickettsia rickettsii the causative agent of Rocky Mountain spotted fever (RMSF) as antigens. The samples positive for endemic typhus were also tested against R. typhi, the agent for endemic typhus by the same method. The aim of this study was to determine the extent of cross-reactivity of R. rickettsii with rickettsial infections in Malaysia. Nine out of 12 tick typhus, 4 out of 10 scrub typhus and 4 out of 12 endemic typhus samples cross reacted with R. rickettsii. Ten out of 12 endemic samples were positive with R. typhi by the same method. From the study, we concluded that the INDX Dip-S-Ticks Kit can be used as a rapid screening test to detect endemic and tick-borne rickettsial infections in Malaysia but a second serological test is strongly recommended on all weakly reactive cases.
Dengue fever/Dengue haemorrhagic fever (DF/DHF) has been a public health problem in Malaysia with an endemic level of about 7 per 100,000 population per year. In 1990, Malaysia experienced its most severe outbreak of DF/DHF with a record total of 5,590 cases referred to the Division of Virology, Institute for Medical Research (IMR). Of these, 1,880 were confirmed serologically to be DF/DHF. The conventional serological procedure, the Haemagglutination Inhibition (HI) test, for the diagnosis of DF/DHF is cumbersome and causes delay in diagnosis. Another problem associated with the HI test has been that it has often been difficult to obtain a second convalescent serum sample for an accurate diagnosis. This has raised an urgent need to establish a "rapid" test for diagnosis of DF/DHF. As such the authors recently carried out an evaluation of a newly available commercial rapid test, namely, the Dengue Blot Assay (Diagnostic Biotechnology Singapore Pte Ltd). The test is intended for use in laboratory confirmation of dengue virus infection. The evaluation was to determine if the test could be utilised as a routine laboratory test and to establish its sensitivity and specificity. Over 400 samples were tested against the Dengue Blot Assay. Results were checked against an in-house Dengue IgM ELISA and HI assay. Preliminary results indicate that the sensitivity and specificity of the Dengue Blot is satisfactory. Our results also indicate that the Dengue Blot has a useful role to play in a routine laboratory especially since it provides rapid results on single serum samples thereby reducing the workload in a busy diagnostic laboratory.
Brugia malayi infection is endemic in several Asian countries. Filaria-specific IgG4 antibody detection based on BmR1 recombinant antigen has been shown to be sensitive and specific for the diagnosis of brugian filariasis. Two formats of the test has been reported ie indirect ELISA (BE) and rapid dipstick test (BR). Since different test formats use different amounts of sample and reagents which may affect its sensitivity and specificity, this study was performed to compare these two test formats in the detection of B. malayi. A total of 264 blinded serum samples from India and Malaysia were employed. Group 1 comprised 164 samples from actively infected individuals and group 2 comprised 100 samples from filaria non-endemic areas. Sensitivity was 96.3% (158/164) and 90.8% (149/164) for rapid test and ELISA respectively; chi-square p=0.00. Both test formats demonstrated 100% specificity. Therefore the rapid test format was equally specific but more sensitive than the ELISA format. The ELISA format would be able to demonstrate decline in IgG4 titer post-treatment while the rapid test would be very useful for screening and diagnosis in the field.
Candida auris is an emerging pathogenic yeast responsible for nosocomial infections with high mortality, on a global scale. A 65-year-old woman with hypovolemic shock and severe metabolic acidosis was intubated and admitted to the intensive care unit (ICU). Shortly after admission, she developed ventilator-associated pneumonia caused by multidrug-resistant Acinetobacter baumannii, which necessitated treatment with high-dose ampicillin-sulbactam. Two weeks later, a yeast was cultured from her blood. It formed pale pink colonies on CHROMagar Candida medium and produced predominantly oval budding yeast cells with the occasional rudimentary pseudohyphae on cornmeal agar. ID 32 C identified the yeast as Candida sake However, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and sequencing of the D1/D2 region of the 28S rRNA gene identified the yeast as C. auris.
Short tandem repeat (STR) analysis is used in chimerism monitoring after allogeneic hematopoietic stem cell transplantation (HSCT) for patients with various hematologic malignancies. Commercial forensic STR kits often contain loci with huge differences in power of discrimination (PD) across populations, causing some loci to be less informative for chimerism analysis in certain populations. This study aimed to construct a new STR multiplex panel with highly informative loci for efficient chimerism analysis. Thirteen STR markers which exhibit high PD (> 0.9) in at least 80% of 50 populations globally were selected to form a new panel and used in STR analysis of 253 Malaysian subjects. Cumulative power of discrimination (CPD) and combined power of exclusion (CPE) were determined from 253 Malaysian individuals. Loci informativity was assessed and compared to the commercial AmpFLSTR Identifiler PCR Amplification kit in 14 donor-recipient pairs. The new panel had detected 202 unique alleles including five novel alleles from the 253 individuals with high CPD and CPE (> 0.99999999999999999 and > 0.999999997 respectively). All loci from the new panel in the donor-recipient pair analysis showed higher than 50% informativity, while five loci from the commercial kit demonstrated lower than 50% informativity. Four loci from the new panel ranked the highest informativity. A sequenced allelic ladder which consists of 202 unique alleles from the 253 subjects was also developed to ensure accurate allele designation. The new 13-loci STR panel, thus, could serve as an additional powerful, accurate, and highly informative panel for chimerism analysis for HSCT patients.
Multidrug resistance (MDR) is believed to be responsible for poor response of patients towards chemotherapy particularly patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). The best-characterized resistance mechanism is the one mediated by permeability-glycoprotein (P-gp) encoded by MDR1 gene, which is responsible for drug efflux. We studied P-gp and multidrug resistance-associated protein 1 (MRP1) expression and functional activities in 43 newly diagnosed acute leukemia cases (19 paediatric ALL cases and 24 adult AML cases). The expression and functional activities were examined using flow cytometry and MultiDrugQuant assay kit (involving calcein AM uptake and efflux). P-gp and MRP1 expression and its functional activities were observed in 68.4% of paediatric ALL. In adult AML cases, all cases expressed MRP1 and its functional activities but only 58.3% were positive for P-gp and its functional activities. We were able to show a significant correlation between the expression of the multidrug resistant protein (P-gp and MRP1) and their functional activity in adult AML and paediatric ALL samples.
The gastric biopsy urease test is an accurate and robust diagnostic test for Helicobacter pylori infection. Large endoscopy units use their own homemade unbuffered ultra-rapid urease test for diagnosis of H. pylori infection but several commercial rapid urease tests are available.