Evaluation of the clinical sensitivity and specificity of the BD Max™ Enteric Bacterial Panel for molecular detection of pathogens for acute gastroenteritis in the Singaporean population.

PURPOSE: Acute gastroenteritis (AGE) is caused by a wide range of pathogens. Culture methods for the detection of bacterial pathogens is time consuming and labour intensive. This study compared a same-day-to-result commercial molecular method using BD Max™ Enteric Bacterial Panel against conventional culture and laboratory-developed PCR assays (LDTs), and characterised the epidemiology of bacterial AGE in Singapore. METHODOLOGY: PCRs for Campylobacter spp., Salmonella spp., Shigella spp./Enteroinvasive Escherichia coli (EIEC) and Shiga toxin-producing E. coli (STEC)/Shigella dysenteriae were performed on the BD Max™ platform. Concurrent routine bacterial culture ("reference standard") was performed for Campylobacter, Salmonella, Shigella, Vibrio and Aeromonas spp. In the event of a discrepancy, an "expanded reference standard" (bacterial culture with LDT) was used. RESULTS: There were 299 stool specimens in the study, with no bacterial pathogens detected in 190 samples (63.5%). The positive samples (n = 109,36.5%) were detected with Salmonella (n = 57,19.1%), Campylobacter (n = 28,9.4%), Vibrio parahaemolyticus (n = 6,2.0%), Shigella/EIEC (n = 6,2.0%), ETEC (n = 4,1.3%), STEC (n = 2,0.7%), Aeromonas (n = 2,0.7%), Plesiomonas shigelloides (n = 1,0.3%) and 3(1.0%) co-infections. Compared to the "expanded reference standard", conventional culture missed 38/112 (33.9%) pathogens. Conversely, testing by BD Max™ alone failed to detect 17 pathogens. BD Max™ reported seven (2.3%) false-positive results. CONCLUSIONS: BD Max™ increased the detection rate of bacterial AGE pathogens in the panel, but was limited by the absence of detection capability for Vibrio and Aeromonas spp.

Evaluation and Validation of the Roche Elecsys SARS-CoV-2 Antigen Electro-Chemiluminescent Immunoassay in a Southeast Asian Region.

INTRODUCTION: SARS-CoV-2 antigen tests can complement and substitute for RT-PCR tests. Centralized laboratory automated SARS-CoV-2 antigen tests that can be scaled to process a large number COVID-19 cases simultaneously are now available. We have evaluated the new Roche Elecsys SARS-CoV-2 antigen electro-chemiluminescent immunoassay. METHODS: The Roche SARS-CoV-2 antigen assay is a double-antibody sandwich electro-chemiluminescent immunoassay, which reports a cut-off index (COI) (COI ≥ 1.0 considered positive). We assessed assay precision and linearity, and confirmed the reactivity limit. We determined the assay sensitivity and specificity with a verification group (289 controls and 61 RT-PCR positive COVID-19 cases). Assay performance was also validated against the consecutive samples we received (7657 controls and 17 cases) for SARS-CoV-2 antigen testing from June to October 2021. RESULT: The assay had a within-run precision CV of 3.0% at COI 0.68, and a CV of 1.5% at COI 3.49. Between-run precision was 3.0% at COI 0.68 and 1.8% at COI 3.49. The assay was linear from COI 0.65 to 7.84. All 35 C50 ± 20% test results performed over 7 days were positive/negative, respectively. In the verification group, overall sensitivity was 42.6% (26/61 positive, 95% CI 30.0-55.9), and specificity was 99.7% (1/289 positive, 95% CI 98.1-100). The agreement between the SARS-CoV-2 antigen and the RT-PCR cycle threshold (Ct) count was good (r = 0.90). In cases with Ct counts ≤ 30, the antigen assay sensitivity improved to 94.7% (18/19 positive, 95% CI 74.0-99.9). In our validation group, antigen sensitivity was 62.5% (5/8 antigen positive, 95% CI 24.5-91.5) within the first week of disease onset, but no cases were reactive after the first week of disease onset. CONCLUSION: The Elecsys SARS-CoV-2 antigen assay has good performance within manufacturer specifications. The sensitivity of the Roche antigen assay was greatest when used in patients with lower RT-PCR Ct values (≤30) and within the first week of disease onset.

A Study of Analytical and Clinical Sensitivity of Aptima SARS-CoV-2 Assay (Hologic) and Proposals of Complementary Tests for SARS-CoV-2 Detection in Low Viral Load Specimens.

Early and accurate detection of SARS-CoV-2 is important for diagnosis and transmission control. The use of high-throughput and automated testing allows laboratories to better deliver diagnostic testing given manpower and resource limitations. We validated the clinical and analytical performance of the Hologic Panther Aptima SARS-CoV-2 assay with an emphasis on detection of specimens with low viral loads. The clinical performance was evaluated using 245 clinical specimens, against a comparator PCR-based laboratory developed test (LDT). The analytical performance was determined by replicate testing of contrived samples in a ten-fold dilution series (CT values 32-42, based on LDT). The Aptima assay had 96.7% overall percent agreement, 100% negative percent agreement and 88.1% positive percent agreement. It was able to consistently detect SARS-CoV-2 in contrived samples with CT = 32 by LDT (calculated 2354 copies/mL). The 95% limit of detection of the Aptima assay was estimated to be at LDT CT = 33 (equivalent to 870 copies/mL). The relative light units (RLU) × 1000 for 52 true positive clinical specimens was 962.2 ± 181.5, and that for the 186 true negative specimens was 264.6 ± 14.3. The Aptima assay was a reliable method with a high overall percent agreement against our comparator LDT. We propose that samples reported as negative by the Aptima assay with RLU > 350 be tested by a secondary method, in order to improve detection of samples with very low viral loads.

Development of a rapid multiplex PCR assay for the detection of common pathogens associated with community-acquired pneumonia.

BACKGROUND: Community-acquired pneumonia (CAP) is one of the most common infectious diseases and is a significant cause of mortality and morbidity globally. A microbial cause was not determined in a sizable percentage of patients with CAP; there are increasing data to suggest regional differences in bacterial aetiology. We devised a multiplex real-time PCR assay for detecting four microorganisms (Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae and Burkholderia pseudomallei) of relevance to CAP infections in Asia. METHODS: Analytical validation was accomplished using bacterial isolates (n=10-33 of each target organism for analytical sensitivity and n=117 for analytical sensitivity) and clinical validation using 58 culture-positive respiratory tract specimens. RESULTS: The qPCR assay exhibited 100% analytical sensitivity and analytical specificity, and 100% clinical sensitivity and 94-100% clinical specificity. The limit of detection and efficiency for the multiplex PCR assay were 3-33 CFU/mL and 93-110%, respectively. The results showed that the PCR-based method had higher sensitivity than traditional culture-based methods. The assay also demonstrated an ability to semiquantify bacterial loads. CONCLUSION: We have devised a reliable laboratory-developed multiplex qPCR assay, with a turnaround time of within one working day, for detection of four clinically important CAP-associated microorganisms in Asia. The availability of a test with improved diagnostic capabilities potentially leads to an informed choice of antibiotic usage and appropriate management of the patient to achieve a better treatment outcome and financial savings.

Sheathless and high-throughput elasto-inertial bacterial sorting for enhancing molecular diagnosis of bloodstream infection.

Purification of bacteria from human blood samples is essential for rapid identification of pathogens by molecular methods, enabling faster and more accurate diagnosis of bloodstream infection than conventional gold standard blood culture methods. The inertial microfluidic method has been broadly studied to isolate biological cells of interest in various biomedical applications due to its label-free and high-throughput advantages. However, because of the bacteria's tininess, which ranges from 0.5 µm to 3 µm, they are challenging to be effectively focused and sorted out in existing inertial microfluidic devices that work well with biological cells larger than 10 µm. Efforts have been made to sort bacterial cells by utilizing extremely small channel dimensions or employing a sheath flow, which thus results in limitations on the throughput and ease of operation. To overcome this challenge, we develop a method that integrates a non-Newtonian fluid with a novel channel design to allow bacteria to be successfully sorted from larger blood cells in a channel dimension of 120 µm × 20 µm without the use of sheath flows. The throughput of this device with four parallel channels is above 400 µL per minute. The real-time polymerase chain reaction (qPCR) analysis indicates that our inertial sorting approach has a nearly 3-fold improvement in pathogen recovery compared with the commonly used lysis-centrifugation method at pathogen abundances as low as 102 cfu mL-1. With the rapid and simple purification and enrichment of bacterial pathogens, the present inertial sorting method exhibits an ability to enhance the fast and accurate molecular diagnosis of bloodstream bacterial infection.

Enhanced Molecular Diagnosis of Bloodstream Candida Infection with Size-Based Inertial Sorting at Submicron Resolution.

Inertial microfluidics has been proven to be a powerful tool for high-throughput, size-based cell sorting in diverse biomedical applications. In the case of Candida-related sepsis, Candida species and major blood cells (i.e., red blood cells and white blood cells) have a size distribution of 3-5 and 6-30 µm, respectively. To effectively retrieve a majority of Candida species and remove most of the interfering blood cells for accurate molecular analysis, inertial sorting of micron-sized biological particles with submicron size difference is highly desired, but far unexplored till now. In this work, we present a new channel design for an inertial microfluidic sorting device by embedding microsquares to construct periodic contractions along a series of repeating curved units. This unique channel design allows us to enhance inertial lift force at the microsquare zone and produce localized secondary Dean flow drag force in addition to global Dean flow drag force. This inertial sorting device has successfully separated 5.5 µm particles from 6.0 µm particles with a recovery ratio higher than 80% and a purity higher than 92%, demonstrating a size-based inertial sorting at submicron resolution (i.e., 0.5 µm). We further applied this inertial sorting device to purify Candida species from whole blood sample for enhanced molecular diagnosis of bloodstream Candida infection and especially compared it with the commonly used lysis-centrifugation-based purification method (STEM method) by recovering two species of Candida (Cornus glabrata and Candida albicans) from Candida-spiked blood samples. Through quantitative polymerase chain reaction (qPCR) analysis, we found that our inertial sorting approach has nearly 3-fold improvement on the pathogen recovery than the STEM method at pathogen abundances of 103 cfu/mL and 102 cfu/mL. The present inertial sorting at submicron resolution provides a simple, rapid, and efficient pathogen purification method for significantly improved molecular diagnosis of bloodstream Candida infection.

Development and validation of a real-time multiplex PCR assay for the detection of dermatophytes and Fusarium spp.

Introduction: Onychomycosis is a debilitating, difficult-to-treat nail fungal infection with increasing prevalence worldwide. The main etiological agents are dermatophytes, which are common causative pathogens in superficial fungal mycoses. Conventional detection methods such as fungal culture have low sensitivity and specificity and are time-consuming.Aim: The main objective of this study was to design, develop and validate a real-time probe-based multiplex qPCR assay for the detection of dermatophytes and Fusarium species.Methodology: The performance characteristics of the qPCR assays were evaluated. The multiplex qPCR assays targeted four genes (assay 1: pan-dermatophytes/Fusarium spp.; assay 2: Trichophyton rubrum/Microsporum spp.). Analytical validation was accomplished using 150 fungal isolates and clinical validation was done on 204 nail specimens. The performance parameters were compared against the gold standard (fungal culture) and expanded gold standard (culture in conjunction with sequencing).Results: Both the single-plex and multiplex qPCR assays performed well especially when compared against the expanded gold standard. Among the 204 tested nail specimens, the culture method showed that 125 (61.3 %) were infected with at least one organism, of which 40 yielded positive results for dermatophytes and Fusarium spp. These target organisms detected include 20 dermatophytes and 22 Fusarium spp. The developed qPCR assays demonstrated excellent limit of detection, efficiency, coefficient of determination, analytical and clinical sensitivity and specificity.Conclusion: The multiplex qPCR assays were reliable for the diagnosis of onychomycosis, with shorter turn-around time as compared to culture method. This aids in the planning of treatment strategies to achieve optimal therapeutic outcome.

Prevalence and antibiotic susceptibility of colistin-resistance gene (mcr-1) positive Enterobacteriaceae in stool specimens of patients attending a tertiary care hospital in Singapore.

OBJECTIVES: The aim of this study was to determine the prevalence of the colistin-resistance gene (mcr-1) and the antibiotic-susceptibility profile of mcr-1 positive, colistin-resistant isolates in stool specimens of patients attending a tertiary care hospital in Singapore. METHODS: 201 diarrheal stool specimens of patients attending the Changi General Hospital between May to August 2017 were collected and screened for the presence of mcr-1 by culture and molecular methods. Antibiotic-susceptibility profile of mcr-1 positive isolates was determined using the polymyxin B and colistin E-tests and the VITEK 2 system. RESULTS: We observed an unexpectedly high prevalence of mcr-1 in patients attending a tertiary care hospital in Singapore, i.e 6.0% and 8.0% estimated by stool culture and direct stool PCR, respectively. The mcr-1 gene was detected predominantly in Escherichia coli. Antibiotic-susceptibility testing on 12 mcr-1 positive Enterobacteriaceae isolates revealed variable susceptibility profiles with no detection of carbapenem-resistant Enterobacteriaceae. CONCLUSIONS: This is the first report of the prevalence of human faecal carriage of mcr-1 in Singapore. Our findings highlight the potential risk of mcr-1 spread among our patient cohort. The mcr-1 gene detection combined with the detection of other resistance gene targets of clinical importance is recommended to pre-empt the spread mcr-1 in our patients.

Development of a real-time assay to determine the frequency of qac genes in methicillin resistant Staphylococcus aureus.

PURPOSE: The emergence of antiseptic resistance and/or antiseptic-resistance genes in methicillin-resistant Staphylococcus aureus (MRSA) may result in failure of decolonization treatments. Plasmid-encoded efflux pump genes qacA/B and qacC (smr) confer tolerance to chlorhexidine and quaternary ammonium compounds. The objective of this study was to develop and validate a multiplex real time-PCR assay for detection of antiseptic-resistance genes, apply the assay on 200 MRSA isolates and explore if carriage of these genes was associated with resistance to topical antibiotics. METHODOLOGY: A SYBR-Green based multiplex real time-PCR assay was developed to detect qacA/B, qacC, and mecA (internal control) simultaneously. The multiplex assay was compared against conventional single-plex PCR followed by agarose gel electrophoresis, using DNA from the first 73 MRSA isolates, followed by multiplex testing of the remaining 127 MRSA isolates. All 200 MRSA isolates were tested for susceptibility to mupirocin, retapamulin, neomycin, bacitracin and octenidine. The genetic diversity of the isolates was investigated by spa-typing. RESULTS: The concordance between multiplex and conventional PCR, in assignments of qacA/B and qacC status were 99%(72/73) and 100%(73/73) respectively. Among 200 MRSA isolates, 48(24%) and 44(23%) were found to harbour qacA/B and qacC genes, respectively. These isolates remained susceptible to many common decolonization agents, except mupirocin. The predominant spa-types were t020 and t1081 (41 and 32 isolates respectively). CONCLUSION: The real-time assay performed acceptably for the detection of qac genes. A high prevalence of antiseptic-resistance genes were detected in the MRSA isolates in our population and appeared to be associated with spa-type t1081.

Impact of microbial Aetiology on mortality in severe community-acquired pneumonia.

BACKGROUND: The impact of different classes of microbial pathogens on mortality in severe community-acquired pneumonia is not well elucidated. Previous studies have shown significant variation in the incidence of viral, bacterial and mixed infections, with conflicting risk associations for mortality. We aimed to determine the risk association of microbial aetiologies with hospital mortality in severe CAP, utilising a diagnostic strategy incorporating molecular testing. Our primary hypothesis was that respiratory viruses were important causative pathogens in severe CAP and was associated with increased mortality when present with bacterial pathogens in mixed viral-bacterial co-infections. METHODS: A retrospective cohort study from January 2014 to July 2015 was conducted in a tertiary hospital medical intensive care unit in eastern Singapore, which has a tropical climate. All patients diagnosed with severe community-acquired pneumonia were included. RESULTS: A total of 117 patients were in the study. Microbial pathogens were identified in 84 (71.8%) patients. Mixed viral-bacterial co-infections occurred in 18 (15.4%) of patients. Isolated viral infections were present in 32 patients (27.4%); isolated bacterial infections were detected in 34 patients (29.1%). Hospital mortality occurred in 16 (13.7%) patients. The most common bacteria isolated was Streptococcus pneumoniae and the most common virus isolated was Influenza A. Univariate and multivariate logistic regression showed that serum procalcitonin, APACHE II severity score and mixed viral-bacterial infection were associated with increased risk of hospital mortality. Mixed viral-bacterial co-infections were associated with an adjusted odds ratio of 13.99 (95% CI 1.30-151.05, p = 0.03) for hospital mortality. CONCLUSIONS: Respiratory viruses are common organisms isolated in severe community-acquired pneumonia. Mixed viral-bacterial infections may be associated with an increased risk of mortality.

Comparison of the Biofire FilmArray Respiratory Panel, Seegene AnyplexII RV16, and Argene for the detection of respiratory viruses.

BACKGROUND: Respiratory infections are common reasons for hospital admission, and are associated with enormous economic burden due to significant morbidity and mortality. The wide spectrum of microbial agents underlying the pathology renders the diagnosis of respiratory infections challenging. Molecular diagnostics offer an advantage to the current serological and culture-based methods in terms of sensitivity, coverage, hands-on time, and time to results. OBJECTIVES: This study aimed to compare the clinical performance of three commercial kits for respiratory viral detection. STUDY DESIGN: The performance of FilmArray Respiratory Panel, AnyplexII RV16, and Argene was compared using clinical respiratory samples (n = 224, comprising 189 nasopharyngeal swabs in Universal Transport Medium (UTM) and 35 endotracheal aspirates), based on common overlapping targets across the platforms. Influenza A "equivocal" and "no-subtype" samples by FilmArray were further compared to a laboratory-developed Influenza A/B test. RESULTS AND CONCLUSIONS: The overall performance of all three platforms appeared to be comparable with regards to sensitivities (95.8-97.9%) and specificities (96.1-98.0%), detection of coinfections, and distinguishment of influenza from non-influenza cases. "Equivocal" and "no-subtype" samples by FilmArray mostly represented weak Influenza A by laboratory-developed test. Lower respiratory tract samples had comparable final-run success-rates and discordant-rates as compared to UTM. Coronavirus HKU1, which was not targeted by AnyplexII RV16, were detected as OC43. The expected test volume would be the main determinant for the selection of platform. Among the platforms, the FilmArray is the most automated but is of the lowest-throughput and has the highest reagent cost.

Faster and economical screening for vancomycin-resistant enterococci by sequential use of chromogenic agar and real-time polymerase chain reaction.

BACKGROUND/PURPOSE: Screening for vancomycin-resistant enterococci (VRE) by culture takes days to generate results, while polymerase chain reaction (PCR) testing directly from clinical specimens lacks specificity. The aims of this study were to develop a real-time PCR to detect and identify Enterococcus faecium, Enterococcus faecalis, and vanA and vanB genes, and to evaluate the impact of this PCR on test-reporting times when performing it directly from suspect VRE isolates present on screening chromogenic media. METHODS: The tetraplex PCR primers were designed to amplify E. faecium, E. faecalis, and vanA and vanB genes, with melt-curve analysis of PCR products. Following analytical and clinical validation of the molecular assay, PCR testing was performed for target colonies present on VRE chromogenic media. PCR results were evaluated against conventional phenotypic identification and susceptibility testing, with the time to result being monitored for both modalities. RESULTS: A total of 519 colonies from clinical specimens were tested concurrently by real-time PCR and phenotypic methods. In all, 223 isolates were identified with phenotypic vancomycin resistance (vanA, n = 108; vanB, n = 105; non-vanA/vanB = 10), with complete agreement between PCR and phenotypic testing for vancomycin-resistant E. faecium and E. faecalis. The majority (88.6%) of PCR results were reported, on average, 24.8 hours earlier than those of phenotypic testing, with 68% reduction in total costs. CONCLUSION: The use of culture on selective media, followed by direct colony PCR confirmation allows faster and economical VRE screening.

Novel cost-effective quality control approach for the Cepheid Xpert CT/NG assay for the detection of Chlamydia Trachomatis and Neisseria Gonorrhoeae.

The Xpert CT/NG is a rapid assay for detection of Neisseria gonorrhoeae and Chlamydia trachomatis. QC materials must be formulated to emulate human specimens, and are prohibitively expensive. A creative, cost-effective QC approach is proposed. The acceptable sample types for the Xpert CT/NG assay were extended to include eye swabs.

Using Pooled Recombinant Plasmids as Control Materials for Diagnostic Real-Time PCR.

BACKGROUND: Despite the potential usefulness of plasmids as reference materials for PCR, there are very few reports documenting the experience with using these materials in quality control (QC) of laboratory developed tests (LDTs), in a clinical diagnostic setting. In this study, an approach to preparing and titering such controls is detailed. The practicality of using plasmids as QC materials was explored and presented. METHODS: Target DNA fragments (n = 11) were amplified from positive samples and cloned using TA-cloning. Identities of the fragments were ascertained using DNA sequencing. Real-time PCR was carried out using TaqMan probes on RGQ or CFX-96 thermal-cyclers. RESULTS: All the 11 targeted DNA fragments were successfully cloned into E. coli vectors. Plasmid pools could be repeatedly reconstituted to generate stable and usable positive controls for multiplex PCR assays in a simple workflow. Importantly, plasmid controls generated meaningful run data that could be used in QC processes for monitoring routine runs. Further, plasmid material could be spiked into clinical specimens for quality assurance (QA) purposes, avoiding the culture of live infectious organisms, showing another routine usefulness of plasmids. CONCLUSIONS: Plasmid pools are useful and inexhaustible sources of reference materials for various routine QC uses in the diagnostic laboratory.

Preimplantation genetic diagnosis of chromosome translocations by analysis of polymorphic short tandem repeats.

INTRODUCTION: We aimed to develop and implement a short tandem repeat (STR) polymerase chain reaction alternative to fluorescence in situ hybridisation (FISH) for the preimplantation genetic diagnosis (PGD) of chromosomal translocations. METHODS: Selected informative STRs located on translocated arms of relevant chromosomes were used to discriminate between normal and unbalanced chromosome states in each embryo. RESULTS: PGD cycles were performed on five couples where one spouse carried a balanced translocation. 27 embryos were analysed, of which 12 were normal/balanced, 12 were abnormal/unbalanced and three were indeterminate. Four PGD cycles proceeded to embryo transfer, of which two led to pregnancy. The first pregnancy showed a normal male karyotype, and a healthy baby was delivered at term. A second pregnancy unexpectedly miscarried in the second trimester from unknown causes. CONCLUSION: STR analysis is a simple and suitable alternative to FISH for detecting unbalanced chromosomal states in preimplantation embryos.

Molecular strategies for pre-implantation genetic diagnosis of single gene and chromosomal disorders.

Pre-implantation genetic diagnosis is used to analyse pre-implantation stage embryos or oocytes for genetic defects, generally for severe Mendelian disorders and chromosome abnormalities. New but controversial indications for pre-implantation genetic diagnosis include identifying human leukocyte antigen compatible embryos suitable as donor, sex selection and adult-onset disorders, particularly cancer. Pre-implantation genetic screening is a variant of pre-implantation genetic diagnosis to improve outcomes of in-vitro fertilisation. Array comparative genomic hybridisation is replacing fluorescence in-situ hybridisation for aneuploidy screening. Besides technical advancement of array platform, the success of pre-implantation genetic screening is strongly related to the embryonic biological nature of chromosomal mosaicism. Having been applied for more than 20 years, pre-implantation genetic diagnosis is recognised as an important alternative to prenatal diagnosis. Diagnosis from a single cell, however, remains a technically challenging procedure, and the risk of misdiagnosis cannot be eliminated.

Transcriptome analysis of amoeboid and ramified microglia isolated from the corpus callosum of rat brain.

BACKGROUND: Microglia, the resident immune cells of the central nervous system (CNS), have two distinct phenotypes in the developing brain: amoeboid form, known to be amoeboid microglial cells (AMC) and ramified form, known to be ramified microglial cells (RMC). The AMC are characterized by being proliferative, phagocytic and migratory whereas the RMC are quiescent and exhibit a slow turnover rate. The AMC transform into RMC with advancing age, and this transformation is indicative of the gradual shift in the microglial functions. Both AMC and RMC respond to CNS inflammation, and they become hypertrophic when activated by trauma, infection or neurodegenerative stimuli. The molecular mechanisms and functional significance of morphological transformation of microglia during normal development and in disease conditions is not clear. It is hypothesized that AMC and RMC are functionally regulated by a specific set of genes encoding various signaling molecules and transcription factors. RESULTS: To address this, we carried out cDNA microarray analysis using lectin-labeled AMC and RMC isolated from frozen tissue sections of the corpus callosum of 5-day and 4-week old rat brain respectively, by laser capture microdissection. The global gene expression profiles of both microglial phenotypes were compared and the differentially expressed genes in AMC and RMC were clustered based on their functional annotations. This genome wide comparative analysis identified genes that are specific to AMC and RMC. CONCLUSIONS: The novel and specific molecules identified from the trancriptome explains the quiescent state functioning of microglia in its two distinct morphological states.

Separation of inorganic anions on hydrophobic stationary phases in ion chromatography.

Inorganic anions were separated on hydrophobic stationary phases such as triacontyl-functionalized silica. Eluent conditions were examined in detail, and iodate, nitrate, iodide, and thiocyanate could be separated by using aqueous solutions. The effect of the eluent concentration on the retention of analyte anions was examined for a wide range of sodium sulfate concentrations of up to 1 M. The retention factor of hydrophobic anions decreased with increasing sodium sulfate concentration in the lower concentration region, while it increased with increasing sodium sulfate concentration in the higher concentration region. The addition of a small amount of an organic substance such as acetonitrile and tetraethylene glycol increased the retention of iodide and thiocyanate, while the addition of alcohols decreased their retention. Operating at lower temperature also increased the retention of analyte anions. It was expected that inorganic anions were retained on the stationary phase via hydrophobic interactions. The retention mechanism was discussed, considering the results obtained.

Molecular and morphological characterization of neural tube defects in embryos of diabetic Swiss Albino mice.

BACKGROUND AND RESULTS: Embryos from diabetic mice exhibit several forms of neural tube defects, including non-closure of the neural tube. In the present study, embryos collected at embryonic day 11.5 from diabetic pregnancies displayed open neural tube with architectural disruption of the surrounding tissues. The percentage of proliferating cells was found to be increased in the dorsal and ventral domains of the spinal neural tube of embryos from diabetic mice, indicating a defect in the proliferation index. We have analyzed the development of various cell types, including motoneurons, interneurons, oligodendrocytes and migrating neurons, as well as radial glial cells in the open neural tube using specific molecular markers. Immunofluorescence results revealed a significantly reduced number of Pax2+ interneurons and increased number of Isl-1+ motoneurons, as well as Olig2+ oligodendrocytes in the neural tube of embryos from diabetic mice as compared to controls. In addition, these embryos exhibited a decreased number of doublecortin positive migrating neurons and Glast/Blbp positive radial glial cells with shortened processes in the neural tube. Expression levels of several developmental control genes involved in the generation of different neuronal cell types (such as Shh, Ngn, Ngn2, Ascl1) were also found to be altered in the neural tube of embryos from diabetic mice. CONCLUSIONS: Overall, the open neural tube in embryos of diabetic mice exhibits defects in the specification of different cell types, including motoneurons and interneurons, as well as glial cells along the dorsoventral axis of the developing spinal cord. Although these defects are associated with altered expression of several development control genes, the exact mechanisms by which maternal diabetes contributes to these changes remain to be investigated.

Simplified PGD of common determinants of haemoglobin Bart's hydrops fetalis syndrome using multiplex-microsatellite PCR.

The high incidence of double-gene deletions in α-thalassaemia increases the risk of having pregnancies with homozygous α(0)-thalassaemia, the cause of the lethal haemoglobin (Hb) Bart's hydrops fetalis syndrome. Preimplantation genetic diagnosis (PGD) has played an important role in preventing such cases. However, the current gap-PCR based PGD protocol for deletional α-thalassaemia requires specific primer design for each specific deletion. A universal PGD assay applicable to all common deletional determinants of Hb Bart's hydrops fetalis syndrome has been developed. Microsatellite markers 16PTEL05 and 16PTEL06 within the α-globin gene cluster were co-amplified with a third microsatellite marker outside the affected region in a multiplex-PCR reaction and analysed by capillary electrophoresis. Eight informed couples at risk of having Hb Bart's hydrops fetalis were recruited in this study and all patients underwent standard procedures associated with IVF. A total of 47 embryos were analysed. Three pregnancies were achieved from three couples, with the births of two healthy babies and one ongoing pregnancy. This work has successfully adapted an earlier protocol and developed a simple and reliable single-cell assay applicable to PGD of Hb Bart's hydrops fetalis syndrome regardless of type of deletion. Alpha-thalassaemia is one of the most common inheritable disorders worldwide. It is a blood disorder that, in its lethal form caused by deletion of all four copies of the α-globin gene, results in the demise of the affected fetus, a condition referred to as haemoglobin (Hb) Bart's hydrops fetalis syndrome. Preimplantation genetic diagnosis (PGD) has played an important role in preventing such cases. Current PGD protocols for deletional α-thalassaemia utilize a strategy called gap-PCR, which requires the different assays for different deletion types. We have developed a universal PGD assay applicable to all common deletional determinants of Hb Bart's hydrops fetalis syndrome based on microsatellite marker analysis. Eight informed couples at risk of having Hb Bart's hydrops fetalis were recruited in this study and all patients underwent standard procedures associated with IVF. Forty-five embryos were analysed in total. Three pregnancies were achieved from three couples, with the births of two healthy babies and one pregnancy still ongoing. We have successfully adapted our earlier protocol and developed a simple and reliable single cell assay applicable to PGD of Hb Bart's hydrops fetalis syndrome regardless of the type of deletion.