Weak Positives Research Articles

Evaluation of HIV-1 rapid tests and identification of alternative testing algorithms for use in Uganda

IntroductionThe World Health Organization recommends that countries conduct two phase evaluations of HIV rapid tests (RTs) in order to come up with the best algorithms. In this report, we present the first ever such evaluation in Uganda, involving both blood and oral based RTs. The role of weak positive (WP) bands on the accuracy of the individual RT and on the algorithms was also investigated.MethodsIn total 11 blood based and 3 oral transudate kits were evaluated. All together 2746 participants from seven sites, covering the four different regions of Uganda participated. Two enzyme immunoassays (EIAs) run in parallel were used as the gold standard. The performance and cost of the different algorithms was calculated, with a pre-determined price cut-off of either cheaper or within 20% price of the current algorithm of Determine + Statpak + Unigold. In the second phase, the three best algorithms selected in phase I were used at the point of care for purposes of quality control using finger stick whole blood.ResultsWe identified three algorithms; Determine + SD Bioline + Statpak; Determine + Statpak + SD Bioline, both with the same sensitivity and specificity of 99.2% and 99.1% respectively and Determine + Statpak + Insti, with sensitivity and specificity of 99.1% and 99% respectively as having performed better and met the cost requirements. There were 15 other algorithms that performed better than the current one but rated more than the 20% price. None of the 3 oral mucosal transudate kits were suitable for inclusion in an algorithm because of their low sensitivities. Band intensity affected the performance of individual RTs but not the final algorithms.ConclusionWe have come up with three algorithms we recommend for public or Government procurement based on accuracy and cost. In case one algorithm is preferred, we recommend to replace Unigold, the current tie breaker with SD Bioline. We further recommend that all the 18 algorithms that have shown better performance than the current one are made available to the private sector where cost may not be a limiting factor.

The development of an in vitro Pig-a assay in L5178Y cells.

A recent flow cytometry-based in vivo mutagenicity assay involves the hemizygous phosphatidylinositol class A (Pig-a) gene. Pig-a forms the catalytic subunit of N-acetylglucosaminyltransferase required for glycophosphatidylinositol (GPI) anchor biosynthesis. Mutations in Pig-a prevent GPI-anchor synthesis resulting in loss of cell-surface GPI-linked proteins. The aim of the current study was to develop and validate an in vitro Pig-a assay in L5178Y mouse lymphoma cells. Ethyl methanesulfonate (EMS)-treated cells (186.24-558.72µg/ml; 24h) were used for method development and antibodies against GPI-linked CD90.2 and stably expressed CD45 were used to determine GPI-status by flow cytometry. Antibody concentration and incubation times were optimised (0.18µg/ml, 30min, 4°C) and Zombie Violet™ (viability marker; 0.5%, 30min, RT) was included. The optimum phenotypic expression period was 8 days. The low background mutation frequency of GPI-deficiency [GPI(-)] in L5178Y cells (0.1%) constitutes a rare event, thus flow cytometry acquisition parameters were optimised; 104 cells were measured at medium flow rate to ensure a CV ≤ 30%. Spiking known numbers of GPI(-) cells into a wild-type population gave high correlation between measured and spiked numbers (R2 0.999). We applied the in vitro Pig-a assay to a selection of well-validated genotoxic and non-genotoxic compounds. EMS, N-ethyl-N-nitrosourea and 4-nitroquinoline-N-oxide dose dependently increased numbers of GPI(-) cells, while etoposide, mitomycin C, and a bacterial-specific mutagen did not. Cycloheximide and sodium chloride were negative. Sanger sequencing revealed Pig-a mutations in the GPI(-) clones. In conclusion, this in vitro Pig-a assay could complement the in vivo version, and follow up weak Ames positives and late-stage human metabolites or impurities.

A Comparative Assessment of the EIA/PCR HIV Test Results among Weakly Reactive Samples on Any of Determine HIV1/2, Statpak and Unigold Rapid Test Kits

Objective: The Government of Uganda recommends the serial HIV rapid testing algorithm where weakly reactive results on any of the three: Determine, Statpak and Unigold are interpreted as positive. We assessed the proportion of weak positive (WP) rapid results against confirmed results. Methods: We generated cross tabulations using Stata 3.0 to compare the WP rapid test results on each kit in the algorithm against final results on EIA/PCR to assess the proportion of samples confirmed to be HIV negative on ELISA and/or qualitative DNA PCR. We also generated Positive Predictive Values (PPV) to find the proportion of results that were true positives. Results: A total of 231 weak positive (WP) results were selected. 71 were WP on Determine HIV1/2, 149 on Statpak and 11 on Unigold. We found that 35.2% of the WP results on Determine were confirmed Negative by EIA/PCR test with a Positive Predictive Value (PPV) of 64.8%, 80.5% of WP on Statpak were confirmed negative with a PPV of 19.5% while 63.6% of the WP on Unigold were confirmed negative with a PPV of 36.4%. When using the National serial rapid HIV testing algorithm, WP on both Determine and Statpak are scored as positive. However, we found that out of the 11 WP on both Determine and Statpak for screening and confirmatory tests respectively, 45.5% returned a final negative result with a PPV of 54.5%. Conclusion: Since point of care results are interpreted following manufacturer’s instructions, a substantial proportion that would have been scored as positive end up with Negative results. WP screening results whether using parallel or serial testing algorithm, need to be confirmed by EIA or PCR before results are finalized. This calls for a reevaluation of the national algorithm that considers WP scores instead of what is instated in the kit insert.

An Accurate and Sensitive RT-qPCR Method That Reliably Detects BCR-ABL1 Beyond MR4.5

▪Introduction: The BCR-ABL1 translocation is a hallmark of chronic myeloid leukemia (CML). As levels of BCR-ABL1 reflect leukemia burden, quantitation of BCR-ABL1 transcripts with reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an essential tool that defines molecular responses in CML monitoring. The prognostic significance of molecular responses has been established by large-scale clinical trials. Achieving designated levels of BCR-ABL1 on the international scale (IS) within specific time frames is an important measure for assessing patient response, probability for relapse and progression, and overall survival. Recent evidence suggests that achievement of MR4.5 at 4 years may portend a better survival than those who achieve 0.1-1% IS (MR3-MR2). With deeper responses generated by second-generation tyrosine kinase inhibitors at a higher frequency, need for detecting a very low level of minimal disease has been increasing. Herein, we evaluate the performance of Asuragen's Quantidex™ (QDX) BCR-ABL IS CMR Kit to determine if the method would meet this need.Materials and Methods: Forty-nine samples with BCR-ABL1 IS values previously determined by a clinically validated BCR-ABL1 RT-qPCR assay (Ipsogen BCR-ABL1 Mbcr Kit) were reevaluated with the Asuragen QDX BCR-ABL assay. Samples were tested in singlet by two operators over four independent runs using an ABI 7500 FAST instrument, software version V2.0.6. The auto threshold setting was used for BCR-ABL1 analysis; ABL1 analysis was performed using a manual threshold. The analytical specificity of the assay was assessed with six non-CML leukemic RNA samples from cell lines. Assay linearity was evaluated using a set of BCR-ABL1 positive cell line dilutions ranging from MR1.0-MR4.0, while precision was established testing this set of cell line dilutions an additional three times each in quadruplicate between two operators. The limit of detection of the assay was determined using four contrived, pre-diluted human-derived specimens with BCR-ABL1 values ranging from MR4.0 to MR 4.9 at 0.1 intervals.Results: There was 96% concordance between the Ipsogen and the QDX BCR-ABL assay results. Two clinical samples that were historically positive for BCR-ABL1 transcripts, and later tested negative using the Ipsogen assay were detected as weak positives by the QDX BCR-ABL assay. Repeat testing of one of the samples with the QDX BCR-ABL assay confirmed the low positive result. All non-CML samples tested negative for BCR-ABL1. One sample yielded a low positive result. Repeat testing yielded the expected negative result. Results were linear between 0.1 and 10% IS. A few outliers were detected above 10% IS, at which point, greater variation is expected. BCR-ABL1 transcripts were detectable down to MR4.9 (0.0013% IS); however, the results were not reproducible for all tested replicates. BCR-ABL1 transcripts were reproducibly detected down to MR4.5. Assay precision was high at 0.1-10%IS values (CV% of 7.5-8.7%), and decreased at %IS values greater than 10% IS and less than 0.1% IS.Conclusions: The QDX BCR-ABL IS CMR Kit is a robust and sensitive assay that generates results sufficient for studies in low-level molecular responses. DisclosuresNo relevant conflicts of interest to declare.

Abstract 570: PD-L1 expression in paired non-small cell lung cancer tumor samples

Abstract Background There is limited information on changes over time in programmed death ligand 1 (PD-L1) expression in non-small cell lung cancer (NSCLC). Methods PD-L1 expression was assessed in paired tumor samples collected at different time points from the same patient using a prototype immunohistochemistry (IHC) assay with the 22C3 antibody. PD-L1 strong and weak positivity were defined to be traceable to the 1% and 50% cutoffs used in the clinical trial version of the assay. Concordance of PD-L1 expression between samples was analyzed by treating the IHC proportional score as a continuous and categorical variable. Results 90 patients were included (73 from Korea, 17 from Denmark). 75% were male, 37% had squamous cell carcinoma, and 83% had stage I-IIIA disease. 97% of first samples and 53% of second samples were from lung. 89% and 63%, respectively, were surgical samples, with a median interval of 20.9 months (range, 0.1-94.0) between collection dates; 91% of paired samples were collected >3 months apart. A significant correlation of the PD-L1 IHC proportional score was observed for the paired samples (Pearson correlation coefficient, 0.62; P < 0.001). At the second time point, scores were identical in 39% of paired samples, higher in 32%, and lower in 29%. Of note, 12% of PD-L1-negative cases became positive and 20% of PD-L1-positive cases become negative at the second time point. The concordance rate was 56% (95% CI, 46%-67%) when PD-L1 expression was categorized as strong or weak positive or negative (Table). Conclusion Although a significant correlation of the PD-L1 IHC proportional score was observed, this preliminary analysis suggested a discordance rate of 44% between initial and subsequent NSCLC tumor samples when patients were categorized by PD-L1 expression level. Similar results were observed when analyzing paired melanoma samples. This discordance should be considered if PD-L1 expression is used for selection of enrollment in clinical trials. Table. Concordance of PD-L1 Expression in Paired NSCLC Samples (N = 90) Later Time PointPD-L1 ExpressionStrong positive, n (%)Weak positive, n (%)Negative, n (%)Earlier Time PointStrong positive3 (3)3 (3)1 (1)Weak positive7 (8)20 (22)17 (19)Negative011 (12)28 (31) Citation Format: Jhingook Kim, Steffen Filskov Sorensen, Yoon-La Choi, Zhen (Adelle) Wang, Jong-Mu Sun, Hyejoo Choi, Jeanette Baehr Georgsen, Marisa Dolled-Filhart, Kenneth Emancipator, Dianna Y. Wu, Peter Meldgaard, Wei Zhou, Henrik Hager. PD-L1 expression in paired non-small cell lung cancer tumor samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 570. doi:10.1158/1538-7445.AM2015-570

Prevalence of Rift Valley fever in domestic ruminants in the central and northern regions of Burkina Faso.

The seroprevalence of Rift Valley fever was determined in cattle, sheep and goats in selected areas of northern and central Burkina Faso. A total of 520 serum samples were screened for anti-Rift Valley fever virus immunoglobulin G (IgG) antibodies using an inhibition enzyme-linked immunosorbent assay (ELISA). An average seroprevalence of 7.67% (range 5% to 20%) was found in ruminants in Seno and Soum provinces, and prevalences of 20% and 22.5% in cattle in Yatenga and Oubritenga provinces, respectively. The location, species and age of the animals were found to influence the seroprevalence. All the ELISA IgG-positive samples were tested for IgM in a competitive ELISA and were found negative, thus ruling out recent infections. The IgG-positive samples, including weak positives, were further tested in a serum neutralisation test for neutralising antibodies and 54.5% of these samples tested positive. The results show that the virus is in circulation in central and northern regions of Burkina Faso, suggesting the need for improved surveillance and control systems to prevent future outbreaks and the consequent economic impact of the disease in Burkina Faso livestock.

Extended result reading window in lateral flow tests detecting exposure to Onchocerca volvulus: a new technology to improve epidemiological surveillance tools.

Onchocerciasis is a neglected tropical disease caused by infection with the parasite Onchocerca volvulus (Ov). An estimated 180 million people are at risk for Ov infection, and 37 million people are infected, mostly in Africa. A lateral flow-based assay to detect human IgG4 antibodies to the Ov-specific antigen Ov-16 was developed as a rapid tool to detect exposure to Ov. The test, when performed on 449 sera specimens from patients with microfiladermia and Ov-negative patients, has a sensitivity of 89.1% (95% confidence interval: 86.2%–92.0%), and specificity of 97% (95% confidence interval: 95.4%–98.6%). Because the intended use of the test is for surveillance, it is highly desirable to have a stable, long-lasting result. An extended read window is thus desirable for a high-volume, busy workflow and facilitates post-surveillance quality assurance. The main restriction on achieving an extended read window for this assay was the erythrocyte lysis that can alter the signal-to-noise ratio, especially in those with low IgG4 levels (weak positives). We describe a test housing that incorporates a user-independent feature driven by assay fluid and an expanding wick that detaches the blood separation membrane from the nitrocellulose used in the assay, but before hemolysis occurs. We demonstrated material functionality at extreme operational conditions (37°C, 80% relative humidity) and a read window of a minimum of 70 days. The fluid-driven assay device performs equally as well with whole blood as with plasma, as demonstrated with 100 spiked clinical specimens (with a correlation coefficient of 0.96). We show a novel, inexpensive, and simple approach to actuating the detachment of the blood separation membrane from the nitrocellulose test with no impact on the performance characteristics of the test.

Comparison of Cytomegalovirus (CMV) Enzyme-Linked Immunosorbent Spot and CMV Quantiferon Gamma Interferon-Releasing Assays in Assessing Risk of CMV Infection in Kidney Transplant Recipients

Assessing cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) represents an appealing strategy for identifying transplant recipients at risk of infection. In this study, we compared two gamma interferon-releasing assays (IGRAs), Quantiferon-CMV and CMV enzyme-linked immunosorbent spot (ELISPOT), to determine the ability of each test to predict protective CMV-specific T-cell responses. Two hundred twenty-one Quantiferon-CMV and ELISPOT tests were conducted on 120 adult kidney transplant recipients (KTRs), including 100 CMV-seropositive transplant recipients (R+) and 20 CMV-seronegative transplant recipients of a CMV-positive donor (D+/R-). As a control cohort, 39 healthy adult subjects (including 33 CMV-seropositive and 6 CMV-seronegative subjects) were enrolled. CMV IgG serology was used as a reference for both tests. In the CMV-seropositive individuals, the ELISPOT and Quantiferon-CMV assays provided 46% concordance with the serology, 12% discordance, 18% disagreement between ELISPOT or Quantiferon-CMV and the serology, and 24% gray areas when one or both tests resulted in weak positives. None of the CMV-seronegative subjects showed detectable responses in the ELISPOT or the Quantiferon-CMV test. In transplant recipients, both the ELISPOT and Quantiferon-CMV assays positively correlated with each other and negatively correlated with CMV DNAemia in a significant way (P<0.05). During the antiviral prophylaxis, all 20 D+/R- KTRs we examined displayed undetectable Quantiferon-CMV and ELISPOT results, and there was no evidence of CMV seroconversion. The receiving operator curve (ROC) statistical analysis revealed similar specificities and sensitivities in predicting detectable viremia (areas under the curve [AUC], 0.66 and 0.62 for Quantiferon-CMV and ELISPOT, respectively). ELISPOT and Quantiferon-CMV values of >150 spots/200,000 peripheral blood mononuclear cells (PBMCs) and >1 to 6 IU gamma interferon (IFN-γ) were associated with protection from CMV infection (odds ratios [OR], 5 and 8.75, respectively). In transplant recipients, the two tests displayed similar abilities for predicting CMV infection. Both the ELISPOT and Quantiferon-CMV assays require several ameliorations to avoid false-negative results.

Transitional Cell Carcinoma of the Ovary is Related to High-grade Serous Carcinoma and is Distinct From Malignant Brenner Tumor

Transitional cell tumors of the ovary include benign, borderline (atypically proliferating), and malignant Brenner tumors (BT), as well as transitional cell carcinoma (TCC). Some TCCs could conceivably be examples of malignant BT where the benign component has been overgrown. Our objectives were: (A) compare the immunophenotypes of BT and TCC and (B) examine a large cohort of ovarian carcinomas for cases with the immunophenotype of BT and transitional features but lacking a benign BT component. Seven BTs (3 benign, 3 borderline/atypically proliferating, 1 malignant) and 7 TCCs were stained for WT1, ER, p53, and p16(INK4a). The BTs were negative for WT1, p53 overexpression, ER (except for weak positivity in 1), and negative or weakly positive for p16(INK4a). In contrast, the TCCs stained as follows: 4/6 positive for WT1, 5/7 positive for ER, 2/7 strongly positive for p16(INK4a), and 6/7 showed abnormal p53, an immunophenotype resembling that of high-grade serous carcinoma. A database of 500 cases of ovarian carcinoma was searched and 116 showed an immunoprofile characteristic of BT: WT1 negative, ER negative, p16(INK4a) or weak positive, p53 negative (77 clear cell carcinoma, 14 endometrioid carcinoma, 12 mucinous carcinoma, 8 high-grade serous carcinoma). None of these tumors showed transitional features on review, indicating that if examples of malignant BT where there has been overgrowth of benign BT components exist, they are rare. Our results suggest that BT and TCC are unrelated, and should not be combined for classification purposes.

High-Content Micronucleus Assay in Genotoxicity Profiling: Initial-Stage Development and Some Applications in the Investigative/Lead-Finding Studies in Drug Discovery

This article describes the first step toward full (that includes conditions for both absence and presence of metabolic activation) validation and drug discovery application of a 96-well, automated, high-content micronucleus (HCMN) assay. The current validation tests were performed using Chinese hamster ovary cells, in the absence of metabolic activation, against three distinct sets of drug-like compounds that represent all stages of a drug discovery pipeline. A compound categorization scheme was created based on quantitative relationships between micronucleus (MN) signals, cytotoxicity, and compound solubility. Results from initial validation compounds (n = 38) set the stage for differentiating overall positive and negative MN inducers. To delve deeper into the compound categorization process, a more extensive validation set, consisting of a larger set (n = 370) of "drug-like but less optimized" early-stage compounds, was used for further refinement of positive and negative compound categories. The predictivity and applicability of the assay for clinical stage compounds was ascertained using (n = 168) clinically developed marketed drugs or well-studied compounds. Upon full validation, a detailed analysis of results established five compound categories--NEG (negative), NEG/xx μM (negative up to the solubility limit of xx μM), WPOS (weak positive), POS (positive), and INCON (inconclusive). Furthermore, examples of lead-finding applications and ongoing investigative HCMN activities are described. A proposal is offered on how the HCMN assay can be positioned in parallel to the overall stage gates (e.g., scaffold selection, lead optimization, late-stage preclinical development) of drug discovery programs. Because of its greater throughput, 1-week turnaround time, and a substantially reduced (1-2 mg) requirement for compound consumption, the HCMN assay is appropriate for developing structure-genotoxicity relationships and for mechanistic genotoxicity studies. The assay does not replace the Organization for Economic Cooperation and Development-compliant, non-good laboratory practice in vitro MN test (e.g., slide-based MN test in TK6 lymphoblastoid cells) that is used for full characterization of lead candidates.

Association between polymorphisms of arachidonate 12-lipoxygenase (ALOX12) and schizophrenia in a Korean population

Arachidonic acid (AA), an essential polyunsaturated fatty acid, is one of the major components of neural membranes, which show an altered phospholipid composition in schizophrenia. Arachidonate 12-lipoxygenase (ALOX12), an important enzyme, metabolizes AA to 12-HPETE, which affects catecholamine synthesis. However, research has yet to show the genetic association between ALOX12 and schizophrenia. Therefore, we investigated single nucleotide polymorphisms (SNP) of the ALOX12 gene in schizophrenia, recruiting patients with schizophrenia (n = 289) and normal controls (n = 306) from a Korean population. We selected three SNPs (rs1126667, rs434473, and rs1042357) of the ALOX12 gene and genotyped them by direct sequencing. We reviewed the schizophrenic patients' medical records and assessed them clinically using the Brief Psychiatric Rating Scale (BPRS), the Scale for the Assessment of Negative Symptoms (SANS), and the Operational Criteria Checklist (OPCRIT). Then we statistically analyzed the genetic associations between the SNPs and schizophrenia, finding a genetic association between both rs1126667 and rs1042357 and schizophrenia, in the recessive model (p = 0.015 and 0.015, respectively). We also found an association between rs434473 and negative symptoms, defined through a factor analysis of the OPCRIT data (p = 0.040). Consequently, we suggest that SNPs of the ALOX12 gene might be associated with schizophrenia and negative symptoms in this Korean population. These weak positives require additional study.

Current and Future Application of Genetic Toxicity Assays: The Role and Value of In Vitro Mammalian Assays

With the advent of new technologies (e.g., genomics, automated analyses, and in vivo monitoring), new regulations (e.g., the reduction of animal tests by the European REACH), and new approaches to toxicology (e.g., Toxicity Testing in the 21st Century, National Research Council), the field of regulatory genetic toxicology is undergoing a serious re-examination. Within this context, Toxicological Sciences has published a series of articles in its Forum Section on the theme, "Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation" (beginning with Goodman et al.). As a contribution to the Forum discussions, we present current methods for evaluating mutagenic/genotoxic risk using standard genotoxicity test batteries, and suggest ways to address and incorporate new technologies. We recognize that the occurrence of positive results in relation to cancer prediction has led to criticism of in vitro mammalian cell genetic toxicity assays. We address criticism of test results related to weak positives, associated only with considerable toxicity, only seen at high concentrations, not accompanied by positive results in the other tests of standard test batteries, and/or not correlating well with rodent carcinogenicity tests. We suggest that the problems pointed out by others with these assays already have been resolved, to a large extent, by international groups working to update assay protocols, and by changes in data interpretation at regulatory agencies. New guidances at the U.S. Environmental Protection Agency and the U.S. Food and Drug Administration improve data evaluation and help refocus risk assessment. We discuss the results of international groups working together to integrate new technologies and evaluate new tests, including human monitoring. We suggest that strategies for identifying human health risks should naturally change to integrate new technologies; however, changes should be made only when justified by strong scientific evidence of improvement in the risk assessment paradigm.

Sensitivity And Specificity Of Hiv Rapid Tests Used For Research And Voluntary Counselling And Testing

HIV rapid tests (RT) are a quick and non-technically demanding means to perform HIV voluntary counselling and testing (VCT) but understanding their limitations is vital to delivering quality VCT. To determine the sensitivity and specificity of HIV rapid tests used for research and voluntary counselling and testing at four sites in East Africa. Cross-sectional study. Masaka District, Uganda; a sugar plantation in Kakira, Uganda; Coastal Villages in the Kilifi District of Kenya; and the Urban slum of Kangemi located West of Nairobi, Kenya. Six thousands two hundred and fifty five consenting volunteers were enrolled into the study, and 675 prevalent HIV infections were identified. The RT sensitivity tended to be high for all assays at all sites (97.63-100%) with the exception of the Uni-Gold assay (90.24% in Kangemi, 96.58% in Kilifi). Twenty four RT results were recorded as 'weak positives', 22 (92%) of which were negative by ELISA. There was a high rate of RT false positives in Uganda (positive predictive values ranging from 45.70% to 86.62%). The sensitivity and specificity of the RT varied significantly across sites. The rate of RT misclassification in Uganda suggests that a multiple test algorithm may be preferable to a single test as screener for HIV VCT.

Genetic association between 5′-upstream single-nucleotide polymorphisms of PDGFRB and schizophrenia in a Korean population

PDGFRB is located on chromosome 5q31–q32, a chromosomal region identified by linkage analyses to contain a susceptibility gene for schizophrenia (SCZ). Recent research has focused on the role of the N-methyl-d-aspartate (NMDA) receptor in the pathogenesis of SCZ. D4 dopamine receptor-mediated transactivation of the gene encoding platelet-derived growth factor receptor beta (PDGFRB) has immediate effects on synaptic neurotransmission via calcium-dependent inactivation of NMDA receptors. In this study, we investigate the association between the PDGFRB gene and SCZ in a Korean population. We screened 6 single-nucleotide polymorphisms (SNPs) in the 5′-upstream region of PDGFRB and conducted a case–control study of 381 SCZ patients and 752 controls. The genotype and haplotype frequencies of 3 of the 6 SNPs [SNP1 (g.−1924T>C, rs3756314), SNP3 (g.−1772A>G, rs3756312) and SNP4 (rs3756311, g.−1658G>A)] were significantly associated with SCZ [SNP1, corrected p=0.012 (co-dominant model), 0.002 (Dominant model), and 0.506 (Recessive model); SNP3 and 4, corrected p=0.003, 0.009, and 0.049]. Haplotype analysis also revealed that ht1 (CGG) and ht2 (TAA) were significantly associated with SCZ (ht1, corrected p=0.018, 0.340, and 0.010; ht2, corrected p=0.002, 0.009, and 0.016). Transient transfection in neuronal cells revealed that ht1 had higher luciferase activity than the vector alone. Furthermore, Pdgfrb expression was increased in the frontal cortex and hippocampus in a mouse model of SCZ induced by MK801. We conclude that SNPs of the 5′-upstream region of PDGFRB are associated with SCZ in a Korean population. These are weak positives that require future studies to confirm these results.

Host range of an NPV and a GV isolated from the common cutworm, Agrotis segetum: pathogenicity within the cutworm complex

The term cutworm covers a range of species with a similar life history that can be very damaging pests on a wide range of crops. Attacks by cutworms are often made up of more than one species; thus, the most cost effective microbial control agent needs to be pathogenic for multiple species within this complex. In this study we investigate the host range of Agrotis segetum NPV and A. segetum GV for other cutworm species and closely related Noctuinae. Eight species, A. segetum, Agrotis ipsilon, Agrotis exclamationis, Agrotis puta, Noctua comes, Peridroma saucia, Xestia sexstrigata, and Xestia xanthographa, were clearly susceptible to AgseNPV, which was confirmed by DNA analysis. Aglais urticae, Diarsia rubi, Noctua pronuba, and Xestia c-nigrum were not susceptible to AgseNPV at the doses used. Noctua fimbriata, Noctua janthina, and Ochroplura plecta gave ambivalent results: larvae died of NPV infection when they were challenged with AgseNPV, but these individuals only produced weak positives in a squash blot analysis and there was insufficient DNA for confirmation by restriction endonuclease profiling. These ambivalent results could suggest either a weak infection by AgseNPV or partial homology between their own virus and AgseNPV. The untreated control insects of several species died of NPV infection, which indicates that these field-collected insects were probably carrying a vertically transmitted NPV. Fewer species were tested with AgseGV and only N. pronuba and N. comes were susceptible. N. fimbriata and Helicoverpa armigera were not susceptible to AgseGV.

Rapid serological profiling by an immunocomb-based dot-enzyme-linked immunosorbent test for three major poultry diseases.

An immunocomb-based dot-ELISA, employing specially designed apparatus, was used to measure the antibody status for the three major poultry diseases--Newcastle disease, infectious bursal disease and infectious bronchitis--in single test sera. Positive samples could be classified into strong, moderate and weak positives by comparison with the colour reaction given by known strong and weak positive serum controls. The simultaneous dot-immunobinding assay gave reproducible results and allowed considerable savings on the cost of reagents compared to liquid ELISA. The antigen-coated immunocomb can be stored under refrigeration and the test can be performed rapidly under field conditions by trained personnel.

First Report of a Member of Aster Yellows Phytoplasma Group and of Clover Proliferation Phytoplasma Group Associated with Onion in Texas.

An unknown disease(s) emerged this spring (2000) in an onion field in southwestern Texas. Infected onion plants exhibited two symptom types, one with shoot proliferation, moderate stunting of plants, and light yellowish discoloration on leaves (A) and the other with only severe stunting of the plants (B). The bulbs of the infected plants collected from both symptom types were smaller than normal. When the aerial shoots were trimmed, the infected (but not asymptomatic or the severely stunted) bulbs produced multiple slender sprouts after storage in room temperature for about a month. These symptoms are characteristic of yellows diseases caused by phytoplasmas. Ten symptomatic (six with symptom type A and four with symptom type B) and ten symptomless onion plants were collected in early May from an onion field about 1 to 2 weeks prior to blooming. Total nucleic acid was extracted from 0.5 g of shoot tissues from each sample. Nested polymerase chain reaction (PCR) using universal primer pairs (P1/P7 followed by R16F2n/R16R2) previously designed based on 16S and 23S rRNA gene sequence (1,2) was employed for the detection of phytoplasma(s) present in the samples. Specific PCR products (all were about 1.2 kb) were heavily amplified from five samples with symptom type A and one with symptom type B. Three of the symptomatic plants showing symptom type B and five of the symptomless samples were scored as weak positives. Restriction fragment length polymorphism (RFLP) analyses of the PCR products obtained from all five symptomatic samples with symptom type A using restriction enzymes including MseI, HhaI, and HpaII revealed that the associated phytoplasmas detected belonged to aster yellows phytoplasma group (16SrI), subgroup A. RFLP analyses of PCR product from the sample with symptom type B indicated that the associated phytoplasma belonged to clover proliferation group (16SrVI), subgroup A (1). Since symptom type A resembles onion yellows reported elsewhere, we propose to adopt "onion yellows" to refer to the new onion disease occurring in Texas. However, correlation between a member of clover proliferation phytoplasma group and onion plants showing severe stunting could not be firmly established. A phytoplasma belonging to 16SrI-B is associated with onion yellows disease reported in Japan (1). This is the first report that onion yellows occurs in the United States.

Relationship between microfilaria count and sensitivity of the direct smear for diagnosis of canine dirofilariosis

Direct blood smear examination (using 0.05 ml of whole blood) detected 168 (80.9%) of 204 microfilaremic canine blood samples as determined by the modified Knott test for microfilariae (mff) of Dirofilaria immitis (using 1 ml of whole blood). Direct smear examination detected all of 134 microfilaremias greater than 50 mff ml −1, but only 31 of 70 (44.3%) microfilaremias having less than 50 mff ml −1. In a separate retrospective query of a database of 963 dogs with necropsy-confirmed heartworm infections, 834 (86.6%) were positive by the DiroCHEK heartworm antigen test, and 504 (52.3%) were microfilaremic by the modified Knott test. Only 2 (0.4%) of the microfilaremic dogs were DiroCHEK negative and another 18 (3.6%) were very weak positives. Although these microfilaremic dogs were not tested by direct smear, only one of the two DiroCHEK-negative and six of 18 weakly DiroCHEK-positive dogs had microfilaremias so low that a direct smear may have given a false negative result. Significant adverse reactions to either diethylcarbamazine or the macrolide endectocides have not been reported for microfilaremias less than 500 mff ml −1, thus substitution of the direct smear for a concentration test for mff, such as the modified Knott test or membrane filtration, does not appear to increase the risk of an unexpected adverse reaction to heartworm prophylactic drugs. Such a substitution results in only a very slight decrease (on the order of 0.1%) in the overall sensitivity of heartworm screening, provided a test for mff is run concurrently with an antigen test. If a test for mff is the only screening test used, then substitution of a direct smear for a concentration test may decrease the sensitivity of heartworm screening by nearly 20%, depending on the prevalence of low level microfilaremias in the population of dogs tested.

Movement and distribution of tomato spotted wilt virus in resistant and susceptible accessions of Capsicum spp.

Resistant Capsicum chinense accessions PI-152225 and PI-159236 and the susceptible cultivar ‘Negral’ of Capsicum annuum were used in three experiments to characterize the resistance to tomato spotted wilt virus (TSWV) in these materials. In the first experiment viral movement in the whole plant was studied at two growth stages (2- and 4-leaf stage). In the second experiment the movement within the inoculated leaf was analysed at three growth stages (2-, 3-, and 4-leaf stage). Two techniques were used in this assay: double antibody sandwich – enzyme-linked immunosorbent assay (DAS–ELISA) and direct tissue blotting (DTB). In the latter experiment viral accumulation in different types of samples was evaluated by DAS–ELISA. The DTB technique showed that viral movement within the inoculated leaf is restricted in the resistant plants. The inoculated area was not totally infected in resistant accessions and slower viral movement within the inoculated area was observed. Detection of weak ELISA positives in inoculated and adjacent areas in resistant plants might not be caused by a lower viral replication, but may result from a low foliar area affected by the virus, as viral movement was restricted in certain areas around the necrotic lesions. However, ELISA absorbance values of the necrotic lesions were similar to the values of infected tissue of ‘Negral’. These observations point to a restricted viral cell-to-cell movement in the resistant accessions.

Quantitative study on expression of p16 multiple tumor suppressor gene in salivary gland neoplasm.

The expression of p16 gene in normal salivary acini, benign tumors and carcinomas were microscopically and quantitatively observed by using immunohistochemical method LSAB and CMIASWIN methods. The expression of p16 gene were found in all 3 groups. The positive unit (PU) was higher in tumor group and cancer group than that in normal group (P < 0.01). Furthermore, the PU of p16 was stronger in cytoplasm than in nucleus. Malignant tumors and acini surrounding the tumor revealed strong positives and weak positives respectively. The PU of p16 gene was higher in deep lobe of recurrent parotid neoplasm with incomplete capsule than that in shallow lobe of primary parotid neoplasm with complete capsule. The findings suggests that p16 gene plays an equally important role in the salivary gland tumors and tumors in other part of the body.