Multicenter Evaluation of the Clinical Performance and the Neutralizing Antibody Activity Prediction Properties of 10 High-Throughput Serological Assays Used in Clinical Laboratories.

As the coronavirus disease 2019 (COVID-19) pandemic second wave is emerging, it is of the upmost importance to screen the population immunity in order to keep track of infected individuals. Consequently, immunoassays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and positive predictive values are needed to obtain an accurate epidemiological picture. As more data accumulate about the immune responses and the kinetics of neutralizing-antibody (nAb) production in SARS-CoV-2-infected individuals, new applications are forecast for serological assays such as nAb activity prediction in convalescent-phase plasma from recovered patients. This multicenter study, involving six hospital centers, determined the baseline clinical performances, reproducibility, and nAb level correlations of 10 commercially available immunoassays. In addition, three lateral-flow chromatography assays were evaluated, as these devices can be used in logistically challenged areas. All assays were evaluated using the same patient panels in duplicate, thus enabling accurate comparison of the tests. Seven immunoassays examined in this study were shown to have excellent specificity (98 to 100%) and good to excellent positive predictive values (82 to 100%) when used in a low (5%)-seroprevalence setting. We observed sensitivities as low as 74% and as high as 95% at ≥15 days after symptom onset. The determination of optimized cutoff values through receiver operating characteristic (ROC) curve analyses had a significant impact on the diagnostic resolution of several enzyme immunoassays by increasing the sensitivity significantly without a large trade-off in specificity. We found that spike-based immunoassays seem to be better correlates of nAb activity. Finally, the results reported here will add to the general knowledge of the interlaboratory reproducibility of clinical performance parameters of immunoassays and provide new evidence about nAb activity prediction.

Differential expression of p16(INK4A) and cyclin D1 in benign and malignant salivary gland tumors: a study of 44 Cases.

Salivary gland tumors (SGT) are a heterogeneous group of lesions. There is conflicting data concerning the molecular events involving the tumour suppressor retinoblastoma protein (pRb) pathway in these tumors. Few studies examined the alterations in components of the Rb pathway by immunohistochemical (IHC) methods in benign and malignant SGTs. Furthermore, recent evidence implicates human papillomavirus (HPV) in mucoepidermoid carcinoma (MEC) carcinogenesis. The purpose of our study is to examine p16(INK4A) and cyclin D1 expression in a variety of benign and malignant salivary gland tumors, and to investigate p16(INK4A) expression as a surrogate marker for HPV infection in MEC. Our series includes 30 malignant tumors [14 MEC, 6 acinic cell carcinomas (ACC), 5 polymorphous low grade adenocarcinomas (PLGA), 5 (AdCC)] and 14 benign tumors (4 benign cysts, 5 Warthin tumors and 5 pleomorphic adenomas (PA). All cases were tested by IHC for p16(INK4A) and cyclin D1. Testing for HPV wide spectrum (HPV-WS) was performed by in situ hybridization in all MEC cases. Staining intensity was recorded semi quantitatively (on a scale from 0 to 4+). Fisher's exact test and Pearson X2 test with a p < 0.05 were used. Cyclin D1 and p16(INK4A) are expressed similarly in malignant and benign tumors (p = 0.146 and p = 0.543, respectively). None of the MEC cases showed nuclear reactivity for HPV-WS. Statistical analysis showed positive correlation between cyclin D1 and p16(INK4A) expression. Our findings suggest that p16(INK4A) overexpression is likely secondary to cyclin D1 gene upregulation or amplification. Further molecular studies are warranted.

Final report of a phase II evaluation of paclitaxel in patients with advanced squamous cell carcinoma of the head and neck: an Eastern Cooperative Oncology Group trial (PA390).

BACKGROUND: A number of single agents have been tested in patients with carcinoma of the head and neck receiving palliative treatment. In general, 15-30% of patients achieve a partial response lasting 3-4 months. Treatment has not been shown to alter survival rates. It is clear that new drugs with potentially greater activity need to be identified. For this purpose, the Eastern Cooperative Oncology Group conducted a Phase II evaluation of paclitaxel. METHODS: Patients with recurrent, metastatic, or locally advanced, incurable squamous cell carcinoma of the head and neck were eligible. The dose and schedule tested was the maximum tolerable dose of 250 mg/m2 determined from Phase I trials using a 24-hour infusion schedule and granulocyte-colony stimulating factor support. Courses were given at 3-week intervals until progression of disease was documented. Dose modifications were specified for hematologic toxicity and for neurotoxicity. RESULTS: Thirty-four patients were registered on study and 30 were eligible. Severe or life-threatening granulocytopenia was the most frequent toxicity observed, occurring in 91% of patients. Prior to response evaluation, one patient died of sepsis and one died of a myocardial infarct. Response was observed in 40% of eligible patients (4 complete and 8 partial responses). The median duration of response was 4.5 months (range, 2-20 months), with a median survival of 9.2 months and a 1-year survival rate of 33%. CONCLUSIONS: These results indicate that paclitaxel is an active agent for the treatment of squamous cell carcinoma of the head and neck. Studies evaluating alternative infusion schedules and combination regimens currently are underway.

Nucleotide sequence and strand displacement amplification of the 70K protein gene from mycobacteria.

We isolated and determined the nucleotide sequence of the 70K gene from nine mycobacteria and from two related non-mycobacteria with the goal of obtaining a region of requisite specificity to serve as a mycobacterial genus-specific probe. Two different primer sets were then designed to amplify the 70K gene using strand displacement amplification. Using one of the primer sets, 10 different mycobacteria were readily detected with sensitivities of 100 molecules DNA, and with only cross-reactivity to two non-mycobacteria. The other set of primers that were tested amplified the same set of mycobacteria, but exhibited no crossreactivity with non-mycobacterial DNAs. By employing one of the primer sets, we were able to successfully amplify with high sensitivity three different target DNA sequences comprised of the 70K mycobacterial genus target, an IS 6110 (M. tuberculosis complex) target, and an internal amplification control using SDA. These results demonstrate the potential of the 70K gene to serve as a mycobacterial genus-specific probe, and demonstrate the first multiplex amplification by SDA of three DNA targets.

Multiplex strand displacement amplification (SDA) and detection of DNA sequences from Mycobacterium tuberculosis and other mycobacteria.

Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying a DNA target sequence prior to detection [Walker et al (1992) Nucleic Acids Res., 20, 1691-1693]. Here we describe a multiplex form of SDA that allows two target sequences and an internal amplification control to be co-amplified by a single pair of primers after common priming sequences are spontaneously appended to the ends of target fragments. Multiplex SDA operates at a single temperature, under the same simple protocol previously developed for single-target SDA. We applied multiplex SDA to co-amplification of a target sequence (IS6110) that is specific to members of the Mycobacterium tuberculosis-complex and a target (16S ribosomal gene) that is common to most clinically relevant species of mycobacteria. Both targets are amplified 10(8)-fold during a 2 hour, single temperature incubation. The relative sensitivity of the system was evaluated across a number of clinically relevant mycobacteria and checked for crossreactivity against organisms that are closely related to mycobacteria.

Chemiluminescent detection of strand displacement amplified DNA from species comprising the Mycobacterium tuberculosis complex.

Strand displacement amplification, a new isothermal in vitro DNA amplification technique, was used to amplify target DNA contained within the IS6110 insertion element of the species within the Mycobacterium complex (Mycobacterium tuberculosis, M. bovis, M. bovis-BCG, M. africanum and M. microti). The target nucleic acid sequence is present in approximately ten, two, one, five and five copies in M. tuberculosis, M. bovis, M. bovis-BCG, M. africanum and M. microti, respectively. Amplified products were detected using a non-isotopic microtitre plate assay employing a biotinylated oligodeoxynucleotide probe and an alkaline phosphatase conjugated oligodeoxynucleotide probe. Lumiphos 530 was the chemiluminescent substrate for alkaline phosphatase. The combination of the strand displacement amplification method with this sensitive and rapid (less than 2 h) detection system resulted in the specific detection of as few as 1-25 initial IS6110 targets in the five Mycobacterium complex species based on signal/noise criteria. Negative results were obtained with eight other Mycobacterium species as well as with 32 non-Mycobacterium species.

Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system.

An isothermal in vitro DNA amplification method was developed based upon the following sequence of reaction events. Restriction enzyme cleavage and subsequent heat denaturation of a DNA sample generates two single-stranded target DNA fragments (T1 and T2). Present in excess are two DNA amplification primers (P1 and P2). The 3' end of P1 binds to the 3' end of T1, forming a duplex with 5' overhangs. Likewise, P2 binds to T2. The 5' overhangs of P1 and P2 contain a recognition sequence (5'-GTTGAC-3') for the restriction enzyme HincII. An exonuclease-deficient form of the large fragment of Escherichia coli DNA polymerase I (exo- Klenow polymerase) [Derbyshire, V., Freemont, P. S., Sanderson, M. R., Beese, L., Friedman, J. M., Joyce, C. M. & Steitz, T. A. (1988) Science 240, 199-201] extends the 3' ends of the duplexes using dGTP, dCTP, TTP, and deoxyadenosine 5'-[alpha-thio]triphosphate, which produces hemiphosphorothioate recognition sites on P1.T1 and P2.T2. HincII nicks the unprotected primer strands of the hemiphosphorothioate recognition sites, leaving intact the modified complementary strands. The exo- Klenow polymerase extends the 3' end at the nick on P1.T1 and displaces the downstream strand that is functionally equivalent to T2. Likewise, extension at the nick on P2.T2 results in displacement of a downstream strand functionally equivalent to T1. Nicking and polymerization/displacement steps cycle continuously on P1.T1 and P2.T2 because extension at a nick regenerates a nickable HincII recognition site. Target amplification is exponential because strands displaced from P1.T1 serve as targets for P2 and strands displaced from P2.T2 serve as targets for P1. A 10(6)-fold amplification of a genomic sequence from Mycobacterium tuberculosis or Mycobacterium bovis was achieved in 4 h at 37 degrees C.

Concurrent neuroborreliosis and Alzheimer's disease: analysis of the evidence.

Several recent reports have claimed a possible association between Borrelia burgdorferi infection and Alzheimer's disease (AD). Herein, we describe our search for additional evidence of neuroborreliosis in AD. Brain tissue from neuropathologically confirmed cases of AD was cultured for B burgdorferi using standard microbiologic methods. Material derived from culture was further examined using electron microscopy, direct immunofluorescence and acridine orange fluorescence. Previous studies have shown high titers of antiborrelia antibodies in CSF in all cases of confirmed neuroborreliosis; therefore, we tested CSF from neuropathologically confirmed cases of AD by indirect immunofluorescence and enzyme-linked immunoassay. In addition, imprint preparations from AD and control brain tissues were studied by direct immunofluorescence using a monoclonal antiborrelia antibody. Finally, a Western blot method was used to analyze protein extracts from cultures and AD brain tissue for the presence of borrelia antigen. Contrary to previous studies, our results do not support an association between infection with B burgdorferi and AD.

Elaborative inferences during reading: do they occur on-line?

Four experiments were conducted to examine the extent to which readers construct elaborative inferences on-line during reading. In Experiment 1, gaze durations were measured while subjects read anaphors to target antecedents that referenced a particular category member either explicitly or implicitly. When the context strongly suggested a particular category member, gaze durations on an anaphor were the same following either an implicit or an explicit antecedent. When the context did not suggest any particular category member, gaze durations were significantly longer following an implicit antecedent. The results confirmed that, with sufficient context, readers will generate a simple elaborative inference on-line. These results were replicated in Experiment 2 in which the materials did not strongly signal the inference but a sentence designed to encourage subjects to infer was included. In Experiment 3, this "demand sentence" was not included, and readers did not appear to construct the targeted inference. The results of Experiment 4 confirmed that once generated, elaborative inferences are stored as part of the long-term-memory representation of a passage.

Colloid (hyaline) inclusion bodies in the central nervous system: their presence in the substantia nigra is diagnostic of Parkinson's disease.

Intracytoplasmic "colloid" inclusions have been described within neurons of several discrete central nervous system nuclei in a variety of entities. Although they lack specificity for any particular disease, they are believed to represent one of the morphologic changes of neuronal aging. Because premature aging of the substantia nigra has been one of the claimed mechanisms occurring in Parkinson's disease, the prevalence of colloid inclusions was studied within the substantia nigra in 15 patients with Parkinson's disease, 15 age-matched controls, 50 "normal" individuals, 10 patients with dementia of Alzheimer's type, and two patients with amyotrophic lateral sclerosis. Colloid bodies were found in the substantia nigra of all patients with Parkinson's disease and were virtually absent in the other populations. Histochemical and ultrastructural analyses showed that colloid bodies differ from early and mature Lewy bodies. They may represent the "pale" inclusions rarely mentioned in the literature and often mistaken for early Lewy bodies. "Colloid" bodies in the substantia nigra are diagnostic of Parkinson's disease. These findings support the theory of "premature" aging of the substantia nigra in this disease.