Average Cycle Threshold Value Research Articles

DROUGHT STRESS EFFECTS ON APETALA2 (AP2) GENE EXPRESSION, PHYSIOLOGICAL, AND YIELD TRAITS OF MAIZE (ZEA MAYS L.)

Maize (Zea mays L.) field experiments commenced during crop season 2022 at the Experimental Farm, Al-Mahnawiya, Extension Training Center, Babylon, Iraq. The study examined the impacts of water stress tolerance on four maize cultivars' AP2 gene expression, physiology, and yield. The experiment used a randomized complete block design (RCBD) with a split-plot arrangement and three replications. Four irrigation treatments applied on the main plots comprised 1) full irrigation (control), 2) withholding irrigation during elongation (Gs-V7), 3) non-irrigation during grain-filling (Gs-R2), and 4) suppressing irrigation during both elongation and grain-filling (Gs-V7+R2). The subplots included four maize cultivars: Furat, Dijlah, ZP, and Konsens. The results showed that withholding water during maize plants' elongation stage (GS-V7) significantly reduced leaf water and chlorophyll content. However, withholding irrigation increased peroxidase and catalase enzyme concentrations significantly. Suppressing irrigation at the elongation stage (GS-V7) and the elongation and grainfilling stages (GS-V7+R2) resulted in a considerable drop in cycle threshold (CT) values, with average CT values of 31.68 and 31.69 cycles, respectively. The AP2 gene expression and CT values indicated a negative correlation. The study found that the AP2 gene increases maize’s water stress tolerance, and the gene expression in maize cultivar "Furat" confirms this. Maize cultivar Furat achieved recommendations for cultivation in desert regions because of its superior ability to survive and adapt.

Diagnostic Agreement of SARS-CoV-2 Lateral Flow Antigen Assay with the Cycle Threshold Values of RT-PCR

COVID-19 detection via lateral flow antigen assays (LFA) are rapid and economically acquiescent to infrastructure facile healthcare settings. Early, prompt identification of cases to facilitate patient isolation and supportive management is the essence of rapid diagnostic tests. Given the backdrop of post COVID-19 pandemic-molecular testing still remains a costly affair. Additionally, molecular assays are incapable of distinguishing remnant RNA from replication competent viruses. In this scenario, we explore the diagnostic consonance of SARS-CoV-2 LFAs with RT-PCR cycle threshold, in a likelihood that it could be used as a surrogate marker for infection transmissibility. Rapid COVID-19 LFA results were compared with Real-time PCR for detection of SARS-CoV-2 in nasopharyngeal swabs. Two hundred rapid antigen positive nasopharyngeal swabs obtained from COVID-19 suspects/contacts/preoperative/screening patients were subjected to RT-PCR to study the correlation with cycle threshold (CT) values obtained for all the antigen positive cases. 200 Rapid COVID-19 LFA positive samples were analyzed in the present study. Amidst the LFA positive samples included in the study 187 (93.5%) were found to have concordant results when subjected to the gold standard Real-time PCR. Discordant results were documented in 13 (6.5%) COVID-19 LFA positive samples which were found to be negative by RT-PCR. The average Cycle threshold values were found to be 23.75 for E gene, 25.36 for N gene and 24.07 for RdRp gene. The average PCR Cycle threshold of LFA positive cases remained significantly undeterred (p<0.5) throughout the time period of the study stipulating the undaunted viral load across the different waves of the pandemic. Maximum association of LFA positivity with symptom-manifestation was seen during the 1st wave of COVID-19 (September-December 2020 in India). The association of symptoms with LFA test positivity reduced to a significant extent during the 3rd wave of the pandemic in January 2022 (p<0.5) indicating the reduced clinical severity but not infectivity of the SARS-CoV-2 infection during the 3rd wave of the pandemic. Lateral flow assay based diagnostic tests are technically & economically convenient modalities with significant interest concordance in comparison with RT-PCR. Definitive advantage in terms of achieving quick patient triage and thereby patient management can be achieved with the use of these tests.

Evaluation of Four Point of Care (POC) Antigen Assays for the Detection of the SARS-CoV-2 Variant Omicron.

ABSTRACTEnsuring SARS-CoV-2 diagnostics that can reliably detect emerging variants has been an ongoing challenge. Due to the rapid spread of the Omicron variant, point-of-care (POC) antigen tests have become more widely used. This study aimed at (i) comparing the analytical sensitivity (LOD) of 4 POC antigen assays, BD Veritor, Abbott BinaxNow, Orasure InteliSwab and Quidel QuickVue, for the Omicron versus the Delta variant and (ii) verifying the reproducible detection of Omicron by the 4 antigen assays. The LOD for all four assays were evaluated using Omicron and Delta virus stocks quantified for infectivity and genome copies. The four assays detected all replicates of Omicron and Delta dilutions at 104 and 105 TCID50/mL, respectively. We quantified both viral stocks using droplet digital PCR (ddPCR), which revealed that the Omicron stock had equivalent copies of the N gene to Delta at a one log lower infectious virus. The Abbott BinaxNow and Orasure InteliSwab had the highest analytical sensitivity for Omicron while the Orasure InteliSwab and the Quidel QuickVue had the highest analytical sensitivity for Delta. When 14 SARS-CoV-2 real-time PCR positive nasal/nasopharyngeal swab samples (12 Omicron and 2 Delta, mean Ct = 19.1), were tested by the four assays, only the QuickVue detected all samples. Antigen test positivity correlated with recovery of infectious virus on cell culture in 9 out of 13 tested specimens from symptomatic, asymptomatic, unvaccinated, and vaccinated individuals. Although our study confirms the reduced analytical sensitivity of antigen testing compared to molecular methods, the Omicron variant was detectable by the four evaluated rapid antigen tests.IMPORTANCE In the manuscript, we report an evaluation of the capability of 4 point of care (POC) antigen assays, the BD Veritor, Abbott BinaxNow, Orasure InteliSwab and Quidel QuickVue to detect the Omicron variant of SARS-CoV-2, and we compared their analytical sensitivity for Omicron versus Delta. In this analysis we found that all four assays detected Omicron and Delta at 104 and 105 TCID50/mL, respectively. We further quantified the viral stocks used by droplet digital (ddPCR) and found that the Omicron stock had equivalent copies of the N gene to Delta at a one log lower infectious virus titer and that an increased RNA to infectious virus ratio may be contributing to discrepancies in limit of detection in Omicron compared to Delta. We evaluated 14 SARS-CoV-2 real-time PCR positive nasal/nasopharyngeal swab samples (12 Omicron and 2 Delta), with an average cycle threshold value of 19.1, and only the QuickVue showed 100% agreement.

Persistent abdominal symptoms in returning travellers: clinical and molecular findings.

Persistent abdominal symptoms (PAS) are the leading cause of post-travel morbidity although there is a paucity of evidence concerning the aetiology of this condition. Recently molecular methods for protozoa detection in stool have been introduced. Herein, we describe the clinical aspects and the prevalence of gastrointestinal protozoa in returning travellers with PAS. From 2017 to 2019, clinical information and stool specimens from returning travellers with PAS were analysed for the presence of parasites using the Allplex-GI-Parasite-assay. Stool findings from symptomatic patients without a travel history were used as a comparator. During the 2-year study, 203 stool specimens from returning travellers were analysed. The median duration of symptoms before seeking care was 6months, the most common symptoms were fatigue (79.2%), abdominal pain (75.7%) and loose stool (70.8%).Most of travellers had returned from Asia (57.6%), mainly from the Indian-subcontinent and only 52.6% were backpackers. Altogether, 36.9% samples were positive for protozoa, with Blastocystis hominis being the most common (26.6%) in samples, followed by Dientamoeba fragilis (18.7%), Giardia lamblia (3.0%) and Cryptosporidium spp (0.5%). The former two were dominant in all regions. In all cases but one, G. lamblia was acquired, but one were acquired in the Indian subcontinent (odds ratios 16.9; 95% confidence intervals: 1.9-148.3). Entamoeba histolytica was not detected. The demographic characterization of the 1359 non-travellers was comparable with the travellers. Among them D. fragilis was the most common followed by B. hominis, which was significantly less frequent compared among the travellers (16.7% vs 26.6%, P < 0.001). Average Cycle threshold values for each stool parasites were comparable between the two groups. Among returning travellers with PAS, more than one-third were positive for gastrointestinal protozoa. A low rate of giardia was found and no E. histolytica while B. hominis followed by D. fragilis were the dominant findings. Further studies are required to better understand the role of these protozoa in PAS.

Urgent care study of the LumiraDx SARS-CoV-2 Ag Test for rapid diagnosis of COVID-19

BackgroundTesting individuals suspected of severe acute respiratory syndrome–like coronavirus 2 (SARS-CoV-2) infection is essential to reduce the spread of disease. The purpose of this retrospective study was to determine the false negativity rate of the LumiraDx SARS-CoV-2 Ag Test when utilized for testing individuals suspected of SARS-CoV-2 infection.MethodsConcurrent swab samples were collected from patients suspected of SARS-CoV-2 infection by their healthcare provider within two different urgent care centers located in Easton, MA, USA and East Bridgewater, MA, USA. One swab was tested using the LumiraDx SARS-CoV-2 Ag Test. Negative results in patients considered at moderate to high risk of SARS-CoV-2 infection were confirmed at a regional reference laboratory by polymerase chain reaction (PCR) using the additional swab sample. The data included in this study was collected retrospectively as an analysis of routine clinical practice.ResultsFrom October 19, 2020 to January 3, 2021, a total of 2241 tests were performed using the LumiraDx SARS-CoV-2 Ag Test, with 549 (24.5%) testing positive and 1692 (75.5%) testing negative. A subset (800) of the samples rendering a negative LumiraDx SARS-CoV-2 Ag Test was also tested using a PCR-based test for SARS-CoV-2. Of this subset, 770 (96.3%) tested negative, and 30 (3.8%) tested positive. Negative results obtained with the LumiraDx SARS-CoV-2 Ag test demonstrated 96.3% agreement with PCR-based tests (CI 95%, 94.7–97.4%). A cycle threshold (CT) was available for 17 of the 30 specimens that yielded discordant results, with an average CT value of 31.2, an SD of 3.0, and a range of 25.2–36.3. CT was > 30.0 in 11/17 specimens (64.7%).ConclusionsThis study demonstrates that the LumiraDx SARS-CoV-2 Ag Test had a low false-negative rate of 3.8% when used in a community-based setting.

May viral load detected in saliva in the early stages of infection be a prognostic indicator in COVID-19 patients?

ObjectiveThis study aimed to investigate the prognostic value of viral load detected in the saliva of COVID-19 patients in the early stage of infection. Study designOro-nasopharyngeal swab and saliva samples were collected from all patients simultaneously in the early stage of COVID-19. Viral loads were determined by extracting viral RNAs from saliva samples of patients whose ONP swabs were positive for SARS-CoV-2 by RT-qPCR. The demographic information, comorbidities, cycle threshold values, and one-month clinical courses were recorded and compared. ResultsThe patients' clinical course was evaluated for one month; 56 % of patients had mild disease, 26.4 % had moderate disease, 9.6 % had severe disease, and 8% had a critical/mortal disease. The average cycle threshold values of SARS-CoV-2 in saliva and ONP samples were measured as 22.28 and 24.19, respectively. Cycle threshold value of saliva was found to be significant in predicting disease severity (Eta coefficient 0.979). A statistically significant relationship was found between the disease's severity and the mean of ONP samples’ Ct-values (p < 0.05). Gender, age, body mass index, and co-morbidities were compared with the severity of the disease; no statistically significant difference was found. ConclusionViral load detected in saliva in the early period of COVID-19 infection may have a prognostic value in showing the disease's course in patients over 45-year-old. Saliva is an easily obtainable, reliable material for COVID-19 screening.

Non-Coding RNA Extraction from Nucleus Pulposus of Intervertebral Discs with Subsequent Expression Profiling

Background. With numerous extraction protocols for total RNA and RNA fractions, like microRNA (miRNA) and long non-coding RNA (lncRNA), available for various cell and tissue types, obtaining a high quantitative and qualitative yield from some special material, such as the nucleus pulposus, remains challenging. Nucleus pulposus is troublesome to manage in common RNA isolation protocols due to low cell content and high biopolymer concentrations, including proteoglycans and glycoproteins, which impair overall purity and yield. A major lack of reproducible methods for total and fraction RNA isolation directly from the nucleus pulposus impedes effective real-time PCR applications for downstream miRNA and lncRNA expression profiling in the course of intervertebral disc degeneration. In this study, we exploit the collagenase type II lytic properties to facilitate extraction of total and fraction RNA from the nucleus pulposus and compare results with the standard RNA isolation method.Materials and methods. Nucleus pulposus samples (n = 8) were obtained from September 2017 to December 2018 from patients with herniated discs in the lumbosacral spine diagnosed during surgery. Equal portions of samples were processed with the standard and original RNA isolation protocols.Results and discussion. Th e enzymatic lysis method for total and fraction RNA isolation from the nucleus pulposus of intervertebral discs demonstrated excellent integrity and high purity. No protein, polysaccharide or collagen contamination was detected.Conclusion. Th e method reported allows an improved quantitative and qualitative total and fraction RNA yield from the nucleus pulposus of intervertebral discs. Th e method can be used in future research on miRNA and lncRNA expression profiling with real-time PCR by improving the average cycle threshold value.

Evaluation of Three Real-Time PCR Methods for Detection of Salmonella from Cloves

The purpose of the study was to evaluate three real-time PCR platforms for rapid detection of Salmonella from cloves and to compare three different DNA extraction methods. Six trials were conducted with two clove cultivars, Ceylon and Madagascar, and three Salmonella serotypes, Montevideo, Typhimurium, and Weltevreden. Each trial consisted of 20 test portions. The preenrichment cultures were used to perform PCR for comparison of the effectiveness of U.S. Food and Drug Administration, Pacific Regional Laboratory Southwest (FDA-PRLSW), Applied Biosystems Inc. (ABI) MicroSEQ, and GeneDisc platforms for detection of Salmonella. Three DNA extraction methods were used: standard extraction method for each PCR platform, boil preparation, and LyseNow food pathogen DNA extraction cards. The results from real-time PCR correlated well with FDA Bacteriological Analytical Manual culture assay results, with a wide range of cycle threshold (CT) values among the three PCR platforms for intended positive samples. The mean CT values for MicroSEQ (16.36 ± 2.78) were significantly lower than for PRLSW (20.37 ± 3.45) and GeneDisc (23.88 ± 2.90) (P < 0.0001). Pairwise comparisons between PCR platforms using different DNA extraction methods indicate that the CT values are inversely proportional to the relative DNA quantity (RDQ) yields by different platform-extraction combinations. The pairing of MicroSEQ and boil preparation generated the highest RDQ of 120 and the lowest average CT value of 14.48, whereas the pairing of GeneDisc and LyseNow generated the lowest RDQ of 0.18 and the highest average CT of 25.97. Boil preparation yielded higher RDQ than the other extraction methods for all three PCR platforms. Although the MicroSEQ platform generated the lowest CT values, its sensitivity was compromised by narrow separations between the positive and negative samples. The PRLSW platform generated the best segregation between positive and negative groups and is less likely to produce false results. In conclusion, FDA-PRLSW was the most efficient PCR assay for Salmonella detection, and boil preparation was the best method for DNA extraction.

Fluorescent quantitative PCR detection of Mycobacterium tuberculosis in tissue sections from granulomatous lesions retrieved using EDTA

AimsThis study aimed to use EDTA to retrieve paraffin-embedded tissue sections of inflammatory granulomatous lesions and increase the detection rate of tuberculosis (TB)/non-tuberculous mycobacteria. Due to the influence of chemical...

Abstract 18427: Impact of Medication on Quantification of Plasma MicroRNAs in Patients With Cardiovascular Disease

Background: MicroRNAs are small non-coding RNAs that are detectable in the blood. Circulating levels of microRNAs have been measured in various epidemiological studies, but the effects of medication are unclear. To address these uncertainties, we assessed how plasma microRNAs change in response to platelet inhibition and heparin administration. We also correlated plasma levels of microRNAs with existing markers of platelet activation. Methods and Results: 92 microRNAs were assessed in a dose-escalation study with aspirin and prasugrel in healthy volunteers (n=11). Plasma levels of platelet microRNAs, such as miR-223, miR-191 and others decreased on platelet inhibition. Findings were confirmed by individual TaqMan qPCR assays and validated in an independent cohort of patients with symptomatic carotid stenosis (n=33). In a cohort of patients on dual anti-platelet therapy 30 days post myocardial infarction (n=129), plasma levels of most platelet microRNAs were positively correlated to the VASP assay. Levels of miR-126 were also significantly associated with results from the VerifyNow P2Y12 aggregation assay. No association was observed with optical aggregometry with arachidonic acid or ADP. When platelet-poor plasma was collected at different time points from patients undergoing PCI (n=20) or diagnostic angiography with (n=7) and without (n=10) heparin administration, heparin was found to have pronounced effects on the assessment of the exogenous C. elegans spike-in control (decrease by &gt;3 cycles), which disappeared six hours after the heparin bolus. Normalisation of individual microRNAs with the average cycle threshold value of all microRNAs provided a suitable alternative to normalization with exogenous C. elegans spike-in control in this setting. Conclusions: Our studies provide evidence for a correlation of plasma microRNAs with commonly used methods in platelet testing. They also highlight that microRNA levels are sensitive to medication, in particular antiplatelet and anticoagulation therapy. This has to be taken into account when designing studies to investigate the relation of circulating microRNAs to cardiovascular disease.

Impact of intravenous heparin on quantification of circulating microRNAs in patients with coronary artery disease.

MicroRNAs are small non-coding RNAs that are detectable in plasma and serum. Circulating levels of microRNAs have been measured in various studies related to cardiovascular disease. Heparin is a potential confounder of microRNA measurements due to its known interference with polymerase chain reactions. In this study, platelet-poor plasma was obtained from patients undergoing cardiac catheterisation for diagnostic coronary angiography, or for percutaneous coronary intervention, both before and after heparin administration. Heparin had pronounced effects on the assessment of the exogenous C. elegans spike-in control (decrease by approx. 3 cycles), which disappeared 6 hours after the heparin bolus. Measurements of endogenous microRNAs were less sensitive to heparin medication. Normalisation of individual microRNAs with the average cycle threshold value of all microRNAs provided a suitable alternative to normalisation with exogenous C. elegans spike-in control in this setting. Thus, both the timing of blood sampling relative to heparin dosing and the normalisation procedure are critical for reliable microRNA measurements in patients receiving intravenous heparin. This has to be taken into account when designing studies to investigate the relation of circulating microRNAs to acute cardiovascular events or coronary intervention.

Real-Time Pcr/rflp Assay to Detect Giardia intestinalis Genotypes in Human Isolates with Diarrhea in Egypt

Two major genotypic assemblages of Giardia intestinalis infect humans; the nested real-time polymerase chain reaction (PCR) was used for targeting the triose phosphate isomerase (tpi) gene to detect and genotype G. intestinalis in human feces in Egypt. Among 97 fecal samples, 30 (31%) were diagnosed as giardiasis by saline wet mount microscopy after staining with Lugol's iodine. The tpi gene was amplified from 41 (42.3%) fecal samples, of which 11 were microscopy-negative specimens. Of the total samples, 24 (58.5%) contained assemblage A group I, and 7 (17.1%) were assemblage A group II from the group of patients complaining of intermittent diarrhea. Eight (19.5%) samples contained assemblage B from patients with persistent diarrhea. Two (5%) samples had a mixture of assemblage A group II and assemblage B. The technique was able to detect as few as 20 trophozoites per PCR on fecal DNA-isolated, microscopy-negative, and quantitative (q)PCR-positive specimens; there was a higher average cycle threshold value than microscopy-positive and qPCR-positive specimens, suggesting that they represented true, low-burden infections. In conclusion, we could genotype G. intestinalis from fresh stool samples in Egypt; in infections commonly presented with intermittent diarrhea, the most prevalent genotype was assemblage A group I. The most vulnerable age group included 10- to 20-yr-old individuals.

Cell Death Regulatory Gene Expression Correlates with MLL Rearrangement Status and Prognostic Clinical Covariates in Acute Leukemia in Infants.

Infants with acute leukemia, especially neonates, comprise a unique high-risk population prone to chemotherapy resistance and treatment complications. Basal levels of expression of cell death and cell survival factors vary greatly in different types of cancer and are key determinants of response to treatment. Until now, expression of these factors had not been evaluated in detail in acute leukemia of infants. In this study the basal expression of pro-death and pro-survival genes in the intrinsic apoptosis pathway, apoptosis execution genes and mitochondrial maintenance genes was quantified in primary leukemia cells in a large cohort of infants, and relationships between gene expression and MLL status and clinical covariates were analyzed.Methods: Bone marrow or peripheral blood samples from the time of diagnosis were examined in 103 infants (age 1–363 d, median 177 d) with acute leukemia (63 ALL, 40 AML). White blood cell (WBC) counts ranged from 4.4–1676×103/μL (median 127.6×103/μL). MLL status was characterized by standard cytogenetics, FISH, Southern blot analysis, reverse transcriptase PCR and/or panhandle PCR approaches. The ALL cases were classified as MLL-AF4+ (n= 31), other MLL rearrangement positive (other MLL+) (n= 22) or MLL rearrangement negative (MLL−) (n= 10). Due to heterogeneity in partner genes, the AML cases were classified as MLL+ (n= 22) or MLL− (n= 18). Quantitative real time PCR analysis of total RNAs was performed using a custom TaqMan® low-density array with one or more assays, each in quadruplicate, for 35 cell death regulatory genes and 3 housekeeping genes, and ΔCT values (average cycle threshold values normalized to 18S rRNA) were computed. The Behrens-Fisher nonparametric multiple comparison test in the npmc package for R was used to determine the significance of the difference in median −ΔCT values among the three ALL groups and between the two AML groups (p<0.001 considered as significant). Relationships were sought between −ΔCT values and clinical covariates (age, sex, WBC count, CNS status, events) by computing Spearman correlation coefficients and their levels of significance.Results: In ALL, expression of the following genes was greater in MLL-AF4+ than MLL− cases: anti-apoptotic BCL-2 and MCL-1; pro-apoptotic BAX and BID; apoptosis execution APAF1, CASP3 and CASP9; and mitochondrial maintenance COX5A, CYC1, UQCR1 and UQCR. For some of these genes, expression was also greater in other MLL+ than MLL− cases. In AML, expression of pro-apoptotic BAK and BIK was higher in MLL+ than MLL− cases. For the sample set as a whole, expression of the following genes was positively correlated with WBC count (r>0.3; p<0.01): anti-apoptotic BCL-2 and BCL-W; pro-apoptotic NOXA; apoptosis execution CASP8; and mitochondrial maintenance HCCS. The correlation between WBC count and BCL-2 was the strongest (r=0.40;p=4E-5). Only the expression of pro-apoptotic BIM was correlated with age at diagnosis and the correlation was not particularly strong (r = −0.246;p=0.01).Conclusions: These results suggest that infant ALL and AML differ with respect to the expression of genes that regulate cell death. Furthermore, cell death regulatory gene expression is affected more by MLL translocations in infant ALL than infant AML. This finding may explain the greater impact of MLL translocations on prognosis in infant ALL than AML. The abundance of specific pro- as well as anti-apoptotic BCL-2 family mRNAs in MLL+ infant ALL is also relevant to the identification of novel therapeutic targets in this pathway. The positive correlation between anti-apoptotic BCL-2 expression and WBC count is biologically plausible and consistent with the aggressive nature of acute leukemia in infants.

Multiplex real-time PCR assay using Scorpion probes and DNA capture for genotype-specific detection of Giardia lamblia on fecal samples.

Two major genotypic assemblages of Giardia lamblia infect humans; the epidemiologic significance of this phenomenon is poorly understood. We developed a single-vessel multiplex real-time PCR (qPCR) assay that genotypes Giardia infections into assemblages A and/or B directly from fecal samples. The assay utilized Scorpion probes that combined genotype-specific primers and probes for the 18S rRNA gene into the same molecule. The protocol was capable of detecting as few as 20 trophozoites per PCR on fecal DNA isolated using a commercial method or 1.25 trophozoites per PCR on fecal DNA isolated using a G. lamblia-specific oligonucleotide capture technique. The assay was specific for fecal specimens, with no amplification of the discordant genotype with the opposite Scorpion probe. When 97 clinical specimens from Bangladesh were used, the multiplex PCR assay detected 95% (21 of 22) of Giardia microscopy-positive specimens and 18% (13 of 74) of microscopy-negative specimens. Microscopy-negative and qPCR-positive specimens had higher average cycle threshold values than microscopy-positive and qPCR-positive specimens, suggesting that they represented true low-burden infections. Most (32 of 35) infections were assemblage B infections. This single-reaction multiplex qPCR assay distinguishes assemblage A Giardia infections from assemblage B infections directly on fecal samples and may aid epidemiologic investigation.