PCR In Samples Research Articles

Evaluation of metagenomic next-generation sequencing (mNGS) combined with quantitative PCR: cutting-edge methods for rapid diagnosis of non-invasive fungal rhinosinusitis.

Fungal rhinosinusitis is a significant and growing health concern in arid regions, with an increasing incidence over recent decades. Without timely and appropriate management, it can lead to severe complications, including potential intracranial spread. This study aims to establish efficient and rapid diagnostics for non-invasive fungal rhinosinusitis (FRS), addressing the challenge of its difficult-to-culture diagnosis. Twenty-eight patients suspected of FRS were studied using endoscopic sinus surgery to obtain tissue samples for histopathology, direct microscopy, fungal culture, quantitative PCR (qPCR) and metagenomic next-generation sequencing (mNGS) detection. A patented qPCR targeting prevalent Aspergillus species was evaluated. The patient cohort had a male-to-female ratio of 9:14, with disease duration up to 50 years. Histopathologically, 23 out of 28 cases were positive. Fungal culture exhibited a sensitivity of 21.74%, with one false positive. qPCR and mNGS showed 100% sensitivity and specificity, with a 100% consistency rate for identification at the species level (23/23), and potential detection of cases with co-infections. The most common pathogen was A. flavus, followed by A. fumigatus and A. niger. Two cases involved mixed infections of A. fumigatus and A. flavus. qPCR and mNGS proved effective in rapidly identifying fungi from fresh sinus tissue that are challenging to culture, surpassing conventional methods. However, further evaluation and optimization with a larger cohort of patients are necessary. Histopathology is still recommended to confirm the clinical significance of the detected fungal species.

Detection of Brucella S19 Vaccine Strain DNA in Domestic and Wild Ungulates from Brazilian Pantanal.

The Pantanal region, the largest floodplain in the world, has a huge biodiversity and is an important livestock center. Bovine brucellosis has been reported in the region over the last three decades, posing implications for cattle industry as well as for the maintenance of biodiversity. We aimed to investigate the presence of B. abortus S19 vaccine strain DNA in unvaccinated domestic and wild ungulates from the Brazilian Pantanal. Fifty-two heifers, 63 ovine, 24 domestic pigs, 28 feral pigs, and three Pampas deer were sampled. Brucella spp. was detected through bcsp31 PCR of blood samples in 45.3% (77/170) of the sampled animals, of which 36.4% (28/77) showed positivity in ery PCR corresponding to B. abortus S19 strain. Feral pigs presented the highest occurrence of positive samples in bcsp31 PCR (75%), followed by ovine (47.6%), domestic pigs (41.7%), and unvaccinated heifers (30.8%). We did not observe positivity in Pampas deer. Our results strongly suggest that vaccination against bovine brucellosis may promote spill-over of B. abortus S19 strain in the Pantanal region. Moreover, our data indicate that wild strains of Brucella circulates in the Pantanal Biome.

Spleen Swabs for Sensitive and High-Throughput Detection of African Swine Fever Virus by Real-Time PCR.

African swine fever (ASF) continues to spread in Africa, Europe, Asia and the island of Hispaniola, increasing the need to develop more streamlined and highly efficient surveillance and diagnostic capabilities. One way to achieve this is by further optimization of already established standard operating procedures to remove bottlenecks for high-throughput screening. Real-time polymerase chain reaction (real-time PCR) is the most sensitive and specific assay available for the early detection of the ASF virus (ASFV) genome, but it requires high-quality nucleic acid extracted from the samples. Whole blood from live pigs and spleen tissue from dead pigs are the preferred samples for real-time PCR. Whole blood can be used as is in nucleic acid extractions, but spleen tissues require an additional homogenization step. In this study, we compared the homogenates and swabs prepared from 52 spleen samples collected from pigs experimentally inoculated with highly and moderately virulent ASF virus strains. The results show that not only are the spleen swabs more sensitive when executed with a low-cell-count nucleic acid extraction procedure followed by real-time PCR assays but they also increase the ability to isolate ASFV from positive spleen samples. Swabbing is a convenient, simpler and less time-consuming alternative to tissue homogenization. Hence, we recommend spleen swabs over tissue homogenates for high-throughput detection of ASFV by real-time PCR.

A COVID-19 Case: Isolated and Persistent Headache

COVID-19 effects many systems such as respiratory, nervous, renal and hematologic system. Third of patients have experienced neurological symptoms such as headache, anosmia, ageusia, and dizziness. Headache has been reported as the most common neurological manifestation but cases just headache are extremely rare. This case is a Covid-19 case with no symptoms other than sudden onset headache. The result of the nasal swab PCR sampling was negative however local infiltration areas with ground glass density in the periphery of the middle lobe, lateral segment of the both lungs were detected in the thorax CT.

The adjunctive effect of frankincense extract gel to nonsurgical treatment of chronic periodontitis: a randomized clinical study

Abstract Aim This study was aimed at assessing the impact of applying the frankincense extract gel subgingivally in addition to scaling and root planing (SRP) of nonsurgically treated moderate chronic periodontitis patients. Patients and methods Twenty cases were randomly chosen and undergo an equal division into two groups: group I underwent only SRP, group II underwent SRP as well as frankincense extract gel. Applying frankincense extract gel subgingivally was carried out after initial SRP (day 1), 7, and 14 days. Clinical measurements involved probing depth, bleeding on probing, as well as clinical attachment level. qRT-PCR was performed to identify the treatment’s effect on Porphyromonas gingivalis. Clinical measurements along with plaque samples for PCR were documented at baseline (prior to the therapy), 1, and 3 months following treatment. Results Group II (frankincense extract gel) exhibited statistically significant enhancement in all clinical parameters involving probing depth, clinical attachment level, as well as bleeding on probing. In addition, P. gingivalis revealed a significant reduction in its load as revealed from qRT-PCR as opposed to the control group. Conclusion Applying frankincense extract gel subgingivally has exhibited potent antibacterial properties. As it is biologically accepted by most cases when applied in periodontal pockets, it is advised to be utilized in addition to SRP for managing moderate chronic periodontitis cases as well as others under the periodontal maintenance phase.

The frequency and associated factors of typhoid carriage in patients undergoing cholecystectomy for gallbladder disease in Pakistan: A cross-sectional study.

Enteric fever is caused by Salmonella enterica serovars Typhi (S. Typhi) and Paratyphi A, B, and C. It continues to be a significant cause of morbidity and mortality worldwide. In highly endemic areas, children are disproportionately affected, and antimicrobial resistance reduces therapeutic options. It is estimated that 2-5% of enteric fever patients develop chronic asymptomatic infection. These carriers may act as reservoirs of infection; therefore, the prospective identification and treatment of carriers are critical for long-term disease control. We aimed to find the frequency of Salmonella Typhi carriers in patients undergoing cholecystectomy. We also compared the detection limit of culturing versus qPCR in detecting S. Typhi, performed a geospatial analysis of the carriers identified using this study, and evaluated the accuracy of anti-Vi and anti-YncE in identifying chronic typhoid carriage. We performed a cross-sectional study in two centers in Pakistan. Gallbladder specimens were subjected to quantitative PCR (qPCR) and serum samples were analyzed for IgG against YncE and Vi by ELISA. We also mapped the residential location of those with a positive qPCR result. Out of 988 participants, 3.4% had qPCR-positive gallbladder samples (23 S. Typhi and 11 S. Paratyphi). Gallstones were more likely to be qPCR positive than bile and gallbladder tissue. Anti-Vi and YncE were significantly correlated (r = 0.78 p<0.0001) and elevated among carriers as compared to qPCR negative controls, except for anti-Vi response in Paratyphi A. But the discriminatory values of these antigens in identifying carriers from qPCR negative controls were low. The high prevalence of typhoid carriers observed in this study suggests that further studies are required to gain information that will help in controlling future typhoid outbreaks in a superior manner than they are currently being managed.

Altered extracellular matrix–related pathways accelerate the transition from normal to prefibroid myometrium in Black women

BackgroundBlack women experience a disproportionate impact of uterine fibroids compared to White women, including earlier diagnosis, higher frequency, and more severe symptoms. The etiology underlying this racial disparity remains elusive. ObjectiveThe aim of this study was to evaluate the molecular differences in normal myometrium (fibroid-free uteri) and at-risk myometrium (fibroid-containing uteri) tissues in Black and White women. Study designWe conducted whole-genome RNA-seq on normal and at-risk myometrium tissues obtained from both self-identified Black and White women (not Hispanic or Latino) to determine global gene expression profiles and to conduct enriched pathway analyses (n=3 per group). We initially assessed the differences within the same type of tissue (normal or at-risk myometrium) between races. Subsequently, we analyzed the transcriptome of normal myometrium compared to at-risk myometrium in each race and determined the differences between them. We validated our findings through real-time PCR (sample size range=5-12), western blot (sample size range=5-6), and immunohistochemistry techniques (sample size range=9-16). ResultsThe transcriptomic analysis revealed distinct profiles between Black and White women in normal and at-risk myometrium tissues. Interestingly, genes and pathways related to extracellular matrix and mechanosensing were more enriched in normal myometrium from Black than White women. Transcription factor enrichment analysis detected greater activity of the serum response transcription factor positional motif in normal myometrium from Black compared to White women. Furthermore, we observed increased expression levels of myocardin-related transcription factor-serum response factor and the serum response factor in the same comparison. In addition, we noted increased expression of both mRNA and protein levels of vinculin, a target gene of the serum response factor, in normal myometrium tissues from Black women as compared to White women. Importantly, the transcriptomic profile of normal to at-risk myometrium conversion differs between Black and White women. Specifically, we observed that extracellular matrix-related pathways are involved in the transition from normal to at-risk myometrium and that these processes are exacerbated in Black women. We found increased levels of Tenascin C, type I collagen alpha 1 chain, fibronectin and, phospho-p38 MAPK (Thr180/Tyr182, active) protein levels in at-risk over normal myometrium tissues from Black women, whereas such differences were not observed in samples from White women. ConclusionsThese findings indicate that the racial disparities in uterine fibroids may be attributed to heightened production of extracellular matrix in the myometrium in Black women, even before the tumors appear. Future research is needed to understand early life determinants of the observed racial differences.

Leucocytozoon Infection Does Not Influence the Survival of Boreal Owl Aegolius funereus Nestlings.

Leucocytozoon infection has been observed to impact the reproductive ecology and physiology of avian hosts, but its influence on nestling survival remains unclear. We investigated the effect of Leucocytozoon infection intensity, determined through triplicate PCR sample analyses, on the survival of 256 boreal owl (Aegolius funereus) nestlings during an 8-yr study. Contrary to our expectations, the survival probability of boreal owl nestlings was not influenced by their Leucocytozoon infection intensity. Nestling age and Leucocytozoon infection intensity in male and female parents also did not impact nestling survival. Instead, food abundance and hatching order were the key factors influencing nestling survival. Additionally, we observed a significantly higher Leucocytozoon infection intensity in male parents compared to female parents and nestlings. We suggest a distinct division of parental roles may lead females and nestlings staying within the nest boxes (cavities) to experience lower exposure to potential vectors transmitting blood parasites than their male counterparts. Our study shows that Leucocytozoon disease may not be lethal for boreal owl chicks, exhibiting a below-average infection intensity compared to their male parents.

Detection of small ruminant Lentivirus proviral DNA in red deer from Poland

Small ruminant lentiviruses (SRLVs) are widespread and infect goats and sheep. Several reports also suggest that SRLVs can infect wild ruminants. The presence of specific antibodies against SRLVs has been identified in wild ruminants from Poland, but no studies have been conducted to detect proviral DNA of SRLVs in these animals. Therefore, the purpose of this study was to examine samples from Polish wild ruminants to determine whether these animals can serve as reservoirs of SRLVs under natural conditions. A total of 314 samples were tested from red deer (n = 255), roe deer (n = 52) and fallow deer (n = 7) using nested real-time PCR. DNA from positive real-time PCR samples was subsequently used to amplify a CA fragment (625 bp) of the gag gene, a 1.2 kb fragment of the pol gene and an LTR-gag fragment. Three samples (0.95%) were positive according to nested real-time PCR using primers and probe specific for CAEV (SRLV group B). All the samples were negative for the primers and probe specific for MVV (SRLV A group). Only SRLV LTR-gag sequences were obtained from two red deer. Phylogenetic analysis revealed that these sequences were more closely related to CAEV than to MVV. Our results revealed that deer can carry SRLV proviral sequences and therefore may play a role in the epidemiology of SRLVs. To our knowledge, this is the first study describing SRLV sequences from red deer.

Transitioning from Triton X-100 to Tergitol 15-S-9: impacts on diagnostic assays using viral PCRsample solution.

In 2019, the European Union banned Triton X-100, a detergent widely used in laboratory diagnostics, including the Viral PCR Sample Solution (VPSS), and urged manufacturers to find environmentally sustainable alternatives. Tergitol 15-S-9 (VPSS2) has been proposed as an alternative surfactant. This multicenter study evaluated the effectiveness of VPSS2, a Tergitol-based viral solution, as a replacement for VPSS. Our results show the equivalent performance of VPSS2 to VPSS for nucleic acid extraction and viral stability over time at different temperatures. The new VPSS formulation was also tested against external quality assurance panels and clinical samples. The results of this work support adopting this modified viral PCR sample solution to replace Triton X-100-containing viral transport solutions.

Abstract PO1-03-10: Differential microbial metabolic pathways associated with resistance to neoadjuvant immunotherapy in locally advanced triple negative breast cancer

Abstract Differential microbial metabolic pathways associated with resistance to neoadjuvant immunotherapy in locally advanced triple negative breast cancer Background: It has previously been shown that microbial metabolic pathways are differentially activated in immunotherapy resistance, hence can serve as potential targets for therapeutic intervention. With the advent of Immune Checkpoint Blockers in the neoadjuvant setting of locally advanced (LA) TNBC, we hypothesized that similar microbial metabolite mediated resistance may be at play. We are presenting the real world data for neoadjuvant treatment in early TNBC along with a subset of tumor metabolite analysis. Method: 35 patients who received neoadjuvant pembrolizumab and chemotherapy for LA TNBC were identified at Moffitt Cancer Center between July 2019 to January 2023. These included 22 Caucasian, 5 African American, 2 Hispanic and 6 patients with undisclosed race. The age range was 33-78 years. The weight and Body Mass Index range was 55.3 kg-127.3 kg and 20.96-45.43 kg/m2 respectively. Nine patients had stage III disease while 24 had stage II breast cancer. The response was assessed in 33 patients; 20 patients (60.6%) achieved pathological complete response (pCR) and 13 (39.4%) had residual disease (RD). 1 patient developed severe immune mediated toxicity with colitis, hepatitis, myasthenia gravis leading to death while 1 patient had disease progression leading to drug discontinuation. Six patients developed immune mediated adverse events. We conducted untargeted metabolomic profiling on 24 patients to identify differentially abundant metabolites associated with pCR (ypT0/is ypN0) vs RD; 16 vs 8 patients respectively. Formalin fixed paraffin embedded (FFPE) slides from tumor tissue vs tumor bed were retrieved and histopathological evaluation of tumor cells was performed. 20 μm sections of FFPE block were used for untargeted metabolomics via liquid chromatography/mass spectrometry. Among the 200 metabolites detected, 63 were differentially abundant between pCR and tumor samples with RD. After correcting for false discovery rate, we observed striking differential activation of two microbial metabolic pathways. Samples with RD had significantly elevated kynurenine (OR-3.64 X1034, P-1.94 X 10-74 and kynurenine/Tryptophan ratio). The tryptophan, kynurenine pathway has been known to be overexpressed in patients resistant to immunotherapy and modulated by the microbiome. Similarly, the purine metabolic pathway, specifically microbiota-derived immunostimulatory metabolites- inosine, hypoxanthine, uric acid were significantly depleted in patients with RD (Inosine OR-0.54, p-0.007, Hypoxanthine OR-0.5, p-0.00039, uric acid OR: 0.41, P-0.003). Previously Inosine has been shown to increase the anti-tumor effects of immune checkpoint blockade by enhancing CD8+ effector T-cell function. Hence depletion of inosine, hypoxanthine and uric acid signals lack of an effector CD8 T cell response. Conclusion: Despite being a small study, it corroborates pCR rates noted in KEYNOTE 522 in the real world setting but shows significant concern for serious immune mediated adverse effects. This highlights the importance of stratifying patients who might be resistant to ICB or develop significant toxicities, thus deriving little to no benefit from standard neoadjuvant treatment. This underscores the need to develop precision-based treatment algorithms. The study represents one of the first investigations of differential abundance of microbial metabolites with immunotherapy resistance in LA TNBC. Our results highlight two microbially mediated mechanisms that could be targeted to improve pCR rates in LA TNBC. We are working to identify microbial metabolic pathways associated with immune mediated toxicity. Citation Format: Humaira Sarfraz, Brian Czerniecki, Zena Jameel, Michael Jaglal, Mostafa Eysha, Turkan Ozkul, Jameel Muzaffar, Aditi Saha, Sara Sarfraz, Fatima Zahra, Min Liu, Khushali Jhaveri, Melissa Sur, Adrianna Bagos-Estevez, Shahla Bari. Differential microbial metabolic pathways associated with resistance to neoadjuvant immunotherapy in locally advanced triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-03-10.

Individualized Cell-Free DNA Monitoring With Chromosomal Junctions for Mesothelioma

IntroductionThe spatially complex nature of mesothelioma and interventions like pleurodesis, surgery, and radiation often complicate imaging-based assessment. Further, cell-free DNA (cfDNA) based monitoring strategies are inadequate for mesothelioma, given the presence of a few recurring nonsynonymous somatic variants. However, patient-specific chromosomal rearrangements are commonly found in mesothelioma. Our study objective was to develop an individualized cfDNA assay to enable blood-based monitoring using circulating tumor DNA (ctDNA) in mesothelioma. We hypothesized that the unique chromosomal rearrangement junctions found in mesothelioma could be employed for individualized ctDNA detection and disease monitoring. MethodsDNA was extracted from tumor specimens for whole genome sequencing. Chromosomal junctions, prioritized by highest allele frequency and low homology to the rest of the genome, were selected for detection. Primers and Taqman probes were designed to span the junctions, forming personalized junction panels. Patient plasma obtained before therapy and at response assessment was tested for the presence of personalized junctions via quantitative PCR. ResultsOur study included nine patients, four with peritoneal and five with pleural mesothelioma. 763 chromosomal junctions were identified in the tumors of all cases. We selected three to five junctions per sample for quantitative PCR. We detected 25/30 (83%) of selected junctions in the plasma of seven out of nine patients (78%). Cell-free junction detection at follow-up was concordant with disease status: cfDNA junctions were detected in three patients with persistent disease, and not detected in a patient with no evidence of disease after surgery. ConclusionsWith further validation, individualized ctDNA junction assays could supplement imaging for disease monitoring in mesothelioma.

Evaluation of Health Literacy and COVID-19 Knowledge Levels of Candidate Soldiers: A Cross-sectional Study from Ankara, Turkey

people’s COVID-19 knowledge and HL levels are thought to be effective in managing the pandemic. This study aimed to evaluate candidate soldiers’ HL and COVID-19 knowledge levels in the Altındağ district of Ankara, Turkey.&#x0D; Material and Methods A questionnaire form containing socio-demographic characteristics, 16-item European HL Survey Questionnaire, and propositions about COVID-19 was applied to candidate soldiers Who were referred for COVID-19 PCR sampling to the Altindg District Health Directorate before enlistment between December 2021_April 2022.&#x0D; Results and Discussion The study was completed with 668 candidate soldiers, most of whom were young adults. HL level of 20.5% was inadequate. The frequency of those who had not been vaccinated against COVID-19 was 16.8%. The COVID-19 knowledge level of those vaccinated was higher (p=0.002). The propositions about COVID-19 symptoms and correct mask use were answered correctly at the highest rate in COVID-19 knowledge level questions. The COVID-19 knowledge level score was significantly higher in those with adequate HL levels, aged 25 and over, non-smokers, and who had university or higher education levels. There was a positive correlation between the COVID-19 knowledge level and HL levels (r=0.108; p&lt;0.001).&#x0D; Conclusion This study reveals the determination of HL and COVID-19 knowledge levels of candidate soldiers and related factors and supports the necessity of proactive participation of individuals with immediate action plans to increase HL and COVID-19 knowledge levels in young adults.&#x0D; Bangladesh Journal of Medical Science Vol. 23 No. 02 April’24 Page: 377-387

Abstract 807: An ethno-phenomenology based approach to study breast cancer disparity in rural India

Abstract There are fundamental lack of right methodologies to study the breast cancer disparity in rural India. Here, we have used an ethnographic-phenomenology approach to study the disparity in breast cancer care in Assam by using retrospective database of a rural cancer care non-profit clinic, the KaviKrishna Telemedicine Care (KTC), set up 1994 to develop a CBPR based cancer care (Abstract 3342, AACR 2019). Methodology: For one and half years, the doctors, researchers, nurses, caregivers, and physician assistants of KaviKrishna Laboratory and KaviKrishna Telemedicine Care were interviewed (total number: 15), and plus literature of the lab and the clinic were studied. Then, the breast cancer patients (n=10) and the families were subjected to ethnographies-phenomenology study to find their experiences of treatment and suffering. Next, thematic analysis and grounded theory were applied to come up with novel approaches to reduce breast cancer disparity. For comparison, 5 patients from rich urban families were interviewed. Results: We found that 10/10 patients found it extremely difficult to navigate the complexity of care in urban hospitals, but found the KTC approachable and comfortable. Importantly, these 10 breast cancer patients were positively responding to our KTC based care services/focussed group discussion (https://zenodo.org/records/8062404). They revealed positive experiences in emotionally and psychologically dealing with diagnostic, surgery, and chemotherapy processes. Next, our analysis of the last 25 years of the care data at KTC showed that in rural population, the loweconomic status, inadequate access to breast cancer detection clinics, lack of awareness,indecisive treatment protocols and social stigma are the main reasons for breast cancer disparity. Grounded theory-based analysis indicates that cancer biomarker study in rural populations can be a suitable method for early detection. Whole genome sequencing of cancer tissue frombiopsy samples shall provide landscape of mutation in the entire coding as well as noncodingregions. Mutation profiling along with correlation studies from patients can lead to discoveryof novel marker(s). Primary health care clinics in rural areas can be trained to collect patientsamples, isolate genomic DNA and perform PCR for the markers (a similar model asfollowed during Covid-19). The PCR samples can be sent to non-profit advanced diagnostic center such as KaviKrishna Lab to perform sequencing to study mutation markers. The method is also cost effective to both thecare givers and the patients as compared to setting up advanced diagnostic lab in every part ofrural area. Conclusion: The community based participatory research based model developed by KTC can be an effective strategy to reduce breast cancer disparity in the rural population since the primary health clinics shall act as a bridge between geologically isolated areas and modern technology. Citation Format: Debduti Dutta, Rupam Das, Chayanika Das, Tulika Sarma, Sonali Das, Anuja Dutta, Tutumoni Baishya, Lekhika Pathak, Bikul Das. An ethno-phenomenology based approach to study breast cancer disparity in rural India [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 807.

Abstract 3672: Development of liquid biopsy HPV (Human Papilloma Virus) cell free DNA (cfDNA) based approach for prognosis of cervical cancer

Abstract Introduction: Recent progress in the analysis of blood samples for circulating tumor cells or cell-free circulating tumor DNA (ctDNA) has shown that liquid biopsies may have potential applications in the detection and monitoring of cancer. Human papillomavirus (HPV) circulating cell-free (ccf) DNA may act as a unique marker for the prognosis of high-risk HPV-related cancers. However, detection of circulating markers for cervical cancer (CC) requires extremely sensitive techniques that can measure circulating human papillomavirus DNA. Here, we used the sensitive droplet digital PCR (dd PCR) based approach for the detection and quantification of circulating human papillomavirus DNA in plasma from patients diagnosed with cervical cancer at baseline and subsequent follow up with intention of validating its utility as a prognostic marker in CC. Method: Blood sera form 34 Patients diagnosed with cervical cancer (Stage 1-1V) at AIIMS Delhi was collected at baseline and after 3 months of the treatment. 10 healthy controls were also recruited in the study after due approval from AIIMS IEC. Plasma samples were stored at -80°C. ccf DNA was isolated from 1 ml of plasma and further processed to detect the presence of circulating high risk Human papilloma virus (HPV) i.e., HPV-16 and HPV18 cf DNA using dd PCR. Result: The median cf DNA concentration was 9.3 ng/uL (range 3-26.6) for all patients while median cf DNA concentration was 7.2 ng/uL (Range 3-57.3) for healthy controls (n=10). Furthermore, the levels of cfDNA decreases post 3 months of treatment with average concentration of 8.07 ng/uL (Range 4.6-19.3). We further processed our samples for dd PCR based screening to detect low copy no HPV cf DNA and ddPCR detection yielded a positive rate of 51% (18/35) for circulating HPV18 DNA while a positivity of 88.5% (31/35) was seen for the circulating HPV16 DNA in the sera of CC patients. By comparing HPV16 and HPV18 genotypes, we observed detection of circulating HPV 16 DNA more frequently than circulating HPV18 DNA in CC patients coming at our center. Furthermore, we found that copies of circulating viral DNA (HPV18 and HPV16) decreased post induction therapy in CC patients indicating progression ability of thid marker. Conclusion: In the current studies we provide evidence that cell free DNA concentration in CC patient can predict tumor burden and detection of low copy no of circulating HPV DNA could be a promising indicator for prognosis in cervical cancer patients and can predict recurrence of the disease. Citation Format: Gunjan Dagar, Ashna Gupta, Ravi Chauhan, Abhishek Shankar, Daya Nand Sharma, Muzafar A. Macha, Ajaz A. Bhat, Rajeev Goyal, Vaishali Suri, Mayank Singh. Development of liquid biopsy HPV (Human Papilloma Virus) cell free DNA (cfDNA) based approach for prognosis of cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3672.

Application of bioluminescence assay to assess PCR carryover contamination in forensic DNA laboratories

In forensic DNA testing, PCR-based multilocus short tandem repeat (STR) profiling kits, which have high sensitivity and discriminatory power, are generally used to analyze autosomal and Y-chromosomal DNA profiles. Forensic DNA laboratories require strict quality control for DNA testing, as contamination during analyses leads to incorrect interpretation of DNA profiles. Here, we aimed to apply bioluminescence assay to detect and monitor residual PCR products on laboratory work area and equipment surfaces by targeting dATP in the PCR product and allelic ladder marker. Two commercially available bioluminescence assay kits (CheckLite HS Plus and UltraSnap™) were examined for their sensitivity after confirming their reactivity to dATP. In the assay using CheckLite HS Plus, the lower detectable sample volumes were calculated as 10 pl of PCR product of GlobalFiler and PowerPlex Fusion and 1 pl of PCR product of Yfiler Plus and the allelic ladder marker of GlobalFiler, whereas those in the assay using UltraSnap™ were calculated as 1 nl of PCR product and allelic ladder marker. The sample volumes of these kits were lower than those detected through electrophoresis. Thus, the sensitivity of these kits was sufficient to control PCR carryover contamination in the post-PCR areas. Furthermore, residual PCR products in the post-PCR areas were continuously monitored using a bioluminescence assay. The results showed that the bioluminescence values increased after handling PCR samples for electrophoresis and decreased after decontamination. Therefore, we concluded that the bioluminescence assay is useful for assessing PCR carryover contamination in post-PCR processes in forensic DNA laboratories.

Results of PCR Pool Testing In Primary and Special Needs Schools In Bavaria For The School Year 2021/2022: Sentinel Surveillance In Face-To-Face Teaching During The Sars-CoV-2 Pandemic

In the school years 2019/20 and 2020/21, children were physically, psychologically, and socially stressed by school closures caused by the SARS-CoV-2 pandemic. To ensure attendance with optimal infection protection, PCR pool testing was conducted during the 2021/22 school year at Bavarian elementary schools and schools for pupils with special needs for timely detection of SARS-CoV-2 infection. This study analyzes the results of PCR pool testing over time stratified by region, school type, and age of children. The data were obtained from classes in elementary and special needs schools, involving pupils aged 6 to 11 years, who participated in the Bavaria-wide PCR pool testing from 09/20/21 to 04/08/22. Samples were collected twice weekly, consisting of PCR pool samples and individual PCR samples, which were only evaluated in case of a positive pool test. A class was considered positive if at least one individual sample from that class was positive within a calendar week (CW). A school (class) was considered to be infection-prone if three or more classes in that school (students in that class) were positive within a CW. The data included 2,430 elementary schools (339 special needs schools) with 23,021 (2,711) classes and 456,478 (29,200) children. A total of 1,157,617 pools (of which 3.37% were positive) and 724,438 individual samples (6.76% positive) were analyzed. Larger schools exhibited higher PR compared to smaller schools. From January 2022, the Omicron variant led to a massive increase in PR across Bavaria. The incidence rates per 100,000 person-weeks within the individual school samples were significantly lower than the concurrently reported age-specific and general infection incidences in the overall Bavarian population. PCR pool testing revealed relatively few positive pools, with an average of four children per one hundred pools testing positive. Schools and classes were rarely considered infection-prone, even during periods of high incidences outside of schools. The combination of PCR pool testing and hygiene measures allowed for a largely safe in-person education for pupils in primary and special needs schools in the school year 2021/22.

Malaria seroepidemiology in very low transmission settings in the Peruvian Amazon

Despite progress towards malaria reduction in Peru, measuring exposure in low transmission areas is crucial for achieving elimination. This study focuses on two very low transmission areas in Loreto (Peruvian Amazon) and aims to determine the relationship between malaria exposure and proximity to health facilities. Individual data was collected from 38 villages in Indiana and Belen, including geo-referenced households and blood samples for microscopy, PCR and serological analysis. A segmented linear regression model identified significant changes in seropositivity trends among different age groups. Local Getis-Ord Gi* statistic revealed clusters of households with high (hotspots) or low (coldspots) seropositivity rates. Findings from 4000 individuals showed a seropositivity level of 2.5% (95%CI: 2.0%-3.0%) for P. falciparum and 7.8% (95%CI: 7.0%-8.7%) for P. vivax, indicating recent or historical exposure. The segmented regression showed exposure reductions in the 40–50 age group (β1 = 0.043, p = 0.003) for P. vivax and the 50–60 age group (β1 = 0.005, p = 0.010) for P. falciparum. Long and extreme distance villages from Regional Hospital of Loreto exhibited higher malaria exposure compared to proximate and medium distance villages (p < 0.001). This study showed the seropositivity of malaria in two very low transmission areas and confirmed the spatial pattern of hotspots as villages become more distant.

Phenotypic and genotypic characterization of Neisseria gonorrhoeae isolates among individuals at high risk for sexually transmitted diseases in Zurich, Switzerland.

While ceftriaxone resistance remains scarce in Switzerland, global Neisseria gonorrhoeae (NG) antimicrobial resistance poses an urgent threat. This study describes clinical characteristics in MSM (men who have sex with men) diagnosed with NG infection and analyses NG resistance by phenotypic and genotypic means. Data of MSM enrolled in three clinical cohorts with a positive polymerase chain reaction test (PCR) for NG were analysed between January 2019 and December 2021 and linked with antibiotic susceptibility testing. Bacterial isolates were subjected to whole genome sequencing (WGS). Of 142 participants, 141 (99%) were MSM and 118 (84%) living with HIV. Participants were treated with ceftriaxone (N = 79), azithromycin (N = 2), or a combination of both (N = 61). No clinical or microbiological failures were observed. From 182 positive PCR samples taken, 23 were available for detailed analysis. Based on minimal inhibitory concentrations (MICs), all isolates were susceptible to ceftriaxone, gentamicin, cefixime, cefpodoxime, ertapenem, zoliflodacin, and spectinomycin. Resistance to azithromycin, tetracyclines and ciprofloxacin was observed in 10 (43%), 23 (100%) and 11 (48%) of the cases, respectively. Analysis of WGS data revealed combinations of resistance determinants that matched with the corresponding phenotypic resistance pattern of each isolate. Among the MSM diagnosed with NG mainly acquired in Switzerland, ceftriaxone MICs were low for a subset of bacterial isolates studied and no treatment failures were observed. For azithromycin, high occurrences of in vitro resistance were found. Gentamicin, cefixime, cefpodoxime, ertapenem, spectinomycin, and zoliflodacin displayed excellent in vitro activity against the 23 isolates underscoring their potential as alternative agents to ceftriaxone.

Implementing L-DNA analogs as mirrors of PCR reactant hybridization state: theoretical and practical guidelines for PCR cycle control.

In previous reports, we described a PCR cycle control approach in which the hybridization state of optically labeled L-DNA enantiomers of the D-DNA primers and targets determined when the thermal cycle was switched from cooling to heating and heating to cooling. A consequence of this approach is that it also "adapts" the cycling conditions to compensate for factors that affect the hybridization kinetics of primers and targets. It assumes, however, that the hybridization state of the labeled L-DNA analogs accurately reflects the hybridization state of the D-DNA primers and targets. In this report, the Van't Hoff equation is applied to determine the L-DNA concentration and ratio of L-DNA strands required by this assumption. Simultaneous fluorescence and temperature measurements were taken during L-DNA controlled cycling, and the optical and thermal switch points compared as a function of both total L-DNA concentration and ratio of strands. Based on the Van't Hoff relationship and these experimental results, L-DNA best mirrors the hybridization of PCR primers and targets when total L-DNA concentration is set equal to the initial concentration of the D-DNA primer of interest. In terms of strand ratios, L-DNA hybridization behavior most closely matches the behavior of their D-DNA counterparts throughout the reaction when one of the L-DNA strands is far in excess of the other. The L-DNA control algorithm was then applied to the practical case of the SARS-CoV-2 N2 reaction, which has been shown to fail or have a delayed Cq when PCR was performed without nucleic acid extraction. PCR Cq values for simulated "unextracted" PCR samples in a nasopharyngeal background and in an NaCl concentration similar to that of viral transport media were determined using either the L-DNA control algorithm (N = 6) or preset cycling conditions (N = 3) and compared to water background controls run in parallel. For preset cycling conditions, the presence of nasopharyngeal background or a high salt background concentration significantly increased Cq, but the L-DNA control algorithm had no significant delay. This suggests that a carefully designed L-DNA-based control algorithm "adapts" the cycling conditions to compensate for hybridization errors of the PCR D-DNA reactants that produce false negatives.