Sensitive Polymerase Chain Reaction Research Articles

Risk-based detection as a cost-effective strategy to reduce foodborne illness due to salmonella

BackgroundSalmonella, a widespread pathogen, poses a significant threat to global food safety, leading to foodborne diseases and substantial economic losses. The timely and accurate detection of foodborne pathogens is pivotal for averting food contamination and outbreaks across the food production chain. This study assesses the cost-effectiveness of traditional culture-based methods versus risk-based approaches, incorporating polymerase chain reaction (PCR) for Salmonella detection. MethodsWe employed a stochastic scenario tree model to simulate scenarios based on the sampling inspection plan for raw aquatic products conducted by the Guangzhou Center for Disease Control and Prevention from 2018 to 2020. Various detection methods (culture or PCR) were applied to these aquatic products based on their categorized risk level. Sensitivity values were derived from published data, and incremental cost-effectiveness ratios were used to compare the different scenarios against the traditional culture method. ResultsA total of 360 samples were collected for analysis. The cost of culture-based detection alone amounted to 125,423.20 Chinese Yuan (CNY) and yielded nine instances of positive Salmonella detections. The risk-based detection strategy, which combined the more sensitive PCR method with high-risk sample characteristics, while employing the culture method for the remaining combinations, imposed a total cost of 128,775.83 CNY and yielded ten positive detections. This approach cost approximately 3391.74 CNY per additional positive sample detected compared to the culture method alone. Meanwhile, PCR-only detection imposed a total cost of 62,960.03 CNY. ConclusionsIn comparison to traditional culture-based methods, both the risk-based detection strategy and the PCR-only approach demonstrated superior capabilities with respect to detecting contaminated aquatic products. Implementing risk-based detection strategies can enhance cost-effectiveness, not only ensuring food safety but also reducing the incidence and economic burden of foodborne diseases.

Investigation of meat species adulteration in beef-based meat products via real-time PCR in Türkiye

The addition of poultry and other meats to products made from 100% beef is prohibited in Türkiye. In addition, in Türkiye, where the majority of the population is Muslim, the use of pork and single-hoofed meat is a matter of concern due to religious reasons. This study utilized a sensitive and specific real-time polymerase chain reaction (PCR) method to identify the different meat species in meat products marketed as 100% beef and sold in the eastern Türkiye provinces of Erzurum, Erzincan, Kars, Ağrı, and Bingöl. The real-time PCR method was used to investigate the presence of DNA specific to six animal species (chicken, turkey, pork, horse, donkey, and camel). The analysis revealed no traces of horse, donkey, camel, or pork meat in any of the 100 samples of Turkish fermented sausage (sucuk), salami, and sausage. Contrary to the product labels claiming 100% beef content, poultry DNA residues were identified in a salami sample from Erzincan, while turkey DNA residues were found in sucuk samples from both Ağrı and Kars. The study’s findings reveal that contrary to label information, various types of meat can be present in meat products sold by both trademarked and local butchers. Consequently, this paper emphasizes the need for routine checks on market-sold products to ensure they comply with legislation and avoid consumer deception. Furthermore, the Real-Time PCR method, with its rapid results and high sensitivity, is deemed beneficial for food safety, consumer rights, and the prevention of unfair competition.

Boron nitride nanoplate-based improvement of the specificity and sensitivity in loop-mediated isothermal amplification for Vibrio parahaemolyticus detection

BackgroundNucleic acid testing based on DNA amplification is gradually entering people's modern life for clinical diagnosis, food safety monitoring and infectious disease prevention. Polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) are the most powerful techniques that have been the gold standard for quantitative nucleic acid analysis. However, the high nonspecific amplification rate caused by the formation of primer dimers, hairpin structures and mismatched hybridization severely restricts their real-world applications. It is highly desirable to explore a way for improving the specificity and sensitivity of PCR and LAMP assays. ResultsIn this work, we demonstrated that a nanomaterial boron nitride nanoplate (BNNP), due to its unique surface properties, can interact with the main components of the amplification reaction, such as single stranded primers and Bst DNA polymerase, and increase the thermal conductivity of the solution. As a result, the presence of BNNPs dramatically improved the specificity of PCR and LAMP. And BNNPs maintained the specificity even after five rounds of PCR. Moreover, the sensitivity of LAMP was also enhanced by BNNPs, and the detection limit of BNNP-based LAMP was two orders of magnitude lower than that of classical LAMP. Then the BNNP-based LAMP was applied to detect Vibrio parahaemolyticus in contaminated seafood samples with high specificity and a 10-fold increase in sensitivity. SignificanceThis is the first systematic demonstration of BNNPs as a promising additive to enhance the efficiency and fidelity of PCR and LAMP amplification reactions, thereby greatly expanding the application of nucleic acid detection in a wide range of laboratory and clinical settings.

B-370 Clinical Utility of microRNA Quantitative Analysis Using Double Quencher Probes and Methylation Analysis by Methylation Sensitive Restriction Enzyme Polymerase Chain Reaction for Bladder Cancer

Abstract Background Histone modifications, DNA methylation, and microRNA (miRNA)-mediated gene silence are examples of epigenetic changes that regulate gene expression without causing sequence changes. The early phases of neoplastic transformation are characterized by the occurrence of these epigenetic changes, which are believed to be crucial for the development and progression of cancer. To increase the sensitivity and practicality of miRNA analysis, we developed a measuring technique using a ZEN double quencher into hydrolysis probe in reverse transcription quantitative polymerase chain reaction (RT-qPCR). Additionally, we developed a method for DNA methylation analysis employing methylation-sensitive restriction enzymes (MSRE) and methylation-insensitive restriction enzymes (MIRE) that can analyze a number of samples quickly. We examined the clinical usefulness of using these systems to understand the level of miRNA expression and methylation in bladder cancer. Methods In this study, 64 biopsied tissues from transurethral bladder tumor resection (TURBT) were used. For miRNA extraction, Cica geniusTM RNA Prep Kit (For Tissue) II (KANTO CHEMICAL CO.,INC.) and a solution that improves miRNA extraction efficiency were used. Using stem-loop RT primers, complementary DNA was created from the isolated miRNA solution. Quantitative analysis was performed by RT-qPCR using a ZEN double quencher probe and miR-21,30a, 200c, and U6 were used as target. Genomic DNA (gDNA) was extracted from the TURBT sample using High Pure PCR Template Preparation Kit (Roche). Restriction enzyme digestion was performed using HapII (Takara Bio) as MSRE and MspI (Takara Bio) as MIRE; the latter has the same recognition site as HapII but is not affected by the presence or absence of methylation. Real-time PCR was performed on the LightCycler 96 (Roche) using Universal ProbeLibrary probes (Roche) targeting ESM1, SFMBT2, and SEPTIN9. From the obtained Cp value, each methylation rate was calculated. The Fisher’s exact test was used to statistically analyze the relationship among miRNA, methylation data and other clinicopathological factors. Results For miR-30a, we observed a higher expression of miRNA was linked to more advanced T stage (P = 0.047). Additionally, ESM1 methylation exhibited a significant correlation with greater grade (P = 0.004) and T stage progress (P = 0.018). Data on miRNA abundance and methylation rate were combined in order to uncover a link with clinical data that was not apparent from either set of data separately. Samples with higher values for both miR-21 and ESM1 methylation rates were shown to have high grade (P = 0.007) and progress to the T stage (P = 0.006). Furthermore, samples with higher values for both miR-200c and SEPTIN9 methylation rates did not progress to the T stage (P = 0.013). Conclusion The combined miRNA and methylation data revealed relationships with clinical results that could not be discovered with either data separately, highlighting the clinical usefulness of the current study’s findings.

First application of a droplet digital PCR to detect Toxoplasma gondii in mussels.

Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is one of the main food-, water- and soil-borne zoonotic disease worldwide. Over the past 20 years many papers were published on the transmission of T. gondii by marine animals, including mollusks, which can concentrate the oocysts and release them. Sporulated oocysts may remain viable and infective for 18 months in seawater. Therefore, raw or undercooked bivalve mollusks pose a risk to humans. This study aimed to apply and validate for the first time a very sensitive digital droplet polymerase chain reaction (ddPCR) protocol to detect and quantify T. gondii DNA in mussels. Four concentration levels: 8000 genomic copies (gc)/μL, 800 gc/μL, 80 gc/μL, 8 gc/μL of a T. gondii reference DNA were tested. DNA was extracted from 80 pools of mussels (Mytilus galloprovincialis). Forty pools were contaminated with T. gondii reference DNA and used as positive controls, while 40 pools were used as negative controls. DdPCR reaction was prepared using a protocol, previously developed by the authors, for detection of T. gondii in meat. Amplification was obtained up 8 gc/μL. All infected replicates resulted positive, as well as no droplets were detected in negative controls. The droplets produced in the reaction ranged from 8,828 to 14,075 (average 12,627 droplets). The sensitivity and specificity of ddPCR were 100% (95%CI = 94.3-99.9). In addition, 100 pools of mussels collected in the Gulf of Naples were used to validate the protocol. Of these 16% were positive (95% CI = 9.7-25.0) for T. gondii. Samples were also tested by real-time PCR and no positive samples were found. Data obtained from ddPCR showed good identification of negative and positive samples with higher specificity and efficiency than real-time PCR. This tool could be very useful for a rapid sensitive detection of low DNA concentrations of T. gondii in mussels, reducing the risk of toxoplasmosis in humans.

The Use of Saliva Samples to Test for Congenital Cytomegalovirus Infection in Newborns: Examination of False-Positive Samples Associated with Donor Milk Use.

A universal screening research study was conducted in six hospitals to identify the clinical sensitivity of polymerase chain reaction (PCR) testing on newborn dried blood spots (DBSs) versus saliva specimens for the diagnosis of congenital cytomegalovirus (cCMV). CMV DNA positive results from DBSs or saliva were confirmed with urine testing. Findings of several false-positive (FP) saliva PCR results prompted an examination of a possible association with donor milk. Documentation of the frequency of positive saliva results, including both true-positive (TP) and FP status from clinical confirmation, occurred. The frequency of donor milk use was compared for TP and FP cases. Of 22,079 participants tested between 2016 and 2022, 96 had positive saliva results, 15 were determined to be FP, 79 TP, and 2 were excluded for incomplete clinical evaluation. Newborn donor milk use was identified for 18 (19.14%) of all the positive saliva screens. Among the 15 FPs, 11 (73.33%) consumed donor milk compared to 7 of the 79 TPs (8.8%) (OR 28.29, 95% CI 7.10-112.73, p < 0.001). While milk bank Holder pasteurization inactivates CMV infectivity, CMV DNA may still be detectable. Due to this possible association, screening programs that undertake testing saliva for CMV DNA may benefit from documenting donor milk use as a potential increased risk for FP results.

Mobile Efficient Diagnostics of Infectious Diseases via On-Chip RT-qPCR: MEDIC-PCR.

The COVID-19 outbreak has caused public and global health crises. However, the lack of on-site fast, reliable, sensitive, and low-cost reverse transcription polymerase chain reaction (RT-PCR) testing limits early detection, timely isolation, and epidemic prevention and control. Here, the authors report a rapid mobile efficient diagnostics of infectious diseases via on-chip -RT-quantitative PCR (RT-qPCR): MEDIC-PCR. First, the authors use a roll-to-roll printing process to accomplish low-cost carbon-black-based disposable PCR chips that enable rapid LED-induced photothermal PCR cycles. The MEDIC-PCR can perform RT (3min), and PCR (9min) steps. Further, the cohort of 89 COVID-19 and 103 non-COVID-19 patients testing is completed by the MEDIC-PCR to show excellent diagnostic accuracy of 97%, sensitivity of 94%, and specificity of 98%. This MEDIC-PCR can contribute to the preventive global health in the face of a future pandemic.

Quantitative Real-Time Polymerase Chain Reaction for Detection of Mycobacterium leprae DNA in Tissue Specimens from Patients with Leprosy.

In leprosy, early diagnosis is crucial to prevent transmission and onset of disabilities of the disease. The purpose of this study was to determine usefulness of quantitative real-time polymerase chain reaction (PCR) in clinically diagnosed cases of leprosy. Thirty-two leprosy cases were included. The real-time PCR was performed using commercial kit targeting Mycobacterium leprae-specific insertion sequence element. The slit skin smear was positive in two (22.2%) borderline tuberculoid (BT) patients, five (83.3%) borderline lepromatous (BL) patients, and seven (50%) lepromatous leprosy (LL). The positivity of quantitative real-time PCR in BT, BL, LL, and pure neuritic leprosy were 77.8%, 83.3%, 100%, and 33.3%, respectively. Using histopathology as the gold standard, sensitivity of quantitative real-time PCR was 93.1%, and specificity was 100%. The DNA load was higher in LL (3,854.29/106 cells), followed by BL (140.37/106 cells), and BT (2.69/106 cells). Because of the high sensitivity and specificity of real-time PCR, our study strongly suggests the use of real-time PCR as a diagnostic tool for leprosy.

Comparative detection efficacy of primers targeting SpeI-AvaI restriction fragment and small subunit ribosomal RNA gene of Babesia bigemina

The objective of the study is to evaluate the comparative detection efficacy of primers targeting SpeI-AvaI restriction fragment and small subunit ribosomal RNA (SSU rRNA) gene of Babesia bigemina by employing conventional polymerase chain reaction (PCR) on 783 animals (296 cattle and 487 buffaloes) of low lying (bet) area of Punjab. The detection rate of SpeI-AvaI and SSU rRNA PCR assays was 3.96% (31/783), and 6.64% (52/783), respectively. Among cattle and buffaloes, prevalence of B. bigemina was higher (P&lt;0.01) in cattle by both the primers. The sensitivity and specificity of SSU rRNA PCR as compared to SpeI-AvaI restriction fragment PCR was 100% and 97.2%, respectively. The blast analysis of the nucleotides of the sequenced amplicons of Ludhiana isolates of SpeI-AvaI and SSU rRNA PCR assay of B. bigemina showed 83 and 100% similarity with available sequence in Genbank. The analysis of evolutionary divergence revealed that range of divergence was lying between 0.000 to 0.011 between SSU rRNA sequence with the other sequences of B. bigemina as well as Babesia species. To conclude, the primers targeting SSU rRNA gene are a better tool for amplification of the B. bigemina.

Comparative detection efficacy of primers targeting SpeI-AvaI restriction fragment and small subunit ribosomal RNA gene of Babesia bigemina

The objective of the study is to evaluate the comparative detection efficacy of primers targeting SpeI-AvaI restriction fragment and small subunit ribosomal RNA (SSU rRNA) gene of Babesia bigemina by employing conventional polymerase chain reaction (PCR) on 783 animals (296 cattle and 487 buffaloes) of low lying (bet) area of Punjab. The detection rate of SpeI-AvaI and SSU rRNA PCR assays was 3.96% (31/783), and 6.64% (52/783), respectively. Among cattle and buffaloes, prevalence of B. bigemina was higher (P&lt;0.01) in cattle by both the primers. The sensitivity and specificity of SSU rRNA PCR as compared to SpeI-AvaI restriction fragment PCR was 100% and 97.2%, respectively. The blast analysis of the nucleotides of the sequenced amplicons of Ludhiana isolates of SpeI-AvaI and SSU rRNA PCR assay of B. bigemina showed 83 and 100% similarity with available sequence in Genbank. The analysis of evolutionary divergence revealed that range of divergence was lying between 0.000 to 0.011 between SSU rRNA sequence with the other sequences of B. bigemina as well as Babesia species. To conclude, the primers targeting SSU rRNA gene are a better tool for amplification of the B. bigemina.

JAK2V617F mutation is highly prevalent in patients with ischemic stroke: a case-control study

AbstractIschemic stroke has a high recurrence rate despite treatment. This underlines the significance of investigating new possible cerebrovascular risk factors, such as the acquired gene mutation JAK2V617F found in 3.1% of the general population. We aimed to investigate the prevalence of the JAK2V617F mutation in a population with ischemic stroke compared with that in matched controls. We enrolled 538 consecutive Danish patients with ischemic stroke (mean age, 69.5 ± 10.9 years; 39.2% female) within 7 days of symptom onset. Using multiple-adjusted conditional logistic regression analysis, we compared the prevalence of JAK2V617F with that in age- and sex-matched controls free of ischemic cerebrovascular disease (ICVD) from the Danish General Suburban Population Study. DNA was analyzed for JAK2V617F mutation using sensitive droplet digital polymerase chain reaction in patients and controls. Of the 538 patients with ischemic stroke, 61 (11.3%) had JAK2V617F mutation. There were no differences in patient demographics or cerebrovascular comorbidities between the patients with and without mutations. Patients with ischemic stroke were more likely to have the JAK2V617F mutation than matched controls, in whom the JAK2V617F prevalence was 4.4% (odds ratio, 2.37; 95% confidence interval, 1.57-3.58; P < .001). A subanalysis stratified by smoking history revealed that the association was strongest in current smokers (odds ratio, 4.78; 95% confidence interval, 2.22-10.28; P < .001). Patients with ischemic stroke were 2.4 times more likely to have the JAK2V617F mutation than matched controls without ICVD when adjusting for other cerebrovascular risk factors. This finding supports JAK2V617F mutation as a novel cerebrovascular risk factor.

Reliability of nasopharyngeal PCR for the detection of otopathogens in children with uncomplicated acute otitis media compared to culture

Otopathogens in acute otitis media (AOM) have implications for care because the likelihood of resolution without antibiotics and optimal antibiotic agent varies by microorganism. We aimed to determine the sensitivity, specificity, positive predictive value, and negative predictive value of nasopharyngeal (NP) qualitative polymerase chain reaction (PCR) for common bacterial otopathogens in children with AOM compared to NP culture. NP flocked swabs collected from enrolled children aged 6 to 35 months with uncomplicated AOM in Denver, CO were tested by culture and multiplex PCR. The sensitivity and negative predictive value of PCR using culture as a reference were high (H. influenzae 93.3%, 98.0%; S. pneumoniae 94.2%, 95.1%; M. catarrhalis 92.3%, 86.4%); whereas the specificity and positive predictive value were lower and varied by organism (54.2%–84.1%, 55.1%–69.2%, respectively). PCR detected 1.5 times more organisms than culture. NP PCR has a high predictive value for excluding otopathogens compared to culture and warrants exploration as a diagnostic tool.

Detection of resistance mutations in patients with anaplastic lymphoma kinase-rearranged lung cancer through liquid biopsy.

Tyrosine kinase inhibitors (TKIs) significantly improve clinical outcomes in patients with non-small cell lung cancer due to anaplastic lymphoma kinase (ALK) gene rearrangement. However, the rate of relapse with TKIs is high owing to the development of resistance mutations during treatment. Repeated biopsies during disease progression are crucial for elucidating the molecular mechanisms underlying the development of resistance to ALK inhibitors. Analysis of cell-free DNA (cfDNA) obtained from plasma is a novel approach for tumor genotyping. In this mixed prospective and retrospective observational cohort study, we investigated the clinical feasibility of continuous quantitative monitoring of ALK-acquired mutations in plasma obtained from patients with ALK+ non-small cell lung cancer by using a highly sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay. We enrolled nine patients, including three treatment-naïve patients recently diagnosed with ALK+ non-small cell lung cancer via tissue biopsy and expected to receive ALK TKIs and six patients already receiving ALK TKIs. Plasma samples were collected from these patients every 3 months. cfDNA was extracted from 66 samples during the study period, and 10 ALK mutations were simultaneously evaluated. The numbers of samples showing the G1202R, C1156Y, G1269A, F1174L, T1151ins, and I1171T mutations were 32, 16, 5, 4, 1, and 1, respectively. The L1196M, L1152R, V1180L, and S1206Y mutations were not detected. Correlation analyses between progression-free survival and the time from treatment initiation (or treatment modification) to the detection of resistance mutations revealed that although resistance mutations may occur before a drug change becomes necessary, there is a duration during which the disease does not progress. Our findings suggest that real-time quantitative monitoring of ALK resistance mutations during the response period could provide a time course of changes while acquiring resistance mutations. This information would be beneficial for designing an appropriate treatment strategy.

Clinical Evaluation of Polymerase Chain Reaction Coupled with Quantum Dot Fluorescence Analysis for Diagnosis of Candida Infection in Vulvovaginal Candidiasis Practice.

Time-consuming culture methods and wet-mount microscopy (WMM) with low sensitivity have difficulties in diagnosing Vulvovaginal candidiasis (VVC). Rapid and highly sensitive polymerase chain reaction coupled with quantum dot fluorescence analysis (PCR-QDFA) for the diagnosis of VVC has not been reported to date. This study was the first to evaluate the performance of PCR-QDFA for diagnosis of Candida strains in the leukorrhea samples from patients with suspected VVC. Leukorrhea samples from all visited patients were taken from the vagina using vaginal swabs by clinicians. We evaluated patients admitted with suspected VVC who completed WMM for diagnosis and reported the diagnostic effectiveness of PCR-QDFA and Candida culture (gold standard) when testing leucorrhea samples. A total of 720 leukorrhea samples from 387 VVC-positive patients and 333 VVC-negative patients were included in this study. Of the 387 leukorrhea samples from the VVC-positive patients, 391 Candida strains were identified by culture. 99.23% (388/391) Candida strains were included in the PCR-QDFA list. The 388 Candida strains belonged to four different species of Candida, including C. albicans (n = 273, 70.36%), C. glabrata (n = 85, 21.91%), C. tropicalis (n = 16, 4.12%), and C. krusei (n = 14, 3.61%). PCR-QDFA diagnosed Candida strains in 340/384 (88.54%) of the leucorrhea samples with Candida strains infection. The sensitivity of PCR-QDFA for C. albicans, C. glabrata, C. tropicalis, and C. krusei was 89.01%, 85.88%, 81.25% and 92.86%, respectively. The specificity of PCR-QDFA for C. albicans, C. glabrata, C. tropicalis and C. krusei was 93.69%, 99.37%, 99.71%, and 99.57%, respectively. The highly sensitive and specific PCR-QDFA technique can be exploited as a rapid (approximately 4 h) diagnostic tool for common Candida strains of leucorrhea samples from patients with suspected VVC.

A specific and sensitive droplet digital polymerase chain reaction assay for the detection of tilapia lake virus in fish tissue and environmental samples.

Tilapia lake virus (TiLV) causes high mortality in farmed and wild tilapia in various countries. We developed a highly specific and sensitive droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify TiLV. The ddPCR assay could detect the virus at a lower threshold than the reverse transcription-quantitative polymerase reaction (RT-qPCR) method, and the sensitivity of the ddPCR assay was 10-fold higher. The diagnostic sensitivity and specificity of the ddPCR assay were 100% and did not cross-react with tilapia tissues infected with Tilapia parvovirus, Infectious spleen and kidney necrosis virus, Aeromonas hydrophila, Streptococcus agalactiae, S. iniae and Francisella noatunensis. The assay reproducibility was demonstrated by a high correlation coefficient of 0.998, and the inter-assay coefficients of variability indicated that the ddPCR assay exhibited low variability within and between measurements. The detection limit of the TiLV ddPCR assay was 100 fg cDNA, which is equal to 3.3 copies of TiLV. Furthermore, the ddPCR assay could detect TiLV in mucus, water and infected tissue samples and the lowest copy number of TiLV detected in water samples by the ddPCR assay was 7.9 ± 0.99 copies/reaction The results of the clinical samples tested for TiLV revealed that the ddPCR assay had a relatively higher detection rate than the RT-qPCR method. Overall, the ddPCR method offers a highly promising approach for the absolute quantification of TiLV in carrier fish and samples from the environment with low viral concentrations.

Comparison of the Performance of Bioresonance, Electrophoresis and RT-PCR in the Diagnosis of Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) continues to be one of the most researched infectious diseases of cats. The diagnosis of FIP is challenging, and diverse techniques have been developed for its accurate diagnosis. However, they have some limitations. The present study was conducted to investigate the efficacy of specific modulation frequency (SMF), compared to other routine diagnostic methods for detecting feline coronavirus. Blood samples were collected from 30 diseased cats suspected of having FIP based on clinical signs. Electrophoresis, polymerase chain reaction (PCR), and SMF tests were performed for each sample. The sensitivity and specificity of each test, as well as the agreement between the tests and the gold standard (the combination of PCR, electrophoresis, and bioresonance results), were calculated using the Kappa coefficient method. The sensitivity and specificity of electrophoresis, PCR, and SMF for the diagnosis of FIP were 70.6%, 70.6%, 100%, and 100%, 72.7%, 81.8%, respectively. According to the findings of the present study, SMF is effective and safe in FIP diagnosis, which is a challenge in veterinary medicine diagnosis.

APOBEC3F Is a Mutational Driver of the Human Monkeypox Virus Identified in the 2022 Outbreak.

On May 6, 2022, a powerful outbreak of monkeypox virus (MPXV) had been reported outside of Africa, with many continuing new cases being reported around the world. Analysis of mutations among the 2 different lineages present in the 2021 and 2022 outbreaks revealed the presence of G->A mutations occurring in the 5'GpA context, indicative of APOBEC3 cytidine deaminase activity. By using a sensitive polymerase chain reaction (differential DNA denaturation PCR) method allowing differential amplification of AT-rich DNA, we analyzed the level of APOBEC3-induced MPXV editing in infected cells and in patients. We demonstrate that G->A hypermutated MPXV genomes can be recovered experimentally from APOBEC3 transfection followed by MPXV infection. Here, among the 7 human APOBEC3 cytidine deaminases (A3A-A3C, A3DE, A3F-A3H), only APOBEC3F was capable of extensively deaminating cytidine residues in MPXV genomes. Hyperedited genomes were also recovered in ∼42% of analyzed patients. Moreover, we demonstrate that substantial repair of these mutations occurs. Upon selection, corrected G->A mutations escaping drift loss contribute to the MPXV evolution observed in the current epidemic. Stochastic or transient overexpression of the APOBEC3F gene exposes the MPXV genome to a broad spectrum of mutations that may be modeling the mutational landscape after multiple cycles of viral replication.

Diagnostic utility of LAMP PCR targeting bcsp-31 gene for human brucellosis infection

PurposeHuman brucellosis is a neglected zoonotic disease of significant public health concern. Molecular diagnosis of brucella remains challenging in low resource settings, due to the high infrastructure and cost involved. Loop-mediated isothermal amplification (LAMP) is a rapid point of care polymerase chain reaction (PCR) with the utility of on-field molecular diagnosis and offers a convenient alternative to conventional PCR. In the present study, we developed and evaluated the diagnostic utility of in house LAMP PCR targeting the Brucella genus-specific bcsp-31 gene in patients having febrile illness. Materials and methodsThe analytical sensitivity and specificity of bcsp-31 LAMP PCR was first evaluated using brucella (n ​= ​8) and non-brucella cultures (n ​= ​5), along with spiked clinical samples. The overall diagnostic utility of developed LAMP PCR was then further evaluated in 393 human samples suspected of brucellosis. ResultsThe developed LAMP PCR could detect as low as 8 ​fg of DNA by visual detection within 35min. We report sensitivity and specificity of the developed LAMP PCR as 90.91% and 99.37%.The accuracy of the developed test assay was found to be 98.60%. In clinical samples, LAMP gave positivity of 20% with the concordance of 89% with conventional PCR. ConclusionTo conclude, a rapid, efficacious, sensitive LAMP PCR targeting the bcsp 31 gene was developed. The existing LAMP PCR can be used as a point of care screening test in various low resource endemic setting in lieu of conventional PCR for estimation of prevalence data, diagnosis and treatment of brucellosis.

Comparison of the Polymerase Chain Reaction Method with Serological Tests in the Diagnosis of Human Brucellosis

Background: Brucellosis is a zoonotic disease with different clinical symptoms. Its early diagnosis is essential to prevent severe complications. Due to the limitations of serological diagnostic methods, the polymerase chain reaction (PCR) method has become important in the diagnosis of the disease. Objectives: Our study aimed to evaluate the PCR method in patients with suspected brucellosis and compare it with serological tests. Methods: This cross-sectional study was performed on 90 febrile patients with clinical features of brucellosis who were examined by an infectious disease specialist. A total of 90 serum samples were collected from the suspected brucellosis patients admitted to the hospital and were analyzed by serological (Rose Bengal) and PCR tests. Then, each method's results were recorded and compared with each other. Results: According to serological test results, 45 samples were negative, and 45 were positive. Then, among the serology-positive patients, all had positive PCR results. However, 40 out of 45 patients had a positive PCR test in serology-negative patients. According to this study, the sensitivity of PCR in diagnosing human brucellosis with the serology-positive test is 100%, and with the negative serology test is 88.9%. Therefore, the sensitivity of PCR is higher than that of serology tests in patients, which was 50% in this study. Conclusions: The PCR test can be a valuable diagnostic method for patients with negative serologic test results.

PCR-dipstick DNA Chromatography for detection of Carbapenemase Producing Carbapenem-Resistant Enterobacteriaceae

Carbapenemase producing carbapenem-resistant Enterobacteriaceae (CP-CRE) are an epidemiologically important emerging public health threat. Early and reliable detection of CP-CRE is important for containment of these pathogens. The aim of this study was to evaluate and observe the sensitivity of polymerase chain reaction (PCR)-dipstick DNA chromatography (GeneFields CPE) for detection of carbapenemase genes (NDM, OXA-48, KPC, IMP) in comparison to conventional PCR. In this cross-sectional study, a total of 145 carbapenem resistant Enterobacteriaceae isolates were subjected to test for carbapenemase by phenotypic mCIM and genotypic- conventional PCR and PCR-dipstick DNA Chromatography method. In PCR dipstick DNA chromatography assay, after multiplex PCR a DNA-DNA hybridization-based detection system is applied where amplified products can be easily interpreted by observing a blue line in DNA strip visually within 15 minutes without gel electrophoresis. All the CP-CRE isolates (100%) were positive for either NDM or OXA-48 by PCR-dipstick DNA Chromatography assay, whereas 142 (97.9%) isolates were detected by conventional PCR. The positivity rate of NDM, OXA-48 and both NDM and OXA-48 among CP-CRE isolates were 73.8%, 51.7% and 25.5% respectively. The sensitivity, specificity of PCR-dipstick DNA Chromatography assay for detection of NDM and OXA-48 were 100.0%, 92.7% and 98.6%, 97.2% respectively. This assay showed excellent agreement with conventional PCR for detection of CP-CRE. PCR-dipstick DNA chromatography may be an excellent and robust diagnostic tool in terms of rapidity and sensitivity for the detection of CP-CRE.