Sensitive Polymerase Chain Reaction Research Articles

Progressive Multifocal Leukoencephalopathy in a Patient with Multifocal Neurological Manifestations Caused by Solitary Brainstem Involvement.

A Japanese man in his 60s on medication for chronic lymphocytic leukemia presented with progressive, multifocal neurological manifestations. Magnetic resonance imaging showed a small, solitary region of brainstem involvement. Sensitive real-time polymerase chain reaction testing detected a small amount of JC virus (JCV) DNA (170 copies/mL) with pathogenic mutation in cerebrospinal fluid. We diagnosed the patient with progressive multifocal leukoencephalopathy (PML). The small PML lesion may have caused multifocal neurological symptoms because of its focal brainstem involvement. This case contributes to knowledge regarding the diagnosis and treatment of brainstem PML in the context of hematologic malignancies and other underlying diseases.

Development of a novel detection technology for drug resistance mutation sites of Mycobacterium tuberculosis using Luminex liquid chip technology

Purpose: To develop a novel detection technology for drug-resistance mutation sites of Mycobacterium tuberculosis (MTB) using a Luminex liquid chip.Methods: Using polymerase chain reaction (PCR) amplification and hybridization analysis, MTB infection and drug-resistant mutation sites of the first-line and second-line anti-MTB drugs were simultaneously identified. A novel detection method was applied to analyze the wild-type standard strains of MTB and 33 clinical samples, and the results were compared with Sanger sequencing results for PCR products.Results: It was revealed that the sensitivity (100 %) and specificity (100 %) of the novel detection method for 31 samples were satisfactory, and all mutation sites were correctly detected. Compared with traditional PCR and culture-based drug sensitivity test, the novel detection method increased the speed of identification of drug-resistant TB, reduced clinicians' workload, and decreased treatment cost. Among 31 samples, 12.90 % were resistant to isoniazid (4/31), 35.48 % to rifampicin (11/31), and 12.90 % to ofloxacin (p < 0.05). Furthermore, 2 (6.45 %) samples were resistant to both isoniazid and rifampicin, 2 (6.45 %) samples to both rifampicin and ofloxacin, and 1 (3.22 %) sample to both isoniazid and ofloxacin, and 1 (3.22%) sample to all the three drugs (p < 0.05).Conclusion: Development and wide application of this novel detection method will facilitate the treatment of MTB, thus reducing the spread of drug-resistant MTB, and improving the prevention and treatment of MTB.

SG-APSIC1123: Diagnostic and infection prevention and control (IPC) performance of rapid polymerase chain reaction (PCR) compared to conventional culture PCR methods for detecting carbapenemase-producing organisms (CPOs)

Objectives: In this study, we compared the performance of a rapid polymerase chain reaction (PCR) method in detecting carbapenemase-producing organisms (CPOs) and its impact on infection prevention and control (IPC) measures compared with a culture PCR method. Methods: All patients requiring CPO screening were included. Rectal swabs were collected with double rayon swabs (Copan 139C). They were simultaneously analyzed for the presence of CPOs using rapid PCR assay (Xpert Carba-R assay, Cepheid, Sunnyvale, CA) and a culture–PCR method (ChromID CARBA-SMART, bioMerieux, Marcy-l’Etoile, France). For CARBA-SMART, only colored colonies (ie, Enterobacterales) were evaluated for CPOs according to the prevailing institutional protocol. We tracked time to CPO detection. Using CPO positivity from either the rapid PCR or the culture PCR method as the gold standard, we calculated the sensitivity and specificity of both tests. We calculated the number of epidemiologically linked contacts generated when the first test results were known. We prospectively followed the ward census to identify the putative additional number of contacts generated by the later known result. Contacts were patients who shared the same ward (with overlapping time) as the CPO patients. Results: Between April 2019 and June 2020, culture PCR method detected CPOs in 316 (1.3%) of 24,514 samples (blaOXA48, N = 211; blaNDM, N = 51; blaIMI, N = 21; blaIMP, N = 10; blaKPC, N = 9; mixed genotypes, N = 14). The rapid PCR test detected CPOs in 605(2.5%) of 24,514 samples (blaOXA48, N = 266; blaNDM, N = 161; blaIMP, N = 99; blaVIM, N = 29; blaKPC, N = 15; mixed genotypes, N = 35). The sensitivity of direct PCR and culture PCR methods were 94.2% (95% CI, 92.1%–95.8%) and 43.5% (95% CI, 39.6%–47.4%), respectively. Both tests had 100% specificity. The median times to detection for the rapid PCR and culture PCR methods were 3–4 hours and 4 days, respectively. Compared with rapid PCR, the culture PCR method generated additional 7,415 contacts when it also tested positive for CPOs and an additional 23,135 contacts when it tested negative for CPOs. Conclusions: In our study, the rapid PCR test was more sensitive, identified CPO faster, and generated fewer epidemiologically linked contacts than the culture PCR method.

Analysis of the reliability of rapid diagnostic tests for varicella, including breakthrough cases.

In the era of universal varicella vaccination, diagnosis of varicella is challenging, especially for breakthrough cases. We sought to clarify the reliability of direct varicella-zoster virus (VZV) loop-mediated isothermal amplification (LAMP) and DermaQuick® VZV using the immunochromatography technique as rapid diagnostic tests for varicella. In addition, the usefulness of saliva as a sample type for direct LAMP was investigated. Among the 46 enrolled patients with suspected VZV infection, 31 patients (67.3%) were positive for the nucleic acid test based on real-time PCR from skin swab samples. Direct LAMP of skin swabs was positive in 29 (63.0%) of 46 patients. DermaQuick® VZV was positive in 25 (54.3%) of 46 patients. VZV DNA was detected in only 48.4% of oral swabs with the direct LAMP method. With real-time polymerase chain reaction (PCR) as the standard for diagnosing varicella, the sensitivity and specificity of DermaQuick® VZV were 80.7% and 100%, respectively. The sensitivity and specificity of direct LAMP from skin swabs were 93.6% and 100%, respectively. The sensitivity and specificity of real-time PCR for DNA extracted from oral swabs were 74.2% and 93.3%, respectively. Thus, oral swab samples are not suitable for breakthrough varicella diagnosis. Although DermaQuick® VZV is considered the most convenient point-of-care test for varicella, its sensitivity and specificity were lower than those of direct VZV LAMP.

Rapid and sensitive detection of Pectobacterium carotovorum subsp. carotovorum in fresh produce using silica-coated magnetic nanoparticles combined with filtration-based real-time PCR

Pectobacterium carotovorum subsp. carotovorum (Pcc) possesses a powerful enzymatic mechanism that destroys plant-cell walls. Therefore, Pcc is the etiology of soft-rot disease in several crops and is recognized as one of the most important pathogens in agriculture. Soft-rot is difficult to control at the source when infection occurs. Thus, it is essential to develop sensitivity monitoring and diagnostic methods for timely control measures. Molecular detection methods, such as real-time polymerase chain reaction (PCR), are widely used for detecting Pcc. However, because Pcc is present at low levels in initial soft-rot infection, enrichment and efficient pathogen-separation methods are needed for sensitive monitoring. In this study, filtration was used as a pretreatment method for pathogen concentration, and silica-coated magnetic nanoparticles (SMN)- based real-time PCR was employed to detect low levels of Pcc in fresh produce. As a result, 102 CFU/10 mL was detected by various DNA-extraction methods in pure culture, whereas SMN detected up to 101 CFU/10 mL. In addition, when DNA was extracted using SMN after filtration using a cellulose acetate filter, the sensitivity of real-time PCR was improved to 10° CFU/10 mL. In conclusion, the pretreatment method including SMN, and concentration procedures increased the detection sensitivity by 100-fold; in potato and chicory, 101 CFU/g was detected at a frequency of 90% of the samples, and 10° CFU/g was detected at 70%; and in Kimchi Cabbage, 101 CFU/g was detected at 100% and 10° CFU/g was detected at 80%.

Сравнительная эффективность лабораторных методов в диагностике различных клинических форм бруцеллеза

Brucellosis is a zoonotic infection characterized by variable clinical manifestations, a variety of transmission routes, chronic inflammation, and risk of disability. Brucellosis remains a problem of practical healthcare in Russia, especially in endemic regions. Laboratory tests for brucellosis diagnosis are rarely used together, which prevents objective comparison of their effectiveness for different clinical forms of the disease. In this study, we analyzed different laboratory methods for comprehensive diagnosis of various clinical forms of brucellosis. Objective. To compare different laboratory methods for the diagnosis of various clinical forms of brucellosis. Materials and methods. We tested serum from 1049 individuals collected between 2005 and 2022. Samples were analyzed using agglutination reaction in tubes (Wright reaction; WR), on slides (Huddleson reaction; HR), Coombs test (CT), enzymelinked immunosorbent assay (ELISA), and real-time polymerase chain reaction (PCR) for BCSP31. We also performed retrospective analysis of 325 patient records. Of them, 110 patients (34%) had acute brucellosis, whereas 215 patients (66%) had chronic brucellosis. The control group comprised 604 individuals (with no clinical, epidemiological signs tested negative for brucellosis). Results. We analyzed 325 brucellosis patients (including 110 with acute disease, 215 with chronic disease, and 120 with no clinical manifestations) and 604 healthy controls. The sensitivity of PCR in acute and chronic disease was 94% and 32%, respectively. The specificity of PCR was 99.7%. ELISA demonstrated 92% and 86% sensitivity in acute and chronic disease, respectively, with a specificity of 98%. The negative and positive prognostic value for ELIZA and PCR were 95% and 96%, 83% and 99% respectively, suggesting similar effectiveness and accuracy of these methods. The sensitivity of traditional serological tests in acute disease was 75%, 62%, and 56.3% for RH, WR, and CT, respectively. In patients with chronic brucellosis, their sensitivity did not exceed 60%. Specificity of WR and CT was 97% in chronic disease, while it of RH dropped to 83%. Conclusion. PCR and ELISA ensured high diagnostic effectiveness and accuracy in assessing the activity of infection and diagnosis of brucellosis clinical forms, as well as in screening among vulnerable individuals. Traditional serological tests, despite their lower sensitivity, confirm acute disease and need for comprehensive laboratory diagnostics of brucellosis patients. Key words: brucellosis, diagnostic methods, enzyme-linked immunosorbent assay, polymerase chain reaction, Wright reaction, Huddleson reaction, Coombs test

Sensitivity of PCR in conjunctival swab samples for the diagnosis of canine visceral leishmaniasis: a systematic review and meta-analysis.

To compare the sensitivity of conjunctival swab (CS) and conventional samples (blood, spleen, liver, lymphoid and cutaneous tissue) in the diagnosis of canine visceral leishmaniasis (CVL) by polymerase chain reaction (PCR), a systematic review and meta-analysis was carried out using PubMed, Science Direct, Scopus, Web of Science, VHL/BVS (Virtual Health Library), CAPES, and Scielo databases. Articles published from 2002 to 2022 were considered and the review was updated in Jul 2023. From the total of 371 identified studies, 8 met all the eligibility criteria and were included in this review. Data from 658 CVL-positive dogs and 2541 PCR results were considered. Using a random effect model, data on the sensitivity of the test was compared between intervention (CS samples) and comparison (all the other samples) groups. Overall, the use of CS in the PCR diagnosis of CVL produced 12% higher sensitivity (p=0.013) in the test than all the other samples in combination. The animals' clinical condition did not influence (p>0.142) this overall result. However, when CS was individually compared to each of the conventional samples, the consistent result was observed (p=0.012) only in the CS versus bone marrow comparison. Given their rapid acquisition, minimal invasiveness, and lower cost relative to conventional samples, CS samples present a promising alternative for the molecular diagnosis of CVL.

Ultra-fast, sensitive and low-cost real-time PCR system for nucleic acid detection.

Nucleic acid detection directly identifies the presence of pathogenic microorganisms and has various advantages, such as high sensitivity, commendable specificity and a short window period, and has been widely used in many fields, such as early tumor screening, prenatal diagnosis and infectious disease detection. Real-time PCR (polymerase chain reaction) is the most commonly used method for nucleic acid detection in clinical practice, but it always takes about 1-3 hours, severely limiting its application in particular scenarios such as emergency testing, large-scale testing and on-site testing. To solve the time-consuming problem, a real-time PCR system based on multiple temperature zones was proposed, which realized the speed of temperature change of biological reagents from 2-4 °C s-1 to 13.33 °C s-1. The system integrates the advantages of fixed microchamber-type and microchannel-type amplification systems, including a microfluidic chip capable of fast heat transfer and a real-time PCR device with a temperature control strategy based on the temperature difference. The detection of HCMV biological samples using the real-time PCR system in this research took only 15 min, which was 75% shorter compared to the commercial qPCR instrument such as BIO-RAD, and the detection sensitivity remained essentially the same. The system could complete nucleic acid detection within 9 min under extreme conditions, characterized by fast detection speed and high sensitivity, providing a promising solution for ultra-fast nucleic acid detection.

Detection of Escherichia coli in Food Samples Using Culture and Polymerase Chain Reaction Methods.

Methods for the rapid detection of Escherichia coli and its related toxins are key to minimizing the risk of exposure to foodborne pathogens. The present study aimed to detect E. coli in food specimens using culture and polymerase chain reaction (PCR) techniques. One hundred and fifty samples from different types of food, comprising beef (n=60), chicken (n=72), and fish (n=18), were analyzed for the identification of E. coli by conventional and PCR methods. The results showed that out of 150 food samples, 44 (29.3%) were positive by culture, and 50 (33.3%) were positive by PCR. Significant differences were detected between sample types with culture (p-value < 0.005). When culture was considered the gold standard, the sensitivity of PCR was 100%, while the specificity was 94.34%. The six-hour pre-enrichment and PCR analysis are reliable in fast detection of E. coli in food samples. Hence, the identification of food pathogens using molecular-based methods would become more useful in routine diagnostic laboratories.

375. Performance Evaluation of RT-qPCR and RT-dPCR Platforms for the wastewater surveillance of SARS-CoV-2

Abstract Background The COVID-19 pandemic is an ongoing global health emergency. Wastewater-based epidemiology is a valuable tool for supplementing clinical testing in identifying infected individuals early thus containing disease transmission. To assess early detection of COVID-19, a building-level wastewater-based surveillance pilot project was implemented within VHA. Here, we report the results from 2 methods of polymerase chain reaction (PCR) testing of 1073 wastewater samples from VHA CLCs (i.e., nursing homes). Methods Daily (Monday-Friday) wastewater samples were collected (January 11, 2021, to July 2, 2021) at eight CLCs located across the US and shipped overnight for processing. The samples were heat inactivated by incubating samples in a 65±1°C heating circulating water bath for 90 minutes. The virus in the wastewater was concentrated using InnovaPrep concentrating pipette select, and RNA was isolated from the concentrate and subjected to reverse transcription quantitative PCR (RT-qPCR) and RT-digital PCR. If SARS-CoV-2 was detected in the wastewater within the prior 10 days of a virus-positive occupant, the wastewater positivity was regarded as an early warning. Results Twenty-seven positives and 7 inconclusive results were reported by RT-qPCR during the surveillance. Among the 27, 15 wastewater positives qualified as early warning and 12 positives were not verified by occupant positivity. Digital PCR with a cutoff value of 0.25 copies/uL of RNA for defining positivity had 28 positives qualifying as early warnings, and 115 positives were not verified by occupant positivity (Figure 1). Figure 1Differences between RT-PCR and Digital PCR Positivity Conclusion The overall viral loads of the wastewater samples were very low corresponding to the dip in cases seen in the US during the pilot period. Although sensitivity of digital PCR appears (based on 0.25 copies/uL of RNA for defining positivity) higher than that of RT-qPCR, there were more occurrences of unverified early warning that could impact precision. The cut-off selected for RT-digital PCR reported here is arbitrary and lacks industry consensus. More controlled studies are needed to determine sensitivity and precision as well as to standardize RT-digital PCR cutoffs to define positivity for routine use. Disclosures Piyali Chatterjee, PhD, AHRQ Grant # 1R03HS027667-01: Grant/Research Support|AHRQ Grant # 1R03HS027667-01: Central Texas Veterans Health Care System Chetan Jinadatha, MD, MPH, AHRQ R01 Grant-5R01HS025598: Grant/Research Support|EOS Surfaces: Copper Coupons and materials for testing.

Gut dysbiosis and bacterial translocation in the aneurysmal wall and blood in patients with abdominal aortic aneurysm.

Inflammation plays a part in the development of abdominal aortic aneurysm (AAA), and the gut microbiota affects host inflammation by bacterial translocation. The relationship between abdominal aortic aneurysm and the gut microbiota remains unknown. This study aimed to detect bacterial translocation in the aneurysmal wall and blood of patients with abdominal aortic aneurysm, and to investigate the effect of the gut microbiota on abdominal aortic aneurysm. We investigated 30 patients with abdominal aortic aneurysm from 2017 to 2019. We analysed the aneurysmal wall and blood using highly sensitive reverse transcription-quantitative polymerase chain reaction, and the gut microbiota was investigated using next-generation sequencing. In the 30 patients, bacteria were detected by reverse transcription- quantitative polymerase chain reaction in 19 blood samples (detection rate, 63%) and in 11 aneurysmal wall samples (detection rate, 37%). In the gut microbiota analysis, the Firmicutes/Bacteroidetes ratio was increased. The neutrophil-lymphocyte ratio was higher (2.94 ± 1.77 vs 1.96 ± 0.61, P < 0.05) and the lymphocyte-monocyte ratio was lower (4.02 ± 1.25 vs 5.86 ± 1.38, P < 0.01) in the bacterial carrier group than in the bacterial non-carrier group in blood samples. The volume of intraluminal thrombus was significantly higher in the bacterial carrier group than in the bacterial non-carrier group in aneurysmal wall samples (64.0% vs 34.7%, P < 0.05). We confirmed gut dysbiosis and bacterial translocation to the blood and aneurysmal wall in patients with abdominal aortic aneurysm. There appears to be a relationship between the gut microbiota and abdominal aortic aneurysm.

Application of Nanomaterials to Enhance Polymerase Chain Reaction.

Polymerase Chain Reaction (PCR) is one of the most common technologies used to produce millions of copies of targeted nucleic acid in vitro and has become an indispensable technique in molecular biology. However, it suffers from low efficiency and specificity problems, false positive results, and so on. Although many conditions can be optimized to increase PCR yield, such as the magnesium ion concentration, the DNA polymerases, the number of cycles, and so on, they are not all-purpose and the optimization can be case dependent. Nano-sized materials offer a possible solution to improve both the quality and productivity of PCR. In the last two decades, nanoparticles (NPs) have attracted significant attention and gradually penetrated the field of life sciences because of their unique chemical and physical properties, such as their large surface area and small size effect, which have greatly promoted developments in life science and technology. Additionally, PCR technology assisted by NPs (NanoPCR) such as gold NPs (Au NPs), quantum dots (QDs), and carbon nanotubes (CNTs), etc., have been developed to significantly improve the specificity, efficiency, and sensitivity of PCR and to accelerate the PCR reaction process. This review discusses the roles of different types of NPs used to enhance PCR and summarizes their possible mechanisms.

Drivers of Deep Molecular Response and Outcomes in Patients with Core Binding Factor Acute Myeloid Leukemia

Introduction: Core-binding factor acute myeloid leukemia (CBF-AML), including t (8;21) and Inv (16), constitutes 15% of all AML cases and generally carries a favorable prognosis. However, relapse rate is ≈ 30% and predictors are not well understood. Although CBF-AML responds to induction chemotherapy, post-remission treatment can be further optimized, particularly with current next generation sequencing (NGS) and measurable residual disease (MRD) detection techniques. Impact of baseline somatic mutations on outcomes remains unclear, as does role of allogeneic stem cell transplant (allo-SCT). In this study, we investigated the potential drivers of treatment response and overall outcomes in CBF-AML. Methods: In this single center retrospective study, clinical, demographic, and molecular data were collected from adult pts with CBF-AML who were treated at Moffitt Cancer Center. We included pts with MRD by polymerase chain reaction (PCR) for CBF abnormality (CBF-MRD) results available from time of induction and consolidation. All statistical analyses were performed in SPSS. Results: Our study included 68 patients (pts), including 26 (38%) t (8;21) AML. The median age at diagnosis was 48 years, and 54% pts were male. Pts with inv (16) AML had significantly more leukocytosis than t (8;21) (10x109/L vs. 23x109/L; p=0.001). Only 5% of pts (n=3) in our cohort had extramedullary disease on presentation. Common mutations identified on baseline NGS (n=60) were KIT (35%), NRAS (27%), KRAS (15%), FLT3-ITD and FLT3-TKD (12% each; 2 pts had both types). Mutation in KIT was more common in t (8;21) AML (50% vs. 25%; p=0.04), whereas inv (16) cases were enriched in NRAS mutations (36% vs. 13%; p=0.04). Overall, 80% of CBF-AML pts had at least one kinase mutation (KIT/RAS/FLT3). Majority of pts were treated with "7+3" (44%) and "7+3+ gemtuzumab ozagamicin (GO)" (40%) as induction regimens (Table 1). Daunorubicin was the predominant anthracycline, used in 80% of pts treated with "7+3" and 48% received dose of 90 mg/m2. Overall, 31 (46%) pts received GO as part of induction treatment, with equal distribution of "day 1 only" and "day 1,4,7" schedules at 3mg/m2. Median number of consolidation cycles was 3, and GO was incorporated in consolidation (commonly with high dose cytarabine) for 29 (43%) pts. After induction, 16% pts experienced CBF-MRD negativity which increased to 73% following consolidation, with no significant difference between t (8;21) and inv (16) AML. Use of GO during induction or consolidation was associated with significantly higher CBF-MRD negativity rate at completion of induction and consolidation (83% vs. 50%; p= 0.021). However, we did not find any significant correlation between groups based on presence of baseline kinase mutation/s and CBF-MRD negativity at end of induction or consolidation. In FLT3-positive cases, 100% of pts (5/5 evaluated;4 ITD and 1 TKD) achieved FLT3-MRD negativity by PCR (sensitivity of 5X10-5) following consolidation. Overall, 35% of pts (n=24) underwent allo-SCT in our cohort, including 15 in first remission (CR1). Kinase mutations were present at baseline in majority of these pts (75%; 87% in CR1 group). Notably, 9 pts (7 in CR1 group) in the allo-SCT cohort had positive CBF-MRD at the end of consolidation. Seven pre-transplant CBF-MRD positive cases turned MRD negative following allo-SCT. Relapse rate was low (n=10; 15%) in our CBF-AML pt population. Among pts with relapse, 6 were CBF-MRD positive post-induction and 3 had MRD even after consolidation. Use of GO in induction was associated with significantly lower relapse rate (0% vs. 28%; p= 0.001). Presence of kinase (KIT/RAS/FLT3) mutation at diagnosis was associated with shorter relapse free survival (RFS) in these pts (median RFS- 7.7 vs. 12.9 months (mo); p= 0.014; Figure 1). After a median follow up of 33.4 mo, median OS was 94.1 mo in our pt cohort. There was no significant OS difference between subgroups based on CBF-AML subtype, baseline mutations, or receipt of GO during treatment. Conclusions: CBF-AML continues to be a favorable risk disease, with a median OS >7 years in our study population. Post-treatment CBF-MRD negativity rate was 73% and was predicted by the incorporation of GO in induction or consolidation. Rate of relapse rate was low at 15%, and all occurred in pts who did not receive GO during induction or consolidation. Baseline kinase mutations (KIT/RAS/FLT3) on NGS was associated with a shorter RFS in our cohort. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Recombinase Polymerase Amplification Combined with Lateral Flow Strip for Rapid Detection of OXA-48-like Carbapenemase Genes in Enterobacterales.

Carbapenem-resistant Enterobacterales (CRE) possessing various carbapenemases, particularly the OXA-48 group, are now rapidly spreading and becoming a major public health concern worldwide. Phenotypic detection of OXA-48-like carbapenemases is still suboptimal due to their weak carbapenemase activity, whereas highly sensitive and specific polymerase chain reaction (PCR)-based methods take at least 3-4 h. We, therefore, developed a recombinase polymerase amplification (RPA) combined with lateral flow (LF) strip assay for the rapid detection of blaOXA-48-like in Enterobacterales. A total of 131 clinical isolates including 61 blaOXA-48-like-carrying Enterobacterales isolates and 70 Gram-negative bacilli isolates containing other bla genes were subjected to the RPA method performed under isothermal conditions at 37 °C within 10 min and visually inspected by LF strip within 5 min. The RPA-LF assay provided 100% sensitivity (95% confidence interval, 92.6-100%) and 100% specificity (93.5-100%) for detecting blaOXA-48-like genes from bacterial colonies. Its detection limit was 100 times less than that of the PCR method. This assay is rapid, easy to perform, and provides excellent performance without any special equipment. It may be applied for directly identifying the blaOXA-48-like genes in Enterobacterales obtained from blood culture. Rapid identification of carbapenemase types is essential for selecting appropriate antimicrobial options, particularly the β-lactams combined with novel β-lactamase inhibitors.

Gauging the skin resident Leishmania parasites through a loop mediated isothermal amplification (LAMP) assay in post-kala-azar dermal leishmaniasis

Despite the availability of highly sensitive polymerase chain reaction (PCR)-based methods, the dearth of remotely deployable diagnostic tools circumvents the early and accurate detection of individuals with post-kala-azar dermal leishmaniasis (PKDL). Here, we evaluate a design-locked loop-mediated isothermal amplification (LAMP) assay to diagnose PKDL. A total of 76 snip-skin samples collected from individuals with probable PKDL (clinical presentation and a positive rK39 rapid diagnostic test (RDT)) were assessed by microscopy, qPCR, and LAMP. An equal number of age and sex-matched healthy controls were included to determine the specificity of the LAMP assay. The LAMP assay with a Qiagen DNA extraction (Q-LAMP) showed a promising sensitivity of 72.37% (95% CI: 60.91–82.01%) for identifying the PKDL cases. LAMP assay sensitivity declined when the DNA was extracted using a boil-spin method. Q-qPCR showed 68.42% (56.75–78.61%) sensitivity, comparable to LAMP and with an excellent agreement, whereas the microscopy exhibited a weak sensitivity of 39.47% (28.44–51.35%). When microscopy and/or qPCR were considered the gold standard, Q-LAMP exhibited an elevated sensitivity of 89.7% (95% CI: 78.83–96.11%) for detection of PKDL cases and Bayesian latent class modeling substantiated the excellent sensitivity of the assay. All healthy controls were found to be negative. Notwithstanding the optimum efficiency of the LAMP assay towards the detection of PKDL cases, further optimization of the boil-spin method is warranted to permit remote use of the assay.

Monitoring of Oie-Listed Parasitic Pathogens in the Pacific Oyster (Crassostrea gigas) on the South Coast of Korea Using Species-Specific PCR

Outbreaks of oyster diseases caused by the World Organization for Animal Health (OIE)-listed protozoan pathogens have been a global concern for several decades. With the wide application of species-specific and sensitive polymerase chain reaction (PCR) techniques, it is now well known that even the Pacific oyster, insusceptible to Perkinsus marinus, Bonamia ostreae, Bonamia exitiosa could act as a carrier host. Furthermore, one of the transmission pathways of oyster pathogens includes anthropogenic activities, such as the discharge of ballast water or hull attachment during international maritime shipping. In this survey, OIE-listed pathogens, including B. ostreae, B. exitiosa, and Marteilia refringens were screened in the Pacific oysters growing in small bays off the south coast of Korea using PCR with OIE-recommended species- or genus-specific (Bonamia) primers. The survey sampled 1980 oysters from 35 sites in small bays in the south, where more than 95% of the national productions are yielded. The PCR screening indicated that none of the oysters screened in this study were positive for B. ostreae, B. exitiosa, P. marinus, and M. refringens. This study first investigated the OIE-listed protozoan pathogens in the Pacific oysters culturing in Korea, and the results indicated that the Pacific oysters raised on the south coast of Korea are free from those OIE-listed pathogens. For secure and sustainable oyster farming on the south coast, however, regular monitoring of such invasive oyster pathogens is recommended, as the international maritime trade activities are increasing in Korea.

Detection of porcine DNA in Korean processed foods by real-time PCR.

In this study, a commercial DNA extraction kit and a real-time Polymerase Chain Reaction (PCR) kit were applied to detect porcine DNA in Korean processed foods. The specificity, sensitivity, applicability and inter-laboratory reproducibility were evaluated for analytical method validation. We observed no false-positive or false-negative results in the assay, confirming its specificity. The sensitivity of the real-time PCR was expressed as the limit of detection (LOD), which was determined to be 0.005ng in 35 cycles. In the applicability test, PCR was performed on 12 types of food matrices, and there were no inhibitor effects. An interlaboratory comparison showed no statistically significant (p > 0.05) differences between the results from two laboratories. We analysed 131 samples to determine the presence of porcine DNA for halal certification: 129 samples were negative, and porcine DNA was detected in one sample each of instant noodles and dumpling. The real-time PCR applied in this study is a reliable analytical method to detect porcine DNA in food and can be used easily, quickly and routinely in food facilities.

Обнаружение особо опасных вирусных болезней рыб семейства лососёвых (Salmo salar) с применением полимеразной цепной реакции

Significant economic damage to aquaculture facilities and marine aquatic organisms is caused by viral diseases of fish, almost a quarter of the detected viruses cause diseases. Pathogens mainly fall during the transportation of infected fish from disadvantaged farms to "clean" aquatic farms, as well as from "escaped" fish from cages to the sea, where contamination of natural salmon and other aquatic organisms occurs. When delivering fish planting material to Russia from countries with different epizootological situations, constant monitoring and forecasting of possible risks is necessary. To prevent the spread of viral infections of fish, diagnostics and prevention occupy a leading place. Laboratory diagnostics of fish diseases of viral etiology is based on the isolation of the pathogen and its identification by serological methods that require a long time and research in specialized specialized laboratories of large research institutes. More effective and highly sensitive are molecular diagnostic methods, in particular, polymerase chain reaction (PCR) with reverse transcription to detect a number of particularly dangerous viral diseases of fish. Infectious necrosis of hematopoietic tissue, infectious pancreatic necrosis, viral hemorrhagic septicemia and infectious anemia of salmon were identified by highly specific and sensitive diagnostic PCR method. The improved technique of PCR with reverse transcription makes it possible to detect pathogens in salmon and other fish both with obvious clinical signs and with hidden virus carrier.

A diagnostic test of real-time PCR detection in the diagnosis of clinical bloodstream infection

Blood culture remains the standard for diagnosing bloodstream infections, but it is difficult to identify bacteria directly and timeliness. The real-time polymerase chain reaction (PCR) has the potential to fill this diagnostic gap. This study intends to explore the sensitivity and specificity of PCR in detecting bloodstream infection pathogens and to compare it with routine blood culture to explore its clinical application value. A total of 126 patients with bloodstream infections collected from various clinical departments of The First Hospital of Hebei Medical University. The patient's sample was divided into two parts. The one for multiplex PCR detection was performed using the Pathogeno Elite Multiplex PCR kit. Another blood culture was a fully automatic blood culture system from Autobio company. Among the 126 patients, a total of 17 pathogens were detected by PCR and blood culture both methods. PCR detected a total of 43 positive samples and 83 negative samples. Five samples were positive with blood culture, and 81 were negative. The negative predictive value of PCR was 0.98, with a sensitivity of 0.71 and a specificity of 0.68. A total of 38 specimens were positive for PCR but negative for blood culture, and 2 samples were positive for blood culture but negative for PCR. The top 5 pathogens with PCR detection were Epstein-Barr virus (27 cases), Human herpes virus 5 (9 cases), Klebsiella pneumoniae (5 cases), Staphylococcus (5 cases), and Stenotrophomonas maltophilia (4 cases). PCR detection can rapidly identify more pathogens and even multi-pathogen infections. Therefore, PCR testing may improve pathogen detection in patients with suspected bloodstream infections, enabling targeted treatment of patients.

Evaluation of Fine-needle Aspiration Cytology (FNAC) Sensitivity Compared to PCR for Diagnosing Tuberculosis Lymphadenitis

Background: Tuberculosis (TB) is a major healthcare burden in Sudan and other developing countries, it is considered the second most common cause of death from infectious diseases after those due to AIDS. In Sudan, TB lymphadenitis (TBLA) remains one of the major health problems. This descriptive cross-sectional study was conducted at the University of Medical Sciences and Technology (UMST) and Total Labcare Diagnostic Center (TDC). The study aims to compare the sensitivity of Fine Needle Aspiration Cytology (FNAC) smears with that of the Polymerase Chain Reaction (PCR) for the diagnosis of TBLA.&#x0D; Materials and Methods: Fifty-five dry smears were obtained using fine-needle aspiration (FNA) from an enlarged lymph node. PCR was applied to detect the target gene (IS6110). May-Grunwald-Giemsa (MGG) or Diff quick stains were used.&#x0D; Results: Two (4%) patients with TBLA were non-necrotic, while fifty-three of them (96%) were necrotic. Moreover, 17 (30%) fine-needle lymph node aspiration specimens were confirmed by PCR to be positive for Mycobacterium tuberculosis complex (MTB complex) while 38 (70%) of them were negative.&#x0D; Conclusion: There was no significant difference between the sensitivity of PCR and that of FNAC (P-value = 0.33).