Comparative assessment of two in-house-built isothermal assays for visual detection of African swine fever virus.

Owing to the lack of effective vaccines, current control measures and eradication strategies for the African swine fever virus (ASFV) rely on early detection and stringent stamping-out procedures. In the present study, we developed two independent isothermal amplification assays, namely, loop-mediated isothermal amplification (LAMP) and polymerase spiral reaction (PSR), for quick visualization of the ASFV genome in clinical samples. Additionally, a quantitative real-time PCR (qRT-PCR)-based hydrolysis probe assay was developed for comparative assessment of sensitivity with the developed isothermal assays. The analytical sensitivity of the LAMP, PSR, and qRT-PCR was found to be 2.64 ×105 copies/µL, 2.64 ×102 copies/µL, and 2.64 ×101 copies/µL, respectively. A total of 165 clinical samples was tested using the developed visual assays. The relative accuracy, relative specificity, and relative diagnostic sensitivity for LAMP vs PSR were found to be 95.37% vs 102.48%, 97.46% vs 101.36%, and 73.33% vs 113.33%, respectively.

Application of LAMP assay for detection of carbapenem-resistant Acinetobacter calcoaceticus-Acinetobacter baumannii complex in ICU admitted sepsis patients: A rapid and cost-effective diagnostic tool.

Carbapenem-resistant significant members of Acinetobacter calcoaceticus-Acinetobacter baumannii (CR-SM-ACB) complex have emerged as an important cause of sepsis, especially in ICUs. This study demonstrates the application of loop-mediated-isothermal-amplification (LAMP) assay for detection of CR-SM-ACB-complex from patients with sepsis. Whole-blood and culture-broths(CB) collected from patients with culture-positive sepsis were subjected to LAMP and compared with PCR, and RealAmp. Vitek-2 system and conventional PCR results were used as confirmatory references. The sensitivity and specificity of LAMP(97 % & 100 %) and RealAmp(100 % & 100 %) for detection of CR-SM-ACB-complex from CB were better than PCR(87 % & 100 %). Diagnostic accuracy of LAMP, RealAmp, and PCR for detection of SM-ACB-complex from CB was 98.5 %, 100 %, and 88.5 % respectively. Turnaround time of Culture, LAMP, PCR, and RealAmp was 28-53, 6-20, 9-23, and 6-20hours, respectively. LAMP is a simple, inexpensive tool that can be applied directly to positive CB and may be customized to detect emerging pathogens and locally-prevalent resistance genes and to optimize antimicrobial use.

Parvalbumin Gene: A Valuable Marker for Pike Authentication and Allergen Risk Assessment.

Fish from the pike (Esox) genus are valued in gastronomy for their superior meat quality. However, they can cause allergic reactions in sensitive consumers. This work aimed to fill the gap in the detection of pike allergens using molecular-biological techniques. New, fast, and accurate loop-mediated isothermal amplification (LAMP) and real-time PCR (qPCR) assays were designed to detect pike DNA using the parvalbumin gene as a marker. LAMP was assessed by electrophoresis, SYBR green optical detection, and real-time fluorescence detection. The latter was the most sensitive, detecting as little as 0.78 ng of pike DNA; the qPCR detection limit was 0.1 ng. The LAMP analysis took 20-70 min, which is significantly faster than qPCR. The study provides reliable detection and quantification of the parvalbumin gene in both fresh and processed samples and further highlights the versatility of the use of the parvalbumin gene for the authentication of food products and consumer protection via refined allergen risk assessment that is independent of the type of tissue or food processing method used.

Development of a new loop-mediated isothermal amplification test for the sensitive, rapid, and economic detection of different genotypes of Classical swine fever virus.

Background: Classical swine fever virus (CSFV) remains one of the most important pathogens in animal health. Pathogen detection relies on viral RNA extraction followed by RT-qPCR. Novel technologies are required to improve diagnosis at the point of care. Methods: A loop-mediated isothermal amplification (LAMP) PCR technique was developed, with primers designed considering all reported CSFV genotypes. The reaction was tested using both fluorometric and colorimetric detection, in comparison to the gold standard technique. Viral strains from three circulating CSFV genotypes were tested, as well as samples from infected animals. Other pathogens were also tested, to determine the LAMP specificity. Besides laboratory RNA extraction methods, a heating method for RNA release, readily available for adaptation to field conditions was evaluated. Results: Three primer sets were generated, with one of them showing better performance. This primer set proved capable of maintaining optimal performance at a wide range of amplification temperatures (60°C - 68°C). It was also able to detect CSFV RNA from the three genotypes tested. The assay was highly efficient in detection of samples from animals infected with field strains from two different genotypes, with multiple matrices being detected using both colorimetric and fluorometric methods. The LAMP assay was negative for all the unrelated pathogens tested, including Pestiviruses. The only doubtful result in both fluorometric and colorimetric LAMP was against the novel Pestivirus italiaense, ovine Italy Pestivirus (OVPV), which has proven to have cross-reaction with multiple CSFV diagnostic techniques. However, it is only possible to detect the OVPV in a doubtful result if the viral load is higher than 10000 viral particles. Conclusion: The results from the present study show that LAMP could be an important addition to the currently used molecular diagnostic techniques for CSFV. This technique could be used in remote locations, given that it can be adapted for successful use with minimal equipment and minimally invasive samples. The joined use of novel and traditional diagnostic techniques could prove to be a useful alternative to support the CSF control.

External quality assessment for molecular detection of Ureaplasma urealyticum in China.

BACKGROUND AND AIMS: A pilot external quality assessment (EQA) scheme for molecular detection of Ureaplasma urealyticum (UU) was conducted by the National Center for Clinical Laboratories (NCCL) to evaluate the testing capabilities of clinical laboratories and the actual performance of DNA-based nucleic acid amplification tests (NAAT) and RNA-based NAATs when applied in clinical settings. MATERIALS AND METHODS: The EQA panel contained twelve lyophilized samples, including positive samples containing inactivated cell culture supernatants of UU at different concentrations and sterile saline for negative samples. The positive samples were further divided into three groups of high, moderate and low concentrations. The panels were distributed to the participants and the datasets were analyzed according to the qualitative results. RESULTS: A total of 365 laboratories participated in the EQA scheme, and 360 results submitted by 338 laboratories were collected, of which 96.11 % (346/360) of the returned results and 95.86 % (324/338) of the laboratories were deemed competent. The positive percentage agreement (PPA) was ≥ 97.5 % for high and moderate concentration samples, but varied significantly for low concentration samples, decreasing from 86.94 % to 51.94 % as the sample concentration decreased. Additionally, for low concentration samples, RNA-based NAAT showed higher PPAs than DNA-based NAATs, but these results were specific to UU supernatants used in this study. CONCLUSION: Most of UU detection assays employed by the participants were generally consistent with their estimated limit of detection (LOD), and the majority of participants can reliably detect UU samples with high and moderate concentrations, while the poor analytical performance for low concentration samples requires further improvement and optimization.

External quality assessment survey for SARS-CoV-2 nucleic acid amplification tests in clinical laboratories in Tokyo, 2021.

INTRODUCTION: Nucleic acid amplification tests (NAATs) play a pivotal role in clinical laboratories for diagnosing COVID-19. This study aimed to elucidate the accuracy of these tests. METHODS: In 2021, an external quality assessment of NAATs for SARS-CoV-2 was conducted in 47 laboratories in Tokyo, Japan. In open testing, where the laboratories knew that the samples were intended for the survey, a simulated nasopharyngeal swab suspension sample was used, featuring a positive sample A with a viral concentration of 50 copies/µL, positive sample B with 5 copies/µL, and a negative sample. Laboratories employing real-time RT-PCR were required to report cycle threshold (Ct) values. In blind testing, where the samples were processed as normal test samples, a positive sample C with 50 copies/µL was prepared using a simulated saliva sample. RESULTS: Of the 47 laboratories, 41 were engaged in open testing. For sample A, all 41 laboratories yielded positive results, whereas for sample B, 36 laboratories reported positive results, 3 laboratories reported "test decision pending", 1 laboratory reported "suspected positive", and 1 laboratory did not respond. All 41 laboratories correctly identified the negative samples as negative. The mean Ct values were 32.2 for sample A and 35.2 for sample B. In the blind test, six laboratories received samples. Sample C was identified as positive by five laboratories and negative by one laboratory. CONCLUSIONS: The nature of the specimen, specifically the saliva, may have influenced the blind test outcomes. The identified issues must be meticulously investigated and rectified to ensure accurate results.

Enhancing malaria detection in resource-limited areas: A high-performance colorimetric LAMP assay for Plasmodium falciparum screening.

Malaria eradication efforts in resource-limited areas require a rapid, economical, and accurate tool for detecting of the low parasitemia. The malaria rapid diagnostic test (mRDT) is the most suitable for on-site detection of the deadliest form of malaria, Plasmodium falciparum. However, the deletions of histidine rich protein 2 and 3 genes are known to compromise the effectiveness of mRDT. One of the approaches that have been explored intensively for on-site diagnostics is the loop-mediated isothermal amplification (LAMP). LAMP is a one-step amplification that allows the detection of Plasmodium species in less than an hour. Thus, this study aims to present a new primer set to enhance the performance of a colorimetric LAMP (cLAMP) for field application. The primer binding regions were selected within the A-type of P. falciparum 18S rRNA genes, which presents a dual gene locus in the genome. The test result of the newly designed primer indicates that the optimal reaction condition for cLAMP was 30 minutes incubation at 65°C, a shorter incubation time compared to previous LAMP detection methods that typically takes 45 to 60 minutes. The limit of detection (LoD) for the cLAMP using our designed primers and laboratory-grown P. falciparum (3D7) was estimated to be 0.21 parasites/µL which was 1,000-fold higher than referencing primers. Under optimal reaction condition, the new primer sets showed the sensitivity (100%, 95% CI: 80.49-100%) and specificity (100%, 95% CI: 94.64-100%) with 100% (95% CI: 95.70-100%) accuracy on the detection of dried blood spots from Malawi (n = 84). Briefly, the newly designed primer set for P. falciparum detection exhibited high sensitivity and specificity compared to referenced primers. One great advantage of this tool is its ability to be detected by the naked eye, enhancing field approaches. Thus, this tool has the potential to be effective for accurate early parasite detection in resource-limited endemic areas.

Assessment of measurement accuracy of amplified DNA using a colorimetric loop-mediated isothermal amplification assay.

A colorimetric loop-mediated isothermal amplification assay detects changes in pH during amplification based on color changes at a constant temperature. Currently, various studies have focused on developing and assessing molecular point-of-care testing instruments. In this study, we evaluated amplified DNA concentrations measured using the colorimetric LAMP assay of the 1POT™ Professional device (1drop Inc, Korea). Results of the 1POT analysis of clinical samples were compared with measurements obtained from the Qubit™ 4 and NanoDrop™ 2000 devices (both from Thermo Fisher Scientific, MA, USA). These results showed a correlation of 0.98 (95% CI: 0.96-0.99) and 0.96 (95% CI: 0.92-0.98) between 1POT and the Qubit and NanoDrop. 1POT can measure amplified DNA accurately and is suitable for on-site molecular diagnostics.

Teste de amplificação de ácidos nucléicos (NAT) para detecção dos vírus da imunodeficiência humana (HIV) e da hepatite (HCV) / Nucleic acid amplification test (NAT) for the detection of human immunodeficiency virus (HIV) and hepatitis (HCV)

O NAT é uma tecnologia desenvolvida para a detecção do ácido nucléico do Vírus da Imunodeficiência Humana - HIV e do Vírus da Hepatite C - HCV, em bolsas de sangue destinadas à transfusão. Os testes desenvolvidos estão sendo implantados com o propósito de identificar os ácidos ribonucléicos (RNA) desses vírus previamente aos testes sorológicos convencionais, além de identificá-los em bolsas doadas com níveis de anticorpos indetectáveis pelos testes sorológicos tradicionais, promovendo a redução do período denominado janela imunológica - período compreendido entre o contato com o antígeno hemotransmissível e a produção de anticorpos em níveis detectáveis pelos testes sorológicos atuais. A implementação da tecnologia NAT para triagem em bancos de sangue reduz o risco de transmissão de agentes virais transmissíveis por transfusão, como HIV e HCV, uma vez que é possível a detecção mais precocemente dos antígenos em doações realizadas durante o período posterior a soro-conversão, porém, ainda em janela imunológica para sorologia. Diferentemente do teste de sorologia, o NAT não detecta a presença de anticorpos e sim do material genético do vírus, reduzindo a janela imunológica no caso do HIV de 19-22 dias para 10 dias e HCV de 60 dias para 11 dias. O NAT é complementar a sorologia e não possui a capacidade de substituí-la uma vez que com a progressão da infecção a carga viral tende a ficar em alguns momentos indetectável. Cabe ressaltar que a total garantia de detecção de agentes infecciosos não é proporcionada por nenhum teste utilizado, devido ao período denominado eclipse viral, ou latência, presente nos ciclos do HIV e HCV, não sendo possível detectar a presença de antígenos e nem anticorpos circulantes, uma vez que o vírus apresenta-se com replicação local intracelular e o organismo ainda não produziu anticorpos para o agente infeccioso. O Kit NAT HIV/HCV Brasileiro, desenvolvido com tecnologia totalmente nacional, tem capacidade de processar 96 reações, destas duas são controles negativos, duas controles positivos, ambos amplificam HIV e HCV, e 92 reações que podem ser processadas em minipool de seis amostras, permitindo assim o processamento de 552 amostras em uma única rotina. Caso algum minipool de seis apresente resultado positivo, as seis amostras que o compõem deverão ser processadas em uma próxima rotina separadamente ("single") para identificação da amostra positiva. Com base nos critérios de: expressivo quantitativo de amostras testadas, maior estrutura da rede de serviços públicos no estado e melhor logística e organização, em relação à centralização dos testes sorológicos no Hemocentro Coordenador, o Ministério da Saúde definiu, visando a economicidade e o melhor aproveitamento dos kits produzidos por Bio-Manguinhos, 14 serviços de hemoterapia listados acima como sítios testadores NAT, os quais centralizarão amostras dos demais Estados, com logística de transporte interestadual de amostras NAT oferecido pelo Ministério da Saúde. Para atender os avanços da tecnologia NAT, a fim de garantir maior segurança tranfusional e a realização dos testes no âmbito nacional, a Coordenação Geral de Sangue e Hemoderivados solicitou a inclusão, na tabela de ressarcimento SIA/SUS, dos custos diretos e indiretos do procedimento, excetuando-se o kit NAT para HIV e HVC fornecidos aos centros testadores por Biomanguinhos. Tais custos referem-se a gastos como: energia elétrica, recursos humanos, logística de transporte de amostras e demais insumos necessários a realização do teste. Os membros da CONITEC presentes na reunião do plenário do dia 07/12/2012 deliberaram, por unanimidade, por recomendar a incorporação do procedimento para possibilitar a testagem de amostra de sangue de doadores pelo teste de amplificação de ácidos nucléicos (NAT) para detecção dos vírus da imunodeficiência humana (HIV) e da hepatite C (HCV) no âmbito do Sistema Nacional de Sangue, Componentes e Hemoderivados. A Portaria CTIE-MS N.º 25, de 12 de junho de 2013 - Toma Decisão de incorporar o procedimento para possibilitar a testagem de amostra de sangue de doadores pelo teste de amplificação de ácidos nucleicos (NAT) para detecção dos vírus da imunodeficiência humana (HIV) e da hepatite C (HCV) no âmbito do Sistema Nacional de Sangue, Componentes e Hemoderivados no Sistema Único de Saúde - SUS.