Specific Real-time Polymerase Chain Reaction Research Articles

TaqMan-based quantitative real-time polymerase chain reaction assay to detect porcine circovirus-like virus.

The aim of this study was to develop a rapid, sensitive and highly specific TaqMan quantitative real-time polymerase chain reaction PCR (qPCR) assay for porcine circovirus-like virus (PCLV). The primers and probe were designed based on the conserved regions of the PCLV ORF4 gene. The assay has a good detection performance (y=-3.3257x+ 1.482, R2=0.9905), with a limit of detection of 10 copies, which was 100 times more sensitive than conventional PCR (cPCR). No cross-reactivity was observed with other common viruses. The intra- and inter-assay coefficients of variation were less than 1.25%. 36 fecal samples were analyzed using this method, detecting a positivity rate of 8.33% (3/36) that was higher than the cPCR detected. In summary, the established assay for PCLV detection has high specificity, sensitivity, and reproducibility and can be used as a tool for clinical diagnosis and epidemiological investigation.

Genomic Characterization of SARS-CoV-2 Isolates Obtained from Antalya, Türkiye

As the number of coronavirus diseases-2019 (COVID-19) cases have decreased and measures have started to be implemented at an individual level rather than in the form of social restrictions, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) still maintains its importance and has already taken its place in the spectrum of agents investigated in multiplex molecular test panels for respiratory tract infections in routine diagnostic use. In this study, we aimed to present mutation analysis and clade distribution of whole genome sequences from randomly selected samples that tested positive with SARS-CoV-2 specific real-time reverse transcription polymerase chain reaction (rRT-PCR) test at different periods of the pandemic in our laboratory with a commercial easy-to-use kit designed for next-generation sequencing systems. A total of 84 nasopharyngeal/oropharyngeal swab samples of COVID-19 suspected patients which were sent for routine diagnosis to the medical microbiology laboratory and detected as SARSCoV-2 RNA positive with rRT-PCR were randomly selected from different periods for sequence analysis. Library preparation for sequencing was performed with the commerical EasySeq SARS-CoV-2 RC PCR kit (Nimagen, the Netherlands). The data generated from the Illumina MiSeq system (Illumina Inc, San Diego, CA, USA) were analysed using CLC Genomics Workbench (CLC, Qiagen, Hilden, Germany). Nextstrain clades detected in order of frequency were 21J (Delta) (25%, n= 21), 21L (Omicron) (23.8%, n= 20), 20B (19%, n= 16), 20A (15.5%, n= 13), 21K (Omicron) (11.9%, n= 10), 19A (3.6%, n= 3), and 22B (Omicron) (1.2%, n= 1). Excluding one patient sample which was identified as 22B (Omicron), a total of 2829 common distinct mutations (2076 missense, 551 synonymous, 192 deletions and 10 insertions) were detected. 100 mutations were observed in the non-coding 5' untranslated region (UTR). The majority of the mutations were located in the Spike gene region (1120 mutations), followed by the ORF1a (624 mutations), nucleocapside (315 mutations) and ORF1b (263 mutations) gene regions. Sampling times of the patients were March 2020 (n= 1), April 2020 (n= 11), May 2020 (n= 1), June 2020 (n= 2), July 2020 (n= 3), August 2020 (n= 1), September 2020 (n= 5), November 2020 (n= 2), December 2020 (n= 6), December 2021 (n= 19), January 2022 (n= 11), March 2022 (n= 16), April 2022 (n= 3), and June 2022 (n= 3). As a result, in this study, SARS-CoV-2 variants and mutations in the Mediterranean Region of Türkiye, Antalya province were analyzed in detail. To the best of our knowledge, no SARS-CoV-2 genome analysis study from the pandemic period has been reported in our province. When the sequences from our study were uploaded to the GISAID Instant Audacity system and the related genomes obtained from different countries in the EpiCoV database metadata were examined, the top two countries in terms of similarity and which could be associated with the main entry route of the virus into Türkiye were Germany and the United Kingdom. In today's world, where it is discussed what can be done to be prepared for possible new pandemics based on the COVID-19 pandemic, the importance of being proactive in molecular surveillance studies is indisputable. Developing countries should be supported and encouraged to research new variants and share data in addition to known variants in pandemic control. At this point, we believe that past pandemic data reported from different geographical regions will also be valuable in terms of predicting the circulation network and taking precautions in a possible new pandemic.

A highly sensitive and specific real-time quantitative polymerase chain reaction assay for Perkinsus marinus detection in oysters

Oyster aquaculture is one of the fastest-growing food production industries worldwide; however, it faces a significant challenge from the protist Perkinsus marinus, particularly in the USA. Although several quantitative molecular diagnostic methodologies are available for identifying diseases caused by P. marinus, the primer pairs used therein led to non-specific identification of other Perkinsus spp. Hence, a quantitative real-time PCR (Pm-qPCR) assay specific for P. marinus was developed using a TaqMan-based probe with the internal quencher in this study. A primer pair and probe specific to P. marinus were designed from a hypothetical protein of P. marinus collected from the whole-genome shotgun sequence database of the National Center for Biotechnology Information (NCBI). In silico analysis using homologous sequences of P. olseni and P. chesapeaki confirmed the high specificity of primers designed in this study. The Pm-qPCR assay was performed using seven different strains of P. marinus, P. olseni, and P. chesapeaki, revealing high specificity and sensitivity for detecting only P. marinus strains. In conclusion, it was demonstrated that Pm-qPCR can effectively and accurately diagnose P. marinus with high specificity and sensitivity. This assay is promising for monitoring oyster health and disease management in ecosystems and aquaculture.

Monkeypoxvirus (MPXV) in a Baby Monkey - Molecular Investigation

Background: Monkeypox is a zoonotic virus that transmits from animal to human and human to human. Despite the fact that the Monkeypox virus (MPXV) was initially isolated in 1958 and the first human case was reported in 1970, thousands of cases have been documented in European countries, the United States, and Turkey in recent years. The goal of this work was to present the results of the first molecular diagnostic analysis of MPXV-induced skin lesions in a baby monkey in Turkey. Case: In 2019, a baby monkey in Antalya Zoo developed skin lesions after being brought from Africa by a female monkey. The baby monkey died within a few weeks due to postnatal respiratory problems. Skin lesions were taken from this case and analyzed molecularly and virologically at the Department of Pathology, Burdur Mehmet Akif Ersoy University Faculty of Veterinary Medicine, and the Department of Virology. To perform molecular diagnosis, skin and pock lesions developed after inoculation on the chorioallantoic membrane (CAM) of the embryonated chicken eggs (ECE) were extracted, and 25 parapoxvirus (orf virus) type-specific primers and 2 MPXV type-specific primers were examined. The inoculum was prepared from the skin lesions for virus isolation, which was carried out in Vero cell culture. Then, the virus titer was determined using the microtitration method. The polymerase chain reaction (PCR) test with 25 parapoxvirus (orf virus) type-specific primers on the extracted skin lesion samples did not detect the presence of viral genome. The presence of viral genome was detected in 1 of the 2 MPXV type-specific primers acidophilic-type inclusion body (ATI gene) in the skin lesion extracts. However, the presence of viral genome could not be determined by the Gabon (1/2) primers. Cytopathological effects (CPE) were observed 72 h. after inoculating the skin lesion inoculum in Vero cells. The virus titer was determined to be 1022 TCID50 /mL. During immunohistochemical examination with orf virus antibodies, positive reaction was observed in the epidermal cells. Discussion: The researchers have been investigated the reservoir or natural hosts of MPXV. The virus has been found in squirrels, rodents, monkeys and chimpanzees. Multiple examinations have revealed that a number of animal species, primarily rodents and nonhuman primates, are susceptible to the virus. The presence of MPXV was detected in the skin lesion of a baby monkey in the study. Capripoxvirus, Cervidpoxvirus, Avipoxvirus, Molluscipoxvirus, Orthopoxvirus, Leporipoxvirus, Suipoxvirus, Yatapoxvirus, and Parapoxvirus are all members of the Chordopoxvirinae subgroup. However, no relationship between MPXV and parapoxvirus (25 type-specific primers) was found in the extracted skin lesion samples. Virus isolation, electron microscopy, (PCR), IgM and IgG ELISA, immunofluorescence assays, and histopathological examination are all laboratory diagnostic procedures that can be used to diagnose monkeypox infections. To detect the MPXV agent and/or specific viral DNA sequences, real-time or traditional PCR techniques should be utilized. Hemagglutinin, ATI gene, and the crmB gene are all MPXV genes which are commonly used for conventional PCR testing. A MPXV type-specific primer (ATI gene) observed the presence of viral genome in the skin lesion extract. After inoculating the skin lesion inoculum in Vero cells, (CPE) were observed. The virus titer was found to be extremely high. Positive reactions were seen in epidermal cells during immunohistochemical testing with orf virus antibodies. Keywords: Monkeypox virus, molecular diagnosis, virological diagnosis, immunohistochemistry, Parapoxvirus.

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.

The association of PON1 and NOS3 genetic variants with the severity of COVID-19

The pandemic of coronavirus disease 2019 (COVID-19) is a global pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The aim of this study was to investigate the association of single nucleotide polymorphisms (SNPs) of paraoxonase 1(PON1) and nitric oxide synthase 3 (NOS3) genes with the severity of COVID-19. Study subjects were divided into mild and severe groups. Genotyping was conducted by allele specific real-time polymerase chain reaction (RT-PCR). Gene-gene interaction was assessed by multifactor dimensionality reduction (MDR) analysis. The results showed a significant association of both PON1 rs662 (A575G), and NOS3 rs2070744 (T786C) with the severity of COVID-19 (p = 0.0026, and p = 0.033, respectively). PON1 575AG (OR = 0.30, 95 % CI [0.13–0.68]) and NOS3 786TC (OR = 0.35, 95 % CI [0.15–0.78]) were associated with mild COVID-19 symptoms. The resulted SNP-SNP interaction model by MDR analysis was statistically significant (p = 0.0003). NOS3 786C * PON1 575G haplotype was significantly associated with mild cases of COVID-19 (p = 0.045).

Development of a specific real-time PCR assay for simultaneous detection and differentiation of Coxiella burnetii strains from environmental soil samples.

Coxiella burnetii, the causative agent of Q fever, is a small, coccoid, Gram-negative strict intracellular pathogen. One of the most common ways of acquiring Q fever is through inhalation of aerosols containing the bacteria. Because C. burnetii is highly infectious, spreads easily through the air, and is very resistant to environmental conditions, it is considered a biological threat. This paper presents the development and validation of a specific real-time polymerase chain reaction (real-time PCR or qPCR) assay for the detection of C. burnetii, based on the amplification of a fragment of the isocitrate dehydrogenase (icd) encoding gene. This real-time PCR is highly specific, reproducible, and sensitive, allowing the detection of as few as 5 genome equivalents (GEs) of C. burnetii per reaction. The method enables a rapid preliminary differentiation among strains, based on a point mutation at nucleotide 745 of the icd gene. The assay was successfully evaluated in environmental soil samples; a limit of detection of 3×104 colony forming units per 0.5g of soil (∼3 GEs per reaction) was achieved. The newly developed real-time PCR offers a valuable tool for differential detection of C. burnetii strains in environmental soil samples.

Distribution of Genotypes of Hepatitis C Virus Studied in a Tertiary Care Hospital in Uttarakhand, India: A Retrospective Study

Introduction: With more than one million cases of Hepatitis C viral infection reported annually, it has become a major global problem. Hepatitis C Virus (HCV) is classified into 7 genotypes and 67 subtypes. Various genotypes show a wide geographical distribution worldwide. Genotypes 1 and 3 are the most common genotypes reported from India. There is minimal data available on the distribution and frequency of HCV genotypes in Uttarakhand. Aim: This three years retrospective study was undertaken to find out the distribution pattern of HCV genotypes among HCV- infected patients attending a tertiary care hospital in this Sub- Himalayan state of Uttarakhand. Materials and Methods: A total of 306 samples were tested for HCV genotyping in 3 years duration i.e., January 2018 to December 2020 at the Molecular Laboratory of Shri Guru Ram Rai Institute of Medical and Health sciences and associated Shri Mahant Indiresh Hospital, Dehradun. Genotyping was performed using HCV genotype specific Real-Time Polymerase Chain Reaction (RT-PCR) assay on Rotor-Gene Q using TRUPCR HCV Genotyping Kit (3B BlackBio Biotech India Ltd.,) as per manufacturer’s instructions. This test kit detects HCV genotypes 1, 1a, 2 (2a/ 2b), 3, 4, 5a, and 6. Results: Genotype 3 (72.7%) was the commonest genotype detected, followed by genotype 1 (13%), genotype 1a (6.25%) and genotype 4 (2.8%). Genotype 3 was the most dominant genotype in all the 3 years of the study. Two cases of genotype 5a (1.13%) and three cases of genotype 6 (1.7%) were also detected in our study. We also detected a case of mixed infection by genotype 1 and genotype 6. Conclusion: The most common genotype detected was three during the study period. Detection of genotype 5a and 6 ascertained the impact of interstate migration of people.

A Rapid and Specific Real-Time PCR Assay for the Detection of Clinically Relevant Mucorales Species

Infections triggered by filamentous fungi placed in the order Mucorales, phylum Zygomycota, can cause serious harm to immunocompromised patients. Since there is lack of a standardized PCR (polymerase chain reaction) assay for early diagnosis of this fungal infection, this work was aimed to develop a new PCR assay able to detect the presence of Mucorales genera in clinical specimens. Here, we describe a novel diagnostic TaqMan MGB probe assay for precise and rapid detection of the most common clinical species of Mucorales. Zygomycete-specific oligonucleotides were designed to specifically amplify and bind highly conserved sequences of fungal 28S rRNA gene. Additionally, we succeeded in differentiating Mucorales species (i.e., Rhizopus, Lichtheimia, Mucor, and Rhizomucor) in artificially infected serum samples, suggesting that the quantitative capability of this real-time PCR assay could potentially optimize the diagnosis of mucormycosis.

Ulceroglandular form of tularemia after squirrel bite: a case report

BackgroundThe diagnosis of tularemia is not often considered in Germany as the disease is still rare in this country. Nonetheless, Francisella tularensis, the causative agent of tularemia, can infect numerous animal species and should, therefore, not be neglected as a dangerous pathogen. Tularemia can lead to massively swollen lymph nodes and might even be fatal without antibiotic treatment. To our knowledge, the case described here is the first report of the disease caused by a squirrel bite in Germany.Case presentationA 59-year-old German woman with a past medical history of hypothyroidism and cutaneous lupus erythematosus presented at the emergency room at St. Katharinen Hospital with ongoing symptoms and a swollen right elbow persisting despite antibiotic therapy with cefuroxime for 7 days after she had been bitten (right hand) by a wild squirrel (Eurasian red squirrel). After another 7 days of therapy with piperacillin/tazobactam, laboratory analysis using real-time polymerase chain reaction (PCR) confirmed the suspected diagnosis of tularemia on day 14. After starting the recommended antibiotic treatment with ciprofloxacin, the patient recovered rapidly.ConclusionThis is the first report of a case of tularemia caused by a squirrel bite in Germany. A naturally infected squirrel has recently been reported in Switzerland for the first time. The number of human cases of tularemia has been increasing over the last years and, therefore, tularemia should be taken into consideration as a diagnosis, especially in a patient bitten by an animal who also presents with headache, increasing pain, lymphadenitis, and fever, as well as impaired wound healing. The pathogen can easily be identified by a specific real-time PCR assay of wound swabs and/or by antibody detection, for example by enzyme-linked immunosorbent assay (ELISA), if the incident dates back longer than 2 weeks.

PMMA Microcapsules for the Inactivation of SARS-CoV-2

Surface disinfection currently plays a decisive role in the epidemiological situation caused by the SARS-CoV-2 coronavirus. However, most disinfection products available on the market have a high evaporation rate and only an immediate action and not continuous, creating the need for a high frequency of disinfection. To overcome this limitation, in the present work, poly(methyl methacrylate) (PMMA) microcapsules were developed with an active agent (hydrogen peroxide) encapsulated, which has the ability to inactivate/neutralize the SARS-CoV-2 virus. PMMA-H2O2 microcapsules have a spherical shape and a smooth structure with low porosity and were successfully attached to nonwoven fabrics, as observed from scanning electron microscopy. The thermogravimetric analysis shows that PMMA-H2O2 microcapsules have high thermal stability and can increase the stability of H2O2. Nonfabric substrates functionalized with PMMA-H2O2 microcapsules were tested by a highly sensitive and specific reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR)-based method to evaluate antiviral activity through the degradation of SARS-CoV-2 deoxyribonucleic acids. The highest percentage of viral nucleic acid elimination was obtained when exposing the viral sample for 1 h to PMMA-H2O2 microcapsules, resulting in an elimination of >97% of the coronavirus. In addition, the microcapsules are stable over a period of three weeks and retain the ability to eliminate SARS-CoV-2. Hence, it is demonstrated that this microcapsule system is efficient for SARS-CoV-2 elimination and inherent surface disinfection.

Profile of the Nasopharyngeal Microbiota Affecting the Clinical Course in COVID-19 Patients.

While populations at risk for severe SARS-CoV-2 infections have been clearly identified, susceptibility to the infection and its clinical course remain unpredictable. As the nasopharyngeal microbiota may promote the acquisition of several respiratory infections and have an impact on the evolution of their outcome, we studied the nasopharyngeal microbiota of COVID-19 patients in association with baseline disease-related clinical features compared to that of patients tested negative. We retrospectively analyzed 120 nasopharyngeal pseudonymized samples, obtained for diagnosis, divided into groups (infected patients with a favorable outcome, asymptomatic, and deceased patients) and patients tested negative for SARS-CoV-2, by using Illumina-16S ribosomal ribonucleic acid (rRNA) sequencing and specific polymerase chain reaction (PCR) targeting pathogens. We first found a depletion of anaerobes among COVID-19 patients, irrespective of the clinical presentation of the infection (p < 0.029). We detected 9 taxa discriminating patients tested positive for SARS-CoV-2 from those that were negative including Corynebacterium propinquum/pseudodiphtericum (p ≤ 0.05), Moraxella catarrhalis (p ≤ 0.05), Bacillus massiliamazoniensis (p ≤ 0.01), Anaerobacillus alkalidiazotrophicus (p ≤ 0.05), Staphylococcus capitis subsp. capitis (p ≤ 0.001), and Afipia birgiae (p ≤ 0.001) with 16S rRNA sequencing, and Streptococcus pneumoniae (p ≤ 0.01), Klebsiella pneumoniae (p ≤ 0.01), and Enterococcus faecalis (p ≤ 0.05) using real-time PCR. By designing a specific real-time PCR, we also demonstrated that C. propinquum is decreased in asymptomatic individuals compared to other SARS-CoV 2 positive patients. These findings indicate that the nasopharyngeal microbiota as in any respiratory infection plays a role in the clinical course of the disease. Further studies are needed to elucidate the potential role in the clinical course of the disease of M. catarrhalis, Corynebacterium accolens, and more specifically Corynebacterium propinquum/diphteriticum in order to include them as predictors of the severity of COVID-19.

The use of combined PCR, fluorescence in situ hybridisation and immunohistochemical staining to diagnose mucormycosis from formalin-fixed paraffin-embedded tissues.

To develop a comprehensive diagnostic system for mucormycosis from formalin-fixed paraffin-embedded tissues, consisting of own-designed real-time polymerase chain reaction (PCR) assays, fluorescence in situ hybridisation, and immunohistochemical staining. We designed 11 primers and probes for specific real-time PCR assays based on genome sequences, and validated the specificity by Aspergillus, Fusarium, Scedosporium, Lomentospora, Cryptococcus and Candida species. Formalin-fixed paraffin-embedded (FFPE) tissues from forty-four mouse model infected by above fungi were collected and extracted DNA by laser capture microdissection (LCM) and direct extraction methods for real-time PCR assays. In addition, seventeen clinical specimens histopathologically proven for mucormycosis were included for specific detection with the new diagnostic system. The real-time PCR assays allowed detection of a minimum of 10CFU/ml equivalent gDNA of each species. No cross-reaction with gDNA among species was noted. From mouse model specimens, the sensitivity of real-time PCR in samples extracted with LCM versus direct extraction method was 100% versus 91.43% at Mucorales level and 80% versus 45.71% at species level, respectively. The specificity was 100%. From clinical samples, LCM combined with real-time PCR can test 88.24% (15/17) of Mucorales. Sensitivities of fluorescence in situ hybridisation (FISH) and immunohistochemical staining (IHC) were 70.59% and 41.18%, respectively. Combined LCM-RT-PCR, FISH and IHC yielded positive results in all samples. The combination diagnostic system we developed is a culture-independent and robust method which enables rapid species identification from FFPE tissues for timely diagnosis of mucormycosis.

Toscana Virus: Ten Years of Diagnostics in Portugal.

Toscana virus (TOSV) is an emerging sandfly-borne virus within the Phlebovirus genus. Although most infections caused by this virus present as asymptomatic or with minimal symptomatology, TOSV may emerge as a febrile disease or sporadic cases of neurological disease such as meningitis or meningoencephalitis. This pathogen is distributed throughout the Mediterranean basin, along with the spatial distribution of its recognized sandfly vector, Phlebotomus perniciosus. Portugal, after Italy, was the second country considered endemic for this virus, with the first case of acquired infection published in 1985. Although little is known about the circulation of this virus in Portugal, the laboratory diagnosis of TOSV is available at the Centre for Vectors and Infectious Diseases Research of the National Institute of Health Dr. Ricardo Jorge (CEVDI/INSA), since 2007. The aim of this study is to report the results of the diagnosis of TOSV at the CEVDI/INSA, between 2009 and 2018. The diagnosis of TOSV in the CEVDI/INSA is included in the arboviruses and vector-borne neurotropic viruses panels or can be performed, when specified, for TOSV only. Direct detection is made in cerebrospinal fluid samples and is available for TOSV by specific real-time reverse transcription polymerase chain reaction followed by conventional real-time reverse transcription polymerase chain reaction for sequencing purposes, if positive. For indirect diagnosis, performed in serum samples, an in-house immunofluorescence assay for the detection of IgM and IgG antibodies against TOSV is used. A commercial immunofluorescence assay consisting in a mosaic of four phleboviruses is also available, in which, in addition to TOSV, antibody detection for sandfly fever Naples virus, sandfly fever Sicilian virus and sandfly fever Cyprus virus can be done. All diagnostic tests requested by clinicians to the CEVDI/INSA for arboviruses, neurotropic viruses and/or TOSV between January 2009 and December 2018, were included in this study. During the study period, the CEVDI/INSA received samples from 608 patients with diagnostic requests for TOSV. Five acute TOSV infections and one acute sandfly fever Sicilian virus infection were confirmed in serum samples. Three other patients had serological evidence of previous contact with the virus. Two of the six patients with acute infection developed febrile syndrome, and the other four presented with neurological disease: meningitis (n = 2), meningoencephalitis (n = 1) and severe depression of consciousness (n =1). These infections were most likely acquired in the districts of Faro (3), Lisbon (2) and Setúbal (1). In Portugal, the number of laboratory diagnostic requests for TOSV is low when compared to the numbers of requests for other less prevalent vector-borne viruses. The Faro district presented the highest number of TOSV-specific diagnostic requests which seems to indicate a higher level of recognition by clinicians in that region. Febrile syndrome and neurological disease were the clinical manifestations that were present in acute cases. In this study, in addition to the Faro district, recent infections were also detected in the districts of Lisbon and Setúbal. It is probable that TOSV may be distributed throughout the mainland territory since its main vector is present from north to south. In 2017, the sandfly fever Sicilian virus was associated for the first time with human disease in our country, thus alerting to the circulation of this phlebovirus. Even though the number of identified cases in Portugal is low, TOSV circulates and causes disease in our country. The diagnosis of this and other phleboviruses should not be neglected in the differential diagnosis of febrile syndrome and viral meningitis and meningoencephalitis, especially during the warmer months, when the vector's activity is higher.

Developing species-specific quantitative real-time polymerase chain reaction primers for detecting &lt;i&gt;Lautropia mirabilis&lt;/i&gt;

This study aimed to develop Lautropia mirabilis -specific quantitative real-time polymerase chain reaction (qPCR) primers based on the sequence of DNA-directed RNA polymerase subunit beta gene. The PrimerSelect program was used in designing of the qPCR primers, RTLam-F4 and RTLam-R3. The specificity of the qPCR primers were performed by conventional PCR with 37 strains of 37 oral bacterial species, including L. mirabilis . The sensitivity of the primers was determined by qPCR with the serial dilution of purified genomic DNA of L. mirabilis KCOM 3484, ranged from 4 ng to 4 fg. The data showed that the qPCR primers could detect only L. mirabilis strains and as little as 40 fg of genome DNA of L. mirabilis KCOM 3484. These results indicate that this qPCR primer pair (RTLam-F4/ RTLam-R3) may be useful for species-specific detection of L. mirabilis in epidemiological studies of oral bacterial infectious diseases such as periodontal disease.

SARS-COV-2 Infection and Specific Antibody Detection on Health Care Workers from a Military Hospital in Madrid, Spain.

This study aims to assess the COVID-19 seroprevalence in HCW at the Hospital Central de la Defensa Gómez Ulla (HCDGU) (Madrid). From 27 April to 10 June 2020 nasopharyngeal swab and serum samples from employees were processed in order to evaluate their seroprevalence and infective situation. Employees were classified according to their exposure to SARS-CoV-2 infection as high, moderate, and low exposure groups (level 1, level 2, and level 3, respectively). A specific real-time polymerase chain reaction (RT-PCR) was run to diagnose each patient, whereas the qualitative detection of IgG antibodies to SARS-CoV-2 was performed by means of an immunoassay. In total, 2781 HCW were screened. From this sample, 30 employees (1.1%) were infected with SARS-CoV-2 and 450 (16.2%) were positive to SARS-CoV-2-IgG antibodies. The seroprevalence was higher in the high exposure group.The seroprevalence of antibodies against SARS-CoV-2 among employees without any COVID-19 training was higher than in those who received COVID-19 training (14.5% vs 18.6%, P = 0.035). The seroprevalence in military and civilian personnel in level 1 was 18.2% and 20.0%, respectively (P = 0.4616), while in level 2 it was 6.0% and 16.0% (P = 0.0008) and in level 3 it was 16.7% and 10.2% (P = 0.0315). The results from the present study have shown that the high exposure group and HCW not receiving specific training against COVID-19 showed higher seroprevalence. Furthermore, the military employees from this hospital presented low percentage of seroprevalence.

In Vivo Validation of Alternative FDXR Transcripts in Human Blood in Response to Ionizing Radiation.

Following cell stress such as ionising radiation (IR) exposure, multiple cellular pathways are activated. We recently demonstrated that ferredoxin reductase (FDXR) has a remarkable IR-induced transcriptional responsiveness in blood. Here, we provided a first comprehensive FDXR variant profile following DNA damage. First, specific quantitative real-time polymerase chain reaction (qPCR) primers were designed to establish dose-responses for eight curated FDXR variants, all up-regulated after IR in a dose-dependent manner. The potential role of gender on the expression of these variants was tested, and neither the variants response to IR nor the background level of expression was profoundly affected; moreover, in vitro induction of inflammation temporarily counteracted IR response early after exposure. Importantly, transcriptional up-regulation of these variants was further confirmed in vivo in blood of radiotherapy patients. Full-length nanopore sequencing was performed to identify other FDXR variants and revealed the high responsiveness of FDXR-201 and FDXR-208. Moreover, FDXR-218 and FDXR-219 showed no detectable endogenous expression, but a clear detection after IR. Overall, we characterised 14 FDXR transcript variants and identified for the first time their response to DNA damage in vivo. Future studies are required to unravel the function of these splicing variants, but they already represent a new class of radiation exposure biomarkers.

Building the National SARS-CoV-2 Laboratory Diagnostic Capacity in Taiwan.

Through early and proactive laboratory testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes novel coronavirus 2019, Taiwan has demonstrated an efficient and rapid control response to contain the outbreak. Two days after the World Health Organization announced the complete viral genome sequence, the national laboratory of the Taiwan Centers for Disease Control developed a specific real-time reverse transcription polymerase chain reaction (real-time RT-PCR) test for SARS-CoV-2. The national laboratory network was further strengthened through the recruitment of medical centers and regional hospitals distributed throughout most geographical regions of the country. Ultimately, a network of 60 laboratories with a capacity of 7,342 real-time RT-PCR tests per day was established. Between January 14 and August 5, 2020, a total of 158,772 tests were conducted, corresponding to 120,487 cases. Test results were obtained within 24 hours, enabling an efficient and rapid control response.

RobHortic: A Field Robot to Detect Pests and Diseases in Horticultural Crops by Proximal Sensing

RobHortic is a remote-controlled field robot that has been developed for inspecting the presence of pests and diseases in horticultural crops using proximal sensing. The robot is equipped with colour, multispectral, and hyperspectral (400–1000 nm) cameras, located looking at the ground (towards the plants). To prevent the negative influence of direct sunlight, the scene was illuminated by four halogen lamps and protected from natural light using a tarp. A GNSS (Global Navigation Satellite System) was used to geolocate the images of the field. All sensors were connected to an on-board industrial computer. The software developed specifically for this application captured the signal from an encoder, which was connected to the motor, to synchronise the acquisition of the images with the advance of the robot. Upon receiving the signal, the cameras are triggered, and the captured images are stored along with the GNSS data. The robot has been developed and tested over three campaigns in carrot fields for the detection of plants infected with ‘Candidatus Liberibacter solanacearum’. The first two years were spent creating and tuning the robot and sensors, and data capture and geolocation were tested. In the third year, tests were carried out to detect asymptomatic infected plants. As a reference, plants were analysed by molecular analysis using a specific real-time Polymerase Chain Reaction (PCR), to determine the presence of the target bacterium and compare the results with the data obtained by the robot. Both laboratory and field tests were done. The highest match was obtained using Partial Least Squares-Discriminant Analysis PLS-DA, with a 66.4% detection rate for images obtained in the laboratory and 59.8% for images obtained in the field.

Performance Comparison of Two Malaria Rapid Diagnostic Test with Real Time Polymerase Chain Reaction and Gold Standard of Microscopy Detection Method

BackgroundThe diagnostic test for malaria is mostly based on Rapid Diagnostic Test (RDT) and detection by microscopy. Polymerase Chain Reaction (PCR) is also a sensitive detection method that can be considered as a diagnostic tool. The outcome of malaria microscopy detection depends on the examiner’s ability and experience. Some RDT has been distributed in Indonesia, which needs to be evaluated for their results.ObjectiveThis study aimed to compare the performance of RightSign RDT and ScreenPlus RDT for detection of Plasmodium in human blood. We used specific real-time polymerase chain reaction abTESTMMalaria qPCRII) and gold standard of microscopy detection method to measure diagnostic efficiency.MethodsBlood specimens were evaluated using RightSign RDT, ScreenPlus RDT, Microscopy detection, and RT-PCR as the protocol described. The differences on specificity (Sp), sensitivity (Sn), positive predictive value (PPV), and negative predictive value (NPV) were analyzed using McNemar and Kruskal Wallis analysis.ResultsA total of 105 subjects were recruited. Based on microscopy test, RightSign RDT had sensitivity, Specificity, PPV, NPV, 100%, 98%, 98.2%, 100%, respectively. ScreenPlus showed 100% sensitivity, 98% specificity, 98.2% PPV, 100% NPV. The sensitivity of both RDTs became lower (75%) and the specificity higher (100 %) when using real-time PCR. Both RDTs showed a 100% agreement. RTPCR detected higher mix infection when compared to microscopy and RDTs.ConclusionRightSign and ScreenPlus RDT have excellent performance when using microscopy detection as a gold standard. Real-time PCR method can be considered as a confirmation tool for malaria diagnosis.