Conventional Polymerase Chain Reaction Tests Research Articles

RELATIONSHIP BETWEEN THE SPECTROMETRIC VALUES OF DNA, RNA, AND THE PCR PRESENCE OF A PATHOGEN IN SINGLE TICK SAMPLES

Aim. Ticks are the vectors of many pathogens, which cause diseases with fatal consequences. Polymerase chain reaction (PCR) was used to detect the presence of these different pathogens in ticks, but there is a need of isolated nucleic acid to conduct the molecular assays. In our previous research, we found that some ticks give huge yield of isolated nucleic acid during spectrometric measurements, therefore aim of this study is to find whether there is any relation between spectrometric values of DNA, RNA and presence of Borrelia burgdorferi as example pathogen in single tick samples. Methods. DNA and RNA were isolated with mini column method from single tick samples. They were run in real time as well as conventional PCR tests for the presence of Borrelia burgdorferi. The nucleic acid yields of isolated nucleic acid samples were measured with a spectrophotometer. Results. It was found that there were 47 ticks positive for Borrelia burgdorferi and 40 were negative. Average isolated DNA and RNA quantity was higher in pathogen positive ticks than those of negative ticks. There was no correlation between the yield of nucleic acid and presence of pathogen in a single tick, but there was tendency that pathogen positive tick gave higher yield of DNA and RNA during the isolation. Conclusions. This study shows some of Borrelia burgdorferi positive ticks give very high yield of DNA and RNA during the isolation. There is no correlation between presence of pathogen and nucleic acid in a single tick, but there is tendency that pathogen positive tick may have higher nucleic acid yield. Therefore, our recommendation is that laboratory should always measure the nucleic acid yield along with conducting the PCR tests.

First case of mpox diagnosed in Queensland, Australia: clinical and molecular aspects.

A man in his thirties presented immediately on return from a one-month trip to Europe with widespread pustular lesions, tender lymphadenopathy, fever, and headache. Initial contact was with his general practitioner, who notified the local Public Health Unit and the Infectious Diseases Unit. He identified as a man who has sex with men. He had a background of human immunodeficiency virus (HIV) infection, which was well controlled (CD4+ cells, 1190 cells/mm3 [reference interval (RI), 560–1580 cells/mm3]; HIV viral load not detected [limit of quantitation, 20 RNA copies/mL]) with bictegravir 50 mg, emtricitabine 200 mg, and tenofovir alafenamide 25 mg daily. He first noticed a lesion resembling a pimple on the forehead while still overseas, which progressed over the six days before his return (Box 1) followed by a clustering of similar lesions over his right buttock, but no mucosal lesions. More lesions then developed over his thigh and hand. He reported intermittent rectal paraesthesia and spasm, in addition to fever and mild generalised headache. Tender inguinal and cervical lymphadenopathy became established, along with fatigue and malaise. He did not report sore throat, cough, diarrhoea, dysuria, or neck stiffness. He had had sexual contact with known cases of mpox (formerly monkeypox) in Europe. He was admitted to hospital in a single negative pressure room, with contact and airborne precautions. Full blood count and chemistry were normal, and C-reactive protein was 17 mg/L (RI, < 5 mg/L). Testing for Chlamydia trachomatis and Neisseria gonorrhoeae was negative. Swabs were taken from the perianal lesion and sent for National Association of Testing Authorities (NATA)-accredited in-house Orthopoxvirus group real-time polymerase chain reaction (PCR) test targeting the OPG105 gene. Positive by real-time PCR, monkeypox virus (MPXV) DNA from both the perianal lesion and throat specimens was subsequently confirmed using two additional conventional PCR tests targeting the OPG105 and OPG185 genes. Whole genome sequencing and phylogenetic analyses (Supporting Information) of a genome sequence obtained from the perianal specimen (MPXV_QLD_MX00001_2022, GenBank accession number OP235282) demonstrated placement within the human MPXV (hMPXV) sublineage B.1 of clade IIb and was most closely related to other recent 2022 MPXV sequences from the United States, Europe, Australia and Canada (Box 2). Additional laboratory methods are included in the Supporting Information. * Alignments were trimmed to remove the 5’ inverted terminal repeat (ITR) sequence and mask several repetitive regions. The newly classified MPXV phylogenetic clades I (formally Central African), IIa and IIb (formally West African) are highlighted, and the human MPXV (h) sublineages A, A.1, A.1.1 and B.1 are named at key nodes and are correspondingly denoted with coloured branches. Clade I includes a collapsed grouping of 34 sequences. The recent Queensland 2022 MPXV sequence (MPXV_QLD_OP235282_2022) is highlighted in bold blue font. No antiviral therapy was initiated, and his systemic symptoms improved rapidly over 24 hours, at which time he was discharged home to self-isolate under the care of the coronavirus disease 2019 (COVID-19) virtual ward. He lived alone and was instructed to stay at home except for essential activities, such as buying groceries or solo outdoor exercise, and to avoid high risk settings and physical and intimate contact with others until all skin lesions had scabbed over. Mpox is caused by MPXV, a double-stranded DNA virus of the genus Orthopoxvirus in the family Poxviridae.1 The incubation period is five to 21 days, and most clinical infections are self-limiting. Symptoms include rash, fever, lethargy and lymphadenopathy, usually lasting two to four weeks.1 By 5 January 2023, in the current global mpox outbreak, 110 countries have reported cases, including 144 laboratory-confirmed cases in Australia.2 An international case series reported that 95% of patients presented with a rash (almost two-thirds having fewer than ten lesions), 62% reported fever, and 56% lymphadenopathy.3 Ninety-eight per cent of infected individuals were found to be gay or bisexual men, 41% had HIV infection, and MPXV was detected in 91% (29/32) of seminal fluid specimens tested. Concomitant sexually transmissible infections were reported in 29% of cases. Although MPXV was thought to be mainly transmitted via respiratory droplets and close contact with skin lesions, the current global outbreak is notable for spreading primarily during sexual contact. Fomites (eg, towels, sex toys) may be also involved in MPXV transmission. Public health interventions including case and close contact management should be provided as per the mpox guidelines from the Communicable Diseases Network Australia (CDNA).4 We were able to repurpose the existing virtual ward and public health capacity developed originally for the COVID-19 pandemic response, demonstrating the legacy of a stronger public health system able to deal with emerging infectious diseases outbreaks. There are no antiviral therapies for mpox approved by the Therapeutic Goods Administration (TGA). However, the antiviral agent tecovirimat, which inhibits the MPXV envelope wrapping protein, is held in the National Medication Stockpile for the treatment of severe disease and for patients at risk of complications, such as severely immunosuppressed individuals and children less than eight years old.5 Vaccinia immune globulin may be an alternative therapy in pregnancy. Management is generally supportive, with careful attention to analgesia for anogenital and oropharyngeal lesions. The replication-incompetent live virus vaccine modified vaccinia Ankara–Bavarian Nordic (MVA-BN, Jynneos [Bavarian Nordic]) prevents mpox infection, although occasional breakthrough cases are described. In these cases, it may reduce the severity of mpox.6 The vaccine is not registered for use in Australia but is available via a special emergency pathway. Genetic classification of MPXV was recently revised, with new naming conventions comprising of clade I (formerly Central African) and clade II (formerly West African, further divided into IIa and IIb).7 The current 2022 human outbreak is caused by the hMPXV sublineage B.1 of clade IIb, which might cause less severe disease. Open access publishing facilitated by The University of Queensland, as part of the Wiley - The University of Queensland agreement via the Council of Australian University Librarians. Krispin Hajkowicz has received speaking fees, honoraria, advisory board fees, and a research grant from Gilead Sciences, and support from Moderna to attend an educational meeting. Adam Stewart has received speaking fees and honoraria from Gilead Sciences, and support from Pfizer for travel and meeting expenses. Not commissioned; externally peer reviewed. Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Evaluation of dry stored disposable sensor strip on rapid SARS-CoV-2 detection platform

This study investigated a SARS-CoV-2 virus detection mechanism using dry-stored disposable strips. The accuracy of this sensing platform is as good as polymerase chain reaction (PCR) with a detection time of fewer than 30 s. SARS-CoV-2 antibodies were biofunctionalized on disposable strips similar to glucose detection strips to detect the presence and concentrations of SARS-CoV-2 in saliva samples. Eight 1 ms electric pulses were sent through the sensor strip with a saliva sample in its microfluidic chamber. A circuit board embedded with MOSFET was also employed to amplify the detected signals and convert the signal to digital readings displayed on an LCD screen. The COV-antibody functionalized disposable strips were stored in a dry condition for at least one day before analyzing clinical human saliva samples with known cycling threshold (Ct) values confirmed with conventional PCR tests. Results demonstrate our system is capable of showing qualitative positive or negative results within 30 s and providing quantitative SARS-CoV-2 concentrations in terms of Ct values in 5 min.

Experimental In vitro Study to Assess the Antibacte-rial Activity of Thymus vulgaris Oil on Streptococ-cus Sanguinis

Background: The Streptococcus genus are the predominant bacteria in the mouth and the Streptococcus sanguinis is one of them which performing a primary function for expansion of dental biofilm. Gingival and periodontal disorders are caused by dental biofilm, today, there is a necessity to discover naturally presenting antibacterial compounds from herbs with less side effects as a substitutive to the commonly handled chlorohexidine. Thus, the purpose of this study was to assess the antibacterial activity of thymus vulgaris oil on Streptococcus sanguinis bacteria In vitro. Materials and Methods: Human supragingival plaque samples were taken from 10 subjects, then morphological and microscopical examination, biochemical tests, Optochin test, hemolytic ability test and conventional polymerase chain reaction test was applied to assure coincidence of Streptococcus sanguinis. The sensitivity of bacteria to Thymus vulgaris oil, the minimum concentration that inhibit the growth and killed the test bacteria were identified a partly as comparable to 0.2% chlorhexidine as a positive control and10 % dimethyl sulphoxide as a negative control. Results: Thymus vulgaris oil presented a significant antibacterial property on Streptococcus sanguinis with several grades of inhibition zones. When compared to Chlorohexidine, Thymus vulgaris oil had stronger antibacterial properties. The minimum concentration that inhibited the growth and killed Streptococcus sanguinis was (0.09%). Conclusion: Thymus vulgaris oil displayed higher antibacterial action with each concentration on Streptococcus sanguinis as compared to Chlorohexidine, therefore; it can be used as a natural substitutive oral health care product to Chlorohexidine.

Efecto del desbridamiento periodontal más azitromicina sistémica en sujetos con periodontitis estadio III: Un ensayo clínico controlado aleatorio

Aim: To evaluate the effect of the systemic administration of azi-thromycin (AZM) as an adjunct to non-surgical periodontal therapy (NSPT) on the clinical and microbiological variables of patients with periodontitis Material and Methods: Eighteen volunteers received NSPT combined with placebo or AZM (500 mg/day) for 3 days (n=9/group). They were monitored clinically for probing pocket depth (PPD), clinical attachment level (CAL), O'Leary index (OI), bleeding on probing (BoP) at baseline and during the first, third and sixth month and microbiologically, at baseline and at 3 and 6 months after therapy, by conventional polymerase chain reaction tests. Results: Fourteen patients completed the study (n=7/group). Differences statistically significant were observed among both groups. The experimental group presented: A PPD mean (p=0.04) significantly lower and PPD reduction (p=0.02), at 6-months post NSPT. Regarding changes (∆), at the third month post NSPT, there was a significant increase in the number of shallow sites (p&lt;0.001) and a decrease in the intermediate sites (p&lt;0.001). In addition, a significant decrease in the mean number of deep sites (p=0.04) was detected at 6 months post treatment. There was also a significant decrease in periodontal index BoP at 1 (p=0.01), 3 (p&lt;0.001) and 6 (p=0.01) months and OI at 3- and 6-months (p&lt;0.001), post treatment. Regarding the presence of periodontal pathogens, no significant differences were observed, intra and inter groups. Conclusion: AZM as an adjuvant to NSPT provides additional beneficial effects for PPD and BoP compared to NSPT alone.

The Impact of Rapid PCR Testing for Viral Respiratory Infections on Acute Admissions From the Emergency Department and Inpatient Length of Stay.

Diagnosing influenza and respiratory syncytial virus (RSV) with conventional multiplex respiratory viral polymerase chain reaction (PCR) takes 24-72 hours compared with under two hours for recently available rapid PCR tests. We investigated the impact of rapid diagnosis of acute respiratory viral infection on admission odds from the emergency department (ED) and acute inpatient length of stay (LOS), as well as concordance between the rapid and conventional PCR tests used at our institution. Single-center retrospective cohort study of patients presenting to the ED with influenza-like illness. We compared the odds of admission and acute LOS in patients investigated with rapid PCR, those investigated with conventional PCR, and those investigated with both tests. Multivariable logistic regression was used to assess the odds of admission, while linear regression was used to assess LOS. There was no significant change in the odds of admission among patients who received the rapid PCR compared to conventional PCR (odds ratio: 1.01, 95% confidence interval [CI]: 0.50-2.02; p = 0.96). There was also no significant difference in LOS of admitted patients who received rapid PCR testing (regression coefficient: -0.32, 95% CI: -1.75 to 1.12; p = 0.66). The rapid PCR test used at our institution yielded fully concordant results with conventional PCR testing. Rapid PCR testing is as sensitive as conventional PCR testing for the diagnosis of influenza and RSV but is neither associated with a significant impact on admission nor inpatient LOS. Further research is needed to assess the impact of rapid testing on isolation room use.

Molecular evidence of Leishmania spp. in spider monkeys (Ateles geoffroyi) from The Tuxtlas Biosphere Reserve, Veracruz, Mexico.

The black-handed spider monkey (Ateles geoffroyi) is a platyrrhine primate distributed in southern Mexico, Central America, and part of South America. Two subspecies inhabit Mexico: Ateles geoffroyi vellerosus and Ateles geoffroyi yucatanensis, both threatened with extinction. Serological evidence of exposure of spider monkeys to various groups of parasites such as Trypanosoma cruzi in México and Leishmania spp. in Brazil has been reported. The genus Leishmania encompasses about 23 species of flagellate protozoa that are transmitted by the bite of females of Phlebotominae sand flies. These parasites cause a zoonotic disease called leishmaniasis, which generates skin, mucocutaneous and/or visceral manifestations. The aim of the present study was to demonstrate the presence of Leishmania sp. in spider monkeys from the Tuxtlas Biosphere Reserve, Veracruz, Mexico. Blood samples from 10 free- ranging specimens of A. geoffroyi yucatanensis and 11 specimens in captivity of A. geoffroyi vellerosus were collected and used. The samples were subjected to a conventional Polymerase Chain Reaction test for the identification of a 116bp fragment of a region from the kinetoplast minicircle of the parasite. Our analyzes showed that 71.4% of the sampled animals had fragment sizes compatible with Leishmania spp. The implications involve the survival of the specimens and the possibility that these primates act as sentinels of the disease. Furthermore, it is the first report suggesting the presence of Leishmania spp. in A. geoffroyi vellerosus and A. geoffroyi yucatanensis in Veracruz, Mexico.

Virologic detection and molecular characterization of canine parvovirus-2 in dogs

This study aimed to search the extent of spread of various canine parvovirus variants in Egypt from year 2019 to year 2020 and the ability of the present used vaccines in the protection .In this study, twenty fecal samples were collected from diarrheic puppies. These samples were tested by rapid CPV Ag test kits for the detection of CPV-2 Ag. Ten samples were found positive for CPV2 by rapid Ag test kit (VDRG CPV Ag rapid kit).Positive samples were propagated on Vero cell culture for 8 passages, CPE appears in the form of cell rounding and detachment within 5 days.  A conventional polymerase chain reaction test was performed on tissue culture isolates. Partial sequencing of five randomly selected positive samples revealed the presence of 2 CPV2C and 3 CPV2B.CPV2B represents the highest level of the variant serotype of CPV circulating in Egypt through records of egyptian isolated cases at the gene bank . One of the isolate has 100% identity with a CPV2B of cat origin. Our isolates CPVS1 and CPVS4 have 100% nucleotide identity with each other and with CPV cairo1-19 and CPV cairo3-19 belonging to CPV2C , this variant is also widespread all over the country. Continuous accurate molecular and epidemiological studies are needed to follow-up the new mutation of the virus genome which may result in vaccination failure(The majority of the used vaccines in Egypt are of CPV2 origin not of variant origin). The use of CPV2b based vaccine gave better protection than cpv2 based vaccine(Wide range protection against CPV2 variants). The obvious spread and emergence of newly introduced CPV variants at different Egyptian governorates as cited at previous study necessitates updated molecular based epidemiological studies of field isolates continuously to follow up the efficacy of the concurrent used vaccines.

ART Initiation for Infants Diagnosed With HIV Through Point of Care and Conventional Polymerase Chain Reaction Testing in Kenya: A Case Series.

We sought to understand the sequence of testing and treatment among nine infants offered both conventional and point-of-care testing and diagnosed as HIV-positive by 6 months of age in Kenya. One infant received per protocol testing and treatment. Patient-level (late presentation and disengagement), provider-level (reluctance and error/oversight) and system-level (stock outs, errors) challenges delayed diagnosis and treatment. Early point-of-care testing can streamline testing; however, challenges mitigate benefits.

Use of Point-of-Care Nucleic Acid Tests Beyond Early Infant Diagnosis of HIV: A Retrospective Case Review in Lesotho.

Rapid diagnostic tests (RDTs) for HIV antibodies remain the primary method of diagnosis of HIV in individuals over age 18 months in Lesotho. Although antibody tests have high sensitivity and specificity, up to 2.3% of serial two-test algorithms can have discrepant results between RDTs. In the case of inconclusive RDT results, Lesotho guidelines at the time of this study recommended either repeat testing with the same RDT algorithm after 14 days or immediately collect a blood sample to be sent for laboratory-based polymerase chain reaction testing. Point-of-care qualitative nucleic acid tests (POC qual NAT) may have benefits in rapidly resolving these inconclusive results, particularly when compared with repeating RDTs later or conventional polymerase chain reaction testing at the National Reference Laboratory. Hospitals and clinics at 29 locations throughout Lesotho that had access to point-of-care nucleic acid testing. Retrospective case review. We identified 100 testing records where POC qual NAT was used to resolve inconclusive RDTs per Lesotho guidelines. Eighty-nine percent of patients received their results in a median of one day from their inconclusive RDT result (interquartile range 0-7 days). Sixty-eight patients (68%) were determined to be HIV positive based on POC nucleic acid tests (NATs), of which 54 (79%) were started on antiretroviral therapy (ART). Median time from inconclusive RDT result to initiation of ART therapy was 2 days (interquartile range 0-14 days). Three patients in this review were pregnant at the time of testing; one was HIV positive by POC qual NAT and was started on ART therapy the same day. As the availability of POC qual NAT platforms increases, they may serve as feasible options for rapid resolution of inconclusive results and initiation of ART, particularly in populations with high risk of imminent transmission.

Developing a Pre-Testing Diagnostic Tool for COVID-19 Using Big Data Predictive Analytics

Background: Standard Polymerase Chain reaction (PCR) tests for SARS-COV-2 are in short supply to meet demand in many countries presenting a need to improve testing efficiency. Pre-testing tools can be used to ensure continued public safety as systems move through the pandemic. In this study we set out to create an instrument based on big data predictive tools to assess pre-test probability for COVID-19. Methods: We analyzed data reported by the Israeli Ministry of Health (IMOH) for standard PCR tests done for SARS-COV-2 from March to April, 2020, overall 108,852 cases. Demographics and symptoms of the patients were collected at time of testing. Four supervised machine learning algorithms were used to analyze 20,537 test results of cases who presented with symptoms. Model results were used to develop efficient pre-test diagnostic tool. Findings: Of symptomatic patients tested, 6,427 (31.3%) tested positive for SARS-COV-2, and 14,110 (68.7%) tested negative. In all models used headache, shortness of breath, sore throat, fever, and having contact with an infected person came up as most predictive of a positive test. The area under the curve of the receiver operating characteristic curve for the test sample was found to be 0.88 and the misclassification rate was between 4.7% and 6.5% for all predictive models, demonstrating effective classification ability. Using our pre-test probability screening tool with conventional PCR testing can potentially increase efficiency by 141%. Interpretation: We suggest a simple diagnostic pre-test tool for assessing the probability of infection can increase efficiency of testing and effectiveness of public health COVID-19 programs. Funding: NoneDeclaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The authors noted that the analysis presented was approved by the IMOH Data Sharing Institutional Review Board.

A Resposta das Tecnologias de Saúde à Pandemia de COVID-19: Uma Luz ao Fundo do Túnel?

The responses to the threats of the COVID-19 pandemic have been based on public health measures that prevent the spread of infection. After months of intense international research, the first vaccines against COVID-19 have already begun to be approved and administered. The first vaccine against COVID-19 was administered in Portugal, on December 27th, 2020, as it happened in all European counterparts. In addition to vaccines, the efficacy and safety of several medicines in the treatment of COVID-19 are still being studied, some of them already approved with other therapeutic indications. The need to generate information that guarantees the safety of the medicines under study, underlines the fundamental role of the pharmacovigilance system in every process. Finally, over time, both the use of tests and the use of medical devices associated with COVID-19 have also evolved very quickly. In testing, in addition to conventional PCR (polymerase chain reaction), rapid antibody and antigen tests have emerged. On the other hand, medical devices and personal protective equipment whose availability, in the first phase, was a serious problem, are now widely available, with emphasis on the greater national offer and technical improvements.

Accuracy of polimerase chain reaction for the diagnosis of pleural tuberculosis

Polymerase chain reaction (PCR)-based techniques to detect Mycobacterium tuberculosis DNA in respiratory specimens have been increasingly used to diagnose pulmonary tuberculosis. Their use in non-respiratory specimens to diagnose extrapulmonary tuberculosis is, however, controversial. In this study, we estimated the accuracy of three in-country commercialized PCR-based diagnostic techniques in pleural fluid samples for the diagnosis of pleural tuberculosis. Patients underwent thoracenthesis for diagnosis purposes; pleural fluid aliquots were frozen and subsequently submitted to two real time PCR tests (COBAS(®)TAQMAN(®)MTB and Xpert(®)MTB/Rif) and one conventional PCR test (Detect-TB(®)). Two different reference standards were considered: probable tuberculosis (based on clinical grounds) and confirmed tuberculosis (bacteriologically or histologically). Ninety-three patients were included, of whom 65 with pleural tuberculosis, 35 of them confirmed. Sensitivities were 29% for COBAS(®)TAQMAN(®)MTB, 3% for Xpert(®)MTB/Rif and 3% for Detect-TB(®); specificities were 86%, 100% and 97% respectively, considering confirmed tuberculosis. Considering all cases, sensitivities were 16%, 3% and 2%, and specificities, 86%, 100%, and 97%. Compared to the 95% sensitivity of adenosine deaminase, the most sensitive test for pleural tuberculosis, the sensitivities of the three PCR-based tests were very low. We conclude that at present, there is no major place for such tests in routine clinical use.

Molecular study on selected vector-borne infections in urban stray colony cats in northern Italy

Feline vector-borne diseases can be caused by a range of pathogens transmitted by arthropods. Many of these infections have zoonotic implications, and stray cats are potential sentinels for human and pet health. This study investigated the prevalence of selected vector-borne infections in stray colony cats in Milan. Blood samples from 260 stray cats were evaluated, using conventional polymerase chain reaction tests (cPCRs), for the presence of DNA associated with Rickettsia species, Anaplasma phagocytophilum and Ehrlichia species. Positive cPCR results occurred in 127/260 subjects (48.9%; 95% confidence interval [CI] = 40.7-58.1), with a prevalence of 31.9% (83/260, 95% CI = 25.4-39.6) for Rickettsia species, 17.7% (46/260, 95% CI= 13.0-23.6) for A phagocytophilum, and 5.4% (14/260, 95% CI = 2.9-9.0) for Ehrlichia species. There was no statistical association between a positive PCR test for vector-borne infections surveyed and colony location, age, gender, body condition score or complete blood count abnormalities, nor feline immunodeficiency virus, feline leukaemia virus or Toxoplasma gondii status. The only variable linked to positive PCR results was detection of signs of ocular infection and PCR positivity for Rickettsia species (P = 0.04, odds ratio [OR] = 2.2, 95% CI = 1.1-4.4, P = 0.02). There is a significant prevalence of vector-borne infections with zoonotic potential in urban stray cats in Milan. Thus, dogs and pet cats with outdoor access should be monitored and treated for ectoparasites on a regular basis to minimise risks of disease and the potential transmission of zoonotic agents to people.

Optimization of a real-time PCR assay for the detection of the quarantine pathogen Melampsora medusae f. sp. deltoidae

Melampsora medusae (Mm), one of the causal agents of poplar rust, is classified as an A2 quarantine pest for European Plant Protection Organization (EPPO) and its presence in Europe is strictly controlled. Two formae speciales have been described within Mm, Melampsora medusae f. sp. deltoidae (Mmd), and Melampsora medusae f. sp. tremuloidae (Mmt) on the basis of their pathogenicity on Populus species from the section Aigeiros (e.g. Populus deltoides) or Populus (e.g. Populus tremuloides), respectively. In this study, a real-time polymerase chain reaction (PCR) assay was developed allowing the detection of Mmd, the forma specialis that is economically harmful. A set of primers and hydrolysis probe were designed based on sequence polymorphisms in the large ribosomal RNA subunit (28S). The real-time PCR assay was optimized and performance criteria of the detection method, i.e. sensitivity, specificity, repeatability, reproducibility, and robustness, were assessed. The real-time PCR method was highly specific and sensitive and allowed the detection of one single urediniospore of Mmd in a mixture of 2 mg of urediniospores of other Melampsora species. This test offers improved specificity over currently existing conventional PCR tests and can be used for specific surveys in European nurseries and phytosanitary controls, in order to avoid introduction and spread of this pathogen in Europe.

The Enhanced Pneumococcal LAMP Assay: A Clinical Tool for the Diagnosis of Meningitis Due to Streptococcus pneumoniae

Background Streptococcus pneumoniae is a leading cause of invasive bacterial disease in developed and developing countries. We studied the loop-mediated isothermal amplification (LAMP) technique to assess its suitability for detecting S. pneumoniae nucleic acid in cerebrospinal fluid (CSF).Methodology/Principal FindingsWe established an improved LAMP assay targeting the lytA gene (Streptococcus pneumoniae [Sp] LAMP). The analytical specificity of the primers was validated by using 32 reference strains (10 Streptococcus and seven non-Streptococcus species) plus 25 clinical alpha-hemolytic streptococcal strains, including four S. pneumoniae strains and 21 other strains (3 S. oralis, 17 S. mitis, and one Streptococcus species) harboring virulence factor-encoding genes (lytA or ply). Within 30 minutes, the assay could detect as few as 10 copies of both purified DNA and spiked CSF specimens with greater sensitivity than conventional polymerase chain reaction (PCR). The linear determination range for this assay is 10 to 1,000,000 microorganisms per reaction mixture using real-time turbidimetry. We evaluated the clinical sensitivity and specificity of the Sp LAMP assay using 106 randomly selected CSF specimens from children with suspected meningitis in Korea, China and Vietnam. For comparison, CSF specimens were also tested against conventional PCR and culture tests. The detection rate of the LAMP method was substantially higher than the rates of PCR and culture tests. In this small sample, relative to the LAMP assay, the clinical sensitivity of PCR and culture tests was 54.5% and 33.3%, respectively, while clinical specificity of the two tests was 100%.Conclusions/SignificanceCompared to PCR, Sp LAMP detected S. pneumoniae with higher analytical and clinical sensitivity. This specific and sensitive LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings.

Direct simultaneous detection of 6 sexually transmitted pathogens from clinical specimens by multiplex polymerase chain reaction and auto-capillary electrophoresis

The availability of a reliable and user-friendly method to identify pathogens causing sexually transmitted diseases (STDs) is essential to reduce the complications and spread of infection. In this study, genital/urinary specimens from 113 patients with STDs were simultaneously tested for 6 pathogens using the automated Seeplex® (Seegene, Seoul, Korea) multiplex polymerase chain reaction (PCR)-based STD6B auto-capillary electrophoresis (ACE) system. The results were compared with conventional reference methods, including culture and PCR tests. The sensitivity of STD6B ACE was found to be 100% for Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, Neisseria gonorrhoeae, and Trichomonas vaginalis, and 98% for genital Ureoplasma (U. urealyticum and U. parvum). Specificity ranged from 97% to 100%. One pathogen was detected in 51 specimens, and 2 or more pathogens were detected in 24. In conclusion, the multiplex PCR and ACE system is highly sensitive and specific for the rapid, simultaneous detection of STD pathogens directly from a single specimen.

Sensitive Detection of Fusarium circinatum in Pine Seed by Combining an Enrichment Procedure with a Real-Time Polymerase Chain Reaction Using Dual-Labeled Probe Chemistry

Fusarium circinatum is the causal agent of pitch canker disease on numerous Pinus spp. This aggressive fungus may infect pine seed cryptically and, therefore, can easily be spread long distances by the seed trade. F. circinatum has recently been listed as a quarantine organism in numerous countries throughout the world, which prompted the development of a specific and sensitive tool for the detection of this pathogen in conifer seed. A new detection protocol for F. circinatum based on a biological enrichment step followed by a real-time polymerase chain reaction (PCR) assay was developed. Several enrichment protocols were compared and a 72-h incubation of the seed with potato dextrose broth was the most efficient technique to increase F. circinatum biomass before DNA extraction. The relative accuracy, specificity, and sensitivity of the real-time PCR assay was evaluated in comparison with a previously published conventional PCR test on 420 seed DNA extracts. The real-time PCR described here proved to be highly specific and significantly more sensitive than the conventional PCR, and enabled the detection of F. circinatum in samples artificially contaminated with less than 1/1,000 infected seed, as well as in naturally infected samples. Last, in order to routinely check the quality of the seed DNA extracts, a primer-probe combination that targets a highly conserved region within the 18S ribosomal DNA in plants or fungi was successfully developed. This assay allows for quick and reliable detection of F. circinatum in seed, which can help to prevent long-distance spread of the pathogen via contaminated seed lots.

Diagnosis of duck circovirus infections by conventional and real-time polymerase chain reaction tests

Development of the first conventional and real-time polymerase chain reaction (PCR) tests for the diagnoses of duck circovirus (DuCV) infections is described. Both tests amplified a 230 bp fragment specific to the DuCV Rep gene. Although both tests had the same detection limit (13×103 target DNA/ml) when the target DNA was diluted in water, the detection limit of the real-time test (13×104 target DNA/ml) was 10-fold less than the conventional test (13×105 target DNA/ml) when the amplifications were performed in the presence of cellular DNA. Using the conventional PCR test, DuCV DNA was detected in 85 (84%) of 101 bursa of Fabricius samples from dead or sick ducks, aged between 1 and 12 weeks, and in samples from 35 (94%) of 37 flocks. Application of the SYBR Green-based real-time PCR test to 54 selected bursa of Fabricius samples indicated that more samples were positive by real-time PCR than by conventional PCR, allowed the numbers of genome copies to be estimated and showed that some bursa of Fabricius samples contained over 1013 genome copies/g tissue. Although DuCV infections were detected in birds aged from 1 to 12 weeks, higher virus DNA levels were detected in ducks aged older than 5 weeks than in ducks younger than 5 weeks. An in situ hybridization method for the detection of DuCV in histological samples was also developed. Additional work is required to determine the clinicopathological significance of DuCV infections. Diagnostic des infections à circovirus du canard par des tests d'amplification en chaîne par polymérase conventionnels et en temps réel Le développement des premiers tests d'amplification en chaîne par polymérase (PCR) conventionnels et en temps réel pour le diagnostic des infections à circovirus du canard (DuCV), sont décrites. Les deux tests amplifient un fragment de 230 pb spécifique du gène Rep du DuCV. Les deux tests ont présenté la même limite de détection (13×103 ADN cible /ml) lorsque l'ADN cible a été dilué dans l'eau. Par contre, la limite de détection du test en temps réel (13×104 ADN cible/ ml) a été 10 fois inférieure à celle du test conventionnel (13×105 ADN cible/ ml) quand les amplifications ont été réalisées en présence d'ADN cellulaire. En utilisant le test PCR conventionnel, l'ADN du DuCV a été détecté à partir de 84% (85/101) des échantillons de bourse de Fabricius (BF) de canards malades ou morts, âgés entre 1 à 12 semaines et à partir de 94% (35/37) des troupeaux. L'application du test PCR en temps réel, basé sur la technologie SYBR Green, à une sélection de 54 échantillons de BF a révélé un plus grand nombre d'échantillons positifs avec ce test qu'avec le test PCR conventionnel et a permis d'estimer le nombre de copies de génome en montrant que quelques échantillons de BF contenaient plus de 1013 copies de génome/g de tissu. Bien que les infections à DuCV aient été détectées chez des animaux âgés de 1 à 12 semaines, des niveaux supérieurs d'ADN viral ont été détectés chez des animaux âgés de plus de 5 semaines comparés à ceux de moins de 5 semaines. Un test de détection du DuCV par hybridation in situ dans des échantillons destinés à l'histologie a également été développé. Des travaux complémentaires sont nécessaires pour déterminer la signification clinicopathologique des infections à DuCV. Diagnose von Entencircovirusinfektionen durch konventionelle und Real Time-Polymerasekettenreaktionstests Die Entwicklung des ersten konventionellen sowie Real Time-Polymerasekettenreaktions (PCR)-Tests zur Diagnose von Entencircovirus (ECV)-Infektionen werden beschrieben. Beide Tests amplifizieren ein für das ECV-Rep-Gen spezifisches 203bp-Fragment. Obwohl beide Tests bei der Verdünnung derr Ziel-DNS in Wasser die gleiche Nachweisgrenze hatten (13×103 Ziel-DNSs/mL), erwies sich die Nachweisgrenze bei der Amplifizierung in Anwesenheit zellulärer DNS in der Real Time-PCR (13×104 Ziel-DNSs/mL) 10fach niedriger als die im konventionellen Test (13×105 ZielDNSs/mL). Mittels dem konventionellen PCR-Test wurde ECV-DNS in 85 (84%) von 101 Bursa Fabricii (BF)-Proben von toten oder kranken 1-12-wöchigen Enten und in Proben von 35 (94%) von 37 Herden festgestellt. Die Anwendung des auf SYBR-Grün basierenden Real Time-PCR-Tests bei 54 ausgewählten BF-Proben ließ erkennen, dass in der Real Time-PCR mehr positive Proben waren als in der konventionellen PCR, und erlaubte die Schätzung der Zahl der Genomkopien, was zeigte, dass einige BF-Proben über 1013 Genomkopien/g Gewebe beinhalteten. Obwohl die ECV-Infektionen in 1-12-wöchigen Enten nachgewiesen worden sind, wurden in Enten, die älter als 5 Wochen waren, höhere Virus-DNS-Gehalte ermittelt als in denen, die jünger als 5 Wochen waren. Eine in situ-Hybridisierungsmethode für den Nachweis von ECV in histologischen Präparaten wurde ebenfalls entwickelt. Weitere Untersuchungen sind notwendig, um die klinisch-pathologische Bedeutung der ECV-Infektionen zu bestimmen. Diagnóstico de infección por circovirus del pato mediante reacción en cadena de la polimerasa convencional y a tiempo real Se describe el desarrollo de la primera técnica de reacción en cadena de la polimerasa convencional y a tiempo real (PCR) para el diagnóstico de infecciones por circovirus del pato (DuCV). Ambas técnicas amplifican un fragmento de 230pb específico del gen Rep del DuCV. Aunque ambas técnicas presentaron el mismo límite de detección (13×103 ADNs diana/ mL) cuando el ADN diana se diluyó en agua, el límite de detección de la técnica a tiempo real (13×104 ADNs diana/ mL) fue 10 veces menor que la técnica convencional (13×105 ADNs diana/ mL) cuando las amplificaciones se realizaron en presencia de ADN celular. Mediante la técnica convencional de PCR, el ADN DuCV fue detectado en 85 (84%) de 101 bolsas de Fabricio (BF) de patos enfermos o muertos, de edades comprendidas entre 1 y 12 semanas y en muestras de 35 (94%) de 37 lotes. La aplicación de la técnica de PCR basada en SYBR Green en 54 muestras seleccionadas de BF indicó que habían más muestras positivas a la PCR a tiempo real que a la PCR convencional y permitió estimar el número de copias de genoma y mostró que algunas muestras de BF contenían más de 1013 copias de genomas/ g tejido. Aunque las infecciones por DuCV se detectaron en aves con edades de entre 1 a 12 semanas, los niveles más altos de ADN vírico se detectaron en patos de más de 5 semanas de edad, más que en los patos de menos de 5 semanas. Se desarrolló un método de hibridación in situ para la detección de DuCV en muestras histológicas. Se necesitan estudios posteriores para determinar la significación clinicopatológica de las infecciones por DuCV.

Comparison of Four Rapid DNA Extraction Techniques for Conventional Polymerase Chain Reaction Testing of Three Mycobacterium spp. that Affect Birds

Mycobacteriosis is an avian disease that is most commonly caused by Mycobacterium avium or Mycobacterium genavense. In order to optimize molecular laboratory tests for diagnosing mycobacteriosis in birds, we compared four methods of rapid DNA extraction with isolates of M. avium, M. genavense, and Mycobacterium fortuitum. DNA extraction methods included enzymatic lysis, boiling for 30 min followed by enzymatic lysis, four cycles of freezing and thawing followed by enzymatic lysis, and bead beating followed by enzymatic lysis. The DNA yield and purity for the four methods were evaluated by spectrophotometry and compared. The bead beating with enzymatic lysis technique yielded significantly purer and higher concentrations of extracted DNA compared with other DNA extraction methods. All four methods yielded extraction products for all three organisms that were successfully amplified by polymerase chain reaction (PCR) for a fragment in the 65-kD heat shock protein gene. Subjectively, the PCR amplification products were most abundant for samples extracted by bead beating with enzymatic lysis.