Rapid Real-time Polymerase Chain Reaction Research Articles

Comparison of Positivity Rates of Rapid Antigen Testing and Real-Time Polymerase Chain Reaction for COVID-19 During the First and Second Waves of the Pandemic in Eastern Uttar Pradesh, India.

BackgroundThe advent of the second wave of coronavirus disease 2019 (COVID-19) in India caused a new range of challenges in diagnosing the virus. Various point-of-care tests have been introduced for rapid diagnosis. Although rapid antigen tests are the most commonly used, the false-negative rates are high. Therefore, the purpose of this study was to compare the positivity rate of real-time polymerase chain reaction (RT-PCR) testing in rapid antigen-negative cases of COVID-19 during the first and second waves of the COVID-19 pandemic.MethodologyThis was an observational study conducted in the Department of Microbiology, All India Institute of Medical Sciences, Gorakhpur.ResultsIn total, 2,168 patients were tested. The percentage positivity rate of the RT-PCR tests among the antigen-negative samples was 4.34% in the first wave of the pandemic whereas it was 8.08% in the second wave.ConclusionsThe main conclusion of this study was that antigen tests should never be used alone for the diagnosis of COVID-19. Instead, they should be confirmed with a RT-PCR test.

Validation of a rapid antigen test as a screening tool for SARS-CoV-2 infection in asymptomatic populations. Sensitivity, specificity and predictive values.

BackgroundEarly diagnosis of SARS-CoV-2 infection is essential to reduce disease spread. Rapid antigen tests have not been sufficiently evaluated in asymptomatic patients to be used as massive population screening tools.MethodsHead-to-head evaluation of Roche SARS-CoV-2 Rapid Antigen Test and real-time reverse transcription polymerase chain reaction (RT-PCR) as SARS-CoV-2 screening tools performed in asymptomatic adults from a semi-closed community in University of Navarra (Spain) from November 2020 to January 2021. Sensitivity, specificity and predictive values were calculated using RT-PCR as reference method.FindingsRoche SARS-CoV-2 Rapid Antigen Test was performed on 2542 asymptomatic adults in a community with a SARS-CoV-2 incidence of 1·93%. It showed a sensitivity of 71·43% (CI 95%: 56·74 – 83·42) and a specificity of 99·68% (CI 95%: 99·37 - 99·86). Positive Predictive Value was 81·4 (CI 95% 66·6 – 91·61) and Negative Predictive Value was 99·44 (CI 95% 99·06 – 99·69). Test sensitivity was related to viral load, with higher sensitivity in RT-PCR cycle threshold (Ct) values under 25 (93·75%, CI 95%: 71·96 – 98·93), that dropped to 29·41% (CI 95%: 10·31- 55·96) in RT-PCR Ct values above 25.InterpretationThis study suggests that rapid antigen tests are less effective in asymptomatic population, when compared with RT-PCR. Further studies are needed to evaluate different options to improve screenings based on rapid antigen test, such as the use of clinical questionnaires to select higher risk-participants, the confirmation of negative results with RT-PCR or the use of repetitive sequential testing.FundingThis research received no external funding.

Prevalence of Malaria at a Tertiary Care Hospital in Mumbai, India

Background: Malaria is caused by parasites of genus Plasmodium. It remains a major public health concern around the world. Though various diagnostic tools are available, there is an urgent need to use a more sensitive diagnostic method for early diagnosis to prevent unwanted outcomes.
 Objectives: (i) To assess the prevalence of malaria in a tertiary care hospital in Mumbai (ii) To detect and speciate Plasmodium by Peripheral Blood Smear (PBS), Rapid Diagnostic Test (RDT) and real-time Polymerase Chain Reaction (PCR) and to compare their performance characteristics.
 Methodology: A hospital-based diagnostic study was carried over a period of 18 months. A total of 550 non-duplicated blood samples from clinically suspected cases of malaria were collected and subjected to three tests- PBS by Field’s staining, RDT and PCR to detect Plasmodium species. The agreement and the differences between the three tests were analyzed and the statistical significance was assessed using Chi-square test.
 Results: Out of the 550 samples, 166 (30.2%) were positive for malaria by either of the three tests, of which 92 (55.42%), 119 (71.69%) and 161 (96.99%) samples were positive by PBS, RDT and PCR, respectively. P. vivax was the predominant Plasmodium species. The most significant finding was PCR detected mixed infections in 8.43% of cases whereas PBS and RDT could detect 0% and 1.20% of mixed infection, respectively. Considering PBS as gold standard, sensitivity of RDT and PCR was 100% and 97.83% and specificity was 94.10% and 84.50% respectively.
 Conclusions: In this study, 30% of the population was infected with malaria with P. vivax being the predominant Plasmodium species. PCR helped identify more mono and mixed infections than conventional methods and would be a helpful adjunct for malaria diagnosis in tertiary care setup.

Pre-Operative SARS CoV-2 Rapid Antigen Test and Reverse Transcription Polymerase Chain Reaction: A conundrum in surgical decision making.

Purpose:Since the eye is in close proximity to the oro-nasal cavity, transmission of SARS CoV-2 is higher during ophthalmic surgical procedures, it is vital to ensure the safety of the healthcare team by pre-operative Rapid Antigen Test (RAT) and real-time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for SARS CoV-2 viral RNA of all patients planned for elective or emergency surgery. Hence, understanding the profile of pre-operative RAT and real-time RT-PCR among patients planned for various ophthalmic procedures, will help us make better decisions for future guidelines, for optimal planning of ophthalmic elective or emergency surgeries, keeping in mind the current scenario.Methods:This is a prospective descriptive study conducted on patients planned for elective or emergency ophthalmic surgeries, who had no COVID-19-related symptoms pre-operatively and were subjected to preoperative RAT and real-time RT-PCR of nasopharyngeal and throat swabs for SARS CoV-2 viral RNA. Data was compiled and statistically analysed.Results:204 patients underwent pre-operative RAT and RT-PCR testing; of which, 85 were females and 119 were males. Mean age of the study population was 51.44 ± 16.501 years. Among the 204, 2 tested positive for SARS CoV-2 with Rapid Antigen testing as well as RT-PCR and 10 patients tested positive via RT-PCR after a negative RAT result of the same sample.Conclusion:Pre-operative testing for COVID-19 disease is indispensable as there is a high chance of transmission from patient to healthcare workers. RAT has the advantage of quick results and may play a role in case of emergency procedures. However, it is prudent to perform the more sensitive real-time RT-PCR before any elective, non-emergency procedures or surgeries to avoid any undue risk to the healthcare team.

The time is “now” for rapid PCR testing for influenza in clinical practice

El Feghaly et al at Children's Mercy Kansas City presented a brief survey to providers in their emergency department and urgent care centers who placed an order for influenza RT-PCR test, asking their estimation of probability of the patient having influenza and their intentions to manage, including therapy. They also captured actual management once the results were available. Overall, about one-half of tests were positive. Clinical suspicion was not a predictor of test results, the positivity rate being 40% if suspicion was low and 57% if high. Test results changed clinicians' management (eg, for further tests/imaging, antibiotic/antiviral therapy) in 44% of influenza PCR-positive cases and in 93% of PCR-negative cases. The net cost savings in avoiding antiviral therapy alone offset the cost of PCR testing compared with rapid antigen testing for influenza. It's a new world. With PCR testing for influenza being the first highly sensitive and specific test, we can accurately discern the etiology of the patient's illness. As the flu season looms, not only are there ramifications of testing for optimizing treatment for influenza and saving resources, but because of pandemic SARS-CoV-2 there is an urgent need to know who has what. With clinical ability to diagnose influenza as shown in this study to be no better than a coin toss, and with most SARS-CoV-2 infections in children not discernible by clinical manifestations, we face a nightmare that includes managing families and teachers and school children, if there is concurrent community activity of both viruses. Can we tamp down the nightmare? Absolutely. 1) Promote, insist, and ensure influenza vaccination (unless a valid contraindication exists) of every person 6 months of age or older – both to reduce illness as well as the seasonal burden of influenza. In another study published in this volume and conducted across 6 countries, Goldman et al showed that there may be increased opportunity. Questioning ∼2400 parents bringing a child to an emergency department earlier in the COVID-19 pandemic about their plan to have their child immunized against influenza in the Fall, almost 30% who had not done so last year planned to do so in 2020. We must capitalize on the moment, offer, and facilitate vaccinations. 2) Continue optimal behaviors to reduce SARS-CoC-2 transmission and acquisition. 3) Sort out the logistics to be able to use the powerful technology of PCR testing in everyday clinical practice in order to accurately determine etiology of respiratory illness at least for these 2 viruses. Articles pages 87 and 271 ▸ Impact of Rapid Influenza Molecular Testing on Management in Pediatric Acute Care SettingsThe Journal of PediatricsVol. 228PreviewTo measure the impact of rapid influenza real-time qualitative reverse transcriptase polymerase chain reaction (RT-PCR) on patient management in busy pediatric emergency department (ED) and urgent care clinic settings. Full-Text PDF Willingness to Vaccinate Children against Influenza after the Coronavirus Disease 2019 PandemicThe Journal of PediatricsVol. 228PreviewTo determine factors associated with parents who plan to vaccinate their children against influenza next year, especially those who did not vaccinate against influenza last year using a global survey. Full-Text PDF

Computer-Aided Detection (CAD) for COVID-19 based on Chest X-Ray Images using Convolutional Neural Network

Covid-19 has spread throughout the world and has been declared as a pandemic by the World Health Organization (WHO). The disease was discovered by the end of 2019 in Wuhan- China. The number of deaths continues to surge sharply and spread to many countries. Covid-19 has sent billions of people on earth into lock-down when health services struggle to cope. A swift and reliable Covid-19 diagnosis system is needed, to direct the patient to the appropriate treatment and prevent the disease dissemination. During this time, we are familiar with rapid tests and Real-Time Polymerase Chain Reaction (RT-PCR) as the procedure of Covid-19 detection. Both of these procedures tend to be impractical and require specialized laboratories that are arranged in such away. It can also take several hours to wait for the amplification process until the results are known. In this study, we introduce a Covid-19 detection system based on Chest C-Ray images using Convolutional Neural Network (CNN). The dataset consists of 1000 images, 500 images each for positive Covid-19, and Pneumonia. The CNN model that was designed consisted of three hidden layers, a fully connected layer with sigmoid activation. The evaluation was conducted to determine the performance of the proposed model using matrices of precision, recall, F1, and accuracy. The experimental results show that the proposed method provides precision, recall, F1 was 1 and 100% accuracy, respectively. This research is expected to be tested in field validation, to help the medical authorities for clinical diagnosis.

Comparison of Patient-collected and Lab Technician-collected Nasopharyngeal and Oropharyngeal Swabs for Detection of COVID-19 by RT-PCR.

Background & Objective:A simple approach to prevent close contact in healthcare settings during the COVID-19 outbreak is to train patients to collect their own nasopharyngeal and oropharyngeal swabs and deliver them to medical laboratories to have them processed. The aim of our study was to compare lab technician- with patient- collected oropharyngeal and nasopharyngeal samples for detection of the coronavirus disease 2019 (COVID 19) using rapid real-time polymerase chain reaction (rRT-PCR).Methods:Fifty adult patients with flu-like symptoms and radiologic findings compatible with atypical pneumonia who were admitted to the infectious diseases ward of Imam Khomeini Hospital Complex, Tehran, Iran, with a clinical diagnosis of COVID-19 from February 28 to April 27 of 2020 were randomly selected and entered in our study. Two sets of naso- and oropharyngeal swabs were collected, one set by a lab technician and the other by the patients, and the COVID-19 rRT-PCR test was performed.Results:Of 50 selected cases, in seven patients all collected naso- and oropharyngeal swabs tested positive, and in 22 patients all samples tested negative for COVID-19 in rRT-PCR. Discrepancies between rRT-PCR results of lab technician- and patient-collected swabs were observed in 12 nasopharyngeal and 13 oropharyngeal specimens. Positive lab technician-collected and negative patient-collected samples were observed in 10 and 5 nasopharyngeal and oropharyngeal specimens, respectively. Negative lab technician-collected and positive patient-collected samples were observed in two and seven nasopharyngeal and oropharyngeal specimens, respectively. The overall percentage of agreement among both nasopharyngeal and oropharyngeal swabs taken by a lab technician and patients was 76% with a kappa value of 0.49 (P=0.001).Conclusion:Based on our findings, lab technician-collected naso- and oropharyngeal swabs cannot be replaced by patient-collected ones with regard to COVID-19 rRT-PCR.

Multiplex Solid-Phase Real-Time Polymerase Chain Reaction without DNA Extraction: A Rapid Intraoperative Diagnosis Using Microvolumes

Current polymerase chain reaction (PCR) methods for the diagnosis of infections are time consuming and require large sample volume and skilled technicians. We developed a novel, easy-to-use, and rapid (processing time, 1 minute; total time, 33 minutes) multiplex real-time PCR test (Direct Strip PCR) that did not require DNA extraction to detect 9 pathogens that could cause uveitis in 20-μl samples. Multicenter prospective evaluation of a diagnostic PCR test. A total of 511 participants (patients with infectious uveitis and controls) were examined at 18 institutes worldwide. After validation, intraocular fluid samples were subjected to etiologic or exclusive diagnosis, including intraoperative rapid diagnosis. The concordance and correlations between Direct Strip PCR and quantitative PCR (qPCR) results. Direct Strip PCR exhibited rapid detection, good repeatability and specificity, long storage stability, and detection ability equal to that of qPCR. It also showed low interinstitutional variability compared with qPCR, even when PCR beginners used various real-time PCR machines. The Direct Strip PCR for 9 pathogens exhibited high concordance against the qPCR (positive concordance rate, 98.8%-100%; negative concordance rate, 99.8%-100%; κ coefficient, 0.969-1.000; P < 0.001-0.031). Additionally, results obtained using Direct Strip PCR and qPCR were highly correlated (ρ= 0.748; P < 0.001). This assay was used for rapid intraoperative diagnosis. The Direct Strip PCR test may improve the prognosis of various infectious diseases because it facilitates rapid etiologic evaluation at the first hospital visit and can be used for intraoperative diagnosis.

Epidemiology of influenza: findings from surveillance system of 40 hospital in Thailand, 2010-2019

Abstract Background Although recent efforts from some Asian countries to describe burden of influenza disease and seasonality, these data are missing for the vast majority, including the private section of Thailand. A near real-time laboratory-based influenza surveillance system, in a network of 40 hospitals was implemented aiming to determine influenza strains circulating in the private hospitals of Thailand and know characteristics, trend and burden of influenza viruses. Methods We obtained the data by monitoring patients with influenza-like illness (ILI) at a network of 40 private hospitals across Thailand. Throat-swab specimens in viral transport media were collected and transported within 24 h of collection using a cold-chain system. The respiratory samples were tested by rapid influenza diagnostic tests and real-time reverse transcription polymerase chain reaction. Results From January 2010 to November 2019, a total of 1,300,594 subjects were tested and 320,499 cases of influenza were identified. Of those positive cases, 116,317(36.3%) were influenza type B, 185,512(57.9%) were influenza A unspecified subtype, 8,833(2.7%) were influenza A(H1N1)pdm2009 and 6,371(1.9%) were seasonal influenza A(H3N2). Positive rate were 50.5 and 49.5 in female and male. Positivity rate was 41.4% in persons 15-49 years followed by 29.1% in 15-14 years, 17.6% in under five children and 11.7% in &amp;gt; 49 years. In 2018-2019 season, the highest positivity rate observed in February and March (39.3%) followed by April (34.2%) and January (32.3%) while the lowest positivity rate was in May (18.1%). Conclusions In Thailand, seasonal Influenza A(H3N2), Influenza A(H1N1)pdm2009 and Influenza B viruses were circulating during 2010-2019. In last season, positivity rate and number of cases peaked in February and March. Key messages Influenza is one of public health problems in Thailand. The need to introduce influenza vaccine and antivirus is important to prevent and treat the disease in future.

Impact of Rapid Influenza Molecular Testing on Management in Pediatric Acute Care Settings

To measure the impact of rapid influenza real-time qualitative reverse transcriptase polymerase chain reaction (RT-PCR) on patient management in busy pediatric emergency department (ED) and urgent care clinic settings. We developed a brief, elective survey that clinicians completed when an influenza RT-PCR order was placed in the ED or urgent care clinic between February 18, 2019, and March 13, 2019. We captured the clinical suspicion for influenza, intended management plans, and actual management plans once influenza RT-PCR results were available. We evaluated 339 encounters, of which 164 (48.4%) had a positive influenza RT-PCR. Clinical suspicion for influenza was a nonsignificant predictor for influenza PT-PCR positivity (P=.126). After rapid influenza RT-PCR results were available, clinicians changed their original plans in 44.5% of influenza RT-PCR positive vs 92.6% of influenza RT-PCR negative cases (P<.0001). Change in plans for antiviral use was observed in 26% of influenza positive vs 77% of influenza negative cases (P<.0001). A total of 135 antiviral prescriptions were avoided in patients with negative influenza RT-PCR. Implementation of a rapid and accurate influenza RT-PCR in the acute care setting is important to systematically diagnose influenza in children and improve outpatient management decisions, because clinical suspicion for influenza is inaccurate. A negative influenza RT-PCR decreases unnecessary antiviral use and has the potential for significant cost savings.

Evaluation of hepatitis B viremia and corresponding antibodies among infected patients attending Abuja Teaching Hospital, Nigeria

In most under-developed and developing countries, diagnosis and treatment of hepatitis B relied mainly on detection of hepatitis B virus (HBV) serological biomarkers. The reliability of these markers in comparison with HBV DNA viral load is required to review their diagnostic value. Thus, this study investigated the serological and HBV viral load profile of persons with hepatitis B attending the University of Abuja Teaching Hospital, Gwagwalada, Nigeria. Attributes of hepatitis B-infected participants (February-May, 2018) were assessed. They included hepatitis B antigens (HBsAg, HBeAg), antibodies (HBsAb, HBcAb, HBeAb) and HBV DNA, using rapid immunichromatigraphical and real-time polymerase chain reaction (qPCR), respectively. Structured questionnaires were used to collate participants biodata. Out of 53 participants, 30 were male and 23 were female. 90.6% (48/53) were positive for HBsAg, 28.3% (15/53) were positive for HBsAb, 60.4% (32/53) were positive for HBcAb, 17.0% (9/53) were positive for HBeAg, while HBeAb was detected in 58.5% (31/53). HBV DNA was significantly associated with HBcAb (χ2=28.622, P=0.000), HBeAg (χ2=11.820, P=0.008), and HBeAb (χ2=16.440, P=0.001). The on-site point of care serological test has significant impact in diagnosis and monitoring Hepatitis B when compared to qPCR.

Guided antibiotic therapy for Mycoplasma genitalium infections: Analysis of mutations associated with resistance to macrolides and fluoroquinolones

IntroductionThe objective of this study was to analyse the susceptibility of Mycoplasma genitalium to macrolides and fluoroquinolones using molecular techniques. MethodsSusceptibility to macrolides was tested (Gipuzkoa, 2014–2017) by a rapid probe-based real-time polymerase chain reaction assay (23S rRNA gene) and to fluoroquinolones by sequencing the parC and gyrA genes. ResultsMutations associated with macrolide resistance were detected in 43/263 (16.3%) cases and potential fluoroquinolone resistance in 21/267 (7.9%). Macrolide resistance was more frequent in patients previously treated with azithromycin (76.5% vs 7.4%, p<0.001) as well as in those treated with a single 1g dose (31.3%) vs the extended regimen (7%, p<0.001). There were 5/245 (2%) cases with mutations probably associated with resistance to both antibiotics. ConclusionsThe technique used for testing M. genitalium susceptibility to azithromycin allowed the rapid implementation of resistance-guided antibiotic therapy. Moxifloxacin could be a good option in cases of macrolide resistance.

Syphilis in female sex workers: an epidemiological study of the highway system of the state of Pará, northern Brazil.

Female sex workers (FSWs) are considered a bridge for transmission of pathogens from high-risk to general populations. We assessed the epidemiological status of syphilis in FSWs along the Pará highway system. Two hundred and twenty-two FSWs were interviewed and samples were analyzed using rapid qualitative tests and real-time polymerase chain reaction. The prevalence of syphilis was high (36.94%). The high rate of prostitution, use of illicit drugs, and search for financial resources increased Treponema pallidum transmission through unprotected sex. Several characteristics of FSWs were identified, which reinforce the need for measures guaranteeing their health and protection.

Salmonella Spp. and Shigella Spp. detection via multiplex real-time PCR and discrimination via MALDI-TOF MS in different animal raw milk samples.

The aim of this study was to provide epidemiological data about the presence of Salmonella spp. and Shigella spp. in raw milk samples collected from different animals. A total of 231 raw milk samples from 48 cows, 65 goats, 65 sheep, and 53 donkeys were studied. The ISO 6579:2002 and ISO 21567:2004 methods, antimicrobial susceptibility tests, and serotyping were performed. Species and subspecies discriminations were made via matrix-assisted laser desorption/ionization-time of flight mass spectrometry. After DNA isolation from all samples, Salmonella spp. and Shigella spp. were detected using real-time polymerase chain reaction (PCR) kits. Five samples (2.16%) showed positivity out of 231 raw milk samples for Salmonella spp., and 2 (0.87%) samples were detected to be positive by multiplex real-time PCR design. We found that raw milk samples were not free of Salmonella spp. and Shigella spp. and need to be tested routinely to avoid public health problems. Rapid and reliable real-time PCR method can be developed and used for this purposes instead of slow bacterial culture processes.

Tratamiento antibiótico dirigido en infecciones por Mycoplasma genitalium: análisis de mutaciones asociadas con resistencia a macrólidos y fluoroquinolonas

IntroductionThe objective of this study was to analyse the susceptibility of Mycoplasma genitalium to macrolides and fluoroquinolones using molecular techniques. MethodsSusceptibility to macrolides was tested (Gipuzkoa, 2014-2017) by a rapid probe-based real-time polymerase chain reaction assay (23S rRNA gene) and to fluoroquinolones by sequencing the parC and gyrA genes. ResultsMutations associated with macrolide resistance were detected in 43/263 (16.3%) cases and potential fluoroquinolone resistance in 21/267 (7.9%). Macrolide resistance was more frequent in patients previously treated with azithromycin (76.5% vs 7.4%, P<.001) as well as in those treated with a single 1g dose (31.3%) vs the extended regimen (7%, P<.001). There were 5/245 (2%) cases with mutations probably associated with resistance to both antibiotics. ConclusionsThe technique used for testing Mycoplasma genitalium susceptibility to azithromycin allowed the rapid implementation of resistance-guided antibiotic therapy. Moxifloxacin could be a good option in cases of macrolide resistance.

A tale of the traded cat: development of a rapid real-time PCR diagnostic test to distinguish between lion and tiger bone

A rapid allelic discrimination real-time polymerase chain reaction (qPCR) assay has been developed for the confirmation of lion (Panthera leo) or tiger (P. tigris) DNA. The method includes a low cost allele specific PCR assay designed for each SNP variant where the genotype is determined by incorporation and subsequent fluorescence of FAM or HEX following PCR. The described method was able to accurately and rapidly distinguish between the two species, and all three mitochondrial regions (16S, 12S and control region) performed equally well. The proposed real-time PCR assay can be applied for the accurate confirmation of either lion or tiger DNA that could be used by law enforcement agencies around the world as a tool to monitor illegal trade of tiger bones.

Development of Visually Improved Loop Mediated Isothermal Amplification for the Diagnosis of Plasmodium vivax Malaria in a Tertiary Hospital in Chandigarh, North India.

More than 80% of the global burden of the Plasmodium vivax is contributed by mainly three countries (India, Indonesia, and Pakistan). Reports from last decades have highlighted the occurrence of severe P. vivax malaria which was earlier considered to be benign. The recent trends of increasing P. vivax-associated morbidity and mortality emphasizes the need for early and accurate diagnosis of P. vivax malaria for the timely management of patients. Microscopy is considered a gold standard but needs experienced laboratory technologists. Over the last few years, Polymerase chain reaction (PCR) is being used as a highly sensitive and specific test but it requires expensive equipment which limits its use in the field. Therefore, in the present study, utility of visually improved loop-mediated isothermal amplification (LAMP) for the detection of P. vivax was evaluated targeting 18SrRNA gene in 145 microscopically confirmed P. vivax and 20 P. vivax negative patients. Sensitivity and specificity of LAMP was assessed with respect to microscopy and multiplex nested PCR (nPCR). Results of the LAMP assay was also correlated with rapid diagnostic test, multiplex nPCR and real-time PCR results. Overall, sensitivity and specificity of P. vivax-specific LAMP compared with microscopy were found to be 100% and 85%, respectively. Furthermore, detection limit for LAMP was found to be 0.8 copies/μL and it was also able to detect three complicated cases of P. vivax which were missed by microscopy. This study showed a LAMP assay to be a rapid and very sensitive method for the early diagnosis of both complicated and uncomplicated P. vivax malaria.

Quantitative assessment of Lactococcus lactis subsp. cremoris present in artisanal raw cow’s milk cheese

Lactococcus lactis subsp. cremoris belongs to lactic acid bacteria that play a crucial role in cheese production and it is known to be beneficial to human health. The aim of the study was to establish a rapid and accurate quantitative real-time polymerase chain reaction (qPCR) method to detect and enumerate L. lactis subsp. cremoris in artisanal raw cow’s milk cheese. Artisanal raw cow’s milk cheese samples were used to check for presence and number of L. lactis subsp. cremoris strains. The method applies a set of target-specific PCR (polymerase chain reaction) primers and a fluorogenic probe, and amplifies a part of the LACR_RS01280 gene that encodes the aminoacetone oxidase family flavin adenine dinucleotide (FAD) binding enzyme. All 5 L. lactis subsp. cremoris strains examined were found to be qPCR positive. There was no signal recorded for 8 strains which belong to closely related species. The limit of detection amounted to ten copies per reaction and the assay indicated a linear dynamic range of seven logs. This method may be applied in detection and enumeration of L. lactis subsp. cremoris in cheese during its ripening. Moreover, it may be applied to examine the distribution of L. lactis subsp. cremoris during the cheese production and ripening.

Development and application of a triplex real-time PCR assay for the simultaneous detection of avian influenza virus subtype H5, H7 and H9

Avian influenza virus (AIV), especially subtypes H5, H7 and H9, has contributed to enormous economic losses and poses a potential pandemic threat to global human public health. Early screening of suspected cases is key to controlling the spread of AIVs. In this study, an accurate, rapid, and triplex real-time polymerase chain reaction (PCR) assay was developed for the simultaneous detection of AIV subtypes H5, H7 and H9. The sensitivity of the real-time PCR was at least 100 times higher than that of the conventional PCR, with a detection limit of 50 copies and an EID50 of 1 (50% egg infections dose) for the H5, H7, and H9 subtypes. The lack of cross-reaction with other avian respiratory viruses suggested that the real-time PCR assay was highly specific. The reproducibility of the assay was confirmed using plasmids containing targets genes. Furthermore, 362 clinical field samples were evaluated. Subtypes H5, H7 and H9 were detected in 102 (28.18%) samples by real-time PCR and in 35 (9.67%) samples by conventional virus isolation. These results indicate that the triplex real-time PCR assay has good sensitivity, specificity and reproducibility and that it might be useful for laboratory surveillance and rapid diagnosis of the H5, H7 and H9 subtypes of influenza A viruses.

SYBR green-based one step quantitative real-time polymerase chain reaction assay for the detection of Zika virus in field-caught mosquitoes

BackgroundThe monitoring of vectors is one of the key surveillance measures to assess the risk of arbovirus transmission and the success of control strategies in endemic regions. The recent re-emergence of Zika virus (ZIKV) in the tropics, including Singapore, emphasizes the need to develop cost-effective, rapid and accurate assays to monitor the virus spread by mosquitoes. As ZIKV infections largely remain asymptomatic, early detection of ZIKV in the field-caught mosquitoes enables timely implementation of appropriate mosquito control measures.ResultsWe developed a rapid, sensitive and specific real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for the detection of ZIKV in field-caught mosquitoes. The primers and PCR cycling conditions were optimized to minimize non-specific amplification due to cross-reactivity with the genomic material of Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex tritaeniorhynchus, Culex sitiens and Anopheles sinensis, as well as accompanying microbiota. The performance of the assay was further evaluated with a panel of flaviviruses and alphaviruses as well as in field-caught Ae. aegypti mosquitoes confirmed to be positive for ZIKV. As compared to a probe-based assay, the newly developed assay demonstrated 100% specificity and comparable detection sensitivity for ZIKV in mosquitoes.ConclusionsBeing a SYBR Green-based method, the newly-developed assay is cost-effective and easy to adapt, thus is applicable to large-scale vector surveillance activities in endemic countries, including those with limited resources and expertise. The amplicon size (119 bp) also allows sequencing to confirm the virus type. The primers flank relatively conserved regions of ZIKV genome, so that, the assay is able to detect genetically diverse ZIKV strains. Our findings, therefore, testify the potential use of the newly-developed assay in vector surveillance programmes for ZIKV in endemic regions.