Nucleic Acid Polymerase Chain Reaction Research Articles

Pupal Exuviae of Culex Pipiens L. (Diptera: Culicidae) Can be Utilised as a Non-Invasive Method of Biotype Differentiation

BackgroundCulex pipiens L. is a principal vector of zoonotic arboviruses in Europe, acting in both an amplification role in enzootic transmission between avian hosts and as a bridge vector between avian hosts and mammals. The species consists of two forms which are indistinguishable using morphological methods but possess varying ecological and physiological traits that influence their vector capacity. In this study we validate methods that can be used to extract trace DNA from single pupal exuviae of Cx. pipiens for use in molecular speciation of samples. These DNA extraction methods are compared using measurement of the total yield and successful identification using a real-time polymerase chain reaction (PCR) assay.ResultsGenomic DNA was initially extracted from colony-derived individuals using an ethanol precipitation method, two commercially available DNA extraction kits: DNeasy® Blood & Tissue Kit (Qiagen, UK) and Wizard® SV Genomic DNA Purification System (Promega, UK) and a direct real-time PCR method. Time elapsed between eclosion and processing of pupae significantly influenced Cx. pipiens form identification as nucleic acid concentration and PCR amplification success decreased with increased time elapsed. Real-time PCR amplification success, however, was not shown to vary significantly between the three extraction methods, with all methods successfully identifying all samples, but the direct real-time PCR method achieved a lesser amplification success rate of 70% (n = 20 for each treatment). More variable results were produced when field-derived exuviae were used, with no significant difference in real-time PCR amplification success found across the four methods and a lower overall rate of successful identification of 55–80%.ConclusionsThis study shows that both colony and field derived Cx. pipiens pupal exuviae can be a useful non-invasive source of trace DNA permitting accurate biotype differentiation for at least twenty-four hours post-eclosion. The significance and utility of this technique in ecological and behavioural studies of Cx. pipiens is discussed and recommendations made for use according to experimental scenario.

Molecular identification of the bacterium acute conjunctivitis by the method of sequensing gen 16S rRNA

Conjunctivitis is the most common eye disease, characterized by contextual inflammation, which can be caused by bacteria. The diagnosis of conjunctivitis is established based various factors, including the patients’s medical history, onset of eye symptoms, non-ocular symptoms, previous disease history, family medical history, allergies, and physical examination of the eyes, which may include assessments of visual acuity and vision field. Some cases of conjunctivitis require laboratory examinations to confirm the diagnosis. These may include cytological examination with Giemsa staining, Chlamydia Diagnostic Test, Nucleic acid amplification tests (NAATs) or Polymerase chain reaction (PCR), and microbiological tests. Microbiological examination helps identify the bacteria responsible for conjunctivitis and aids in treatment by prescribing antibiotics to suppress the growth of the infecting bacteria. While identification tests are not routinely performed, researchers often seek to determine the specific type of bacteria causing conjunctivitis infections, which may require several laboratory tests. Bacterial virulence plays a significant role, with genetic mutations potentially leading to severe infections of varying severity. Virulence genes encode proteins that express pathogenic properties. The species responsible for conjunctivitis can be definitively identified definitively through microbiological examination, utilizing methods such as the 16S Ribosomal RNA Sequencing (rRNA) technique, known for its accuracy and speed. This study aims to analyze the results of rRNA sequencing in cases of acute bacterial conjunctivitis caused by 16S rRNA genotyping. The research employed an exploratory metholodolot, with the results analyzed using the The Basic Local Alignment Search Tool (BLAST) tracking program database on the National Center for Biotechnology Information (NCBI) website. The findings revealed that Sphingomonas paucimobilis encoded the 16S rRNA using Universal Primary 27 F and 1492 R, obtained in a sequence size of 1351bp. The isolate demonstrated similarities to Sphingomonas paucimobilis.

Disposable impedance sensors based on novel hybrid MoS2 nanosheets and microparticles to detect Escherichia Coli DNA.

The rapid and accurate detection of pathogenic bacteria is essential for food safety and public health. Conventional detection techniques, such as nucleic acid sequence-based amplification and polymerase chain reaction, are time-consuming and require specialized equipment and trained personnel. Here, we present quick, disposable impedance sensors based on the novel hybrid MoS2 nanomaterial for detecting Escherichia coli DNA. Our results indicate that the proposed sensors operate linearly between 10- 20 and 10-15 M concentrations, achieving an impressive detection limit of 10-20 M with the highest sensitivity observed at a 0.325 nM probe concentration sensor. Furthermore, the electrochemical impedance spectroscopy biosensors exhibited potential selectivity for Escherichia coli DNA over Bacillus subtilis and Vibrio proteolyticus DNA sequences. The findings offer a promising avenue for efficient and precise DNA detection, with potential implications for broader biotechnology and medical diagnostics applications.

Characteristics and prognostic analysis of patients with detected KRAS mutations in resected lung adenocarcinomas by peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp method.

Kirsten rat sarcoma virus (KRAS) gene mutations are a type of driver mutation discovered in the 1980s, but for a long time no molecular targeted drugs were available for them. Recently, sotorasib was developed as a molecular targeted drug for KRAS mutations. It is therefore necessary to identify the characteristics of patients with KRAS mutations. This was the single-institution retrospective study. Surgically resected tumors from lung adenocarcinoma patients were collected at a single institution from June 2016 to September 2019. Peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamp analysis of KRAS G12X mutations was compared with analysis by therascreen KRAS RGQ kit. The association between KRAS mutation status and patient characteristics and prognosis was assessed. Among 499 lung adenocarcinomas, KRAS mutations were evaluated in 197 cases, excluding stage IV lung cancer and tumors with epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutations. KRAS G12X mutations were detected in 59 cases (29.9%). The highest frequency by gene mutation subtype was G12V in 23 cases (39.0%), followed by G12C in 16 cases (27.1%), G12D in 12 cases (20.3%), G12S in 4 cases (6.8%) and G12A in 2 cases. For the G12C mutation, the PNA-LNA PCR clamp and therascreen methods were consistent, but for the G12D and G12S mutations, the PNA-LNA PCR clamp method showed higher detection rates. In operable tumors, G12C mutations were more frequent in males, smokers, and patients with high expression of programmed death-ligand 1 (PD-L1), and had no correlation with prognosis. By the PNA-LNA PCR clamp method, G12C mutation of surgical specimens was detected successfully. The PNA-LNA PCR clamp method is expected to be applied to the detection of druggable G12C mutations.

Identification of animal species by nucleic acid chromatography of hair samples and investigation of its applicability to human biological samples

GeneFields®-Hair is a simple analysis kit that uses nucleic acid chromatography and polymerase chain reaction (PCR) to identify animal species of hair-like food contaminants. In this study, we evaluated GeneFields®-Hair as a simple and rapid method for identifying animal species from hair-like materials collected in forensic science, such as at crime scenes. The use of this kit with other human biological materials (whole blood, head dandruff, nails, saliva, oral mucosa, sebum, and urine) was also investigated. Animal body hair samples were pretreated by grinding in a buffer solution, centrifuged, and the supernatant was used for PCR. Nucleic acid chromatography of the PCR products allowed the identification of the animal species by the presence or absence of coloration on the decision line. For human biological materials, nucleic acid chromatography was performed after the appropriate pretreatment like body hair material. The determination of some animal species was difficult, even if they had a dedicated DNA Strip determination line. Furthermore, animals from the same family but different genera were sometimes detected on the same determination line. All the human biological samples were correctly identified. Smartphone photographs of the coloration of the judgment line were processed using the ImageJ software for quantitative determination.

Development of a Detection System for ESR1 Mutations in Circulating Tumour DNA Using PNA-LNA-Mediated PCR Clamping.

Although circulating tumour DNA (ctDNA)-based next-generation sequencing (NGS) is a less invasive method for assessing ESR1 mutations that are essential mechanisms of endocrine therapy resistance in patients with oestrogen receptor-positive breast cancer, adequate amounts of DNA are required to assess polyclonal ESR1 mutations. By combining a peptide nucleic acid and locked nucleic acid polymerase chain reaction (PNA-LNA PCR) clamping assay, we have developed a novel detection system to screen for polyclonal ESR1 mutations in ctDNA. A validation assay was prospectively performed on clinical samples and compared with the NGS results. The PNA-LNA PCR clamp assay was validated using six and four blood samples in which ESR1 mutations were detected by NGS and no mutations were detected, respectively. The PNA-LNA assay results were comparable with those of NGS. We prospectively assessed the concordance between the PNA-LNA PCR clamp method and NGS. Using the PNA-LNA PCR clamp method, ESR1 mutations were detected in 5 out of 18 samples, including those in which mutations were not detected by NGS due to small amounts of ctDNA. The PNA-LNA PCR clamping method is a highly sensitive and minimally invasive assay for polyclonal ESR1 mutation detection in the ctDNA of patients with breast cancer.

Research advances in the CRISPR-Cas13 system in the field of nucleic acid detection

Nucleic acid detection plays an important role in environmental monitoring, food safety, pathogen detection, genetic disease detection and so on. At present, nucleic acid detection methods mainly include nucleic acid molecular hybridization and polymerase chain reaction. Nucleic acid detection of Corona Virus Disease 2019 has become an indispensable part of our daily life. With the establishment of new detection methods, nucleic acid detection is developing towards single molecule sensitivity and single base specificity. Colloidal gold method and magnetic particle chemiluminescence method have been widely used in the autonomous detection of COVID-19，while both approaches have their limitations. There is a more efficient and specific approach: CRISPR-Cas13 system

Hundreds-Dollar-Level Multiplex Integrated RT-qPCR Quantitative System for Field Detection.

The COVID-19 pandemic poses a threat to global health. Due to its high sensitivity, specificity, and stability, real-time fluorescence quantitative (real-time PCR) detection has become the most extensively used approach for diagnosing SARS-CoV-2 pneumonia. According to a report from the World Health Organization, emerging and underdeveloped nations lack nucleic acid detection kits and polymerase chain reaction (PCR) instruments for molecular biological detection. In addition, sending samples to a laboratory for testing may result in considerable delays between sampling and diagnosis, which is not favorable to the timely prevention and control of new crown outbreaks. Concurrently, there is an urgent demand for accurate PCR devices that do not require a laboratory setting, are more portable, and are capable of completing testing on-site. Hence, we report on HDLRT-qPCR, a new, low-cost, multiplexed real-time fluorescence detection apparatus that we have developed for on-site testing investigations of diverse diseases in developing nations. This apparatus can complete on-site testing rapidly and sensitively. The entire cost of this instrument does not exceed USD 760. In order to demonstrate the applicability of our PCR instrument, we conducted testing that revealed that we achieved gradient amplification and melting curves comparable to those of commercially available equipment. Good consistency characterized the testing outcomes. The successful detection of target genes demonstrates the reliability of our inexpensive PCR diagnostic technique. With this apparatus, there is no need to transport samples to a central laboratory; instead, we conduct testing at the sampling site. This saves time on transportation, substantially accelerates overall testing speed, and provides results within 40 min.

Effective hybrid deep learning model for COVID-19 patterns identification using CT images.

Coronavirus disease 2019 (COVID‐19) has attracted significant attention of researchers from various disciplines since the end of 2019. Although the global epidemic situation is stabilizing due to vaccination, new COVID‐19 cases are constantly being discovered around the world. As a result, lung computed tomography (CT) examination, an aggregated identification technique, has been used to ameliorate diagnosis. It helps reveal missed diagnoses due to the ambiguity of nucleic acid polymerase chain reaction. Therefore, this study investigated how quickly and accurately hybrid deep learning (DL) methods can identify infected individuals with COVID‐19 on the basis of their lung CT images. In addition, this study proposed a developed system to create a reliable COVID‐19 prediction network using various layers starting with the segmentation of the lung CT scan image and ending with disease prediction. The initial step of the system starts with a proposed technique for lung segmentation that relies on a no‐threshold histogram‐based image segmentation method. Afterward, the GrabCut method was used as a post‐segmentation method to enhance segmentation outcomes and avoid over‐and under‐segmentation problems. Then, three pre‐trained models of standard DL methods, including Visual Geometry Group Network, convolutional deep belief network, and high‐resolution network, were utilized to extract the most affective features from the segmented images that can help to identify COVID‐19. These three described pre‐trained models were combined as a new mechanism to increase the system's overall prediction capabilities. A publicly available dataset, namely, COVID‐19 CT, was used to test the performance of the proposed model, which obtained a 95% accuracy rate. On the basis of comparison, the proposed model outperformed several state‐of‐the‐art studies. Because of its effectiveness in accurately screening COVID‐19 CT images, the developed model will potentially be valuable as an additional diagnostic tool for leading clinical professionals.

CSGBBNet: An Explainable Deep Learning Framework for COVID-19 Detection.

The COVID-19 virus has swept the world and brought great impact to various fields, gaining wide attention from all walks of life since the end of 2019. At present, although the global epidemic situation is leveling off and vaccine doses have been administered in a large amount, confirmed cases are still emerging around the world. To make up for the missed diagnosis caused by the uncertainty of nucleic acid polymerase chain reaction (PCR) test, utilizing lung CT examination as a combined detection method to improve the diagnostic rate becomes a necessity. Our research considered the time-consuming and labor-intensive characteristics of the traditional CT analyzing process, and developed an efficient deep learning framework named CSGBBNet to solve the binary classification task of COVID-19 images based on a COVID-Seg model for image preprocessing and a GBBNet for classification. The five runs with random seed on the test set showed our novel framework can rapidly analyze CT scan images and give out effective results for assisting COVID-19 detection, with the mean accuracy of 98.49 ± 1.23%, the sensitivity of 99.00 ± 2.00%, the specificity of 97.95 ± 2.51%, the precision of 98.10 ± 2.61%, and the F1 score of 98.51 ± 1.22%. Moreover, our model CSGBBNet performs better when compared with seven previous state-of-the-art methods. In this research, the aim is to link together biomedical research and artificial intelligence and provide some insights into the field of COVID-19 detection.

Opportunities for Closing the Gap in HIV Diagnosis, Treatment, and Viral Load Suppression in Children in Malawi: Results From a 2015-2016 Population-based HIV Impact Assessment Survey.

Control of the pediatric HIV epidemic is hampered by gaps in diagnosis and linkage to effective treatment. The 2015-2016 Malawi Population-based HIV impact assessment data were analyzed to identify gaps in pediatric HIV diagnosis, treatment, and viral load suppression. In half of the surveyed households, children ages ≥18 months to <15 years were tested using the national HIV rapid test algorithm. Children ≤18 months reactive by the initial rapid test underwent HIV total nucleic acid polymerase chain reaction confirmatory testing. Blood from HIV-positive children was tested for viral load (VL) and presence of antiretroviral drugs. HIV diagnosis and antiretroviral treatment (ART) use were defined using guardian-reporting or antiretroviral detection. Of the 6166 children tested, 99 were HIV-positive for a prevalence of 1.5% (95% confidence intervals [CI]: 1.1-1.9) and 8.0% (95% CI: 5.6-10.5) among HIV-exposed children. The prevalence of 1.5% was extrapolated to a national estimate of 119,501 (95% CI: 89,028-149,974) children living with HIV (CLHIV), of whom, 30.7% (95% CI: 20.3-41.1) were previously undiagnosed. Of the 69.3% diagnosed CLHIV, 86.1% (95% CI: 76.8-95.6) were on ART and 57.9% (95% CI: 41.4-74.4) of those on ART had suppressed VL (<1000 HIV RNA copies/mL). Among all CLHIV, irrespective of HIV diagnosis or ART use, 57.7% (95% CI: 45.0-70.5) had unsuppressed VL. Critical gaps in HIV diagnosis in children persist in Malawi. The large proportion of CLHIV with unsuppressed VL reflects gaps in diagnosis and need for more effective first- and second-line ART regimens and adherence interventions.

Immunoaffinity-Based Graphene Field-Effect Transistor Sensor Platform for Fast Detection of S1 Subunit Protein from COVID-19

The epidemic of the novel coronavirus (COVID-19) pneumonia touches the nerve of every person. To prevent and control the epidemic, early diagnosis must be addressed. Nucleic acid polymerase chain reaction (PCR) is currently used worldwide to identify positive carriers of COVID-19 when the virus attacks. However, it requires sophisticated biological design, trained personnel, bulky instruments, and is impossible or inaccurate to detect infected but recovered people or asymptomatic carriers. Here, in an effort towards expanding the screening coverage, we combined the extremely sensitive graphene field-effect transistor (Gr-FET) with highly selective antibody-antigen interaction to develop a COVID-19 immuno-sensor. The Gr-FET immunosensors can fast identify and accurately capture the subunit S1 protein from COVID-19 at a limit of detection down to 0.2 pM. Further results suggest that antigen S1 functionalized Gr-FET can be applied to screen neutralizing-antibodies with high efficiency. Thus, Gr-FET immunosensors provide an appealing opportunity to address the earlier diagnosis as well as the analysis and design of neutralizing-antibody locking methods of this ongoing public health crisis.

Fast and Accurate Control Strategy for Portable Nucleic Acid Detection (PNAD) System Based on Magnetic Nanoparticles.

Portable nucleic acid detection (PNAD) systems are performed for sample processing, amplification and detection automatically in an individual device realizing "sample in, answer out." For this goal, numerous function modules should be integrated in a diminutive device, in which temperature controller is one of the most important modules. In a nucleic acid detection process, both sample processing and polymerase chain reaction (PCR) require fast and accurate temperature control to increase concentration and purity of the extraction product and to improve amplification efficiency. In this paper, a dual-channel temperature controller for PNAD systems is developed, which contains a printed circuit board (PCB) and an integrated control program with a fast and accurate control strategy. According to the principle of nucleic acid detection based on magnetic nanoparticles, the controller can work in different modes such as high-precision heating control for nucleic acid extraction, rapid thermal cycle control for PCR, and rate adjustable constant heating/cooling control for melting curve. Evaluatively, the average heating/cooling rate of the module can exceed about 6 C/s, while the temperature fluctuation was less than ± 0.1°C, which can meet the demands of PNAD systems very well.

Value of next-generation sequencing in early diagnosis of patients with tuberculous meningitis

ObjectivesTo assess the value of next-generation sequencing (NGS) technology in early diagnosis of patients with tuberculous meningitis (TBM). Methods56 patients with clinically suspected TBM who came to Shandong Provincial Chest Hospital from February 2, 2018 to August 2, 2018 were prospectively included, and the clinical diagnosis and treatment outcomes were followed up. NGS was performed for the cerebrospinal fluid specimens submitted for test on the BGISEQ-100 platform of Tianjin Huada Gene Research Institute and the obtained pathogen sequences were compared with the pathogen data to get the final results. The NGS results were positive for detecting the unique matching sequence of the Mycobacterium tuberculosis (MTB) complex and negative for no unique matching sequence. Patients confirmed with TBM should have at least one of the following four items: cerebrospinal fluid MTB culture positive, smear positive, Xpert MTB/RIF test positive, or MTB nucleic acid polymerase chain reaction (PCR) test positive; clinically diagnosed patients were those with clinically suspected TBM and effective anti-tuberculosis treatment; non-TBM patients were those with other pathogenic basis or clinical exclusion of TBM. The sensitivity and specificity of NGS in early diagnosis of TBM were analyzed. Results22 patients were confirmed with TBM, of which 13 were positive for Xpert MTB/RIF test, 6 were positive for cerebrospinal fluid MTB culture, 5 were positive for MTB nucleic acid PCR test, 12 patients were clinically diagnosed with TBM, and there were 16 cases of non-TBM patients. Among confirmed and clinically diagnosed patients, 20 cases of MTB complex were detected by NGS technology, with a sensitivity of 58.8% (20/34) and specificity of 100% (16/16). Among confirmed patients, the sensitivity of NGS was 63.6% (14/22). Of the 50 specimens that were simultaneously subjected to traditional methods, Xpert MTB/RIF test and NGS, the specificity of the three methods was 100% (16/16) based on clinical diagnosis, and the sensitivity was 29.4% (10/34), 38.2% (13/34), and 58.8% (20/34) respectively. The difference of sensitivity between the first two detection methods and NGS was statistically significant (McNemar test, p = 0.013, x2 = 5.786 and p = 0.065, x2 = 3.273). The sensitivity of traditional methods combined with NGS was as high as 82.4% (28/34). ConclusionsNGS technology could rapidly detect the MTB complex in cerebrospinal fluid with significant sensitivity and specificity, which could be used as an early diagnosis index of TBM. NGS combined with MTB culture could increase the detection rate.

Molecular bacterial load assay versus culture for monitoring treatment response in adults with tuberculosis.

The lack of rapid, sensitive, and deployable tuberculosis diagnostic tools is hampering the early diagnosis of tuberculosis and early detection of treatment failures. The conventional sputum smear microscopy or Xpert MTB/RIF assay cannot distinguish between alive and dead bacilli and the culture method delays providing results. Tuberculosis molecular bacterial load assay is a reverse transcriptase real-time quantitative polymerase chain reaction that quantifies viable tuberculosis bacillary load as a marker of treatment response for patients on anti-tuberculosis therapy. However, results are not synthesized enough to inform its comparative advantage to tuberculosis culture technique which is yet the gold standard of care. With this review, we searched electronic databases, including PubMed, Embase, and Web of Science, from March 2011 up to February 2021 for clinical trials or prospective cohort studies that compared tuberculosis molecular bacterial load assay with tuberculosis culture in adults. We included eight studies that meet the inclusion criteria. Tuberculosis molecular bacterial load assay surpasses culture in monitoring patients with tuberculosis during the first few weeks of anti-tuberculosis treatment. It is more desirable over culture for its shorter time to results, almost zero rates of contamination, need for less expertise on the method, early rate of decline, lower running cost, and reproducibility. Its rapid and specific tuberculosis treatment monitoring competency benefits patients and healthcare providers to monitor changes of bacillary load among isolates with drug-susceptible or resistance to anti-tuberculosis regimens. Despite of the high installing cost of the tuberculosis molecular bacterial load assay method, molecular expertise, and a well-equipped laboratory, tuberculosis molecular bacterial load assay is a cost-effective method with comparison to culture in operational running. To achieve maximum utility in high tuberculosis burden settings, an intensive initial investment in nucleic acid extraction and polymerase chain reaction equipment, training in procedures, and streamlining laboratory supply procurement systems are crucial. More evidence is needed to demonstrate the potential large-scale and sustainable use of tuberculosis molecular bacterial load assay over culture in resource-constrained settings.

Sample pooling is a viable strategy for SARS-CoV-2 detection in low-prevalence settings

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has significantly increased demand on laboratory throughput and reagents for nucleic acid extraction and polymerase chain reaction (PCR). Reagent shortages may limit the expansion of testing required to scale back containment measures. The aims of this study were to investigate the viability of sample pooling as a strategy for increasing test throughput and conserving PCR reagents; and to report our early experience with pooling of clinical samples.A pre-implementation study was performed to assess the sensitivity and theoretical efficiency of two, four, and eight-sample pools in a real-time reverse transcription PCR-based workflow. A standard operating procedure was developed and implemented in two laboratories during periods of peak demand, inclusive of over 29,000 clinical samples processed in our laboratory.Sensitivity decreased (mean absolute increase in cycle threshold value of 0.6, 2.3, and 3.0 for pools of two, four, and eight samples, respectively) and efficiency increased as pool size increased. Gains from pooling diminished at high disease prevalence. Our standard operating procedure was successfully implemented across two laboratories. Increased workflow complexity imparts a higher risk of errors, and requires risk mitigation strategies. Turnaround time for individual samples increased, hence urgent samples should not be pooled.Pooling is a viable strategy for high-throughput testing of SARS-CoV-2 in low-prevalence settings.

Salivary Detection of COVID-19

Salivary Detection of COVID-19

Maternal death due to COVID-19

BackgroundDespite 2.5 million infections and 169,000 deaths worldwide (as of April 20, 2020), no maternal deaths and only a few pregnant women afflicted with severe respiratory morbidity have been reported to be related to COVID-19 disease. Given the disproportionate burden of severe and fatal respiratory disease previously documented among pregnant women following other coronavirus-related outbreaks (SARS-CoV in 2003 and MERS-CoV in 2012) and influenza pandemics over the last century, the absence of reported maternal morbidity and mortality with COVID-19 disease is unexpected.ObjectiveTo describe maternal and perinatal outcomes and death in a case series of pregnant women with COVID-19 disease.Study DesignWe describe here a multiinstitution adjudicated case series from Iran that includes 9 pregnant women diagnosed with severe COVID-19 disease in their second or third trimester. All 9 pregnant women received a diagnosis of SARS-CoV-2 infection by reverse transcription polymerase chain reaction nucleic acid testing. Outcomes of these women were compared with their familial/household members with contact to the affected patient on or after their symptom onset. All data were reported at death or after a minimum of 14 days from date of admission with COVID-19 disease.ResultsAmong 9 pregnant women with severe COVID-19 disease, at the time of reporting, 7 of 9 died, 1 of 9 remains critically ill and ventilator dependent, and 1 of 9 recovered after prolonged hospitalization. We obtained self-verified familial/household cohort data in all 9 cases, and in each and every instance, maternal outcomes were more severe compared with outcomes of other high- and low-risk familial/household members (n=33 members for comparison).ConclusionWe report herein maternal deaths owing to COVID-19 disease. Until rigorously collected surveillance data emerge, it is prudent to be aware of the potential for maternal death among pregnant women diagnosed as having COVID-19 disease in their second or third trimester.

Prevalence of endometrial tuberculosis-polymerase chain reaction (TB PCR) positive (Latent TB) in a population referred for in vitro fertilisation(IVF) in a developing country

SettingStudies have suggested a possible role of antitubercular treatment (ATT) for treating infertility in women with latent or preclinical tubeculosis (TB). ObjectiveThis study investigates the effect of latent TB on IVF outcome. Study designThis was a prospective cohort study which included women referred for assisted reproductive technique between December 2013 to February 2015. Two hundred forty-five women who consented for an endometrial biopsy for TB DNA PCR (Deoxy-ribo nucleic acid-polymerase chain reaction) in the premenstrual phase were included in this study. MethodsNested TB DNA PCR for IS6110 and MPB64 was performed on endometrial samples of the women. The effect of latent TB (positive endometrial DNA PCR) was estimated in terms of statistical difference in clinical pregnancy rate. ResultsEndometrial TB DNA PCR was positive for 19 women (7.7%). The clinical pregnancy rate was similar in both groups (28.16 vs. 26.3%; p = 0.5) without ATT. Patients with both positive and negative polymerase chain reaction (PCR) were similar in age, duration and type of infertility, and the infertility diagnosis. ConclusionPositive endometrial TB DNA PCR (latent TB) alone does not require antitubercular drugs for treatment of infertility. TB DNA PCR should not be the sole criterion for diagnosis of genital TB and starting or concluding treatment.

Anion exchange chromatography of oligonucleotides under denaturing conditions

Denaturing anion exchange HPLC simplifies the chromatographic profiles of self-complementary sequences such as d(CG)6, d(CG)6 with locked nucleic acid modifications, d(AT)15, and polymerase chain reaction mixtures. These chromatographic conditions use eluents containing up to 4 M urea at pH 12.4, and lead to the abolishment of secondary structures and meaningful chromatographic patterns of self-complementary sequences. Similarly, PCR template, FAM-labelled primer and FAM-labelled PCR products were resolved, making interpretation of PCR reaction products possible.