Simultaneous Detection of Seven Sexually Transmitted Agents in Human Immunodeficiency Virus–Infected Brazilian Women by Multiplex Polymerase Chain Reaction

We determined the prevalence of seven clinically important pathogens that cause sexually transmitted infections (STIs) (Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, herpes simplex virus 1 [HSV-1], HSV-2, and Treponema pallidum), by using a multiplex polymerase chain reaction (M-PCR) in samples from Brazilian woman infected with human immunodeficiency virus 1 (HIV-1) and uninfected Brazilian women (controls). The M-PCR assay identified all STIs tested for and surprisingly, occurred association between the control and STIs. This association was probably caused by excellent HIV infection control and regular monitoring in these women established by public health strategies in Brazil to combat HIV/acquired immunodeficiency syndrome. Studies using this M-PCR in different populations may help to better elucidate the roles of STIs in several conditions.It is estimated that annually more than 340 million new cases of sexually transmitted infections (STIs) occur throughout the world, and the highest incidence is in developing countries. Pelvic inflammatory disease, infertility, ectopic pregnancy, chronic pelvic pain, neonatal morbidity and mortality, and genital cancer all have been assumed to be associated with STIs, although in many cases the exact etiologic agents remain unknown.¹ Furthermore, an increasing risk of acquisition of these infections may have a substantial impact on global human immunodeficiency virus 1 (HIV-1) transmission and have the potential to cause serious health risks in HIV-infected persons.^2–4 However, few studies in Latin America have examined the reciprocal effect of HIV-1 on susceptibility and prevalence to STIs.Over the past three decades, diagnostics for STIs depended on traditional methods such as culture, enzyme immunoassay, and fluorescent antibody staining. However, in recent years several molecular methods have become available. These newer methods have advantages because they are more sensitive, enabling detection in symptomatic and asymptomatic patients, including cases of viral infections.⁵ The multiplex PCR has an additional advantage in screening because it enables simultaneous detection of multiple pathogens, reducing costs and time.⁶The purpose of this study was to determine the prevalence of seven clinically important STIs: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, herpes simplex virus 1 (HSV-1), HSV-2, and Treponema pallidum by using multiplex PCR (M-PCR) in samples from HIV-1-infected Brazilian women and those from HIV1-uninfected Brazilian women.A total of 556 women were randomly selected for this study: 178 HIV-infected (HIV group) and 378 HIV-uninfected women (control group) registered in the Public Specialized Assistance Service for HIV/acquired immunodeficiency syndrome (AIDS) for detection and control in Maringá/Paraná, Brazil, during April 2011–May 2012. Women spontaneously sought testing for HIV detection. Inclusion criteria were HIV-infected or uninfected women independent of signs and symptoms for STIs determined by a gynecologist; and HIV-infected or uninfected women who had two negative or positive diagnoses for HIV/AIDS by different methods, respectively. Exclusion criteria were pregnancy, age < 18 years, and no history of sexual activity. Data regarding HIV infection and treatment were obtained from Public Specialized Assistance Service medical records. This study was approved by the Committee for Ethics in Research Involving Humans at the State University of Maringá, Paraná, Brazil (no. 085/2011), and each woman involved signed a consent form.Cervical, endocervical and vaginal samples were collected by using a cytobrush and an Ayre''s spatula, transferred to tubes containing 1.0 mL of sterile 0.9% NaCl solution, and stored at −20°C. DNA was extracted by using an AxyPrep™ Body Fluid Viral DNA/RNA Miniprep Kit (Axygen, Union City, CA) according to the manufacturer''s instructions. The quality and quantity of purified DNA were measured by spectrophotometry.We made selected adaptations to a previously designed M-PCR to achieve simultaneous detection of the seven selected STIs.^5,6 Primers were characterized by compatible melting temperatures and yielded amplicons with sizes easily separable by agarose gel electrophoresis (7 All clinical samples were also tested by using specific primers GH20/ PC04 for the human β-globin gene as an internal control of amplification and DNA integrity under the same conditions of M-PCR or single-target PCR.

Table 1

Nucleotide sequences of amplification primers used in the multiplex polymerase chain reaction^*

Detection of influenza A virus RNA in birds by optimized Real—Time PCR system

ObjectiveTo evaluate the use of Real-Time PCR system based on specific amplification of matrix protein gene fragment for influenza A virus RNA detection in cloacal swabs from wild birds.MethodsSensitivity, specificity and reproducibility of analysis results were identified. Study of cloacal swabs from wild birds for influenza A virus presence was performed.ResultsReproducibility of low concentrations of virus detection in samples by Real-Time PCR was significantly higher than that of detection based on cytopathic effect of viruses grown on MDCK cell culture.ConclusionsReal-Time PCR system for influenza A virus RNA detection is developed and applied for virus surveillance study.     

Toward the Operationalization and Examination of “Hitting Bottom” for Problematic Alcohol Use: A Literature Review

“Hitting bottom” among individuals with alcohol use disorders (AUDs) has empirical and theoretical support; however, it has never been operationalized and, therefore, remains largely unexplored. To operationalize and evaluate hitting bottom, it is important to understand the emergence of the construct, how it is used by researchers, and how it is perceived by individuals recovering from AUDs. Accordingly, the authors review extant literature on hitting bottom for individuals with AUDs. Specifically, the authors discuss historical evolution of “hitting bottom” to inform future research efforts to operationalize and evaluate the concept of hitting bottom for individuals with problematic alcohol use.     

Measurement of mRNA by Solution Hybridization with 32P-Labelled Single Stranded cRNA Probe (“SP6 TEST”). Comparison with A 32P-Labelled Single Stranded cDNA as Hybridization Probe (“S1 Test”) for Measurement of AVP mRNA

Radioactively labelled cRNA for the rat AVP gene exon C was synthetized from a pSP64-vector and used for solution hybridization measurement of AVP mRNA (“SP6 test”). For comparison hybridization was carried out with a gel-purified radioactively labelled cDNA probe synthetized by primer extension of AVP gene exon C cloned into an M13mp9 phage vector DNA (“S1 test”). Both tests had a comparable sensitivity of up to 0.2 pg AVP mRNA. Under optimal hybridization conditions kinetics were similar in both tests. The fast and easy preparation of large amounts of labelled cRNA probe and simple determination of absolute amounts of mRNA by saturation kinetics without need of a mRNA standard makes the “SP6 test” an attractive alternative to the known “S1 test”. The “SP6 test” should be applicable for a wide variety of genes.     

A Review of: “RNA Methodologies,a Laboratory Guide for Isolation and Characterization (third edition)”

A new hyperunstable hemoglobin was found in a Japanese girl who had very severe, chronic hemolytic anemia. Her parents and siblings were hematologically normal. The abnormal hemoglobin comprised a very small proportion of the total hemoglobin, although it was produced almost at the same rate as normal hemoglobin. Sequencing of an abnormal peptide which was liberated from the 8 chain by hydrolysis with a protease from Staphylococcus aureus V8 disclosed the tandem insertion of a five-residue segment which included the proximal histidine at β92(F8).     

Detection of the tick-borne encephalitis virus (TBEV) in ticks in several federal “Länder” of Germany by means of the polymerase chain reaction (PCR) — Characterization of the virus

Summary The aim of the present study was to analyse the current epidemiological situation with respect to TBE in the new federal Länder of Germany and in Saarland through detection of the TBEV genome in unengorged ticks using an RT-PCR technique. 22,273 ticks (Ixodes ricinus) were collected in the five new Länder (and some in Bavaria and Baden-Württemberg) and divided into 294 pools. It was possible to detect TBEV RNA in six pools of ticks from Mecklenburg Western-Pomerania [4], Brandenburg [1], Thuringia [1] (and in three pools from Bavaria and Baden-Württemberg). The nucleotide sequence data of the PCR products were analysed and compared. In Saarland 8,780 ticks were collected in 70 habitats from all the geographic regions and analysed using the PCR in 21 pools; two pools produced positive PCR signals (Saarlouis, Perl). We cannot as a result make a general recommendation that TBE-immunization be introduced in Saarland and in the new federal Länder of Germany. In Germany, however, TBE immunoprophylaxis in Bavaria and Baden-Württemberg is very important.

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Nachweis des Virus der Frühsommer-Meningoenzephalitis in Zecken einiger Bundesländer mittels Polymerase-Kettenreaktion und nähere Charakterisierung des Virus

Zusammenfassung Das Ziel der vorgelegten Studie ist die Einschätzung der aktuellen epidemiologischen Situation der Frühsommer-Meningoenzephalitis in den fünf neuen Bundesländern und im Saarland mit Hilfe des Nachweises von FSMEV-RNA in ungesogenen Zecken (Ixodes ricinus) durch eine RT-PCR-Technik. 22 273 Zecken wurden in den fünf neuen Ländern (einige auch in Bayern und Baden-Württemberg) gesammelt und in 294 Pools untersucht. Der spezifische RNA-Nachweis gelang viermal in Zecken aus Mecklenburg-Vorpommern, einmal in solchen aus Brandenburg und einmal aus Thüringen. In Bayern und Baden-Württemberg gelang der Virus-RNA-Nachweis dreimal. Die Sequenzdaten der PCR-Amplifikate zeigten, auch im Vergleich mit denen des Prototypstammes Neudoerfl, den hohen Grad der Konservierung im Bereich der 5 NCR. 8780 saarländische Zecken aus allen Gebieten des Bundeslandes wurden in 21 Pools untersucht, positive PCR-Signale konnten in zwei Pools aus Saarlouis und Perl und Umgebung gefunden werden. Der relativ seltene FSMEV-RNA-Nachweis in den neuen Ländern und im Saarland berechtigt nicht, eine generelle Impfempfehlung für diese Gebiete zu geben. Ein Impfschutz sollte jedoch vor Einreise in die Endemiegebiete Bayerns und Baden-Württembergs bestehen.

     

“Diabetes care at doorsteps”: A customised mobile van for the prevention,screening, detection and management of diabetes in the urban underprivileged populations of Delhi

Diabetes is on the rise in India and recently shown to be increasing in the urban underprivileged. Lack of awareness of the disease, its complications, combined with lack of financial resources among the underprivileged, often results in late detection and more complications in them. To combat this, healthcare delivered at the doorstep through the use of a customised mobile medical van is a potentially attractive option.We used a customized mobile van (included trained personnel, glucose meters, fundus evaluation camera, apparatus for detection of neuropathy and foot circulation and net enabled Skype calling for remote consultation) for educating general population regarding healthy lifestyle and screening, management and intervention in patients with diabetes.The project covered 10 underprivileged areas (n, 2,31,000 people) in Delhi. Total of 24,072 individuals (10.9% of total population) attended 352 awareness sessions. A total 3,12,347 visits (included repeat visits) were carried out for screening, education and management for obesity and diabetes. During screening (n, 16,834), 2933 subjects (18.7%) had high random blood glucose levels (>200 mg/dL) and had a blood pressure averaging 127.1 ± 23.6/81.3 ± 16.6 mm of mercury (n, 16,339). A pre-post intensive lifestyle counselling for 6 months in a subset of 352 diabetic patients (of which 77.8% i.e. n, 274 were overweight/obese) showed a significant lowering in weight (p < 0.001). In addition, 292 frontline workers and 256 paramedical workers were given training regarding lifestyle and diabetes, over 20 sessions.Based on achievements of this project of spreading awareness, screening, and management of diabetes and obesity in the large number of individuals in urban underprivileged colony, we believe this project could be extended to other cities and rural areas of India, and to other developing countries as well.     

One-stop procedure of “Atrial fibrillation radiofrequency ablation + left atrial appendage closure” guided by 3D printing technology: A case report

Left atrial appendage occlusion (LAAO) in the treatment of atrial fibrillation (AF) has become a hot topic in clinical research in recent years. We report a 68-year-old female with a 3-year history of paroxysmal atrial fibrillation refractory to antiarrhythmic therapy and unable to tolerate anticoagulation therapy who underwent successful atrial fibrillation radiofrequency ablation combined with left atrial appendage occlusion guided by 3D printing technology. There was no recurrence of her atrial fibrillation and there was continued complete occlusion of her left atrial appendage at 3-month and 1-year follow-ups.This case supports the potential advantage of 3D printing technology to guide a “one-stop combined AF radiofrequency ablation and left atrial appendage occlusion procedure.” But whether it can improve the prognosis and quality of life of patients, further multi-center research and large data statistics are required.     

Additional methodological considerations regarding optimization of the dose of intracerebroventricular streptozotocin A response to: “Optimization of intracerebroventricular streptozotocin dose for the induction of neuroinflammation and memory impairments in rats” by Ghosh et al., Metab Brain Dis 2020 July 21

Metabolic Brain Disease - A recent article by Ghosh et al. entitled "Optimization of intracerebroventricular&nbsp;streptozotocin dose for the induction of neuroinflammation and memory...