Molecular detection of Anaplasma, Babesia, Theileria, and Trypanosoma infection in cattle and buffaloes in India.

Tick-borne pathogens pose a significant global threat, causing substantial economic losses to the dairy industry. In India, tropical theileriosis, anaplasmosis, babesiosis, and trypanosomiasis are major hemo-parasitic diseases affecting bovines. A cross-sectional study was conducted to determine the prevalence of hemo-parasites in different farms in India. PCR assays were employed to detect carrier status, using gene targets msp1b, tams1, rap-1, ama1, and ITS1 for A. marginale, T. annulata, B. bovis, B. bigemina, and Trypanosoma species, respectively. Out of the 578 apparently healthy animals screened, 30.45% (95% CI: 26.84-34.32%) were infected with at least one hemo-parasite. Cattle showed an overall positivity of 32.87%, while buffaloes had a prevalence of 15.19%, which was statistically significant (p < 0.001). Interestingly, prevalence was higher in indigenous cattle (47.81%) compared to cross-breeds (25.53%) and exotics (14.62%), with a statistically significant difference (p < 0.001). The prevalence of hemo-parasites varied widely among the farms, ranging from 5.77 to 100%. A. marginale was the most prevalent parasite (23.70% of animals), followed by T. annulata (13.67%), Babesia species (1.90%), and Trypanosoma species (1.56%). Enzootic instability was observed in six of the eight farms, indicating a potential for future outbreaks. Co-infection was detected in 60 out of 176 animals positive for hemo-parasites, with 59 animals co-infected with A. marginale and T. annulata, and only one cross-breed cattle infected with both Anaplasma marginale and Babesia bigemina. The findings highlight the prevalence of hemo-parasites in farms, underscoring the need for whole-herd screening, treatment of infected animals, and improvement in farm management practices to prevent production losses caused by these pathogens. Supplementary Information: The online version contains supplementary material available at 10.1007/s12639-024-01673-3.

Rectal swab DNA collection protocol for PCR genotyping in rats.

DNA collection is essential for genotyping laboratory animals. Common collection methods require tissue amputation, causing discomfort and injury. Rectal swabbing has been proposed as an effective, minimally invasive alternative, but an evidence-backed protocol for the technique remains unavailable. This report evaluates the effects of collection parameters on the quality of PCR results and presents a protocol for genotyping a litter of rats within 3-5 h. Samples with 2-8 scrapes produced enough DNA to amplify targets up to ∼1800 bp long using PCR. Rectal swabbing produced PCR results with similar utility as ear clip samples, and results were unaffected by residual fecal matter or cell debris. The protocol enables fast, minimally invasive and repeatable genotyping using commercial PCR reagents.

DNA collection for genotyping laboratory rodents commonly requires tissue amputation, leading to injury and discomfort. Rectal swabbing can be an effective, minimally invasive alternative, but there is a lack of information on how to apply the technique and the necessary parameters involved. This report assesses collection and processing parameters while combining the rectal swab collection method with commercial PCR reagents to allow for fast genotyping with minimally processed DNA samples. Findings were used to design a protocol for minimally invasive DNA collection and genotyping.

Rectal Swab DNA Collection Protocol for PCR Genotyping in Rats.

DNA collection is essential for genotyping laboratory animals. However, common collection methods require tissue amputation, causing discomfort and injury. Rectal swabbing has been proposed as an effective non-invasive alternative, but an evidence-backed protocol for the technique remains unavailable. We evaluate the effect of collection parameters on PCR result quality and present a genotyping protocol that can yield results for a litter of rats within 3-5 hours. We found that samples with 2-8 scrapes produced enough DNA to amplify targets up to ~1800bp long using PCR. Rectal swabbing produced PCR results with similar quality to ear clip samples, and results were unaffected by residual fecal matter or cell debris. Our protocol enables fast, non-invasive, and repeatable genotyping using commercial PCR reagents.

Asociación de marcadores inflamatorios y de disfunción endotelial con la depresión en la enfermedad cerebrovascular isquémica

Fundamento: la depresión es una de las complicaciones no neurológicas más frecuentes en la enfermedad cerebrovascular isquémica. Objetivo: determinar la asociación de marcadores inflamatorios y de disfunción endotelial con la depresión en pacientes con enfermedad cerebrovascular isquémica. Métodos: se realizó un estudio analítico, prospectivo de corte transversal en pacientes con enfermedad cerebrovascular isquémica en fase aguda (N=22) y no aguda (N=37); atendidos en el Instituto de Neurología y Neurocirugía y el Hospital Manuel Fajardo, de La Habana, Cuba. Se recogieron variables demográficas, factores de riesgo, etiología y localización del infarto, deficiencia neurológica, discapacidad para las actividades de la vida diaria (índice de Barthel), neuropsicológicas (depresión por inventario de Beck y test de Hamilton). Se determinó proteína C-reactiva, alfa-1-antitripsina, complementos C3 y C4 y microalbuminuria. Resultados: las puntuaciones de las pruebas neuropsicológicas no tuvieron diferencias significativas entre la fase aguda y no aguda, pero hubo un aumento estadístico de la frecuencia de pacientes sin depresión y con ligera depresión en la fase no aguda. En la fase aguda, el complemento C4 y en la fase no aguda el complemento C3, la proteína C-reactiva y el alfa-1-antitripsina se correlacionaron directamente con la puntuación del inventario de Beck. La proteína C-reactiva y C3 se correlacionaron estadísticamente con la puntuación del test de Hamilton. En el análisis multivariado, la proteína C-reactiva mostró asociación independiente con el grado de depresión por el test de Hamilton. Conclusiones: la proteína C-reactiva pudiera estar relacionada con la severidad de la depresión, quizás por asociación con la discapacidad para las actividades de vida diaria.

Foundation: depression in ischemic cerebrovascular disease is one of the most frequent non-neurological complications. Objective: to determine the association of inflammatory markers and endothelial dysfunction with depression in patients with ischemic cerebrovascular disease. Methods: an analytical, prospective, cross-sectional study was carried out in patients with acute (N=22) and non-acute (N=37) ischemic cerebrovascular disease; treated at the Institute of Neurology and Neurosurgery; and the Manuel Fajardo Hospital, in Havana, Cuba. Demographic variables, risk factors, etiology and location of the infarction, neurological deficiency, disability for activities of daily living (Barthel index), neuropsychological (depression by Beck inventory and Hamilton test) were collected. C-reactive protein, alpha-1-antitrypsin, C3 and C4 complements, and microalbuminuria were determined. Results: the scores of the neuropsychological tests did not have significant differences between the acute and non-acute phase, but there was a statistical increase in the frequency of patients without depression and with slight depression in the non-acute phase. In the acute phase, C4, and in the non-acute phase, C3, C-reactive protein and alpha-1-antitrypsin were directly correlated with the Beck inventory score. C-reactive protein and C3 were statistically correlated with the Hamilton test score. In the multivariate analysis, C-reactive protein showed an independent association with the degree of depression by the Hamilton test. Conclusions: C-reactive protein could be related to the severity of depression, perhaps by association with the disability for activities of daily living.

Rapid-Onset Temporal Encephalitis With Negative Cerebrospinal Fluid Polymerase Chain Reaction Testing.

Herpes simplex encephalitis is a rare disease presentation that is usually characterized by its temporal involvement and positive cerebrospinal fluid (CSF) polymerase chain reaction (PCR) for the herpes simplex virus (HSV). HSV PCR has a sensitivity of 96% and specificity of 99%. Even when the test is negative, if clinical suspicion is high, acyclovir therapy should be continued with a repeated PCR within a week. In this case, we report a 75-year-old female patient who presented with signs of hypertensive emergency with rapid deterioration to seizure-like activity on electroencephalogram (EEG) and signs of temporal encephalitis on magnetic resonance imaging (MRI). The patient did not respond to the initial regimen of antibiotics but did show significant clinical response to acyclovir though she had a negative CSF PCR for HSV ten days after the start of her neurological symptoms. In this case, we argue that alternative methods of diagnosis should be considered in cases of acute encephalitis. Our patient had negative PCR but her computerized tomography (CT), EEG, and MRI results pointed to temporal encephalitis caused by HSV.

Why Is Whipple's Disease Still a Challenging Diagnosis? A Case Report and Brief Review of Literature.

Whipple's disease (WD) is a rare multisystemic infectious disease caused by Tropheryma whipplei. The pathogenesis of Whipple's disease remains unknown and clinical experience relies solely on various case reports published in the literature. The disease may occur at any age, with most studies describing patients in their fifth decade. Classic WD mainly affects the gastrointestinal tract, but extraintestinal commitment can occur, with the most common manifestations being arthralgias, lymphadenopathy, fever, and neurological symptoms. We present a case of a 69-year-old woman who presented with fever, macular rash, abdominal pain, lymphadenopathy, pleural and pericardial effusion, weight loss, and severely altered mental status over seven days. Initial workup tests only revealed leucopenia, thrombocytopenia, and hyperferritinemia. Since the fever persisted despite antibiotic treatment, an extensive workup was required until the final diagnosis of classic WD through histological examination of duodenal biopsies. Treatment with ceftriaxone was implemented for two weeks, followed by trimethoprim-sulfamethoxazole 160/800mg bid for 12 months. The patient presented full recovery and no recurrence after three years of follow-up. Even though WD was first described more than a century ago, WD is an elusive disease with a wide variety of clinical findings, leading to a still significant delay in diagnosis. WD should be considered in the differential diagnosis of rheumatologic disorders, chronic abdominal pain or diarrhea, neurological manifestations not suggestive of any other specific disease, non-caseating granulomatous diseases, and cases of lymphadenopathies. The authors aim to add additional clinical data and raise awareness for a rare condition that can be lethal if not timely treated. More studies and recommendations are needed concerning screening patients and treatment, with an urgent need to improve the delay in diagnosis.

Purpura fulminans due to Vibrio vulnificus severe infection.

Purpura fulminans is a life-threatening disease, characterized by disseminated intravascular coagulation and endovascular thrombosis; can often occur secondary to heterogeneous etiologies, such as sepsis, and to a lesser extent, secondary to sepsis due to halophilic bacteria, such as V. vulnificus, found in marine environments. Patients with specific comorbidities are at the highest risk of worst scenarios, without prompt treatment, infection can rapidly evolve to fatal, with a mortality rate close to 100 %. We present a case of Purpura fulminans due to V. vulnificus septicemia.

SAMBA: A Multicolor Digital Melting PCR Platform for Rapid Microbiome Profiling.

During the past decade, breakthroughs in sequencing technology have provided the basis for studies of the myriad ways in which microbial communities in and on the human body influence human health and disease. In almost every medical specialty, there is now a growing interest in accurate and quantitative profiling of the microbiota for use in diagnostic and therapeutic applications. However, the current next-generation sequencing approach for microbiome profiling is costly, requires laborious library preparation, and is challenging to scale up for routine diagnostics. Split, Amplify, and Melt analysis of BActeria-community (SAMBA), a novel multicolor digital melting polymerase chain reaction platform with unprecedented multiplexing capability is presented, and the capability to distinguish and quantify 16 bacteria species in mixtures is demonstrated. Subsequently, SAMBA is applied to measure the compositions of bacteria in the gut microbiome to identify microbial dysbiosis related to colorectal cancer. This rapid, low cost, and high-throughput approach will enable the implementation of microbiome diagnostics in clinical laboratories and routine medical practice.

mRNA expression of the severe acute respiratory syndrome-coronavirus 2 angiotensin-converting enzyme 2 receptor in the lung tissue of Wistar rats according to age.

Background and Aim: Angiotensin-converting enzyme 2 (ACE2) is expressed and plays functional and physiological roles in different tissues of the body. This study aimed to distinguish the levels of expression of ACE2 in the lung tissue at different ages of rats. Materials and Methods: In this study, 18 male rats were used and divided into three groups according to age. Real-time quantitative polymerase chain reaction (RT-qPCR) was conducted to determine the levels of the quantification of eosinophil cationic protein mRNA transcript. In addition, tissue specimens of the lung were stained with routine hematoxylin and eosin stains. Results: This study confirmed that RT-qPCR amplification plots of ACE2 gene exhibited clearly expression of the lung tissue of rats in the different groups and there are strong different threshold cycles numbers according to the age at 2 weeks, 2 months, and 6-8 months. Consequently, the expression of ACE2 was completely different between groups depending on the age of the rats. The RT-qPCR results showed that the older animal group (age of 6-8 months) had a significantly higher expression of ACE2 than the other animal groups (ages of 2 weeks and 2 months). In the same way, the second group (age of 2 months) had a significantly higher expression of ACE2 than the first group (age of 2 weeks). This study confirmed that the ACE2 expression is influenced by the age of rats. Conclusion: This study concluded that the expression of the ACE2 receptor of coronavirus disease 2019 would be different according to the age of rats, and this result suggested that expression of ACE2 in lung tissue could determine infection and pathogenesis of COVID-19 during different ages of rats or some individual differences.

A rapid near-patient RT-PCR test for suspected COVID-19: a study of the diagnostic accuracy.

BACKGROUND: Management of large numbers of reverse transcriptase-polymerase chain reactions (RT-PCR) for diagnosis of coronavirus 2019 disease (COVID-19) requires robust infrastructures, located in dedicated premises with a high standard of biosafety procedures, and well-trained personnel. The handling of a "run-of-river sample" to obtain rapid reporting of results is challenging. METHODS: We studied the clinical performance of the Idylla™ SARS-CoV-2 Test (index test) on a platform capable of fully automated nucleic acid testing including extraction, amplification, and detection in a single-use cartridge to establish the diagnosis of COVID-19. The study was conducted on a prospective cohort of 112 volunteers with recent symptoms and an unknown SARS-CoV-2 status who came to free screening centers of the Nice metropolitan area. All subjects underwent bilateral nasopharyngeal sampling. One sample was processed using the index test, the other using the standard of care RT-PCR. Samples were treated blind. RESULTS: Most of the participants (70%) were sampled within 4 days of symptom onset. Forty-five (40.2%) were positive for COVID-19. No clinical symptoms were distinguished between SARS-CoV-2 RT-PCR positive and negative subjects except anosmia and dysgeusia. Positive and negative agreement between the index and the standard of care test was 100%. CONCLUSIONS: The Idylla™ SARS-CoV-2 Test is very sensitive, specific, rapid and easy to use in a near-patient RT-PCR approach to distinguish between symptomatic SARS-CoV-2 positive and negative patients in selected settings.

Comparison of High-Resolution Computed Tomography and Real-Time Reverse Transcriptase Polymerase Chain Reaction in Diagnosis of COVID-19 Pneumonia in Intensive Care Unit Population.

Introduction The efficacy of high-resolution computed tomography (HRCT) chest in common respiratory infections is well-established; however, its use in the diagnosis of COVID-19 pneumonia is less popular. The previous studies have failed to establish the efficacy of HRCT in the diagnosis of COVID-19 pneumonia. Objective The current study aimed to assess the efficacy of HRCT as compared to a polymerase chain reaction (PCR) in diagnosing COVID-19 pneumonia in patients in our setting. Methodology A prospective observational study was conducted at the Department of Chest Medicine, Shifa International Hospital from April 2020 to December 2020. A total of 250 patients were admitted to medical intensive care units. Findings of HRCT and PCR were documented. The accuracy of HRCT compared with PCR was assessed. Data were analyzed using SPSS version 24 (IBM Corp., Armonk, NY). Results COVID-19 infection was more prevalent in male patients (62.8% vs 37.2%). The mean age was 60 years (interquartile range, IQR, 49-72). Sensitivity and specificity of HRCT segregated into typical, indeterminate, and atypical HRCT were (94.8%, 56.8%), (92.7%, 47.2%), and (91.7%, 76.8%), respectively. The positive predictive value for typical HRCT was 84.3% (p≤0.001). Conclusion We concluded that typical HRCT findings have diagnostic utility in the diagnosis of COVID pneumonia. Similarly, a negative HRCT chest reliably excludes the possibility of COVID pneumonia. HRCT chest is a reliable alternative to RT-PCR.

Detection of SARS-CoV-2 in Clinical Samples: Target-specific Analysis of Qualitative Reverse Transcription-Polymerase Chain Reaction(RT-PCR) Diagnostic Kits.

INTRODUCTION: The Coronavirus disease 2019 pandemic caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the rise of many available modalities for diagnosis. One such modality is the Reverse Transcription-Polymerase Chain Reaction (RT-PCR) kits which require evaluation amongst the many available commercial kits in the market. METHODS: We conducted a performance evaluation of twelve RT-PCR SARS-CoV-2 commercial kits. A total of 75 nasopharyngeal and oropharyngeal clinical samples were selected with their cycling threshold (Ct) values. Inclusion of 5 gene targets: E gene, N gene, S gene, RdRp and ORF1ab were assessed. Data was analyzed using R software version 4.1.1 and Microsoft Excel. RESULTS: We observe that, the positive sample's Ct values differs significantly across the 12 diagnostic kits. However, for gene-specific analysis, we observe that, positive sample's Ct values does not differ significantly across gene targets. There is significant difference in Ct values in Commercial kits targeting all genes except S-gene. All the commercial kits Altona (E and S genes), Thermo (ORF1ab and N genes), Multiplex (E, ORF1ab, RdRdp genes), Meril (N and ORF1ab genes), S D Biosensor (E and ORF1ab genes), Lab Gun (RdRp and N genes) and Lab systems (ORF1ab and E genes) scored a sensitivity of 100%. All other kits scored sensitivity above 95% and lowest sensitivity with the Genes2me (E gene) and Genes2me (RdRp) at 95.08% each. All kits were 100% specific. CONCLUSION: This study provides an accurate comprehensive assessment of the different kits in the detection of SARS-CoV-2 which may promote standardization of testing across laboratories.

The 2014 autochthonous dengue fever outbreak in Tokyo: A case series study and assessment of the causes and preventive measures.

OBJECTIVE: In 2014, an autochthonous dengue fever outbreak occurred around the Yoyogi Park in Japan for the first time in 70 years. Despite no local cases reported since then, the risk of another outbreak remains high. This study reviews the autochthonous dengue fever cases of the outbreak, investigates its causes, and delineates preventive measures against autochthonous dengue epidemics. METHODS: We conducted a case series study of 15 patients who visited our institution during the 2014 outbreak. We collected and evaluated data on the surveillance of vector mosquitoes, weather, pest control, travelers' origins and destinations, and imported dengue fever cases using reports made by public institutions. RESULTS: All patients recovered with supportive treatments and none met the diagnostic criteria for severe dengue infection. Twelve patients with positive real-time polymerase chain reactions were confirmed as having dengue virus-1 infections. We found no obvious associations between the number of mosquitoes and the weather, or between the number of imported dengue fever cases and that of travelers. Insect growth regulator (IGR) against vector mosquitoes has been used since 2014 for pest control, but the number of larvae has not declined in the Yoyogi Park, although that of imagoes has been relatively suppressed. CONCLUSION: The 2014 outbreak emerged without particularly favorable climate conditions for vector mosquitoes. We found no obvious associations between the number of travelers or the imported dengue fever cases and the outbreak, but the increasing number of travelers may contribute to another outbreak. Pest control, including IGR, remains essential for infection control.

Molecular capsular typing and multi locus sequence typing of invasive, non-invasive and commensal Streptococcus pneumoniae isolates from North India.

Purpose: The purpose of this study is to determine the antimicrobial susceptibility pattern, serotype distribution and sequence type (ST) of Streptococcus pneumoniae isolates from invasive and non-invasive infection and correlate it with isolates from commensal nasopharyngeal flora to ascertain their role in infection. Materials and Methods: S. pneumoniae isolates from blood, cerebrospinal fluid, pleural fluid and respiratory secretions (sputum, bronchoalveolar lavage and nasopharyngeal swab/throat swab) were analysed to determine ST, serotype and antimicrobial susceptibility pattern. Serotyping was performed by multiplex polymerase chain reactions as well as by quellung reaction. Antimicrobial susceptibility testing was determined using Kirby Bauer's disc diffusion method as per the Clinical Laboratory Standards Institute guidelines. Minimum inhibitory concentration was determined using E-test for penicillin. Multilocus sequence typing (MLST) was done to understand genetic relatedness and evolutionary relationship among strains. Results: A total of 125 S. pneumoniae isolates were collected, including 25 from invasive pneumococcal disease, 25 from non-invasive and 75 from nasopharyngeal swab of healthy children (Commensal). Resistance to penicillin, erythromycin, and co-trimoxazole was observed in 14.4%, 12% and 81.6% of the isolates, respectively, by KirbyBauer's disc diffusion method. Serotype 14 was found to be the most prevalent in invasive and non-invasive isolates, while serotype 6 was the most common in commensal isolates. New STs were found among invasive (ST13826, ST13827), non-invasive (ST13823, ST13824, and ST13961) and commensal (ST13825) isolates. Conclusion: MLST sequence analysis shows that invasive isolates were found to be clustered with non-invasive and commensal isolates. Analysis of MLST suggests the possibility of genetic relatedness and exchange of genetic material between invasive, non-invasive and commensal isolates.

An insulin-inducible transcription factor, SHARP-1, represses transcription of the SIRT1 longevity gene.

The rat enhancer of split- and hairy-related protein (SHARP)-1 genes encode insulin-inducible transcriptional repressors. A longevity gene, sirtuin 1 (SIRT1) encodes protein deacetylase. These play an important role in regulating hepatic glucose metabolism. In this study, to evaluate a correlation with these gene expressions, we examined whether SIRT1 effects on expression of the SHARP-1 gene by a treatment with a SIRT1 inhibitor or activator in rat H4IIE hepatoma cells. Whereas the SIRT1 inhibitor increased the level of SHARP-1 mRNA, the SIRT1 activator decreased it. Next, whether SHARP-1 effect on the transcriptional activity of the human SIRT1 gene using luciferase reporter assays was determined. Promoter activity of the SIRT1 gene was specifically repressed by SHARP-1. Further reporter analysis using 5'- deleted or mutated constructs revealed that an E box sequence (5'-CACGTG-3') of the SIRT1 gene promoter was required for the inhibitory effect of SHARP-1. Thus, we conclude that expressions between the SHARP-1 and the SIRT1 genes show a negative correlation and that SHARP-1 represses transcription of the SIRT1 gene.

Comparación de métodos de extracción de ADN de Giardia spp. medidos por PCR convencional / Comparison of methods of DNA extraction from Giardia spp. measured by conventional PCR

RESUMEN Objetivos . Comparar diferentes métodos de extracción de ADN a partir de quistes y trofozoítos de Giardia spp. mediante la técnica de reacción en cadena de la polimerasa (PCR) convencional. Materiales y métodos. Se aislaron quistes de Giardia spp. a partir de 65 muestras coprológicas procedentes de hospitales de referencia nacional, obteniéndose una carga promedio de 5x104 parásitos. Asimismo, se cultivaron trofozoítos de Giardia intestinalis (ATCC® 30957™) obteniéndose una carga parasitaria de 5x106. Se compararon once métodos de extracción para quistes y seis para trofozoítos. La concentración y pureza del ADN extraído se determinó por espectrofotometría y el rendimiento de la extracción se evaluó mediante la amplificación de los genes beta giardina (bg) y glutamato deshidrogenasa (gdh) por PCR semi-anidada. Resultados. Se observó que el método I mostró la mayor concentración de ADN a partir de quistes (12,24 ng/µL), pureza (1,4) y mejor rendimiento (100% amplificación bg, 60% gdh) en comparación con los otros métodos evaluados. En el caso de los trofozoítos el método que no tuvo pretratamientos presentó la mayor concentración de ADN, pureza y rendimiento (26,56 ng/µL; 1,85; 100% amplificación bg y gdh). Conclusiones. Los pretratamientos mecánicos, de choque térmico y enzimáticos son necesarios para la ruptura de la pared quística de Giardia spp., siendo el marcador molecular bg de mayor rendimiento para detección de ADN de quistes. Los trofozoítos no requieren pretratamientos para lograr resultados satisfactorios. Se cuenta con una metodología reproducible para la extracción de ADN de Giardia spp. a partir de cualquier estadio evolutivo.

ABSTRACT Objectives. To compare different methods of DNA extraction from cysts and trophozoites of Giardia spp. using the conventional polymerase chain reaction (PCR) technique. Materials and Methods. Cysts of Giardia spp. were isolated from 65 coprological samples from national reference hospitals, obtaining an average load of 5x104 parasites. In addition, Giardia intestinalis trophozoites (ATCC® 30957™) were cultured obtaining a 5x106 parasitic load. Eleven extraction methods for cysts and six for trophozoites were compared. The concentration and purity of the extracted DNA were determined by spectrophotometry and the extraction yield was assessed by amplification of the ß-giardin (bg) and glutamate dehydrogenase (gdh) genes with a semi nested PCR assay. Results. It was observed that method 1 showed the highest concentration of DNA from cysts (12.24 ng/µL), purity (1.4) and best performance (bg: 100% amplification; gdh: 60% amplification) compared to the other methods evaluated. In the case of trophozoites, the method without pre treatment showed the highest level of DNA concentration, purity, and yield (26.56 ng/µL; 1.85; 100% amplification of bg and gdh, respectively). Conclusions . Mechanical, thermal shock, and enzymatic pre-treatments are necessary for the rupture of the cystic wall of Giardia spp. making it the highest-yielding bg molecular marker for detecting cyst DNA. Trophozoites do not require pre-treatment to achieve satisfactory results. A reproducible methodology for the extraction of DNA from Giardia spp. from any evolutionary stage is available.

Unprecedented green-emissive boranyl-hydrazone supramolecular assemblies and their in vitro diagnostic applications.

This paper describes a novel symmetric N,N'-diethylsalicylaldehyde boranyl hydrazone (1) and its in situ-generated assemblies displaying opto-analytical capabilities for the diagnosis of nucleic acids under physiological conditions. The sensing capabilities of these unprecedented supramolecular assemblies were characterized using UV-Vis spectroscopy, fluorescence spectroscopy, 1D and 2D NMR spectroscopy, dynamic light scattering, and zeta potential measurements. Model compounds lacking boranyl units (2, 3) were prepared to correlate and evaluate the sensing mechanism. The rationally designed probe 1 displays unusual aggregation-induced emissive (AIE) properties, with an average particle size of 1096 nm exhibiting green emission upon excitation at 377 nm in pH-7.2 TRIZMA buffer. A selective switch on response toward organic PO43- accompanied through specific nano-aggregations patterns and sizes, thereby causing a significant red-shift through AIE. Exploiting such switch on in green channel behavior has allowed the monitoring of DNase I activities and polymerase chain reactions.

Polymerase Chain Reaction Assay Using the Restriction Fragment Length Polymorphism Technique in the Detection of Prosthetic Joint Infections: A Multi-Centered Study.

BACKGROUND: PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) techniques have been used for the diagnosis of bacteria in some infections. In this study, we aimed to evaluate the diagnostic accuracy of PCR for the diagnosis of prosthetic joint infections (PJI) and to identify isolated microorganisms, using the RFLP method. METHODS: During January 2015 to January 2018, patients who were suspected of having PJI after arthroplasty surgery or were candidates for revision surgery due to loosening of implant entered the study. Patients who had 1 major criterion or 3 minor criteria for PJI based on the Philadelphia Consensus Criteria (PCC) on Periprosthetic Joint Infection were considered as cases of PJI. Both culture results and PCR findings, were cross compared with results of the PCC (as the gold standard criteria). RESULTS: Overall, 76 samples were included in the study. Mean (standard deviation) age of patients was 66.72 ± 11.82 years. Overall, 57.9% of patients were females. Prevalence of PJI was 50% based on the PCC. Sensitivity, specificity, positive predictive value, negative predictive value, and general efficacy of PCR for detection of PJI was 97.4%, 100%, 100%, 97.4%, and 98.7%, respectively. Sensitivity, specificity, positive predictive value, negative predictive value, and general efficacy of culture was 31.6%, 100%, 65.7%, 100%, and 59.4%, respectively. We isolated a broad range of bacteria using PCR-RFLP including Gram-positive cocci such as Staphylococcus sp., Streptococcus sp., and Enterococcus sp., and Gram-negative bacilli such as Enterobacteriaceae sp., Pseudomonas sp. Citrobacter sp., as well as Chlamydophila pneumonia, Stenotrophomonas maltophilia, Brucella melitensis, non-gonococcal Neisseria, Kingella kingae, Bacteroides ovatus, and Proteus mirabilis from PJI patients. CONCLUSION: Inhere, for the first time, we showed that PCR-RFLP is a powerful tool for identifying the type of bacteria involved in PJI, and can be used for follow-up of patients suspected of PJI and those with a history of antibiotic use. PCR-RFLP may be able to substantially decrease detection time of PJI among PCR-based methods, while allowing more accurate identification of the bacteria involved.

While it is not deliberate, much of today's biomedical research contains logical and technical flaws, showing a need for corrective action.

Biomedical research has advanced swiftly in recent decades, largely due to progress in biotechnology. However, this rapid spread of new, and not always-fully understood, technology has also created a lot of false or irreproducible data and artifacts, which sometimes have led to erroneous conclusions. When describing various scientific issues, scientists have developed a habit of saying "on one hand… but on the other hand…", because discrepant data and conclusions have become omnipresent. One reason for this problematic situation is that we are not always thoughtful enough in study design, and sometimes lack enough philosophical contemplation. Another major reason is that we are too rushed in introducing new technology into our research without assimilating technical details. In this essay, we provide examples in different research realms to justify our points. To help readers test their own weaknesses, we raise questions on technical details of RNA reverse transcription, polymerase chain reactions, western blotting and immunohistochemical staining, as these methods are basic and are the base for other modern biotechnologies. Hopefully, after contemplation and reflection on these questions, readers will agree that we indeed know too little about these basic techniques, especially about the artifacts they may create, and thus many conclusions drawn from the studies using those ever-more-sophisticated techniques may be even more problematic.

Transcriptional-Readthrough RNAs Reflect the Phenomenon of "A Gene Contains Gene(s)" or "Gene(s) within a Gene" in the Human Genome, and Thus Are Not Chimeric RNAs.

Tens of thousands of chimeric RNAs, i.e., RNAs with sequences of two genes, have been identified in human cells. Most of them are formed by two neighboring genes on the same chromosome and are considered to be derived via transcriptional readthrough, but a true readthrough event still awaits more evidence and trans-splicing that joins two transcripts together remains as a possible mechanism. We regard those genomic loci that are transcriptionally read through as unannotated genes, because their transcriptional and posttranscriptional regulations are the same as those of already-annotated genes, including fusion genes formed due to genetic alterations. Therefore, readthrough RNAs and fusion-gene-derived RNAs are not chimeras. Only those two-gene RNAs formed at the RNA level, likely via trans-splicing, without corresponding genes as genomic parents, should be regarded as authentic chimeric RNAs. However, since in human cells, procedural and mechanistic details of trans-splicing have never been disclosed, we doubt the existence of trans-splicing. Therefore, there are probably no authentic chimeras in humans, after readthrough and fusion-gene derived RNAs are all put back into the group of ordinary RNAs. Therefore, it should be further determined whether in human cells all two-neighboring-gene RNAs are derived from transcriptional readthrough and whether trans-splicing truly exists.