Tuberculosis – still a diagnostic challenge

Before the COVID pandemic, tuberculosis (TB) was the leading cause of death among infectious diseases. As TB can affect different organs, diagnosis sometimes remains difficult. Here, we present a series of three cases where TB was detected in three different systems after a comprehensive workup. The first case was diagnosed as abdominal TB. She had prolonged fever, weight loss, pain abdomen, shortness of breath with mild ascites, jejunal thickening, and bilateral pleural effusion. The initial workup failed to establish a diagnosis. She was put for diagnostic laparoscopy which aided in diagnosis. The second case was confirmed as nasopharyngeal TB. The patient had recurrent upper respiratory symptoms but relevant investigations including imaging studies and nasal endoscopy were normal. Lung parenchyma was not involved. Nucleic acid amplification test confirmed the diagnosis. The third case was diagnosed as pulmonary TB which was detected only after repeated sputum test. The patient had a history of recurrent hemoptysis with weight loss with a cavitary lesion in the left lung. His consecutive three sputum samples were negative even by nucleic acid amplification test before being positive in the 4th time. Extensive workup and repeated test are required in suspected cases of TB to minimize false negative results.

Primary Multidrug-resistant Tuberculosis of the Breast: A Rare Entity

A 41-year-old female patient presented with a painful swelling in right breast for 6 months. Pus was aspirated and subjected to cartridge-based nucleic acid amplification test (CBNAAT) and line probe assay (LPA) which confirmed it as a case of multidrug-resistant tuberculosis. Isolates were resistant to rifampicin (R) and second-line injectables (SLI). The patient improved on bedaquiline (BDQ)-containing regimen.

Comparative assessment of two in-house-built isothermal assays for visual detection of African swine fever virus

Owing to the lack of effective vaccines, current control measures and eradication strategies for the African swine fever virus (ASFV) rely on early detection and stringent stamping-out procedures. In the present study, we developed two independent isothermal amplification assays, namely, loop-mediated isothermal amplification (LAMP) and polymerase spiral reaction (PSR), for quick visualization of the ASFV genome in clinical samples. Additionally, a quantitative real-time PCR (qRT-PCR)-based hydrolysis probe assay was developed for comparative assessment of sensitivity with the developed isothermal assays. The analytical sensitivity of the LAMP, PSR, and qRT-PCR was found to be 2.64 ×105 copies/µL, 2.64 ×102 copies/µL, and 2.64 ×101 copies/µL, respectively. A total of 165 clinical samples was tested using the developed visual assays. The relative accuracy, relative specificity, and relative diagnostic sensitivity for LAMP vs PSR were found to be 95.37% vs 102.48%, 97.46% vs 101.36%, and 73.33% vs 113.33%, respectively.

Misdiagnosis of Bacterial Pathogens by the Diagnostic Centers: A Potential Route for Antibiotic Resistance

Successful treatment against infectious agents depends on rapid and accurate detection of the causative organisms. Lack of proper identification may facilitate improper antibiotic recommendations. Apart from a few advanced diagnostic facilities in developing countries, most facilities identify pathogens through culture-based methods and suggest antibiotics based solely on the results of disk-diffusion tests. In this pilot study, we tried to validate the identity of the clinical isolates precharacterized by diagnostic facilities. One hundred precharacterized clinical isolates were collected and analyzed phenotypically, biochemically, and genotypically. We employed random amplified polymorphic DNA-polymerase chain reaction (PCR), rcsA, and phoA genes-based PCR and loopmediated isothermal amplification (LAMP) methods to validate the identification of Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli), respectively. Further validation through phylogenetic analysis based on 16S ribosomal deoxyribonucleic acid (rDNA) sequencing was also performed. Phenotypical, biochemical, and phylogenetic analyses found that 30% and 46% misidentification among the diagnostic center identified E. coli and Klebsiella spp., respectively. Moreover, 16S rDNA sequencing confirmed that the representatives of the misidentified organisms belonged to Enterobacter, Acinetobacter, and Pseudomonas genus. Furthermore, LAMP successfully detected the clinical E. coli within 60 minutes. In this study, we recommend proper monitoring and validation of different tests performed in clinical facilities to avoid misidentification, thus facilitating the avoidance of possible routes responsible for developing antimicrobial resistance.

Predictive value of serum glycoprotein non-metastatic melanoma protein B in patients with non-small cell lung cancer accompanied by epidermal growth factor receptor amplification with mutation / 中国医科大学学报

Objective To evaluate the clinical value of free glycoprotein non-metastatic melanoma protein B(GPNMB)as a drug resis-tance and prognostic marker for non-small cell lung cancer(NSCLC)patients with epidermal growth factor receptor(EGFR)amplifica-tion accompanied by mutations.Methods Fifty-five cases of NSCLC patients with EGFR amplification associated with mutations who received treatment from March 2018 to September 2019 were included as the observation group.All patients received an EGFR-tyrosine kinase inhibitor(EGFR-TKI)as the first-line treatment;67 blood samples from the physical examination center during the same period were randomly included as healthy control.We compared the expression levels of free GPNMB between the two groups,explored the correlation between GPNMB expression and the clinicopathological information in the observation group;and combined the clinical efficacy to evaluate its value as a drug resistance marker.Through follow-up,the progress free survival(PFS)of patients was statistically analyzed,and through multivariate Cox regression analysis,independent risk factors affecting the survival in the observation group were explored.Results Compared with that in the control group,the expression level of free GPNMB in the observation group was signi-ficantly up-regulated.The expression level of free GPNMB in the observation group is significantly related to the clinical efficacy of EGFR-TKI(P = 0.016).Patients with high GPNMB expression have significantly stronger drug resistance,and patients with high GPNMB expression have significantly shorter PFS duration(P = 0.032).A high free GPNMB expression(HR = 4.029,95%CI:1.942-8.358,P＜0.001)is also an independent risk factor affecting patient survival.Conclusion The expression level of free GPNMB in patients with EGFR amplification accompanied by mutant NSCLC is significantly up-regulated,and its high expression is significantly related to the enhancement of the patient's drug resistance.High GPNMB expression is significantly related to the poor prognosis of patients and is an independent risk factor affecting patient survival.

Feasible analysis of DR long board detector in clinical application / 中国医学装备

Objective:To explore the feasibility of long board detector of digital radiography(DR)in clinical application.Methods:The long board detector(detector)was erected and placed upright.The scale long ruler with marked metal lead wire was placed at 20 cm in front of the center of long axis of the board of detector,which paralleled medial axis.Three test cards of spatial resolution were respectively placed at three positions(upper,middle and lower)of detector,and they were stuck on the board of detector as 30cm intervals between each other and 45° position.The exposures were conducted at 100,150,and 200 cm of source image distance(SID).The incident doses were tested,which obtained from different SID spots of upper,middle and lower positions of detector.The spatial resolutions of 3 positions were determined through observed the images of cards.The ratio of the marked scale length with metal lead wire to actual length of lead wire was measured through the projection of the scale length,so as to obtain the amplification rate of different spot positions.The spatial distribution of effective focal plane on the direction of long axis of detector,and the morphological change of that were observed.Results:When SID spots were respectively 100,150 and 200cm,the amplification rates of images decreased with increasing SID.The difference of amplification rates among three SID spots was significant(F=223.80,P<0.001).There was significant difference in the corresponding radiation doses among different SID spots(F=7.57,P<0.05).The spatial resolution was constantly 1.8 LP/mm.There was heel effect along with the direction of short axis of detector.The effective focal spot on the direction of long axis of detector appeared up-down symmetrical display.Conclusion:The long board detector of DR equipment has realized the capture for the images of the overall length of spine or the overall length of lower limbs in one exposure,which can meet the clinical requirement,and improve the detection efficiency of X-ray.

Prevalence of Neisseria meningitidis among healthy children and adolescents aged 3-18 in Anhui Province / 中国学校卫生

Objective@#To monitor the prevelance of Neisseria meningitidis among healthy children and adolescents aged 3-18 in Anhui Province, so as to provide support for epidemic cerebrospinal meningitis prevention and control.@*Methods@#From September to October in 2021 and 2022, 4 033 healthy children and adolescents aged 3-18 were selected by stratified random sampling from Anhui Province, including areas north of the Yangtze River (Bozhou, Fuyang, Bengbu, Huainan, Chuzhou, Hefei) and areas south of the Yangtze River (Wuhu, Maanshan, Tongling, Anqing, Chizhou, Xuancheng) and the secretions of children and adolescents were collected from the posterior pharyngeal wall above the uvula, and the strains were identified by bacterial culture and nucleic acid detection. McNemar test and Kappa consistency test were used for statistical analysis. Chisquare test and Chisquare trend test were used to compare the rates.@*Results@#The carrier rates of Neisseria meningitidis were 0.47% and 1.07%, respectively. The sensitivity of nucleic acid detection was higher. Among the detected bacteria, group B accounted for the largest proportion of 76.74%. It was followed by group C (4.65%), group Y (4.65%), group W 135 (2.33%) and group X (2.33%). The bacteria bearing rate of male students was higher than that of female students (χ2=11.44); with the increase of age, the bacteria bearing rate of children and adolescents showed an increasing trend (χ2trend=42.69); the bacterial bearing rate of children and adolescents in the north of the Yangtze River was higher than that in the south of the Yangtze River (χ2=23.19); the bacteria bearing rate of children and adolescents without immune history was higher than that with immune history (χ2=11.02)(P<0.01).@*Conclusions@#Neisseria meningitidis group B has become an epidemic strain in Anhui Province, and senior children and adolescents have become the key population of epidemic prevention and control. It is necessary to continue to do a good job in the surveillance of epidemic cerebrospinal meningitis disease and focuse on group B disease cases, so as to prevent the occurrence of cluster outbreaks in schools.

Clinical application of LAMP in rapid detection of pathogens in lower respiratory tract infections / 中国现代医生

@#Objective Loop mediated isothermal amplification(LAMP)was used to detect the distribution of pathogens in sputum samples,namely combined nucleic acid detection of respiratory pathogens(13 pairs),so as to provide provide reference for clinical accurate diagnosis and treatment.Methods A total of 1642 patients with lower respiratory tract infection admitted to Tongren City People's Hospital from January to December in 2022 were selected.Each patient collected sputum specimens/bronchoalveolar for detection by using LAMP(13 pairs).The detection of pathogenic bacteria in respiratory tract and the relationship with sex,age and season were analyzed.Results The overall detection rate of 13 respiratory pathogens was significantly higher in males than in females(P<0.01);In different age groups,the detection rates of Haemophilus influenzae and Streptococcus pneumoniae in patients aged 3-6,Mycoplasma pneumoniae in patients aged 6-18,and methicillin-resistant Staphylococcus aureus,Streptococcus maltophilus,and Klebsiella pneumoniae in patients aged over 60 were significantly higher than those in other groups(P<0.05);In different seasonal groups,Streptococcus pneumoniae and Chlamydia pneumoniae were more prevalent in spring,while Mycoplasma pneumoniae had the highest infection rate in autumn,and the differences were statistically significant(P<0.05).Conclusion LAMP can be used to detect the pathogen rapidly and provide the basis for clinical diagnosis and treatment.

A Precise and Portable Detection System for Infectious Pathogens Based on CRISPR/Cas Technology / 分析化学

Nucleic acid-based molecular diagnostic methods are considered the gold standard for detecting infectious pathogens.However,when applied to portable or on-site rapid diagnostics,they still face various limitations and challenges,such as poor specificity,cumbersome operation,and portability difficulties.The CRISPR(Clustered regularly interspaced short palindromic repeats)/CRISPR-associated protein(Cas)-fluorescence detection method holds the potential to significantly enhance the specificity and signal-to-noise ratio of nucleic acid detection.In this study,we developed a portable grayscale reader detection system based on loop-mediated isothermal amplification(LAMP)-CRISPR/Cas.On one hand,in the presence of CRISPR RNA(crRNA),the CRISPR/Cas12a system was employed to achieve precise fluorescent detection of self-designed LAMP amplification reactions for influenza A and influenza B viruses.This further validated the high selectivity and versatility of the CRISPR/Cas system.On the other hand,the accompanying independently developed portable grayscale reader allowed for low-cost collection of fluorescence signals and high-reliability visual interpretation.At the end of the detection process,it directly provided positive or negative results.Practical sample analyses using this detection system have verified its reliability and utility,demonstrating that this system can achieve highly sensitive and highly specific portable analysis of influenza viruses.

Establishment of rapid detection method for zika virus based on direct amplification RT-PCR technique / 国际检验医学杂志

Objective To establish a rapid detection method for zika virus based on direct amplification re-al-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-PCR)technique.Methods A direct amplification RT-PCR technique for the rapid detection of zika virus in 5 samples(whole blood,serum,saliva,throat swab and urine)was established by using a special function DNA polymerase and a preferred PCR enhancer.Results The detection limits of the 5 samples were 103 PFU/mL in serum,102 PFU/mL in urine,throat swab,and saliva,and 104 PFU/mL in whole blood.The coefficient of goodness-fit of stand-ard curves was above 0.98,and the amplification efficiency was 90％-110％.Zika virus nucleic acid was suc-cessfully amplified,but non-zika virus nucleic acid was not amplified.Based on the repeatable detection of sam-ples from urine,whole blood,and saliva,the variation coefficient of 6 repeated Ct values at 106 PFU/mL and 102 PFU/mL concentrations were all＜5％.The zika virus detection method established by the direct amplifi-cation RT-PCR technique was consistent with the detection results of conventional RT-PCR technique.Only two serum samples were detected in eight zika virus samples,and the remaining 62 non-zika virus samples and 12 negative samples were not amplified.Conclusion A rapid detection method for zika virus based on direct ampli-fication RT-PCR technique is successfully established.The method is simple,rapid,sensitive and specific.

Amplificación de sondas múltiples dependientes de ligación: actualización y revisión del principio, variantes y aplicaciones / Multiplex ligation-dependent probe amplification: update and review of principles, variants, and applications / Amplificação de múltiplas sondas dependentes de ligação: atualização e revisão do princípio, variantes e aplicações

Resumen La amplificación de sondas múltiples dependientes de ligación (MLPA) es una valiosa herramienta en el estudio de alteraciones en el número de copias para distintas patologías de origen genético. La existencia de una amplia oferta de kits comerciales, el fácil y rápido procesamiento de laboratorio, su alta sensibilidad y, en general, los buenos resultados informados han permitido que su uso se encuentre expandido en muchos laboratorios de biología molecular alrededor del mundo. El principio de esta técnica ha sido adaptado para distintas aplicaciones como la MLPA metilación específica para el estudio de enfermedades relacionadas con la impronta epigenética y la MLPA digital que se acopla a equipos de secuenciación de nueva generación para aumentar la cantidad de regiones analizadas en un solo ensayo. Al contar con 10 años de experiencia en el uso de esta técnica en el Laboratorio Nacional de Tamizaje Neonatal y Alto Riesgo se realiza esta revisión con el fin de analizar los principios, variantes de la técnica, análisis de los datos, algunas aplicaciones, ventajas y desventajas de la MLPA en comparación con otras tecnologías disponibles.

Abstract Multiplex ligation-dependent probe amplification (MLPA) is a valuable tool in the study of copy number alterations for different pathologies of genetic origin. The availability of a wide range of commercial kits, the easy and fast laboratory processing, its high sensitivity and, in general, the good results reported have allowed its use to be expanded in many molecular biology laboratories around the world. The basic principle of this technique has been adapted for different applications such as specific methylation MLPA for the study of diseases related to epigenetic imprinting and digital MLPA that is coupled to next-generation sequencing equipment to increase the number of regions analysed in a single trial. With 10 years of experience in the use of this technique, the National Laboratory for Neonatal and High Risk Screening performs this review in order to analyse the principles, variants of the technique, data analysis, some applications, and advantages and disadvantages of MLPA compared to other available technologies.

Resumo A amplificação de múltiplas sondas dependentes de ligação (MLPA) é uma ferramenta valiosa no estudo das alterações do número de cópias para diferentes patologias de origem genética. A existência de uma ampla gama de kits comerciais, processamento laboratorial fácil e rápido, alta sensibilidade e, em geral, os bons resultados relatados, permitiram que seu uso se encontre expandido em muitos laboratórios de biologia molecular ao redor do mundo. O princípio desta técnica foi adaptado para diferentes aplicações como a MLPA metilação específica para o estudo de doenças relacionadas com o imprinting epigenético e a MLPA digital que é acoplada a equipamentos de sequenciamento de nova geração para aumentar o número de regiões analisadas em um único ensaio. Com 10 anos de experiência no uso da técnica, o Laboratório Nacional de Triagem Neonatal e de Alto Risco realiza esta revisão visando a analisar os princípios, variantes da técnica, análise dos dados, algumas aplicações, vantagens e desvantagens da MLPA comparado com outras tecnologias disponíveis.

Vocal Demand Response to the Educational Setting Based Communication Scenarios: A Single Subject Study

Objectives: This was a single-subject study, aimed to demonstrate different vocal demand situations that are typical for primary school and teacher's vocal demand response under two acoustical conditions, with and without voice amplification, during five working days. Methods: The long-term voice dosimetry with Vocal Holter Med (PR.O. Voice Srl) was carried out on a 49-year-old female teacher with voice disorders during daily teaching activities. A sound field amplification system (SFAS) PentaClass Runa was installed in the classroom. Voice dosimetry was provided under two different acoustical conditions: without SFAS (2 days) and with SFAS (3 days). Results: Phonation time percentage, sound pressure level (SPL), SPL SD, fundamental frequency (F0), F0 SD, cycle, and distance doses were investigated in seven communication scenarios (lessons, group/individual classes, sports lessons in the gym and schoolyard, breaks, lunch breaks, and other activities). The median scores of all voice parameters differed significantly between different vocal demand contexts. The significant statistical difference in the vocal demand response was in the communication situations with and without SFAS. In addition, the number of children, reverberation time, and ambient air relative humidity impacted voice SPL and the cycle dose. Conclusions: Lessons, sports lessons held in the gym or schoolyard, breaks, and lunch breaks were considered as high vocal demand communication situations requiring higher voice intensity and fundamental frequency, higher phonation time percentage, cycle, and distance doses. Group/individual work and other teacher activities during the day, unrelated to direct work with students, were categorized as low vocal demand communication scenarios.

Objetivos: Este fue un estudio de sujeto único, cuyo objetivo fue demostrar diferentes situaciones de demanda vocal típicas de la escuela primaria y la respuesta vocal de los docentes bajo dos condiciones acústicas, con y sin amplificación de voz, durante cinco días laborables. Métodos: Se llevó a cabo dosimetría vocal a largo plazo con Vocal Holter Med (PR.O. Voice Srl) durante las actividades diarias de enseñanza en una docente de 49 años con trastornos de la voz. Se instaló un sistema de amplificación de campo sonoro (SFAS) PentaClass Runa en el aula. La dosimetría vocal se realizó bajo dos condiciones acústicas diferentes: sin SFAS (2 días) y con SFAS (3 días). Resultados: Se investigaron el porcentaje de tiempo de fonación, el nivel de presión sonora (SPL), SPL SD, la frecuencia fundamental (F0), F0 SD, ciclos y dosis de distancia en siete escenarios de comunicación diferentes (clases, clases grupales/individuales, clases de educación física en el gimnasio y el patio de la escuela, recreos, almuerzos y otras actividades). Las puntuaciones medias de todos los parámetros vocales diferían significativamente entre los diferentes contextos de demanda vocal. La diferencia estadísticamente significativa en la respuesta a la demanda vocal se observó en las situaciones de comunicación con y sin SFAS. Además, el número de niños, el tiempo de reverberación y la humedad relativa del aire ambiente afectaron al SPL de la voz y la dosis de ciclo. Conclusiones: Las lecciones, las clases de educación física en el gimnasio o el patio de la escuela, los recreos y los almuerzos se consideraron situaciones de comunicación de alta demanda vocal, que requerían una mayor intensidad y frecuencia fundamental de la voz, un mayor porcentaje de tiempo de fonación y dosis de ciclo y distancia más altas. El trabajo grupal/individual y otras actividades del profesor durante el día no relacionadas con el trabajo directo con los estudiantes se categorizaron como escenarios de comunicación de baja demanda vocal.

Correlation of serological and molecular markers in the screening for hepatitis B virus in blood bank in northern Brazil

ABSTRACT Introduction: In Brazil, the blood donor screening for hepatitis B virus (HBV) includes laboratory testing for serological (HBsAg and Anti-HBc) and molecular (HBV DNA) markers. This study aims to correlate serology reactive results with HBV DNA detection among blood donors with at least one HBV infection marker detected in a blood bank in northern Brazil. Method: A retrospective search for HBV reactive blood donor data from January 2017 to December 2019 was performed. Serological screening was performed by chemiluminescent microparticle immunoassays Architect HBsAg and Architect Anti-HBc, whereas molecular screening was performed by the HBV nucleic acid test (HBV NAT). Main results: A total of 556 HBsAg reactive results were detected, between positive (47.66%) and inconclusive (52.34%). A total of 3,658 Anti-HBc reactive results were detected, between positive (83.71%) and inconclusive (16.29%). None of the inconclusive results were associated with HBV DNA detection. The HBV DNA detection rates were 47.55% among HBsAg positive samples and 4.08% among Anti-HBc positive samples. The signal-to-cutoff (S/CO) ratio median of HBV NAT positive samples was superior in comparison to HBV NAT negative samples (p < 0.0001). The thresholds found to optimize sensitivity and specificity were 404.15 for Architect HBsAg and 7.77 for Architect Anti-HBc. Three blood donors were in the window period and 1 occult HBV infection case was detected. Conclusion: High S/CO ratios were more predictive of HBV DNA detection. However, a number of HBV NAT positive samples gave low values, while some HBV NAT negative samples showed high values, reaffirming the significance of molecular testing to enhance transfusion safety.

Comparison of Microscopy, Culture and Molecular Methods for Diagnosing Gonorrhea

Gonorrhea, a sexually transmitted infection caused by Neisseria gonorrhoeae, continues to be a significant global public health concern. The timely and accurate diagnosis of this infectious disease is crucial for its effective management. Traditional methods, especially culture, were historically considered the gold standard for diagnosing gonorrhea. However, the introduction of nucleic acid amplification tests (NAATs), such as Real-Time PCR, has revolutionized diagnostic approaches. Currently, the Center for Disease Control and Prevention (CDC) recommends NAAT as the primary diagnostic method, with culture reserved for specific cases, particularly for testing antimicrobial susceptibility in instances of suspected treatment failure. The International Union against Sexually Transmitted Infections (IUSTI) provides guidelines for the use of NAAT or culture, depending on clinical scenarios. This study conducted a retrospective comparative analysis of various diagnostic methods at the Apex Regional STD Centre in New Delhi, India, spanning from January 1, 2022, to December 31, 2022. Culture, Real-Time PCR, and smear examination were compared for the diagnosis of gonorrhea. A total of 33 samples were included in the analysis, with the following percentages: culture (92.02%), PCR (100%), and smear examination (100%). An intriguing finding was that 7.98% of samples were culture-negative but PCR-positive, highlighting a significant disparity between the two methods. This observation underscores the limitations of relying solely on culture for gonorrhea diagnosis and the potential consequences, including treatment delays, disease transmission, and the development of antibiotic-resistant strains. In summary, this study underscores the critical need for accurate and reliable diagnostic methods for gonorrhea. It emphasizes the evolving diagnostic landscape, with NAATs emerging as essential tools. The findings from multiple studies stress the complementary roles of different diagnostic methods and the necessity of adapting to evolving diagnostic techniques. This research highlights the importance of collaborative approaches to enhance accuracy and address the evolving challenges of gonorrhea diagnosis. Ultimately, the significance of laboratory testing extends beyond individual patient care to broader public health goals and the prevention of sexually transmitted infections.

Molecular detection of SARS-CoV-2 in Argentina: Evaluation of alternative diagnostic tools for the decentralization of the diagnosis

Abstract The rocketing number of COVID-19 cases highlighted the critical role that diagnostic tests play in medical and public health decision-making to contain and mitigate the SARS-CoV-2 pandemic. This study reports the evaluation and implementation of different tests for the molecular detection of SARS-CoV-2 in the central region of Argentina. We evaluated 3 real time RT-PCR kits (GeneFinder COVID-19 Plus RealAmp Kit, DisCoVery SARS-CoV-2 RT-PCR Detection Kit and WGene SARS-CoV-2 RT Detection), 2 nucleic acid extraction methods [MagaBio plus Virus DNA/RNA Purification Kit II (BioFlux), 35-min vs. 9-min, a pre-analytical reagent (FlashPrep®) and 2 isothermal amplification tests (Neokit Plus and ELA CHEMSTRIP®). The order according to the best performance of the 3 real-time RT-PCR kits evaluated was: DisCoVery > GeneFinderTM> WGene. The 2 RNA extraction methods showed similar good results: MagaBio plus Virus RNA Purification Kit II (BioFlux) 9-min was selected due to its faster performance. FlashPrep® reagent showed excellent results to perform direct RNA detection. Isothermal amplification assays showed acceptable sensitivity and specificity values (>80%), except in samples with Ct> 30. Our data show optimal real time RT-PCR kits and alternative molecular methods for SARS-CoV-2 diagnostic. These alternative assays proved to be aceptable.

Resumen La explosión de casos de COVID-19 resaltó el papel fundamental que desempeñan las pruebas de diagnóstico en la toma de decisiones médicas y de salud pública para contener y mitigar la pandemia de SARS-CoV-2. Este estudio reporta la evaluación y la implementación de diferentes test para la detección molecular de SARS-CoV-2 en la región central de Argentina. Evaluamos tres kits de RT-PCR en tiempo real (GeneFinder COVID-19 Plus RealAmp Kit, DisCoVery SARS-CoV-2 RT-PCR Detection Kit y WGene SARS-CoV-2 RT Detection), dos métodos de extracción de ácidos nucleicos (MagaBio plus Virus DNA/RNA Purification Kit II [BioFlux, 35-min vs. 9-min), un reactivo pre-analítico (FlashPrep®) y dos test de amplificación isotérmica (Neokit Plus and ELA CHEMSTRIP®). El orden de rendimiento de los tres kits de RT-PCR en tiempo real evaluados fue el siguiente: DisCoVery GeneFinder™ WGene. Los dos métodos de extracción de RNA mostraron buenos y similares resultados; se seleccionó MagaBio plus Virus RNA Purification Kit II (BioFlux) 9-min debido a su rápido tiempo de procesamiento. El reactivo FlashPrep® mostró excelentes resultados para realizar detección directa de RNA. Los ensayos de amplificación isotérmica mostraron valores de sensibilidad y de especificidad aceptables (80%), excepto en muestras con Ct 30. Nuestros resultados muestran kits de RT-PCR en tiempo real óptimos, como así también métodos moleculares alternativos para el diagnóstico de SARS-CoV-2 que resultan aceptables para su uso en contextos adversos, de descentralización y en diferentes escenarios epidemiológicos, para la detección rápida y precisa del SARS-CoV-2.

Long-range PCR amplification-based targeted enrichment & next generation sequencing: A cost-effective testing strategy for lysosomal storage disorders

Background & objectives: Lysosomal storage disorders (LSDs) are genetic metabolic disorders which result from deficiency of lysosomal enzymes or defects in other lysosomal components. Molecular genetic testing of LSDs is required for diagnostic confirmation when lysosomal enzyme assays are not available or not feasible to perform, and for the identification of the disease causing genetic variants. The aim of this study was to develop a cost-effective, readily customizable and scalable molecular genetic testing strategy for LSDs. Methods: A testing method was designed based on the in-house creation of selective amplicons through long range PCR amplification for targeted capture and enrichment of different LSD genes of interest, followed by next generation sequencing of pooled samples. Results: In the first phase of the study, standardization and validation of the study protocol were done using 28 samples of affected probands and/or carrier parents (group A) with previously identified variants in seven genes, and in the second phase of the study, 30 samples of enzymatically confirmed or biopsy-proven patients with LSDs and/or their carrier parents who had not undergone any prior mutation analysis (group B) were tested and the sequence variants identified in them through the study method were validated by targeted Sanger sequencing. Interpretation & conclusions: This testing approach was found to be reliable, easily customizable and cost-effective for the molecular genetic evaluation of LSDs. The same strategy may be applicable, especially in resource poor settings, for developing cost-effective multigene panel tests for other conditions with genetic heterogeneity.

Screening of haemoglobinopathies by high performance liquid chromatography and its molecular characterization using amplification refractory mutation system and direct DNA sequencing techniques among college students in central Gujarat.

Background: Haemoglobinopathies are inherited blood disorders that can have significant health implications and are common in certain populations. Aim and Objectives: To investigate the prevalence of haemoglobinopathies among college students in central Gujarat, India. Material and Methods: A combination of techniques were employed to identify haemoglobinopathies. Chromatograms were examined to identify variant haemoglobin based on characteristics like proportion, retention time, and peak features. Prior to DNA analysis, prevalent Indian thalassemia mutations, as well as HbS and HbE, were identified using the Amplification Refractory Mutation System (ARMS) Polymerase Chain Reaction (PCR) technique. DNA sequencing was performed at SN Gene Lab, Surat. Results: The findings revealed that HbS codon 6 (A?T) heterozygosity, Hb S Codon 6 (A?T) double heterozygosity, and HbE: codon 26 (G? A) were relatively common in the population. Among carrier students screened for haemoglobino- pathies, IVS-1, nt5 (G?C), and Codon41/42 (-CTTT) were the most prevalent beta-thalassemia mutations in India. Codon 88 (C-T) and other less common mutations were also detected. Conclusion: The study underscores the importance of raising awareness and providing counselling for individuals affected by haemoglobinopathies. By utilizing High Performance Liquid Chromatography (HPLC) screening and molecular techniques for characterization, the study contributes to the understanding of mutation patterns in central Gujarat. This knowledge is crucial for the effective detection of mutations in screening programs and prenatal diagnostics, ultimately enhancing the quality of life for affected individuals and preventing further transmission of these disorders.

Auramine-O Staining vs Ziehl Neelsen Staining: Advantages and Disadvantages

Background: Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is still a major public health concern around the world. Prompt detection of active tuberculosis cases helps in timely therapeutic intervention and reduces community transmission. Despite limited sensitivity, conventional microscopy is still used to diagnose pulmonary tuberculosis in high-burden nations such as India. This study, therefore, was aimed at assessing the diagnostic performance of microscopy by Ziehl Neelsen (ZN) and auramine (AO) staining in the diagnosis of pulmonary tuberculosis. Materials and methods: A prospective comparative study was done on the sputum samples of 2,395 adult patients from November 2018 to May 2020 suspected of having pulmonary tuberculosis visiting the Designated Microscopic Centre of SGT Medical College, Budhera, Gurugram. Each sample was subjected to ZN staining, and AO staining as per NTEP guidelines. Results: Out of the 2,395 samples studied, 161 (6.76%) and 224 (9.35%) were positive by ZN and AO staining methods respectively. Pauci-bacillary cases detected by AO were more than ZN staining. There were 63 more sputum samples detected by AO staining which were missed by ZN microscopy. Conclusion: When compared to conventional ZN staining, the auramine staining technique is more sensitive and takes less time to diagnose pulmonary tuberculosis

Detection and species determination of malaria parasites by microscopy, antigen detection, polymerase chain reaction, and loop-mediated isothermal amplification assay in a tertiary care hospital

Background: Malaria is a major health issue in tropical and subtropical areas. Out of all subtypes, Plasmodium falciparum (Pf) is the most dangerous form accounting for high mortality and morbidity. It is transmitted by infected female anopheles mosquitoes and infected blood transfusions. Aims and Objectives: The aim of the study is to establish correct diagnosis by direct microscopy, Immunochromatographic test (ICT), and molecular studies. Materials and Methods: This prospective study was conducted in the PG Department of Microbiology, SCB Medical College, Cuttack. Thick blood smears were drawn and then stained with Leishman’s stain to visualize falciparum rings. DNA was extracted from infected blood samples by phenol chloroform method with some modification as described by Sambrook and Russel for molecular analysis. Results: In the present study, 150 cases of malaria were analyzed. The male: female ratio was 1.7:1 and age ranged from 0 to 56 years. The Plasmodium vivax positivity was compared with thin smear to 21 (84%) in ICT, 100% both polymerase chain reaction (PCR) and loop mediated isothermal amplification assay (LAMP) assays followed by the Pf positivity as 76 (92.7%) in ICT, 82 (100%) both PCR and LAMP assays, respectively. The results obtained were statistically significant with P < 0.001. The PCR and LAMP showed 100% response to specificity and positive predictive value. Conclusion: The present study established the role of molecular tests such as PCR and LAMP are highly specific for diagnosis of Plasmodium species whereas they are more or less similar in sensitivity as compared to other diagnostic methods such as ICT and microscopy.

An overview of mycetoma and its diagnostic dilemma: Time to move on to advanced techniques

The neglected tropical disease mycetoma can become extremely devastating, and can be caused both by fungi and bacteria; these are popularly known as eumycetoma and actinomycetoma respectively. The classical triad of the disease is subcutaneous swelling, multiple discharging sinuses and the presence of macroscopic granules. The present study aims to highlight the existing diagnostic modalities and the need to incorporate newer and more advanced laboratory techniques like pan fungal/pan bacterial 16S rRNA gene polymerase chain reaction (PCR) and sequencing, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). It is important for the medical team to be aware of the various diagnostic options (both existing and future), so that diagnosis of such a debilitating disease is never missed, both by clinicians and microbiologists/pathologists. The newer diagnostic methods discussed in this article will help in rapid, accurate diagnosis thus facilitating early treatment initiation, and decreasing the overall morbidity of the disease. In the Indian context, newer technologies need to be made available more widely. Making clinicians aware and promoting research and development in mycetoma diagnostics is the need of the hour.