Novel Synthesis and Phenotypic Analysis of Mutant Clouds for Hepatitis E Virus Genotype 1.

Many RNA viruses exist as an ensemble of genetically diverse, replicating populations known as a mutant cloud. The genetic diversity (cloud size) and composition of this mutant cloud may influence several important phenotypic features of the virus, including its replication capacity. We applied a straightforward, bacterium-free approach using error-prone PCR coupled with reverse genetics to generate infectious mutant RNA clouds with various levels of genetic diversity from a genotype 1 strain of hepatitis E virus (HEV). Cloning and sequencing of a genomic fragment encompassing 70% of open reading frame 1 (ORF1) or of the full genome from variants in the resultant clouds showed the occurrence of nucleotide mutations at a frequency on the order of 10-3 per nucleotide copied and the existence of marked genetic diversity, with a high normalized Shannon entropy value. The mutant clouds showed transient replication in cell culture, while wild-type HEV did not. Cross-sectional data from these cell cultures supported the existence of differential effects of clouds of various sizes and compositions on phenotypic characteristics, such as the replication level of (+)-RNA progeny, the amounts of double-stranded RNA (a surrogate for the rate of viral replication) and ORF1 protein, and the expression of interferon-stimulated genes. Since mutant cloud size and composition influenced the viral phenotypic properties, a better understanding of this relationship may help to provide further insights into virus evolution and prediction of emerging viral diseases.IMPORTANCE Several biological or practical limitations currently prevent the study of phenotypic behavior of a mutant cloud in vitro We developed a simple and rapid method for synthesizing mutant clouds of hepatitis E virus (HEV), a single-stranded (+)-RNA [ss(+) RNA] virus, with various and controllable levels of genetic diversity, which could then be used in a cell culture system to study the effects of cloud size and composition on viral phenotype. In a cross-sectional analysis, we demonstrated that a particular mutant cloud which had an extremely high genetic diversity had a replication rate exceeding that of wild-type HEV. This method should thus provide a useful model for understanding the phenotypic behavior of ss(+) RNA viruses.

Role of acoustic radiation force impulse and shear wave velocity in prediction of preterm birth: a prospective study.

Background Preterm birth is one of the important causes of neonatal morbidity where we rely on subjective criteria such as modified Bishop's scoring and contemporary sonographic measurement of cervical length. Acoustic radiation force impulse (ARFI) is a technological advancement in elastography that can be employed in prediction of cervical softening and preterm labor. Purpose To evaluate the role of ARFI technique and shear wave velocity (SWV) estimates as a predictor of preterm birth and its comparison with other clinical and sono-elastographic measures. Material and Methods Thirty-four pregnant women (gestation age = 28-37 weeks age) showing features suggestive of preterm labor were included and evaluated with modified Bishop's score, cervical length by ultrasound (US), ARFI to derive Elastography index (EI), and SWV of the cervix. The patients were later divided into two groups, using the clinical outcome of preterm or term delivery. Results Twenty patients delivered at term (gestational age > 37 weeks) and 14 were preterm. Receiver operating characteristics (ROC) curves showed SWV with highest sensitivity and specificity (93% and 90%, respectively) for the prediction of preterm birth at a cutoff value of 2.83 m/s. EI and modified Bishop's score were comparable to each other, but were less sensitive techniques. Conclusion Elastographic assessment of antenatal cervix is a novel technique of virtual palpation of internal os and can be utilized as an objective criterion for preterm birth prediction.

Comparison of Visual Inspection Methods with Pap Smear as Screening Test for Premalignant Lesions of the Cervix.

Aim and Objectives: The present study was done to compare the visual inspection methods with Pap smear as a screening test for premalignant lesions of the cervix. Materials and Methods: The present observational prospective study was done at the Department of Obstetrics and Gynecology at Teerthanker Mahaveer Medical College and Research Center for 18 months. All study subjects were subjected to Papanicolaou smear, VIA, and VILI examination. If any of these tests were found positive, then a colposcopy and cervical biopsy were done. Results: The sensitivity of Pap, VIA, VILI, and colposcopy was 52.63%, 84.21%, 73.68%, and 84.21%, respectively. The specificity of Pap, VIA, VILI, and colposcopy was 60.0%, 80.0%, 60.0%, and 80.0%, respectively. The accuracy of Pap, VIA, VILI, and colposcopy was 54.17%, 83.33%, 70.83%, and 83.33%, respectively. Conclusion: It is well known that VIA and VILI are very easy to carry out and apply. Even technically they do not cost much, consume less tax and can be applied to all the patients. Even the results are calculated in a faster manner which helps in chalking out the plan in an easy way.

Long Story of Short Femur: A Single-Center Study with Step-Wise Imaging Approach.

PURPOSE: To evaluate the possible outcomes of fetuses diagnosed with short femur length (FL) and to guide diagnosis through a step-wise imaging algorithm. METHODS: This was a prospective cohort study of 42 pregnancies with fetal femur length (FL) below the 5th centile for gestational age. The cases were divided into two categories of isolated short FL & non-isolated short FL and followed up to determine the etiology. RESULTS: There were 11 cases of non-isolated short FL with skeletal dysplasia observed in 7 and chromosomal abnormalities in 4 cases. There were 31 cases with isolated short FL in which fetal growth restriction (FGR) occurred in 14/31 (45%) cases; 13 out of 31 (42%) were constitutional (short for gestational age, SGA) whereas 4/31(13%) showed normal interval growth on follow up (false positive). CONCLUSION: Short femur can be isolated or non-isolated. Short femur length can be a good predictor and early sign of FGR. Serial follow up scan of the all cases of isolated short FL is important since a majority of them are normal and not require any further intervention. Cases of non-isolated short FL require step-wise approach to differentiate into dysplasia or aneuploidy.

Anterior chamber depth and its associations with ocular and general parameters in adults.

BACKGROUND: To investigate the normative data of anterior chamber depth and its associations in adults in rural Central India. DESIGN: Population-based study. PARTICIPANTS: The Central India Eye and Medical Study included 4711 subjects aged 30+ years (response rate 80.1%). METHODS: The subjects underwent an ophthalmological examination including measurement of the anterior chamber depth by sonography. MAIN OUTCOME MEASURE: Anterior chamber depth. RESULTS: Mean anterior chamber depth was 3.22 ± 0.34 mm. In multivariate analysis, a shallow chamber depth was significantly associated with higher age (P < 0.001), female gender (P < 0.001), shorter body stature (P = 0.003), hyperopic refractive error (P < 0.001), higher lens thickness (P < 0.001) and shorter axial length (P < 0.001). CONCLUSIONS: In the rural Central Indian population, a shallow anterior chamber was associated with higher age, female gender, short body stature, hyperopia, thick lens and shorter axial length. Compared with population-based data from East Asia (China), the anterior chamber was markedly deeper in India. The data may be helpful to explain anatomic relationships of the anterior segment of the eye, to elucidate risk factors of angle-closure glaucoma and to explain ethnic differences in the prevalence of angle-closure glaucoma.

Reverse Genetics to Engineer Positive-sense RNA Virus Variants.

The lack of a convenient method for the iterative generation of diverse full-length viral variants has impeded the study of directed evolution in RNA viruses. By integrating a full RNA genome error-prone PCR and reverse genetics, random genome-wide substitution mutagenesis can be induced. We have developed a method using this technique to synthesize diverse libraries to identify viral mutants with phenotypes of interest. This method, called full-length mutant RNA synthesis (FL-MRS), offers the following advantages: (i) the ability to create a large library via a highly efficient one-step error-prone PCR; (ii) the ability to create groups of libraries with varying levels of genetic diversity by manipulating the fidelity of DNA polymerase; (iii) the creation of a full-length PCR product that can directly serve as a template for mutant RNA synthesis; and (iv) the ability to create RNA that can be delivered into host cells as a non-selected input pool to screen for viral mutants of the desired phenotype. We have found, using a reverse genetics approach, that FL-MRS is a reliable tool to study viral-directed evolution at all stages in the life cycle of the hepatitis C virus, JFH1 isolate. This technique appears to be an invaluable tool to employ directed evolution to understand adaptation, replication, and the role of viral genes in pathogenesis and antiviral resistance in positive-sense RNA viruses.

Role of placenta accreta index in prediction of morbidly adherent placenta: A reliability study.

OBJECTIVE: To estimate the level of interobserver agreement in the calculation of placenta accreta index (PAI) as well as to evaluate the accuracy of PAI in prediction of morbidly adherent placenta. MATERIALS AND METHODS: This was a prospective study where 45 pregnant women (from 28 to 37 weeks of gestational age) with at least one previous Caesarean section and ultrasound-proven placenta previa were included. A known and previously published scoring system, the PAI, was evaluated independently by two radiologists and the cases were followed for the delivery and histopathology outcome. The accuracy of the PAI and the level of interrater agreement was analysed using cross-table analysis, intraclass correlation efficient and Cohen's kappa as statistical variables. RESULTS: Adherent placenta was found in 15 patients accounting for 33% of cases. The PAI showed nearly 90% sensitivity, specificity and the predictive values. Interrater agreement in calculation of PAI by the two radiologists was perfect with an intraclass correlation efficient of 0.959. An easy-to-use morbid adherent placenta score was also predicted to simplify the results of PAI, which showed moderate agreement (κ = 0.746). CONCLUSIONS: The PAI can be helpful in stratifying the individual risk of placental invasion above the baseline risk. The PAI-derived, simplified scoring system called morbid adherent placenta score can be used as a simple tool to interpret and convey the results of PAI.

Molecular mechanism of anti-SARS-CoV2 activity of Ashwagandha-derived withanolides.

COVID-19 caused by SARS-CoV-2 corona virus has become a global pandemic. In the absence of drugs and vaccine, and premises of time, efforts and cost required for their development, natural resources such as herbs are anticipated to provide some help and may also offer a promising resource for drug development. Here, we have investigated the therapeutic prospective of Ashwagandha for the COVID-19 pandemic. Nine withanolides were tested in silico for their potential to target and inhibit (i) cell surface receptor protein (TMPRSS2) that is required for entry of virus to host cells and (ii) viral protein (the main protease Mpro) that is essential for virus replication. We report that the withanolides possess capacity to inhibit the activity of TMPRSS2 and Mpro. Furthermore, withanolide-treated cells showed downregulation of TMPRSS2 expression and inhibition of SARS-CoV-2 replication in vitro, suggesting that Ashwagandha may provide a useful resource for COVID-19 treatment.

Fetal bradyarrhythmia causing hydrops fetalis: A journey from fetal echo to autopsy.

Introduction: Non-immune hydrops fetalis is a condition with poor fetal prognosis. The incidence of this clinical condition is increasing as compared to its iso-immune variant. The diagnosis of hydrops fetalis is straightforward; however, delineating the primary cause of non-immune hydrops fetalis requires a holistic approach and background knowledge of the entity. Case report: We present a case of non-immune hydrops fetalis due to a rare functional cardiac disorder demonstrated by features of cardiac failure in the form of clinically significant tricuspid regurgitation detected on echocardiography. Fetal autopsy supported the diagnosis by excluding any structural anomaly. Discussion: Non-immune hydrops fetalis may be due to structural and non-structural cardiac anomalies. Meticulous work-up is required to establish the diagnosis in such cases. Conclusion: The case also highlights the systematic approach as well as the series of investigations required for the early diagnosis and management of such cases.

Fetal micromelia, thoracic dysplasia and polydactyly revisited: A case-based antenatal sonographic approach.

INTRODUCTION: Skeletal dysplasia is a condition associated with various abnormalities of the skeleton and comprises multiple groups of disorders. Antenatal ultrasonographic assessment of the skeletal dysplasia requires a robust and systematic assessment of the long bones, fetal thorax, skull, spine, pelvis, hands and the feet. Large number of diseases, their overlapping phenotypic features and the lack of systematic approach lead to diagnostic inefficiency. A precise molecular diagnosis also requires an elaborate antenatal sonographic assessment to reach a final diagnosis. CASE REPORT: A fetus with micromelia, thoracic dysplasia and polydactyly was detected on prenatal sonography. An algorithmic approach of this rare combination on prenatal sonography is highlighted. DISCUSSION: Fetal micromelia is a relatively common entity which can be subclassified into mild and severe types. The lethal nature of the condition requires assessment of the thoracic biometry which may further narrow down the diagnostic possibilities. The red flags or highlighting features of various conditions like polydactyly, hitch-hiker thumb deformity, ovoid tibia and absent fibula may lead to a specific diagnosis. CONCLUSION: A background knowledge of various types of micromelia, their lethal nature, associations and specific features of various differential skeletal dysplasia will always be useful, if employed in a systematic manner.

Fetal adrenal gland biometry and cervical elastography as predictors of preterm birth: A comparative study.

BACKGROUND: Preterm birth is a major health problem in developing and developed countries leading to rising health care costs and long-term neurodevelopmental disability. The study aims to evaluate the role of new quantitative markers, like the elastography of cervix (shear wave speed estimation), fetal adrenal zone enlargement, and corrected fetal adrenal gland volume; in preterm birth prediction and analyze their relative importance. Thus, these markers may be beneficial in early preterm birth detection and prevent the related morbidities. METHODS: Thirty pregnant females (from 28 to 37 weeks of gestational age), showing clinical signs and delivery outcome of preterm birth were included in the study with an equal number of not-in-labor antenatal females at ≥37 weeks as controls. These patients were categorized as preterm and term groups. Both the groups were subjected to trans-abdominal ultrasonography where cervical length, cervical shear wave speed (dynamic elastography) and fetal adrenal gland parameters were measured. RESULTS: Shear wave speed estimation of the antenatal cervix showed the highest sensitivity and specificity (96.7% and 87% respectively) in the prediction of preterm birth and also showed a strong correlation with fetal adrenal gland enlargement. Fetal adrenal zone enlargement was also shown to be a reliable marker of preterm birth, however, with reduced sensitivity and specificity than shear wave speed. CONCLUSION: The elastographic advancement and fetal adrenal biometry derived quantitative markers can be used as an objective and standard criterion for accurate prediction of preterm birth.

Swyer Syndrome With Gonadoblastoma: A Clinicoradiological Approach.

Primary amenorrhea is a common diagnostic challenge in the gynecology department, wherein there are numerous causes that need to be approached in a systematic manner. However, when a case with a pelvic lump or a solid pelvic mass presents to a gynecologist or a radiologist, the approach becomes difficult to justify amenorrhea and pelvic mass as a single entity. We present the case of a 36-year-old female with the complaints of primary amenorrhea with a pelvic mass. The case was approached keeping in view the diagnostic possibilities and applying the role of clinical, radiological, and laboratory analyses. The final diagnosis of Swyer syndrome with gonadoblastoma was made, and she was further subjected to operative resection and hormonal therapy. This study stresses on the approach to a case, wherein the diagnosis was based only on the clinician's acumen and the radiologist's expertise, providing a way to simplify the protocol in the evaluation of such types of cases.

Microdeletion of Y chromosome as a cause of recurrent pregnancy loss.

CONTEXT: In majority of couples experiencing recurrent pregnancy loss (RPL), etiology is still unknown. Two genetic factors have been suggested to underlie miscarriage in a subset of patients, namely skewed X chromosome inactivation in females and Y chromosome microdeletions in their partners. In males, microdeletions of the Y chromosome are known to cause spermatogenetic failure and male infertility. AIMS: The aim of the study was to find out the role of Y chromosome microdeletion in male partners of couples experiencing RPL. SETTINGS AND DESIGN: University hospital and genetic laboratory. Prospective case-control study. SUBJECTS AND METHODS: 59 couples with a history of RPL and 20 fertile controls (FC) with no miscarriage were included in the study. The study subjects were divided into male partners of RPL couples with abnormal semen parameters (AS) (n = 8), and couples with normal semen parameters (NS) (n = 51). Fertile controls with normal semen parameters were (FC) (n = 20). Y chromosome microdeletion was performed on 40 male partners of RPL and 20 FC. STATISTICAL ANALYSIS USED: Chi-square test. P <0.05 were considered statistically significant. RESULTS: 13 of the 40 RPL cases showed deletion in three azoospermia factor loci on the long arm of Y chromosome. The P value was significant with Y chromosome microdeletion in RPL cases as compared to 20 FC where no Y chromosome microdeletion was present. CONCLUSIONS: Y chromosome microdeletion may be an important hidden cause of recurrent pregnancy miscarriage and can be offered to couples with the undiagnosed cause of miscarriage.

Prevalence of myelinated retinal nerve fibres in adult Indians: the Central India Eye and Medical Study.

PURPOSE: To determine the prevalence of myelinated retinal nerve fibers in the adult Indian population. METHODS: The Central India Eye and Medical Study performed in rural Central India included 4711 participants aged 30+ years. The participants underwent a detailed ophthalmic and medical examination. RESULTS: Readable fundus photographs were available for 8645 eyes of 4485 (95.2%) subjects. Myelinated retinal nerve fibers were detected in 52 eyes (46 subjects) with a prevalence rate of 0.58±0.08 per 100 eyes [95% confidence interval (CI): 0.42, 0.74] and 1.03±0.15 per 100 subjects (95%CI: 0.73, 1.32). Prevalence of myelinated retinal nerve fibers was significantly associated hyperopic refractive error (p=0.008; OR: 1.31; 95%CI: 1.07, 1.59). It was not significantly associated with age (p=0.11), best corrected visual acuity (logMAR; p=0.33), intraocular pressure (p=0.09), amount of nuclear cataract (p=0.93), optic disc area (p=0.60), presence of glaucomatous optic nerve atrophy (p=0.62), and early age-related macular degeneration (p=0.53). CONCLUSIONS: Myelinated retinal nerve fibers are present in about 10 out of 1000 adult Indians in rural Central India, with a higher prevalence in hyperopic eyes. Prevalence of myelinated retinal nerve fibers was not associated with age, visual acuity, glaucoma and macular degeneration.

Antral follicle count in normal (fertility-proven) and infertile Indian women.

BACKGROUND: Antral follicle count (AFC) has been labeled as the most accurate biomarker to assess female fecundity. Unfortunately, no baseline Indian data exists, and we continue using surrogate values from the Western literature (inferred from studies on women, grossly different than Indian women in morphology and genetic makeup). AIMS: (1) To establish the role of AFC as a function of ovarian reserve in fertility-proven and in subfertile Indian women. (2) To establish baseline cut-off AFC values for Indian women. SETTINGS AND DESIGN: Prospective observational case-control study. MATERIALS AND METHODS: Thirty patients undergoing workup for infertility were included and compared to equal number of controls (women with proven fertility). The basal ovarian volume and AFC were measured by endovaginal. USG the relevant clinical data and hormonal assays were charted for every patient. STATISTICAL ANALYSIS USED: SPSS platform was used to perform the Student's t-test and Mann-Whitney U-test for intergroup comparisons. Correlations were determined by Pearson's ranked correlation coefficient. RESULTS: Regression analysis revealed the highest correlation of AFC and age in fertile and infertile patients with difference in mean AFC of both the groups. Comparison of the data recorded for cases and controls showed no significant difference in the mean ovarian volume. CONCLUSIONS: AFC has the closest association with chronological age in normal and infertile Indian women. The same is lower in infertile women than in matched controls. Baseline and cut-off values in Indian women are lower than that mentioned in the Western literature.

Retinal nerve fibre layer cross-sectional area, neuroretinal rim area and body mass index.

PURPOSE: To examine associations between body mass index, neuroretinal rim area and retinal nerve fibre layer cross-sectional area (RNFLCRA) in a population-based setting. METHODS: The Central India Eye and Medical Study is a population-based study performed in a rural region of Central India. The study included 4711 subjects (aged 30 + years). A detailed ophthalmic and medical examination was performed. Inclusion criteria for this study were an intra-ocular pressure ≤21 mmHg, open anterior chamber angles and an axial length ≤26.5 mm. Neuroretinal rim area and RNFLCRA were measured by confocal laser scanning tomography. RESULTS: The study included 3959 subjects fulfilling the inclusion criteria. Larger neuroretinal rim area (mean: 1.63 ± 0.32 mm(2) ) was significantly (p = 0.004) associated with a higher body mass index after adjusting for larger disc area (p < 0.001), younger age (p < 0.001), lower intraocular pressure (p < 0.001), and hyperopic refractive error (p < 0.001). Larger retinal nerve fibre layer cross-sectional area (mean:1.29 ± 0.39 mm(2) ) was significantly associated with higher body mass index (p = 0.001) after adjusting for younger age (p < 0.001), shorter axial length (p < 0.001), larger optic disc area (p < 0.001), taller body height (p < 0.001) and male gender (p = 0.04). CONCLUSIONS: In a rural Central Indian population, neuroretinal rim area and RNFLCRA as surrogates of the optic nerve fibres were related to a higher body mass index. Because body mass index is associated with cerebrospinal fluid pressure, the higher cerebrospinal fluid pressure may be associated with a larger neuroretinal rim area. It may vaguely point towards an association between cerebrospinal fluid pressure and glaucomatous optic neuropathy.

Cysticercosis: hooked by a hooklet on fine needle aspiration cytology-a case report.

Cysticercosis is a systemic parasitic disease caused by the larval form of cestode T. solium. It has a worldwide distribution and is potentially harmful with variable clinical manifestations. The patient most commonly presents with subcutaneous and muscle involvement in the form of nodular lesions. The other most commonly involved sites include eye, brain, bladder wall, and heart. Cysticercosis can be diagnosed on serology, and radiologically but confirmatory diagnosis is based on histopathological examination of the involved tissue biopsy specimen. Fine needle aspiration cytology is a useful low-cost outpatient procedure tool for preoperative diagnosis of cysticercosis and is absolutely essential for diagnosis of the parasitic lesions in a peripheral hospital, one like ours.

Pseudoexfoliation: normative data and associations. The Central India Eye and Medical Study.

PURPOSE: To assess the prevalence of pseudoexfoliation (PEX) and its associations in a population-based setting. DESIGN: Population-based, cross-sectional study. METHODS: The Central India Eye and Medical Study included 4711 individuals. All study participants underwent a detailed ophthalmological examination. After medical pupil dilation, PEX was assessed by an experienced ophthalmologist using slit-lamp based biomicroscopy. RESULTS: Slit lamp examination results were available for 4646 (98.6%) study participants with a mean age of 49.3 ± 13.3 years (range: 30-100 years). PEX was detected in 87 eyes (prevalence: 0.95 ± 0.10% (95%CI: 0.75, 1.15) of 69 subjects (prevalence: 1.49 ± 0.18% (95%CI: 1.14, 1.83). PEX prevalence increased significantly (P<0.001) from 0% in the age group of 30-39 years, to 2.85 ± 0.56% in the age group of 60-69 years, to 6.60 ± 1.21% in the age group of 70-79 years, and to 12.3 ± 4.11% in the age group of 80+ years. In multivariate analysis, PEX prevalence was associated with higher age (P<0.001; regression coefficient B:0.11; odds ratio (OR): 1.11 (95%CI: 1.09, 1.13)), lower body mass index (P = 0.001; B: -0.12; OR: 0.88 (95CI: 0.82, 0.95)) and higher diastolic blood pressure (P = 0.002; B: 0.02; OR: 1.03 (95%CI: 1.01, 1.04)). In the multivariate analysis, PEX was not associated with retinal nerve fiber layer cross section area (P = 0.76) and presence of open-angle glaucoma (P = 0.15). CONCLUSIONS: In a rural Central Indian population aged 30+ years, PEX prevalence (mean: 1.49 ± 0.18%) was significantly associated with older age, lower body mass index and higher diastolic blood pressure. It was not significantly associated with optic nerve head measurements, refractive error, any ocular biometric parameter, nuclear cataract, early age-related macular degeneration and retinal vein occlusion, diabetes mellitus, smoking, and dyslipidemia.