Clinical, laboratory profile and molecular characterization of Orientia tsutsugamushi among fatal scrub typhus patients from Karnataka, India.

BACKGROUND: Scrub typhus is a vector-borne infection caused by the obligate intracellular organism Orientia tsutsugamushi. In some cases, scrub typhus can result in severe complications, multiorgan failure and death. OBJECTIVE: To study the clinical and laboratory profiles of patients who succumbed to scrub typhus. METHODS: A prospective cohort study was conducted from August 2019 through April 2023 on scrub typhus patients admitted to our hospital. Clinical and laboratory parameters of all the patients were recorded, and blood samples were drawn. To confirm scrub typhus, a nested polymerase chain reaction (nPCR) was performed in collected samples. Viable amplicons were sequenced, and phylogenetic analyses were performed to identify infecting genotypes. RESULTS: A total of 261 patients were enrolled. Of these, nine (3.45%) patients succumbed at a median (Interquartile Range) duration of 5 (1.5, 10.5) days after admission. Sepsis with septic shock (9, 100%) and acute kidney injury (AKI) (6, 66%) were noted among the succumbed patients. All the succumbed patients (100%) required intensive care admission, inotropic and ventilatory support. While 5 (55%) patients required dialysis, two (22%) required blood transfusion. Three (33%) patient samples were co-positive for Leptospira IgM, and four (44%) patients had superinfection with Candida tropicalis, multi-drug-resistant (MDR) E. Coli sepsis, pan drug-resistant (PDR) Acinetobacter Baumanii, and Klebsiella pneumoniae. Phylogenetic analysis revealed Orientia tsutsugamushi Japanese Gilliam-variant (JG-v) like (50%), Karp-like (37.5%), and Japanese Gilliam (JG) like (12.5%) strains among succumbed patients. CONCLUSION: Delay in scrub typhus diagnosis can result in severe complications, septic shock, and multisystem organ failure, culminating in death.

Clinical, laboratory, and molecular epidemiology of Orientia tsutsugamushi infection from Southwestern India.

Scrub typhus is a vector borne disease which in a proportion of patients causes multiorgan involvement and death if untreated. Infecting genotype and virulence factors play a role in severity of infection and outcome. The current prospective cohort study was undertaken to elucidate the severity of illness in scrub typhus patients and to identify the circulating genotypes in Karnataka, India. A total of 214 patients of either gender from 9 districts of Karnataka and one patient each from Andhra Pradesh and Kerala, India were enrolled in the study. With a predefined severity criterion, 132 patients were segregated to the severe group. Multi organ involvement was seen in 59 (44.69%) patients. Phylogenetic analysis revealed JG-v like (48.97%), Karp-like (26.53%), JG-like (22.44%), and Kato-like (2.04%) strains in Karnataka. Patients infected with Orientia tsutsugamushi Karp-like strains had respiratory involvement (69.2%), cardiovascular involvement (46.2%) and thrombocytopenia (23.1%) and required higher hospital resource utilization.

Association of MTHFR gene polymorphism C677T (rs1801133) studies with early primary knee osteoarthritis in a South Indian population: a hospital-based study.

Osteoarthritis (OA) is the most commonly occurring disease of middle and elderly population, which is characterized by focal loss of joint articular cartilage, osteophyte formation and sub chondral bone remodeling. Classical risk factors of OA include age, gender, weight, joint injury, trauma, however hereditary component is one of the main crucial factors. Several genome wide association studies and candidate gene approaches have identified genetic variants involved in the influence and association of OA. In the current study influence of Methylene tetra hydro folate reductase MTHFR C677T (rs1801133) gene with early primary knee OA was evaluated. In this study 400 samples were included (200 cases & 200 controls). DNA was extracted & processed for PCR- RFLP evaluation and genotype analysis. Statistical analysis was performed & results indicated a lack of association between MTHFR gene polymorphism and early primary KOA. The stratification was done based on age & gender and also both. Individual's i.e females below the age of 40 years are more prone to the disease when compared with males. MTHFR gene polymorphism showed a lack of association with early primary knee osteoarthritis. To the best of our knowledge this is the first study from south India.

Utility of next-generation sequencing in genetic testing and counseling of disorders involving the musculoskeletal system-trends observed from a single genetic unit.

BACKGROUND: Disorders involving the musculoskeletal system are often identified with short stature and a range of orthopedic problems. The clinical and genetic heterogeneity of these diseases along with several characteristic overlaps makes definitive diagnosis difficult for clinicians. Hence, using molecular testing in addition to conventional tests becomes essential for appropriate diagnosis and management. METHODS: Comprehensive clinical examination, detailed pretest and posttest counseling, molecular diagnosis with next-generation sequencing (NGS), genotype-phenotype correlation and Sanger sequencing for targeted variant analysis. RESULTS: This manuscript reports a molecular spectrum of variants in 34 orthopedic cases referred to a single genetic unit attached to a tertiary care hospital. The diagnostic yield of NGS-based tests coupled with genetic counseling and segregation analysis was 79% which included 7 novel variants. In about 53% (i.e. 18/34 cases), molecular testing outcome was actionable since 8 of the 18 underwent prenatal diagnosis, as they were either in their early gestation or had planned a pregnancy subsequent to molecular testing, while ten cases were premaritally/prenatally counseled for the families to take informed decisions as they were in the reproductive age. CONCLUSIONS: The report highlights the importance of NGS-based tests even in a low resource setting as it helps patients, families and healthcare providers in reducing the economic, social and emotional burden of these disorders.

Chromosomal Abnormalities in Couples with Primary and Secondary Infertility: Genetic Counseling for Assisted Reproductive Techniques (ART).

BACKGROUND: World Health Organization estimates that 60-80 million couple worldwide currently suffer from infertility. Recurrent pregnancy loss (RPL) is also another major concern. Chromosomal rearrangements play a crucial role in primary and secondary infertility and RPL. Underlying genetic abnormalities like chromosomal abnormalities contribute to 5-10% of the reproductive failures. The aim of the study was to evaluate the chromosomal abnormalities in infertility and RPL cases to help obstetrician/fertility experts to carry out risk assessment and provide appropriate assisted reproductive techniques for better management of the problem. METHODS: Karyotyping was performed for 414 cases with the history of infertility and RPL over a period of one year. Samples were processed according to procedures of AGT cytogenetic laboratory manual. RESULTS: Chromosomal abnormalities were observed in 15% of cases. Robertsonian translocation, reciprocal translocation, inversion, derivatives, marker chromosomes, mosaics, aneuploidy and polymorphic variants each contributed 2%, 3%, 3%, 13%, 2%, 10%, 6% and 61%, respectively. CONCLUSION: Evaluation of chromosomal abnormalities in couple is warranted prior to planning pregnancy especially for assisted reproductive management cases. Chromosomal analysis can be used as one of the diagnostic tools by OBG/IVF specialists in association with geneticist/genetic counselor for proper reproductive counseling and management.

Robertsonian and Balanced Reciprocal Translocation in Both Child and Mother with a History of Recurrent Abortions.

BACKGROUND: Similar rare Robertsonian and balanced reciprocal translocation in both child and mother with a history of multiple miscarriages in the first trimester was the motive to write this case report. Cytogenetic analysis helps in genetic counselling of infertility, BOH and dysmorphology which in turn helps in pre implantation genetic testing. Although many case reports have already been published about Robertsonian and balanced translocations, this is the first case report in India which showed both types of translocation in the same patient, rob (13;14) and t (4;7). Interestingly, in the same patient, same translocations were also identified in the mother and father having no chromosomal abnormalities. CASE PRESENTATION: Proband with dysmorphology was refered first for karyotyping and later parental karyotyping was performed. CONCLUSION: Cytogenetic analysis plays an important role in the diagnosis and management of disease along with prenatal screening.

Role of SREBP2 gene polymorphism on knee osteoarthritis in the South Indian Hyderabad Population: A hospital based study with G595C variant.

INTRODUCTION: Osteoarthritis (OA) is a multifactorial disease with genetic factors playing a crucial role, and it has been associated with a family history of obesity. G595C polymorphism in the sterol regulatory element-binding protein 2 (SREBP2) gene has demonstrated an association with knee osteoarthritis (KOA) patients. However, this polymorphism has been never explored in an Indian population. Hence, the current study aimed to examine whether G595C (rs2228314) polymorphism in SREBP2 gene was associated with KOA susceptibility in the South Indian Hyderabad population. METHODS: G595C polymorphism was genotyped with 200 KOA cases and 200 healthy controls using polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: A significant association was observed between age, body mass index (BMI), and family histories in KOA cases and controls (p < 0.05). The current allele (C vs G; OR-2.8 [95%CI = 2.1-3.7]; p < 0.0001) and genotype analysis confirms the significant association with (GC + CC vs GG; OR-3.5 [95%CI = 2.3-5.3]; p < 0.0001 & GC vs GG + CC; OR-1.7 [95%CI = 1.0-2.9]; p = 0.02) KOA vs. control subjects. On stratification analysis, genotype CC and C allele were associated with KOA. Gender association failed to demonstrate positive genotype frequencies (p > 0.05). Multifactor-dimensionality reduction (MDR) analysis showed a positive association with BMI and G595C genotypes (p < 0.05); 51% of the homozygous variant CC genotypes were present in obesity subjects. CONCLUSION: In conclusion, our findings suggest that G595C polymorphism in SREBP2 gene is associated with KOA in the South Indian Hyderabad population and presents scope for further investigation of the gene's function in KOA.

Amplification of specific chromosomal regions assessed by fluorescent in situ hybridization on Pap smears to be added as screening tool for identifying women at risk of progressing to cervical cancer.

Cervical carcinoma is a frequent malignancy in developing countries despite being a preventable disease. For the first time, four screening tests were used simultaneously for identifying women with a risk of developing cervical cancer, to help clinicians and policy makers to implement the best strategy for reducing the burden of this disease. Women visiting a hospital in India were enrolled after institutional ethics clearance and informed consent. Visual inspection using acetic acid and Pap smear tests were performed on 2683 women, and 104 had abnormal cytology: atypical squamous cells of undetermined significance (n = 29), low-grade squamous intraepithelial lesion (n = 41), high-grade squamous intraepithelial lesion (n = 17), and squamous cell carcinoma (n = 17). These and 96 samples, with normal cytology, were subjected to high-risk human papilloma virus testing and fluorescent in situ hybridization evaluation. Women with abnormal cytology were followed for 5 years and evaluated with colposcopy-guided biopsy. Three accepted methods of screening and one novel fluorescent in situ hybridization assay were carried out in 200 cases. Cutoffs for fluorescent in situ hybridization were established. The screening methods had 88%-96% negative predictive value, while positive predictive value was low (20%) for visual inspection using acetic acid, 47% for fluorescent in situ hybridization, 56% for high-risk human papilloma virus, and 73% for combined high-risk human papilloma virus and fluorescent in situ hybridization. Combined high-risk human papilloma virus and fluorescent in situ hybridization had 94% sensitivity, specificity, and negative predictive value, suggesting that simultaneous screening with these two tests is appropriate for identifying women progressing to cervical cancer and not visual inspection using acetic acid, which has low positive predictive value and Pap cytology which requires to be repeated. Policy makers and clinicians can assess feasibility of incorporating this screening strategy to prevent cervical cancer.

Pedigree and BRCA gene analysis in breast cancer patients to identify hereditary breast and ovarian cancer syndrome to prevent morbidity and mortality of disease in Indian population.

Global burden of breast cancer is expected to increase to >2 million new cases every year by 2030 and 10% of these are likely to have hereditary breast and ovarian cancer syndrome. Identifying these individuals by pedigree and BRCA1/2 mutation analyses will enable us to offer targeted mutation testing and appropriate counseling. This study from a tertiary care hospital showed that of the 127 breast cancer patients on treatment during 2014-2015, 24 of them fulfilled the criteria of hereditary breast and ovarian cancer syndrome after detailed verbal autopsy and pedigree analysis, and BRCA1 and 2 next-generation sequencing done after pre-test counseling revealed mutations in 13 cases (54%), these included 9 BRCA1 mutations (69%) and 4 BRCA2 mutation (31%). Subsequent post-test counseling recommended targeted mutation analysis for 64 high-risk members in these 13 families with pathogenic mutations, which will help in surveillance for early detection, appropriate management, and prevention of the disease by decreasing the burden to both family and nation. Results from this preliminary study highlight the importance of genetic counseling, pedigree analysis, and genetic testing. It can be recommended that all oncology units should have a genetic counseling service for providing appropriate support to oncologists, patients, and families to prevent unnecessary testing; however, breast cancer screening program is incomplete without evaluating for hereditary breast and ovarian cancer syndrome.

Type 2 Diabetes Mellitus and the Association of Candidate Genes in Asian Indian Population from Hyderabad, India.

INTRODUCTION: Genetic and environmental factors play an important role in susceptibility to type 2 diabetes mellitus (T2DM). Several genes have been implicated in the development of T2DM. Genetic variants of candidate genes are, therefore, prime targets for molecular analysis. AIM: In this study, we have selected 3 candidate genes, namely, TCF7L2, SLC30A8, and IGF2, for assessing their association with T2DM in an Indian population. MATERIALS AND METHODS: Five hundred individuals were enrolled in this case-control study- 250 T2DM patients and 250 healthy control individuals. Clinical characteristics were obtained for all subjects, and genotype analysis was performed by PCR-RFLP analysis. RESULTS: Allele and genotyping frequencies, odds ratios, and 95% confidence intervals were calculated for 3 single nucleotide polymorphisms (SNPs), 1 each from TCF7L2 (rs7903146), SLC30A8 (rs13266634), and IGF2 (rs680) in T2DM patients. The rs7903146 and rs680 polymorphisms were found to be significantly associated with T2DM (p < 0.05), whereas the rs13266634 polymorphism was not (p > 0.05). The multifactor dimensionality reduction method identified the particular polymorphisms associated with an increased risk of disease. CONCLUSION: The present study indicated that the gene-gene interaction model successfully predicted T2DM risk based on TCF7L2 and SLC30A8 polymorphisms. These results provide strong evidence of independent association between T2DM and the 3 SNPs analysed herein.

Degenerated intervertebral disc prolapse and its association of collagen I alpha 1 Spl gene polymorphism: A preliminary case control study of Indian population.

BACKGROUND: Degenerated disc disease (DDD) is a common disorder responsible for increased morbidity in a productive age group. Its etiology is multifactorial and genetic factors have been predominantly implicated. Disc prolapse results due to tear in the annulus, which is a fibrous structure composed largely of type I collagen. Functional polymorphism at the Sp1 site of the collagen I alpha 1 (COL1A1) gene has shown a positive association with DDD in Dutch and Greek populations. The purpose of this study was to assess COL1A1 Sp1 gene polymorphism in the Indian population. MATERIALS AND METHODS: Fifty clinically and radiologically proven patients with disc prolapse requiring surgery were included as cases and 50 healthy, age-matched volunteers served as controls. After isolating DNA from their blood sample, genotyping for COL1A1 polymorphism (rs1800012) was performed and identified as GG, GT, and TT. RESULTS: The mean age and body mass index in cases and controls were similar. 76% of the patients were males. The most common site of disc degeneration was L4-L5 (36%), followed by L5-S1 (34%). Homozygous-GG, heterozygous GT, and homozygous TT genotypes were seen in 38 (76%), 10 (20%) and 2 (4%) cases respectively, controls had similar percentage of genotypes as well. The alleles in cases and the control group showed no significant difference (P = 0.6744) and followed the Hardy-Weinberg Equilibrium in the study population. CONCLUSION: The COL1A1 (rs1800012) is in Hardy-Weinberg equilibrium in the present subset of Indian population. But taken as a single factor, it was not found to be associated with DDD in this preliminary study. Disc degeneration is multifactorial and also anticipated to be a result of multiple genes involvement and gene-gene interaction.

The insertion and deletion (I28005D) polymorphism of the angiotensin I converting enzyme gene is a risk factor for osteoarthritis in an Asian Indian population.

INTRODUCTION: Angiotensin I converting enzyme (ACE) insertion and deletion (I/D) polymorphism has been implicated in the pathogenesis of osteoarthritis (OA). In recent years, numerous genetic factors have been identified and implicated in OA. In this Asian Indian population-based study, we aimed to evaluate the relationship between ACE (I28005D) gene polymorphism and primary OA. We performed a case-control association study to identify and explore the correlation between clinically, radiologically diagnosed individuals with primary knee OA and the ACE I/D polymorphism. METHODS: Genomic DNA was isolated from 200 samples, including 100 OA cases and 100 healthy volunteers. DNA was amplified by polymerase chain reaction (PCR) using I and D allele-specific primers. PCR products were assessed via UV visualization of products electrophoresed on 2% agarose gels. RESULTS: The groups differed significantly in genotype distributions (p < 0.05). The primary knee OA group showed a considerably higher incidence of the DD genotype and the D allele compared to the control group (OR = 2.14, 95% CI: 1.10-4.15, p = 0.02 and OR = 2.08, 95% CI: 1.39-3.10, p = 0.0003). CONCLUSION: The ACE gene polymorphism I28005D was found to be associated with primary knee OA in Asian Indian populations. This is the first study in India to report that the ACE gene polymorphism is a risk factor for early onset primary knee OA.

Role of dynamic and mitochondrial mutations in neurodegenerative diseases with ataxia: lower repeats and LNAs at multiple loci as alternative pathogenesis.

Spinocerebellar ataxia is a growing group of hereditary neurodegenerative diseases for which ≥30 different genetic loci have been identified. In this study, we assessed the repeats at eight spinocerebellar ataxia (SCA) loci in 188 clinical SCA patients and 100 individuals without any neurological signs. Results from the present study were able to identify 16/188 (8.5%) clinical ataxia patients with repeat expansions in the pathological range of SCA genes, with the majority having expansion at the SCA1, 2, and 3 loci. The present study further evaluated two mitochondrial mutations associated with ataxia, i.e., T8993G and A8344G. Six patients were identified with A8344G mutation and none had the mutation in ATPase 6 gene; however, G8994A variation was found in three cases. Overall, three cases had triplet repeat expansions as well as mitochondrial (mt) mutations, which indicates potential association of triplet repeat expansions and mitochondrial mutations. Both the molecular analysis of several SCA loci and two relevant mt mutations indicated that the majority of ataxia cases were still undiagnosed; hence, the following hypotheses were proposed and tested based on available data: (i) lower repeats than normal range and (ii) large normal alleles (LNAs) at multiple loci may be an alternative basis for disease pathogenesis.