Molecular-based study of scrub typhus in Kerala, South India from 2014 to 2021: a laboratory-based study.

Scrub typhus (ST) is a neglected acute, febrile, infectious disease caused by the intracellular parasite Orientia tsutsugamushi, a gram-negative coccobacillus of the family Rickettsiaceae. Early and precise diagnosis is crucial to reduce the risk of developing disease complications. However, IgM antibody enzyme-linked immunosorbent assay (IgM ELISA) and indirect immunofluorescence assay (IFA) remain essential for diagnosis. However, it could be more helpful for early diagnosis due to the need for uniformity of approach in the diagnostic accuracy studies to determine appropriate ELISA cut-offs for various geographic locations. Hence, we aim to study the O. tsutsugamushi type-specific 56 kilodalton (kDa) protein gene using nested PCR (nPCR) and DNA sequence analysis as a molecular marker for early diagnosis. Out of 10,439 suspected cases, 1147/10,439 (11%) patients were positive for IgM ELISA. A total of 1044/10,439 (10%) samples were randomly tested after nPCR and compared with IgM ELISA results and DNA sequence analysis. Using nested PCR and IgM ELISA methods, 13% (134/1044) and 12% (125/1044) of the samples were positive, respectively. The serology method could not replicate the substantial number of positive cases demonstrated by nPCR; therefore, significant mutual exclusivity of the two techniques requires further investigation. Furthermore, our phylogenetic analysis revealed a clustering of isolates with Karp-related strains, providing insight into the transmission dynamics. Therefore, molecular diagnostic methods may aid in the early diagnosis of infection and enable prompt treatment of ST in endemic regions. Our results show that IgM ELISA can provide complete diagnostic advantages in conjunction with nPCR and can be an essential tool for accurate diagnosis. In addition, the DNA sequencing analysis of the samples showed that Karp-related strains were the main strains. Furthermore, research with samples from various regions in combination with the entire genome sequencing of O. tsutsugamushi is required to understand the infection mechanism better and develop robust early detection methods. Supplementary Information: The online version contains supplementary material available at 10.1007/s00580-023-03443-8.

Comparative performance of CDC-modified SARS-CoV-2 real-time PCR assay with four different commercial assays: laboratory-based study.

The coronavirus infectious disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses. The pandemic has emerged as a global public health crisis, and the threat of fast-spreading of the latest variants of the coronavirus (such as omicron, delta) is rampant. Therefore, a fast and reliable diagnostic assay is needed to make the clinical decision for further treatment. The study aims to develop a Centers for Disease and Prevention (CDC)-modified qualitative real-time reverse transcriptase PCR (RT-qPCR) assay and parallel assessment of commercially available RT-qPCR assay (Altona, Seegene, BD, and GBC) to detect SARS-CoV-2. Two hundred nine samples were chosen randomly out of around two hundred thousand samples. The panel consisted of SARS-CoV-2-positive (n = 156) and SARS-CoV-2-negative (n = 52) nasopharyngeal swab specimens for a primary clinical evaluation. Furthermore, 29 positive samples were sequenced using Oxford Nanopore Minion technology. Two hundred nine patient sample data of the cycle threshold (Ct) readings for target genes of five assays are 100% sensitive for Ct values. Mean Ct values for N1, N2, RdRp, S, and E of the positive controls in CDC assay, RealStar®, Allplex, GBC, and SD Biosensor were 17.5 ± 0.49, 16.9 ± 0.51, 20 ± 0.49, 21.7 ± 0.38, and 23.1 ± 0.43, respectively. F test value shows ≥ 1, which was statistically significant. All assays showed an efficiency of < 120% and R squares were < 0.99, which is well above the required threshold value. Thus, when taking the CDC-modified assay as a gold standard, the other four assays demonstrated a p value of 0.0000, concordance at 100%, and a Kappa at 1.000. A maximum-likelihood (ML) tree was constructed and compared based on full-length SARS-CoV-2 with Wuhan isolate. These isolates are closely related to the B.1.617 lineage and reference sequences. Therefore, we conclude that all RT-PCR kits assessed in this study shall be used for routine diagnostics of COVID-19 in patients. Supplementary information: The online version contains supplementary material available at 10.1007/s00580-022-03356-y.

Pharmacogenetics Based Dose Prediction Model for Initial Tacrolimus Dosing in Renal Transplant Recipients.

Tacrolimus, an immunosuppressant used in solid organ transplantation, has a narrow therapeutic index and exhibits inter-individual pharmacokinetic variability. Achieving and maintaining a therapeutic level of the drug by giving appropriate doses is crucial for successful immunosuppression, especially during the initial post-transplant period. We studied the effect of CYP3A5, CYP3A4, and ABCB1 gene polymorphisms on tacrolimus trough concentrations in South Indian renal transplant recipients from Kerala to formulate a genotype-based dosing equation to calculate the required starting daily dose of tacrolimus to be given to each patient to attain optimal initial post-transplant period drug level. We also investigated the effect of these genes on drug-induced adverse effects and rejection episodes and looked into the global distribution of allele frequencies of these polymorphisms. One hundred forty-five renal transplant recipients on a triple immunosuppressive regimen of tacrolimus, mycophenolate mofetil, and steroid were included in this study. Clinical data including tacrolimus daily doses, trough levels (C0) and dose-adjusted tacrolimus trough concentration (C0/D) in blood at three time points (day 6, 6 months, and 1-year post-transplantation), adverse drug effects, rejection episodes, serum creatinine levels, etc., were recorded. The patients were genotyped for CYP3A5*3, CYP3A4*1B, CYP3A4*1G, ABCB1 G2677T, and ABCB1 C3435T polymorphisms by the PCR-RFLP method. We found that CYP3A5*3 polymorphism was the single most strongly associated factor determining the tacrolimus C0/D in blood at all three time points (p < 0.001). Using multiple linear regression, we formulated a simple and easy to compute equation that will help the clinician calculate the starting tacrolimus dose per kg body weight to be administered to a patient to attain optimal initial post-transplant period tacrolimus level. CYP3A5 expressors had an increased chance of rejection than non-expressors (p = 0.028), while non-expressors had an increased risk for new-onset diabetes mellitus after transplantation (NODAT) than expressors (p = 0.018). Genotype-guided initial tacrolimus dosing would help transplant recipients achieve optimal initial post-transplant period tacrolimus levels and thus prevent the adverse effects due to overdose and rejection due to inadequate dose. We observed inter-population differences in allele frequencies of drug metabolizer and transporter genes, emphasizing the importance of formulating population-specific dose prediction models to draw results of clinical relevance.

Shear Stress Alterations Activate BMP4/pSMAD5 Signaling and Induce Endothelial Mesenchymal Transition in Varicose Veins.

Chronic venous diseases, including varicose veins, are characterized by hemodynamic disturbances due to valve defects, venous insufficiency, and orthostatism. Veins are physiologically low shear stress systems, and how altered hemodynamics drives focal endothelial dysfunction and causes venous remodeling is unknown. Here we demonstrate the occurrence of endothelial to mesenchymal transition (EndMT) in human varicose veins. Moreover, the BMP4-pSMAD5 pathway was robustly upregulated in varicose veins. In vitro flow-based assays using human vein, endothelial cells cultured in microfluidic chambers show that even minimal disturbances in shear stress as may occur in early stages of venous insufficiency induce BMP4-pSMAD5-based phenotype switching. Furthermore, low shear stress at uniform laminar pattern does not induce EndMT in venous endothelial cells. Targeting the BMP4-pSMAD5 pathway with small molecule inhibitor LDN193189 reduced SNAI1/2 expression in venous endothelial cells exposed to disturbed flow. TGFß inhibitor SB505124 was less efficient in inhibiting EndMT in venous endothelial cells exposed to disturbed flow. We conclude that disturbed shear stress, even in the absence of any oscillatory flow, induces EndMT in varicose veins via activation of BMP4/pSMAD5-SNAI1/2 signaling. The present findings serve as a rationale for the possible use of small molecular mechanotherapeutics in the management of varicose veins.

Molecular-genetic characterization of human parvovirus B19 prevalent in Kerala State, India.

BACKGROUND: Human parvovirus B19V is a DNA virus, and a member of the family Parvoviridae, that causes various clinical manifestations, from asymptomatic to persistent infection that is associated with different autoimmune diseases. The parvovirus B19 evolves with a very high mutation rate that is closer to those of existing RNA viruses. Globally circulating B19V is currently classified into three genotypes, but their distribution is not spatially and temporally correlated. Except for a few recent reports on B19V entry into the human host and its genetic diversity, there is a lack of sufficient studies on this virus from distinct geographical locations and no clear understanding of its evolution has been documented. METHODS: To better understand the evolution of the Human parvo B19V virus from India's southern part, a geographically distinct location with no reports of B19V genomes, we have screened for B19V in 456 suspected cases using VP1/2 surface marker genes, and its characteristics were studied in detail. Amongst 456 clinically suspected B19V samples, 7.2% (33/456) were found positive by nested PCR (nPCR) were subsequently validated by real-time PCR, Sanger sequencing, and metagenome analysis. RESULTS: Human parvovirus B19 infection was shown among 33 of 456 patients when tested by nPCR; 30 among these were also positive by qPCR and were subsequently confirmed by sequencing 75% nPCR positive samples and 76% qPCR positive samples were from patients with age. ≥ 50 years respectively (Additional file 1: Table S1). The complete VP1/2 gene assembly from the South Indian strain showed three novel mutations (T122A, V128I, I283V), which might significantly impact the stability and virulence of the B19V virus circulating in this part of the world. These mutations might be crucial for its adaptive evolutionary strategies facilitating the spread and infectivity potential of the virus. In maximum likelihood phylogeny of VP1/2 sequences, the South Indian B19V strain forms a separate clade closer to the existing genotype two strains circulating worldwide. CONCLUSION: Our study contributes to a better understanding of the human parvovirus's genetic and evolutionary characteristics in South India. Also, it highlights the possibility that a positive selection pressure acting on VP1/2 could increase the survival and replication capabilities of the viruses.

Complete Genome Analysis of Influenza A(H1N1) Viruses Isolated in Kerala, India.

Here, we report the complete sequence of four influenza A(H1N1) virus samples isolated from cases that occurred during the 2017 epidemic season in Kerala in South India. Sequence analysis showed mutations that differentiate this strain from the reference strain A/California/07/2009 virus.

Evolutionary, genetic, structural characterization and its functional implications for the influenza A (H1N1) infection outbreak in India from 2009 to 2017.

Influenza A (H1N1) continues to be a major public health threat due to possible emergence of a more virulent H1N1 strain resulting from dynamic changes in virus adaptability consequent to functional mutations and antigenic drift in the hemagglutinin (HA) and neuraminidase (NA) surface proteins. In this study, we describe the genetic and evolutionary characteristics of H1N1 strains that circulated in India over a period of nine years from 2009 to 2017 in relation to global strains. The finding is important from a global perspective since previous phylogenetic studies have suggested that the tropics contributed substantially to the global circulation of influenza viruses. Bayesian phylogenic analysis of HA sequences along with global strains indicated that there is a temporal pattern of H1N1 evolution and clustering of Indian isolates with globally circulating strains. Interestingly, we observed four new amino acid substitutions (S179N, I233T, S181T and I312V) in the HA sequence of H1N1 strains isolated during 2017 and two (S181T and I312V) were found to be unique in Indian isolates. Structurally these two unique mutations could lead to altered glycan specificity of the HA gene. Similarly, sequence and structural analysis of NA domain revealed that the presence of K432E mutation in H1N1 strains isolated after 2015 from India and in global strains found to induce a major loop shift in the vicinity of the catalytic site. The findings presented here offer an insight as to how these acquired mutations could be associated to an improved adaptability of the virus for efficient human transmissibility.

Deciphering foot-and-mouth disease (FMD) virus-host tropism.

The pattern of interactions between foot and mouth disease (FMD) viral protein 1 (VP1) with susceptible and resistant host integrins were deciphered. The putative effect of site-directed mutation on alteration of interaction is illustrated using predicted and validated 3D structures of VP1, mutated VP1 and integrins of Bos taurus, Gallus and Canis. Strong interactions were observed between FMDV-VP1 protein motifs at conserved tripeptide, Arg-Gly-Asp 143RGD145 and at domain 676SIPLQ680 in alpha-integrin of B. taurus. Notably, in-silico site-directed mutation in FMDV-VP1 protein led to complete loss of interaction between FMD-VP1 protein and B. taurus integrin, which confirmed the active role of arginine-glycine-aspartic acid (RGD) domain. Interestingly, in-vitro analysis demonstrates the persistence of the putative tropism site 'SIPLQ' in different cattle breeds undertaken. Thus, the attempt to decipher the tropism of FMDV at host receptor level interaction might be useful for future FMD control strategies through development of mimetic marker vaccines and/or host receptor manipulations. Communicated by Ramaswamy H. Sarma.

Comparative Study of Molecular Approaches for the Detection of Influenza Virus from Patient Samples Using Real-time PCR: Prospective Disease Burden Study in Kerala (India) from 2010 to 2016.

PURPOSE OF REVIEW: Acute respiratory infections caused by influenza virus are a major cause of viral respiratory diseases globally. Surveillance of circulating subtypes and estimation of disease burden is of utmost clinical importance. Molecular surveillance and proper disease burden estimates are scarce in India although clinical influenza infections are on the rise. Our study aims to delineate the prevalent influenza subtypes in a South Indian population from cases requiring hospital visits. Using real-time polymerase chain reaction (RT-PCR), 2154 throat/nasopharyngeal swabs from patients attending Government Medical College, Thiruvananthapuram, Kerala, India, with suspected influenza-like illness, were tested for the presence of different influenza subtypes. RESEARCH FINDINGS: Forty-three percent of specimens were positive for the influenza virus. Among these, prevalence of influenza A(H3N2), influenza B, and H1N1pdm09 was 26.7, 6.3, and 10%, respectively. Nominal co-infections were detected. An easy to use commercial kit was used for the majority of the study after proper evaluation for sensitivity and specificity against a gold standard protocol. Specific diagnosis using molecular tools caters to the urgency, and a precise measure of the disease burden and management actions are needed, especially in developing countries like India. Infection rate estimation using a sensitive RT-PCR assay signified that influenza was highly prevalent in the region. The study data generated will help understand the epidemiology of influenza in India as well as generate information for global influenza surveillance and disease burden.

Whole-Genome Sequences of Influenza A(H1N1)pdm09 Virus Isolates from Kerala, India.

We report here the whole-genome sequence of six clinical isolates of influenza A(H1N1)pdm09, isolated from Kerala, India. Amino acid analysis of all gene segments from the A(H1N1)pdm09 isolates obtained in 2014 and 2015 identified several new mutations compared to the 2009 A(H1N1) pandemic strain.

Development of a multiplex RT-PCR for simultaneous diagnosis of human metapneumovirus (HMPV) and human respiratory syncytial virus (HRSV) from clinical specimens.

Human metapneumovirus (HMPV) and human respiratory syncytial virus (HRSV) are ubiquitous respiratory viral pathogens. They belong to the family Paramyxoviridae (subfamily Pneumovirinae) and is responsible for acute respiratory tract infections in children, elderly and immunocompromised patients. We designed and tested a multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) as a cost-effective alternative to real-time PCR and cell culture based detection for HMPV and HRSV. The newly developed PCR was used to screen nasal/throat swab samples from 356 patients with suspected acute respiratory infection attending the Government Medical College, Thiruvananthapuram, Kerala, India. The method was compared with a commercially available kit employing real time PCR, for its sensitivity and specificity. 53 (14.9 %) samples were positive for at least one tested pathogen by mRT-PCR. All except one among the positive samples showed similar pathogen profile when tested using real time PCR. 8 (15.1 %) among these 53 were positive for HRSVA, 33 (62.3 %) positive for HRSVB and 12 (22.6 %) were positive for HMPV. 17 (32.7 %) samples showed co-infections in them. Sensitivity and specificity of the mRT-PCR was comparable to that of the commercial kit. Our findings indicate that this newly developed mRT-PCR can be used as a cost-effective alternative for laboratory diagnosis of HMPV/HRSV infection and will significantly reduce diagnostic costs for these viruses in clinical settings.

Correlation of phylogenetic clade diversification and in vitro infectivity differences among Cosmopolitan genotype strains of Chikungunya virus.

Cosmopolitan genotypes of Chikungunya virus caused the large-scale febrile disease outbreaks in the last decade in Asian and African continents. Molecular analyses of these strains had revealed significant genetic diversification and occurrence of novel mosquito-adaptive mutations. In the present study we looked into whether the genetic diversification has implications in the infectivity phenotype. A detailed sequence and phylogenetic analyses of these virus strains of Indian Ocean lineage from Kerala, South India from the years 2008 to 2013 identified three distinct genetic clades (I, II and III), which had presence of clade-specific amino acid changes. The E2 envelope protein of the strains from the years 2012 to 2013 had a K252Q or a novel K252H change. This site is reported to affect mosquito cell infectivity. Most of these strains also had the E2 G82R mutation, a mutation previously identified to increase mammalian cell infectivity, and a novel mutation E2 N72S. Positive selection was identified in four sites in the envelope proteins (E1 K211E, A226V and V291I; E2 K252Q/H). In infectivity analysis, we found that strains from clade III had enhanced cytopathogenicity in HEK293 and Vero cells than by strains representing other two clades. These two strains formed smaller sized plaques and had distinctly higher viral protein expression, infectious virus production and apoptosis induction in HEK293 cells. They had novel mutations R171Q in the nsP1; I539S in nsP2; N409T in nsP3; and N72S in E2. Our study identifies a correlation between phylogenetic clade diversification and differences in mammalian cell infectivity phenotype among Cosmopolitan genotype CHIKV strains.

Interdisciplinary Approach to a Tooth with Open Apex and Persistent Sinus.

Traumatic injuries in childhood may disrupt root development leading to a tooth with open apex. Apexification procedures in such cases aim at root end closure after reasonable period of time. In some chronic cases, complete healing of the periapical area does not occur resulting in development of a nonhealing sinus. Failure of nonsurgical approach in such cases needs surgical intervention permitting thorough periapical curettage. In the present case, apexification procedure with MTA achieved root end closure but failed to heal the sinus for which surgical treatment was completed with thorough periapical curettage and application of platelet rich fibrin (PRF) and a combination of ß-tricalcium phosphate and hydroxyapatite resulted in healing.

Dengue vaccine development: strategies and challenges.

Infection with dengue virus may result in dengue fever or a more severe outcome, such as dengue hemorrhagic syndrome/shock. Dengue virus infection poses a threat to endemic regions for four reasons: the presence of four serotypes, each with the ability to cause a similar disease outcome, including fatality; difficulties related to vector control; the lack of specific treatment; and the nonavailability of a suitable vaccine. Vaccine development is considered challenging due to the severity of the disease observed in individuals who have acquired dengue-specific immunity, either passively or actively. Therefore, the presence of vaccine-induced immunity against a particular serotype may prime an individual to severe disease on exposure to dengue virus. Vaccine development strategies include live attenuated vaccines, chimeric, DNA-based, subunit, and inactivated vaccines. Each of the candidates is in various stages of preclinical and clinical development. Issues pertaining to selection pressures, viral interaction, and safety still need to be evaluated in order to induce a complete protective immune response against all four serotypes. This review highlights the various strategies that have been employed in vaccine development, and identifies the obstacles to producing a safe and effective vaccine.

Comparative performance of the probable case definitions of dengue by WHO (2009) and the WHO-SEAR expert group (2011).

BACKGROUND: The primary objective of this study was to find the performance of the 2009 probable case definition of dengue and compare it with the definition given by the WHO-SEAR expert group in 2011. METHODS: A cross-sectional study was conducted in Thiruvananthapuram district of Kerala, which is hyperendemic for dengue. A consecutive series of 851 participants defined by the selection criteria were recruited from the primary, secondary, and tertiary health care settings. Sensitivity, specificity, predictive values, and likelihood ratios of the clinical case definitions were calculated using reverse transcriptase-polymerized chain reaction (RT-PCR) as gold standard in case of fever less than or equal to 5 days and serology (IgM positivity) for fever >5 days. Diagnostic odds ratio (DOR) was also calculated as a single indicator of performance of the case definition. RESULTS: The 2009 World Health Organization (WHO) case definition had a sensitivity of 76·4% (69·6-82·1) and negative predictive value of 87·5%. The 2011 WHO-SEAR expert group case definition had a higher sensitivity of 87·9% (82·2-91·9) but lower negative predictive value of 86·6%. The three independent criteria which were significantly associated with dengue were thrombocytopenia less than 150,000 (OR 2·80), leukopenia (OR 2·28), and absence of backache (OR 2·68). The performance of 2009 case definition was better (DOR 2·4) than the 2011 WHO-SEAR expert group case definition. This was further enhanced when thrombocytopenia was specified as platelet count less than 150,000 (DOR2·7). When 'no backahe' was added as an additional criteria, the performance of both definitions improved. CONCLUSIONS: The 2009 WHO case definition has better discriminatory power than the 2011 WHO-SEAR expert group case definition. The performance of 2009 WHO case definition is enhanced by specifying thrombocytopenia as platelet count less than 150,000. The inclusion of 'no backache' further improves the discriminatory power. This may be more useful in primary care settings, to rule out dengue.

Forkhead box C2 promoter variant c.-512C>T is associated with increased susceptibility to chronic venous diseases.

Chronic venous disease (CVD) is one of the most prevalent yet underrated disorders worldwide. High heritability estimates of CVD indicate prominent genetic components in its etiology and pathology. Mutations in human forkhead box C2 (FoxC2) gene are strongly associated with valve failure in saphenous and deep veins of lower extremities. We explored the association of genetic variants of FoxC2 as well as FoxC2 mRNA and protein expression levels with CVD of lower limbs. We systematically sequenced the single coding exon, 5' and 3' flanking regions of FoxC2 gene in 754 study subjects which includes 382 patients with CVD and 372 healthy subjects. Four novel and three reported polymorphisms were identified in our cohort. Three variants in 5' flanking region and one in 3' flanking region of FoxC2 gene were significantly associated with CVD risk. FoxC2 mRNA in vein tissues from 22 patients was 4±1.42 fold increased compared to saphenous veins from 20 normal subjects (p<0.01). FoxC2 protein was also significantly upregulated in varicose veins compared to control samples. The c.-512C>T (rs34221221: C>T) variant which is located in the FoxC2 putative promoter region was further analyzed. Functional analysis of c.-512C>T revealed increased mRNA and protein expression in patients with homozygous TT genotype compared to heterozygous CT and wild CC genotypes. Luciferase assay indicated higher transcriptional activity of mutant compared to wild genotype of this variant. These findings suggested that c.-512C>T variant of FoxC2 was strongly associated with susceptibility to CVD and also that this variant resulted in FoxC2 overexpression. To obtain a mechanistic insight into the role of upregulated FoxC2 in varicosities, we overexpressed FoxC2 in venous endothelial cells and observed elevated expression of arterial markers Dll4 and Hey2 and downregulation of venous marker COUP-TFII. Our study indicates altered FoxC2-Notch signaling in saphenous vein wall remodeling in patients with varicose veins.

ECG discrimination between right and left circumflex coronary arterial occlusion in patients with acute inferior myocardial infarction: value of old criteria and use of lead aVR.

STUDY OBJECTIVES: Prior studies have proposed several ECG criteria for identifying the culprit artery in patients with acute inferior myocardial infarction (MI). We applied each criterion to our patients to assess its utility. In doing so, we discovered a previously unreported, but highly useful, criterion utilizing lead aVR. STUDY DESIGN: Retrospective review. PATIENTS: Thirty consecutive patients with symptoms of acute MI, ST-segment elevation in the inferior ECG leads, an appropriate rise and fall of creatine kinase and troponin I levels, and coronary arteriography within 7 days of the onset of symptoms. MEASUREMENTS: The ECG recorded within 24 h of the onset of symptoms that had the most prominent ST-segment changes was analyzed. In the 12 standard leads and in lead V(4)R, ST-segment elevation or depression was measured 0.06 s after the J point. RESULTS: Four previously described criteria were useful in identifying the right coronary artery (RCA) or the left circumflex coronary artery (LCX) as the culprit: ST-segment elevation in lead I, ST-segment more or less elevated in lead II than in lead III, ST-segment elevation >or= 0.5 mm in lead V(4)R, and various combinations of ST-segment elevation or depression in leads V(1) and V(2). A new criterion was found to be at least as useful as any previously described: the presence and amount of ST-segment depression in lead aVR. CONCLUSIONS: At least five different ST-segment criteria help to identify the RCA or the LCX as the culprit artery in patients with acute inferior MI. One of these, the amount of ST-segment depression in lead aVR, has not been reported previously and needs validation in a larger study.