Objective: To evaluate the value of next-generation sequencing (NGS) in the prevention and management of thalassemia. Methods: A systematic search was performed in eight databases including China Biomedical Literature Database, Chinese National Knowledge Infrastructure, Chinese Scientific Journals Database, Wanfang database, PubMed, EMBASE, Web of Science, and Cochrane Library from the inception to 1 June 2022. Stata 17.0 and Review Manager 5.4 were used for the meta-analysis. Results: Nine studies containing 14 794 participants were included in the meta-analysis. Compared with the routine genetic testing (including Gap-PCR and reverse dot blot), NGS had higher detection rates in screening thalassemia (RR 1.22, 95% CI 1.13-1.31, P<0.01), particularly for the α-thalassaemia mutation carriers (RR 1.24, 95% CI 1.07-1.44, P<0.01). However, no significant difference was found in the screening of β-thalassemia (RR 1.10, 95% CI 0.99-1.23, P>0.05). Conclusions: Compared with routine genetic testing, NGS had a higher detection rate in general, particularly in the detection of α-thalassemia.

Objective: To assess the effectiveness and adverse drug reactions of all-oral regimens for patients with multidrug-resistant tuberculosis. Methods: This retrospective study was conducted at 10 Programmatic Management of Drug Resistant Tuberculosis sites in Punjab province of Pakistan. Patients receiving treatment for drug resistant tuberculosis from July 2019 to December 2020 with at least interim result i.e. 6th month culture conversion or final outcomes (cured, complete, lost to follow-up, failure, death) available, were included in the study. Data was extracted from electronic data management system. For the reporting and management of adverse drug events, active tuberculosis drug safety monitoring and management was implemented across all sites. All the data was analyzed using SPSS version 22. Results: Out of 947 drug resistant tuberculosis patients included in this study, 579 (68%) of the patients had final outcomes available. Of these, 384 (67.9%) successfully completed their treatment. Out of 368 (32%) patients who had their interim results available, all had their 6th month culture negative. Combining new medications was thought to result in serious adverse outcomes such as QT prolongation. However, this study did not record any severe adverse events among patients. Conclusions: All-oral regimens formulation guided by overall treatment effectiveness resulted in treatment outcomes comparable to those obtained with traditional injectable treatment.

Objective: To evaluate coagulation abnormalities and their relationship with bleeding manifestations among patients with dengue. Methods: This observational study was conducted on 292 adult dengue patients who were admitted to a tertiary care hospital of Western India from July 2021 to June 2022. Coagulation tests including prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (aPTT), fibrinogen, and D-dimer were performed. Patients were monitored for bleeding manifestations. Results: Coagulation abnormalities were reported in 42.8% of the patients. Overall, prolonged aPTT was the most common coagulation abnormality (40.8%), followed by low fibrinogen (38.7%), raised D-dimer (31.2%), raised INR (26.0%) and prolonged PT (19.2%). Bleeding manifestations were present in 19.9% patients. PT, INR, aPTT and D-dimer levels were significantly higher (P<0.01) and fibrinogen level was significantly lower (P<0.001) in patients with bleeding compared to patients without bleeding. Patients with bleeding had a significantly higher rate of all coagulation abnormalities than patients without bleeding (P<0.01). Conclusions: Patients with bleeding showed a significantly higher frequency of coagulation abnormalities compared to patients without bleeding. Patients with dengue should be assessed for coagulation abnormalities.

Objective: To determine the genetic diversity, natural selection and mutations in Plasmodium ( P.) knowlesi drug resistant molecular markers Kelch 13 and dhps gene in clinical samples of Malaysia. Methods: P. knowlesi full-length gene sequences Kelch 13 gene (PkK13) from 40 samples and dhps gene from 30 samples originating from Malaysian Borneo were retrieved from public databases. Genetic diversity, natural selection, and phylogenetic analysis of gene sequences were analysed using DNAsp v5.10 and MEGA v5.2. Results: Seventy-two single nucleotide polymorphic sites (SNPs) across the full-length PkK13 gene (63 synonymous substitutions and 9 non-synonymous substitutions) with nucleotide diversity of π~0.005 was observed. Analysis of the full-length Pk dhps gene revealed 73 SNPs and π~0.006 (44 synonymous substitutions and 29 non-synonymous substitutions). A high number of haplotypes (PkK13; H=37 and Pk dhps; H=29) with haplotype diversity of Hd ~0.99 were found in both genes, indicating population expansion. Nine mutant alleles were identified in PkK13 amino acid alignment of which, 7 (Asp³Glu, Lys⁵⁰Gln, Lys⁵³Glu, Ser¹²³Thr, Ser¹²⁷Pro, Ser¹⁴⁹Thr and Ala¹⁶⁹Thr) were within the Plasmodium specific domain, 2 (Val³⁷²Ile and Lys⁴²⁴Asn) were in the BTB/POZ domain and no mutation was observed within the kelch propeller domain. The 29 non-synonymous mutations in the Pk dhps gene were novel and only presented in exon 1 and 2. Conclusions: Monitoring the mutations from clinical samples collected from all states of Malaysia along with clinical efficacy studies will be necessary to determine the drug resistance in P. knowlesi.

Objective: To characterise a collection of pili-carrying and none pili-carrying pneumococcal isolates of clinical origin for serotypes, antibiotic resistance and genotype. Methods: In total, 42 clinical isolates were collected between October 2017 and December 2019. Those isolates were analysed for antimicrobial susceptibility, serotype distribution, detection of pneumococcal virulence and pilus genes. Multilocus sequence typing was performed only for piliated isolates, followed by phylogenetic analysis. Results: The common isolation sites among the pneumococcal isolates were tracheal aspirate (28.6%), blood (26.2%), and sputum (23.8%). Fifty percent isolates were resistant to erythromycin, tetracycline (50.0%) and trimethoprim-sulfamethoxazole (43.0%). The most frequent were serotypes 19F (28.6%), 6A/B (23.8%) and 19A (14.3%). Piliated isolates were detected in a small proportion (33.3%); 64.3% were multidrug-resistant. ST320 was the prevalent sequence type among the piliated isolates and genetically related to the Pneumococcal Molecular Epidemiology Network clones Taiwan^19F-14 (CC271). In the phylogenetic analysis, some piliated isolates showed a close association having similar ST320, carrying serotype 19A and both pilus genes indicating their clonal spread. Conclusions: Pneumococcal lineages of piliated isolates have been globally disseminated and pili could have played a role in the spread of antibiotic resistant clones.

Rationale: Neurocysticercosis is a neglected tropical disease caused by the larvae of Taenia solium and may occur in immunocompetent people. We report a case of diffuse parenchymal neurocysticercosis mimicking tuberculous meningitis in an immunocompetent patient. Patient concerns: A 24-year-old immunocompetent woman presented with serial focal to generalized seizure, left body weakness, left abducens palsy, chronic cough, and body weight loss. She came from an area where she was unlikely to have contact with Taenia solium. Diagnosis: Diffuse parenchymal neurocysticercosis. Intervention: Valproic acid, dexamethasone and 15 months of albendazole therapy. Outcome: Complete recovery without any neurological sequelae. Lessons: Prolonged anthelminthic treatment may benefit neurocysticercosis patients with multiple cerebral parenchymal lesions.

Mutations in the receptor binding domain (RBD) of SARS-CoV-2 spike protein have given birth to the new variants of concern viz. Delta and Omicron strain. As we are using vaccines which are either mRNA of spike protein of Wuhan strain or its whole virion vaccine, these vaccines will trigger antibodies to be able to bind with the epitopes of spike protein of Wuhan strain. There appears a need to study whether vaccine generated circulating neutralizing antibodies in vaccinated people are able to neutralize the receptor binding domain of spike protein of currently circulating Delta and Omicron strains as well. We need to address this contention in two phases: first to undertake docking studies of vaccine generated antibodies with Wuhan, Delta and Omicron strains and second to conduct the wet lab studies and observe antigen-antibody bindings employing Immunofluorescence and Cryo-Electron Microscopic studies. The present paper reports results of docking studies using bioinformatic softwares to provide the baseline information for wet lab studies. The structures of two types of vaccine produced antibodies (Ab) were obtained from Research Collaboratory for Structural Bioinformatics Database Protein Data Bank (RCSB PDB). For whole virus inactivated vaccine (Vaccine 1), PDB ID 7WE9 and for mRNA vaccine (Vaccine 2), PDB ID 6XCA were used. Antibody of Vaccine 1 was in complex with spike protein of Omicron variant. The antibody structure was constructed using ABody Builder-ML tool. After downloading amino acid sequence of light chain and heavy chain in Fasta format and by using tool ABody Builder-ML, the antibody structure was built and further downloaded. The PDB Format of spike antigen of the 3 strains were also obtained viz. Wuhan strain (PDB ID 6VXX), Delta strain (PDBID 7W92) and Omicron strain (PDBID 7QO7). Docking was performed in Cluspro app in antibody mode in which antibody was used as receptor and antigen as ligand. After docking, the model structures were analyzed and the lowest energy score was selected. To analyze the antigen-antibody (Ag-Ab) interactions, Pymol software was used. In this, the Ag-Ab structure was drawn from the software and then values of site of binding of Ag-Ab were recorded and analyzed. The antibody generated for Vaccine 1 when docked with Wuhan strain PDB ID 6VXX showed the lowest energy score of -309.7 (Figure 1A). The same when done for Delta strain PDB ID 7W92 and Omicron strain PDB ID 7QO7 showed the lowest energy score of -317.3 (Figure 1B) and -956.4 respectively (Figure1C). In the Ag-Ab interaction studies, it was observed that Wuhan strain showed good binidng with vaccine generated antibodies; however, both Delta and Omicron strains did not show any binidng (Figure 1A-1C). The antibody generated by Vaccine 2 when docked with Wuhan strain PDB ID 6VXX showed the lowest energy score of -341.0 (Figure 1D). The same when done for Delta strain PDB ID 7W92 and Omicron strain PDB ID 7QO7 showed the lowest energy score of -374.2 (Figure 1E) and -375.0 respectively (Figure 1F). Vaccine generated antibodies also did not show binding with Delta and Omicron strains (Figure 1D-1F).

Dengue virus, a member of the Flavivirus genus in the Flaviviridae family, has four serotypes (DEN-1 to DEN-4), and is responsible for both mild and severe dengue fever (dengue hemorrhagic fever and dengue shock syndrome). Dengue cases are under reported in India and the cases are treated clinically based on the clinical triage of symptoms. Nonstructural protein (NS1) antigen is a highly conserved glycoprotein found in Flavivirus group such as Dengue, Zika, and Japanese encephalitis virus. Simultaneous detection of NS1 antigen, IgM and IgG antibody in a single cassette is used for testing primary and secondary dengue infection. Early diagnosis of disease progression associated with severe dengue and accessibility to appropriate medical care reduces severe dengue fatality rates to less than 1%. This retrospective study, conducted from January 2021 to December 2021 in a tertiary care hospital, South India, was aimed to determine the seroprevalence of dengue adult patients with acute febrile illness. The study was approved by Institutional Human Ethics committee (IHEC-II/0092/21). During the study period, blood samples were collected from the male and female patients aged ≥18 years with history of fever after obtaining informed consent from them. A total of 5 mL blood was collected in a plain vacutainer (red topped) for the serological tests. Serum was separated by centrifugation and was used for the rapid card or ELISA. In case the sample could not be processed on the same day, the serum samples were aliquoted into Eppendorf vials and stored in the deep freezer at -20 ℃. The samples were subjected to immunochromatography (ICT) and ELISA for detecting NS1 antigen, IgM and IgG antibodies. ICT card used was SD Biosensor, cassette duo rapid card test and for ELISA Microlisa J Mithra testing was used. The procedures were followed according to the manufacturer instructions. Of 1 220 samples tested by ICT, 82 (6.7%) were found to be reactive, and 65.9% were female, 80.5% were outpatient. Among the total samples, the NS1 antigen were detected in 46 (3.8%) samples and the IgM, IgG were positive in 32 (2.6%) and 4 (0.3%) samples, respectively. IgM with NS1 antigen positivity was seen in 10 (0.8%) samples and the combined IgM with IgG positivity was found in 3 (0.2%) samples. Combined NS1 antigen with IgG positivity was not found in any of the tested samples. Among the non-reactive samples (1 138), 61.0% were female and 80.0% were outpatient. By ELISA, of 114 samples, 22 (19.3%) were found to be positive for NS1 antigen, 24 (17.5%) out of 137 were positive for IgM, 13 (15.5%) out of 84 samples were positive for IgG. Combined IgM with NS1 antigen positivity and IgM with IgG positivity were found in 10 and 5 samples, respectively. Four samples were positive for NS1 antigen as well as both IgM and IgG antibodies. None of the samples showed the combined NS1 antigen positivity with IgG antibodies. Seropositivity was found to be equal among males and females and it was higher among inpatient patients compared to outpatient patients (Supplementary Table 1). Increase in the number of cases was noted in the end of monsoon period (June-September) and post monsoon period (October-December) (Figure 1A). In ICT and ELISA, the maximum number of reactive cases were noted in the age group of 19-29 years, followed by 30-39 years (Figure 1B). The present study shows that simultaneous detection of NS1 antigen and IgM antibody would be useful for the diagnosis of acute dengue infection, suggesting that the ICT can be used as primary screening test. For increasing the sensitivity and specificity, ELISA testing can be used in conjugation which will exclude the false positive or negative result.