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Rotavirus is an important cause of severe diarrhea requiring hospitalization, accounting for approximately 78,000 deaths annually in Indian children below 5 years of age. We present epidemiological data on severe rotavirus disease collected during hospital-based surveillance in India before the introduction of the oral rotavirus vaccine into the national immunization schedule. The National Rotavirus Surveillance Network was created involving 28 hospital sites and 11 laboratories across the four geographical regions of India. From September 2012 to August 2016 children less than 5 years of age hospitalized for diarrhea for at least 6 h, were enrolled. After recording clinical details, a stool sample was collected from each enrolled child, which was tested for rotavirus antigen using enzyme immunoassay (EIA). Nearly 2/3rd of EIA positive samples were genotyped using reverse transcription polymerase chain reaction to identify the G and P types. Of the 21,421 children enrolled during the 4 years surveillance, 36.3% were positive for rotavirus. The eastern region had the highest proportion of rotavirus associated diarrhea (39.8%), while the southern region had the lowest (33.8%). Rotavirus detection rates were the highest in children aged 6–23 months (41.8%), and 24.7% in children aged < 6 months. Although rotavirus associated diarrhea was seen throughout the year, the highest positivity was documented between December and February across all the regions. The most common rotavirus genotype was G1P[8] (52.9%), followed by G9P4 (8.7%) and G2P4 (8.4%). There is high burden of rotavirus gastroenteritis among Indian children below 5 years of age hospitalized for acute diarrhea thereby highlighting the need for introduction of rotavirus vaccine into the national immunization program and also for monitoring circulating genotypes.

•Study identify regionally strains, human-animal reassortment, untypeable and mixed genotypes.•Overall, G1P[8] (23.8%) was the most predominant strain detected during the six-year period.•G2P[4] (11.8%), G9P[8] (10.4%), G12P[8] (4.9%), G2P[6] (4.2%) and G3P[6] (3.7%) were circulating.•Data from 6 countries showed no difference in strains pre and post vaccine introduction. The African Rotavirus Surveillance Network has been detecting and documenting rotavirus genotypes in the African sub-continent since 1998 in anticipation of the rollout of rotavirus vaccination in routine Expanded Programme on Immunisation. This paper reports distribution of the rotavirus strains circulating in 15 Eastern and Southern African (ESA) countries from 2010-2015 as part of active World Health Organization (WHO) rotavirus surveillance, and investigates possibility of emergence of non-vaccine or unusual strains in six selected countries post-vaccine introduction. Stool samples were collected from children <5 years of age presenting with acute gastroenteritis at sentinel hospitals pre- and post-rotavirus vaccine introduction. Samples were tested for group A rotavirus using an enzyme immunoassay by the national and sentinel laboratories. At the WHO Rotavirus Regional Reference Laboratory in South Africa, molecular characterisation was determined by PAGE (n = 4186), G and P genotyping (n = 6447) and DNA sequencing for both G and P types (n = 400). The six-year surveillance period demonstrated that 23.8% of the strains were G1P[8], followed by G2P[4] (11.8%), G9P[8] (10.4%), G12P[8] (4.9%), G2P[6] (4.2%) and G3P[6] (3.7%) in 15 ESA countries. There was no difference in circulating strains pre- and post-rotavirus vaccine introduction with yearly fluctuation of strains observed over time. Atypical rotavirus G and P combinations (such as G1P[4], G2P[8], G9P[4] and G12P[4]) that might have arisen through inter-genogroup or inter-genotypes reassortment were detected at low frequency (2%). Close genetic relationship of African strains were reflected on the phylogenetic analysis, strains segregated together to form an African cluster in the same lineages/sub-lineage or monophyletic branch. There has been considerable concern about strain replacement post-vaccine introduction, it was not clear at this early stage whether observed cyclical changes of rotavirus strains were due to vaccine pressure or this was just part of natural annual fluctuations in the six ESA countries, long-term surveillance is required.

Ghana introduced the monovalent rotavirus vaccine (Rotarix) into its national paediatric vaccination programme in May2012. Vaccine introduction was initiated nationwide and achieved >85% coverage within a few months. Rotavirus strain distribution pre- and post-RV vaccine introduction is reported. Stool samples were collected from diarrhoeic children <5 years of age hospitalized between 2009 and 2016 at sentinel sites across Ghana and analyzed for the presence of group A rotavirus by enzyme immunoassay. Rotavirus strains were characterized by RT-PCR and sequencing. A total of 1363 rotavirus EIA-positive samples were subjected to molecular characterization. These were made up of 823 (60.4%) and 540 (39.6%) samples from the pre- and post-vaccine periods respectively. Rotavirus VP7 genotypes G1, G2 and G3, and VP4 genotypes P[6] and P[8] constituted more than 65% of circulating G and P types in the pre–vaccine period. The common strains detected were G1P[8] (20%), G3P[6] (9.2%) and G2P[6] (4.9%). During the post-vaccine period, G12, G1 and G10 genotypes, constituted more than 65% of the VP7 genotypes whilst P[6] and P[8] made up more than 75% of the VP4 genotypes. The predominant circulating strains were G12P[8] (26%), G10P[6] (10%) G3P[6] (8.1%) and G1P[8] (8.0%). We also observed the emergence of the unusual rotavirus strain G9P[4] during this period. Rotavirus G1P[8], the major strain in circulation during the pre-vaccination era, was replaced by G12P[8] as the most predominant strain after vaccine introduction. This strain replacement could be temporary and unrelated to vaccine introduction since an increase in G12 was observed in countries yet to introduce the rotavirus vaccine in West Africa. A continuous surveillance programme in the post-vaccine era is necessary for the monitoring of circulating rotavirus strains and the detection of unusual/emerging genotypes.

Ferritin, widely present in liver and spleen tissue, is considered as a serological biomarker for liver diseases and cancers. The detection of ferritin may be an important tool in health diagnosis. In this study, 14 non-immunized chicken spleens were utilized to construct a single-chain fragment (scFv) phage library. After 4 rounds of panning, 7 unique clones were obtained. The optimal clone was further screened and combined with NanoLuc luciferase (Nluc) as a dual functional immunoprobe to bioluminescent enzyme immunoassay (BLEIA), which was twice as sensitive as its parental scFv-based double-sandwich enzyme-linked immunoassay (ds-ELISA). The cross-reactivity analysis revealed that the proposed methods were highly selective and suitable for clinical detection. To further verify the performance of the immunoassays, serum samples were tested by the proposed methods and a commercial ELISA kit, and there was a good correlation between the results. These results suggested that scFv fused with Nluc might be a powerful dual functional tool for rapid, practically reliable, and highly sensitive ferritin detection.

The quantification of serum/plasma estradiol (E2) is useful for the diagnosis, pathological analysis, and monitoring of the therapeutic efficacy of estrogen-dependent diseases. In this study, an improved derivatization method using 1-(2,4-dinitro-5-fluorophenyl)-4,4-dimethylpiperazinium iodide (MPDNP-F) was developed and combined with liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) for the sensitive and specific quantification of the serum/plasma E2. In the new method, the reaction time was reduced to 15 min from 90 min (two-step reaction in the previous method) by the direct reaction of MPDNP-F with E2 at 60°C in the presence of 4-dimethylaminopyridine (DMAP). DMAP served as the organic catalyst and had a less negative effect on the LC/ESI-MS/MS instrument compared to the non-volatile inorganic salt (NaHCO3), which was used in the previous method. The collision-induced dissociation of the molecular cation ([M]+) of the resulting derivative provided a product ion containing the E2-skeleton ([M–NO2–H]+), which significantly enhanced the assay sensitivity and specificity; compared to the dansyl chloride derivatization, which is the currently most-used derivatization procedure for the LC/ESI-MS/MS assays of E2, the MPDNP-F derivatization had significantly fewer interfering peaks and a clear and flat baseline in the serum sample analysis. The MPDNP-F derivatization–LC/ESI-MS/MS method enabled the precise and accurate quantification of E2 even at a 5.0 pg/mL concentration (lower limit of quantification) with a small sample volume (100 μL of serum/plasma) and had a tolerance for the matrix effect. This method was also proven to serve as a more sensitive and specific alternative to the clinically used chemiluminescence enzyme immunoassay.

The transition from radioimmunoassay (RIA) to chemiluminescent enzyme immunoassay (CLEIA) for plasma aldosterone concentration (PAC) assays has raised concerns over its impact on primary aldosteronism (PA) diagnosis. This study investigated the correlation between PAC and renin values using RIA, CLEIA, and liquid chromatography/mass spectrometry/mass spectrometry (LC–MS/MS), established cutoff values for PA diagnosis using the aldosterone-to-renin ratio (ARR) with PAC_CLEIA, and assessed the differences in PAC values by measuring weak mineralocorticoids (WMs). This retrospective study evaluated 312 serum PAC samples using RIA, CLEIA, and LC–MS/MS, and analyzed 315 plasma renin samples. Method correlations were assessed through Passing-Bablok regression. Receiver operating characteristic curves determined ARR cutoffs for PA diagnosis. WMs were quantified to evaluate their impact on ΔPAC (RIA-LC–MS/MS) through multiple regression analysis. PAC_CLEIA and PAC_LC-MS/MS values were highly correlated. ARRs derived from PAC_RIAs demonstrated more false positives and lower specificity than ARRs using PAC_CLEIA or PAC_LC-MS/MS. WMs significantly influenced ΔPAC in both the PA and non-PA groups. ARRs using PAC_CLEIA are valuable for determining PA cutoffs in clinical practice. The transition to PAC using CLEIA may enhance PA detection rates. WMs were found to interfere with PAC measurements in the RIA method, affecting outcomes.

Background Homocysteine (HCY) is a sulfur-containing amino acid that is an independent or important risk factor for the occurrence of many chronic diseases and is one of the most important indicators for determining health risks. However, existing HCY detection methods do not meet the requirements of clinical diagnosis. Therefore, there is an urgent need to establish new detection methods to meet the needs of clinical detection. Results In this study, we used the principle of competitive method to establish a new method for the determination of HCY in human serum using a chemiluminescent enzyme immunoassay in conjunction with a chemiluminescent assay instrument that uses magnetic microparticles as the solid phase of the immunoreaction. The established method achieved satisfactory results in terms of minimum detection limit, specificity, accuracy, and clinical application. The limit of detection was 0.03 ng/mL. The intra-assay coefficient of variation (CV) was 1.94–5.05%, the inter-assay CV was 2.29–6.88%, and the recovery rate was 88.60–93.27%. Cross-reactivity with L-cysteine ranged from 0.0100 to 0.0200 μmol/L, and cross-reactivity with glutathione ranged from 0.0100 to 0.200 μmol/L, all of which were less than the limit of detection (LoD) of this method. The linear factor R of this method was greater than 0.99. Conclusions In summary, the developed method showed a good correlation with the product from Abbott. A total of 996 clinical patients with cardiovascular diseases were evaluated using the method developed in this study.

Objective This study aims to determine the diagnostic utility of galactomannan enzyme immunoassay (GM EIA) in invasive aspergillosis (IA) in children with hematological malignancy (high risk population) in terms of sensitivity, specificity, negative predictive value (NPV) and positive predictive values (PPV) at various cut offs while validating the revised EORTC/MSG 2019 criteria in order to obtain the best cut-off. Material and Methods For 100 pediatric patients, serum and respiratory samples were collected. Clinical, mycological workup (potassium-hydroxide mount, fungal culture) and GM EIA was done to classify proven, probable, and possible IA as per EORTC-MSG guidelines,2019. Sensitivity, specificity, PPV and NPV were calculated of GM indices at cut-off 0.5, 0.7 and 1, and validated with revised EORTC -MSG, 2019. Results Of 100 patients enrolled, 75 were diagnosed with ALL, 14 with AML, two with Hodgkin's, three had non-Hodgkin lymphoma, and six had undifferentiated leukemia. With routine mycological findings, 51 were classified as probable IA, 11 as possible IA, and 38 as no IA. Aspergillus flavus was the most prevalent on culture (56.9%, 29/51) followed by A. fumigatus (29%, 15/51) A. niger (7.8%, 4/51), A . terreus (3.9%, 2/51) and A. nidulans (2%, 1/51). GM EIA demonstrated sensitivity 82.3%, specificity 97.4%, PPV 98.1%, and NPV 77.1% at cut-off 0.67 when comparing probable/possible IA v/s no IA groups. The GM EIA had the best sensitivity (82.4%), specificity (81.8%), PPV (95.5%), and NPV (50%) at cut off 0.78 when the probable IA group was compared to the possible IA. Seven patients succumbed of whom 5 had GMI ≥ 2. Conclusion This study deduces the optimal cut-off for serum GM EIA to be 0.67 obtained by ROC analysis when comparing possible and probable IA versus no IA and reinforces the definition of probable category of EORTC-MSG criteria, 2019. At 0.5 ODI the sensitivity (87.1%) and NPV (80.5%) are high, thus making it the most suitable cut-off for detecting true positive and ruling out IA respectively, in pediatric patients with hematological malignancy. GM EIA when performed adjunctive to clinico-radiological findings can prove to be screening, diagnostic and prognostic test for IA in pediatric hematological malignancy patients.

•Anti-SP antibody titer compared after second and third doses of the BNT162b2 vaccine.•Study conducted on healthcare workers directly exposed to COVID-19 patients.•Anti-SP IgG titer decreased slowly after second dose of the vaccine.•Peak IgG titer 4.1-fold higher after the third dose than after first and second doses.•Adverse events following immunization were generally mild to moderate. Administration of three doses of Pfizer-BioNTech BNT162b2 COVID-19 mRNA vaccine was completed in Japan in the spring of 2022. This study aimed to evaluate the antibody responses, and kinetics of three doses of vaccine in healthcare workers (HCWs). We conducted a longitudinal cohort study with HCWs, who had no history of COVID-19 or serologic evidence of SARS-CoV-2 infection, from a single hospital. Immunoglobulin G (IgG) titers of anti-SARS-CoV-2 spike protein (SP) and nucleocapsid protein (NP) titers were measured using an automated chemiluminescent enzyme immunoassay system. A total of 636 HCWs participated in the study. The anti-SP IgG titers decreased slowly after the second dose of the BNT162b2 vaccine in all participants, and robust antibody response was observed after the third dose of the vaccine. The peak anti-SP IgG titer after the third dose was approximately 4.1-fold higher than that after the first and second doses, and the rate of decrease in the anti-SP IgG titer after the third dose was significantly more gradual, than that after the second dose. After the second dose of vaccine, the antibody response was weaker in older participants than in younger participants, and in males than in females respectively, whereas the response to the third dose of vaccine did not differ significantly by sex or age. Adverse events following immunization were generally mild to moderate. The third dose of the BNT162b2 vaccine induced a significant and sustained increase in anti-SP IgG titers, and was generally safe and well-tolerated.

Background Blastocystis is a common anaerobic colonic protist in humans with controversial pathogenicity. Clostridium difficile ( C. difficile ) is the commonest cause of infectious diarrhea in healthcare settings. The prevalence and subtype (ST) characteristics of Blastocystis in patients with C. difficile infection (CDI) are rarely documented. Therefore, the present study was conducted to investigate the prevalence and subtype characteristics of Blastocystis in patients with suspicion of CDI in Singapore. Methods Fecal samples were collected from 248 patients presenting with suspected CDI from a single tertiary hospital in Singapore. C. difficile was diagnosed through positive glutamate dehydrogenase (GDH) with or without toxin A/B using enzyme immunoassay methods. The prevalence and subtype genetic characteristics of Blastocystis were determined by polymerase chain reaction (PCR) amplification and analysis of the barcode region of the SSU rRNA gene. Results The proportion of C. difficile in patients with healthcare-associated diarrhea in this study was 44% (109/248). Among the 109 C. difficile -positive patients, 59 (54.1%, 59/109) tested positive for toxigenic C. difficile , which was considered CDI. Based on the sequence analyses of the barcode region of the SSU rRNA gene, 10.1% (25/248) of the patients were found to be Blastocystis -positive, and three subtypes were identified: ST7 (64%, 16/25), ST1 (20%, 5/25), and ST3 (16%, 4/25). Remarkably, we found five patients with Blastocystis and C. difficile coinfection, and further subtype analysis showed two with ST7, two with ST1, and one with ST3. Conclusions To the best of our knowledge, this is the first study to investigate the subtype distributions of Blastocystis in patients with CDI in Singapore. We found ST7 to be the predominant subtype in diarrheal patients. The pathogenicity of ST7 has been strongly suggested in previous in vitro and mouse model experiments, further confirming its potential pathogenicity to humans. Graphical Abstract