Immunogenicity of convalescent and vaccinated sera against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron variants

The omicron variant of concern (VOC) of SARS-CoV-2 was first reported in November 2021 in Botswana and South Africa. Omicron variant has evolved multiple mutations within the spike protein and the receptor binding domain (RBD), raising concerns of increased antibody evasion. Here, we isolated infectious omicron from a clinical specimen obtained in Canada. The neutralizing activity of sera from 65 coronavirus disease (COVID-19) vaccine recipients and convalescent individuals against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron VOCs was assessed. Convalescent sera from unvaccinated individuals infected by the ancestral virus during the first wave of COVID-19 in Canada (July, 2020) demonstrated reduced neutralization against beta, delta and omicron VOCs. Convalescent sera from unvaccinated individuals infected by the delta variant (May-June, 2021) neutralized omicron to significantly lower levels compared to the delta variant. Sera from individuals that received three doses of the Pfizer or Moderna vaccines demonstrated reduced neutralization of both delta and omicron variants relative to ancestral SARS-CoV-2. Sera from individuals that were naturally infected with ancestral SARS-CoV-2 and subsequently received two doses of the Pfizer vaccine induced significantly higher neutralizing antibody levels against ancestral virus and all VOCs. Importantly, infection alone, either with ancestral SARS-CoV-2 or the delta variant was not sufficient to induce high neutralizing antibody titers against omicron. This data will inform current booster vaccination strategies and we highlight the need for additional studies to identify longevity of immunity against SARS-CoV-2 and optimal neutralizing antibody levels that are necessary to prevent infection and/or severe COVID-19.

An in vitro and in vivo approach for the isolation of Omicron variant from human clinical specimens

Due to failure of virus isolation of Omicron variant in Vero CCL-81 from the clinical specimens of COVID-19 cases, we infected Syrian hamsters and then passage into Vero CCL-81 cells. The Omicron sequences were studied to assess if hamster could incorporate any mutation to changes its susceptibility. L212C mutation, Tyrosine 69 deletion, and C25000T nucleotide change in spike gene and absence of V17I mutation in E gene was observed in sequences of hamster passage unlike human clinical specimen and Vero CCL-81 passages. No change was observed in the furin cleavage site in any of the specimen sequence which suggests usefulness of these isolates in future studies.

Nipah virus outbreak in Kerala state, India amidst of COVID-19 pandemic

BackgroundWe report here a Nipah virus (NiV) outbreak in Kozhikode district of Kerala state, India which had caused fatal encephalitis in an adolescent male and the outbreak response which led to the successful containment of the disease and the related investigations.MethodsQuantitative real-time RT-PCR, ELISA based antibody detection and whole genome sequencing were performed to confirm the Nipah virus infection. Contacts of the index case were traced and isolated based on risk categorization. Bats from the areas near the epicenter of the outbreak were sampled for throat swabs, rectal swabs and blood samples for Nipah virus screening by real time RT-PCR and anti-Nipah virus bat IgG ELISA. Plaque reduction neutralization test was performed for the detection of neutralizing antibodies.ResultsNipah viral RNA and anti-NiV IgG antibodies were detected in the serum of the index case. Rapid establishment of an onsite NiV diagnostic facility and contact tracing helped in quick containment of the outbreak. NiV sequences retrieved from the clinical specimen of the index case formed a sub-cluster with the earlier reported Nipah I genotype sequences from India with more than 95% similarity. Anti-NiV IgG positivity could be detected in 21% of Pteropus medius and 37.73% of Rousettus leschenaultia. Neutralizing antibodies against NiV could be detected in P.medius.ConclusionsStringent surveillance and awareness campaigns needs to be implemented in the area to reduce human-bat interactions and minimize spill over events which can lead to sporadic outbreaks of NiV.

ANALISIS KELENGKAPAN STANDAR INSTALASI LABORATORIUM DI RSIA ERIA BUNDA PEKANBARU TAHUN

Laboratorium klinik adalah laboratorium kesehatan yang melaksanakan pelayanan, pemeriksaan specimen klinik untuk mendapatkan informasi tentang kesehatan perorangan terutama untuk menunjang upaya diagnosis penyakit, penyembuhan penyakit, dan pemulihan kesehatan.Tujuan penelitian ini secara umum untuk mengetahui Analisi Kelengkapan Standar Instalasi Laboratorium di RSIA Eria Bunda Pekanbaru Tahun 2020.Dilakukan wawancara mendalam dan observasi kepada 5 informan.Lokasi penelitian dilakukan di RSIA Eria Bunda Pekanbaru di jln.K.H.Ahmad Dahlan No.163 Sukajadi Pekanbaru, Riau, khususnya dibagian laboratorium pada bulan Maret-April 2020.Hasil penelitian menunjukkan bahwa SDM di laboratorium belum sesuai dengan standar, Sarana di laboratorium belum sesuai dengan standar, Prasarana di laboratorium belum sesuai dengan standar, dan SOP di laboratorium belum sesuai dengan standarnya. Dari hasil penelitian dapat disimpulkan bahwa SDM di laboratorium masih perlu penambahan SDM atau memaksimalkan SDM yang ada dengan menggatur ulang jadwal shiff, Sarana di laboratorium harus dilengkapi dari ruang-ruangan sampai alat laboratorium, Prasarana menyediakan KM/WC untuk pasien dan petugas secara terpisah disekitar laboratorium, dan SOP di laboratorium perlu adanya pengawasan untuk staf analis dalam menerapkan SOP di RSIA Eria Bunda Pekanbaru Tahun 2020. A clinical laboratory is a health laboratory that provides service, examines clinical specimen to obtain information on individual health, especially to support effort to diagnose disease, cures disease and restores health. The purpose of this study generally was to determine the analysis of standard completeness of laboratory installation at RSIA Eria Bunda Pekanbaru in 2020. In-depth interview and observation were conducted with 5 informants. The location of the research was carried out at RSIA Eria Bunda Pekanbaru on KH. Ahmad Dahlan street No.163 Sukajadi Pekanbaru, Riau, especially in the laboratory section on March-April 2020. The result showed that the human resources in the laboratory were not in accordance with standard, the facility in the laboratory was not in accordance with the standard, the infrastructure in the laboratory was not in accordance with the standard, and SOP in the laboratory was not in accordance with the standard. From the result of the study it can be concluded that the human resources in the laboratory still need to add more human resources or maximize existing human resources by resetting the shiff schedule, the facility in the laboratory must be equipped from rooms to laboratory equipment, infrastructure provides bathroom /toilet for patient and staff separately around the laboratory , and SOP in the laboratory needs supervision for analizing staff in implementing SOP at RSIA Eria Bunda Pekanbaru in 2020.

Melioidosis: A Neglected Infection in Bangladesh

Bangladesh is an example of a highly populous, agricultural country where melioidosis may be a significantly under diagnosed cause of infection and death. A recent regression model predicted 16,931 cases annually in Bangladesh with a mortality rate of 56%. However, we only manage to confirm (culture) around 80 cases in last 60 years. A lack of awareness among microbiologists and clinicians and a lack of diagnostic microbiology infrastructure are factors that are likely to lead to the underreporting of melioidosis. Melioidosis transmits through inoculation, inhalation and ingestion. Diabetes mellitus is the most common risk factor (12 times higher chance of getting the infection) predisposing individuals to melioidosis and is present in >50% of all patients. The clinical presentation is widely varied and can be mistaken for other diseases such as tuberculosis or more common forms of pneumonia giving rise to its nickname as the “great mimicker”. Disease manifestations vary from pneumonia or localized abscess to acute septicemias, or may present as a chronic infection. Culture is considered the current gold-standard for diagnosis and culture-confirmation should always be sought in patients where disease is suspected. It is strongly recommended that any non–Pseudomonas aeruginosa, oxidase-positive, Gram-negative bacillus isolated from any clinical specimen from a patient in an endemic area should be suspected to be Burkholderia pseudomallei (BP). In addition, based on antibiogram, any Gramnegative bacilli that are oxidase-positive, typically resistant to aminoglycosides (e.g., gentamicin), colistin, and polymyxin but sensitive to amoxicillin/clavulanic acid should be considered as BP. This bacteria is inherently resistant to penicillin, ampicillin, first generation and second-generation cephalosporins, gentamicin, tobramycin, streptomycin, and polymyxin. For intensive phase (10 to 14 days), ceftazidime or carbapenem is the drug of choice. For eradication phase (3 to 6 months), oral trimethoprim/ sulfamethoxazole is the drug of choice. Surgery (drainage of abscess) has an important role in the management of melioidosis. Preventive measures through protective gears could be useful particularly for the risk groups. J MEDICINE 2021; 22: 139-145

Prevalence of Malignancy in Solitary Thyroid Nodule-A Retrospective Study

Background: Solitary thyroid nodule is defined as discrete mass palpable in an otherwise apparently normal thyroid gland. Solitary nodule is the common presentation of thyroid disorders. Objective: This study aimed to look into the prevalence of malignancy in clinico-radiologically detected solitary thyroid nodule and to correlate the findings in pre-operative fine needle aspiration cytology(FNAC) and post-operative histopathological examination(HPE). Materials and Methods: A retrospective study was carried out in our Institute for a period of 6 months using the data obtained between 2018-2020 of patients who were clinically and radiologically diagnosed as solitary thyroid nodule in the Department of General Surgery. Results: Out of 30 cases of clinically detected solitary thyroid nodule 7(23.3%)cases was found to be malignant. The mean age of presentation was 41.2 years with male female ratio of 1:9. 25(83.3%)cases was reported as benign nodules according to pre-operative FNAC out of these 2(6.6%)cases turned out to be malignant on post-operative histopathological examination. Conclusion: It is concluded that from the present study the prevalence of malignancy in clinically detected solitary thyroid nodule is 23.3%. FNAC being sensitive, cost effective and reliable tool in the preoperative assessment of solitary thyroid nodules and HPE in post operative evaluation of clinical specimen both playing a vital role in management of solitary thyroid nodule thus helping in early diagnosis and proper surgical intervention.

Evaluation of antigen-detecting and antibody-detecting diagnostic test combinations for diagnosing melioidosis

Background Melioidosis, an infectious disease caused by Burkholderia pseudomallei, is endemic in many tropical developing countries and has a high mortality. Here we evaluated combinations of a lateral flow immunoassay (LFI) detecting B. pseudomallei capsular polysaccharide (CPS) and enzyme-linked immunosorbent assays (ELISA) detecting antibodies against hemolysin co-regulated protein (Hcp1) or O-polysaccharide (OPS) for diagnosing melioidosis. Methodology/Principal findings We conducted a cohort-based case-control study. Both cases and controls were derived from a prospective observational study of patients presenting with community-acquired infections and sepsis in northeast Thailand (Ubon-sepsis). Cases included 192 patients with a clinical specimen culture positive for B. pseudomallei. Controls included 502 patients who were blood culture positive for Staphylococcus aureus, Escherichia coli or Klebsiella pneumoniae or were polymerase chain reaction assay positive for malaria or dengue. Serum samples collected within 24 hours of admission were stored and tested using a CPS-LFI, Hcp1-ELISA and OPS-ELISA. When assessing diagnostic tests in combination, results were considered positive if either test was positive. We selected ELISA cut-offs corresponding to a specificity of 95%. Using a positive cut-off OD of 2.912 for Hcp1-ELISA, the combination of the CPS-LFI and Hcp1-ELISA had a sensitivity of 67.7% (130/192 case patients) and a specificity of 95.0% (477/502 control patients). The sensitivity of the combination (67.7%) was higher than that of the CPS-LFI alone (31.3%, p<0.001) and that of Hcp1-ELISA alone (53.6%, p<0.001). A similar phenomenon was also observed for the combination of CPS-LFI and OPS-ELISA. In case patients, positivity of the CPS-LFI was associated with a short duration of symptoms, high modified Sequential (sepsis-related) Organ Failure Assessment (SOFA) score, bacteraemia and mortality outcome, while positivity of Hcp1-ELISA was associated with a longer duration of symptoms, low modified SOFA score, non-bacteraemia and survival outcome. Conclusions/Significance A combination of antigen-antibody diagnostic tests increased the sensitivity of melioidosis diagnosis over individual tests while preserving high specificity. Point-of-care tests for melioidosis based on the use of combination assays should be further developed and evaluated.

355. A Novel Likelihood-Based Model to Estimate SARS-CoV-2 Viral Titer from Next-Generation Sequencing Data

Background The quantitative level of pathogens present in a host is a major driver of infectious disease (ID) state and outcome. However, the majority of ID diagnostics are qualitative. Next-generation sequencing (NGS) is an emerging ID diagnostics and research tool to provide insights, including tracking transmission, evolution, and identifying novel strains. Methods We built a novel likelihood-based computational method to leverage pathogen-specific genome-wide NGS data to detect SARS-CoV-2, profile genetic variants, and furthermore quantify levels of these pathogens. We used de-identified clinical specimens tested for SARS-CoV-2 using RT-PCR, SARS-CoV-2 NGS Assay (hybrid capture, Twist Bioscience), or ARTIC (amplicon-based) platform, and COVID-DX software. A training (n=87) and validation (n=22) set was selected to establish the strength of our quantification model. We fit non-uniform probabilistic error profiles to a deterministic sigmoidal equation that more realistically represents observed data and used likelihood maximized over several different read depths to improve accuracy over a wide range of values of viral load. Given the proportion of the genome covered at varying depths for a single sample as input data, our model estimated the Ct of that sample as the value that produces the maximum likelihood of generating the observed genome coverage data. Results The model fit on 87 SARS-CoV-2 NGS Assay training samples produced a good fit to the 22 validation samples, with a coefficient of correlation (r2) of ~0.8. The accuracy of the model was high (mean absolute % error of ~10%, meaning our model is able to predict the Ct value of each sample within a margin of ±10% on average). Because of the nature of the commonly used ARTIC protocol, we found that all quantitative signals in this data were lost during PCR amplification and the model is not applicable for quantification of samples captured this way. The ability to model quantification is a major advantage of the SARS-CoV-2 NGS assay protocol. The likelihood-based model to estimate SARS-CoV-2 viral titer Left Observed genome coverage (y-axis) plotted against Ct value (x-axis). The best-fitting logistic curve is demonstrated with a red line with shaded areas above and below representing the fitted error profile. RIGHT: Model-estimated Ct values (y-axis) compared to laboratory Ct values (x-axis) with grey bars representing estimated confidence intervals. The 1:1 diagonal is shown as a dotted line. Conclusion To our knowledge, this is the first model to incorporate sequence data mapped across the genome of a pathogen to quantify the level of that pathogen in a clinical specimen. This has implications in ID diagnostics, research, and metagenomics. Disclosures Heather L. Wells, MPH, Biotia, Inc. (Consultant) Joseph Barrows, MS, Biotia (Employee) Mara Couto-Rodriguez, MS, Biotia (Employee) Xavier O. Jirau Serrano, B.S., Biotia (Employee) Marilyne Debieu, PhD, Biotia (Employee) Karen Wessel, PhD, Labor Zotz/Klimas (Employee) Christopher Mason, PhD, Biotia (Board Member, Advisor or Review Panel member, Shareholder) Dorottya Nagy-Szakal, MD PhD, Biotia Inc (Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder)

Cohnella cholangitidis sp. nov., a novel species of the genus Cohnella isolated from a clinical specimen in Korea

A Gram-positive, aerobic, rod-shaped bacterium, designated as strain 1605-214T, was isolated from the blood sample of a patient with cholangitis. Based on its 16S rRNA gene sequence, the strain 1605-214T belonged to the genus Cohnella and exhibited 97.9% sequence identity with Cohnella luojiensis DSM 24270T (GQ214052). DNA–DNA hybridization, digital DNA–DNA hybridization, and average nucleotide identity values between the two species were 23% ± 1.9, 21.1%, and 77.2%, respectively. The cellular fatty acids of strain 1605-214T were mainly comprised of anteiso-C15:0 (36.1%), iso-C16:0 (16.5%), and C16:0 (15.1%). The predominant quinone was menaquinone-7; predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, and aminophospholipid-1. The cell wall peptidoglycan of strain 1605-214T contained meso-diaminopimelic acid. DNA G + C content of strain 1605-214T was 50.6 mol%. 5187 genes out of a total of 5413 (94.6%) were assigned putative functions using eggNOG v5.0. Based on genotypic characteristics and genomic sequence analysis results, strain 1605-214T was confirmed to represent a novel species of genus Cohnella, for which the name Cohnella cholangitidis sp. nov., was proposed.

Emerging cases of mucormycosis in post Covid-19 disease patients

Mucormycosis (MC) is a serious threat in this covid-19 pandemic situation; cases of MC continue to increases as post covid-19 disease affliction in India. It is a life-threatening disease that happens due to black mold. Mucormycosis is described as a potentially lethal infection amongst immune-compromised (IC) hosts, particularly in those with diabetes, leukemia, and lymphoma. Mortality rate of MC just doubles in IC host, delayed diagnosis increase the rate of mortality. Mucormycosis is difficult to diagnose which affects outcomes and results in a poor prognosis. The main objective of the study was to detect MC in the clinical species received during or post covid-19 treatment in our laboratory.Basic microbiological methods such as gram stain and KOH smear were used for the detection of MC in the received clinical specimen and morphology was seen in the microscope. The is a cross-sectional observational study conducted in our microbiology laboratory for one month periods during May 15, 2021 to June 15, 2021 in a tertiary care hospital of central India. During study period our microbiology lab received n=35 suspected clinical specimens from N= 27 post covid-19 patients for MC diagnosis over one month period. Out of n=35 specimens, n=8 specimens (obtained from N=5 patients) were positive for MC by gram and KOH smear method and we saw filamentous fungi by conventional microscopic method. The present study concluded that the cases of life threatening MC increase day by day in central India as post complication of covid-19 disease.