Phylogenetic, Sequencing, and Mutation Analysis of SARS-CoV-2 Omicron (BA.1) and Its Subvariants (BA.1.1, BA.2) During the Fifth Wave of the COVID-19 Pandemic in the Iraqi Kurdistan Region.

Introduction In December 2019, a global outbreak of SARS-CoV-2 occurred in Wuhan, China, resulting in the COVID-19 pandemic. Since then, the virus has spread to all countries, necessitating a worldwide initiative to create effective treatments and vaccines. Methods The RNA of samples QIAamp Viral RNA Mini Kit (Qiagen, MD). SARS-CoV-2 RNA was reverse transcribed with SuperScript IV VILO (ThermoFisher Scientific, Waltham, MA). The virus cDNA was amplified in two multiplexed PCR reactions using Q5 DNA High-fidelity Polymerase (New England Biolabs, Ipswich, MA). The genome was entirely sequenced from 40 samples at the Scripps Research Institute (TSRI) in California, USA. The samples were sequenced using a NovaSeq 6000 SP Reagent Kit v1.5 (Illumina, USA). The TSRI then entered these sequences into the GISAID database. The virus sequence was matched to the SARS-COV-2 virus identified in Wuhan, China (accession number: NC 045512.2) using Illumina sequencing technology (Illumina, CA), finding 95 different changes. The NextClade (clades.nextstrain.org) and Mega 11 (https://www.megasoftware.net) software tools were used to analyze SARS-CoV-2 genome sequence alignment and mutation studies. Results Following a sequencing analysis, it was determined that the spike glycoprotein (S) included a total of 38 mutations. Thirty of these mutations were found in the ORF1a gene. Additionally, 11 mutations were found in the ORF1b gene, with the remaining mutations found in the nucleocapsid (N), membrane protein (M), open reading frames 6 (ORF6), open reading frames 9 (ORF9), and envelope (E) genes. The phylogenetic analysis and transmission studies indicated that the isolates discovered in Iraq had separate infection origins and were closely linked to those discovered in other nations and states. Conclusion According to the findings of this study, a new vaccine can be developed based on identifying new Omicron variant mutations and subvariants such as BA.2, which were identified for the first time in Iraq.

The effect of erythroferrone suppression by transfusion on the erythropoietin-erythroferrone-hepcidin axis in transfusion-dependent thalassaemia: A pre-post cohort study.

To track post-transfusion changes on the erythropoietin (EPO)-erythroferrone (ERFE)-hepcidin axis, we collected blood samples from 82 regularly transfused patients with ß-thalassaemia major (ß-TM) immediately before and 4-6 days after transfusion. The post-transfusion haemoglobin, hepcidin, and ferritin levels were increased, while the EPO, ERFE, and soluble transferrin receptor were suppressed. In addition, hepcidin change was inversely associated with erythropoietic change, which was confirmed by an increase in the hepcidin-to-ERFE ratio after transfusion. Age was the main predictor of serum ERFE, followed by EPO, transfusion frequencies, and ferritin. We found ERFE to be a highly sensitive indicator of erythroid activity in ß-TM and that the hepcidin-to-ERFE ratio after transfusion may be used as an appropriateness index of serum hepcidin regulation relative to the degree of erythropoiesis.

Cryptosporidiosis in Children in Duhok City / Kurdistan Region / Iraq.

This is the first study to detect cryptosporidiosis among children in Duhok city/Kurdistan region of Iraq. The study included 332 stool samples of children using Modified Ziehl Neelsen Method (MZNM) and 122 of these were randomly selected to detect Cryptosporidium by ELISA. By MZNM, the infection rate of Cryptosporidium oocysts was 66.95% in children; 44.68% in immunocompromised (ICD) and 22.27% in immunocompetent (ICT) group. Among ICT children, the infection rate was highest among less than one year of age (39.34%) in diarrheoic group, while in non-diarrheoic group, it was highest among 1-4 years age (28.57%). Among ICD children, the relations were non-significant among ages. Out of 122 stool samples of children, 26 (21.31%), and 30 (24.59%) were positive by MZNM and ELISA, respectively. In conclusion, ELISA was more sensitive and specific than MZNM which were 82.5% and 90.91% respectively. This study indicates that asymptomatic infection is common among different age groups of children.

Development and laboratory evaluation of a lateral flow device (LFD) for the serodiagnosis of Theileria annulata infection.

Several DNA-based and serological tests have been established for the detection of Theileria annulata infection, including polymerase chain reaction, reverse line blot and loop-mediated isothermal amplification, indirect enzyme-linked immunosorbent assay (ELISA), and competitive ELISA. In this study, we have applied knowledge from the development and application of a recombinant protein-based indirect ELISA and competitive ELISA to establish a rapid test for point-of-care diagnosis of T. annulata infection in the field to be used by the veterinarian. For the development of a lateral flow test, the recombinantly expressed T. annulata surface protein (TaSP) was applied as the test antigen and anti-TaSP antiserum as the control line. TaSP antigen conjugated to colloidal gold particles was used as the detection system for visualization at the test line for the binding of anti-TaSP antibody present in the serum of infected animals. The developed test specifically detected antibodies in the serum of animals experimentally infected with T. annulata and showed no cross-reactivity with serum from animals infected with other tested bovine pathogens (Trypanosoma brucei, Anaplasma marginale, Babesia bigemina, Babesia bovis, and Theileria parva). Testing of field samples was compared to results obtained by other serological tests, resulting in a sensitivity and specificity of 96.3% and 87.5% compared to indirect fluorescence antibody test, 98.7% and 81.8% compared to indirect ELISA, and 100% and 47.6% compared to competitive ELISA. In conclusion, a rapid test for the detection of T. annulata infection (T. annulata lateral flow device, Ta-LFD) has been developed, which is easy to perform, delivers results to be read by the naked eye within 10 min, and is suitable for the detection of infection in field samples.

Identification of clone-9 antigenic protein of Theileria uilenbergi and evaluation of its application for serodiagnosis.

The pathogenic protozoan parasite Theileria uilenbergi is one of the causative agents of theileriosis in small ruminants in China. The infection results in great economical losses in the northwest part of China. Efforts are underway to establish an enzyme-linked immunosorbent assay (ELISA) based on a T. uilenbergi immunodominant recombinantly expressed protein using different approaches in order to perform epidemiological studies in the area. In this study, we describe the possible use of the clone-9 protein for this purpose, which was identified as a potential immunogenic piroplasm protein by random sequencing of cDNA library clones followed by bioinformatic analyses. The clone-9 gene was partially recombinantly expressed and used for the development of an indirect ELISA for the detection of circulating antibodies in sera of T. uilenbergi-infected sheep. No cross-reactivity was observed in serum from animals infected with Theileria lestoquardi. The cut-off was calculated at 48.6% positivity using 25 serum samples from uninfected animals. A total of 101 field samples collected from an endemic area in China were used to evaluate the clone-9 ELISA for its use in the field.

Epidemiological studies on tropical theileriosis (Theileria annulata infection of cattle) in Kurdistan Region, Iraq.

In an ad hoc survey conducted during 2006, the epidemiology of tropical theileriosis in Kurdistan Region, Iraq, was addressed. For this purpose, a total of 299 blood samples were collected from female cattle older than 1 year reared under open system management in Duhok (n = 99), Sulaimanyia (n = 100) and Erbil (n = 100) governorates. The samples were subjected to TaSP indirect ELISA as well as polymerase chain reaction (PCR) and nested PCR assays. The results indicated that the seroprevalence was 77.9%, and PCR reported an infection rate of 68.9% in the Kurdistan Region of Iraq. The implication of the results in the epidemiology of tropical theileriosis in the region is discussed with emphasis on comparisons between the two tests used and recommendations for the future work are outlined.

Identification of Theileria uilenbergi immunodominant protein for development of an indirect ELISA for diagnosis of ovine theileriosis.

Theileriosis of small ruminants in the northwest of China is a protozoan disease that restricts the development of the livestock industry. The disease is caused by infection with Theileria uilenbergi and Theilerialuwenshuni, both of which are transmitted by ixodid Heamaphysalis ticks. The development of serological tools as a means of integrated control of the disease is an urgent and important requirement. Here we describe the identification and partial recombinant expression of a T.uilenbergi immunodominant protein (TuIP), which was identified by immunoscreening of a merozoite cDNA library. Using the recombinant TuIP (rTuIP), a novel indirect ELISA was established using 329 negative serum samples to determine the cut-off value. The internal quality control revealed satisfactory stability and repeatability of the assay. Preliminary validation using 128 positive and 48 negative reference samples demonstrated that the rTuIP ELISA is able to detect T. uilenbergi infection with high sensitivity and specificity. No cross-reactivity was found in sera from animals infected with Theileria lestoquardi, Babesia sp. China or Anaplasma ovis. Furthermore, circulating antibodies were detected in sera collected from endemic regions in China. Analyses of the antibody responses of experimentally infected animals demonstrated that tick infestation resulted in a sharply rising and stronger production of specific antibodies against TuIP while inoculation with infected blood induced an earlier production of TuIP-specific antibodies. The persistence of the TuIP-specific antibodies lasted more than 100days p.i. These data indicate the usefulness of the TuIP antigen for the development of diagnostic methods and as a potential candidate for vaccine design.

Field validation of a competitive ELISA for detection of Theileria annulata infection.

In previous studies, Theileria annulata surface protein (TaSP) was identified as an immunodominant antigen and successfully used to develop a recombinant-protein-based indirect enzyme-linked immunosorbent assay (ELISA) for the detection of circulating antibodies in serum of T. annulata-infected animals. To increase the specificity, a competitive ELISA (cELISA) was developed using recombinant TaSP antigen and a monoclonal antibody (1C7) specifically binding to TaSP. Since the cELISA accurately differentiated T. annulata-infected from uninfected animals, a study was performed to analyse the suitability of the cELISA in the field. For this, 230 sera with unknown status from different governorates in the north of Iraq were analysed using both the indirect and competitive ELISA and were compared. There was a significant (p < 0.5 x 10(-19)) correlation (r = 0.556) between the tests, whereby the cELISA detected more sera as negative (44/230) compared to the indirect ELISA (21/270). Accordingly, less sera were determined to be positive in the competitive (186/230) than in the indirect ELISA (209/230). Sensitivity and specificity of the cELISA taking the indirect ELISA as a reference were 84.2% and 52.4%, respectively. Accordingly, the calculated prevalence of T. annulata infection was 90.9%, and the positive predictive value was determined to be 94.6%. Taken together, the cELISA proved its suitability for field application and was found qualified for use in serological surveys to monitor the prevalence of T. annulata infection and to identify carrier animals.