Assessing the clinical severity of the Omicron variant in the Western Cape Province, South Africa, using the diagnostic PCR proxy marker of RdRp target delay to distinguish between Omicron and Delta infections: a survival analysis

Background Emerging data suggest that SARS-CoV-2 Omicron variant of concern (VOC)is associated with reduced risk of severe disease. The extent to which this reflects a difference in the inherent virulence of Omicron, or just higher levels of population immunity, is currently not clear. Methods RdRp target delay (RTD: a difference in cycle threshold value of RdRp - E > 3.5) in the Seegene AllplexTM 2019-nCoV PCR assay is a proxy marker for the Delta VOC. The absence of this proxy marker in the transition period was used to identify suspected Omicron VOC infections. Cox regression was performed for the outcome of hospital admission in those who tested positive for SARS-CoV-2 on the Seegene AllplexTM assay from 1 November to 14 December 2021 in the Western Cape Province, South Africa, public sector. Vaccination status at time of diagnosis, as well as prior diagnosed infection and comorbidities, were adjusted for. Results 150 cases with RTD (proxy for Delta) and 1486 cases without RTD (proxy for Omicron) were included. Cases without RTD had a lower hazard of admission (adjusted Hazard Ratio [aHR] of 0.56, 95% confidence interval [CI] 0.34-0.91). Complete vaccination was protective of admission with an aHR of 0.45 (95%CI 0.26-0.77). Conclusion Omicron has resulted in a lower risk of hospital admission, compared to contemporaneous Delta infection in the Western Cape Province, when using the proxy marker of RTD. Under-ascertainment of reinfections with an immune escape variant like Omicron remains a challenge to accurately assessing variant virulence.

SARS-CoV-2 variants of concern dominate in Lahore, Pakistan in April 2021

The SARS-CoV-2 pandemic continues to expand globally, with case numbers rising in many areas of the world, including the Indian sub-continent. Pakistan has one of the world’s largest populations, of over 200 million people and is experiencing a severe third wave of infections caused by SARS-CoV-2 that began in March 2021. In Pakistan, during the third wave until now only 12 SARS-CoV-2 genomes have been collected and among these nine are from Islamabad. This highlights the need for more genome sequencing to allow surveillance of variants in circulation. In fact, more genomes are available among travellers with a travel history from Pakistan, than from within the country itself. We thus aimed to provide a snapshot assessment of circulating lineages in Lahore and surrounding areas with a combined population of 11.1 million. Within a week of April 2021, 102 samples were sequenced. The samples were randomly collected from two hospitals with a diagnostic PCR cutoff value of less than 25 cycles. Analysis of the lineages shows that the Alpha variant of concern (first identified in the UK) dominates, accounting for 97.9 % (97/99) of cases, with the Beta variant of concern (first identified in South Africa) accounting for 2.0 % (2/99) of cases. No other lineages were observed. In depth analysis of the Alpha lineages indicated multiple separate introductions and subsequent establishment within the region. Eight samples were identical to genomes observed in Europe (seven UK, one Switzerland), indicating recent transmission. Genomes of other samples show evidence that these have evolved, indicating sustained transmission over a period of time either within Pakistan or other countries with low-density genome sequencing. Vaccines remain effective against Alpha, however, the low level of Beta against which some vaccines are less effective demonstrates the requirement for continued prospective genomic surveillance.

Genome-wide association study reveals 14 new SNPs and confirms two structural variants highly associated with the horned/polled phenotype in goats

Background There is a long-term interest in investigating the genetic basis of the horned/polled phenotype in domestic goats. Here, we report a genome-wide association study (GWAS) to detect the genetic loci affecting the polled phenotype in goats. Results We obtained a total of 13,980,209 biallelic SNPs, using the genotyping-by-sequencing data from 45 Jintang Black (JT) goats, which included 32 female and nine male goats, and four individuals with the polled intersex syndrome (PIS). Using a mixed-model based GWAS, we identified two association signals, which were located at 150,334,857–150,817,260 bp (P = 5.15 × 10− 119) and 128,286,704–131,306,537 bp (P = 2.74 × 10− 15) on chromosome 1. The genotype distributions of the 14 most significantly associated SNPs were completely correlated with horn status in goats, based on the whole-genome sequencing (WGS) data from JT and two other Chinese horned breeds. However, variant annotation suggested that none of the detected SNPs within the associated regions were plausible causal mutations. Via additional read-depth analyses and visual inspections of WGS data, we found a 10.1-kb deletion (CHI1:g. 129424781_129434939del) and a 480-kb duplication (CHI1:150,334,286–150,818,098 bp) encompassing two genes KCNJ15 and ERG in the associated regions of polled and PIS-affected goats. Notably, the 10.1-kb deletion also served as the insertion site for the 480-kb duplication, as validated by PCR and Sanger sequencing. Our WGS genotyping showed that all horned goats were homozygous for the reference alleles without either the structural variants (SVs), whereas the PIS-affected goats were homozygous for both the SVs. We also demonstrated that horned, polled, and PIS-affected individuals among 333 goats from JT and three other Chinese horned breeds can be accurately classified via PCR amplification and agarose gel electrophoresis of two fragments in both SVs. Conclusion Our results revealed that two genomic regions on chromosome 1 are major loci affecting the polled phenotypes in goats. We provided a diagnostic PCR to accurately classify horned, polled, and PIS-affected goats, which will enable a reliable genetic test for the early-in-life prediction of horn status in goats.

Genotyping of potato samples from the GenAgro ICG SB RAS collection using DNA markers of genes conferring resistance to phytopathogens

Wart (a disease caused by Synchytrium endobioticum) and golden cyst potato nematode (Globodera rostochiensis), which parasitize the roots of the host plant, cause significant damage to potato crop. Both of these disease factors are quarantined in the Russian Federation, and each registered variety is tested for resistance to their most common races and pathotypes. The main method of opposing such diseases is by the development of resistant varieties. An important step in this process is the selection of resistant genotypes from the population and the estimation of the resistance of hybrids obtained by crosses during the breeding process. Conducting a permanent phenotypic evaluation is associated with difficulties, for example, it is not always possible to work with pathogens, and phenotypic evaluation is very costly and time consuming. However, the use of DNA markers linked to resistance genes can significantly speed up and reduce the cost of the breeding process. The aim of the study was to screen the GenAgro potato collection of ICG SB RAS using known diagnostic PCR markers linked to golden potato cyst nematode and wart resistance. Genotyping was carried out on 73 potato samples using three DNA markers 57R, CP113, Gro1-4 associated with nematode resistance and one marker, NL25, associated with wart resistance. The genotyping data were compared with the data on the resistance of the collection samples. Only the 57R marker had a high level of correlation (Spearman R = 0.722008, p = 0.000000, p < 0.05) between resistance and the presence of a diagnostic fragment. The diagnostic efficiency of the 57R marker was 86.11 %. This marker can be successfully used for screening a collection, searching for resistant genotypes and marker-assisted selection. The other markers showed a low correlation between the presence of the DNA marker and resistance. The diagnostic efficiency of the CP113 marker was only 44.44 %. Spearman’s correlation coefficient (Spearman R = –0.109218, p = 0.361104, p < 0.05) did not show significant correlation between resistance and the DNA marker. The diagnostic efficiency of the NL25 marker was 61.11 %. No significant correlation was found between the NL25 marker and resistance (Spearman R = –0.017946, p = 0.881061, p < 0.05). The use of these markers for the search for resistant samples is not advisable.

Personal observations on COVID-19 and the conduct and application of biomedical science

We begin by describing our observations of the ways in which the conduct of research has changed during the COVID-19 pandemic and go on to comment on the quality of the scientific advice that is provided to UK citizens, and especially to schools. Researchers, like many, have suffered from the effects of the pandemic. Those hardships notwithstanding, we suggest that research into COVID-19 has benefitted from a ‘seed corn’ of discovery science that has provided the basis for routine diagnostic PCR and antibody tests; for structural analyses of the way in which the SARS-CoV-2 virus interacts with cells; for the development of new treatments (and the debunking of ineffective ones); for studies of the genetics of susceptibility to SARS-CoV-2; and for the development of vaccines. The speed of dissemination of research has benefitted from the widespread use of pre-prints, and researchers and funders have become more nimble in their approaches to research and more willing to change their priorities in the face of the pandemic. In our experience, the advice provided to schools on the basis of this research was, however, often published at the last minute and was frequently flawed or inconsistent. This has led to a widening of the attainment gap between children from disadvantaged backgrounds and their peers and it has exacerbated the digital divide and holiday hunger. The consequences will be felt for many years to come and will jeopardize diversity in research and other careers.

Rapid Detection and Typing of Actinobacillus pleuropneumoniae Serovars Directly From Clinical Samples: Combining FTA® Card Technology With Multiplex PCR

Actinobacillus pleuropneumoniae (APP), the causative agent of porcine pleuropneumonia, is highly contagious and responsible for high morbidity, mortality, and economic losses in the swine industry worldwide, but quick serotyping and diagnosis are still not widely available. In this study, we sought to validate the use of Whatman FTA® cards for collection and processing of A. pleuropneumoniae isolates, or porcine lung tissue samples, for direct use in diagnostic multiplex PCRs. We have optimized the processing of 3-mm discs punched from FTA® cards loaded with cultured A. pleuropneumoniae, or imprinted on lesioned regions of lung tissue, with only three distilled water washes before addition into our APP-multiplex PCR (mPCR) assay for rapid, low-cost identification and serotyping. DNA captured on FTA® cards generated the same diagnostic PCR results as DNA extracted using commercial kits for 85 A. pleuropneumoniae clinical isolate cultures and 22 lung samples. Additionally, bacterial DNA bound to FTA® cards was detectable by PCR after 6 months of storage at 37°C. This study provides simple, efficient, rapid, and practical sample processing for detection and molecular serotyping of A. pleuropneumoniae.

Are members of the Anopheles fluviatilis complex conspecific?

Anopheles fluviatilis sensu lato, a primary malaria vector in India, was identified to be comprised of four cryptic species, provisionally designated as species S, T, U and V. However, Kumar et al. (Mol Ecol Resour, 2013;13:354-61) considered all of the then known three members of this species complex (S, T and U) conspecific. The specific status of species S and T was refuted based on the lack of sufficient barcode gap in mitochondrial-CO1 and the perceived presence of heterozygotes in populations as detected through one of the two species-specific PCR assays employed for the cryptic species identification. The existence of species U was refuted claiming that earlier investigations have already refuted their existence. This conclusion is concerning because of the differential public health implications of members of the Fluviatilis Complex. Here we discuss problems associated with the CO1-based barcode approach for delimitation of cryptic species, the perceived heterozygosity between species S and T based on a species-specific PCR assay, and interpretation of published reports. We demonstrated that fixed differences do exist in the ITS2-rDNA sequence of species S and T with no evidence of heterozygotes in sympatric populations and, that the observed heterozygosity by Kumar et al. in the ITS2-based species diagnostic PCR is due to the high mispriming tendency of the T-specific primer with species S. We infer that mitochondrial DNA-based barcoding-gap, an arbitrary threshold recommended for species delimitation, alone, is inadequate to delimit the members of An. fluviatilis complex.

Bulinus senegalensis and Bulinus umbilicatus Snail Infestations by the Schistosoma haematobium Group in Niakhar, Senegal

Thorough knowledge of the dynamics of Bulinus spp. infestation could help to control the spread of schistosomiasis. This study describes the spatio-temporal dynamics of B. senegalensis and B. umbilicatus infestation by the Schistosoma haematobium group of blood flukes in Niakhar, Senegal. Molecular identification of the S. haematobium group was performed by real-time PCR, targeting the Dra 1 gene in 810 samples of Bulinus spp. collected during the schistosomiasis transmission season in 2013. In addition to Dra 1 PCR, a rapid diagnostic-PCR was performed on a sub-group of 43 snails to discriminate S. haematobium, S. bovis, and S. mattheei. Out of 810 snails, 236 (29.1%) were positive for Dra 1 based on the PCR, including 96.2% and 3.8% of B. senegalensis and B. umbilicatus, respectively. Among the sub-group, 16 samples were confirmed to be S. haematobium while one was identified as a mixture of S. haematobium and S. bovis. Snails infestations were detected in all villages sampled and infestation rates ranged from 15.38% to 42.11%. The prevalence of infestation was higher in the north (33.47%) compared to the south (25.74%). Snail populations infestations appear early in the rainy season, with a peak in the middle of the season, and then a decline towards the end of the rainy season. Molecular techniques showed, for the first time, the presence of S. bovis in the Bulinus spp. population of Niakhar. The heterogeneity of snail infestations at the village level must be taken into account in mass treatment strategies. Further studies should help to improve the characterizations of the schistosome population.

New Developments in PCR-Based Diagnostics for Bacterial Pathogens Causing Gastrointestinal Infections—A Narrative Mini-Review on Challenges in the Tropics

The application of modern PCR approaches for the diagnosis of bacterial gastrointestinal pathogens is on the rise due to their rapidly available results combined with high sensitivity. While multiple studies describe the ongoing implementation of this technique for routine diagnostic purposes in laboratories in Western industrialized countries, reports on successful and also sustainable respective approaches in resource-poor tropical settings are still scarce. In order to shed light on potential reasons for this marked discrepancy, this narrative review summarizes identified challenges for the application of diagnostic PCR targeting bacterial gastrointestinal pathogens from stool samples in the tropics. The identified and discussed issues comprise the lack of generally accepted definitions for (1) minimum standards regarding sample acquisition, storage and transport time for diagnostic PCR analyses in the tropics, (2) nucleic acid extraction standards allowing an optimum detection of all types of pathogens which may be responsible for gastroenteritis in the tropics, (3) validation standards to ensure comparable quality of applied diagnostic assays, and (4) cut-offs for a reliable discrimination of infection and mere colonization in areas where semi-immunity due to repeated exposition associated with poor hygiene conditions has to be expected. Further implementation research is needed to solve those issues.

Resource-efficient internally controlled in-house real-time PCR detection of SARS-CoV-2

Background The reliable detection of SARS-CoV-2 has become one of the most important contributions to COVID-19 crisis management. With the publication of the first sequences of SARS-CoV-2, several diagnostic PCR assays have been developed and published. In addition to in-house assays the market was flooded with numerous commercially available ready-to-use PCR kits, with both approaches showing alarming shortages in reagent supply. Aim Here we present a resource-efficient in-house protocol for the PCR detection of SARS-CoV-2 RNA in patient specimens (RKI/ZBS1 SARS-CoV-2 protocol). Methods Two duplex one-step real-time RT-PCR assays are run simultaneously and provide information on two different SARS-CoV-2 genomic regions. Each one is duplexed with a control that either indicates potential PCR inhibition or proves the successful extraction of nucleic acid from the clinical specimen. Results Limit of RNA detection for both SARS-CoV-2 assays is below 10 genomes per reaction. The protocol enables testing specimens in duplicate across the two different SARS-CoV-2 PCR assays, saving reagents by increasing testing capacity. The protocol can be run on various PCR cyclers with several PCR master mix kits. Conclusion The presented RKI/ZBS1 SARS-CoV-2 protocol represents a cost-effective alternative in times of shortages when commercially available ready-to-use kits may not be available or affordable.