Shortening turnaround time for high-priority patients during the COVID-19 epidemic: evaluation of the Xpert Xpress SARS-CoV-2 test

Background: Although several molecular tests are now available for detecting SARS-CoV-2 RNA in nasopharyngeal swab samples, the number of requested tests exceeds the capacity of many diagnostic laboratories. Unfortunately, the available high-throughput platforms exhibit longer turnaround times than those required for management of high-priority patients. Methods: The aim of this study was to evaluate the performance and possible benefits of the Cepheid Xpert Xpress SARS-CoV-2 test, focusing mainly on turnaround time when applied to high-priority patients. We evaluated the Xpert Xpress SARS-CoV-2 test in comparison to the Roche’s cobas 6800 SARS-CoV-2 test by monitoring turnaround times and by retrospectively testing 20 nasopharyngeal swabs from COVID-19 patients with various viral loads. In addition, 50 patients were tested by both methods prospectively. Results: We observed a lower limit of detection of one SARS-CoV-2 genome equivalent/µL and 100% (95% CI, 92.6−100%) specificity and 95.5% (95% CI, 77.2−99.9%) sensitivity in comparison to the cobas SARS-CoV-2 test. When applying the Xpert Xpress SARS-CoV-2 test for high-priority patients the turnaround time could be greatly reduced, i.e. from 3 - 5 hours that take our routine diagnostics methods to about 1 hour. Conclusion: The novel Xpert Xpress SARS-CoV-2 test is a useful, easy to perform tool, valuable for rapid and reliable diagnosis of COVID-19, especially in high-priority patients when a short turnaround time is of key importance for further patient management.

Pedigree analysis of a red fox (Vulpes vulpes) population

Fur animal breeding has a long history. In many countries several fur animal species (including the red fox) have been recognized as livestock. The aim of this study was to estimate the pedigree parameters in the population of red fox on a Polish breeding farm. The data set consisted of information on 39 434 individuals, including 18 697 females and 20 004 males (733 animals were of unknown sex), from the years 1956–2016. The following pedigree parameters were estimated: average number of discrete generation equivalents, individual inbreeding coefficient, total and effective number of founders, effective population size, average relationship, founder genome equivalent, effective number of non-founders, and genetic diversity coefficient. The population size changed in successive years. The average inbreeding level was 5.34% for the population as a whole, and 6.04% for the inbred population. The estimated effective number of founders of the population was 84.18. The founder genome equivalent, which indicates the anticipated loss of genetic diversity caused by genetic drift, reached 9.59 in 2016 from an initial value of 34.22 in 1956. The loss of genetic diversity caused by the unequal contribution of the founder alleles did not change significantly over the years. Generally, the results indicate the good pedigree structure (including pedigree completeness) of the population studied. This implies reliable estimation of the inbreeding level, as one of the most important parameters in the genetic improvement programme.

Wide Range of the Prevalence and Viral Loads of Porcine Circovirus Type 3 (PCV3) in Different Clinical Materials from 21 Polish Pig Farms

Porcine circovirus type 3 (PCV3) was described in different clinical cases and healthy pigs. However, little is known about its circulation in pig farms. In order to assess PCV3 prevalence in 21 Polish farms, serum, feces, and oral fluid samples were examined by quantitative real-time PCR. In total, 1451 pairs of serum and feces from the same animals, as well as 327 samples of oral fluids were analyzed. The results showed that PCV3 is more commonly detected in oral fluids (37.3% positives) than in serum (9.7% positives) or feces (15.0% positives) samples. The viral loads detected in these materials ranged from 102.5–107.2 genome equivalent copies/mL. Although in most farms PCV3 was detected post weaning, in nine farms, the virus was also found in groups of suckling piglets, and in six of them viremia was detected. In four farms with reproductive failure, fetal materials were also obtained. PCV3 was detected in 36.0% of fetuses or stillborn piglets (9/25) with viral loads of 103.1–1010.4 genome equivalent copies/mL. In summary, the virus circulation may show different patterns, and congenital or early infection is not uncommon. Precise quantification of PCV3 loads in clinical materials seems to be necessary for the study and diagnosis of the infection.

Genetic analysis of the Hungarian population of endangered Hucul horses

The population genetic evaluation of the Hungarian Hucul horse population was performed based on pedigree records. Herd book data of registered Hucul horses available up to 2016 were analysed using ENDOG (Gutierrez and Goyache 2005) and POPREP (Groeneveld et al. 2009) on the whole population (WP) as well as on the reference stock (RS) (breeding stock registered in 2016). Inbreeding coefficients were 5.57% (WP) and 7.18% (RS). Average relatedness was 10.39% in WP and higher in RS (12.67%). Effective population size was 52.32. Generation interval was 13.01 years for WP and 10.99 years for RS. The values for equivalent complete generations were 6.07 and 8.75, for the maximum number of generations 14.11 and 19.16, and for the number of full generations traced 3.77 and 5.50 for WP and RS, respectively. The effective number of founders (f_e) was 23 both for WP and RS. The effective number of ancestors (f_a) was 20 in WP and lower in RS (16). The f_a/f_e ratio was 0.869 in WP and 0.696 in RS. Founder genome equivalent (f_g) was 9.618 in WP and 5.790 in RS. The f_g/f_e ratio was 0.481 in WP and 0.361 in RS. The study revealed that both the inbreeding coefficient and the average relatedness were high. The above mentioned ratios indicated loss of genetic diversity in the Hungarian Hucul population.

Analysis of population structure and genetic variability in Iranian buffaloes (Bubalus bubalis) using pedigree information

The objective of this study was to use pedigree analysis to evaluate the population structure, genetic variability and inbreeding in Iranian buffaloes. The analysis was based on the pedigree information of 42 285 buffaloes born from 549 sires and 6376 dams within 1697 herds. Pedigree information used in this study was collected during 1976 to 2012 by the Animal Breeding Centre of Iran. The CFC program was applied to calculate pedigree statistics and genetic structure analysis of the Iranian buffaloes. Also, the INBUPGF90 program was used for calculating regular inbreeding coefficients for individuals in the pedigree. The analysis of pedigree indicated that inbreeding coefficient ranged from 0% to 31% with an average of 3.42% and the trend of inbreeding was significantly positive over the years (P < 0.0001). Average coancestry was increased in recent years and overall generation interval was 6.62 years in Iranian buffaloes. Founder genome equivalent, founder equivalent, effective number of founders and effective number of non-founders were increased from 1976 to 2002, but their values decreased from 2002 onwards. A designed mating system to avoid inbreeding may be applied to this population of buffalo to maintain genetic diversity.

Construction of a Llama Bacterial Artificial Chromosome Library with Approximately 9-Fold Genome Equivalent Coverage

The Ilama is an important agricultural livestock in much of South America. The llama is increasing in popularity in the United States as a companion animal. Little work has been done to improve llama production using modern technology. A paucity of information is available regarding the llama genome. We report the construction of a llama bacterial artificial chromosome (BAC) library of about 196,224 clones in the vector pECBAC1. Using flow cytometry and bovine, human, mouse, and chicken as controls, we determined the llama genome size to be2.4×109 bp. The average insert size of the library is 137.8 kb corresponding to approximately 9-fold genome coverage. Further studies are needed to further characterize the library and llama genome. We anticipate that this new library will help facilitate future genomic studies in the llama.

Circulating tumor DNA in plasma of breast cancer patients from India

e22213 Background: Recently, breast cancer has become the most common cancer among women in all urban population in India. Annually about 80000 new cases and 40000 deaths occur and majority of breast cancers are pre-menopausal. Conventional diagnostic methods are not very sensitive especially in early stages of cancer. This necessitated a more sensitive and reliable method for early diagnosis leading to effective treatment, better prognosis and survival. Recently, the level of cell free circulating tumor DNA in blood plasma or serum of patients with variety of tumors are being considered as reliable non-invasive diagnostic tool but no study has been done in India. The present study has therefore been undertaken to evaluate clinical utility of cell free DNA as potential biomarkers for early diagnosis and management of breast cancer. Methods: 25 newly diagnosed untreated breast cancer patients and 25 healthy subjects having no sign of significant medical illness with informed consent were enrolled for the study. 9 patients after chemotherapy were also included in the study. Blood plasma collected from both patients and controls were employed for DNA isolation, using Qiagen kit. Concentration of cell free plasma DNA was analyzed by 3 methods viz. nanodrop spectro-photometry, integrated density value (IDV) of PCR products of Exon 7 of p53 gene and quantitative real time PCR (cycles threshold converted to genome equivalent). All values of DNA concentration obtained by three methods used as continuous variables and receiver operating characteristic (ROC) were plotted and the cut-of value was determined at 90% sensitivity and 100% specificity level of ROC. Results: Mean free plasma DNA concentration as determined by both Q-RT PCR and IDV in cancer patients was found to be significantly higher in advanced stage breast cancer patients than in controls (genome equivalent 18850 vs 431; IDV 17912 vs 4197; p=0.001). However, no significant difference could be observed in early stage disease as compared to controls possibly due small sample size. Conclusions: Free Plasma DNA concentration is a reliable molecular marker for detection of breast cancer and can serve as a prognostic indicator leading to its potential clinical application either alone or in combination with other conventional methods. No significant financial relationships to disclose.