Specific Polymerase Chain Reaction Assay Research Articles

A highly sensitive and specific real-time quantitative polymerase chain reaction assay for Perkinsus marinus detection in oysters

Oyster aquaculture is one of the fastest-growing food production industries worldwide; however, it faces a significant challenge from the protist Perkinsus marinus, particularly in the USA. Although several quantitative molecular diagnostic methodologies are available for identifying diseases caused by P. marinus, the primer pairs used therein led to non-specific identification of other Perkinsus spp. Hence, a quantitative real-time PCR (Pm-qPCR) assay specific for P. marinus was developed using a TaqMan-based probe with the internal quencher in this study. A primer pair and probe specific to P. marinus were designed from a hypothetical protein of P. marinus collected from the whole-genome shotgun sequence database of the National Center for Biotechnology Information (NCBI). In silico analysis using homologous sequences of P. olseni and P. chesapeaki confirmed the high specificity of primers designed in this study. The Pm-qPCR assay was performed using seven different strains of P. marinus, P. olseni, and P. chesapeaki, revealing high specificity and sensitivity for detecting only P. marinus strains. In conclusion, it was demonstrated that Pm-qPCR can effectively and accurately diagnose P. marinus with high specificity and sensitivity. This assay is promising for monitoring oyster health and disease management in ecosystems and aquaculture.

Development and evaluation of a multi-target droplet digital PCR assay for highly sensitive and specific detection of Yersinia pestis.

Plague, caused by the bacterium Yersinia pestis, is a zoonotic disease that poses considerable threats to human health. Nucleic acid tests are crucial for plague surveillance and the rapid detection of Y. pestis. However, inhibitors in complex samples such as soil and animal tissues often hamper nucleic acid detection, leading to a reduced rate of identifying low concentrations of Y. pestis. To address this challenge, we developed a sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay for detecting Y. pestis DNA from soil and animal tissue samples. Three genes (ypo2088, caf1, and pla) from Y. pestis were used to develop a multi-target ddPCR assay. The limits of detection (LoD), reproducibility, and specificity were assessed for bacterial genomic DNA samples. The ability of the assay to detect low concentrations of Y. pestis DNA from simulated soil and mouse liver tissue samples was respectively evaluated and compared with that of quantitative real-time PCR (qPCR). The results showed that the ddPCR LoDs ranged from 6.2 to 15.4 copies/reaction for the target genes, with good reproducibility and high specificity for Y. pestis. By testing 130 soil and mouse liver tissue samples spiked with Y. pestis, the ddPCR assay exhibited a better sensitivity than that of the qPCR assay used in the study, with LoDs of 102 colony forming units (CFU)/100 mg soil and 103 CFU/20 mg liver. Moreover, the assay presented good quantitative linearity (R2 = 0.99) for Y. pestis at 103-106 CFU/sample for soil and liver samples. The ddPCR assay presented good performance for detecting Y. pestis DNA from soil and mouse tissue samples, showing great potential for improving the detection rate of low concentrations of Y. pestis in plague surveillance and facilitating the early diagnosis of plague cases.

Development and evaluation of specific polymerase chain reaction assays for detecting Theileria equi genotypes

BackgroundTheileria equi causes equine piroplasmosis, an economically significant disease that affects horses and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classified into five genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However, no conventional polymerase chain reaction (PCR) assays are available to differentiate the genotypes of T. equi. To overcome this limitation, we developed and evaluated PCR assays specific for the detection of each T. equi genotype.MethodsA pair of forward and reverse primers, specifically targeting the 18S rRNA sequence of each genotype, was designed. The genotype-specific PCR assays were evaluated for their specificity using plasmids containing inserts of the 18S rRNA sequence of each genotype. Subsequently, the assays were tested on 270 T. equi-positive equine blood DNA samples (92 from donkeys in Sri Lanka and 178 from horses in Paraguay). 18S rRNA sequences derived from the PCR amplicons were analyzed phylogenetically.ResultsEach genotype-specific PCR assay accurately targeted the intended genotype, and did not produce any amplicons when 18S rRNA from other T. equi genotypes or genomic DNA of Babesia caballi or uninfected horse blood was used as the template. Previous studies employing PCR sequencing methods identified T. equi genotypes C and D in the Sri Lankan samples, and genotypes A and C in the Paraguayan samples. In contrast, our PCR assay demonstrated exceptional sensitivity by detecting four genotypes (A, C, D, and E) in the Sri Lankan samples and all five genotypes in the Paraguayan samples. All the Sri Lankan samples and 93.3% of the Paraguayan samples tested positive for at least one genotype, further emphasizing the sensitivity of our assays. The PCR assays also had the ability to detect co-infections, where multiple genotypes in various combinations were detected in 90.2% and 22.5% of the Sri Lankan and Paraguayan samples, respectively. Furthermore, the sequences obtained from PCR amplicons clustered in the respective phylogenetic clades for each genotype, validating the specificity of our genotype-specific PCR assays.ConclusionsThe genotype-specific PCR assays developed in the present study are reliable tools for the differential detection of T. equi genotypes.Graphical abstract

THE FIRST SURVEY OF BOVINE BABESIA SPECIES INFECTING YAKS (BOS GRUNNIENS) IN MONGOLIA.

Yak (Bos grunniens) farming is an important part of Mongolia's livestock industry. Yaks survive in harsh mountain environments; provide meat, milk, and wool; and serve as a mode of transportation. In Mongolia, yaks are frequently raised alongside other livestock animals such as cattle, Bactrian camels, sheep, goats, and horses. Recently, we demonstrated that Babesia bovis, Babesia bigemina, and Babesia naoakii-parasites with the potential to cause clinical bovine babesiosis-infect not only cattle but also Bactrian camels in Mongolia. However, yaks have never been surveyed for Babesia infections in this country. In the present study, we surveyed yaks in 8 Mongolian provinces: Bayankhongor, Bayan-Ulgii, Khovd, Khovsgol, Omnogovi, Ovorkhangai, Uvs, and Zavkhan. Blood samples were taken and deoxyribonucleic acid (DNA) was extracted from 375 yaks. Furthermore, Giemsa-stained thin smears were prepared from 315 of the 375 blood samples and then examined for the microscopic detection of Babesia parasites. Microscopy revealed that 34 (10.8%) of 315 blood smears were positive for Babesia parasites. All 375 DNA samples were then tested for B. bovis, B. bigemina, and B. naoakii infection using specific polymerase chain reaction assays. We observed that 238 (63.5%) yaks in all surveyed provinces and 8 (2.1%) yaks in 3 provinces (Bayankhongor, Bayan-Ulgii, and Omnogovi) were positive for B. bovis and B. bigemina, respectively. However, all yaks tested were negative for B. naoakii. This epidemiological survey, the first to report Babesia infection in Mongolian yaks, suggests that disease management strategies for yaks in this country should further address bovine babesiosis.

Biosensor for PCR amplicons by combining split trehalase and DNA-binding proteins: A proof of concept study

The use of polymerase chain reaction (PCR) technology in low-cost settings has gained significant attention due to its ability to amplify and detect specific bacterial pathogen genes, aiding in the diagnosis of infectious diseases. PCR amplicons can be visualized by conventional endpoint agarose gel electrophoresis and fluorochrome-enabled real-time PCR. However, this is not practical in on-field tests due to cumbersome instrumentation, labor-intensive reaction preparation, and long time-to-results. Many studies have combined microfluidic devices or electrochemical dyes with PCR technology to enhance in-field operability. However, the high cost of manufacturing high-precision microfluidic chips and the dependence on non-portable readout equipment limit their further development. In this paper, we present a proof-of-principle study of a novel method combining split enzyme technology and DNA-binding proteins for the convenient and efficient detection of amplified genetic material from bacterial pathogens. The amplicon binding split trehalase assay (ABSTA) relies on incorporating specific recognition sequences of DNA-binding protein SpoIIID in tandem within one of the PCR primers. Applied by a Gram-type specific PCR assay, ABSTA was capable of discriminating Staphylococcus devriesei and Escherichia coli in <90 min after colony PCR amplicons bound split trehalase fragments-fused SpoIIID and triggered split enzyme complementation. The salt concentration, protein reagents versus DNA substrate ratio, direction and linker length of tandem recognition sites required for the complementation were optimized. The glucose produced by restored enzymatic activity was detectable by glucometer. With limited requirements for reaction preparation and the compatibility of ABSTA with commercially available handheld glucometers, this test platform has substantial potential to be implemented into a future point-of-care (POC) diagnostic tool for detecting pathogen specific genes with further improvement.

Detection of resistance mutations in patients with anaplastic lymphoma kinase-rearranged lung cancer through liquid biopsy.

Tyrosine kinase inhibitors (TKIs) significantly improve clinical outcomes in patients with non-small cell lung cancer due to anaplastic lymphoma kinase (ALK) gene rearrangement. However, the rate of relapse with TKIs is high owing to the development of resistance mutations during treatment. Repeated biopsies during disease progression are crucial for elucidating the molecular mechanisms underlying the development of resistance to ALK inhibitors. Analysis of cell-free DNA (cfDNA) obtained from plasma is a novel approach for tumor genotyping. In this mixed prospective and retrospective observational cohort study, we investigated the clinical feasibility of continuous quantitative monitoring of ALK-acquired mutations in plasma obtained from patients with ALK+ non-small cell lung cancer by using a highly sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay. We enrolled nine patients, including three treatment-naïve patients recently diagnosed with ALK+ non-small cell lung cancer via tissue biopsy and expected to receive ALK TKIs and six patients already receiving ALK TKIs. Plasma samples were collected from these patients every 3 months. cfDNA was extracted from 66 samples during the study period, and 10 ALK mutations were simultaneously evaluated. The numbers of samples showing the G1202R, C1156Y, G1269A, F1174L, T1151ins, and I1171T mutations were 32, 16, 5, 4, 1, and 1, respectively. The L1196M, L1152R, V1180L, and S1206Y mutations were not detected. Correlation analyses between progression-free survival and the time from treatment initiation (or treatment modification) to the detection of resistance mutations revealed that although resistance mutations may occur before a drug change becomes necessary, there is a duration during which the disease does not progress. Our findings suggest that real-time quantitative monitoring of ALK resistance mutations during the response period could provide a time course of changes while acquiring resistance mutations. This information would be beneficial for designing an appropriate treatment strategy.

A specific and sensitive droplet digital polymerase chain reaction assay for the detection of tilapia lake virus in fish tissue and environmental samples.

Tilapia lake virus (TiLV) causes high mortality in farmed and wild tilapia in various countries. We developed a highly specific and sensitive droplet digital polymerase chain reaction (ddPCR) assay to detect and quantify TiLV. The ddPCR assay could detect the virus at a lower threshold than the reverse transcription-quantitative polymerase reaction (RT-qPCR) method, and the sensitivity of the ddPCR assay was 10-fold higher. The diagnostic sensitivity and specificity of the ddPCR assay were 100% and did not cross-react with tilapia tissues infected with Tilapia parvovirus, Infectious spleen and kidney necrosis virus, Aeromonas hydrophila, Streptococcus agalactiae, S. iniae and Francisella noatunensis. The assay reproducibility was demonstrated by a high correlation coefficient of 0.998, and the inter-assay coefficients of variability indicated that the ddPCR assay exhibited low variability within and between measurements. The detection limit of the TiLV ddPCR assay was 100 fg cDNA, which is equal to 3.3 copies of TiLV. Furthermore, the ddPCR assay could detect TiLV in mucus, water and infected tissue samples and the lowest copy number of TiLV detected in water samples by the ddPCR assay was 7.9 ± 0.99 copies/reaction The results of the clinical samples tested for TiLV revealed that the ddPCR assay had a relatively higher detection rate than the RT-qPCR method. Overall, the ddPCR method offers a highly promising approach for the absolute quantification of TiLV in carrier fish and samples from the environment with low viral concentrations.

Molecular Detection and Genetic Characterization of Bovine Kobuvirus (BKV) in Diarrhoeic Calves in a Central Italy Herd

Bovine kobuvirus (BKV) is an infectious agent associated with neonatal calf diarrhoea (NCD), causing important economic losses to dairy and beef cattle herds worldwide. Here, we present the detection rate and characterize the genome of BKV isolated from diarrhoeic calves from a Central Italy herd. From January to December 2021, we collected blood samples and nasal and rectal swabs from 66 calves with severe NCD between 3 and 20 days of age. After virological (bovine coronavirus, bovine viral diarrhoea virus, and bovine rotavirus), bacteriological (Escherichia coli spp. and Salmonella spp.), and parasitological (Cryptosporidium spp., Eimeria spp., and Giardia duodenalis) investigations, we detected BKV using the metagenomic analysis. This result was confirmed using a specific polymerase chain reaction assay that revealed the number of BKV-positive nasal (24.2%) and rectal swabs (31.8%). The prevalence of BKV was higher than that of BCoV. Coinfection with BKV and BCoV was detected in 7.5% of the rectal swabs, highlighting the involvement of another infectious agent in NCD. Using next generation sequencing (NGS) approach, it was possible to obtain the complete sequence of the BKV genome from other two rectal swabs previously analysed by real-time PCR. This is the first report describing the whole genome sequence (WGS) of BKV from Italy. The Italian BKV genomes showed the highest nucleotide sequence identity with BKV KY407744.1, identified in Egypt in 2014. The sequence encoding VP1 best matched that of BKV KY024562, identified in Scotland in 2013. Considering the small number of BKV WGSs available in public databases, further studies are urgently required to assess the whole genome constellation of circulating BKV strains. Furthermore, pathogenicity studies should be conducted by inoculating calves with either only BKV or a combination with other enteric pathogens for understanding the probable role of BKV in NCD.

Development of a specific real-time PCR assay for simultaneous detection and differentiation of Coxiella burnetii strains from environmental soil samples.

Coxiella burnetii, the causative agent of Q fever, is a small, coccoid, Gram-negative strict intracellular pathogen. One of the most common ways of acquiring Q fever is through inhalation of aerosols containing the bacteria. Because C. burnetii is highly infectious, spreads easily through the air, and is very resistant to environmental conditions, it is considered a biological threat. This paper presents the development and validation of a specific real-time polymerase chain reaction (real-time PCR or qPCR) assay for the detection of C. burnetii, based on the amplification of a fragment of the isocitrate dehydrogenase (icd) encoding gene. This real-time PCR is highly specific, reproducible, and sensitive, allowing the detection of as few as 5 genome equivalents (GEs) of C. burnetii per reaction. The method enables a rapid preliminary differentiation among strains, based on a point mutation at nucleotide 745 of the icd gene. The assay was successfully evaluated in environmental soil samples; a limit of detection of 3×104 colony forming units per 0.5g of soil (∼3 GEs per reaction) was achieved. The newly developed real-time PCR offers a valuable tool for differential detection of C. burnetii strains in environmental soil samples.

Evaluating outpatient diagnostic stewardship of comprehensive polymerase chain reaction Clostridioides difficile testing in a regional health system.

We examined the use of comprehensive and targeted polymerase chain reaction (PCR) of Clostridioides difficile infection (CDI) among immunocompetent patients with and without CDI risk factors across different outpatient settings. A priori, we expected patients with higher CDI risk to be associated with targeted testing to reflect providers incorporating pretest risk factors in their choice of test assay. Retrospective analysis of adult patients from clinic, emergency room, and non-medically acute inpatient settings. A tertiary academic medical center offering inpatient and outpatient medical, surgical, mental health, and rehabilitation services to Veterans across the Puget Sound region. Immunocompetent adult patients with ≥1 stool PCR assay performed between January 2016 and December 2019. Patients were tested with either a specific tcdB PCR assay or a comprehensive gastrointestinal PCR panel that tests for 22 pathogens. A total of 2,717 tests (74% targeted, 26% comprehensive) were obtained from 2,156 patients, among which 13% detected C. difficile and 7% detected other organisms. The proportion of comprehensive PCR tests increased nearly four-fold from 2016 to 2019 in clinic and emergency room settings, independent of CDI risk factors. Only two CDI risk factors (prior history of CDI and antibiotic use within three months before testing) were associated with increased targeted testing. The use of comprehensive GI PCR among immunocompetent adults with diarrhea is increasing in the outpatient setting. There may be an opportunity for diagnostic stewardship by nudging providers to consider all CDI risk factors at the time of test selection.

Distribution of Genotypes of Hepatitis C Virus Studied in a Tertiary Care Hospital in Uttarakhand, India: A Retrospective Study

Introduction: With more than one million cases of Hepatitis C viral infection reported annually, it has become a major global problem. Hepatitis C Virus (HCV) is classified into 7 genotypes and 67 subtypes. Various genotypes show a wide geographical distribution worldwide. Genotypes 1 and 3 are the most common genotypes reported from India. There is minimal data available on the distribution and frequency of HCV genotypes in Uttarakhand. Aim: This three years retrospective study was undertaken to find out the distribution pattern of HCV genotypes among HCV- infected patients attending a tertiary care hospital in this Sub- Himalayan state of Uttarakhand. Materials and Methods: A total of 306 samples were tested for HCV genotyping in 3 years duration i.e., January 2018 to December 2020 at the Molecular Laboratory of Shri Guru Ram Rai Institute of Medical and Health sciences and associated Shri Mahant Indiresh Hospital, Dehradun. Genotyping was performed using HCV genotype specific Real-Time Polymerase Chain Reaction (RT-PCR) assay on Rotor-Gene Q using TRUPCR HCV Genotyping Kit (3B BlackBio Biotech India Ltd.,) as per manufacturer’s instructions. This test kit detects HCV genotypes 1, 1a, 2 (2a/ 2b), 3, 4, 5a, and 6. Results: Genotype 3 (72.7%) was the commonest genotype detected, followed by genotype 1 (13%), genotype 1a (6.25%) and genotype 4 (2.8%). Genotype 3 was the most dominant genotype in all the 3 years of the study. Two cases of genotype 5a (1.13%) and three cases of genotype 6 (1.7%) were also detected in our study. We also detected a case of mixed infection by genotype 1 and genotype 6. Conclusion: The most common genotype detected was three during the study period. Detection of genotype 5a and 6 ascertained the impact of interstate migration of people.

Non-invasive prenatal screening & diagnosis of β-thalassaemia in an affected foetus.

Non-invasive prenatal testing (NIPT) of maternally inherited alleles of β-thalassaemia (MIB) remains to be a challenge. Furthermore, current techniques are not available for use as routine tests. NIPT for β-thalassaemia disease was developed by using a specific droplet digital polymerase chain reaction (ddPCR) assay to analyze the cell-free foetal DNA (cffDNA) derived from maternal plasma. Pregnant women and their spouses who are at risk of bearing an offspring with β-thalassaemia disease from common MIB mutations (CD 41/42-TCTT, CD17A>T, IVS1-1G>T and CD26G>A) were enrolled. The ddPCR assay sets were constructed for each of the four mutations. All cell-free DNA samples were first screened for the paternally inherited β-thalassaemia (PIB) mutation. The PIB-negative samples were considered as non-disease and were not further analyzed. For PIB-positive samples, DNA fragments of 50-300 base pairs in size were isolated and purified, and further analyzed for MIB mutation. The allelic ratio between the mutant and the wild-type was used to determine the presence of MIB in cffDNA. All cases underwent a prenatal diagnosis by amniocentesis for a definite diagnosis. Forty two couples at risk were enrolled. Twenty two samples were positive for PIBs. Among these 22 samples, there were 10 cases with allelic ratio >1.0 (MIB positive). All foetuses with over-represented mutant alleles were further diagnosed with β-thalassaemia disease; eight with compound heterozygous and two with homozygous mutations. The 20 PIB-negative and 12 MIB-negative foetuses were non-affected. The results of this study suggest that NIPT utilizing the ddPCR assay can be effectively used for the screening and diagnosis of foetal β-thalassaemia in at risk pregnancies.

A Rapid and Specific Real-Time PCR Assay for the Detection of Clinically Relevant Mucorales Species

Infections triggered by filamentous fungi placed in the order Mucorales, phylum Zygomycota, can cause serious harm to immunocompromised patients. Since there is lack of a standardized PCR (polymerase chain reaction) assay for early diagnosis of this fungal infection, this work was aimed to develop a new PCR assay able to detect the presence of Mucorales genera in clinical specimens. Here, we describe a novel diagnostic TaqMan MGB probe assay for precise and rapid detection of the most common clinical species of Mucorales. Zygomycete-specific oligonucleotides were designed to specifically amplify and bind highly conserved sequences of fungal 28S rRNA gene. Additionally, we succeeded in differentiating Mucorales species (i.e., Rhizopus, Lichtheimia, Mucor, and Rhizomucor) in artificially infected serum samples, suggesting that the quantitative capability of this real-time PCR assay could potentially optimize the diagnosis of mucormycosis.

Isolation and genetic analysis of Porcine circovirus 2 in southern India evidences high circulation of Porcine circovirus 2d genotype.

Porcine circovirus 2 is globally noted swine pathogen with multiple genotypes associated with vast clinical and subclinical outcomes. This study aimed to isolate and characterize PCV2 genotypes circulating in southern states of India. A total of 434 field samples comprising of serum (n = 273), tissues (n = 109) and swabs (n = 52) collected from swine during 2019 to 2021 from southern states of India were screened for PCV2 by specific polymerase chain reaction (PCR) assay. Molecular prevalence of PCV2 in southern India was found to be 12.21% (n = 53). All the 53 PCV2 positive samples were further subjected to the PCR assay with designed primers targeting full length amplification of ORF2 gene of PCV2 for molecular characterization. Randomly 32 positive samples by full length PCV2-ORF2 gene PCR were sequenced for genotyping. Signature motif and phylogenetic analysis of 32 PCV2 sequences revealed 62.5% (n = 20) prevalence of PCV2d genotype followed by 21.8% (n = 7) of PCV2h or PCV2-IM1 and 15.6% (n = 5) of PCV2b genotypes. Twenty five PCR positive field samples were subjected for virus isolation in PK15 cells and characterized. Out of 25 samples processed 5 (20%) PCV2 isolates obtained in this study were confirmed by PCR and immune fluorescence assay. Molecular characterization of PK15 adapted five PCV2 isolates confirmed circulation of PCV2d, PCV2h and PCV2b genotypes in pigs under field conditions in southern India. Isolation and molecular epidemiological study of PCV2 in southern states of India evidences high circulation of PCV2d genotypes in field conditions in comparison to other genotypes.

Orthopoxvirus Testing Challenges for Persons in Populations at Low Risk or Without Known Epidemiologic Link to Monkeypox - United States, 2022.

Since May 2022, approximately 20,000 cases of monkeypox have been identified in the United States, part of a global outbreak occurring in approximately 90 countries and currently affecting primarily gay, bisexual, and other men who have sex with men (MSM) (1). Monkeypox virus (MPXV) spreads from person to person through close, prolonged contact; a small number of cases have occurred in populations who are not MSM (e.g., women and children), and testing is recommended for persons who meet the suspected case definition* (1). CDC previously developed five real-time polymerase chain reaction (PCR) assays for detection of orthopoxviruses from lesion specimens (2,3). CDC was granted 510(k) clearance for the nonvariola-orthopoxvirus (NVO)-specific PCR assay by the Food and Drug Administration. This assay was implemented within the Laboratory Response Network (LRN) in the early 2000s and became critical for early detection of MPXV and implementation of public health action in previous travel-associated cases as well as during the current outbreak (4-7). PCR assays (NVO and other Orthopoxvirus laboratory developed tests [LDT]) represent the primary tool for monkeypox diagnosis. These tests are highly sensitive, and cross-contamination from other MPXV specimens being processed, tested, or both alongside negative specimens can occasionally lead to false-positive results. This report describes three patients who had atypical rashes and no epidemiologic link to a monkeypox case or known risk factors; these persons received diagnoses of monkeypox based on late cycle threshold (Ct) values ≥34, which were false-positive test results. The initial diagnoses were followed by administration of antiviral treatment (i.e., tecovirimat) and JYNNEOS vaccine postexposure prophylaxis (PEP) to patients' close contacts. After receiving subsequent testing, none of the three patients was confirmed to have monkeypox. Knowledge gained from these and other cases resulted in changes to CDC guidance. When testing for monkeypox in specimens from patients without an epidemiologic link or risk factors or who do not meet clinical criteria (or where these are unknown), laboratory scientists should reextract and retest specimens with late Ct values (based on this report, Ct ≥34 is recommended) (8). CDC can be consulted for complex cases including those that appear atypical or questionable cases and can perform additional viral species- and clade-specific PCR testing and antiorthopoxvirus serologic testing.

Ulceroglandular form of tularemia after squirrel bite: a case report

BackgroundThe diagnosis of tularemia is not often considered in Germany as the disease is still rare in this country. Nonetheless, Francisella tularensis, the causative agent of tularemia, can infect numerous animal species and should, therefore, not be neglected as a dangerous pathogen. Tularemia can lead to massively swollen lymph nodes and might even be fatal without antibiotic treatment. To our knowledge, the case described here is the first report of the disease caused by a squirrel bite in Germany.Case presentationA 59-year-old German woman with a past medical history of hypothyroidism and cutaneous lupus erythematosus presented at the emergency room at St. Katharinen Hospital with ongoing symptoms and a swollen right elbow persisting despite antibiotic therapy with cefuroxime for 7 days after she had been bitten (right hand) by a wild squirrel (Eurasian red squirrel). After another 7 days of therapy with piperacillin/tazobactam, laboratory analysis using real-time polymerase chain reaction (PCR) confirmed the suspected diagnosis of tularemia on day 14. After starting the recommended antibiotic treatment with ciprofloxacin, the patient recovered rapidly.ConclusionThis is the first report of a case of tularemia caused by a squirrel bite in Germany. A naturally infected squirrel has recently been reported in Switzerland for the first time. The number of human cases of tularemia has been increasing over the last years and, therefore, tularemia should be taken into consideration as a diagnosis, especially in a patient bitten by an animal who also presents with headache, increasing pain, lymphadenitis, and fever, as well as impaired wound healing. The pathogen can easily be identified by a specific real-time PCR assay of wound swabs and/or by antibody detection, for example by enzyme-linked immunosorbent assay (ELISA), if the incident dates back longer than 2 weeks.

MicroRNA-126 expression in the peripheral white blood cells of patients with breast and ovarian cancer is a potential biomarker for the early prediction of cancer risk in the carriers of methylated BRCA1.

Constitutive breast cancer type 1 gene (BRCA1) promoter methylation is associated with increased cancer risk, but its role in cancer-free (CF) female carriers is incompletely understood. MicroRNA (miR) is modulated during early tumorigenesis. The present study assessed the modulation of miR-126 expression in the peripheral white blood cells (WBC) of patients with breast cancer (BC) and ovarian cancer (OC) as a biomarker of cancer risk in BRCA1 methylation carriers. A total of 1,114 female subjects [502 patients with BC, 187 patients with OC and 425 CF volunteers] were involved. Screening for BRCA1 promoter methylation in WBC was performed using the methylation–specific polymerase chain reaction (PCR) assay, BRCA1 mRNA was analyzed using a reverse transcription-quantitative PCR assay and miR-126 expression was analyzed using a stem-loop RT-qPCR assay. WBC BRCA1 promoter methylation status was significantly associated with OC (P=0.0266), early-onset BC (P=0.0003) and triple-negative BC (P=0.0066). Notably, 9.4% of the CF group exhibited WBC BRCA1 promoter methylation. In addition, high levels of miR-126 in WBCs were detected in all three groups. The increased level of miR-126 was significantly associated with a lower risk of distant metastasis (P=0.045) in BC, but a higher risk of disease progression and death (P=0.0029) in OC. There was a positive correlation between BRCA1 mRNA and miR-126 levels in the WBCs of all three groups, regardless of BRCA1 promoter methylation status. Notably, circulating miR-126 level was decreased in the BC and OC groups, but not in the CF group. Together, these results suggest the likely involvement of miR-126 in the constitutional methylation of BRCA1 promoter-related malignancies. Therefore, miR-126 may be a candidate biomarker for the early prediction of BC and OC risk in CF BRCA1 methylation carriers.

Highly prevalent MDR, frequently carrying virulence genes and antimicrobial resistance genes in Salmonella enterica serovar 4,[5],12:i:- isolates from Guizhou Province, China

Salmonella enterica serovar 4,[5],12:i:-, a monophasic variant of Salmonella Typhimurium lacking the phase 2 flagellin, is one of the common serotypes causing Salmonellosis worldwide. However, information on Salmonella serovar 4,[5],12:i:- from Guizhou Province has lacked so far. This study aimed to investigate the antimicrobial resistance, the presence of antimicrobial resistance genes and virulence genes, and characterize the MLST genotypes of Salmonella serovar 4,[5],12:i:- isolates from Guizhou province, China. We collected 363 non-typhoid Salmonella (NTS) isolates of Guizhou from 2013 to 2018. Biochemical identification, serogroups testing, and specific multiplex polymerase chain reaction (mPCR) assay were conducted to identify Salmonella 4,[5],12:i:- isolates. Isolates were determined the antimicrobial resistance by the micro broth dilution method, detected the presence of antimicrobial resistance genes and virulence genes by PCR, and examined the molecular genotyping by Multilocus sequence typing (MLST). Eighty-seven Salmonella 4,[5],12:i:- isolates were detected, accounting for 23.9% (87/363) of the total NTS isolates. All Salmonella 4,[5],12:i:- isolates showed highly resistant to sulfaoxazole (93.1%), streptomycin (90.8%), ampicillin (88.5%), tetracycline (86.2%) and doxycycline (86.2%). A high proportion (94.2%) of multi-drug resistance (MDR) isolates were found. Most (83.9%) Salmonella 4,[5],12:i:- isolates carried four antimicrobial resistance genes, especially blaTEM-1, strA-strB, sul2, and tetB genes. Salmonella 4,[5],12:i:- isolates showed a high rate of invA, sseL, mgtC, siiE, sopB, gipA, gtgB, sspH1, and sspH2 (72.4%~98.9%). On the contrary, none of the isolates were detected the spvC and pefA genes. MLST analysis revealed three sequence types (STs), and ST34 (97.7%) was the dominant sequence type. This study is the first report of Salmonella 4,[5],12:i:- in humans from Guizhou province, China. The data might be useful for rational antimicrobial usage against Salmonella 4,[5],12:i:- infections, risk management, and public health strategies in Guizhou.

EP009: Identification of a novel exonic deletion in the GALNS gene causing Morquio syndrome

Mucopolysaccharidosis (MPS) IVA or Morquio syndrome is a rare lysosomal storage disorder caused by N-acetylgalactosamine-6-sulfatase deficiency. The condition is characterized by intracellular accumulation of the glycosaminoglycans keratan sulfate and chondroitin-6-sulfate, which classically leads to progressive skeletal and joint abnormalities, short stature, cardiorespiratory compromise, impaired vision, hearing loss, and hepatomegaly. The diagnosis is made by the identification of reduced N-acetylgalactosamine-6-sulfatase activity as well as detection of compound heterozygous or homozygous pathogenic variants in GALNS. We present a case of two sisters with the severe classic phenotype of Morquio syndrome who were born to healthy non-consanguineous parents of East Indian background. Patient 1 first presented at age 1.5 years with trouble walking and abnormal gait with a subsequent skeletal survey suggestive of dysostosis multiplex. She was diagnosed at age 2 years with Morquio syndrome based on reduced leukocyte N-acetylgalactosamine-6-sulfatase activity of 2.8 nmol/17hr/mg protein (reference range 45-443 nmol/17hr/mg protein). Medical history for patient 1 includes normal cognition, truncal shortening, scoliosis, distal joint laxity, cervical arthropathy, gibbus deformity, corneal clouding, and obstructive sleep apnea. Patient 2, the younger sister, was diagnosed with MPS IV at age 3 following a history of frequent falls and identification of genu valgum and early dysostosis. Given the family history, she was tested for Morquio syndrome and N-acetylgalactosamine-6-sulfatase leukocyte activity was <0.062nmol/17hr/mg protein (reference range 45-433 nmol/17hr/mg protein). Patient 2 has a history of cervical C1-C2 instability and occipitocervical stenosis, severe bilateral genu valgum, hip dysplasia, leg length discrepancy, pectus carinatum and corneal clouding. Both children, now at age 11 and 10 years respectively, need to use a wheelchair for any distance outside the home, despite orthopedic procedures. Prior genetic testing for MPS IVA, which utilized next generation sequencing (NGS), did not identify sequence variants associated with MPS IVA, however it was noted that a large region of the GALNS gene corresponding to exon 9 was not covered in the sequencing data. After establishing care with our center, repeat enzyme testing identified absent N-acetylgalactosamine-6 sulfatase activity for patient 1 and significantly reduced activity of 0.18 nmol/17hr/mg protein for patient 2 (reference range 49-255 nmol/17hr/mg protein; affected range < 7 nmol/17hr/mg protein). Subsequent polymerase chain reaction (PCR) amplification of the coding exons for GALNS produced an appropriate product for all exons with the exception of exon 9 and we hypothesized that a deletion encompassing exon 9 was present in our patients. Therefore, an allele specific PCR assay was designed to confirm the exon 9 deletion and determine the precise deletion breakpoints (c.899-397_1003-18632del) for our patients. The reference mRNA sequence was NM_000512.4 with codon 1 corresponding to the start ATG. To our knowledge, the pathogenic deletion identified in our patients (c.899-397_1003-18632del) has not been reported in the literature. Various deletions encompassing at least one exon have been reported in GALNS, however none encompassing only exon 9. NGS is increasingly utilized as a first-tier test for lysosomal storage diseases via targeted gene panels or exome/genome sequencing. The inability of this testing to detect large deletions or rearrangements, especially in the heterozygous state, could lead to a delayed or even missed diagnosis. For example, if the deletion described in this case was in cis with missense change in GALNS, NGS would label the patient as a heterozygote rather than affected. Our case highlights the benefit of biochemical analysis for lysosomal storage disorders in cases with equivocal or incomplete molecular analysis. Molecular analysis is useful to confirm a biochemical diagnosis and assist with genetic counseling and future prenatal testing and carrier testing. Recognizing limitations of molecular testing is important to ensure accurate diagnosis and treatment for individuals with Morquio syndrome in a timely manner.

Identification and Characterisation of Stripe Rust Resistance Genes Yr66 and Yr67 in Wheat Cultivar VL Gehun 892

Wheat cultivar VL Gehun 892 has shown a high level of resistance against Australian Puccinia striiformis f. sp. tritici (Pst) pathotypes. In this study, it was crossed with Westonia, a susceptible wheat cultivar, and digenic segregation was observed in the derived population against Pst pathotype 134 E16A+Yr17+Yr27+. Single-gene recombinant inbred line (RIL) populations were developed from F3 families (VL Gehun 892/Westonia#1 and VLGehun 892/Westonia#4) that showed monogenic segregations with two distinct phenotypes. Single-gene segregation against Pst pathotype 134 E16A+Yr17+Yr27+ was confirmed in both F6 RIL populations. Bulked segregant analysis using a 90K Infinium SNP array placed YrVL1 in the short arm of chromosome 3D and YrVL2 in the long arm of chromosome 7B. Kompetitive allele specific polymerase chain reaction (KASP) assays were developed for the SNPs linked with YrVL1 and YrVL2 and were mapped onto the respective populations. KASP_48179 (0.6 cM proximal) and KASP_18087 (2.1 cM distal) flanked YrVL1, whereas YrVL2 was mapped between KASP_37096 (1.2 cM proximal) and KASP_2239 (3.6 cM distal). Based on their pathotypic specificities, map locations, and stages of expression, YrVL1 and YrVL2 were demonstrated to be unique loci and named Yr66 and Yr67, respectively. Markers linked with these genes showed more than 85% polymorphism when tested on a set of 89 Australian cultivars and hence could be used for the marker-assisted selection of these genes in wheat breeding programs, following checks of parental polymorphisms.