Polymerase Chain Reaction-based DNA Research Articles

Tapeworm Infection Diagnosed after Campylobacter jejuni-induced Enteritis.

A 35-year-old man with fever and diarrhea visited our hospital because of white string-like fecal excretion. Based on a morphological examination of the excreted object, a Diphyllobothrium infection was suspected. Additionally, Gram staining of a fecal sample revealed Campylobacter infection. After the intraduodenal administration of meglumine/diatrizoate sodium, the tapeworm was excreted. A polymerase chain reaction-based DNA sequence analysis demonstrated that the tapeworm excreted in this case was Diphyllobothrium nihonkaiensis. This report presents a rare case of coinfection with Diphyllobothrium nihonkaiensis and Campylobacter jejuni. Therefore, it is important to consider the coexistence of other intestinal infections when diagnosing parasitic infections in patients with fever.

Inherited Thrombophilia and Risk of Thrombosis in Children with Cancer: a Single-center Experience.

Thrombosis is an increasingly recognized complication of childhood malignancy and its treatment. The incidence and etiology of pediatric cancer-related thrombosis is still not well understood. The aim of this study was to evaluate the prevalence of common prothrombotic genetic conditions in children with cancer, the frequency of thrombosis, and the role of inherited thrombophilia in the development of thrombosis in a pediatric oncology population. Forty-seven children (36 treated for hematological malignancies and 11 for solid tumors) with a median age of 8.8. years (range 0.4 - 19.3 years) were included in the study. Genetic polymorphisms of Factor V Leiden (G1691A), prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T were determined by real-time polymerase chain reaction-based DNA analysis. Four (8.5%) patients were heterozygous for Factor V Leiden, 3 (6.4%) were heterozygous for prothrombin G20210A mutation, and 3 (6.4%) were homozygous for MTHFR C677T mutation. All patients had implanted central venous catheters. Four (8.5%) children had documented thrombosis, three of which were in the upper venous system. Two of the four patients with thrombosis had Factor V Leiden heterozygosity. Thrombosis is an important complication of childhood cancer. The risk of thrombosis may be increased in patients with Factor V Leiden. In the absence of consensus guidelines, our results support the recommendation for thrombophilia screening in children with cancer.

Assessment of crustacean allergen detection methods: cross reactivity with edible insect samples

Increased interest in consumption of insects in recent years has led to an increased focus on associated food safety concerns, and allergy is one of the most relevant. In the United States, crustacean shellfish are regulated as a major allergenic food group per the Food, Drug, and Cosmetic (FD&C) Act. Insects and crustacean shellfish are both arthropods, and clinical cross-reactivity between the two groups has been demonstrated. The goal of this work was to establish whether that clinical cross-reactivity translates into analytical cross-reactivity with detection assays targeting crustacean shellfish allergens. Edible insect samples were analyzed using four different crustacean allergen detection methods: Multi-Analyte Profiling Food Allergen Detection Assay (xMAP FADA), enzyme-linked immunosorbent assay (ELISA), western blot, and real-time polymerase chain reaction (PCR). Results indicate that the immunoassay-based xMAP FADA, ELISA, and western blot were susceptible to cross-reactivity, while the DNA-based PCR methods had minimal reactivity with insect samples. These results confirm that edible insects show analytical cross-reactivity with the immunoassays which may result in false positive detection of crustacean allergens in insect samples. Confirmation using DNA-based PCR, which shows little to no cross-reactivity, clarifies ambiguous results.

Molecular understanding of unusual HbE-β+-thalassemia with Hb phenotype similar to HbE heterozygote: simple and rapid differentiation using HbE levels

Background Low HbF expression in HbE-β+-thalassemia may lead to misdiagnosis of HbE heterozygosity. We aimed to characterize the β- and α-globin genes and the modifying factors related to HbF expression in patients with an Hb phenotype similar to that of HbE heterozygotes. Furthermore, screening tools for differentiating HbE-β+-thalassemia from HbE heterozygotes have been investigated. Participants and Methods A total of 2133 participants with HbE and HbA with varying HbF levels were recruited. Polymerase chain reaction-based DNA analysis and sequencing were performed to characterize β- and α-globin genes. DNA polymorphism at position −158 nt 5′ to Gγ-globin was performed by XmnI restriction digestion. Receiver operating characteristic (ROC) curves were constructed using the area under the curve (AUC). Cutoff values of HbA2, HbE, and HbF levels for the differentiation of HbE-β+-thalassemia from HbE heterozygotes were determined. Results Five β+-thalassemia mutations trans to βE-gene (β−87(C>A), β−31(A>G), β−28(A>G), β19(A>G), and β126(T>G)) were identified in 79 patients. Among these, 54 presented with low HbF levels, and 25 presented with high HbF levels. ROC curve analysis revealed an excellent AUC of 1.000 (95% confidence interval:1.000–1.000) for HbE levels, and a cut-off point of ≥35.0% had 100.0% sensitivity, specificity, and Youden’s index for differentiating HbE-β+-thalassemia from HbE heterozygotes. The proportion of α-thalassemia mutations was 46.3 and 8.0% among HbE-β+-thalassemia patients with low and high HbF levels, respectively. Two rare α-thalassemia mutations (Cap +14(C>G) and initiation codon (ATG>-TG)) of α2-globin genes were identified. The genotype and allele of the polymorphism at −158 nt 5′ to Gγ-globin was found to be negatively associated with HbF expression. Conclusions HbE-β+-thalassemia cannot be disregarded until appropriate DNA analysis is performed, and the detection of α-thalassemia mutations should always be performed under these conditions. An HbE level ≥35.0% may indicate screening of samples for DNA analysis for HbE-β+-thalassemia diagnosis.

Prevalence of human papillomavirus in eyelid carcinoma among Koreans: a clinicopathological study

BackgroundHuman papillomavirus (HPV) has been detected in eyelid sebaceous gland carcinoma (SGC) and squamous cell carcinoma (SCC), and detection rates greatly varied across studies. This study aimed to investigate the presence of HPV in eyelid SGC and SCC among Koreans and its correlation with clinicopathological characteristics.MethodsSurgically resected eyelid samples diagnosed as SGC or SCC from January 1999 to June 2011 were identified from the pathology database of three referral centres in Korea. Clinicopathological information including origin (skin vs. tarsal conjunctiva) and treatment outcomes were retrospectively reviewed. Immunohistochemistry (IHC) for p16, HPV DNA in situ hybridisation (ISH), and polymerase chain reaction-based DNA microarray were performed in paraffin-embedded tissue sections.ResultsOur cohort included 34 SGC and 12 SCC cases with Asian ethnicity. HPV was detected in 4 SGC and 6 SCC by DNA microarray, while 2 SCC (16.7%) showed positivity in ISH. SCC of tarsal conjunctival origin was significantly more common in HPV-positive SCC than in HPV-negative SCC (5 of 6 vs. 0 of 6, P = 0.015, Fisher’s exact test). Among samples showing positive staining in p16 IHC, HPV positivity rates were 0.0% (0/19) in SGC and 100% (3/3) in SCC. There was no significant difference in overall and local recurrence rate in eyelid SGC and SCC according to the HPV status (P > 0.99).ConclusionsHPV was found in a subset of eyelid SGC and SCC among Koreans and might be aetiologically related to SCC of tarsal conjunctival origin. Overexpression of p16 is considered to be inappropriate as an indicator of HPV infection in eyelid SGC. Further investigation is required to elucidate the transmission route and pathogenic roles of HPV.

Investigating Metabolic and Molecular Ecological Evolution of Opportunistic Pulmonary Fungal Coinfections: Protocol for a Laboratory-Based Cross-Sectional Study.

Fungal-bacterial cocolonization and coinfections pose an emerging challenge among patients suspected of having pulmonary tuberculosis (PTB); however, the underlying pathogenic mechanisms and microbiome interactions are poorly understood. Understanding how environmental microbes, such as fungi and bacteria, coevolve and develop traits to evade host immune responses and resist treatment is critical to controlling opportunistic pulmonary fungal coinfections. In this project, we propose to study the coexistence of fungal and bacterial microbial communities during chronic pulmonary diseases, with a keen interest in underpinning fungal etiological evolution and the predominating interactions that may exist between fungi and bacteria. This is a protocol for a study aimed at investigating the metabolic and molecular ecological evolution of opportunistic pulmonary fungal coinfections through determining and characterizing the burden, etiological profiles, microbial communities, and interactions established between fungi and bacteria as implicated among patients with presumptive PTB. This will be a laboratory-based cross-sectional study, with a sample size of 406 participants. From each participant, 2 sputa samples (one on-spot and one early morning) will be collected. These samples will then be analyzed for both fungal and bacterial etiology using conventional metabolic and molecular (intergenic transcribed spacer and 16S ribosomal DNA-based polymerase chain reaction) approaches. We will also attempt to design a genome-scale metabolic model for pulmonary microbial communities to analyze the composition of the entire microbiome (ie, fungi and bacteria) and investigate host-microbial interactions under different patient conditions. This analysis will be based on the interplays of genes (identified by metagenomics) and inferred from amplicon data and metabolites (identified by metabolomics) by analyzing the full data set and using specific computational tools. We will also collect baseline data, including demographic and clinical history, using a patient-reported questionnaire. Altogether, this approach will contribute to a diagnostic-based observational study. The primary outcome will be the overall fungal and bacterial diagnostic profile of the study participants. Other diagnostic factors associated with the etiological profile, such as incidence and prevalence, will also be analyzed using univariate and multivariate schemes. Odds ratios with 95% CIs will be presented with a statistical significance set at P<.05. The study has been approved by the Mbarara University Research Ethic Committee (MUREC1/7-07/09/20) and the Uganda National Council of Science and Technology (HS1233ES). Following careful scrutiny, the protocol was designed to enable patient enrollment, which began in March 2022 at Mbarara University Teaching Hospital. Data collection is ongoing and is expected to be completed by August 2023, and manuscripts will be submitted for publication thereafter. Through this protocol, we will explore the metabolic and molecular ecological evolution of opportunistic pulmonary fungal coinfections among patients with presumptive PTB. Establishing key fungal-bacterial cross-kingdom synergistic relationships is crucial for instituting fungal bacterial coinfecting etiology. ISRCTN Registry ISRCTN33572982; https://tinyurl.com/caa2nw69. DERR1-10.2196/48014.

Diagnosis and biomarkers for ocular tuberculosis: From the present into the future.

Tuberculosis is an airborne disease caused by Mycobacterium tuberculosis (Mtb) and can manifest both pulmonary and extrapulmonary disease, including ocular tuberculosis (OTB). Accurate diagnosis and swift optimal treatment initiation for OTB is faced by many challenges combined with the lack of standardized treatment regimens this results in uncertain OTB outcomes. The purpose of this study is to summarize existing diagnostic approaches and recently discovered biomarkers that may contribute to establishing OTB diagnosis, choice of anti-tubercular therapy (ATT) regimen, and treatment monitoring. The keywords ocular tuberculosis, tuberculosis, Mycobacterium, biomarkers, molecular diagnosis, multi-omics, proteomics, genomics, transcriptomics, metabolomics, T-lymphocytes profiling were searched on PubMed and MEDLINE databases. Articles and books published with at least one of the keywords were included and screened for relevance. There was no time limit for study inclusion. More emphasis was placed on recent publications that contributed new information about the pathogenesis, diagnosis, or treatment of OTB. We excluded abstracts and articles that were not written in the English language. References cited within the identified articles were used to further supplement the search. We found 10 studies evaluating the sensitivity and specificity of interferon-gamma release assay (IGRA), and 6 studies evaluating that of tuberculin skin test (TST) in OTB patients. IGRA (Sp = 71-100%, Se = 36-100%) achieves overall better sensitivity and specificity than TST (Sp = 51.1-85.7%; Se = 70.9-98.5%). For nuclear acid amplification tests (NAAT), we found 7 studies on uniplex polymerase chain reaction (PCR) with different Mtb targets, 7 studies on DNA-based multiplex PCR, 1 study on mRNA-based multiplex PCR, 4 studies on loop-mediated isothermal amplification (LAMP) assay with different Mtb targets, 3 studies on GeneXpert assay, 1 study on GeneXpert Ultra assay and 1 study for MTBDRplus assay for OTB. Specificity is overall improved but sensitivity is highly variable for NAATs (excluding uniplex PCR, Sp = 50-100%; Se = 10.5-98%) as compared to IGRA. We also found 3 transcriptomic studies, 6 proteomic studies, 2 studies on stimulation assays, 1 study on intraocular protein analysis and 1 study on T-lymphocyte profiling in OTB patients. All except 1 study evaluated novel, previously undiscovered biomarkers. Only 1 study has been externally validated by a large independent cohort. Future theranostic marker discovery by a multi-omics approach is essential to deepen pathophysiological understanding of OTB. Combined these might result in swift, optimal and personalized treatment regimens to modulate the heterogeneous mechanisms of OTB. Eventually, these studies could improve the current cumbersome diagnosis and management of OTB.

Development of a Multiplex Polymerase Chain Reaction-Based DNA Lateral Flow Assay as a Point-of-Care Diagnostic for Fast and Simultaneous Detection of MRSA and Vancomycin Resistance in Bacteremia.

To reduce high mortality and morbidity rates, timely and proper treatment of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection is required. A multiplex polymerase reaction (mPCR)-based DNA lateral flow assay (MBDLFA) was developed as a point-of-care diagnostic for simultaneous identification of S. aureus, methicillin resistance, and vancomycin resistance directly from blood or blood cultures. A mPCR was developed to detect nuc, mecA, and vanA/B; its sensitivity, specificity, and limit of detection (LOD) were determined. The developed reaction was further modified for use in MBDLFA and its sensitivity for detection of target genes from artificially inoculated blood samples was checked. The optimized mPCR successfully detected nuc, mecA, and vanA/B from genomic DNA of bacterial colonies with LODs of 107, 107, and 105 CFU/mL, respectively. The reaction was sensitive and specific. The optimized mPCR was used in MBDLFA that detected nuc, mecA, and vanA/B with LODs of 107, 108, and 104 CFU/mL, respectively, directly from artificially inoculated blood. The developed MBDLFA can be used as a rapid, cheap point-of-care diagnostic for detecting S. aureus, MRSA, and vancomycin resistance directly from blood and blood cultures in ~2 h with the naked eye. This will reduce morbidity, mortality, and treatment cost in S. aureus bacteremia.

AAV-p40 Bioengineering Platform for Variant Selection Based on Transgene Expression.

The power of adeno-associated viral (AAV)-directed evolution for identifying novel vector variants with improved properties is well established, as evidenced by numerous publications reporting novel AAV variants. However, most capsid variants reported to date have been identified using either replication-competent (RC) selection platforms or polymerase chain reaction-based capsid DNA recovery methods, which can bias the selection toward efficient replication or unproductive intracellular trafficking, respectively. A central objective of this study was to validate a functional transduction (FT)-based method for rapid identification of novel AAV variants based on AAV capsid mRNA expression in target cells. We performed a comparison of the FT platform with existing RC strategies. Based on the selection kinetics and function of novel capsids identified in an in vivo screen in a xenograft model of human hepatocytes, we identified the mRNA-based FT selection as the most optimal AAV selection method. Lastly, to gain insight into the mRNA-based selection mechanism driven by the native AAV-p40 promoter, we studied its activity in a range of in vitro and in vivo targets. We found AAV-p40 to be a ubiquitously active promoter that can be modified for cell-type-specific expression by incorporating binding sites for silencing transcription factors, allowing for cell-type-specific library selection.

Prevalence and Genetic Analysis of β-Thalassemia in the Dali Bai Autonomous Prefecture of the Yunnan Province, China.

Background: β-Thalassemia is the most common monogenetic hemolytic hemoglobin-associated disease in the south of China; the distribution of genetic mutations associated with this condition varies according to geographic regions. This study investigated the prevalence and distribution of β-thalassemia-associated mutations across different ethnic groups in the Dali Bai Autonomous Prefecture of the Yunnan Province, China. Methods: This cross-sectional study included 4723 participants (15-45 years old) who volunteered for thalassaemia screening from the Dali Bai Autonomous Prefecture from May 2017 to October 2020. Cellulose acetate membrane electrophoresis was used to screen for β-thalassemia carriers. Genotypic analyses was performed using polymerase chain reaction-based reverse dot blotting and DNA sequencing. Results: The overall prevalence of β-thalassemia in the study population was 2.01%. The genotypic analyses showed the presence of four types of mutations in the β-globin gene: CD26 (GAG→AAG), CD56 (GGC→GAC), IVS-II-81 (C→T), and CD121 (GAA→CAA). In contrast to previous studies from other regions of Yunnan Province, our results showed that the prevalence of CD26 mutations was significantly higher than that of the other mutations. Conclusion: Our data suggests that the Dali Autonomous Prefecture is an area with a high prevalence of β-thalassemia. Moreover, CD26 was the only β-thalassemia mutation that we have detected. Moreover, the vast majority of the β-thalassemia mutations observed were CD26.

&lt;em&gt;Scedosporium&lt;/em&gt; species and &lt;em&gt;Lomentospora&lt;/em&gt; prolificans in Italian cystic fibrosis patients: prevalence and distribution in seven centers using a selective medium

Background and aims. Scedosporium species and Lomentospora prolificans are the filamentous fungi isolated more frequently from the cystic fibrosis (CF) lower airways, after Aspergillus fumigatus. Previous studies showed that, in CF patients, Scedosporium species and Lomentospora prolificans are responsible for airways colonization/ infection, enhancing pulmonary deterioration, and for severe invasive infections in CF patients undergoing lung transplantation. Aims of our project were to evaluate the prevalence of Scedosporium and Lomentospora species in Italian patients with CF, and to evaluate the interest of the Scedosporium-selective colture medium SceSel compared to routinely used agar-based culture media, in order to improve laboratory diagnostic performances.Materials and methods. A total of 1977 sputum samples from 1154 CF patients were collected and processed according to CF Italian recommendations in the year 2017 (January 1st to December 31st). The SceSel agar was used in addition to the routine culture procedures to burst growth and isolation of Scedosporium/ Lomentospora species. The fungal isolates were identified by classical mycological methods and polymerase chain reaction-based DNA sequencing of ITS1 and ITS4 regions. Susceptibility of the isolates to antifungal drugs was investigated by E-test method. Results. Among the 1154 enrolled patients, 62 (5.3%) were positive for Scedosporium/Lomentospora species (median age: 27y; range 8-64y; 33 male). Out of 1977 samples, 93 were positive for Scedosporium/Lomentospora species, and 13 (13.9%) were recovered only on SceSel agar. According to molecular analyses, isolation rates of each species were: S. apiospermum 53.1%, S. boydii 37.5%, L. prolificans 6.3% and S. aurantiacum 3.1%. Amphotericin B minimum inhibitory concentration (MIC) values were above or equal to 2 mg/L in all strains. Voriconazole MIC values were below or equal to 0.38 mg/L in almost all strains, proving this drug to be the most effective antimycotic for members of Scedosporium species. Otherwise, Lomentospora prolificans showed to be resistant to all considered antifungal drugs, only Voriconazole seems to be active on some Lomentospora prolificans isolates. Conclusions. Our results suggest that SceSel agar should be used in combination with routine media as standard microbiological protocol and procedures are not always adequate to isolate Scedosporium and Lomentospora species in CF respiratory samples. Molecular identification and susceptibility tests are needed, especially for strains isolated from critical patients and those considered for transplantation, as they might develop invasive scedosporiosis.

Discrepancy of p16 immunohistochemical expression and HPV RNA in penile cancer. A multiplex in situ hybridization/immunohistochemistry approach study

BackgroundThe high-risk human papillomavirus (HPV) infection represents one of the main etiologic pathways of penile carcinogenesis in approximately 30–50 % of cases. Several techniques for the detection of HPV are currently available including Polymerase chain reaction-based techniques, DNA and RNA in situ hybridization (ISH), p16 immunohistochemistry (IHC). The multiplex HPV RNA ISH/p16 IHC is a novel technique for the simultaneous detection of HPV E6/E7 transcripts and p16INK4a overexpression on the same slide in a single assay. The main aim of this study was to evaluate the discrepancy of p16 IHC expression relatively to HPV RNA ISH in penile cancer tissue.MethodsWe collected a series of 60 PCs. HPV has been analysed through the RNA ISH, p16 IHC and the multiplex HPV RNA ISH/p16 IHC.ResultsThe multiplex HPV RNA ISH /p16 IHC results in the series were in complete agreement with the previous results obtained through the classic p16 IHC and HPV RNA scope carried out on two different slides. The multiplex HPV RNA ISH /p16 IHC showed that HPV positivity in our series is more frequently in usual squamous cell carcinoma than in special histotypes (19 out of 60 − 15 %- versus 6 out of 60 − 10 %-), in high-grade than in moderate/low grade carcinomas (6 out of 60 − 10 %- versus 4 out of 60 − 6.7 %-). In addition, our data revealed that in 5 out of 20 cases with p16 high intensity expression is not associated with HPV RNA ISH positivity.ConclusionsOur findings emphasize that the use of p16 as a surrogate of HPV positivity was unsuccessful in approximatively 8 % of cases analysed in our series. Indeed, p16 IHC showed a sensitivity of 100 % and a specificity of 71 %, with a positive predictive value (PPV) of 54 % and a negative predictive value of 100 %; when considering high intensity, p16 IHC showed a sensitivity of 100 %, a specificity of 89 %, with a PPV of 75 % and NPV of 100 %.Since HPV positivity could represent a relevant prognostic and predictive value, the correct characterization offered by this approach appears to be of paramount importance.

Clinical Implications of Minimal Residual Disease Detection in Infants With KMT2A-Rearranged Acute Lymphoblastic Leukemia Treated on the Interfant-06 Protocol.

PURPOSEInfant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of KMT2A gene rearrangements and poor outcome. We evaluated the value of minimal residual disease (MRD) in infants with KMT2A-rearranged ALL treated within the Interfant-06 protocol, which compared lymphoid-style consolidation (protocol IB) versus myeloid-style consolidation (araC, daunorubicin, etoposide/mitoxantrone, araC, etoposide).MATERIALS AND METHODSMRD was measured in 249 infants by DNA-based polymerase chain reaction of rearranged KMT2A, immunoglobulin, and/or T-cell receptor genes, at the end of induction (EOI) and end of consolidation (EOC). MRD results were classified as negative, intermediate (< 5 × 10−4), and high (≥ 5 × 10−4).RESULTSEOI MRD levels predicted outcome with 6-year disease-free survival (DFS) of 60.2% (95% CI, 43.2 to 73.6), 45.0% (95% CI, 28.3 to 53.1), and 33.8% (95% CI, 23.8 to 44.1) for infants with negative, intermediate, and high EOI MRD levels, respectively (P = .0039). EOC MRD levels were also predictive of outcome, with 6-year DFS of 68.2% (95% CI, 55.2 to 78.1), 40.1% (95% CI, 28.1 to 51.9), and 11.9% (95% CI, 2.6 to 29.1) for infants with negative, intermediate, and high EOC MRD levels, respectively (P < .0001). Analysis of EOI MRD according to the type of consolidation treatment showed that infants treated with lymphoid-style consolidation had 6-year DFS of 78.2% (95% CI, 51.4 to 91.3), 47.2% (95% CI, 33.0 to 60.1), and 23.2% (95% CI, 12.1 to 36.4) for negative, intermediate, and high MRD levels, respectively (P < .0001), while for myeloid-style–treated patients the corresponding figures were 45.0% (95% CI, 23.9 to 64.1), 41.3% (95% CI, 23.2 to 58.5), and 45.9% (95% CI, 29.4 to 60.9).CONCLUSIONThis study provides support for the idea that induction therapy selects patients for subsequent therapy; infants with high EOI MRD may benefit from AML-like consolidation (DFS 45.9% v 23.2%), whereas patients with low EOI MRD may benefit from ALL-like consolidation (DFS 78.2% v 45.0%). Patients with positive EOC MRD had dismal outcomes. These findings will be used for treatment interventions in the next Interfant protocol.

Significance of telomerase reverse-transcriptase promoter mutations in differentiated thyroid cancer

Background: Telomerase reverse-transcriptase (TERT) encodes the reverse transcriptase of telomerase, and TERT promoter mutations are enriched in advanced thyroid cancer. In this study, we aimed to characterize the clinicopathologic biology of differentiated thyroid cancer harboring TERT promoter mutations in Taiwan. Materials and Methods: Consecutive adult patients treated with differentiated thyroid cancer between 2017 and 2019 were included in this study. Mutational testing for the TERT promoter was performed by DNA-based polymerase chain reaction. Results: Among 389 patients included in the analysis, 22 (5.7%) had papillary or follicular thyroid cancer harboring TERT promoter mutations, including C228T (n = 18), C250T (n = 3), and CC242TT (n = 1). The frequency of BRAF V600E mutation was 73%. TERT promoter mutations were significantly associated with older age, tall cell variant of papillary thyroid cancer, extrathyroidal extension, positive surgical margins, lymphovascular invasion, perineural invasion, and distant metastasis. The generalized additive model showed that patient age was positively and almost linearly correlated with the likelihood of the presence of TERT promoter mutations. Conclusion: The frequency of TERT promoter mutations is relatively low in patients with differentiated thyroid cancer in Taiwan. These tumors carry unfavorable clinicopathologic features, present in a more advanced stage, and probably predict worse prognosis.

Incidence and Outcomes of Pneumocystis pneumonia When Utilizing PCR-Based Diagnosis in Patients with Hodgkin Lymphoma Receiving Intensive Chemotherapy

Background: The overall incidence of Pneumocystis Pneumonia (PJP) in lymphoma varies according to diagnosis, host factors and treatment rendered.(Carmona, et al. 2011) A meta-analysis suggested that prophylaxis is appropriate when the chance of incurring PJP is 3.5% or greater.(Green, et al. 2007) Accurately characterizing the incidence of PJP is further complicated by a transition in diagnostic methods from direct immunofluorescence assay (DFA) to a DNA-based polymerase chain reaction (PCR) technique.(Caliendo et al., 1998, Wilson JW, et al., 2011) The incidence of PCR-based diagnosis of PJP and the need for anti-Pneumocystis prophylaxis in patients diagnosed with Hodgkin lymphoma receiving chemotherapy requires further investigation. Methods: This single center, retrospective, observational study was approved by the Mayo Clinic Institutional Review Board and performed in accordance with the ethical standards of the 1964 Declaration of Helsinki. Consecutive adult patients with a diagnosis of Hodgkin lymphoma receiving chemotherapy between January 2006 and August 2018 were evaluated. Patients with history of bone marrow transplant were excluded. Kaplan-Meier estimation was used to calculate the cumulative incidence of PJP with a 95% confidence interval. Cox proportional hazard models were used to demonstrate the strength of association between the independent variables and PJP rate. Results: The 506 included patients had a median (IQR) age of 40.3 (28.6, 59.7) years, 287 (56.7%) were male, and 470 (92.9%) were Caucasian. Further baseline demographics and clinical characteristics are described in table 1 with no significant differences between those diagnosed and those free from PJP. Chemotherapy combined with radiation therapy was prescribed for 163 (32.3%) patients while 342 (67.7%) patients received chemotherapy alone. Chemotherapy regimen distribution included 356 patients (70.4%) receiving bleomycin-containing chemotherapy. Median (IQR) total Bleomycin dose was 122 (IQR: 78, 178) Units. Bleomycin-containing combination chemotherapy regimen was not significantly associated with a higher risk for PJP when compared to other combination regimens (HR=1.59, 95% CI 0.55-4.62 p=0.40). Bleomycin-related lung compromise was suspected in 130 (25.7%) patients. Anti-pneumocystis prophylaxis was provided for 97 patients (n=39 a priori, 58 after chemotherapy initiation) and this was not significantly associated with a decreased incidence of PJP (HR=0.51, 95% CI 0.15-1.71, p=0.28). The cumulative incidence of PJP 1 year after start of chemotherapy was 6.2% (95% CI 3.8%-8.5%) and is shown in figure 1. Diagnosis of PJP occurred most commonly during the 2nd (n=8, 30.7%), 3rd (n=8, 30.7%), and 4th (n=7, 26.9%) cycles of chemotherapy. Trimethoprim-sulfamethoxazole was the only agent prescribed as PJP management. There were 10 (38%) patients with PCP that required ICU admission with 9 needing mechanical ventilation, 4 necessitating vasopressor support, and none requiring dialysis. Mortality 30-days from PJP diagnosis was 8% (n=2) with one death attributable to PJP infection. Conclusion: Cumulative incidence of PJP was 6.2% in patients receiving intensive chemotherapy for Hodgkin lymphoma. As the incidence is above the 3.5% threshold previously described, the clinician should consider the potential value of prophylaxis. Defining the overall utility of universal vs. targeted anti-pneumocystis prophylaxis requires further prospective, randomized investigation. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Trimethoprim/Sulfamethoxazole, Pentamidine isethionate, and dapsone will be discussed as prevention of Pneumocystis Pneumonia in the HIV-negative population

Assessing the risk of Babesia to the United States blood supply using a risk-based decision-making approach: Report of AABB's Ad Hoc Babesia Policy Working Group (original report).

Recognizing the increasing threat of transfusion-transmitted babesiosis to the US blood supply, the AABB Board of Directors tasked an Ad Hoc Babesia Policy Working Group (the Working Group) to use the Alliance of Blood Operators' risk-based decision-making (RBDM) framework to assess the risks and benefits of introducing Babesia donation testing in the United States. The regional nature of the Babesia microti risk added complexity to the RBDM assessment because of the unique operational and financial considerations for operators and hospitals located in endemic states. Therefore, the assessment considered safety, product availability, sector sustainability, and technology availability. After assessing safety risk, economic and operational impact, reimbursement equity, ethical considerations, and stakeholder feedback from two consultations, the Working Group concluded that a regional approach to donor screening in endemic states was appropriate because it applied the intervention where the risk was highest and appropriately allocated cost to the risk. Nucleic acid testing using a ribosomal RNA template was the recommended intervention because it was the most cost-effective, resulted in no wasted units, and captured similar numbers of infections as antibody plus DNA-based polymerase chain reaction. The current model for blood reimbursement was maintained but AABB was encouraged to facilitate collection of data to identify threats to sector sustainability in endemic states. Babesia expansion was acknowledged with a mechanism to regularly reevaluate what are "endemic states." Finally, given that public awareness of the Babesia threat is the first line of defense, AABB should work with appropriate agencies for general education about the health risk from B. microti.

A Simple Whole-Blood Polymerase Chain Reaction without DNA Extraction for Thalassemia Diagnosis

Polymerase chain reaction (PCR) diagnosis of thalassemia usually relies on using genomic DNA. Preparing the genomic DNA can lead to sample-to-sample contamination. This report was aimed to establish the PCR protocol using whole-blood for detecting mutations of α- and β-globin genes causing the thalassemia syndrome. First, the PCR facilitators, betaine and bovine serum albumin (BSA), were tested, simultaneously with an adjustment of PCR thermal cycler and of whole-blood volume. Thereafter, the established whole-blood PCR was applied for detecting, in both known and unknown samples, the HBA1 Southeast Asian (– –SEA) (NG_000006.1: g.26264_45564del19301) deletion, Hb Constant Spring (Hb CS, HBA2: c.427T>C, αCSα), codon 17 (A>T) (HBB: c.52A>T), codons 41/42 (–TTCT) (HBB: c.126_129delCTTT) deletion, –28 (A>G) (HBB: c.-78A>G) and codon 26 (G>A) (Hb E or HBB: c.79G>A). It was shown that the whole-blood PCR worked successfully in 9.0% (w/v) betaine, with 1 μL of EDTA whole blood and with addition of 10 heat-cool steps (3 min. at 94 °C, followed by 3 min. at 55 °C) prior to the typical thermal cycles for the mutations. The capability of the new whole-blood PCR was similar to that of the typical DNA-based PCR. Therefore, the newly established whole-blood PCR could be performed for PCR diagnosis of thalassemia. Using this platform, sample-to-sample contamination should be eliminated.

Using Translation Elongation Factor Gene to Specifically Detect and Diagnose Fusarium xylaroides, a Causative Agent of Coffee Wilt Disease in Ethiopia, East and Central Africa.

The present study presents the first report on the application of DNA-based polymerase chain reaction (PCR) for the specific detection and diagnosis of Fusarium xylarioides (anamorph: Gibberrela xylarioides). Fusarium xylarioides is the causative agent of Coffee wilt disease (Tracheomycosis), and the disease is the most important economic constraint in Robusta coffee production in Uganda. The pathogen has two races, one pathogenic to Robusta coffee and the other to Arabica coffee, and not vice versa. Its laboratory diagnosis has been mainly based on microscopy, which is slow, has poor discriminative power, requires high expertise, only applicable on host plants with symptoms, and has since failed to detect the pathogen from the soil. Translation Elongation factor-1α (TEF-1α) gene from a F. xylarioides isolated from infected Robusta coffee plant was amplified by Fusarium genus specific primer then the PCR product sequenced. The sequence data was then used to design the specific primer. The primer-BLAST product was found to match only F. xylarioides sequences comprising 75% of the race pathogenic to Robusta and 25% to Arabica coffee. In vitro test by PCR showed the primer to be specific to only F. xylarioides amplifying a 284bp product and was able to differentiate F. xylarioides from all closely related species of Fusarium and other plant pathogens tested. More so it was able to amplify DNA from all the F. xylarioides isolates from different regions of Uganda, and amplified DNA concentrations as minute as 0.78 ng/μL.

Potential role of polymorphisms in the transporter genes ENT1 and MATE1/OCT2 in predicting TAS-102 efficacy and toxicity in patients with refractory metastatic colorectal cancer

BackgroundTrifluridine (FTD) is an active cytotoxic component of the metastatic colorectal cancer (mCRC) drug TAS-102, and thymidine phosphorylase inhibitor (TPI) inhibits the rapid degradation of FTD. We tested whether single nucleotide polymorphisms (SNPs) in genes involved in FTD metabolism and TPI excretion could predict outcome in patients with mCRC treated with TAS-102. Patients and methodsWe investigated three different cohorts: a training cohort (n = 52) and a testing cohort (n = 129) both receiving TAS-102 and a control cohort (n = 52) receiving regorafenib. SNPs of TK1, ENT1, CNT1, MATE1, MATE2 and OCT2 were analysed by polymerase chain reaction-based direct DNA sequencing. ResultsIn the training cohort, patients with any ENT1 rs760370 G allele had a significantly longer progression-free survival (PFS; 3.5 versus 2.1 months, respectively, hazard ratio [HR] 0.44, P = 0.004) and overall survival (OS; 8.7 versus 5.3 months, respectively, HR 0.27, P = 0.003) than the A/A genotype. These findings were validated in the testing cohort (P = 0.021 and 0.009 for PFS and OS, respectively). In addition, the combination of ENT1 rs760370, MATE1 rs2289669 and OCT2 rs316019 SNPs significantly stratified patients with the risk of PFS and OS in both cohorts (P < 0.001 for PFS and OS in the training cohort; P = 0.053 and 0.025 for PFS and OS, respectively, in the testing cohort). No significant differences were observed in the control group. ConclusionsThe combination of ENT1, MATE1 and OCT2 SNPs may serve as a predictive and prognostic marker in mCRC patients treated with TAS-102.

The specific host plant DNA detection suggests a potential migration of Apolygus lucorum from cotton to mungbean fields

The polyphagous mirid bug Apolygus lucorum (Heteroptera: Miridae) has more than 200 species of host plants and is an insect pest of important agricultural crops, including cotton (Gossypium hirsutum) and mungbean (Vigna radiata). Previous field trials have shown that A. lucorum adults prefer mungbean to cotton plants, indicating the considerable potential of mungbean as a trap crop in cotton fields. However, direct evidence supporting the migration of A. lucorum adults from cotton to mungbean is lacking. We developed a DNA-based polymerase chain reaction (PCR) approach to reveal the movement of A. lucorum between neighboring mungbean and cotton fields. Two pairs of PCR primers specific to cotton or mungbean were designed to target the trnL-trnF region of chloroplast DNA. Significant differences in the detectability half-life (DS50) were observed between these two host plants, and the mean for cotton (8.26 h) was approximately two times longer than that of mungbean (4.38 h), requiring weighted mean calculations to compare the detectability of plant DNA in the guts of field-collected bugs. In field trials, cotton DNA was detected in the guts of the adult A. lucorum individuals collected in mungbean plots, and the cotton DNA detection rate decreased successively from 5 to 15 m away from the mungbean-cotton midline. In addition to the specific detection of cotton- and mungbean-fed bugs, both cotton and mungbean DNA were simultaneously detected within the guts of single individuals caught from mungbean fields. This study successfully established a tool for molecular gut-content analyses and clearly demonstrated the movement of A. lucorum adults from cotton to neighboring mungbean fields, providing new insights into understanding the feeding characteristics and landscape-level ecology of A. lucorum under natural conditions.