Polymerase chain reaction in meat species identification

Lebensmittel gelten als authentisch, wenn das Produkt oder sein Inhalt mit dem Originalzustand und den Angaben auf der Deklaration übereinstimmt. Abweichungen von den Behauptungen und Angaben auf der Deklaration werden als Verstöße gegen die Lebensmittelvorschriften angesehen. Aufgrund des gestiegenen Bewusstseins für das Problem und die negativen Auswirkungen von Lebensmittelbetrug wird der Fleischkonsum maßgeblich von der Wahrnehmung der Verbraucher in Bezug auf die Lebensmittelqualität und Sicherheit beeinflusst. Daher ist eine genaue Etikettierung einer der wichtigsten Faktoren, die die Verbraucherpräferenzen bei der Auswahl und dem Kauf von Fleisch und Fleischerzeugnissen beeinflussen. Aus diesem Grund sind Analysemethoden zur Überprüfung der Echtheit von Fleisch und Fleischerzeugnissen wichtig, um die Produktqualität, Lebensmittelsicherheit und den Verbraucherschutz zu gewährleisten. Die Substitution von Fleischsorten ist nicht das einzige Kriterium für die Bestimmung der Echtheit von Fleisch und Fleischerzeugnissen, sondern auch die Herkunft des Fleisches, die Behandlung des Fleisches und der Zusatz von sonstigen Zutaten. Die Polymerase-Kettenreaktion (PCR) und die davon abgeleiteten Technologien haben sich als die am besten geeigneten Methoden zur Identifizierung von Arten in rohem und technologisch verarbeitetem Fleisch erwiesen. Die PCR-Methoden basieren hauptsächlich auf der Identifizierung der Zielregion der mitochondrialen DNA, was den Nachweis von Arten in einer breiten Palette von Fleischerzeugnissen ermöglicht, einschließlich aller Haustiere und des für den menschlichen Verzehr bestimmten Wildfleischs. Sie haben jedoch auch einige Nachteile. So eignet sich beispielsweise die zufällig amplifizierte polymorphe DNA (PCR-RAPD) nicht für den Artennachweis in Fleischmischungen und in thermisch verarbeitetem Fleisch. Andererseits sind einige Methoden teuer und zeitaufwendig, und die Ergebnisse sind schwer zu interpretieren. In diesem Artikel werden die wichtigsten PCR-basierten Methoden zur Identifizierung von Fleischarten vorgestellt und beschrieben.

Accuracy of High-risk HPV DNA PCR, p16INK4a Immunohistochemistry or the Combination of Both to Diagnose HPV-driven Oropharyngeal Cancer

BACKGROUNDThe incidence of high-risk human papillomavirus (hrHPV)-driven head and neck squamous cell carcinoma, in particular oropharyngeal cancers (OPC), is increasing in high-resource countries. Patients with HPV-induced cancer respond better to treatment and consequently have lower case-fatality rates than patients with HPV-unrelated OPC. These considerations highlight the importance of reliable and accurate markers to diagnose truly HPV-induced OPC. METHODSThe accuracy of three possible test strategies, i.e. a) hrHPV DNA PCR (DNA), b) p16INK4a immunohistochemistry (IHC) (p16), and c) the combination of both tests (considering joint DNA and p16 positivity as positivity criterion), was analysed in tissue samples from 99 Belgian OPC patients enrolled in the HPV-AHEAD study. Presence of HPV E6*I mRNA (mRNA) was considered as the reference, indicating HPV etiology. RESULTSNinety-nine OPC patients were included, for which the positivity rates were 36.4%, 34.0% and 28.9% for DNA, p16 and mRNA, respectively. Ninety-five OPC patients had valid test results for all three tests (DNA, p16 and mRNA). Using mRNA status as the reference, DNA testing showed 100% (28/28) sensitivity, and 92.5% (62/67) specificity for the detection of HPV-driven cancer. p16 was 96.4% (27/28) sensitive and equally specific (92.5%; 62/67). The sensitivity and specificity of combined p16+DNA testing was 96.4% (27/28) and 97.0% (65/67), respectively. In this series, p16 alone and combined p16+DNA missed 1 in 28 HPV driven cancers, but p16 alone misclassified 5 in 67 non-HPV driven as positive, whereas combined testing would misclassify only 2 in 67. CONCLUSIONSSingle hrHPV DNA PCR and p16INK4a IHC are highly sensitive but less specific than using combined testing to diagnose HPV-driven OPC patients. Since HPV-driven OPC cancer can be treated less aggressively, combined testing can reduce treatment sequelae at the expense of a small number of patients that may receive less effective therapy.

A case of primary varicella meningoencephalitis in the absence of cutaneous lesions in a 4-month-old infant

Primary varicella infection has typical cutaneous lesions which aid in clinical diagnosis. Infants with transplacental transfer of varicella antibody can have varied cutaneous lesions. We report a 4-month-old infant with primary varicella meningoencephalitis without cutaneous lesions whose mother had no history of varicella during antenatal or post-natal period. Diagnosis was made possible by CSF DNA PCR. Infants with encephalitis pose diagnostic challenge to clinicians in resource limited settings. Varicella encephalitis is one such aetiology for which definitive therapy with Acyclovir is available. CSF PCR is the definitive and cost-effective test for the diagnosis varicella encephalitis. In children with meningoencephalitis it is prudent to add Acyclovir empirically pending CSF viral PCR results.

Outcomes of Cytomegalovirus Monitoring in Autologous Transplantation: A Single Institution Experience

Introduction: Identification of patients seropositive for cytomegalovirus (CMV) prior to stem cell transplant (SCT) is a well-accepted practice across institutions designed to reduce a known cause of morbidity and mortality in this population, but the role of monitoring and preemptive approaches to CMV identification and treatment are controversial and not standardized in autologous transplantation. The preemptive approach necessitates the use of significant resources and requires persistent patient involvement. Patients undergoing autologous SCT are at a relatively low risk for CMV reactivation, especially those seronegative for CMV at the time of transplant. Here, we show that the necessity of routine monitoring of autologous transplant patients is of minimal clinical value. Methods: To determine the efficacy of the CMV monitoring protocol currently in place at our institution in detecting patients who would later develop CMV reactivation and disease following autologous SCT, we retrospectively analyzed the charts of 218 adult patients between 11/1/14 and 8/1/19 who underwent transplant at St. Louis University Hospital. No patients underwent CD34 selected stem cell infusions. The protocol stipulated the following: CMV IgG/IgM and CMV DNA PCR prior to preparative regimen followed by weekly CMV DNA PCR to day +30 . We correlated the predictive ability of positive results on any of these screening tests to identify whether patients would later develop quantifiable CMV DNA PCR positivity, clinical manifestations of CMV disease, and/or require pharmacologic treatment for CMV. Results: Quantifiable pre-BMT DNA PCR was positive in only 0.46% of patients, and 97.79% of patients were DNA PCR negative prior to transplant. CMV IgG was positive in 56.4% patients, and only 22.1% of patients in this group went on to develop a quantifiable post-transplant PCR. Of the remaining 43.6% of patients initially testing negative for CMV IgG, no patients went on to develop a quantifiably positive post-transplant PCR. Regardless of seropositivity, only 0.08% of the 1,191 PCRs performed during the study period were found to be quantifiable. Further, no patients in our cohort developed CMV disease or required CMV treatment during the monitoring period. This trend persisted despite stratification by age, diagnosis, transplant number, and preparative regimen. Conclusion: When clinically-significant CMV is defined by cases requiring treatment or the development of end-organ disease, no screening tests performed elicited clinical action. Laboratory-based CMV surveillance, based on our data, has minimal diagnostic implications and represents an overly-stringent practice in a set of patients already utilizing a substantial share of healthcare resources. We believe that pre-transplant screening for CMV IgM serology and CMV DNA PCR can be safely eliminated in the autologous SCT population at our institution while CMV IgG still plays a role in determining candidacy for CMV-negative blood products. We also propose the elimination of serial post-transplant monitoring with DNA PCR in patients without clinical signs, symptoms, or pathologic findings suggestive of CMV disease. We have changed the protocol to test for CMV PCR only if there are clinical scenarios that indicate a utility, such as prolonged fever post-transplant, unexplained cytopenias, or unexplained pneumonitis, colitis, or hepatitis. By extension, other centers should consider determining the necessity of CMV screening in their autologous transplant population given the potential resource conservation and reduction in healthcare expenditures. Disclosures Fesler: abbvie: Consultancy, Speakers Bureau; incyte: Consultancy, Speakers Bureau; sanofi: Speakers Bureau; morphosys: Speakers Bureau; epizyme: Consultancy; jazz: Consultancy; Skipta: Consultancy; Best Doctors: Consultancy; Aptitude Health: Consultancy; Care Dx: Consultancy; Opinionsite: Consultancy. Goldenberg: Seattle Genetics: Membership on an entity's Board of Directors or advisory committees.

924. Cytomegalovirus (CMV) Retinitis during Maintenance Chemotherapy for Acute Lymphoblastic Leukemia

Background Acute leukemia patients are at risk for cytomegalovirus (CMV) retinitis following hematopoietic stem cell transplantation, though the disease can also occur in non-transplant adult leukemia patients. Emerging data suggest a shift to pediatric-inspired chemotherapy regimens in adults with acute lymphoblastic leukemia (ALL) can lead to increasing cytopenias and impaired functional immunity, placing these patients at risk for this opportunistic infection. Here we describe a case of CMV retinitis in an ALL patient following a lower-intensity regimen during maintenance chemotherapy. Methods Chart review. Results A 55-year-old male with ALL presented to his optometrist with complaints of visual changes including “fogginess” and “floaters”. The patient had completed 8 cycles of dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (EPOCH-R) and achieved complete remission. He had been on maintenance chemotherapy with 6-mercaptopurine, vincristine, methotrexate, and prednisone (POMP) for 2 months at the time of symptom onset. He was referred to his local ophthalmologist who had concerns for bilateral, zone 1 CMV retinitis based on fundoscopic exam (Figure 1). Vitreous aspiration was performed and CMV DNA PCR returned positive at 1.6 million IUs/ml. Peripheral blood CMV DNA PCR was also positive at 1133 IU/ml. He was started on combination therapy with intravitreal ganciclovir injections and oral valganciclovir 900 mg twice daily (Figure 2). The patient received 14 intravitreal injections with resultant stability of his eye exam, though he remained on induction valganciclovir for 5 months due to persistent blood CMV DNAemia. Letermovir was added to help suppress his peripheral CMV DNAemia and he attained partial vision recovery. Figure 1. Fundoscopic images Conclusion CMV retinitis is an uncommon and highly morbid infection that can occur during maintenance chemotherapy in adult non-transplant ALL patients. Early identification of the disease is imperative as delay can result in blindness or further systemic CMV disease. Treatment is challenging, involving systemic and intravitreal antiviral therapy, serial ophthalmologic exams, serum CMV monitoring, and close coordination with the treating hematologist. Disclosures Michael Boeckh, MD PhD, AlloVir (Consultant)Ansun Biopharma (Grant/Research Support)Astellas (Grant/Research Support)EvrysBio (Consultant, Other Financial or Material Support, Options to acquire equity, but have not exercised them)Gilead Sciences (Consultant, Grant/Research Support)GlaxoSmithKline (Consultant)Helocyte (Consultant, Other Financial or Material Support, Options to acquire equity, but have not exercised them)Janssen (Grant/Research Support)Kyorin (Consultant)Merck (Consultant, Grant/Research Support)Moderna (Consultant)Symbio (Consultant)Takeda (formerly known as Shire) (Consultant, Grant/Research Support)VirBio (Consultant, Grant/Research Support) Ryan Cassaday, MD, Amgen (Grant/Research Support, Advisor or Review Panel member)Kite/Gilead (Grant/Research Support, Advisor or Review Panel member)Merck (Grant/Research Support)Pfizer (Grant/Research Support, Advisor or Review Panel member)Seagen (Other Financial or Material Support, Spouse is employee and hold stock)Vanda Pharmaceuticals (Grant/Research Support)

Factors Associated with HIV prevalence among HIV-exposed Infants with HIV DNA Polymerase Chain Reaction Tests in Malawi: 2013-2020

Background: Despite the high availability of individual-level data of infants accessing HIV DNA polymerase chain reaction (DNA-PCR) testing service, there has been little in-depth analysis of such data. Therefore, we describe spatial and temporal trends in risk of HIV infection among Malawi HIV-exposed infants (HEI) with DNA-PCR HIV test result from 2013 to 2020. Methods: This is an implementation study using routinely collected patient-level HIV DNA-PCR test result data extracted from the national Laboratory Management Information System database managed by the Department of HIV/AIDS between 1 January 2013 and 30 June 2020. We calculated frequencies, proportions and odds ratios (OR) with their associated 95% confidence intervals (95%CI). We performed a random-effects logistic regression to determine the risk factors associated with HIV infection in infants, controlling for the spatial autocorrelation between districts and adjusting for other variables. Results: We evaluated 255,229 HEI across 750 facilities in 28 districts. The overall risk of HIV infection among all tested HEI between 2013 and 2020 was 7.2% (95%CI: 7.1-7.3). We observed a decreasing trend in the proportion of HEI that tested HIV positive from 7.0% (95%CI: 6.6-7.4) in 2013 to 5.7% (95%CI: 5.4-5.9) in 2015 followed by an increase to 9.9% (95%CI: 9.6-10.2) in 2017 and then a decreasing trend to 4.2% (95%CI: 3.7-4.6) in 2020. The risk of HIV infection increased by age of the HEI. There was spatial heterogeneity of HIV prevalence between districts of Malawi. Conclusion: We summarised spatial and temporal trends of risk of HIV infection amongst HEI in Malawi between 2013 and 2020. There is need for further strengthening of EID program to ensure that all the HEI are enrolled in care by eight weeks of age in order to further reduce mother-to-child transmission of HIV.

New record of species Monascus purpureus in Iraq

This study was conducted in the Laboratory of Fungus in the Department of Biology / College of Science / University of Qadisiyah to isolate and diagnose some insulation from fungi isolated from imported dried fruits (raisins) in Qadisiyah province, Iraq. The isolations were diagnosed both morphologically and microscopically using the classification keys and to confirm the appearance and microscopic diagnosis diagnosed using polymerase chain reaction(PCR), And determine the sequence of nitrogen bases (Nucleotide sequence(of compound DNA products using ITS1 and ITS4. The results of the nucleotide sequence analysis of DNA (PCR product) compounding innate isolation and using BLAST to compare with data available at the U.S. National Center for Biotechnology Information (NCBI) have shown that this isolation belongs to the type Monascus purpureus. By comparing the sequence of nitrogen bases of isolated M. purpureus fungus in this study, it was found that there was a 100% similarity to many of the M. purpureus fungus isolates previously registered at the National Center for Biotechnology Information (NCBI), including those diagnosed in China (MT361825, MK359689, MW581230) and Japan (AB477248).

HPV Status as Prognostic Biomarker in Head and Neck Cancer—Which Method Fits the Best for Outcome Prediction?

The incidence of human papillomavirus (HPV)-related head and neck cancer (HNSCC) is rising globally, presenting challenges for optimized clinical management. To date, it remains unclear which biomarker best reflects HPV-driven carcinogenesis, a process that is associated with better therapeutic response and outcome compared to tobacco/alcohol-induced cancers. Six potential HPV surrogate biomarkers were analyzed using FFPE tissue samples from 153 HNSCC patients (n = 78 oropharyngeal cancer (OPSCC), n = 35 laryngeal cancer, n = 23 hypopharyngeal cancer, n = 17 oral cavity cancer): p16, CyclinD1, pRb, dual immunohistochemical staining of p16 and Ki67, HPV-DNA-PCR, and HPV-DNA-in situ hybridization (ISH). Biomarkers were analyzed for correlation with one another, tumor subsite, and patient survival. P16-IHC alone showed the best performance for discriminating between good (high expression) vs poor outcome (low expression; p = 0.0030) in OPSCC patients. Additionally, HPV-DNA-ISH (p = 0.0039), HPV-DNA-PCR (p = 0.0113), and p16-Ki67 dual stain (p = 0.0047) were significantly associated with prognosis in uni- and multivariable analysis for oropharyngeal cancer. In the non-OPSCC group, however, none of the aforementioned surrogate markers was prognostic. Taken together, P16-IHC as a single biomarker displays the best diagnostic accuracy for prognosis stratification in OPSCC patients with a direct detection of HPV-DNA by PCR or ISH as well as p16-Ki67 dual stain as potential alternatives.

Molecular identification of pathogenic bacteria causing foodborne disease in Caulerpa racemosa

Caulerpa racemosa is a green algae consumed by people in northern coastal areas. C. racemosa has a habitat attached to the shallow seabed. C. racemosa usualy consumed fresh without any cooking process so that the contamination of microorganisms can be eaten. Molecular identification using 16S rRNA is needed to determine the type of bacterial contaminants in C. racemosa. The isolates of C. racemosa were cultured in HIA, BAP, and BHI media. Bacteria from BHI media were isolated by DNA, PCR for 16S rRNA gene, and sequencing. Bacteria isolate C. racemosa was found to have the α-hemolytic ability in BAP media. The sequencing analysis showed that the three bacterial colonies of C. racemosa isolate had high similarity with V. parahemolyticus, Caldalkalibacillus mannanilyticus, and Exiguobacterium profundum.

Comparison of Three In-House Real PCR Assays Targeting Kinetoplast DNA, the Small Subunit Ribosomal RNA Gene and the Glucose-6-Phosphate Isomerase Gene for the Detection of Leishmania spp. in Human Serum

To perform PCR from serum for the diagnosis of visceral leishmaniasis is convenient and much less invasive than the examination of deeper compartments such as bone marrow. We compared three Leishmania-specific real-time PCRs with three different molecular targets (kinetoplast DNA, the small subunit-ribosomal RNA-(ssrRNA-)gene, the glucose-6-phosphate isomerase-(gpi-)gene) regarding their sensitivity and specificity in human serum. Residual sera from previous diagnostic assessments at the German National Reference Center for Tropical Pathogens Bernhard Nocht Institute for Tropical Medicine Hamburg and the Swiss Tropical and Public Health Institute were used. The sensitivities of kinetoplast DNA-PCR, ssrRNA-gene PCR, and gpi-PCR were 93.3%, 73.3%, and 33.3%, respectively, with 15 initial serum samples from visceral leishmaniasis patients, as well as 9.1%, 9.1%, and 0.0%, respectively, with 11 follow-up serum samples taken at various time points following anti-leishmanial therapy. Specificity was 100.0% in all assays as recorded with 1.137 serum samples from deployed soldiers and migrants without clinical suspicion of visceral leishmaniasis. Kinetoplast-DNA PCR from serum was confirmed as a sensitive and specific approach for the diagnosis of visceral leishmaniasis. The results also indicate the suitability of serum PCR for diagnostic follow-up after therapy, in particular regarding therapeutic failure in case of persisting positive PCR results.