71. Diagnostic Stewardship of Clostridioides difficile Testing

Background C. difficile (CD) testing is frequently ordered inappropriately. Highly sensitive polymerase chain reaction (PCR) tests can detect CD colonization leading to misdiagnosis. Providers often overlook other causes of diarrhea, notably laxatives. To improve diagnostic stewardship, our hospital introduced an electronic medical record (EMR)-based order set (OS). Methods In a 926-bed, teaching hospital, we conducted a 3-step intervention to improve CD diagnostic stewardship. (1) A retrospective analysis of CD orders before and after OS implementation was done to assess its impact on inappropriate orders. The OS included two questions: (a) Did patient have ≥ 3 loose bowel movements in past 24 hours? and (b) No laxatives in past 24 hours? An appropriate order was defined if “yes” to both questions. It was still appropriate if “no” to either question but ≥ 2 unexplained following features: fever > 100.4 F, abdominal pain, megacolon, ileus or leukocytosis > 11,000 cells/mm3 in prior day. (2) After implementation of OS, house staff compliance with OS was surveyed via email. (3) Rationale for inappropriate orders was discussed with providers. Results Of 238 patients in retrospective analysis, 44% were ≥ 65 years and 37% had other potential causes of diarrhea. Common clinical features were leukocytosis (40%) and fever (31%). There was no significant difference in inappropriate testing: pre-OS 27/99 (27%) vs post-OS 44/139 (32%) (p=0.47). Of 43 house officers who participated in the survey, 75% indicated they over rode the OS. When asked to provide rationale of inappropriate CD testing, providers acknowledged inappropriate ordering but did not want to miss a CD diagnosis and frequently overlooked other causes of diarrhea. Conclusion Appropriate CD testing relies on providers’ appreciation of a clinical picture consistent with CD infection, confirmation of clinically significant diarrhea, and consideration of other causes of diarrhea. Providers order inappropriate tests, not due to lack of knowledge, but likely fear of missing diagnosis and overlooking other causes of diarrhea. Disclosures All Authors: No reported disclosures

Application of PCR for the detection of tubercle bacilli from retropharyngeal and thyroid abscess: case reports

The diagnosis of extra-pulmonary tuberculosis by Mycobacterium tuberculosis infection in retro­pharyngeal and thyroid abscess is challenging due to a lack of rapid, sensitive and specific diag­nostic assay. Hence, we evaluated the performance of highly sensitive polymerase chain reaction (PCR) to detect the M. tuberculosis in the retropharyngeal and thyroid abscess. The microscopy and culture were negative test result however; PCR result had demonstrated the presence of M. tuberculosis in the same specimens with retropharyngeal and thyroid abscess. Therefore, it is rec­ommended to perform PCR to detect undiagnosed cases, which were not detected by convention­al approaches for the diagnosis, of extra-pulmonary tuberculosis.

MGMT promoter methylation in patients with glioblastoma: is methylation-sensitive high-resolution melting superior to methylation-sensitive polymerase chain reaction assay?

OBJECTIVEThe methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter is a prognostic factor in adults with glioblastoma (GBM); it also yields information that is useful for clinical decision-making in elderly GBM patients. While pyrosequencing is the gold standard for the evaluation of the methylation status of MGMT, methylation-sensitive polymerase chain reaction (MS-PCR) assay continues to be used widely. Although MS-PCR results exhibited a good correlation with the prognosis of patients with GBM treated under the Stupp protocol, interpretation of the bands is based on subjective judgment, and the assay cannot be used to analyze heterogeneously methylated samples. We assessed whether methylation-sensitive high-resolution melting (MS-HRM) is an alternative to MS-PCR.METHODSThe authors prepared 3 primer sets that covered CpG 72–89 for MS-HRM analysis to determine the methylation levels of 6 human glioma cell lines. The results were validated by bisulfite sequencing of cloned alleles. The authors also subjected surgical samples from 75 GBM patients treated with temozolomide (TMZ) to MS-HRM to assess the MGMT methylation status and compared the findings with MS-PCR results using receiver operating characteristic (ROC), univariate, and multivariate analyses.RESULTSThere was a strong correlation between the methylation levels of the 6 glioma cell lines evaluated by MS-HRM and by bisulfite sequencing; with primers 1 and 2, the correlation was significant (r = 0.959 and r = 0.960, respectively, p < 0.01). Based on log-rank analysis, MS-HRM was significantly better than MS-PCR for predicting progression-free survival (PFS) and overall survival (OS) based on the methylation status of the MGMT promoter (PFS predicted by MS-HRM and MS-PCR = 0.00023 and 0.0035, respectively; OS = 0.00019 and 0.00028, respectively). ROC analysis showed that the area under the curve was larger with MS-HRM than with MS-PCR (PFS: 0.723 vs 0.635; OS: 0.716 vs 0.695). Based on multivariate Cox regression analysis, MS-HRM was significantly better than MS-PCR for predicting the treatment outcome (MS-HRM vs MS-PCR: PFS, p = 0.001 vs 0.207; OS, p = 0.013 vs 0.135).CONCLUSIONSThe authors’ findings show that MS-HRM is superior to MS-PCR for the detection of MGMT promoter methylation. They suggest MS-HRM as an alternative to MS-PCR for assessing the prognosis of patients with GBM.

High Frequency of MYD88 L265P Mutation in Primary Ocular Adnexal Marginal Zone Lymphoma and Its Clinicopathologic Correlation: A Study From a Single Institution

Context.— The pathogenesis of primary ocular adnexal marginal zone lymphoma (POAMZL) remains unclear. The reported associations with Chlamydia psittaci infection and MYD88 mutations are highly variable. Objective.— To examine MYD88 L265P mutation in ocular marginal zone lymphomas and correlate with clinicopathologic features and Chlamydia infection. Design.— Presence of MYD88 L265P mutation and Chlamydia infection in lymphoma was analyzed by using sensitive polymerase chain reaction (PCR) methods. Results.— The MYD88 L265P mutation was identified in 8 of 22 POAMZLs (36%), including 2 of 3 cases in which PCR failed to detect clonal IGH gene rearrangement; none of the 4 secondary marginal zone lymphomas were positive. Test results for Chlamydia were negative in all cases. Patients with and without the MYD88 mutation had similar clinicopathologic features. Conclusions.— The MYD88 mutational analysis provides important information in diagnostic workup of POAMZL. The frequent MYD88 mutation suggests a critical role of this aberration in the pathogenesis of POAMZL and may serve as a therapeutic target for patients with progressive disease.

RESULTS OF THE USE OF SOFOSBUVIR IN COMBINATION WITH LEDIPASVIR OR DACLATASVIR FOR CHRONIC HEPATITIS C TREATMENT IN THE REPUBLIC OF BELARUS

To evaluate the efficacy of therapy with sofosbuvir in combination with ledipasvir or daclatasvir, the results of treatment of 299 patients with chronic hepatitis C, including 128 non-responders to combined interferon plus ribavirin therapy, who have prognostically unfavorable single nucleotide polymorphisms 39743165T> G (rs8099917) and 39738787C> T (rs12979860) of interleukin-28B gene, were analyzed. 57 people had liver cirrhosis. 80,9% (242) had genotype 1 of hepatitis C virus, 5% (15) – genotype 2, 13,7% (41) – genotype 3, and 0,4% (1) – genotype 4.All 299 patients, who adhered to the recommendations of the European Association for the Study of the Liver 2016 and the American Association for the Study of the Liver Disease 2017, achieved a sustained virologic response 12 weeks after the end of therapy. The clinical case of treatment failure, associated with the lack of confirmation of the elimination of hepatitis C virus by means of highly sensitive polymerase chain reaction methods and with the later identified amino acid substitution in position Y93H of NS5A (resistance to NS5A inhibitors), is shown.It is necessary to carry out monitoring of effectiveness of therapy only by means of highly sensitive polymerase chain reaction (from 10 ME/ml). If the virus elimination delays in patients with advanced stages of liver fibrosis it is needed to use the prolonged schemes of treatment. Repeated treatment of patients with existence of a mutation of Y93H requires the use of new NS5A inhibitors or combined drugs.

Elsődleges genetikai vizsgálat Prader–Willi-szindróma igazolására

Introduction: According to the international literature, DNA methylation analysis of the promoter region of SNRPN locus is the most efficient way to start genetic investigation in patients with suspected Prader–Willi syndrome. Aim: Our aim was to develop a simple, reliable first-tier diagnosis to confirm Prader–Willi syndrome, therefore to compare our self-designed simple, cost-efficient high-resolution melting analysis and the most commonly used methylation-specific multiplex ligation-dependent probe amplification to confirm Prader–Willi syndrome. Method: We studied 17 clinically suspected Prader–Willi syndrome children and their DNA samples. With self-designed primers, bisulfite-sensitive polymerase chain reaction, high-resolution melting analysis and, as a control, methylation-specific multiplex ligation-dependent probe amplification were performed. Results: Prader–Willi syndrome was genetically confirmed in 6 out of 17 clinically suspected Prader–Willi syndrome patients. The results of high-resolution melting analysis and methylation-specific multiplex ligation-dependent probe amplification were equivalent in each case. Conclusion: Using our self-designed primers and altered bisulfite-specific PCR conditions, high-resolution melting analysis appears to be a simple, fast, reliable and effective method for primarily proving or excluding clinically suspected Prade-Willi syndrome cases. Orv Hetil. 2018; 159(2): 64–69.