Allelic dropout in PAH affecting the results of genetic diagnosis in phenylketonuria

Objectives Phenylketonuria (PKU) is an inherited autosomal recessive disorder of phenylalanine metabolism. It is mainly caused by a deficiency in phenylalanine hydroxylase (PAH) and frequently diagnosed with Sanger sequencing. To some extent, allelic dropout can explain the inconsistency in genotype and phenotype. Methods Three families were evaluated through DNA sequence analysis, multiplex ligation-dependent probe amplification (MLPA) and prenatal diagnosis technologies. The possibility of inconsistency in phenotype and genotype with c.331C>T variant was analysed. Results Through pedigree analysis, three mothers carried a homozygous c.331C>T variant, which was a false-positive result. New primers were used, and this error was caused by allelic dropout. In this case, c.158G>A was likely a benign variant. Conclusions Sequence variants in primer-binding regions could cause allelic dropout, creating unpredictable errors in genotyping. Our results emphasised the need for careful measures to treat genotype–phenotype inconsistencies.

RapidResa Polymyxin Acinetobacter NP® Test for Rapid Detection of Polymyxin Resistance in Acinetobacter baumannii

A homemade and culture-based test, relying on the visual detection of the reduction of the resazurin reagent (a cell viability indicator), has been developed for the rapid detection of polymyxin resistance in Acinetobacter baumannii. Here, we evaluated the industrial version of this test, the RapidResa Polymyxin Acinetobacter NP® test (Liofilchem, Italy). A well-characterized panel of 68 clinical A. baumannii strains (36 polymyxin-susceptible, 26 polymyxin-resistant A. baumannii, and 6 colistin-heteroresistant isolates) of worldwide origin was tested. All the colistin-susceptible A. baumannii isolates gave negative results according to the RapidResa Polymyxin Acinetobacter NP® test, except for a single isolate that gave a false-positive result. Out of the 26 colistin-resistant A. baumannii strains, 25 were correctly identified as colistin resistant using the RapidResa Polymyxin Acinetobacter NP® test. Only a single colistin-resistant A. baumannii strain gave a false-negative result. Additionally, the six colistin-heteroresistant isolates tested gave positive results. Altogether, the sensitivity and the specificity of the test were found to be 96% and 97%, respectively. The turn-around-time of this easy-to-perform test was 3-4h, which showed excellent reliability for identification of polymyxin resistance in A. baumannii. The RapidResa Polymyxin Acinetobacter NP® test allows a rapid differentiation between polymyxin-susceptible and -resistant A. baumannii isolates, which may contribute to optimization of the use of polymyxins for treating infections due to multidrug-resistant A. baumannii.

A case of syphilis with an extremely high RPR titer

It remains unidentified why this patient has an extremely high Rapid Plasma Reagin Titer (RPR) 1: 2048. A false positive result was ruled out by a Treponemal test. To ensure accuracy, repeat tests were requested for both the Non-Treponemal and Treponemal Tests in the same laboratory, and the results came back the same. In the Traditional Algorithm, Positive Nontreponemal Syphilis screening tests are confirmed by Treponemal tests, although the latter is not quantitative. The lack of full sensitivity in the Traditional Algorithm Testing has led the CDC and some other centers to adopt Reverse Sequence Testing. The resurgence of syphilis after 20 years could lead to mutagenic strain thus making it relevant for a need for heightened awareness.

Subsequent attendance in a breast cancer screening program after a false-positive result in the Local Health Authority of Bologna (Italy)

We conducted a cross-sectional study to assess the likelihood of returning for routine breast cancer screening among women who have experienced a false-positive result (FPR) and to describe the possible individual and organizational factors that could influence subsequent attendance to the screening program. Several information were collected on demographic and clinical characteristics data. Electronic data from 2014 to 2016 related to breast screening program of the Local Health Authority (LHA) of Bologna (Italy) of women between 45 and 74 years old were reviewed. A total of 4847 women experienced an FPR during mammographic screening and were recalled to subsequent round; 80.2% adhered to the screening. Mean age was 54.2 ± 8.4 years old. Women resulted to be less likely to adhere to screening if they were not-Italian (p = 0.001), if they lived in the Bologna district (p < 0.001), if they had to wait more than 5 days from II level test to end of diagnostic procedures (p = 0.001), if the diagnostic tests were performed in a hospital with the less volume of activity and higher recall rate (RR) (p < 0.001) and if they had no previous participation to screening tests (p < 0.001). Our results are consistent with previous studies, and encourages the implementation and innovation of the organizational characteristics for breast cancer screening. The success of screening programs requires an efficient indicators monitoring strategy to develop and evaluate continuous improvement processes.

Crispr-cas 12a combination to alleviate the false-positive in loop-mediated isothermal amplification-based diagnosis of Neisseria meningitidis

Loop mediated Isothermal amplicafication (LAMP) was recently suggested as a diagnostic tool for the identification of Neisseria meningitides. Howevere, this isothermal amplification is challenged by the fact its amplification leads to risks of obtaining false-positive results. Whereas, with abilities to accurately recognize specific sequence, the CRISPR/Cas12a can forms complexes with cognate RNA sensors and cleave pathogen’s DNA targets complimerntary to its cognate RNA and acquires collateral activity to unbiasedly cut nearby off-target fragments. Therefore, if relevant fluorescent-quencher-nucleic probes are present in the reaction, the non-specific cleavage of probes releases fluorescences and establish diagnostic read-outs. In this study, we demonstrate a proof-of-concept that in relevant biochemical conditions, CRISPR/Cas12a and LAMP can work synchronously to identify genetics materials of Nesseria menitigistis at the level of 0.00004% in less than 2 h. Additionally, our clinical data also showed that the combinatory of CRISPR/Cas12a help to alleviate false positive result therefore enhance the specificity gained by the LAMP assays.