A surrogate molecular approach for the detection of Philadelphia chromosome-like B-acute lymphoblastic leukemia.

BACKGROUND: Philadelphia chromosome (Ph)-like B-acute lymphoblastic leukemia (B-ALL) is a clinically significant, high-risk genetic subtype of B-ALL cases. There are few data on the incidence, characterization, and treatment outcomes of Ph-like ALL cases from low- and middle-income countries. There is a pressing need to establish a well-organized/cost-effective approach for identifying Ph-like ALL instances. METHODS: Multiplex reverse transcriptase polymerase chain reaction, nCounter NanoString, and fluorescence in situ hybridization were used to detect and characterize Ph-like ALL cases among recurrent genetic abnormalities (RGA)neg B-ALL cases. At the end of induction therapy, flow cytometry-minimal residual disease (MRD) assay was used to quantify MRD positivity in Ph-like ALL cases. RESULTS: Of 130 newly diagnosed B-ALL cases, 25% (BCR::ABL1), 4% (ETV6::RUNX1), 5% (TCF3::PBX1), 2% (KM2TA::AFF1), and 65% RGAneg B-ALL cases were revealed by multiplex reverse transcriptase polymerase chain reaction. Among RGAneg B-ALL cases, 24% Ph-like ALL cases using nCounter NanoString were identified, with 48% CRLF2high cases with 45% CRLF2::P2RY8 and 18% CRLF2::IGH rearrangements(∼r) revealed by fluorescence in situ hybridization. In 52% of CRLF2low cases, 17% ABL1 and JAK2∼r 8% EPOR::IGH & PDGRFB∼r were identified. Ph-like ALL cases had higher total leukocyte count (p < .05), male preponderance (p < .05), and high MRD-positivity/induction failure compared with RGAneg B-ALL cases. Furthermore, in Ph-like ALL cases, 11 significant genes using quantitative polymerase chain reaction were identified and validated. CRLF2, IGJ, CEACAM6, MUC4, SPATS2L and NRXN3 genes were overexpressed and show statistical significance (p < .05) in Ph-like ALL cases. CONCLUSIONS: This study showed the high incidence of Ph-like ALL cases with kinase activating alterations and treatment outcomes from low- and middle-income region. Furthermore, a surrogate cost-effective multiplex panel of 11 overexpressed genes for the prompt detection of Ph-like ALL cases is proposed. PLAIN LANGUAGE SUMMARY: Identification of recurrent gene abnormalities (RGA)neg B-acute lymphoblastic leukemia (B-ALL) cases using multiplex-reverse transcriptase polymerase chain reaction. Identification and characterization of Philadelphia (Ph)-like ALL cases using nCounter NanoString gene expression profiling and fluorescence in situ hybridization. Furthermore, Ph-like ALL cases were characterized according to CRLF2 expression and kinase-activating genomic alterations. Minimal residual disease of Ph-like ALL cases were quantified using flow cytometry-minimal residual disease assay. A surrogate molecular approach was established to detect Ph-like ALL cases from low- and middle-income countries.

Analytical evaluation of the automated genotyping system (GenPlex) compared to a traditional real-time PCR assay for the detection of high-risk human papillomaviruses.

The detection of high-risk human papillomaviruses (HPVs) is crucial for early screening and preventing cervical cancer. However, the substantial workload in high-level hospitals or the limited resources in primary-level hospitals hinder widespread testing. To address this issue, we explored a sample-to-answer genotyping system and assessed its performance by comparing it with the traditional real-time polymerase chain reaction (PCR) method conducted manually. Samples randomly selected from those undergoing routine real-time PCR detection were re-analyzed using the fully automatic GenPlex® system. This system identifies 24 types of HPV through a combination of ordinary PCR and microarray-based reverse hybridization. Inconsistent results were confirmed by repeated testing with both methods, and the κ concordance test was employed to evaluate differences between the two methods. A total of 365 samples were randomly selected from 7259 women. According to real-time PCR results, 76 were high-risk HPV negative, and 289 were positive. The GenPlex® system achieved a κ value greater than 0.9 (ranging from 0.920 to 1.000, p < 0.0001) for 14 types of high-risk HPV, except HPV 51 (κ = 0.697, p < 0.0001). However, the inconsistent results in high-risk HPV 51 were revealed to be false positive in real-time PCR by other method. When counting by samples without discriminating the high-risk HPV type, the results of both methods were entirely consistent (κ = 1.000, p < 0.0001). Notably, the GenPlex® system identified more positive cases, with 73 having an HPV type not covered by real-time PCR, and 20 potentially due to low DNA concentration undetectable by the latter. Compared with the routinely used real-time PCR assay, the GenPlex® system demonstrated high consistency. Importantly, the system's advantages in automatic operation and a sealed lab-on-chip format respectively reduce manual work and prevent aerosol pollution. For widespread use of GenPlex® system, formal clinical validation following international criteria should be warranted.

Smart nano generation of transgenic algae expressing white spot syndrome virus in shrimps for inner ear-oral infection treatments using the spotted hyena optimizer (SHO)-Long short-term memory algorithm.

Nanotechnology offers a promising avenue to amplify the effectiveness and precision of using transgenic algae in managing WSSV in shrimp by possibly crafting nano-carriers for targeted therapeutic agent delivery or modifying algae cells at a molecular level. Leveraging the capabilities of nano-scale interventions, this study could explore innovative means to manipulate cellular processes, control biological interactions, and enhance treatment efficacy while minimizing undesirable impacts in aquatic environments. The White Spot Syndrome Virus (WSSV) is a double-stranded DNA virus with a tail and rod form that belongs to theNimaviridaefamily. There is no workable way to manage this illness at the moment. This research proposes a new model based on the Long Short-Term Memory (LSTM) and Spotted Hyena Optimizer (SHO) method to control the inner ear-oral infection, utilizing transgenic algae (Chlamydomonas reinhardtii). It is pretty tricky to modify the weight matrix in LSTM. The output will be more accurate if the weight of the neurons is exact. Histological examinations and nested polymerase chain reaction (PCR) testing were performed on the challenged shrimp every 4 h to assess the degree of white spot disease. The SHO-LSTM has shown the highest accuracy and Roc value (98.12% and 0.93, respectively) and the lowest error values (MSE = 0.182 and MAE = 0.48). The hybrid optimized model improves the overall inner ear-oral linked neurological diseases detection ratio. Additionally, with the slightest technical complexity, it effectively controls the forecast factors required to anticipate the ENT. Algal cells were found to be particularly well-suited for inner ear-oral infections, and shrimps fed a transgenic line had the best survival ratio in WSSV infection studies, with 87% of the shrimp surviving. This shows that using this line would effectively stop the spread of WSSV in shrimp populations.

Development of Rapid Detection Methods for Fusarium oysporum f. sp. melonis in Melon Seeds.

Melon (Cucumis melo L.) is a global commercial crop that is sensitive to seed-borne wilt infections caused by Fusarium oxysporum f. sp. melonis (Fom). To address the challenge of detecting Fom contamination, we designed a probe-based real-time PCR method, TDCP2, in combination with rapid or column-based DNA extraction protocols to develop reliable molecular detection methods. Utilizing TDCP2, the detection rate reached 100% for both artificially Fom-inoculated (0.25-25%) and pod-inoculated melon seeds in conjunction with DNA samples from either the rapid or column-based extraction protocol. We performed analyses of precision, recall, and F1 scores, achieving a maximum F1 score of 1 with TDCP2, which highlights the robustness of the method. Additionally, intraday and interday assays were performed, which revealed the high reproducibility and stability of column-based DNA extraction protocols combined with TDCP2. These metrics confirm the reliability of our developed protocols, setting a foundation for future enhancements in seed pathology diagnostics and potentially broadening their applicability across various Fom infection levels. In the future, we hope that these methods will reduce food loss by improving the control and management of melon diseases.

Microsporidial keratoconjunctivitis - first outbreak in Japan.

BACKGROUND: Most cases of microsporidial keratoconjunctivitis are found in the Southern hemisphere. Our purpose was to investigate the first outbreak of microsporidial keratoconjunctivitis in Japan among healthy, immunocompetent soccer players from the same team during a 1-month period. CASE PRESENTATION: This study is an observational case series. The medical records were analyzed for five cases with microsporidial keratoconjunctivitis who presented within September 2022. All five cases were males between 28 and 36 years old. These previously healthy individuals belonged to the same football team. Their eyes were considered susceptible to contaminated water or dirt from the turf at game and practice sites. All cases involved unilateral conjunctivitis, with scattered round white lesions that showed positive fluorescein staining in the corneal epithelium. All cases experienced diminution of vision in the affected eye. In three cases, direct smears showed spores of approximately 2-3 µm in diameter. Polymerase chain reaction (PCR) analysis of corneal scrapes revealed partial amplification of microsporidial 18 S ribosomal RNA gene in four cases. Sequences of PCR products from all four cases showed 100% identity with strains of Vittaforma corneae previously reported from an outbreak in Singapore. All cases were treated with topical therapy, including voriconazole, fluorometholone, and levofloxacin. Four eyes underwent corneal scraping. After treatment, all eyes healed without residual opacities. CONCLUSIONS: Only a few sporadic case reports of this disease have previously been reported in Japan. We detected V. corneae in our case series, representing what appears to be the first outbreak of microsporidial keratoconjunctivitis in Japan. Exposure to contaminated water or soil, in addition to inadequate sanitary facilities, represents a potential source of infection. Further investigations to clarify the characteristics of microsporidia seem warranted.

Cost-effectiveness analysis of diagnostic strategies for COVID-19 in Iran.

BACKGROUND: Since 2020, COVID-19 has become a global public health issue and has caused problems worldwide. This infection can lead to a fever and respiratory problems. Asymptomatic carriers of the virus are a significant part of the spread of the disease, so early screening and diagnosis of suspected cases of COVID-19 are essential. Generally, standard diagnostic methods include lung imaging (CT), polymerase chain reaction (PCR), and corona antibody (IgM&IgG) testing. However, the costs of the above tests for the healthcare system cannot be ignored, and evaluating the incremental costs against the additional benefit is necessary. Therefore, this study aimed to determine the cost-effectiveness of diagnostic methods for COVID-19 patients. MATERIALS AND METHODS: In this research, an economic evaluation analysis was conducted to reveal the cost-effectiveness of the diagnostic strategies for COVID-19 from the service provider's perspective. Basic information about the costs of CT, serology (IgG&IgM), and molecular (PCR) tests were collected from the Ministry of Health of Iran. The effectiveness data were calculated according to the sensitivity and specificity of the diagnostic tests for COVID-19. In this study, the incremental cost-effectiveness ratio (ICER) of the diagnostic strategies for COVID-19 was estimated, and the most cost-effective diagnostic strategy was determined. In calculating ICER and analyzing the sensitivity of the results, Treeage software was used. RESULTS: According to the calculated incremental effectiveness cost ratio for scenarios with 5, 10, and 50% prevalence of COVID-19 and according to the threshold defined by the World Health Organization, in the study, PCR, PCR, and IgG&IgM strategies are the most cost-effective diagnostic methods of the corona. Also, the results were not sensitive to the desired parameters based on the results of one-way sensitivity analysis. CONCLUSION: Nowadays there are various tests with different levels of accuracy in the diagnosis of COVID-19. In general, PCR tests are more cost-effective for low prevalence of Covid-19, while IgM&IgG tests are more cost-effective for high estimated prevalence. The results of this research can help policymakers and health system managers to validate the most accurate diagnostic method for COVID-19, considering the prevalence of the disease.

Identification of 'Candidatus Liberibacter asiaticus' the Huanglongbing Bacterium in citrus from Colombia.

Candidatus Liberibacter spp is the most prevalent microorganism in the citrus plant, associated with Citrus Huanglongbing (HLB), which is transmitted by the psyllid vector. In Colombia, the vector Diaphorina citri Kugayama has been reported in different regions, but "Ca. Liberibacter asiaticus" (CLas) has only been detected in insect vectors, not in citrus host plants. To identify the presence and quantify the pathogen in citrus tissues, we employed a combined strategy that involved three techniques based on polymerase chain reaction (PCR). First, we used endpoint PCR with specific primers for CLas (OI1-OI2c) to confirm the infection. Second, we used qPCR with specific primers CIT295a - CIT298 designed on 16S rDNA gene regions to quantify the pathogen load. Finally, we employed droplet digital PCR (ddPCR) to determine the copy number of the pathogen in citrus tissues using the ß-subunit of ribonucleotide reductase (RNR) gene (nrdB) that is specific to CLas. We identified the presence of CLas in citrus plants for the first time in Colombia and quantified its titer in the plant tissue. We employed ddPCR and qPCR to provide crucial information for the country's disease management, control strategies, and general crop health.

Low-Cost, Real-Time Polymerase Chain Reaction System with Integrated RNA Extraction.

Rapid, easy-to-use, and low-cost systems for biological sample testing are important for point-of-care diagnostics and various other health applications. The recent pandemic of Coronavirus Disease 2019 (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) showed an urgent need to rapidly and accurately identify the genetic material of SARS-CoV-2, an enveloped ribonucleic acid (RNA) virus, in upper respiratory specimens from people. In general, sensitive testing methods require genetic material extraction from the specimen. Unfortunately, current commercially available extraction kits are expensive and involve time-consuming and laborious extraction procedures. To overcome the difficulties associated with common extraction methods, we propose a simple enzymatic assay for the nucleic acid extraction step using heat mediation to improve the polymerase chain reaction (PCR) reaction sensitivity. Our protocol was tested on Human Coronavirus 229E (HCoV-229E) as an example, which comes from the large coronaviridae family of viruses that affect birds, amphibians, and mammals, of which SARS-CoV-2 is a member. The proposed assay was performed using a low-cost, custom-made, real-time PCR system that incorporates thermal cycling and fluorescence detection. It had fully customizable reaction settings to allow versatile biological sample testing for various applications, including point-of-care medical diagnosis, food and water quality testing, and emergency health situations. Our results show that heat-mediated RNA extraction is a viable extraction method when compared to commercial extraction kits. Further, our study showed that extraction has a direct impact on purified laboratory samples of HCoV-229E, but no direct impact on infected human cells. This is clinically relevant, as it allows us to circumvent the extraction step on clinical samples when using PCR.

α- and ß-Globin Gene Mutations in Individuals with Hemoglobinopathies in the Chattogram and Sylhet Regions of Bangladesh.

Hemoglobinopathies, including α- and ß-thalassemias and sickle cell disease, are among the most widely disseminated hereditary blood disorders worldwide. Bangladesh is considered a hotspot for hemoglobinopathies, and these diseases cause a significant health concern in the country. However, the country has a dearth of knowledge on the molecular etiology and carrier frequency of thalassemias, primarily due to a lack of diagnostic facilities, limited access to information, and the absence of efficient screening programs. This study sought to investigate the spectrum of mutations underlying hemoglobinopathies in Bangladesh. We developed a set of polymerase chain reaction (PCR)-based techniques to detect mutations in α- and ß-globin genes. We recruited 63 index subjects with previously diagnosed thalassemia. Along with age- and sex-matched control subjects, we assessed several hematological and serum indices and genotyped them using our PCR-based methods. We identified that parental consanguinity was associated with the occurrence of these hemoglobinopathies. Our PCR-based genotyping assays identified 23 HBB genotypes, with the codons 41/42 (-TTCT) (HBB: c.126_129delCTTT) mutation leading the spectrum. We also observed the presence of cooccurring HBA conditions, of which the participants were not aware. All index participants in this study were on iron chelation therapies, yet we found they had very high serum ferritin (SF) levels, indicating inefficient management of the individuals undergoing such treatments. Overall, this study provides essential information on the hemoglobinopathy mutation spectrum in Bangladesh and highlights the need for nationwide screening programs and an integrated policy for diagnosing and managing individuals with hemoglobinopathies.

Cost-effective, high-yield production of Pyrobaculum calidifontis DNA polymerase for PCR application.

Polymerase Chain Reaction (PCR) is widely used for cloning, genetic engineering, mutagenesis, detection and diagnosis. A thermostable DNA polymerase is required for PCR. Here we describe low-cost and high-recovery production of Pyrobaculum calidifontis DNA polymerase (Pca-Pol). The gene was cloned in pET-28a and expressed in Escherichia coli BL21CodonPlus. Gene expression conditions were optimized. Eventually, gene expression was induced with 0.1 mM IPTG for 3 hours at 37 °C. Recombinant Pca-Pol produced was purified to homogeneity by immobilized metal-ion affinity chromatography yielding around 9000 U of Pca-Pol per liter of the culture with a recovery of 92%. Stability and PCR amplification efficiency of Pca-Pol was tested under various storage conditions with highest efficiency in 25 mM Tris-Cl buffer (pH 8.5) containing 0.1% Tween 20, 0.2 mg/mL BSA and 20% glycerol. Under this condition, no loss in PCR activity of Pca-Pol was observed, even after one year of storage. Repeated freeze-thaw, however, deteriorated enzyme activity of Pca-Pol. 55% PCR amplification activity retained after 7 prolong freeze-thaw cycles (freezing overnight at -20 °C and thawing for 45 minutes at 28 °C). Purified Pca-Pol possessed 3'-5' exonuclease (proofreading) activity and is expected to have greater fidelity as compared to Taq polymerase which does not have proofreading activity.

High incidence of Pneumocystis jirovecii pneumonia in oncological patients: a 19-year study.

AIM: In the past, Pneumocystis jirovecii belonged to the Protozoa group, but is currently taxonomically included in the kingdom Fungi. P. jirovecii is an opportunistic pathogen, responsible for pneumocystis pneumonia with frequent complications of immunocompromised patients. Delayed initiation of appropriate therapy increases the risk of death in immunocompromised patient. The aim of this work was to determine and evaluate the reliability of methods of laboratory diagnosis of pneumocystosis used in routine laboratories as well as the occurrence of this disease in patients from Slovakia during 19 years. MATERIAL AND METHODS: The diagnosis is based on microscopic examination (Giemsa- and Gram-Weigert-staining) and detection of parasite DNA by classical or real-time PCR in bronchoalveolar lavage and sputum. RESULTS: Pneumocysts were detected in 190 persons (5.7%) from the whole group of patients. Cancer patients represented the riskiest group in terms of pneumocystosis, which was confirmed by the highest percentage (57.9%) of individuals infected with P. jirovecii. Compared with the PCR, 33.7% sensitivity and 100% specificity of microscopy was calculated by using a binary classification test. Molecular methods are more sensitive in the detection of P. jirovecii compared to microscopic evidence and currently represent a reliable detection system in the diagnosis of pneumocystosis. CONCLUSION: In view of the increasing number of immunocompromised persons, diagnostics of P. jirovecii in patients with pulmonary complications is essential. This was also confirmed in our study, where the number of examinations and detection of this opportunistic pathogen increased over the years.

Designing a sustainable-resilient-responsive supply chain network considering uncertainty in the COVID-19 era.

Effective supply chain management is crucial for economic growth, and sustainability is becoming a key consideration for large companies. COVID-19 has presented significant challenges to supply chains, making PCR testing a vital product during the pandemic. It detects the presence of the virus if you are infected at the time and detects fragments of the virus even after you are no longer infected. This paper proposes a multi-objective mathematical linear model to optimize a sustainable, resilient, and responsive supply chain for PCR diagnostic tests. The model aims to minimize costs, negative societal impact caused by shortages, and environmental impact, using a scenario-based approach with stochastic programming. The model is validated by investigating a real-life case study in one of Iran's high-risk supply chain areas. The proposed model is solved using the revised multi-choice goal programming method. Lastly, sensitivity analyses based on effective parameters are conducted to analyze the behavior of the developed Mixed-Integer Linear Programming. According to the results, not only is the model capable of balancing three objective functions, but it is also capable of providing resilient and responsive networks. To enhance the design of the supply chain network, this paper has considered various COVID-19 variants and their infectious rates, in contrast to prior studies that did not consider the variations in demand and societal impact exhibited by different virus variants.

Cerebrospinal fluid findings in COVID-19: a multicenter study of 150 lumbar punctures in 127 patients.

BACKGROUND: Comprehensive data on the cerebrospinal fluid (CSF) profile in patients with COVID-19 and neurological involvement from large-scale multicenter studies are missing so far. OBJECTIVE: To analyze systematically the CSF profile in COVID-19. METHODS: Retrospective analysis of 150 lumbar punctures in 127 patients with PCR-proven COVID-19 and neurological symptoms seen at 17 European university centers RESULTS: The most frequent pathological finding was blood-CSF barrier (BCB) dysfunction (median QAlb 11.4 [6.72-50.8]), which was present in 58/116 (50%) samples from patients without pre-/coexisting CNS diseases (group I). QAlb remained elevated > 14d (47.6%) and even > 30d (55.6%) after neurological onset. CSF total protein was elevated in 54/118 (45.8%) samples (median 65.35 mg/dl [45.3-240.4]) and strongly correlated with QAlb. The CSF white cell count (WCC) was increased in 14/128 (11%) samples (mostly lympho-monocytic; median 10 cells/µl, > 100 in only 4). An albuminocytological dissociation (ACD) was found in 43/115 (37.4%) samples. CSF L-lactate was increased in 26/109 (24%; median 3.04 mmol/l [2.2-4]). CSF-IgG was elevated in 50/100 (50%), but was of peripheral origin, since QIgG was normal in almost all cases, as were QIgA and QIgM. In 58/103 samples (56%) pattern 4 oligoclonal bands (OCB) compatible with systemic inflammation were present, while CSF-restricted OCB were found in only 2/103 (1.9%). SARS-CoV-2-CSF-PCR was negative in 76/76 samples. Routine CSF findings were normal in 35%. Cytokine levels were frequently elevated in the CSF (often associated with BCB dysfunction) and serum, partly remaining positive at high levels for weeks/months (939 tests). Of note, a positive SARS-CoV-2-IgG-antibody index (AI) was found in 2/19 (10.5%) patients which was associated with unusually high WCC in both of them and a strongly increased interleukin-6 (IL-6) index in one (not tested in the other). Anti-neuronal/anti-glial autoantibodies were mostly absent in the CSF and serum (1509 tests). In samples from patients with pre-/coexisting CNS disorders (group II [N = 19]; including multiple sclerosis, JC-virus-associated immune reconstitution inflammatory syndrome, HSV/VZV encephalitis/meningitis, CNS lymphoma, anti-Yo syndrome, subarachnoid hemorrhage), CSF findings were mostly representative of the respective disease. CONCLUSIONS: The CSF profile in COVID-19 with neurological symptoms is mainly characterized by BCB disruption in the absence of intrathecal inflammation, compatible with cerebrospinal endotheliopathy. Persistent BCB dysfunction and elevated cytokine levels may contribute to both acute symptoms and 'long COVID'. Direct infection of the CNS with SARS-CoV-2, if occurring at all, seems to be rare. Broad differential diagnostic considerations are recommended to avoid misinterpretation of treatable coexisting neurological disorders as complications of COVID-19.

The evaluation of potential global impact of the N501Y mutation in SARS-COV-2 positive patients.

Rapid and reliable detection of severe acute respiratory syndrome coronavirus 2 mutations are significant to control the contagion and spread rate of the virus. We aimed to evaluate the N501Y mutation rate in randomly chosen positive patients with the polymerase chain reaction (PCR). The evaluation and analysis of the data with a retrospective approach in cases with mutations, in terms of public health, will contribute to the literature on the global pandemic that affects our society. Public health authorities will take the necessary precautions and evaluate the current situation. The N501Y mutation was detected in patients with positive Covid-19 PCR test results. The positive samples were examined based on the 6-carboxy-fluorescein (FAM) channel in reverse transcription PCR (RT-PCR) quantitation cycle (Cq) values as low Cq (<25), medium Cq (25-32), and high Cq (32-38) groups. In the study, 2757 (19.7%) of 13 972 cases were detected as mutation suspects and 159 (5.8%) of them were found to have mutations. The ages of the cases with mutations ranged from 1 to 88 years (mean age of 40.99 ± 17.55). 49.7% (n = 79) of the cases with mutations were male, and 50.3% (n = 80) were female. When the RT-PCR-Cq results were examined, it was seen that it varied between 11.3 and 35.03, with an average of 20.75 ± 3.32.

Clinical profile of children with parechovirus meningitis in Singapore.

Human parechovirus (HPeV) is one of the most common causes of aseptic meningitis in children worldwide. This study aims to review the epidemiology, clinical presentation, and cerebrospinal fluid (CSF) findings in HPeV meningitis and compare these with Enterovirus (EV) meningitis. This is a retrospective study of children aged ≤ 1 year admitted for HPeV meningitis between November 2015 and July 2017, with positive CSF HPeV PCR and negative blood and CSF bacterial cultures. The clinical findings were compared with a historical cohort of children with EV meningitis admitted between July 2008 and July 2011. There were 71 children with HPeV meningitis, aged between 2 and 127 days, with the majority (96%) being ≤ 90 days old. The most common symptoms reported were poor feeding (42%), tachycardia out of proportion to fever (27%), and lethargy (20%). Only 2 patients (3%) had CSF pleocytosis. Cerebral spinal fluid white blood cell counts ranged from 0 to 28 cells/mm3, with a median of 3 cells/mm3 [interquartile range (IQR) 1-6 cells/mm3]. When compared to our historical cohort of EV meningitis ≤ 90 days old, children with HPeV meningitis ≤ 90 days old were less likely to have CSF pleocytosis (OR 0.008, 95% CI 0.001-0.057). HPeV and EV meningitis are known to cause sepsis-like illness in infants < 90 days old. This study further supports this, with the requirement for fluid bolus therapy for tachycardia or poor perfusion noted to be higher in children with HPeV meningitis ≤ 90 days old (OR 6.3, 95% CI 2.7-14.2).

Risk stratification by anamnesis increases SARS-CoV-2 test efficiency in cancer patients.

PURPOSE: To evaluate the impact of testing asymptomatic cancer patients, we analyzed all tests for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) before and during radiotherapy at a tertiary cancer center throughout the second wave of the pandemic in Germany. METHODS: Results of all real-time polymerase chain reaction (RT-PCR) tests for SARS-CoV­2 performed at our radio-oncology department between 13 October 2020 and 11 March 2021 were included. Clinical data and anamnestic information at the time of testing were documented and examined for (i) the presence of COVID-19-related symptoms and (ii) virus-related anamnesis (high-risk [prior positive test or contact to a positive tested person within the last 14 days] or low-risk [inconspicuous anamnesis within the last 14 days]). RESULTS: A total of 1056 SARS-CoV­2 tests in 543 patients were analyzed. Of those, 1015 tests were performed in asymptomatic patients and 41 tests in patients with COVID-19-associated symptoms. Two of 940 (0.2%) tests in asymptomatic patients with low-risk anamnesis and three of 75 (4.0%) tests in asymptomatic patients with high-risk anamnesis showed a positive result. For symptomatic patients, SARS-CoV­2 was detected in three of 36 (8.3%) low-risk and three of five (60.0%) high-risk tests. CONCLUSION: To the best of our knowledge, this is the first study evaluating the correlation between individual risk factors and positivity rates of SARS-CoV­2 tests in cancer patients. The data demonstrate that clinical and anamnestic assessment is a simple and effective measure to distinctly increase SARS-CoV­2 test efficiency. This might enable cancer centers to adjust test strategies in asymptomatic patients, especially when test resources are scarce.

Convective polymerase chain reaction in standard microtubes.

Polymerase chain reaction (PCR) is the most widely used method for nucleic acids amplification. To date, a huge number of versatile PCR techniques have been developed. One of the relevant goals is to shorten PCR duration, which can be achieved in several ways. Here, we report on the results regarding nucleic acids amplification by convective PCR (cPCR) in standard 0.2 ml polypropylene microtubes. The following conditions were found to be optimal for such amplification: 1) 70 µl reaction volume, 2) the supply of external temperature 145°Ð¡ for the denaturation zone and 0°Ð¡ for the annealing zone, 3) ∼30° inclination of the microtube main axis, 4) the use of nearby primers, and 5) duration of the reaction 15-20 min. At these conditions, the amplification products are accumulated in an amount sufficient to be registered by gel electrophoresis, and high sensitivity of the reaction comparable to that of conventional PCR is achieved. cPCR provided the reliable detection of SARS-CoV-2 coronavirus RNA isolated from nasopharyngeal swabs of COVID-19 patients.

Pre-treatment CT-based radiomics nomogram for predicting microsatellite instability status in colorectal cancer.

OBJECTIVES: Stratification of microsatellite instability (MSI) status in patients with colorectal cancer (CRC) improves clinical decision-making for cancer treatment. The present study aimed to develop a radiomics nomogram to predict the pre-treatment MSI status in patients with CRC. METHODS: A total of 762 patients with CRC confirmed by surgical pathology and MSI status determined with polymerase chain reaction (PCR) method were retrospectively recruited between January 2013 and May 2019. Radiomics features were extracted from routine pre-treatment abdominal pelvic computed tomography (CT) scans acquired as part of the patients' clinical care. A radiomics nomogram was constructed using multivariate logistic regression. The performance of the nomogram was evaluated using discrimination, calibration, and decision curves. RESULTS: The radiomics nomogram incorporating radiomics signatures, tumor location, patient age, high-density lipoprotein expression, and platelet counts showed good discrimination between patients with non-MSI-H and MSI-H, with an area under the curve (AUC) of 0.74 [95% CI, 0.68-0.80] in the training cohort and 0.77 [95% CI, 0.68-0.85] in the validation cohort. Favorable clinical application was observed using decision curve analysis. The addition of pathological characteristics to the nomogram failed to show incremental prognostic value. CONCLUSIONS: We developed a radiomics nomogram incorporating radiomics signatures and clinical indicators, which could potentially be used to facilitate the individualized prediction of MSI status in patients with CRC. KEY POINTS: • There is an unmet need to non-invasively determine MSI status prior to treatment. However, the traditional radiological evaluation of CT is limited for evaluating MSI status. • Our non-invasive CT imaging-based radiomics method could efficiently distinguish patients with high MSI disease from those with low MSI disease. • Our radiomics approach demonstrated promising diagnostic efficiency for MSI status, similar to the commonly used IHC method.

Molecular investigation of association between common IL-6 polymorphism with cytomegalovirus (CMV) infection and recurrent miscarriage in Iranian women.

BACKGROUND: Recurrent pregnancy loss (RPL) is described as two or more spontaneous abortions. To date, scientists in various fields of knowledge, such as genetics, endocrinology, anatomy, immunology, and microbiology, have identified some important factors that affect abortions; nonetheless, the precise basic etiology is not determined in up to 50% of RPL cases. Human cytomegalovirus (CMV) infection and host genetic background, like IL-6 SNP polymorphisms, play important roles in RPL etiology. OBJECTIVE: This study aimed to evaluate the relationships among single nucleotide polymorphisms (-634C/G and -174 G/C) in the IL-6 gene with CMV infection and the risk of RPL for early detection and treatment. MATERIALS AND METHODS: This case-control study was carried on 80 Iranian females with RPL and 80 healthy females as controls. DNA was extracted from samples and CMV and IL6 SNPs were detected using Tetra ARMS-PCR. Statistics were analyzed by Epi Info TM and SPSS software by X2 test for the roles of CMV detection and two polymorphisms in RPL. RESULTS: The results indicated an increased rate of CMV infection in the RPL group (44%) compared to the control group (25.45%). The prevalence of IL-6-634C/G genotype among RPL patients with CMV infection was 80%, while the frequency of this genotype among RPL patients without CMV infection was 50%. Furthermore, no substantial relation was found between IL-6-174 G/C genotypes and RPL (p = 0.005). CONCLUSION: This study not only indicated a significant role for CMV in RPL, but also showed an association between CMV and allele G in IL6-634 among Iranian women. In addition, the findings suggested the use of CMV and IL-6-634 GG genotypes as diagnostic and prognostic biomarkers for RPL in the Iranian population.

Is the Medium Still the Message? Culture-Independent Diagnosis of Gastrointestinal Infections.

Infectious diarrhea is caused by a variety of pathogens, including viruses, bacteria, and parasitic organisms. Though the causative agent of diarrhea has historically been evaluated via stool cultures, recently, culture-independent diagnostic tests (CIDT) have been developed and utilized with increasing frequency. Current practice guidelines recommend their use as adjuncts to stool cultures for diagnosing acute and chronic diarrhea. The three principal CIDT are microscopy, enzyme-based immunoassays (EIAs), and molecular based polymerase chain reaction (PCR). This review explores the common causes of infectious diarrhea, the basics of stool culture, the diagnostic utility of these three culture-independent modalities, and the strengths and weaknesses of all currently available clinical techniques. It also outlines considerations for specific populations including returning travelers and those with inflammatory bowel disease.