Multiplex Reverse Transcriptase-polymerase Chain Reaction Research Articles

Prevalence of the Potato virus Y strain composition impacting Michigan seed potato production

Potato virus Y (PVY) is a complex of multiple strains that cause a variety of foliar and tuber symptoms. Several current strains have potential to result in variety-dependent yield and quality losses. Understanding the local PVY strain population and variety responses are critical for detection and management. From 2019-2023, PVY strain composition was monitored across seven Michigan seed potato-producing counties growing prevalent chip, russet, and tablestock market classes. Using multiplex reverse-transcriptase polymerase chain reaction methods, PVY strains were identified from potato tuber or leaf samples collected through the certification program (N = 78 positive lots). PVYN-Wi remained most prevalent in Michigan, however, tuber necrotic strains PVYNTN and PVYE also continued to be detected. These, as well as some PVYN-Wi isolates, are known to cause necrotic symptoms in tubers, causing yield and quality reductions for susceptible potato cultivars. As in other U.S. potato-growing regions, PVYN-Wi and PVYNTN were most prevalent in Michigan seed potato lots. Of note, mild to severe tuber necrosis was observed in diagnostic samples positive with local PVYN-Wi strains. Parental strain PVYO appeared to re-emerge in 2023 and should be considered in certification and management. Mild or asymptomatic reactions may still lead to yield or quality losses and may pose a risk to more susceptible cultivars. Due to the diversity of strains and varieties prevalent in the Great Lakes region, continued uniform certification is recommended. Information gathered from this and future survey efforts will be valuable for growers, inspectors, and future breeding and selection efforts.

Prevalence and molecular characterization of rotavirus strains circulating among children with gastroenteritis in Egypt

Background and objectives Human rotavirus (RV) is the main cause of diarrheal disease among children younger than 5 years old, worldwide. The aim of the current study was to investigate the prevalence of RV infections and the genotype distribution of RV in Egypt. Materials and methods A total of 642 fecal samples were collected from children younger than 5 years of age, suffering from acute diarrhea and attending ten regional public hospitals (n=585) and five private clinics (n=57) located in 6 Egyptian cities between February and June 2021. All samples were screened by immunochromatographic assay to determine RV prevalence. Then RV-positive samples were further subjected for detection of G (VP7) and P (VP4) genotypes by seminested multiplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Results Out of the 642 children, RV was detected in 268 (41.7%). Inpatients were more likely to be RV-positive (43.2%) than outpatients (26.3%) and most of the positive samples 215/268 (80.2%) were found in children less than 1 year of age. RV infections were more common in males than females (65.3% vs. 34.7%). The VP7 predominant G type was G3 (31.3%), followed by G8 (20.5%), G1 (7%), mixed G infections (6.3%), G2 (1.9%), G9 (1.9%), G4 (1.5%), and G10 (0.4%). The VP4 predominant P type was P[8] (53.7%), followed by P[4] (16%), P[6] (9.3%), P[9] (6.3%), P[11] (4.5%), P[10] (2.6%), and mixed P infections (1.9%). The dominant VP7/VP4 combination was G3P[8] (24.2%), G8P[8] (10%), G8P[4] (5.2%), G1P[8] (4.5%), G8P[6] (3.3%), and G3P[4] (2.2%). Several other combinations were also identified with detection rates less than 2% of positive RV samples. Mixed genotype combinations and partially typed strains were detected in 31.7% and 7.5%, respectively. Conclusion This study highlights the necessity for continuous epidemiology and surveillance of RVA infection to improve our control and management of RVA infection. Furthermore, due to the lack of a national anti-RV vaccination program, RV remains the main causative agent for acute gastroenteritis in Egyptian children. Therefore, it is important to introduce RV vaccine into the national immunization program in Egypt free of charge to all infants to reduce the burden of RV gastroenteritis.

Nationwide data from comprehensive genomic profiling assays for detecting driver oncogenes in non-small cell lung cancer.

Driver oncogenes are investigated upfront at diagnosis using multi-CDx systems with next-generation sequencing techniques or multiplex reverse-transcriptase polymerase chain reaction assays. Additionally, from 2019, comprehensive genomic profiling (CGP) assays have been available in Japan for patients with advanced solid tumors who had completed or were expected to complete standard chemotherapy. These assays are expected to comprehensively detect the driver oncogenes, especially for patients with non-small cell lung cancer (NSCLC). However, there are no reports of nationwide research on the detection of driver oncogenes in patients with advanced NSCLC who undergo CGP assays, especially in those with undetected driver oncogenes at diagnosis. In this study, we investigated the proportion of driver oncogenes detected in patients with advanced NSCLC with undetectable driver oncogenes at initial diagnosis and in all patients with advanced NSCLC who underwent CGP assays. We retrospectively analyzed data from 986 patients with advanced NSCLC who underwent CGP assays between August 2019 and March 2022, using the Center for Cancer Genomics and Advanced Therapeutics database. The proportion of driver oncogenes newly detected in patients with NSCLC who tested negative for driver oncogenes at diagnosis and in all patients with NSCLC were investigated. Driver oncogenes were detected in 451 patients (45.7%). EGFR was the most common (16.5%), followed by KRAS (14.5%). Among the 330 patients with undetected EGFR, ALK, ROS1, and BRAF V600E mutations at diagnosis, 81 patients (24.5%) had newly identified driver oncogenes. CGP assays could be useful to identify driver oncogenes in patients with advanced NSCLC, including those initially undetected, facilitating personalized treatment.

Challenges in Diagnosing and Treating Acutely Febrile Children with Suspected Malaria at Health Care Facilities in the Lake Mwanza Region of Tanzania.

Acute febrile diseases transmitted by mosquitos are a diagnostic challenge for pediatricians working in sub-Saharan Africa. Misclassification due to the lack of rapid, reliable diagnostic tests leads to the overuse of antibiotics and antimalarials. Children presenting with acute fever and suspected of having malaria were examined at health care facilities in the Mwanza Region of Tanzania. The sensitivity and specificity of blood smear microscopy and malaria rapid diagnostic tests that targeted histidine-rich protein 2 and Plasmodium lactate dehydrogenase were compared with a multiplex reverse transcriptase-polymerase chain reaction (PCR)-ELISA. Six hundred ninety-eight children presented with acute fever and met the criteria for inclusion; 23% received antibiotics and 23% received antimalarials prior to admission. Subsequently, 20% were confirmed by PCR to have Plasmodium falciparum infection. Blood smear microscopy exhibited 33% sensitivity and 93% specificity. The malaria rapid test provided 87% sensitivity and 98% specificity in detecting acute malaria infections. Only 7% of malaria-negative children received antimalarials at Sengerema Designated District Hospital when treatment was guided by the results of rapid testing. In contrast, 75% of malaria-negative patients were treated with antimalarial drugs at health facilities that used blood smears as the standard diagnostic test. Misclassification and premedication of nonmalarial, febrile illnesses contribute to the emergence of antimalarial and antimicrobial resistance. The incorporation of malaria rapid diagnostic tests into the clinical routine translated into improved treatment and a significant reduction in antimalarial drug prescriptions.

One-Year Investigation of Hepatitis A Virus and Rotavirus in Two Waste Water Treatment Plants in Egypt

Background: Waterborne enteric viruses are evolving source of disease outbreaks and represent a major threat to global public health. The repetitive assessment of water environments is necessary to manage enteric virus-mediated fecal infection and the probable emergence of different variants. Methods: Here, we detected human rotavirus and hepatitis A virus circulating in two wastewater treatment plants, for one year in sewage and sludge samples from two largest wastewater treatment plant (El-Gabal El-Asfar and Zenin) located in Egypt. For this purpose, we examine predefined viruses' prevalence in 72 samples (24 raw sewage samples, 24 treated effluent samples, and 24 sludge) collected from August 2022 to July 2023. Viral RNA was extracted and detected by nested Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) in case of HAV and by multiplex semi nested RT-PCR In case of rotavirus with the Results: Percentage of detection rate 58.3%, 25% 41.6%, 50%, 16.6%58.6%, 41.6%, 8.3%, 41.6%41.6%, 8.3% and 41.6% for inlet, outlet, sludge in WWTP zenin and WWTP El-Gabal El-Asfar for HAV, RV respectively. Using generic primer result indicated G1P8 was the most common although G2P [4], G1, G2, G3P? G9P [8], G9P [11], G1 P [4] were detected with different percentage. Conclusion: These results demonstrated that HAV and RV still remains in the environment after sewage treatment and could play an important role in maintaining the endemicity of HAV and RV infection. Routine and continuous monitoring of waterborne viruses are most useful approach to understand disease occurrence in communities.

Detection of Enteropathogens in Human Immunodeficiency Virus and Non-Human Immunodeficiency Virus-Infected Children with Acute Diarrhea in an Indonesian Tertiary Hospital Using Multiplex Real-Time Polymerase Chain Reaction.

Diarrhea is one of the leading causes of mortality in children living in developing countries. The etiology of acute diarrhea in each healthcare center varies depending on place, time, and population. This study aimed to identify pathogen patterns in human immunodeficiency virus (HIV)-infected and non-HIV children suffering from acute diarrhea, using multiplex real time reverse transcriptase polymerase chain reaction (RT-PCR), in an Indonesian tertiary hospital. This cross-sectional study was conducted at Dr. Cipto Mangunkusumo National Hospital from March 2019 to April 2020. The study showed that multiplex RT-PCR results were positive in 58.9% of the specimens, with more positive results in HIV-infected children than in non-HIV-infected children (70% vs. 54.7%). Altogether 72 enteropathogens were detected from all specimens. Enteropathogens in non-HIV children with acute diarrhea consisted of bacteria (70.6%) and viruses (29.4%) with a predominance of enteroaggregative Escherichia coli (25.4%), followed by Campylobacter spp. (11.8%), enteropathogenic E. coli (9.8%), Norovirus GII (7.8%), and Clostridium difficile (7.8%). Enteropathogens in HIV-infected children consisted of viruses (57.1%), bacteria (28.6%), and parasites (14.3%) comprising Norovirus GII (24%), Cryptosporidium spp. (14.3%), Campylobacter spp. (14.3%), Norovirus GI (14.3%), and Astrovirus (14.3%). Cryptosporidium spp. was the only parasite found in this study and was found only in HIV-infected children. In non-HIV children with acute diarrhea, most pathogens were invasive bacteria, while in HIV-infected children, more viral and parasite infections occurred, primarily caused by opportunistic pathogens. The pattern of enteropathogens can help clinicians determine further examinations and appropriate empirical antimicrobial therapy for the patient.

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

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. 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. 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. 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. 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.

Comparison of performance of LIAISON SARS-CoV-2 antigen assay with RT-PCR during the Omicron wave.

Due to the newly emerging Omicron variant, there is a need to re-evaluate the performance of automated antigen tests. Our study aim was to evaluate the performance of the automated Liaison SARS-CoV-2 antigen assay against reverse transcriptase polymerase chain reaction (RT-PCR) in samples with Omicron variant.A prospective study was performed on 373 combined oro-nasopharyngeal samples (NPS) randomly collected from symptomatic patients. NPS were tested with Liaison SARS-CoV-2 Ag test (DiaSorin, Italy) and DS Coronex COVID-19 Multiplex RT-PCR Diagnosis Kit (DS BioTechnology, Ankara, Turkey).Of 373 samples, 124 (33.2%) were found to be RT-PCR positive and 249 (66.8%) RT-PCR negative. Taking RT-PCR as a reference, the sensitivity and specificity of the Liaison SARS-CoV-2 Ag assay were found as 84.6% (95%CI 77.3%-90%) and 100% (95%CI 98.5%-100%), respectively. For samples with a cycle threshold (Ct) value <25 (high viral load), the sensitivity increased to 100%. When antigen concentration and Ct values were compared, a strong negative correlation between antigen and Ct values was determined (P < 0.001).The Liaison antigen test met the performance criteria recommended by the WHO for samples with the Omicron variant. In addition, it showed excellent sensitivity and specificity in patients with high viral load. Therefore, Liaison antigen test can be a reliable and useful alternative in the diagnosis of SARS-CoV-2 infection, particularly in resource-constrained laboratories.

Diagnostic and prognostic value of circulating tumor cells in Indian women with suspected ovarian cancer.

"Liquid biopsy," where body fluids are screened for biomarkers, is gathering substantial research. We aimed to examine women with suspected ovarian cancer for the presence of circulating tumor cells (CTCs) and study its role in prediction of chemoresistance and survival. Magnetic powder labeled monoclonal antibodies for epithelial cell adhesion molecule (EpCAM), mucin 1 cell surface associated, mucin 16 cell surface associated, or carbohydrate antigen 125 (CA125), were prepared according to the manufacturer's protocol. Expression of three ovarian cancer related genes was detected in CTCs using multiplex reverse transcriptase-polymerase chain reaction. CTCs and serum CA125 were measured in 100 patients with suspected ovarian cancer. Correlations with clinicopathological parameters and treatment were analyzed. CTCs were detected in 18/70 (25.7%) of women with malignancy compared to 0/30 (0.0%) in those with benign gynecologic diseases (P = 0.001). The sensitivity and specificity of the CTC test for predicting a malignant histology in pelvic masses were 27.7% (95% CI: 16.3%, 37.7%) and 100% (95% CI: 85.8%, 100%), respectively. The number of CTCs correlated with stage of ovarian cancer (P = 0.030). The presence of EpCAM + CTC at primary diagnosis in ovarian cancer was found to be an independent predictor of a poor progression free survival (HR, 3.3; 95% CI, 1.3-8.4; P = 0.010), overall survival (HR, 2.6; 95% CI,1.1-5.6; P = 0.019), and resistance to chemotherapy (OR 8.6; 95% CI, 1.8-43.7; P = 0.009). Expression of EpCAM + CTC in ovarian cancer predicts platinum resistance and poor prognosis. This information could be further used in investigating anti-EpCAM-targeted therapies in ovarian cancer.

Ultrafast Gene Fusion Assessment for Nonsquamous NSCLC

IntroductionGene fusion testing of ALK, ROS1, RET, NTRK, and MET exon 14 skipping mutations is guideline recommended in nonsquamous NSCLC (NS-NSCLC). Nevertheless, assessment is often hindered by the limited availability of tissue and prolonged next-generation sequencing (NGS) testing, which can protract the initiation of a targeted therapy. Therefore, the development of faster gene fusion assessment is critical for optimal clinical decision-making. Here, we compared two ultrafast gene fusion assays (UFGFAs) using NGS (Genexus, Oncomine Precision Assay, Thermo Fisher Scientific) and a multiplex reverse-transcriptase polymerase chain reaction (Idylla, GeneFusion Assay, Biocartis) approach at diagnosis in a retrospective series of 195 NS-NSCLC cases and five extrapulmonary tumors with a known NTRK fusion. MethodsA total of 195 NS-NSCLC cases (113 known gene fusions and 82 wild-type tumors) were included retrospectively. To validate the detection of a NTRK fusion, we added five NTRK-positive extrathoracic tumors. The diagnostic performance of the two UFGFAs and standard procedures was compared. ResultsThe accuracy was 92.3% and 93.1% for Idylla and Genexus, respectively. Both systems improved the sensitivity for detection by including a 5′-3′ imbalance analysis. Although detection of ROS1, MET exon 14 skipping, and RET was excellent with both systems, ALK fusion detection was reduced with sensitivities of 87% and 88%, respectively. Idylla had a limited sensitivity of 67% for NTRK fusions, in which only an imbalance assessment was used. ConclusionsUFGFA using NGS and reverse-transcriptase polymerase chain reaction approaches had an equal level of detection of gene fusion but with some technique-specific limitations. Nevertheless, UFGFA detection in routine clinical care is feasible with both systems allowing faster initiation of therapy and a broad degree of screening.

Clinical features and viral etiology of acute respiratory infection in an outpatient fever clinic during COVID-19 pandemic in a tertiary hospital in Nanjing, China.

Clinical feature and viral etiology for acute respiratory infection (ARI) in the community was unknown during coronavirus disease 2019 (COVID-19) pandemic. In a retrospective study, we aimed to characterize the clinical feature and etiology for the ARI patients admitted to the outpatient fever clinic in Nanjing Drum Tower Hospital between November 2020 and March 2021. Fifteen common respiratory pathogens were tested using pharyngeal swabs by multiplex reverse transcriptase-polymerase chain reaction assays. Of the 242 patients, 56 (23%) were tested positive for at least one viral agent. The predominant viruses included human rhinovirus (HRV) (5.4%), parainfluenza virus type III (PIV-III) (5.0%), and human coronavirus-NL63 (HCoV-NL63) (3.7%). Cough, sputum, nasal obstruction, and rhinorrhea were the most prevalent symptoms in patients with viral infection. Elderly and the patients with underlying diseases were susceptible to pneumonia accompanied with sputum and chest oppression. Three (5.4%) patients in virus infection group, whereas 31 (16.7%) in non-viral infection group (p=0.033), were empirically prescribed with antiviral agents. Among 149 patients who received antibiotic therapy, 30 (20.1%) patients were later identified with viral infection. Our study indicated the importance of accurate diagnosis of ARI, especially during the COVID-19 pandemic, which might facilitate appropriate clinical treatment.

The application of a novel 5-in-1 multiplex reverse transcriptase–polymerase chain reaction assay for rapid detection of SARS-CoV-2 and differentiation between variants of concern

To rapid detect for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and immediately distinguish whether positive samples represent variants of concern (VOCs), we established a novel 5-in-1 VOC assay. This assay could distinguish among five VOCs: Alpha, Beta, Gamma, Delta, and Omicron, in a single reaction tube. The five variants exhibit different single nucleotide polymorphisms (SNPs) in their viral genome, which can be exploited to distinguish them. We selected target SNPs in the spike gene, including N501Y, P681R, K417N, and deletion H69/V70 for the assay. The limit of detection of each gene locus was 80 copies per PCR reaction. We observed a high consistency among the results when comparing the performance of our 5-in-1 VOC assay, whole gene sequencing, and the Roche VirSNiP SARS-CoV-2 test in retrospectively analyzing 150 clinical SARS-CoV-2 variant positive samples. The 5-in-1 VOC assay offers an alternative and rapid high-throughput test for most diagnostic laboratories in a flexible sample-to-result platform. The assay can also be applied in a commercial platform with completion of the SARS-CoV-2 confirmation test and identification of its variant within 2.5 hours.

Effect of the incremental protection of previous infection against Omicron infection among individuals with a hybrid of infection- and vaccine-induced immunity: a population-based cohort study in Canada

ObjectivesWe examined the incremental protection and durability of infection-acquired immunity against Omicron infection in individuals with hybrid immunity in Ontario, Canada. MethodsWe followed up 6 million individuals with at least one multiplex reverse transcriptase–polymerase chain reaction test before November 21, 2021, until an Omicron infection. Protection via infection-acquired immunity was assessed by comparing Omicron infection risk between previously infected individuals and those without documented infection under different vaccination scenarios and stratified by time since the last infection or vaccination. ResultsA previous infection was associated with 68% (95% CI 61-73) and 43% (95% CI 27-56) increased protection against Omicron infection in individuals with two and three doses, respectively. Among individuals with two-dose vaccination, the incremental protection of infection-induced immunity decreased from 79% (95% CI 75-81) within 3 months after vaccination or infection to 27% (95% CI 14-37) at 9-11 months. In individuals with three-dose vaccination, it decreased from 57% (95% CI 50-63) within 3 months to 37% (95% CI 19-51) at 3-5 months after vaccination or infection. ConclusionPrevious SARS-CovV-2 infections provide added cross-variant immunity to vaccination. Given the limited durability of infection-acquired protection in individuals with hybrid immunity, its influence on shield-effects at the population level and reinfection risks at the individual level may be limited.

A Clinical Laboratory-Oriented Targeted RNA-Seq System Accurately and Efficiently Detected Various Fusion Gene Mutations in Philadelphia Chromosome-like Acute Lymphoblastic Leukemia

Introduction: Genetic mutations that occur in blood cancer can be broadly classified into nucleotide sequence mutations including duplications or deletions, fusion gene mutations, and abnormal gene overexpression. Among these genetic mutations, gene fusion not only plays an important role in the development of blood cancers, such as leukemia, but is also important as an essential marker for the diagnosis, risk assessment, optimal treatment selection, and residual lesion detection. Most clinical laboratories use conventional cytogenetic testing, fluorescence in situ hybridization, and multiplex reverse transcriptase-polymerase chain reaction (PCR) to detect fusion gene mutations. However, the currently used methods have great limitations in detecting various gene fusions occurring in blood cancer. To overcome these shortcomings, RNA sequencing (RNA-seq) methods are being introduced in clinical laboratories. The authors developed a targeted RNA-seq panel, a bioinformatics pipeline used for targeted RNA-seq to detect gene fusions, sequence variants, and altered gene expression, which can be used more conveniently in clinical laboratories. Methods: The targeted RNA-seq panel and analysis pipeline developed based on information technology with an automated reporting system and commercialized by the authors was applied to a comparative evaluation with the commercialized targeted RNA-seq panel (Archer™ FusionPlex™ Heme Panel version 2; Archer DX, Boulder, CA, USA) using samples of acute leukemia patients. A laboratory-oriented targeted RNA-seq system consisting of 84 genes was developed in consideration of the test reporting time and operational efficiency in clinical laboratories (KBB-RNAseq NGS-Leukemia-PHB; KBlueBio Inc., Hwasun, Korea). For comparison with the existing analysis system, the Archer FusionPlex Heme v2 panel (ArcherDx) based on the cDNA library for specific genes using anchored multiplex PCR was used. To evaluate the test performance of these systems, bone marrow specimens were used while diagnosing 93 patients with leukemia (15 cases of acute myeloid leukemia, 35 cases of adult B-acute lymphoid leukemia, 30 cases of childhood B-acute leukemia, and 13 cases of T-acute lymphoid leukemia). Results: Among all 93 leukemia patients, tier 1 or tier 2 gene fusions were observed in 72 (77%) patients. Gene fusions were detected in 83% (25/30) of patients with pediatric B-acute lymphoid leukemia (B-ALL), and in 94% (33/35) of patients with adult B-ALL. In patients with acute myeloid leukemia (AML) and T-ALL, the proportions of fusion gene mutations were 53% (8/15) and 46% (6/13), respectively. For the comparative evaluation, 4 cases of B-ALL, 2 cases of AML, and 1 case of acute promyelocytic leukemia and T-ALL were used. The results of the two analysis systems were consistent in 7 of the 8 comparatively evaluated patients, but an IGH-CRLF2 fusion in a B-ALL case was detected only with the panel developed by the authors. It is known that the CRLF2 gene is a causative gene in half of the Philadelphia chromosome (Ph)-ALL cases, which account for 20-25% of all B-ALL cases and are classified as a new type of ALL showing a very poor prognosis. Conclusions: Our targeted RNA-seq system accurately and efficiently detected various fusion gene mutations that occur in acute leukemia. Importantly, our targeted RNAseq panel detected fusion gene mutations that cause Ph-like B-ALL more accurately than the c-ommercialized test system. This targeted RNA-seq system showed a reliable and stable performance in detecting various gene fusions occurring in blood cancers, such as leukemia, in clinical laboratories. Also, our targeted RNA-seq system was found to be very useful in diagnosing Ph-like B-ALL compared to the commercial RNA-seq panel. Keywords: Targeted RNA-seq, Clinical Laboratory, Ph-like B-ALL, Acute leukemia Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Constitutional pericentric inversion of chromosome 16, inv(16)(p13.1q22), mimicking acute myeloid leukemia.

An acquired abnormality, inv(16)(p13q22), is a hallmark of acute myeloid leukemia with eosinophilia (AML M4Eo), which leads to leukemogenesis by forming the CBFB::MYH11 fusion gene [1]. We experienced a case of inv(16)(p13.1q22) discovered incidentally during bone marrow examination without evidence of AML. A 64-year-old woman was referred to our hospital because of hypergammaglobulinemia. She was being treated for rheumatoid arthritis, but had no past and family history of AML. Laboratory examination showed polyclonal gammopathy but no significant abnormalities in complete blood count with differential. Bone marrow picture showed a normocellular marrow and mild increase in plasma cells to 3.8% (reference range, 0.2%–1.7%), but no evidence of hematological malignancy. Surprisingly, chromosome analysis of the bone marrow cells revealed that all 20 cells had inv(16)(p13.1q22) mimicking AML M4Eo (Figure 1A). We therefore performed fluorescence in situ hybridization (FISH) analysis of the bone marrow cells by using the Vysis CBFB Dual Color Break Apart Rearrangement Probe (Abbott Molecular Diagnostics, Des Plaines, IL, USA). The splitting of the red and green signals reflecting truncation of the CBFB locus on chromosome 16q22 was not observed (Figure 1B). In addition, a multiplex reverse transcriptase-polymerase chain reaction assay covering 28 leukemia-specific fusion transcripts including CBFB::MYH11 was performed by using the HemaVision-28Q kit (VERITAS, Tokyo, Japan), but none of them was detected. Therefore, it was suggested that the chromosome inversion of this patient did not disrupt the CBFB gene. High-resolution chromosome analysis of the phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes also revealed inv(16)(p13.1q22) in all 20 cells, indicating a constitutional abnormality of chromosome 16 (Figure 1C). Thus, this patient was not misdiagnosed as AML M4Eo and has been followed up without treatment. To the best of our knowledge, only a few cases with inv(16)(p13q22) unrelated to AML have been reported [2-5]. All of these were concluded to be constitutional abnormalities, and some cases have been confirmed as familial onset. In these reports, there was no misdiagnosis of AML M4Eo in individuals without hematological malignancy, and in the case of chronic myeloid leukemia with t(9;22)(q34;q11) and inv(16)(p13q22), FISH analysis showed no abnormality of the MYH11 gene and inv(16)(p13q22) was also found in her healthy father, which avoided misdiagnosis as an additional cytogenetic abnormality in chronic phase [4]. Therefore, although extremely rare, we should be aware of the presence of constitutional chromosome abnormalities mimicking hematological malignancies, and it is important to perform further tests such as FISH and genomic analysis for differential diagnosis. We thank Yoshiko Sato, MT (LSI Medience Corporation, Tokyo, Japan), for her technical support. The authors declare that there is no conflict of interest. All procedures in this study were performed in accordance with the principles of the Declaration of Helsinki and the institutional guidelines. Informed consent was obtained from the patient and her family. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Plant Virome Analysis by the Deep Sequencing of Small RNAs of Fritillaria thunbergii var. chekiangensis and the Rapid Identification of Viruses.

Thunberg fritillary (Fritillaria thunbergii), a perennial used in traditional Chinese herbal medicine, is a members of the family Liliaceae. The degeneration of germplasm is a severe problem in the production of Fritillaria thunbergii var. chekiangensis. However, no information about viral infections of F. thunbergii var. chekiangensis has been reported. In this study, we sequenced the small RNAs of F. thunbergii var. chekiangensis from leaves and bulbs, and viruses were identified using a phylogenetic analysis and BLAST search for sequence. In addition, multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) was used to rapidly detect viruses in this variety. Our study first reported that five viruses infected F. thunbergii var. chekiangensis. Among them, fritillary virus Y (FVY), lily mottle virus (LMoV), Thunberg fritillary mosaic virus (TFMV), and hop yellow virus (HYV) had been reported in F. thunbergii, while apple stem grooving virus was first reported in the genus Fritillaria. A multiplex RT-PCR method was developed to rapidly test the four viruses FVY, LMoV, TFMV, and HYV in F. thunbergii var. chekiangensis. Our results provide a better understanding of the infection of F. thunbergii var. chekiangensis by viruses and a basic reference for the better design of suitable control measures.

Epidemiology and Clinical Characteristics of Human Coronaviruses-Associated Infections in Children: A Multi-Center Study.

BackgroundHuman coronaviruses (HCoVs) are associated with upper respiratory tract infections. Although studies have analyzed the clinical and epidemiological characteristics of HCoV-associated infections, no multi-center studies have been conducted in Korean children. We aimed to describe the epidemiology and clinical characteristics of HCoV-associated infection in children.MethodsWe retrospectively reviewed medical records of children in whom HCoVs were detected using multiplex reverse transcriptase-polymerase chain reaction amplification in five centers from January 2015 to December 2019.ResultsOverall, 1,096 patients were enrolled. Among them, 654 (59.7%) patients were male. The median age was 1 year [interquartile range (IQR), 0–2 years]. HCoVs were identified mainly in winter (55.9%). HCoV-229E, HCoV-OC43, and HCoOV-NL63 were detected mainly in winter (70.9, 55.8, and 57.4%, respectively), but HCoV-HKU1 was mainly identified in spring (69.7%). HCoV-OC43 (66.0%) was detected most frequently, followed by HCoV-NL63 (33.3%), and HCoV-229E (7.7%). Two different types of HCoVs were co-detected in 18 samples, namely. Alphacoronavirus-betacoronavirus co-infection (n = 13) and, alphacoronavirus-alphacoronavirus co-infection (n = 5). No betacoronavirus-betacoronavirus co-infection was detected. Patients were diagnosed with upper respiratory tract infection (41.4%), pneumonia (16.6%), acute bronchiolitis (15.5%), non-specific febrile illness (13.1%), croup (7.3%), and acute gastroenteritis (5.1%). There were 832 (75.9%) hospitalized patients with a median duration of hospitalization of 4 days (IQR, 3–5 days); 108 (9.9%) patients needed supplemental oxygen with 37 (3.4%) needing high-flow nasal cannula or mechanical ventilation. There were no deaths.ConclusionHCoV-associated infections exhibit marked seasonality with peaks in winter. Patients with lower respiratory tract infection, a history of prematurity, or underlying chronic diseases may progress to a severe course and may need oxygen therapy.

Genotypic characterization of group A rotavirus in children < 5 years of age at tertiary care hospital in North India

PurposeThis study was undertaken to find out the positivity of rotavirus associated diarrhoea in children <5 years of age in Meerut district and to determine the genomic diversity of the circulating rotavirus strains in this geographical area. MethodsA cross-sectional study was conducted in a tertiary care hospital for a period of one year. A total of 240 stool samples were collected from children suffering from acute diarrhoea and subjected for rotavirus antigen detection by enzyme immunoassay (EIA). The EIA-positive samples were further genotyped to determine the G and P types using semi nested multiplex reverse transcriptase-polymerase chain reaction (RT-PCR). Demographic and clinical parameters were recorded in a pre-designed proforma. ResultsThe rotavirus antigen positivity rate was 14.58% in Meerut district. There was male predominance and highest positivity rate was seen in children 13–24 months of age, in winter months and majority of cases belonged to moderate degree of severity. A total of 64% children in the study area were vaccinated and positivity was low in the vaccine group. G9P[4] was the predominant genotype followed by G3P[8]. Uncommon strains G12P[6] and G12P[8] were also reported as circulating genotypes in this study. ConclusionsThe positivity rate of rotavirus associated diarrhoea has reduced in Meerut district post vaccination. G9P[4] was the most common circulating RV genotype in Meerut. Our study highlights the importance of including the emerging rotavirus strains in future multivalent RV vaccines to make the vaccines regionally more specific.

Spatial-temporal distribution and sequence diversity of group a human respiratory syncytial viruses in Kenya preceding the emergence of ON1 genotype.

BackgroundHuman respiratory syncytial virus (HRSV) is a major cause of severe viral acute respiratory illness and contributes significantly to severe pneumonia cases in Africa. Little is known about its spatial–temporal distribution as defined by its genetic diversity.MethodsA retrospective study conducted utilizing archived nasopharyngeal specimens from patients attending outpatient clinics in hospitals located in five demographically and climatically distinct regions of Kenya; Coast, Western, Highlands, Eastern and Nairobi. The viral total RNA was extracted and tested using multiplex real time RT‐PCR (reverse transcriptase polymerase chain reaction). A segment of the G‐gene was amplified using one‐step RT‐PCR and sequenced by Sanger di‐deoxy method. Bayesian analysis of phylogeny was utilized and subsequently median joining methods for haplotype network reconstruction.ResultsThree genotypes of HRSVA were detected; GA5 (14.0%), GA2 (33.1%), and NA1 (52.9%). HRSVA prevalence varied by location from 33% to 13.2% in the Highlands and the Eastern regions respectively. The mean nucleotide diversity (Pi[π]) varied by genotype: highest of 0.018 for GA5 and lowest of 0.005 for NA1. A total of 58 haplotypes were identified (GA5 10; GA2 20; NA1 28). These haplotypes were introduced into the population locally by single haplotypes and additional subsidiary seeds amongst the GA2 and the NA1 haplotypes.ConclusionsHRSVA was found across all the regions throughout the study period and comprised three genotypes; GA5, GA2, and NA1 genotypes. The genotypes were disproportionately distributed across the regions with GA5 gradually increasing toward the Western zones and decreasing toward the Eastern zones of the country.

Detection of dengue virus serotypes by single-tube multiplex RT-PCR and multiplex real-time PCR assay

BackgroundAll four dengue serotypes cause infection, with one of them predominantly reported from a particular geographical region. Coinfection by more than one serotype is reported from hyperendemic regions. These coinfections are clinically more severe than infection with a single serotype. This study was carried out to detect the predominant dengue serotype and presence of coinfections. MethodsAcute-phase serum samples of patients suffering from dengue infection were collected. They were screened for the presence of IgM, IgG and NS1Ag by a rapid test. Conventional multiplex reverse transcriptase polymerase chain reaction (RT-PCR) and multiplex real-time RT-PCR assays were carried out for detection and serotyping of the dengue virus. ResultsA total of 196 samples were positive by the rapid card test. Of these, 139 were NS1Ag positive, 40 were positive only for IgM, 5 were positive only for IgG and 12 samples were positive for different combinations of antigen and antibodies. All four serotypes were detected in these samples by PCR. DENV-3 was found to be most common circulating serotype. A total of 22 cases were found to have coinfection with more than one dengue serotypes. Samples having only antibodies and no antigen on rapid card test were also positive for virus by PCR. ConclusionPrevalence of dengue co-infections is increasing. Moreover, it is important to screen for dengue virus in those samples also which do not show NS1Ag on rapid tests and have either one or both the antibodies. Real-time multiplex RT-PCR is found to be more sensitive in detecting coinfection than conventional multiplex RT-PCR.