Postmortem genetic analysis of 17 sudden cardiac deaths identified nonsense and frameshift variants in two cases of arrhythmogenic cardiomyopathy.

Sudden death, or unexpected natural death of a healthy individual, is a serious problem in all nations. Sudden cardiac death (SCD) mainly due to ischemic heart diseases is the top cause of sudden death. However, there are pathophysiological conditions, referred to as sudden arrhythmic death syndrome, in which no apparent lesion can be identified even after complete conventional or ordinary autopsy. While postmortem genetic analyses have accumulated evidence about underlying genetic abnormality in such cases, the precise relationships between genetic background and the phenotype have been largely elusive. In this study, a retrospective investigation of 17 autopsy cases in which lethal arrhythmia was suspected to be the cause of death was carried out. Genetic analysis focusing on 72 genes reported to be associated with cardiac dysfunctions was performed, in combination with detailed histopathological and postmortem imaging examination, and a family study. As a result, in two cases of suspected arrhythmogenic cardiomyopathy (ACM), we found a nonsense variant in PKP2 and frameshift variant in TRPM4 gene. In contrast, the other 15 cases showed no morphological changes in the heart despite the presence of a frameshift variant and several missense variants, leaving the clinical significance of these variants obscure. The findings of the present study suggest that nonsense and frameshift variants could be involved in the morphological abnormality in cases of SCD due to ACM, while missense variants alone rarely contribute to massive structural changes in the heart.

Acute subdural hematoma caused by rupture of a mycotic aneurysm due to meningitis associated with infectious endocarditis: comparison of autopsy findings with postmortem computed tomography.

Forensic pathologists often encounter cases of acute subdural hematoma (SDH) due to trauma, whereas those attributable to endogenous causes are rare. Here, we report a case of the latter type in a 42-year-old man who was found dead at home after several months of fever and malaise. Postmortem computed tomography (PMCT) and autopsy were undertaken to clarify the cause of death. PMCT images revealed a fatal SDH and a localized hyper-density area in the right parietal lobe; macroscopic and microscopic examinations revealed SDH due to rupture of a mycotic aneurysm (MA) associated with meningitis. The PMCT images also indicated thickening and calcification of the mitral valve, while autopsy demonstrated infective endocarditis (IE). In addition, PMCT demonstrated a low-density area in the spleen, which was shown to be a splenic abscess at autopsy. PMCT also demonstrated tooth cavities. Based on the findings of autopsy, the cause of death was considered to be SDH due to rupture of the MA resulting from meningitis with IE and splenic abscess. Although PMCT was unable to clarify the significance of any individual feature, a retrospective review of the PMCT images might have suggested IE, bacteremia, or ruptured MA leading to SDH. This case suggests that, instead of interpreting individual features demonstrated on PMCT images, integrated interpretation of overall PMCT findings might provide clues for identifying causes of death, despite the fact that PMCT lacks diagnostic accuracy for infectious diseases such as IE and meningitis.

Single molecule in situ hybridization reveals distinct localizations of schizophrenia risk-related transcripts SNX19 and AS3MT in human brain.

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) associated with schizophrenia risk. Integration of RNA-sequencing data from postmortem human brains with these risk SNPs identified transcripts associated with increased schizophrenia susceptibility, including a class of exon 9-spliced isoforms of Sorting nexin-19 (SNX19d9) and an isoform of Arsenic methyltransferase (AS3MT) splicing out exons 2 and 3 (AS3MTd2d3). However, the biological function of these transcript variants is unclear. Defining the cell types where these risk transcripts are dominantly expressed is an important step to understand function, in prioritizing specific cell types and/or neural pathways in subsequent studies. To identify the cell type-specific localization of SNX19 and AS3MT in the human dorsolateral prefrontal cortex (DLPFC), we used single-molecule in situ hybridization techniques combined with automated quantification and machine learning approaches to analyze 10 postmortem brains of neurotypical individuals. These analyses revealed that both pan-SNX19 and pan-AS3MT were more highly expressed in neurons than non-neurons in layers II/III and VI of DLPFC. Furthermore, pan-SNX19 was preferentially expressed in glutamatergic neurons, while pan-AS3MT was preferentially expressed in GABAergic neurons. Finally, we utilized duplex BaseScope technology, to delineate the localization of SNX19d9 and AS3MTd2d3 splice variants, revealing consistent trends in spatial gene expression among pan-transcripts and schizophrenia risk-related transcript variants. These findings demonstrate that schizophrenia risk transcripts have distinct localization patterns in the healthy human brains, and suggest that SNX19 transcripts might disrupt the normal function of glutamatergic neurons, while AS3MT may lead to disturbances in the GABAergic system in the pathophysiology of schizophrenia.

Reduction of blood group A antigen on erythrocytes in a patient with myelodysplastic syndrome harboring somatic mutations in RUNX1 and GATA2.

BACKGROUND: Reduction of blood group ABO antigens on red blood cells (RBCs) is well known in patients with leukemias, and this reduction of ABO expression is strongly associated with DNA methylation of the ABO promoter. Previously, we reported a two-nucleotide deletion in RUNX1 encoding an abnormally elongated protein lacking the trans-activation domain in a patient with myelodysplastic syndrome (MDS) showing A-antigen loss on RBCs. This prompted us to investigate the underlying mechanism responsible for A-antigen reduction on RBCs in another patient with MDS. STUDY DESIGN AND METHODS: Screening of somatic mutations was carried out using a targeted sequencing panel with genomic DNA from peripheral blood mononuclear cells from the patient and eleven MDS controls without A- or B-antigen loss. DNA methylation of the ABO promoter was examined by bisulfite genomic sequencing. Transient transfection assays were performed for functional evaluation of mutations. RESULTS: Screening of somatic mutations showed missense mutations in RUNX1 and GATA2 in the patient, while no mutation was found in exons of those genes in the controls. There was no significant difference in ABO promoter methylation between the patient and the controls. Transient transfection experiments into COS-7 and K562 cells suggested that the amino acid substitutions encoded by those mutations reduced or lost the trans-activation potential of the ABO expression. CONCLUSION: Considering the discrepancy between the variant frequencies of these mutations and the ratios of the RBCs with A-antigens loss, the antigen reduction might be associated with these somatic mutations and hypermethylation of the ABO promoter.

dotdotdot: an automated approach to quantify multiplex single molecule fluorescent in situ hybridization (smFISH) images in complex tissues.

Multiplex single-molecule fluorescent in situ hybridization (smFISH) is a powerful method for validating RNA sequencing and emerging spatial transcriptomic data, but quantification remains a computational challenge. We present a framework for generating and analyzing smFISH data in complex tissues while overcoming autofluorescence and increasing multiplexing capacity. We developed dotdotdot (https://github.com/LieberInstitute/dotdotdot) as a corresponding software package to quantify RNA transcripts in single nuclei and perform differential expression analysis. We first demonstrate robustness of our platform in single mouse neurons by quantifying differential expression of activity-regulated genes. We then quantify spatial gene expression in human dorsolateral prefrontal cortex (DLPFC) using spectral imaging and dotdotdot to mask lipofuscin autofluorescence. We lastly apply machine learning to predict cell types and perform downstream cell type-specific expression analysis. In summary, we provide experimental workflows, imaging acquisition and analytic strategies for quantification and biological interpretation of smFISH data in complex tissues.

RUNX1 mutation in a patient with myelodysplastic syndrome and decreased erythrocyte expression of blood group A antigen.

BACKGROUND: Loss of blood group ABO antigens on red blood cells (RBCs) is well known in patients with leukemias, and such decreased ABO expression has been reported to be strongly associated with hypermethylation of the ABO promoter. We investigated the underlying mechanism responsible for A-antigen reduction on RBCs in a patient with myelodysplastic syndrome. STUDY DESIGN AND METHODS: Genetic analysis of ABO was performed by PCR and sequencing using peripheral blood. RT-PCR were carried out using cDNA prepared from total bone marrow (BM) cells. Bisulfite genomic sequencing was performed using genomic DNA from BM cells. Screening of somatic mutations was carried out using a targeted sequencing panel with genomic DNA from BM cells, followed by transient transfection assays. RESULTS: Genetic analysis of ABO did not reveal any mutation in coding regions, splice sites, or regulatory regions. RT-PCR demonstrated reduction of A-transcripts when the patient's RBCs were not agglutinated by anti-A antibody and did not indicate any significant increase of alternative splicing products in the patient relative to the control. DNA methylation of the ABO promoter was not obvious in erythroid cells. Targeted sequencing identified somatic mutations in ASXL1, EZH2, RUNX1, and WT1. Experiments involving transient transfection into K562 cells showed that the expression of ABO was decreased by expression of the mutated RUNX1. CONCLUSION: Because the RUNX1 mutation encoded an abnormally elongated protein without a transactivation domain which could act as dominant negative inhibitor, this frame-shift mutation in RUNX1 may be a genetic candidate contributing to A-antigen loss on RBCs.

DNA methylation of the NR3C1 promoter region in brains of pediatric victims of physical abuse.

Early life stress (ELS) has been suggested to cause epigenetic changes to genes in the brain, such as the Nuclear Receptor Subfamily 3, Group C, Member 1 gene (NR3C1). Conversely, evaluation of the epigenetic status in the postmortem brain might provide clues to the antemortem ELS experience. We examined DNA methylation of the 1F promoter region of NR3C1 in the postmortem brains of eight children including four ELS cases. As a result, DNA methylation was evident in ELS cases due to severe physical abuse. Epigenetic status may have potential application as a biomarker for clarifying the antemortem experiences of deceased.

Epithelial Expression of Human ABO Blood Group Genes Is Dependent upon a Downstream Regulatory Element Functioning through an Epithelial Cell-specific Transcription Factor, Elf5.

The human ABO blood group system is of great importance in blood transfusion and organ transplantation. The ABO system is composed of complex carbohydrate structures that are biosynthesized by A- and B-transferases encoded by the ABO gene. However, the mechanisms regulating ABO gene expression in epithelial cells remain obscure. On the basis of DNase I-hypersensitive sites in and around ABO in epithelial cells, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays and histone modifications indicated a novel positive regulatory element, designated the +22.6-kb site, downstream from ABO, and this was shown to enhance ABO promoter activity in an epithelial cell-specific manner. Expression of ABO and B-antigen was reduced in gastric cancer KATOIII cells by biallelic deletion of the +22.6-kb site using the CRISPR/Cas9 system. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that the site bound to an epithelial cell-specific transcription factor, Elf5. Mutation of the Ets binding motifs to abrogate binding of this factor reduced the regulatory activity of the +22.6-kb site. Furthermore, ELF5 knockdown with shRNA reduced both endogenous transcription from ABO and B-antigen expression in KATOIII cells. Thus, Elf5 appeared to be involved in the enhancer potential of the +22.6-kb site. These results support the contention that ABO expression is dependent upon a downstream positive regulatory element functioning through a tissue-restricted transcription factor, Elf5, in epithelial cells.

Histone deacetylase inhibitors suppress ABO transcription in vitro, leading to reduced expression of the antigens.

BACKGROUND: The ABO system is of fundamental importance in the fields of transfusion and transplantation and has apparent associations with certain diseases, including cardiovascular disorders. ABO expression is reduced in the late phase of erythroid differentiation in vitro, whereas histone deacetylase inhibitors (HDACIs) are known to promote cell differentiation. Therefore, whether or not HDACIs could reduce the amount of ABO transcripts and A or B antigens is an intriguing issue. STUDY DESIGN AND METHODS: Quantitative polymerase chain reactions were carried out for the ABO transcripts in erythroid-lineage K562 and epithelial-lineage KATOIII cells after incubation with HDACIs, such as sodium butyrate, panobinostat, vorinostat, and sodium valproate. Flow cytometric analysis was conducted to evaluate the amounts of antigen in KATOIII cells treated with panobinostat. Quantitative chromatin immunoprecipitation (ChIP) assays and luciferase assays were performed on both cell types to examine the mechanisms of ABO suppression. RESULTS: HDACIs reduced the ABO transcripts in both K562 and KATOIII cells, with panobinostat exerting the most significant effect. Flow cytometric analysis demonstrated a decrease in B-antigen expression on panobinostat-treated KATOIII cells. ChIP assays indicated that panobinostat altered the modification of histones in the transcriptional regulatory regions of ABO, and luciferase assays demonstrated reduced activity of these elements. CONCLUSION: ABO transcription seems to be regulated by an epigenetic mechanism. Panobinostat appears to suppress ABO transcription, reducing the amount of antigens on the surface of cultured cells.

Bevacizumab safety in Japanese patients with colorectal cancer.

BACKGROUND: Bevacizumab (Avastin(®)) was approved in Japan in April 2007 for patients with advanced or metastatic colorectal cancer. To address the limited clinical experience in Japanese patients, a post-approval surveillance study was undertaken in bevacizumab-treated patients in Japan. METHODS: Bevacizumab (5 or 10 mg/kg every 2 weeks) was administered with chemotherapy; patients were observed for 26 weeks from initiation of treatment. The primary objective was to investigate the incidence of adverse drug reactions, particularly those of interest for bevacizumab. Univariate and multivariate analyses were performed to identify potential risk factors for adverse drug reactions. RESULTS: In total, 2712 patients were registered and 2696 patients were included in the safety analysis. Hypertension (13.1%), hemorrhage (10.5%) and proteinuria (4.5%) were the most common adverse drug reaction. The incidences of serious adverse drug reactions were low: gastrointestinal perforation occurred in 0.9% of patients, hemorrhage in 1.3%, arterial thromboembolic events in 0.3%, venous thromboembolic events in 1.3% and wound-healing complications in 0.4%. The incidence of bevacizumab-specific adverse drug reactions was not influenced by the bevacizumab dose. Multivariate analyses identified risk factors for the following adverse drug reactions: hypertension (prior/concurrent hypertension); tumor-associated bleeding (performance status, prior/concomitant anticoagulant or nonsteroidal anti-inflammatory drug use); proteinuria (sex, performance status, prior/concurrent diabetes and proteinuria); gastrointestinal perforation (primary tumor in situ, concurrent nonsteroidal anti-inflammatory drug use); venous thromboembolic event (treatment stage, port insertion). CONCLUSIONS: The safety profile of bevacizumab-containing regimens in this Japanese population was comparable with studies performed in Western countries. Bevacizumab is generally well tolerated in Japanese patients with advanced or metastatic colorectal cancer.

Intestinal obstruction in a mentally retarded patient due to pica.

A 40-year-old mentally retarded Japanese man was admitted at rehabilitation facility for handicapped persons and found dead in his bed. His neonatal period was complicated by seizures, and he had a medical history of schizophrenia. A postmortem computed tomography scan suggested an intestinal obstruction, but the cause was unknown. To clarify the cause of death, a medicolegal autopsy was carried out. The gastrointestinal tract was found to contain copious amounts of cloth pieces. A diagnosis of intestinal obstruction secondary to pica of clothes was made. Despite still being an essentially neglect condition; mental retardation is cause to significant burden to the patient, his relatives and caregivers and the whole society. Moreover, people with mental retardation may be at increased risk for potentially self-injury due to ingestion of non-eating substance or incongruent intake of eating substances, which may on turn lead to severe or even life-threatening medical and surgical complications as herein reported. Specific attention also to pica in mentally-retarded patients with sudden, severe, gastrointestinal events, should therefore be placed in order to prevent potential death or otherwise severe chronic consequences, ideally aiming at enhancing the early recognition and multi-disciplinary management of those psychological stressors or triggers potentially responsible for pica too.

Detection Method for Gene Doping in a Mouse Model Expressing Human Erythropoietin from Adeno-Associated Virus Vector-9.

With the rapid development of gene therapy technology in recent years, its abuse as a method of sports doping in athletics has become a concern. However, there is still room for improvement in gene-doping testing methods, and a robust animal model needs to be developed. Therefore, the purposes of this study were to establish a model of gene doping using recombinant adeno-associated virus vector-9, including the human erythropoietin gene (rAAV9-hEPO), and to establish a relevant testing method. First, it was attempted to establish the model using rAAV9-hEPO on mice. The results showed a significant increase in erythrocyte volume accompanied by an increase in spleen weight, confirming the validity of the model. Next, we attempted to detect proof of gene doping by targeting DNA and RNA. Direct proof of gene doping was detected using a TaqMan-qPCR assay with certain primers/probes. In addition, some indirect proof was identified in RNAs through the combination of a TB Green qPCR assay with RNA sequencing. Taken together, these results could provide the foundation for an effective test for gene doping in human athletes in the future.

Mutation of the GATA site in the erythroid cell-specific regulatory element of the ABO gene in a Bm subgroup individual.

BACKGROUND: The ABO blood group is important in blood transfusion. Recently, an erythroid cell-specific regulatory element has been identified in the first intron of ABO using luciferase reporter assays with K562 cells. The erythroid cell-specific regulatory activity of the element was dependent upon GATA-1 binding. In addition, partial deletion of Intron 1 including the element was observed in genomic DNAs obtained from 111 Bm and ABm individuals, except for one, whereas the deletion was never found among 1005 individuals with the common phenotypes. STUDY DESIGN AND METHODS: In this study, further investigation was performed to reveal the underlying mechanism responsible for reduction of B antigen expression in the exceptional Bm individual. Peptide nucleic acid-clamping polymerase chain reaction was carried out to amplify the B-related allele, followed by sequence determination. Electrophoretic mobility assays and promoter assays were performed to examine whether a nucleotide substitution reduced the binding of a transcription factor and induced loss of function of the element. RESULTS: Sequence determination revealed one point mutation of the GATA motif in the element. The electrophoretic mobility shift assays showed that the mutation abolished the binding of GATA transcription factors, and the promoter assays demonstrated complete loss of enhancer activity of the element. CONCLUSION: These observations suggest that the mutation in the GATA motif of the erythroid-specific regulatory element may diminish the binding of GATA transcription factors and down regulate transcriptional activity of the element on the B allele, leading to reduction of B antigen expression in erythroid lineage cells of the Bm individual.

[Retrospective analysis of the effectiveness of Yokukansan(Japanese Herbal Medicine, TJ-54)in the treatment of delirium following acute stroke].

The aim of our retrospective study was to investigate the efficacy and safety of Yokukansan(TJ-54)in treating delirium during hospitalization following acute stroke. We retrospectively analyzed the patients 1)who were admitted to our single stroke center from January 2010 to December 2011 due to acute stroke within four days from onset, 2)who presented with delirium after admission, which was diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders-IV-Text Revision(DSM-IV-TR)criteria, and 3)who received 2.5g of Yokukansan(TJ-54)three times a day for treating delirium and which was continued until discharge or until onset of adverse effects. We investigated the patient's baseline characteristics, the period of Yokukansan treatment, history of using of another psychotropic drug and complications. We used the Delirium Rating Scale(DRS)to assess delirium state before and at five days after initiation of Yokukansan(TJ-54). We analyzed 77 patients retrospectively. Their mean age was 79.3 years old, and 48 patients(62%)were female. Median DRS score was improved from 15(interquartile range;14-18)to 8(interquartile;6-13), statistically significant(p<0.01)at five days after the initiation of Yokukansan(TJ-54). The median period of Yokukansan(TJ-54)treatment was seven days. There were no patients who received psychotropic drugs or who were suspected of developing oversedation due to Yokukansan(TJ-54). In our study, delirium symptoms following acute stroke improved after Yokukansan(TJ-54)treatment. A randomized controlled trial is needed to establish the effectiveness of Yokukansan(TJ-54)in delirium after stroke.

Usefulness of virtual reassembly of the skull and spine in cases of fragmentation due to high-energy trauma: A feasibility study.

With the widespread use of postmortem computed tomography (PMCT) beside forensic autopsies for investigation of causes of death, three-dimensional (3D) reconstruction and fusion imaging using PMCT data are now becoming common. In the present study, the applicability of virtual reassembly from PMCT data was investigated in three cases involving fragmentation of the skull or spine due to high-energy trauma, as in such cases it is sometimes difficult to obtain detailed information on fractures using macroscopic observation alone. In the first case, virtual reassembly of the skull provided more information about the fractures than conventional reconstruction with adhesive. In the second case, although the skull was severely fractured and could not be examined macroscopically, virtual reassembly allowed detailed visualization of the fractures. In the last case, virtual reassembly of the spine helped to clarify that the 6th-8th thoracic vertebrae had been run over by a vehicle at the scene. Thus, virtual reassembly was shown to be useful for assessment of injury patterns, and event reconstruction.

Establishment of a random forest regression model to estimate the age of bloodstains based on temporal colorimetric analysis.

Bloodstain age estimation is important in forensic science. Although several studies have used spectroscopy to estimate bloodstain ages, this method has not yet been practically applied due to the need for expensive equipment and low reproducibility. Thus, we aimed to develop a bloodstain age estimation model that can be easily performed using a spectrophotometric colorimeter. First, bloodstains were prepared by placing blood obtained from five healthy volunteers on a plastic plate. The bloodstains were kept on conditions with various brightness and temperatures. Then, each bloodstain was dissolved in saline every 24 h to a final concentration of 1%, measured with a spectrophotometric colorimeter, and subjected to machine learning to generate a random forest regression (RFR) model, and finally, the prediction accuracy of the bloodstain age was verified. We also elucidated the mechanism of the color changes utilizing aminoguanidine, which is an inhibitor of Maillard reaction. Finally, we measured the time-dependent color changes of the blood fluids obtained from healthy volunteers and examined if the method could be potentially applied to estimate postmortem interval (PMI). Our results showed that the RFR model estimated the bloodstain age with no substantial assessment, and it was applicable to bloodstains, regardless of the brightness or temperature. The color changes were affected by the addition of aminoguanidine. Furthermore, the method could be applied to blood fluids, suggesting its potential usefulness for PMI estimation. Considering its feasibility, the present method could potentially be introduced to practical forensic sciences in the near future.

Hypothermic death resulting from extreme freezing with characteristic postmortem computed tomography findings: A case report and review of the literature.

We report a case of hypothermic death that resulted from extreme freezing, with characteristic postmortem computed tomography (PMCT) findings. A 75-year-old man died in a deeply frozen state. In PMCT, there was a lack of increase in the bilateral lung-field attenuation. Urinary retention, with a hypodense area of frozen urine, was observed in the bladder. Changes that appeared to involve the crystallization of serum in frozen blood were observed in the aorta. Based on the scene and his circumstances, it was speculated that he died of hypothermia. Present case and our review revealed that although PMCT findings from hypothermic death that resulted from deep freezing are very rare, the characteristic PMCT findings may help determine the cause of death.

Establishing a Sequencing Method for the Whole Mitochondrial DNA of Domestic Dogs.

In human beings, whole mitochondrial DNA (mtDNA) sequencing has been widely used in many research fields, including medicine, forensics, and genetics. With respect to the domestic dog (Canis lupus familiaris), which is commonly recognized as being an additional member of the traditional human family structure, research studies on mtDNA should be developed to expand and improve our collective knowledge of dog medicine and welfare as it seems that there is still room for further development in these areas. Moreover, a simple and robust method for sequencing whole mtDNA that can be applied to various dog breeds has not yet been described in the literature. In the present study, we aim to establish such a method for the whole mtDNA sequencing of the domestic dog. In the experiments we conducted, oral mucosa DNA samples obtained from six Japanese domestic dogs were used as a template. We designed four primer pairs that could amplify approximately 5 kbp from each region of the mtDNA and validated several PCR conditions. Subsequently, the PCR amplicons were pooled and subjected to library preparation. The sequencing of the libraries was performed using next-generation sequencing (NGS), followed by bioinformatics analysis. Our results demonstrate that the proposed method can be used to perform highly accurate resequencing. We believe that this method may be useful for future research conducted to better understand dog medicine and welfare.

Development of a detector tube for screening tadalafil and its analogues in adulterated sexual enhancement products.

Sexual enhancement products adulterated with phosphodiesterase 5 inhibitors (PDE-5i) pose a serious public health concern. Tadalafil and its analogues (Tds) are PDE-5i frequently detected as adulterants. In this study, a Td detector tube for the rapid detection of Tds was developed based on the color change reaction between sulfuric acid and Tds. The specificity of this test method was evaluated using 13 Tds, all of which elicited positive results. Additionally, 30 commonly found adulterants in dietary supplements, 11 active pharmaceutical ingredients of psychotropic drugs and 18 food ingredients were tested and obtained no false-positive results, except levomepromazine. The test tube accurately detected the presence or absence of Tds in 54 commercially available products. The visual detection limit was 2-50 and 5-20 µg/ml for Tds and tadalafil-spiked samples with matrix, respectively. The applicability of the developed detector tube to a semiquantitative test using digital image analyses were investigated using red, green, and blue color values. The results of the recovery test suggested that the tube test was affected by the dark-colored matrix. The results of semiquantitative analyses of tadalafil for five marketed products were consistent with the liquid chromatographic quantification results, except for the blue value. The detector tube developed in this study can facilitate with the rapid screening of Tds in adulterated sexual enhancement products.