Clinical outcomes of coronavirus disease in patients with breast cancer treated with granulocyte colony-stimulating factor following chemotherapy: Triangulation of evidence using population-based cohort and Mendelian randomization analyses.

Recombinant human granulocyte colony-stimulating factor (G-CSF) administration in patients with cancer and coronavirus disease (COVID-19) remains controversial. Concerns exist that it may worsen COVID-19 outcomes by triggering an inflammatory cytokine storm, despite its common use for managing chemotherapy-induced neutropenia (CIN) or febrile neutropenia post-chemotherapy. Here, we determined whether prophylactic or therapeutic G-CSF administration following chemotherapy exacerbates COVID-19 progression to severe/critical conditions in breast cancer patients with COVID-19. Between December 2022 and February 2023, all 503 enrolled breast cancer patients had concurrent COVID-19 and received G-CSF post-chemotherapy, with most being vaccinated pre-chemotherapy. We prospectively observed COVID-19-related adverse outcomes, conducted association analyses, and subsequently performed Mendelian randomization (MR) analyses to validate the causal effect of genetically predicted G-CSF or its associated granulocyte traits on COVID-19 adverse outcomes. Only 0.99% (5/503) of breast cancer patients experienced COVID-19-related hospitalization following prophylactic or therapeutic G-CSF administration after chemotherapy. No mortality or progression to severe/critical COVID-19 occurred after G-CSF administration. Notably, no significant associations were observed between the application, dosage, or response to G-CSF and COVID-19-related hospitalization (all p >.05). Similarly, the MR analyses showed no evidence of causality of genetically predicted G-CSF or related granulocyte traits on COVID-19-related hospitalization or COVID-19 severity (all p >.05). There is insufficient evidence to substantiate the notion that the prophylactic or therapeutic administration of G-CSF after chemotherapy for managing CIN in patients with breast cancer and COVID-19 would worsen COVID-19 outcomes, leading to severe or critical conditions, or even death, especially considering the context of COVID-19 vaccination.

The secondary outbreak risk and mechanisms of Microcystis aeruginosa after H2O2 treatment.

The secondary outbreak of cyanobacteria after algicide treatment has been a serious problem to water ecosystems. Hydrogen peroxide (H2O2) is an algaecide widely used in practice, but similar re-bloom problems are inevitably encountered. Our work found that Microcystis aeruginosa (M. aeruginosa) temporarily hibernates after H2O2 treatment, but there is still a risk of secondary outbreaks. Interestingly, the dormant period was as long as 20 and 28 days in 5 mg L-1 and 20 mg L-1 H2O2 treatment groups, respectively, but the photosynthetic activity was both restored much earlier (within 14 days). Subsequently, a quantitative imaging flow cytometry-based method was constructed and confirmed that the re-bloom had undergone two stages including first recovery and then re-division. The expression of ftsZ and fabZ genes showed that M. aeruginosa had active transcription processes related to cell division protein and fatty acid synthesis during the dormancy stat. Furthermore, metabolomics suggested that the recovery of M. aeruginosa was mainly by activating folate and salicylic acid synthesis pathways, which promoted environmental stress resistance, DNA synthesis, and cell membrane repair. This study reported the comprehensive mechanisms of secondary outbreak of M. aeruginosa after H2O2 treatment. The findings suggest that optimizing the dosage and frequency of H2O2, as well as exploring the potential use of salicylic acid and folic acid inhibitors, could be promising directions for future algal control strategies.

Polo-like kinase 1 inhibitor NMS-P937 represses nasopharyngeal carcinoma progression via induction of mitotic abnormalities.

Polo-like kinase 1 (PLK1) inhibitor NMS-P937 is a targeted therapeutic agent with good preclinical efficacy in various human cancers, and its therapeutic effect on nasopharyngeal carcinoma (NPC) remains to be determined. Here, to explore biological activity of NMS-P937 in NPC, multiple types of NPC cells were utilized. We tested IC50 values, carried out flow cytometry, western blot analysis analysis, immunofluorescence, and constructed subcutaneous xenograft mouse models. We found that treatment with NMS-P937 increased the proportion of G2/M phase NPC cells, where CyclinB1 expression was upregulated and CyclinE1 expression was downregulated. Besides, NMS-P937 treatment-induced NPC cell apoptosis with increased cleavage of PARP and caspase-3. Mechanistically, NMS-P937 treatment led to aberrant mitosis, causing increased reactive oxygen species (ROS) levels. ROS scavenger N-acetylcysteine partially reversed ROS levels induced by NMS-P937. Furthermore, NMS-P937 administration restrained NPC xenografts growth in nude mice. Overall, NMS-P937 suppressed NPC cell proliferation and increased ROS levels, causing cell cycle abnormalities and apoptosis. NMS-P937 holds great promise as a therapeutic agent for treating nasopharyngeal carcinoma.

Prognostic Value of Immunohistochemistry-based Subtyping Before and After Neoadjuvant Chemotherapy in Patients with Triple-negative Breast Cancer.

To assess the predictive and prognostic value of a subtyping method based on immunohistochemistry in patients with triple-negative breast cancer (TNBC) treated with neoadjuvant chemotherapy (NAC). This study included patients with TNBC treated with anthracycline- and taxane-based NAC and curative surgery. Immunohistochemical (IHC) subtyping was performed using core needle biopsy specimens before NAC (pre-NAC) and residual tumors after NAC (post-NAC). Logistic regression was performed to identify predictive biomarkers of pathological complete response (pCR). Invasive disease-free survival (iDFS), distant disease-free survival (DDFS), and overall survival (OS) were assessed using the log-rank test and Cox proportional hazards regression. A total of 230 patients were followed up for a median of 59 months. Clinical lymph node status and the pre-NAC subtype were independent predictors of pCR (P=0.006 and 0.005, respectively). The pre-NAC subtype was an independent prognostic factor for long-term survival (iDFS: P < 0.001, DDFS: P=0.010, and OS: P=0.044). Among patients with residual disease (RD) after NAC, approximately 45% of tumors changed their IHC subtype. Furthermore, the post-NAC subtype, but not the pre-NAC subtype, was strongly associated with the survival of patients with RD (iDFS: P < 0.001, DDFS: P=0.005, and OS: P=0.006). The IHC subtype predicted response to NAC and long-term survival in patients with early TNBC. In patients with RD, almost 45% of the tumors changed subtype after NAC. The IHC subtype should be considered when planning additional therapies pre- and post-NAC.

Solar/periodate-triggered rapid inactivation of Microcystis aeruginosa by interrupting the Calvin-Benson cycle.

Frequent outbreak of cyanobacteria is a serious problem for drinking water treatment. The microcystins released from Microcystis aeruginosa (M. aeruginosa) could cause irreversible damage to human health. Catalyst-free solar/periodate (PI) system has recently presented great potential for bacterial inactivation, whereas the application potential and underlying mechanisms of the effective M. aeruginosa control remain unclear. Our work delineated the key role of ROS that inactivating/harmless disposing M. aeruginosa in the simulated sunlight (SSL)/PI system. Singlet oxygen may specifically cause DNA damage but maintain membrane integrity, preventing the risk of microcystins leakage. The SSL/PI 300 µM system could also effectively inhibit M. aeruginosa recovery for >7 days and completely degrade microcystin-LR (50.0 µg/L) within 30 min. Non-targeted metabolomic analysis suggested that the SSL/PI system inactivated M. aeruginosa mainly by interrupting the Calvin-Benson cycle, which damaged the metabolic flux of glycolysis and its downstream pathways such as the oxidative PPP pathway and glutathione metabolism. Furthermore, the activated PI system exhibited an even better algal inhibition under natural sunlight irradiation, evidenced by the seriously damaged cell membrane of M. aeruginosa. Overall, this study reported the comprehensive mechanisms of algal control and application potentials of solar/PI systems. The findings facilitated the development of emerging algicidal technology and its application in controlling environmental harmful algae.

High leukocyte mitochondrial DNA copy number contributes to poor prognosis in breast cancer patients.

BACKGROUND: Compelling evidence has indicated a significant association between leukocyte mitochondrial DNA copy number (mtDNAcn) and prognosis of several malignancies in a cancer-specific manner. However, whether leukocyte mtDNAcn can predict the clinical outcome of breast cancer (BC) patients has not been well investigated. METHODS: The mtDNA copy number of peripheral blood leukocytes from 661 BC patients was measured using a Multiplex AccuCopy™Kit based on a multiplex fluorescence competitive PCR principle. Kaplan-Meier curves and Cox proportional hazards regression model were applied to investigate the association of mtDNAcn with invasive disease-free survival (iDFS), distant disease-free survival (DDFS), breast cancer special survival (BCSS), and overall survival (OS) of patients. The possible mtDNAcn-environment interactions were also evaluated by the Cox proportional hazard regression models. RESULTS: BC patients with higher leukocyte mtDNA-CN exhibited a significantly worse iDFS than those with lower leukocyte mtDNAcn (5-year iDFS: fully-adjusted model: HR = 1.433[95%CI 1.038-1.978], P = 0.028). Interaction analyses showed that mtDNAcn was significantly associated with hormone receptor status (adjusted p for interaction: 5-year BCSS: 0.028, 5-year OS: 0.022), so further analysis was mainly in the HR subgroup. Multivariate Cox regression analysis demonstrated that mtDNAcn was an independent prognostic factor for both BCSS and OS in HR-positive patients (HR+: 5-year BCSS: adjusted HR (aHR) = 2.340[95% CI 1.163-4.708], P = 0.017 and 5-year OS: aHR = 2.446 [95% CI 1.218-4.913], P = 0.011). CONCLUSIONS: For the first time, our study demonstrated that leukocyte mtDNA copy number might influence the outcome of early-stage breast cancer patients depending on intrinsic tumor subtypes in Chinese women.

Onvansertib inhibits the proliferation and improves the cisplatin-resistance of lung adenocarcinoma via ß-catenin/c-Myc signaling pathway.

Polo-like kinase 1 (PLK1) is a key regulator of cell division, and its abnormal expression is related to the progression and prognosis of cancers. However, the effect of PLK1 inhibitor onvansertib on the growth of lung adenocarcinoma (LUAD) has not been explored. In this study, we performed a series of bioinformatics and experimental analyses to comprehensively investigate the role of PLK1 in LUAD. We used CCK-8 assay and colony formation assay to evaluate the growth inhibitory ability of onvansertib. Furthermore, flow cytometry was applied to exploit the effects of onvansertib on cell cycle, apoptosis, and mitochondrial membrane potential. Moreover, the therapeutic potential of onvansertib was assessed in vivo by using xenograft tumor and patient-derived xenograft (PDX) models. We found that onvansertib significantly induced the apoptosis and inhibited the proliferation and migration of LUAD cells. Mechanistically, onvansertib arrested the cells at G2/M phase and enhanced the levels of reactive oxidative species in LUAD. Accordingly, onvansertib regulated the expression of glycolysis-related genes and improved the cisplatin resistance in LUAD. Notably, the protein levels of ß-catenin and c-Myc were affected by onvansertib. Taken together, our findings provide insight into the function of onvansertib and shed light on the potential clinical application of onvansertib for the treatment of patients with LUAD.

Uncovering novel disinfection mechanisms of solar light/periodate system: The dominance of singlet oxygen and metabolomic insights.

Disinfection plays an essential role in waterborne pathogen control and disease prevention, especially during the COVID-19 pandemic. Catalyst-free solar light/periodate (PI) system has recently presented great potential in water disinfection, whereas the in-depth chemical and microbiological mechanisms for efficient bacterial inactivation remain unclear. Our work delineated firstly the critical role of singlet oxygen, instead of reported hydroxyl radicals and superoxide radicals, in dominating bacterial inactivation by the PI/simulated sunlight (SSL) system. Multi-evidence demonstrated the prominent disinfection performance of this system for Staphylococcus aureus in terms of culturability (> 6 logs CFU), cellular integrity, and metabolic activity. Particularly, the excellent intracellular DNA removal (> 95%) indicated that PI/SSL system may function as a selective disinfection strategy to diminish bacterial culturability without damaging the cell membrane. The PI/SSL system could also effectively inhibit bacterial regrowth for > 5 days and horizontal gene transfer between E. coli genera. Nontargeted metabolomic analysis suggested that PI/SSL system inactivated bacteria by triggering the accumulation of intracellular reactive oxygen species and the depletion of reduced glutathione. Additionally, the PI/SSL system could accomplish simultaneous micropollutant removal and bacterial inactivation, suggesting its versatility in water decontamination. Overall, this study deciphers more comprehensive antibacterial mechanisms of this environmentally friendly disinfection system, facilitating the technical development and application of the selective disinfection strategy in environmental pathogen control.

Clinical application of artificial neural network (ANN) modeling to predict BRCA1/2 germline deleterious variants in Chinese bilateral primary breast cancer patients.

BACKGROUND: Bilateral breast cancer (BBC), as well as ovarian cancer, are significantly associated with germline deleterious variants in BRCA1/2, while BRCA1/2 germline deleterious variants carriers can exquisitely benefit from poly (ADP-ribose) polymerase (PARP) inhibitors. However, formal genetic testing could not be carried out for all patients due to extensive use of healthcare resources, which in turn results in high medical costs. To date, existing BRCA1/2 deleterious variants prediction models have been developed in women of European or other descent who are quite genetically different from Asian population. Therefore, there is an urgent clinical need for tools to predict the frequency of BRCA1/2 deleterious variants in Asian BBC patients balancing the increased demand for and cost of cancer genetics services. METHODS: The entire coding region of BRCA1/2 was screened for the presence of germline deleterious variants by the next generation sequencing in 123 Chinese BBC patients. Chi-square test, univariate and multivariate logistic regression were used to assess the relationship between BRCA1/2 germline deleterious variants and clinicopathological characteristics. The R software was utilized to develop artificial neural network (ANN) and nomogram modeling for BRCA1/2 germline deleterious variants prediction. RESULTS: Among 123 BBC patients, we identified a total of 20 deleterious variants in BRCA1 (8; 6.5%) and BRCA2 (12; 9.8%). c.5485del in BRCA1 is novel frameshift deleterious variant. Deleterious variants carriers were younger at first diagnosis (P = 0.0003), with longer interval between two tumors (P = 0.015), at least one medullary carcinoma (P = 0.001), and more likely to be hormone receptor negative (P = 0.006) and HER2 negative (P = 0.001). Area under the receiver operating characteristic curve was 0.903 in ANN and 0.828 in nomogram modeling individually (P = 0.02). CONCLUSION: This study shows the spectrum of the BRCA1/2 germline deleterious variants in Chinese BBC patients and indicates that the ANN can accurately predict BRCA deleterious variants than conventional statistical linear approach, which confirms the BRCA1/2 deleterious variants carriers at the lowest costs without adding any additional examinations.

Development and validation of a predictive model for peripherally inserted central catheter-related thrombosis in breast cancer patients based on artificial neural network: A prospective cohort study.

BACKGROUND: Peripherally inserted central catheters have been extensively applied in clinical practices. However, they are associated with an increased risk of thrombosis. To improve patient care, it is critical to timely identify patients at risk of developing peripherally inserted central catheter-related thrombosis. Artificial neural networks have been successfully used in many areas of clinical events prediction and affected clinical decisions and practice. OBJECTIVE: To develop and validate a novel clinical model based on artificial neural network for predicting peripherally inserted central catheter-related thrombosis in breast cancer patients who underwent chemotherapy and determine whether it may improve the prediction performance compared with the logistic regression model. DESIGN: A prospective cohort study. SETTING: A large general hospital in Fujian Province, China. PARTICIPANTS: One thousand eight hundred and forty-four breast cancer patients with peripherally inserted central catheters placement for chemotherapy were eligible for the study. METHODS: The dataset was divided into a training set (N = 1497) and an independent validation set (N = 347). The synthetic minority oversampling technique (SMOTE) was used to handle the effect of imbalance class. Both the artificial neural network and logistic regression models were then developed on the training set with and without SMOTE, respectively. The performance of each model was evaluated on the validation set using accuracy, sensitivity, specificity, and area under the receiver operating characteristic curve (AUC). RESULTS: Of the 1844 enrolled patients, 256 (13.9%) were diagnosed with peripherally inserted central catheter-related thrombosis. Predictive models were constructed in the training set and assessed in the validation set. Eight factors were selected as input variables to develop the artificial neural network model. Without SMOTE, the artificial neural network model (AUC = 0.725) outperformed the logistic regression model (AUC = 0.670, p = 0.039). SMOTE improved the performance of both two models based on AUC. With the SMOTE sampling, the artificial neural network model performed the best across all evaluated models, the AUC value remained statistically better than that of the logistic regression model (0.742 vs. 0.675, p = 0.004). CONCLUSION: Artificial neural network model can effectively predict peripherally inserted central catheter-related thrombosis in breast cancer patients receiving chemotherapy. Identifying high-risk groups with peripherally inserted central catheter-related thrombosis can provide close monitoring and an opportune time for intervention.

Optimization of the extract from flower of Citrus aurantium L. var. amara Engl. and its inhibition of lipid accumulation.

Flower of Citrus aurantium L. var. amara Engl. (CAVA) has been confirmed to have promising anti-obesity effects. However, the regulation of alkaloid extracts from flower of CAVA (Al) on lipid metabolism remain unknown. In this study, Al was optimized by ultrasound-assisted extraction using response surface methodology. The optimal conditions were ultrasonic time 72 min, ethanol concentration 78% and liquid/solid ratio 30 ml/g with the maximum alkaloid yield 5.66%. LC-MS assay indicated that the alkaloid compounds were enriched in Al after optimization. Nine alkaloid compounds were identified in Al by LC-MS assay and stachydrine, caffeine and cathine appeared as the major alkaloid compounds. Bioactivity assay showed that Al treatment significantly increased superoxide dismutase (SOD) activity, and reduced malonaldehyde (MDA) and reactive oxygen species (ROS) levels. Al administration also reversed oleic acid-induced hepatic steatosis in Hep G2 cells by inhibiting the expression of lipogenesis-signaling genes including fatty acid synthase (FAS), peroxisome proliferator-activated receptor subtype γ (PPARγ), uncoupling protein 2 (UCP2), and retinol binding protein (RBP4). However, OA-induced reduction of lipolysis-related gene carnitine palmitoyl transferase 1A (CPT1A) in Hep G2 cells was not improved by Al supplementation. Moreover, the increased SOD activity and decreased MDA and ROS contents were also observed in Caenorhabditis elegans by Al addition. Al intervention exhibited the ability to inhibit lipid accumulation in C. elegans by suppressing expression of lipid metabolism-related genes. These results suggested that the alkaloid extracts from the flower of CAVA showed great potential to regulate lipid metabolism. PRACTICAL APPLICATIONS: The extraction of alkaloid extracts from the flower of CAVA was optimized with a maximum yield of 5.66%. The regulatory effects and mechanisms of Al on lipid metabolism of Hep G2 cells and Caenorhabditis elegans were also investigated. More clinical studies are required to evaluate the potential of using alkaloids from the flower of CAVA as therapeutic agents against lipid metabolic disorders.

Association of Adjuvant Hormone Therapy Timing With Overall Survival Among Patients With Hormone Receptor-Positive Human Epidermal Growth Factor Receptor-2-Negative Early Breast Cancer Without Chemotherapy.

Importance: Studies have shown that delayed initiation of surgery and adjuvant chemotherapy is associated with lower rates of breast cancer survival. However, it remains unclear whether delayed initiation of adjuvant hormone therapy (AHT) is associated with survival. Objective: To assess the association of time to adjuvant hormone therapy (TTH) with breast cancer survival and evaluate the factors associated with AHT. Design, Setting, and Participants: This cohort study examined data from the National Cancer Database from 2004 through 2014 to assess the association of TTH (stratified as ≤150 and >150 days) with cancer survival. All patients included were diagnosed with stage I to stage III hormone receptor-positive, human epidermal growth factor receptor-2 (ERBB2; formerly HER2)-negative invasive breast cancer and underwent AHT without chemotherapy. Data were analyzed from April 2019 to May 2020. Exposures: AHT was administered at different time points following surgical procedures for breast cancer treatment. Main Outcomes and Measures: An inverse probability of treatment weighting (IPTW) model was constructed to evaluate overall survival by adjusting for treatment facility, patient demographics, tumor characteristics, and treatment; multivariable logistic regression was conducted to assess factors associated with delayed treatment. Results: A total of 144 103 patients (median [IQR] follow-up, 36.6 months [25.5-49.2 months]; mean [SD] age, 63.7 [11.6] years) were identified, which included 142 916 (99.2%) women, 11 574 (8.0%) Black patients, and 126 013 (87.4%) White patients. Of these, 134 873 patients (93.6%) had a TTH of 150 days or less and 9230 patients (6.4%) had a TTH longer than 150 days. The IPTW-based Cox model demonstrated that patients with delayed AHT (ie, a TTH past 150 days) were associated with decreased survival (hazard ratio [HR], 1.31; 95% CI, 1.26-1.35; P < .001) compared with those receiving the timely treatment (TTH ≤150 days). Several sensitivity analyses (including IPTW with stabilized weight [HR, 1.31; 95% CI, 1.19-1.45; P < .001], propensity score matching [HR, 1.41; 1.13-1.76; P = .002], and propensity score regression adjustment [HR, 1.29; 95% CI, 1.16-1.43; P < .001]) and exploratory subgroup analyses yielded similar trends. Factors associated with delayed AHT included Black racial identity (OR, 1.66; 95% CI, 1.55-1.77), nonprivate insurance (eg, no insurance: OR, 1.46; 95% CI, 1.26-1.70), living in large metropolitan or metropolitan areas (reference vs urban, less urban, or rural: OR, 0.82; 95% CI, 0.76-0.87), treatment in a community hospital (reference vs academic or research: OR, 0.91; 95% CI, 0.84-0.98), Charlson-Deyo Comorbidity Index score 2 or higher (OR, 1.17; 95% CI, 1.04-1.32), poor grade differentiation (OR, 1.42; 95% CI, 1.32-1.53), II and III pathological stage (stage III: OR, 3.13; 95% CI, 2.76-3.54), estrogen receptor-positive (ER+)/progesterone receptor-negative (PR-) or ER-/PR+ (OR, 1.22; 95% CI, 1.13-1.31), receiving breast conservation surgery (reference vs mastectomy: OR, 0.87; 95% CI, 0.79-0.94), and radiotherapy (reference vs no radiotherapy: OR, 0.56; 95% CI, 0.52-0.61). Conclusions and Relevance: The delay of the initiation of AHT past 150 days was associated with diminished survival in hormone receptor-positive, ERBB2-negative patients with breast cancer who did not receive chemotherapy. Efforts should be made to address factors associated with delayed treatment to improve survival.

Prognostic Value of Programmed Cell Death-Ligand 1 Expression in Tumor-Infiltrating Lymphocytes and Viral Load in Peripheral Blood Mononuclear Cells for Epstein-Barr Virus-Positive Nasopharyngeal Carcinoma.

BACKGROUND: Epstein-Barr virus (EBV) infection has a role in the development and progression of nasopharyngeal carcinoma (NPC); however, it is unclear whether EBV load correlates with tumor prognosis or the need for immunotherapy. This study evaluated whether the EBV DNA concentration in peripheral blood mononuclear cells (PBMC) or programmed cell death-ligand1 (PD-L1) expression in tumor-infiltrating lymphocytes (TIL) could predict the clinical outcomes of patients with NPC. METHODS: Clinicopathological parameters of 198 patients with NPC were analyzed retrospectively from June 2012 to May 2018. Patients' EBV loads were determined by droplet digital PCR. TIL PD-L1 was analyzed by immunohistochemistry. RESULTS: A log value of 1.98 log IU/mL for PBMC EBV DNA and a percentage of PD-L1 expression of 15% in TILs marked distinguishing cutoffs in NPC prognosis. The 5-year progression-free survival (PFS) rates in patients with high vs low log (PBMC EBV DNA) were 68.2% and 93.1%, respectively (P = 0.002). The 5-year PFS rates in patients with high vs low TIL PD-L1 expression were 66.3% and 33.7%, respectively (P = 0.03). The 5-year PFS rates of the high-risk group (high log [PBMC EBV DNA] and low TIL PD-L1), low-risk group (low log [PBMC EBV DNA] and high TIL PD-L1), and those in between (intermediate group) were 0%, 91.9%, and 71.4%, respectively (P < 0.001). CONCLUSION: Concentrations of PBMC EBV DNA and TIL PD-L1 expression can be used as prognostic markers in NPC. The combination of both an increased EBV DNA concentration and suppressed TIL PD-L1 expression is associated with metastasis or relapse.

PCR assay for the cell-free copro-DNA detection of Angiostrongylus cantonensis in rat faeces.

To facilitate improved detection of the first stage larvae (L1) of Angiostrongylus cantonensis from rat faeces, a TaqMan(®) probe real-time PCR method for the detection in situ was developed targeting the second internal transcribed region of the ribosomal DNA (ITS2) of A. cantonensis. The assay was capable of detecting a single L1 in a grain of fresh faeces (weight 320 ± 125 mg) from the experimental infected Sprague-Dawley rats, and the method can also detect cell-free copro-DNA from positive faeces placed for up to 12 months at ambient environment. The present study exhibited a high level of specificity for A. cantonensis, with no fluorescence signals were observed in reference control consisting of four parasite species commonly found in the intestine of rat. This approach can overcome the limitations of DNA-based identification that faecal materials should be stored in 70% ethanol or kept as frozen samples for further tests, and thus it might be suitable and feasible for the detection of target DNA in faecal materials preserved at ambient temperature, but the detecting efficiency will depend on the amount of DNA in the samples and the time placed for the samples due to DNA degradation.