A Customized Microfluidic Paper-Based Platform for Colorimetric Immunosensing: Demonstrated via hCG Assay for Pregnancy Test

Over the past decades, paper-based lateral flow immunoassays (LFIAs) have been extensively developed for rapid, facile, and low-cost detection of a wide array of target analytes in a point-of-care manner. Conventional home pregnancy tests are the most significant example of LFAs, which detect elevated concentrations of human chorionic gonadotrophin (hCG) in body fluids to identify early pregnancy. In this work, we have upgraded these platforms to a higher version by developing a customized microfluidic paper-based analytical device (μPAD), as the new generation of paper-based point-of-care platforms, for colorimetric immunosensing. This will offer a cost-efficient and environmentally friendly alternative platform for paper-based immunosensing, eliminating the need for nitrocellulose (NC) membrane as the substrate material. The performance of the developed platform is demonstrated by detection of hCG (as a model case) in urine samples and subsequently indicating positive or negative pregnancy. A dual-functional silane-based composite was used to treat filter paper in order to enhance the colorimetric signal intensity in the detection zones of μPADs. In addition, microfluidic pathways were designed in a manner to provide the desired regulated fluid flow, generating sufficient incubation time (delays) at the designated detection zones, and consequently enhancing the obtained signal intensity. The presented approaches allow to overcome the existing limitations of μPADs in immunosensing and will broaden their applicability to a wider range of assays. Although, the application of the developed hCG μPAD assay is mainly in qualitative (i.e., positive or negative) detection of pregnancy, the semi-quantitative measurement of hCG was also investigated, indicating the viability of this assay for sensitive detection of the target hCG analyte within the related physiological range (i.e., 10–500 ng/mL) with a LOD value down to 10 ng/mL.

Emerging Trends in the Epidemiology of COVID-19: The Croatian ‘One Health’ Perspective

During the four pandemic waves, a total of 560,504 cases and 10,178 deaths due to COVID-19 were reported in Croatia. The Alpha variant, dominant from March 2021 (>50% of positive samples), was rapidly replaced by Delta variants (>90%) by August 2021. Several seroprevalence studies were conducted in different populations (general population, children/adolescents, professional athletes, healthcare workers, veterinarians) and in immunocompromised patients (hemodialysis patients, liver/kidney transplant recipients). After the first pandemic wave, seroprevalence rates of neutralizing (NT) antibodies were reported to be 0.2–5.5%. Significantly higher seropositivity was detected during/after the second wave, 2.6–18.7%. Two studies conducted in pet animals (February-June 2020/July–December 2020) reported SARS-CoV-2 NT antibodies in 0.76% of cats and 0.31–14.69% of dogs, respectively. SARS-CoV-2 NT antibodies were not detected in wildlife. Environmental samples taken in the households of COVID-19 patients showed high-touch personal objects as most frequently contaminated (17.3%), followed by surfaces in patients’ rooms (14.6%), kitchens (13.3%) and bathrooms (8.3%). SARS-CoV-2 RNA was also detected in 96.8% affluent water samples, while all effluent water samples tested negative. Detection of SARS-CoV-2 in humans, animals and the environment suggests that the ‘One Health’ approach is critical to controlling COVID-19 and future pandemics.

Diagnostic Value of Color Doppler Ultrasound Combined with Superb Microvascular Imaging in the Detection of Small Renal Tumors Less than 3 cm Treated with Jinkui Shenqi Pills

The purpose of this study was to investigate the diagnostic value of color Doppler ultrasound combined with superb microvascular imaging (SMI) in the detection of small renal tumors less than 3 cm treated with Jinkui Shenqi pills. 50 cases were randomly selected from the patients with angioleiomyoma (a kind of small renal tumor) less than 3 cm confirmed by pathological examination and treated in our hospital from January 2018 to January 2020. All patients were treated with Jinkui Shenqi pills. All patients were first detected by color Doppler ultrasound and then by SMI. The results of color Doppler ultrasound were used as the control group, while those of color Doppler ultrasound combined with SMI were used as the experimental group. After that, the specificity, sensitivity, positive and negative detection results, and detection accuracy were compared between the two groups. The specificity and sensitivity in the experimental group were significantly higher than those in the control group, with statistical significance ( P < 0.05 ). The cases of positive and negative detection results in the experimental group were significantly higher than those in the control group, with statistical significance ( P < 0.05 ). The detection accuracy in the experimental group was significantly higher than that in the control group, with statistical significance ( P < 0.05 ). The specificity, sensitivity, positive and negative detection results, and detection accuracy of color Doppler ultrasound combined with SMI in the detection of small renal tumors less than 3 cm treated with Jinkui Shenqi pills were all significantly higher than those of color Doppler ultrasound; therefore, the application of color Doppler ultrasound combined with SMI for the diagnosis of small renal tumors is of high value.

Gadoxetic Acid-Enhanced and Diffusion-Weighted Magnetic Resonance Imaging of Histologically Defined Early Hepatocellular Carcinoma

Purpose: To describe the imaging features of histologically defined early hepatocellular carcinoma (eHCC) on gadoxetate disodium-enhanced MRI (EOB-MRI) and diffusion-weighted imaging (DWI).Materials and Methods: We enrolled 173 surgically confirmed eHCCs in 119 patients examined by preoperative EOB-MRI and DWI between January 2006 and September 2017. The imaging features of preoperatively detected eHCCs were retrospectively analyzed by two radiologists. The clinical and imaging characteristics associated with false-negative detection were evaluated.Results: Of the 173 eHCCs, 118 (68%) in 78 patients were prospectively reported on preoperative EOB-MRI. After retrospective review, 17 eHCCs in 13 patients were additionally detected, with a per-lesion detection sensitivity of 78% (135/173). Thus, the imaging features of 135 eHCCs in 91 patients were analyzed. Most eHCCs exhibited hepatobiliary hypointensity (90%, 122/135). Arterial phase hyperenhancement, washout, and capsule appearance were seen in 68 (50%), 79 (59%), and 11 (8%) detected lesions, respectively. Diffusion restriction and fatty change were noted in 30 (22%) and 39 (29%) lesions, respectively; most eHCCs exhibited T1 and T2 isointensity (80 [59%] and 89 [66%], respectively). False-negative detection was associated with small lesion size (< 1 cm), history of HCC treatment (odds ratio, 0.34 [95% confidence interval, 0.13-0.92]), number of HCC lesions (≥ 2; odds ratio, 0.08 [0.01-0.66]), and poor functional liver imaging score (< 4; odds ratio, 0.13 [0.04-0.51]).Conclusions: Histologically defined eHCCs typically appear as hepatobiliary phase hypointensity. Detection sensitivity of eHCC may be affected by lesion size, history of HCC treatment, number of HCCs, and hepatobiliary enhancement.

Diagnosis of SARS-CoV-2 infection

The most important use of serology in the COVID-19 diagnostics is for determination of the extent of disease in the population. However, immunoassays could represent an additional diagnostic method, especially in patients with exposure history and clinical symptoms compatible with COVID-19 who failed to be confirmed by RT-PCR. We analyzed the preliminary results of six serology tests for the diagnosis of SARS-CoV-2. Three point-of-care lateral flow chromatographic immunoassays (POC): ACRO, AMP and ENCODE and three enzyme immunoassays (ELISA): DiaPro, Vircell and Euroimmun were used. A total of 15 serum samples from COVID-19 patients and 15 serum samples from asymptomatic persons were tested. Time of sampling for COVID-19 patients was 4 – 10 days (N=4), 11 – 19 days (N=6) and 20 – 34 days (N=5) after disease onset. Initially reactive results were confirmed using a virus neutralization test (VNT). In COVID-19 patients (N=15), IgM/IgA positive detection rates were 9/60.0% (ACRO), 11/73.3% (AMP, ENCODE, Euroimmun), 12/80.0% (DiaPro) and 13/86.6% (Vircell). Overall IgG detection rates were 10//66.6% (AMP, Euroimmun) and 11/73.3% (other tests). According to the sampling time, positive detection rates were as follows: a) days 4 – 10: 1/25.0% and 2/50.0% (IgM/IgA and IgG); b) days 11 –19: 4/66.6%-6/100% (IgM/IgA), 4/66.6% and 5/83.3% (IgG); c) days 20 – 34: 4/80.0% and 5/100% (IgM/IgA), 5/100% (IgG). One asymptomatic participant tested IgM/IgA positive using ACRO, DiaPro and Vircell was confirmed seropositive using a VNT. In a group of asymptomatic persons detected seronegative using a VNT (N=14), IgM/IgA negative detection rates were 12/85.7% (ACRO), 13/92.8% (DiaPro, Vircell) and 14/100% (AMP, ENCODE, Euroimmun). IgG negative detection rates were 13/92.8% (ACRO) and 14/100% (other tests). ELISA tests showed a higher overall IgM/IgA sensitivity compared to POC tests in patients with COVID-19, while the IgG sensitivity was similar in both POC and ELISA.

Development of High Resolution DNA Melting Analysis for Simultaneous Detection of Potato Mop-Top Virus and Its Vector, Spongospora subterranea, in Soil

In this study, a set of duplex reverse transcription (RT)-PCR-mediated high resolution DNA melting (HRM) analyses for simultaneous detection of potato mop-virus (PMTV) and its protist vector, Spongospora subterranea f.sp. subterranea (Sss), was developed. The infestation of soil by PMTV was detected by using a tobacco-based baiting system. Total RNA extracted from the soil led to successful RT-PCR gel-electrophoresis detection of both PMTV and Sss. To facilitate more efficient detection, newly designed primer pairs for PMTV RNA species (i.e., RNA-Rep, -CP, and -TGB) were analyzed together with the existing Sss primers using real-time RT-PCR. The resulting amplicons exhibited melting profiles that could be readily differentiated. Under duplex RT-PCR format, all PMTV and Sss primer combinations led to successful detection of respective PMTV RNA species and Sss in the samples by high resolution DNA melting (HRM) analyses. When the duplex HRM assay was applied to soil samples collected from six fields at four different sites in New Brunswick, Canada, positive detection of PMTV and/or Sss was found in 63-100% samples collected from fields in which PMTV-infected tubers had been observed. In contrast, the samples from fields where neither PMTV- nor Sss-infected tubers had been observed resulted in negative detection by the assay. Bait tobacco bioassay for PMTV and Sss produced similar results. Between 63%-83% and 100% of the soil samples collected from PMTV-infested fields led to PMTV and Sss infections in the bait tobacco plants, respectively; whereas no PMTV or Sss infected plants were obtained from soil samples collected from PMTV/Sss-free fields.

SARS-CoV-2 Translocate from Nasopharyngeal to Bronchoalveolar Site: A Case Presentation

The nasopharyngeal swab is commonly used for the diagnosis of SARS-CoV-2 infection. Since the swab is performed in this site, of course, it cannot detect the presence of the virus in other tissue districts such as the lung, brain, or bowel. In the present case report, the nasopharyngeal swab was negative twice. From this, the patient discontinued antiviral therapy. Nasopharyngeal swabs were maintained negative until five days later, when we recorded a severe impairment of the patient’s clinical condition. At this time, the bronchoalveolar lavage was positive for SARS-CoV-2. The purpose of the case herein described is to suggest paying attention to the nasopharyngeal swab result. A negative detection in nasopharyngeal swab could not be indicative of COVID-19 recovery.

Long-Term Monitoring of Dynamic Changes in Plasma EBV DNA For Improved Prognosis Prediction of Nasopharyngeal Carcinoma

Background: This study was performed to investigate whether long-term monitoring of dynamic changes in plasma Epstein-Barr virus (EBV) DNA could improve prognosis prediction of nasopharyngeal carcinoma (NPC).Methods: 1077 non-metastatic NPC patients were recruited to retrospectively analyze the prognostic value of plasma EBV DNA load pre-treatment and 3, 12, 24, and 36 months post-treatment. We also examined the prognostic value of dynamic changes in plasma EBV DNA at various time points.Results: Patients with plasma EBV DNA load above optimal pre- and post-treatment cut-offs had significantly worse five-year progression-free survival, distant metastasis-free survival, locoregional relapse-free survival, and overall survival(OS) at all time points, excluding only OS at 36 months post-treatment due to limited mortalities. Patients with persistently undetectable plasma EBV DNA at the first four time points had the best prognosis, followed by those with positive detection pre-treatment and consistently negative detection post-treatment, those with negative detection pre-treatment and positive detection at one time point post-treatment, and those with positive detection pre-treatment and at one time point post-treatment, whereas patients with positive detection at ≥2 time points post-treatment had the worst prognosis. Cox proportional hazard models identified the dynamic change pattern as an independent prognostic factor, and ROC curve analysis demonstrated that the dynamic change at four time point was more valuable than any single time point for predicting disease progression, distant metastasis, locoregional relapse, and mortality.Conclusions: Dynamic changes in plasma EBV DNA pre- and post-treatment could predict the long-term survival outcome and provide accurate risk stratification in NPC.