Search

Nucleic acid extraction (NAE) is crucial for molecular diagnostics but presents challenges in point-of-care testing (POCT) and decentralized settings. We developed a streamlined, paper-based NAE method for hepatitis C virus (HCV) RNA amplification, suitable for integration into POCT and lab-on-a-chip systems. This method uses Fusion 5 paper discs, completing extraction in under 30 min without centrifugation. The nucleic acids on the disc can be directly used or eluted for amplification. We validated this method's compatibility with reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR (RQ-PCR), and loop-mediated isothermal amplification (LAMP), demonstrating versatility across amplification platforms. Clinical evaluation (n = 60) showed 100% sensitivity and specificity with a low detection limit of ~10 1  IU/mL. Results matched those from standard HCV RQ-PCR, confirming accuracy. Additionally, incorporating polyethylene glycol (PEG) improves extraction efficiency, eliminating the need for ethanol treatment and washing/drying steps. This modification enhances performance and suitability for field applications. Our paper-based HCV amplification is affordable and user-friendly, making it valuable for decentralized HCV detection and supporting global health initiatives., (© 2024 Wiley Periodicals LLC.)

The incidence of arbovirus infections has increased dramatically in recent decades, affecting hundreds of millions of people each year. The Togaviridae family includes the chikungunya virus (CHIKV), which is typically transmitted by Aedes mosquitoes and causes a wide range of symptoms from flu-like fever to severe arthralgia. Although conventional diagnostic tests can provide early diagnosis of CHIKV infections, access to these tests is often limited in developing countries. Consequently, there is an urgent need to develop efficient, affordable, simple, rapid, and robust diagnostic tools that can be used in point-of-care settings. Early diagnosis is crucial to improve patient management and to reduce the risk of complications. A glass-fiber laser-cut microfluidic device (paper-based analytical device [PAD]) was designed and evaluated in a proof of principle context, for the analysis of 30 µL of patient serum. Biological raw materials used for the functionalization of the PAD were first screened by MAC-ELISA (IgM capture enzyme-linked immunosorbent assay) for CHIKV Immunoglobulin M (IgM) capture and then evaluated on the PAD using various human samples. Compared with viral lysate traditionally used for chikungunya (CHIK) serology, CHIKV pseudo-particles (PPs) have proven to be powerful antigens for specific IgM capture. The PAD was able to detect CHIKV IgM in human sera in less than 10 minutes. Results obtained in patient sera showed a sensitivity of 70.6% and a specificity of around 98%. The PAD showed few cross-reactions with other tropical viral diseases. The PAD could help health workers in the early diagnosis of tropical diseases such as CHIK, which require specific management protocols in at-risk populations., (© 2019 Wiley Periodicals, Inc.)

Dried blood spots collected on filter paper are considered potential clinical specimens for measles surveillance because of their ease of collection, storage, and transport. The usefulness of these samples for surveillance of measles was evaluated in a field setting. Blood spots were collected by finger-prick from 316 clinically diagnosed measles patients in suburban Khartoum, mostly within a week after onset of the rash. Samples were collected between October, 2000 and April, 2003, and stored at 4 degrees C. Measles virus-specific IgM antibodies were detected in 200 (63%) of the samples using an "in-house" IgM capture ELISA. For 201 samples reconstitution and IgM measurement was repeated 1 year after initial testing with essentially the same results, showing the stability of IgM in the filter paper under these conditions. In a limited number of samples (n = 38) measles virus-specific IgM was also tested with a commercial indirect IgM ELISA. Although the results of the two assays correlated well, the "in-house" IgM capture ELISA proved slightly more sensitive. Measles virus-specific reverse transcriptase polymerase chain reaction (RT-PCR) amplicons were obtained from 16 of 57 (28%) samples tested. Sequencing of the 3' 456 nucleotides of the nucleoprotein gene showed the continued endemic circulation of genotype B3 viruses identified previously in this region. Although problems related to limited sample quantities were encountered, the present study confirms the usefulness of dried blood spots for measles surveillance. The results also demonstrate that measles continues to be endemic in the Sudan., (Copyright 2004 Wiley-Liss, Inc.)

We compared the use of serum and filter paper blood spots as specimen sources for the detection of measles- and rubella-specific IgM and IgG. We collected capillary blood into microtainer tubes and onto filter paper spots from 60 children and 60 healthy adults. The blood was collected from 12-15-month-old children approximately 3 weeks after primary vaccination with measles, mumps, rubella vaccine, and the sample-pairs were tested for measles-specific IgM and IgG antibodies by using a capture antibody EIA and an indirect EIA, respectively. We tested sample-pairs from a subset of participants for rubella- specific IgM and IgG antibodies by using commercially available capture IgM (Captia) and indirect IgG (Wampole) assays. The concordance of results from serum and filter paper blood spots was high for all assays: 98% for measles IgM, 93% for measles IgG, 94% for rubella IgM, and 93% for rubella IgG, and increased to between 96-100% for all four assays when indeterminate samples were excluded. The correlation coefficients for EIA signals were 0.99 and 0.77 for measles IgM and IgG, respectively, and 0.92 and 0.94 for rubella IgM and IgG, respectively. The cut-off values used for filter paper samples were the same as those used for serum samples for all tests except for the rubella IgM assay. The use of filter paper blood spots is a promising future option for the detection of measles- and rubella-specific antibodies., (Copyright 2001 Wiley-Liss, Inc.)

A thermophilic xylanase from Bacillus strain D3 suitable for use as a bleach booster in the paper pulping industry has been identified and characterized. The enzyme is suited to the high temperature and alkaline conditions needed for using xylanases in the pulp industry. The xylanase is stable at 60 degrees C and relatively stable at high temperatures, with a temperature optimum of 75 degrees C. The pH optimum is 6, but the enzyme is active over a broad pH range. The xylanase has been cloned and sequenced, and the crystal structure has been determined. The structure of Bacillus D3 xylanase reveals an unusual feature of surface aromatic residues, which form clusters or "sticky patches" between pairs of molecules. These "sticky patches" on the surface of the enzyme are responsible for the tendency of the protein to aggregate at high concentrations in the absence of reagents such as ethylene glycol. The formation of dimers and higher order polymers via these hydrophobic contacts may also contribute to the thermostability of this xylanase.

This article describes two cases in which the advantages of the ESEM have been exploited in unanticipated ways. First, we have found that etching occurs as the electron beam scans the surface of uncoated polymers in the ESEM. The surface topography caused by this etching, as seen in ESEM images, reflects the morphology of crystalline structures in the polymers. This technique has been valuable in the study of such textures in polymers. The second application is related to our use of the ESEM in support of research on the deinking of paper. In this effort we have learned that an unconventional contrast mechanism can be used during ESEM imaging to distinguish between inked and non-inked areas of newsprint. Under usual operating conditions, ESEM imaging does not distinguish between inked and non-inked areas. However, at relatively low sample chamber pressures the non-inked areas appear brighter than inked areas in ESEM images.

In order to determine the relationship between hepatitis B virus (HBV) infection of human white blood cells and different forms of HBV-associated liver diseases, we tested for HBV DNA in the sera and leukocytes of 11 healthy individuals without any serological markers of HBV infection and 91 patients with HBV infection and other gastrointestinal and urinary diseases by dot and Southern blot hybridization. HBV DNA was found in leukocytes of chronic HBV carriers, in acute and chronic hepatitis, and in patients with liver cirrhosis and hepatocellular carcinoma. Between 27 and 50% of individuals in different categories of patients examined were positive for leukocyte HBV DNA. HBV DNA was also detected in the sera of some of these patients but was absent in others. Serum HBV DNA-positive rates seemed to be highest in hepatitis B e antigen-positive asymptomatic carriers (8/10, 80%), and tended to drop to lower levels as the disease progressed to liver cirrhosis (0/8) while leukocyte HBV DNA-positive rates were highest in patients with cirrhosis (4/8, 50%). The results also show that in individuals who were serologically negative for hepatitis B surface antigen (HBsAg) and positive for antibodies to HBsAg and/or HBcAg, HBV DNA was absent in most of the sera (27/28, 96%) but it was present in leukocytes of some of these patients (7/28, 25%). In control experiments with 11 healthy individual, HBV DNA was not detected in either sera or leukocytes. In all the cases with leukocyte HBV DNA, the HBV DNA molecules were present in free forms with discrete sizes. The exceptions were a case of liver cirrhosis and a case of chronic hepatitis with possible HBV sequence integration into high molecular weight cellular DNA. Since HBV does infect human leukocytes, it may perhaps interfere with the immunological functions of the white blood cells, and thus play an important role in the pathogenesis of HBV-induced liver disease.

In Sapporo City, the mass-screening for neuroblastoma concerning the urine of infants at about 6 months of age, started in 1981. After 1984, the quantitative determination of VMA, HVA in the urine was carried out using high performance liquid chromatography from the first step. About 104,000 babies have received the screening during the last 7 years, and 19 patients were found (incidence of detection 1:5,500). Except for one, who died of the duodenal perforation shortly after the surgery, all other patients are surviving free from disease. Among 10,000 neonates, we determined urinary concentration of VMA, HVA, and no positive cases were found. However, from this group, 2 patients we detected by the mass-screening at 6 months of age (1:5,000). After the start of mass-screening, the number of the patients with this tumor in much earlier stages and younger ages increased, clarifying that the main cause for improvement of the survival rates of neuroblastoma in Sapporo.