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In the last few years, next-generation sequencing (NGS) has emerged as a technology for laboratory diagnosis of many culture-negative infections and slow-growing microorganisms. In this study, we describe the use of metagenomic NGS (mNGS) for rapid diagnosis of T. marneffei infection in a 37-year-old renal transplant recipient who presented with chronic pneumonia syndrome. Bronchoalveolar lavage for mNGS was positive for T. marneffei sequence reads. Prolonged incubation of the bronchoalveolar lavage revealed T. marneffei colonies after 6 days of incubation. Analysis of 23 cases of T. marneffei infections in renal transplant recipients from the literature revealed that the number of cases ranged from 1 to 4 cases per five years from 1990 to 2020; but increased rapidly to 9 cases from 2021 to 2023, with 7 of them diagnosed by NGS. Twenty of the 23 cases were from T. marneffei-endemic areas [southern part of mainland China (n = 9); Hong Kong (n = 4); northeastern India (n = 2); Indonesia (n = 1) and Taiwan (n = 4)]. For the 3 patients from non-T. marneffei-endemic areas [United Kingdom (n = 2) and Australia (n = 1)], they had travel histories to China and Vietnam respectively. The time taken for diagnosis by mNGS [median 1 (range 1 to 2) day] was significantly shorter than that for fungal culture [median 6 (range 3 to 15) days] (P = 0.002). mNGS is useful for picking up more cases of T. marneffei infections in renal transplant recipients as well as providing a rapid diagnosis. Talaromycosis is an emerging fungal infection in renal transplant recipients. [ABSTRACT FROM AUTHOR]

Cassava brown streak disease (CBSD) caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) is the most economically important viral disease of cassava. As cassava is a vegetatively propagated crop, the development of rapid and sensitive diagnostics would aid in the identification of virus-free planting material and development of effective management strategies. In this study, a rapid, specific and sensitive real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed for real-time detection of CBSV and UCBSV. The RT-RPA was able to detect as little as 2 pg/µl of purified RNA obtained from infected cassava leaves, a sensitivity equivalent to that obtained by quantitative real-time reverse transcription PCR (qRT-PCR), within 20 min at 37 °C. Further, the RT-RPA detected each target virus directly from crude leaf and stem extracts, avoiding the tedious and costly isolation of high-quality RNA. The developed RT-RPA assay provides a valuable diagnostic tool that can be adopted by cassava seed certification and virus resistance breeding programs to ensure distribution of virus-free cassava planting materials to farmers. This is the first report on the development and validation of crude sap-based RT-RPA assay for the detection of cassava brown streak viruses (UCBSV and CBSV) infection in cassava plants. [ABSTRACT FROM AUTHOR]

Familial hemophagocytic lymphohistiocytosis (FHLH) is a fatal hyperinflammation syndrome arising from the genetic defect of perforin-mediated cytolysis. Curative hematopoietic cell transplantation (HCT) is needed before development of central nervous system (CNS) disease. We studied treatment outcomes of 13 patients (FHLH2 n = 11, FHLH3 n = 2) consecutively diagnosed from 2011 to 2022 by flow cytometric screening for non-myeloablative HCT in a regional treatment network in Kyushu, Japan. One patient with a novel PRF1 variant escaped screening, but all patients with FHLH2 reached diagnosis and 8 of them received HCT until 3 and 9 months of age, respectively. The earliest HCT was conducted 65 days after birth. Three pretransplant deaths occurred in newborns with liver failure at diagnosis. Ten posttransplant patients have remained disease-free, 7 of whom had no neurological involvement. Time from first etoposide infusion to HCT was shorter in patients without CNS disease or bleeding than in patients with those factors (median [range] days: 62 [50–81] vs. 122 [89–209], p = 0.016). Six of 9 unrelated patients had a PRF1 c.1090_1091delCT variant. These results suggest that the critical times to start etoposide and HCT are within 3 months after birth and during etoposide control, respectively. Newborn screening may increase the percentage of disease-free survivors without complications. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (MTB), the etiological agent of tuberculosis (TB), is the leading cause of death due to a single infectious agent worldwide. Rapid and accurate diagnosis of MTB is critical for controlling TB especially in resource-limited countries, since any diagnosis delay increases the chances of transmission. Here, a real-time recombinase-aided amplification (RAA) assay targeting conserved positions in IS1081 gene of MTB, is successfully established to detect MTB. The intact workflow was completed within 30 min at 42 °C with no cross-reactivity observed for non-tuberculous mycobacteria and other clinical bacteria, and the detection limit for recombinant plasmid of MTB IS1081 was 163 copies/reaction at 95% probability, which was approximately 1.5-fold increase in analytical sensitivity for the detection of MTB, compared to conventional quantitative real-time PCR (qPCR; 244 copies/reaction). Furthermore, the result of clinical performance evaluation revealed an increased sensitivity of RAA assay relative to qPCR was majorly noted in the specimens with low bacteria loads. Our results demonstrate that the developed real-time RAA assay is a convenient, sensitive, and low-cost diagnostic tool for the rapid detection of MTB. [ABSTRACT FROM AUTHOR]

Septic arthritis is a serious condition with significant morbidity and mortality, routinely diagnosed using culture. The FDA has recently approved the rapid molecular BioFire® Joint Infection Panel (BJIP) for synovial fluid. We aimed to evaluate the BJIP compared to culture and its potential use in patient management. A multicentre retrospective evaluation of BJIP was conducted in the UK and Ireland. Positive percent agreement (PPA) and negative percent agreement (NPA) were calculated between the BJIP and routine culture. A multidisciplinary team (MDT) discussion addressing the optimal or potential case use of the assay practice was facilitated. Three hundred ninety-nine surplus synovial fluid samples (~ 70% from native joints) from eight centres were processed using BJIP in addition to routine culture. An increased yield of positive results was detected using BJIP compared to routine culture (98 vs 83), giving an overall PPA of 91.6% and overall NPA of 93% for the BJIP compared to culture results. The BJIP detected resistant markers and additional organisms that could influence antibiotic choices including Neisseria gonorrhoeae and Kingella kingae. The MDT agreed that the assay could be used, in addition to standard methods, in adult and children patients with specialist advice use based on local needs. Rapid results from BJIP were assessed as having potential clinical impact on patient management. Organisms not included in the panel may be clinically significant and may limit the value of this test for PJI. [ABSTRACT FROM AUTHOR]

We introduce a lateral flow assay (LFA) integrated with a modified isothermal nucleic acid amplification procedure for rapid and simple genetic testing. Padlock probes specific for the target DNA were designed for ligation, followed by rolling circle amplification (RCA) using capture ligand-modified oligonucleotides as primers. After hybridization with detection linker probes, the amplified target DNA is flowed through an LFA membrane strip for binding of gold nanoparticles as the substrate for colorimetric detection. We established and validated the "RCA-LFA" method for detection of mecA, the antibiotic resistance gene for methicillin-resistant Staphylococcus aureus (MRSA). The assay was optimized using various concentrations of primers and probes for RCA and LFA, respectively. The sensitivity was determined by performing RCA-LFA using various amounts of mecA target DNA, showing a detection limit of ~ 1.3 fmol. The specificity of the assay was examined using target DNAs for other resistance genes as the controls, which demonstrated positive detection signals only for mecA DNA, when added either individually or in combinations with the control targets. Furthermore, applying the RCA-LFA method using specifically designed probes for RNA-dependent RNA polymerase (RdRp) and receptor binding domain (RBD) gene for SARS-CoV-2, which demonstrated feasibility of the method for viral gene targets. The current method suggests a useful platform which can be universally applied for various nucleic acid targets, allowing rapid and sensitive diagnosis at point-of-care. [ABSTRACT FROM AUTHOR]

Several studies have examined the informational needs of patients undergoing the breast diagnostic process where needs are highest during testing and prior to receiving a diagnosis. To aid in the development of an education pathway, we identified patient information needs. A multi-method approach to identify areas of need and to understand when and how information should be provided to patients was undertaken. The methods included an environmental scan of consumer health information, ethnographic observation of the patient clinical experience, key informant interviews, and a needs assessment survey. The data collected from the environmental scan, ethnography, and interviews were used to develop the items in the survey. The survey was developed around four domains: (1) Medical Procedures and Tests, (2) Understanding the Rapid Diagnostic Process, (3) Breast Cancer and Other Breast Conditions, and (4) Support and Coping. A total of 101 patients completed the survey. Mean importance scores were significantly different between domains of information need (p <.0001) and significantly higher for the 'Medical Procedures and Tests' domain compared with all others. Multivariate analysis suggested that participants with higher levels of education (p =.02) and a preference to speak English at home (p =.009) tended to rate the importance of 'Support and Coping' information lower than other participants. Information about medical procedures and tests are most important for the patients undergoing rapid diagnostic testing in our sample. Education materials that are tailored to patient needs should be provided to patients during this stage of the cancer journey to help meet informational needs. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a common clinical bacterial pathogen that can cause a diverse range of infections. The establishment of a rapid and reliable assay for the early diagnosis and detection of S. aureus is of great significance. In this study, we developed a closed-tube loop-mediated isothermal amplification (LAMP) assay for the visual detection of S. aureus using the colorimetric indicator hydroxy naphthol blue (HNB). The LAMP reaction was optimized by adjusting the amplification temperature, the concentrations of Mg2+, dNTP, and HNB, and the incubation time. In the optimized reaction system, the specificity of LAMP for S. aureus was 100%. The results established that this method accurately identified S. aureus, with no cross-reactivity with 14 non-S. aureus strains. The limit of detection (LOD) of LAMP was 8 copies/reaction of purified plasmid DNA or 400 colony-forming units/reaction of S. aureus. Compared with conventional PCR, LAMP lowered the LOD by tenfold. Finally, 220 clinically isolated strains of S. aureus and 149 non-S. aureus strains were used to evaluate the diagnostic efficacy of LAMP (test accuracy, 99.46%). The findings indicated that LAMP is a reliable test for S. aureus and could be a promising tool for the rapid diagnosis of S. aureus infections. [ABSTRACT FROM AUTHOR]

Rapid diagnostics provide actionable information for patient care at the time and site of an encounter with the health care system. The mainstay of infectious diseases care is early detection (case finding) and treatment completion, but for many, it is hard to identify positive individuals, as is the case of infection with low burden in schistosomiasis, a parasitic disease common in the tropics and subtropics. We developed a new, accurate, and fast Dot blot methodology (iDot) to indirectly detect Schistosoma mansoni in individuals with very low parasite burden using urine samples. Accuracy of 0.74 was obtained with a significant difference between negative and positive patients and a substantial agreement was found when iDot was compared with five available methods. Our analysis also revealed the superiority of iDot in detecting negative individuals from non-endemic sites, thus, presenting the lowest rate of false positives. This new method called iDot is convenient and suitable for qualitative and quantitative detection of schistosomiasis in individuals with low parasite burden. [ABSTRACT FROM AUTHOR]

Purposes: The diagnosis of strangulated bowel obstruction (SBO) is sometimes difficult. We attempted to create and verify a discriminant formula for use as a diagnostic aid for the early diagnosis of SBO. Methods: This retrospective study included 97 patients who underwent an operation for SBO from January 2007 to September 2018. First, a discriminant analysis was performed for 73 patients who underwent an operation from January 2007 to December 2015 in order to obtain a formula. Next, we analyzed 34 patients who underwent an operation from January 2016 to September 2018 to verify the formula. Results: The risk factors for SBO included ascites, signs of preperitoneal irritation, and lactate > 1.16 mmol/L. The discriminant formula is as follows: 1.954 × collection of ascites (1 or 0) + 1.239 × peritoneal irritation sign (1 or 0) + 0.378 × lactate − 2.331 (1: positive, 0: negative). The predictive value was as follows: sensitivity, 87.5%; specificity, 64.7%; and predictive accuracy, 73.5%. In patients who presented within 24 h of the onset, the sensitivity was 92.3%, the specificity was 75.0%, and the predictive accuracy was 85.7%. Conclusion: Our discriminant formula seems useful for the rapid diagnosis of SBO. [ABSTRACT FROM AUTHOR]

Objective: The diagnosis of superficial tuberculous lymphadenitis (TBLN) remains difficult due to low detection rate of etiology. To increase the diagnostic value for TBLN, contrast-enhanced ultrasound (CEUS) guided core biopsy was introduced to obtain the specimen followed by Xpert MTB/RIF (Xpert) and other methods testing and to explore the optimum diagnostic pattern for TBLN in China. Methods: A prospective study was performed on patients with suspected superficial TBLN. All patients underwent CEUS-guided core biopsy from which specimens were tested by histopathology, Xpert, acid-fast bacilli (AFB), and MGIT960 culture (MGIT960), respectively. The diagnostic values were calculated and compared. Results: A total of 328 patients were included the study, 272 were diagnosed as TBLN (254 definite TB, 18 probable TB) and 56 cases with Non-TBLN, and 100% (272/272) of TBLN patients obtained diagnosis sampled by CEUS-guided core biopsy. The overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of comprehensive diagnosis on the specimens by CEUS-guided core biopsy for TBLN were 100% (272/272, 95% CI 98.26–100.00), 94.64% (53/56, 95% CI 84.20–98.61), 98.91% (272/275, 95% CI 96.58–99.72), and 100% (53/53, 95% CI 91.58–100%), respectively. Xpert obtained 93.31% (237/254) of etiology detection rate on the specimens sampling by CEUS-guided biopsy. The etiology detection rate was associated with histopathological caseous necrosis. Conclusions: Current examinations on specimens by CEUS-guided core biopsy can achieve a high diagnostic efficacy for TBLN. Pathological differentiation of CEUS-guided biopsy tissue, then followed by Xpert, may be the best pattern for the diagnosis of TBLN in high TB burden areas. [ABSTRACT FROM AUTHOR]

Nowadays, the diagnosis of viral infections is receiving broad attention. We have developed a non-competitive fluorescence polarization immunoassay (NC-FPIA), which is a separation-free immunoassay, for a virus detection. H5 subtype avian influenza virus (H5-AIV) was used as a model virus for the proof of concept. The fluorescein-labeled Fab fragment that binds to H5 hemagglutinin was used for NC-FPIA. The purified H5-AIV which has H5 hemagglutinin was mixed with the fluorescein-labeled Fab fragment. After that, the degree of fluorescence polarization was measured with a portable FPIA analyzer. H5-AIV was successfully detected with an incubation time of 15 min. In addition, the portable FPIA analyzer enables performance of on-site NC-FPIA with a sample volume of 20 μL or less. This is the first research of detecting a virus particle by FPIA. This NC-FPIA can be applied to rapid on-site diagnosis of various viruses. [ABSTRACT FROM AUTHOR]

Infection with Helicobacter pylori is a global health issue, and rapid and accurate testing is a key to diagnosis. We aimed to assess the performance of two novel enzyme immunoassays (EIA), the H. PYLORI QUIK CHEK™ and the H. PYLORI CHEK™ assays, for the detection of H. pylori antigen in stool. Patients from five geographically diverse sites across the USA, Germany, and in Bangladesh were tested for infection with Helicobacter pylori with the two novel stool antigen tests and two commercially available stool antigen assays. All patients provided a stool sample and underwent esophagogastroduodenoscopy for biopsy. Results were compared to a clinical diagnosis using a composite reference method consisting of histological analysis and rapid urease testing of the biopsy. A total of 271 patients, 68.2% female and mean age of 46 years, were included. The overall prevalence of H. pylori infection was 24.1%. The sensitivity of the H. PYLORI QUIK CHEK™ and H. PYLORI CHEK™ was 92% and 91%, respectively. The specificity of H. PYLORI QUIK CHEK™ and H. PYLORI CHEK™ was 91% and 100%, respectively. No significant cross-reactivity against other gut pathogens was observed. The H. PYLORI QUIK CHEK™ and H. PYLORI CHEK™ assays demonstrate excellent clinical performance compared the composite reference method. [ABSTRACT FROM AUTHOR]

Rapid and accurate identification of respiratory tract infection pathogens is of utmost importance for clinical diagnosis and treatment, as well as prevention of pathogen transmission. To meet this demand, a microfluidic chip-based PCR-array system, Onestart, was developed. The Onestart system uses a microfluidic chip packaged with all the reagents required, and the waste liquid is also collected and stored on the chip. This ready-to-use system can complete the detection of 21 pathogens in a fully integrated manner, with sample lysis, nucleic acid extraction/purification, and real-time PCR sequentially implemented on the same chip. The entire analysis process is completed within 1.5 h, and the system automatically generates a test report. The lower limit-of-detection (LOD) of the Onestart assay was determined to be 1.0 × 103 copies·mL−1. The inter-batch variation of cycle threshold (Ct) values ranged from 0.08% to 0.69%, and the intra-batch variation ranged from 0.9% to 2.66%. Analytical results of the reference sample mix showed a 100% specificity of the Onestart assay. The analysis of batched clinical samples showed consistency of the Onestart assay with real-time PCR. With its ability to provide rapid, sensitive, and specific detection of respiratory tract infection pathogens, application of the Onestart system will facilitate timely clinical management of respiratory tract infections and effective prevention of pathogen transmission. [ABSTRACT FROM AUTHOR]

Novel rapid diagnostic tests (RDTs) offer huge potential to optimise clinical care and improve patient outcomes. In this study, we aim to assess the current patterns of use around the world, identify issues for successful implementation and suggest best practice advice on how to introduce new tests. An electronic survey was devised by the International Society of Antimicrobial Chemotherapy (ISAC) Rapid Diagnostics and Biomarkers working group focussing on the availability, structure and impact of RDTs around the world. It was circulated to ISAC members in December 2019. Results were collated according to the UN human development index (HDI). 81 responses were gathered from 31 different countries. 84% of institutions reported the availability of any test 24/7. In more developed countries, this was more for respiratory viruses, whereas in high and medium/low developed countries, it was for HIV and viral hepatitis. Only 37% of those carrying out rapid tests measured the impact. There is no 'one-size fits all' solution to RDTs: the requirements must be tailored to the healthcare setting in which they are deployed and there are many factors that should be considered prior to this. [ABSTRACT FROM AUTHOR]

We aimed to report a 32-month laboratory experience with the eazyplex® CSF direct panel assay for the rapid diagnosis of meningitis due to six most common bacterial species (Escherichia coli, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, and Streptococcus pneumoniae). We included all cerebrospinal fluid (CSF) samples from patients admitted with a clinical suspicion of meningitis/encephalitis between May 2016 and December 2018 at our hospital. In addition to the eazyplex® assay, both Gram stain microscopy and culture were performed, and results were confirmed with 16S rRNA PCR/sequencing. Patients' demographics and relevant clinical information were collected. Of 135 studied patients, 44 (32.6%) had a microbiologically documented diagnosis of meningitis. Overall, we identified 21 S. pneumoniae, 10 N. meningitidis, 6 L. monocytogenes, 3 E. coli, 2 Streptococcus pyogenes, 1 S. agalactiae, and 1 Citrobacter koseri as aetiological agents. The eazyplex® assay allowed identification in 40 (90.9%) cases, with four not identified cases due to microorganisms not included in the panel at the time of testing. Thirty-two (72.7%) cases had positive culture results, whereas 28 (63.6%) cases had positive Gram stain results. Notably, combining Gram stain and eazyplex® assay allowed identification in 100% of cases. After notification of rapid results, physicians modified the empiric antibiotic therapy, which became appropriate in three patients (all with L. monocytogenes meningitis). The eazyplex® CSF panel assay worked better than culture in detecting the most common agents of bacterial meningitis and accelerated the diagnosis leading to timely initiation or continuation of appropriate antibiotic therapy. [ABSTRACT FROM AUTHOR]

Gold nanoparticles of different sizes have been synthesized and surface-functionalized with selected RNA probes in order to develop a rapid, low-cost and sensitive method for detection of microRNA146a (miR146a). The strategy is based on the change of colour that can be observed visually after aggregation of the RNA modified-gold nanoparticles (AuNPs) in presence of miR146a. Experimental conditions have been carefully selected in order to obtain a good sensitivity that allows to perform visual detection of microRNA at the nM level, achieving a detection limit of 5 nM. Good repeatability and selectivity versus other sequences that only differ from miR146a in 3 bases was achieved. miR146a has been described as one of the main microRNA involved in the immune response of bovine mastitis, being expressed in tissue, blood and milk samples. The method was successfully applied to the detection of miR146a in raw cow milk samples. The present scheme constitutes a rapid and low-cost alternative to perform highly sensitive detection of microRNA without the need of instrumentation and amplification steps for the early detection of bovine mastitis in the agrofood industry. [ABSTRACT FROM AUTHOR]

Bloodstream infection (BSI) is defined by positive blood cultures in a patient with systemic signs of infection and may be either secondary to a documented source or primary-that is, without identified origin. Community-acquired BSIs in immunocompetent adults usually involve drug-susceptible bacteria, while healthcare-associated BSIs are frequently due to multidrug-resistant (MDR) strains. Early adequate antimicrobial therapy is a key to improve patient outcomes, especially in those with criteria for sepsis or septic shock, and should be based on guidelines and direct examination of available samples. Local epidemiology, suspected source, immune status, previous antimicrobial exposure, and documented colonization with MDR bacteria must be considered for the choice of first-line antimicrobials in healthcare-associated and hospital-acquired BSIs. Early genotypic or phenotypic tests are now available for bacterial identification and early detection of resistance mechanisms and may help, though their clinical impact warrants further investigations. Initial antimicrobial dosing should take into account the pharmacokinetic alterations commonly observed in ICU patients, with a loading dose in case of sepsis or septic shock. Initial antimicrobial combination attempting to increase the antimicrobial spectrum should be discussed when MDR bacteria are suspected and/or in the most severely ill patients. Source identification and control should be performed as soon as the hemodynamic status is stabilized. De-escalation from a broad-spectrum to a narrow-spectrum antimicrobial may reduce antibiotic selection pressure without negative impact on mortality. The duration of therapy is usually 5-8 days though longer durations may be discussed depending on the underlying illness and the source of infection. This narrative review covers the epidemiology, diagnostic workflow and therapeutic aspects of BSI in ICU patients and proposed up-to-date expert statements. [ABSTRACT FROM AUTHOR]

Objective: To determine the diagnostic accuracy of pneumococcal antigen detection in diagnosis of pneumococcal meningitis in children. Methods: Purulent meningitis was diagnosed according to European Society for Clinical Microbiology and Infectious Diseases (ESCMID) guideline between July 2014 and June 2016. Along with a cerebrospinal fluid (CSF) culture, pneumococcal antigen detection in cerebrospinal fluid (CSF) was performed, and further identification of pathogens was done with 16S rDNA-PCR and high-throughput sequencing. Results: CSF samples collected from 184 children (median age of 1.92 mo). CSF culture was used as the gold standard. 46 (25%) had positive results for culture and 10 (5.4%) were pneumococci; 34 (18.5%) were pneumococcal antigen positive. The sensitivity and specificity of pneumococcal antigen detection were 100% (95% CI: 89.4%–100%) and 86.2% (95% CI: 96.4%–99.9%), respectively. 92.3% (12/13) were confirmed by nucleic acid detection to be pneumococci. Conclusions: Pneumococcal antigen detection in CSF has adequate sensitivity and specificity in diagnosing pneumococcal meningitis. [ABSTRACT FROM AUTHOR]

In this study, we develop a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid and easy detection of goose astroviruses (GAstVs) in clinical samples. The specific LAMP primer sets were designed targeting the ORF2 gene of GAstV. The conditions of LAMP amplification were optimized in terms of reaction time and temperature. The optimal conditions are 60 min in a 60 °C water bath. No cross-reactivity was noted with fowl adenovirus serotype 4 (FAdV-4), duck tembusu virus (DTMUV), goose parvovirus (GPV), avian infectious bronchitis virus (IBV), or chicken anemia virus (CAV). The proposed RT-LAMP method was compared with conventional RT polymerase chain reaction (RT-PCR) and with nested RT-PCR. The results showed that the sensitivity of the proposed method was comparable to that of nested RT-PCR and tenfold higher than that of the conventional RT-PCR. Clinical samples (N = 129) of the liver and kidney from sick geese collected from six commercial goose farms were tested. The positive rate was 39.5% (51/129), 38.8% (50/129), and 34.9% (45/129) using RT-LAMP, nested RT-PCR and conventional RT-PCR, respectively. The developed RT-LAMP diagnostic method is not only simple, rapid, and highly specific, but also portable for use on the field. It may be used in epidemiological investigation to detect GAstVs. [ABSTRACT FROM AUTHOR]

Background: Steroid-resistant nephrotic syndrome (SRNS) has a heterogeneous spectrum of monogenic causes that substantially differ among populations. The aim of this study was to analyse the genetic aetiology of SRNS in Czech and Slovak paediatric patients.Methods: We analysed clinical data from 74 patients (38 boys) with congenital (15%), infant (14%), and childhood-onset (71%) SRNS collected from the Czech Republic and Slovakia from 2000 to 2017 (inclusive). The DNA samples were first analysed by Sanger sequencing (genes NPHS2, NPHS1, and WT1) and then by next generation sequencing (NGS) using a targeted panel of 48 genes previously associated with SRNS. Family segregation of the causative variants was confirmed by Sanger sequencing when possible.Results: Genetic diagnosis was established in 28/74 patients (38%) based on findings of pathogenic or likely pathogenic causative variants in genotypes conforming to the expected mode of inheritance. Sanger sequencing diagnosed 26% of patients, whereas second-tier testing by a targeted NGS panel diagnosed a further 12%. Frequent causative genes were NPHS2 (15%), WT1 (9.5%), and surprisingly NUP93 with four (5.4%) unrelated cases. Additional causative genes included COQ2 (two patients), NPHS1, INF2, DGKE, and LMX1B (one patient each).Conclusions: Compared with outright use of NGS, our tiered genetic testing strategy was considerably more rapid and marginally less expensive. Apart from a high aetiological fraction of NPHS2 and WT1 genes, our study has identified an unexpectedly high frequency of a limited set of presumably ancestral causative mutations in NUP93. The results may aid in tailoring testing strategies in Central European populations. [ABSTRACT FROM AUTHOR]

Objectives: To systematically analyze the diagnostic accuracy of Raman spectroscopy system (RAS) in the rapid diagnosis of gastric cancer with histopathology as the reference standard. Methods: We searched a wide range of electronic databases for all published researches that assessed the diagnostic accuracy of RAS to detect gastric carcinoma. Full papers were obtained for potentially eligible studies and evaluated according to predefined criteria. The Quality Assessment of Diagnostic Accuracy Studies checklist was used to assess the quality of included studies. From each study, we extracted information on diagnostic performance of RAS. After exploring heterogeneity, we adopted a random effects model to pool related effect sizes. Results: The initial literature search identified 257 reference articles in which 15 relevant articles with 15 data sets were selected and reviewed. The pooled sensitivity and specificity of RAS in diagnosing gastric cancer were 0.89 (95 % CI 0.84-0.92) and 0.92 (95 % CI 0.88-0.95), respectively. The positive likelihood ratio, the negative likelihood ratio, and the area under the curve were 10 (95 % CI 6.5-15.3), 0.13 (95 % CI 0.08-0.22), and 0.96 (95 % CI 0.94-0.97), respectively. All the pooled estimates, calculated by random and fixed effect models, were similar. There was no evidence of considerable publication bias. Conclusions: RAS is an objective and sensitive optical diagnostic technology for detecting gastric cancer and has advantages of being noninvasive to the body, real-time diagnosis, and ease of use. Consequently, it does deserve to be recommended. [ABSTRACT FROM AUTHOR]

Rapid diagnostic assays for Fusarium root rot in soybean, caused by Fusarium equiseti or Fusarium graminearum were developed using the target gene CYP51C. Both assays amplified the target gene in 60 min at 62 °C, after which they were assessed for specificity and sensitivity. Specificity was evaluated against other Fusarium spp., other fungal species, and oomycetes. A positive yellow-green color (by the naked eye) or intense green fluorescence (under ultraviolet light) was observed only in the presence of F. equiseti or F. graminearum after adding SYBR Green I, whereas other strains showed either no color change or weak fluorescence. The detection limit of the CYP51C-Fe-LAMP assay was 10 pg. μL genomic DNA per reaction, and as few as four conidia per gram of soil could be detected; and the detection limit of the CYP51C-Fg-LAMP assay was 100 pg. μL genomic DNA per reaction, and 40 conidia per gram of soil could be identified. The assays also detected F. equiseti or F. graminearum from inoculated soybean tissues and diseased plants in the field. These results suggest that CYP51C-Fe-LAMP assay and CYP51C-Fg-LAMP assay are effective in rapidly diagnosing soybean root rot caused by F. equiseti and F. graminearum. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the role and reliability of rapid bedside diagnostic test in early diagnosis and treatment of bacterial meningitis in children using reagent strips. Methods: This prospective, single blinded study was conducted in the Department of Pediatrics of VMMC & Safdarjung Hospital, New Delhi in collaboration with the Department of Microbiology of VMMC & Safdarjung Hospital, New Delhi, over a period of 15 mo (August 2009 to Nov 2010). Seventy-five children aged 3 mo to 12 y admitted in the pediatric ward with suspected diagnosis of acute meningitis were included. All enroled patients underwent lumbar puncture. CSF samples were taken and divided in 2 parts for laboratory evaluation and rapid strip analysis. The sensitivity, specificity, positive predictive value and the negative predictive values of the reagent strips for the diagnosis of bacterial meningitis were calculated. Accuracy of the reagent strips was established using kappa statistics. Latex agglutination for antigen detection and microbiological culture were also done. Results: Highly significant association was observed between CSF examination in routine laboratory method and dipstick method. The number of laboratory values that correlated were- for cells 71(94.63 %), for protein 68 (90.67 %), for glucose 68(90.67 %) out of total 75 cases. The sensitivity and specificity of reagent strip in diagnosing acute bacterial meningitis were 96.7 % and 97.8 % respectively. The positive predictive and negative predictive values of reagent strip in diagnosing acute bacterial meningitis were 96.7 % and 97.8 % respectively. Staphylococcus aureus was found to be the most common organism isolated (50 %). Conclusions: Thus reagent strip analysis is a very rapid, reliable and effective method for diagnosis of acute bacterial meningitis in children. Staphylococcus aureus was the most common organism isolated. [ABSTRACT FROM AUTHOR]

Purpose: When lung cancer (LC) is suspected in chest radiography, an adequate interpretation and management by experts would improve the selection, the access to rapid diagnostic units, the diagnostic effectiveness and prevent the loss of patients with suspected LC. To ensure this, we planned a system for alerting pulmonologists by radiologists to radiological suspicion of LC. Methods: This system consists of an alert from radiologists to pulmonologists through a specific email. The pulmonologists alerted has to contact the study doctor petitioner who must refer the patient for study to the Lung Cancer Rapid Diagnostic Unit (LCRDU). We have prospectively analyzed all patients studied in a 2-year period including clinical variables, time invested in the different diagnostic steps and the degree of collaboration and satisfaction among the involved professionals. Results: Of 118 alerts received, 84 (71 %) were studied in our LCRDU. The median of days until petitioner contact, patient consulted at LCRDU and to obtain a diagnosis was 1 (IQR 0-1.5), 2 (IQR 1-5) and 13 (IQR 7.5-30), respectively. In 45 cases (53 %), the suspicion of malignancy was confirmed (LC 84.4 % and metastasic 10.1 %). After staging was complete, 33 % of non-small cell lung cancer was potentially resectable (clinical TNM stage I-II). The level of satisfaction was high so that only one of the petitioner's studies chose other diagnostic pathways. Conclusion: This strategy for radiological suspicion of LC ensures the communication between general practitioners, radiologists and pulmonologist improving the LC diagnostic effectiveness. This system can be easily implemented in health care systems. [ABSTRACT FROM AUTHOR]

It is not known that saliva is useful to diagnose Mycoplasma pneumoniae ( M. pneumoniae) infection by PCR. We evaluated prospectively whether crude saliva samples without the DNA extraction process were useful for the detection of M. pneumoniae DNA in a locked nucleic acid (LNA) probe-based real-time PCR assay. Fifty-one clinical specimens (29 sputum, 22 saliva) that were positive by conventional M. pneumoniae-specific PCR were evaluated in this study. We designed an LNA probe-based real-time PCR that could discriminate the mutant strain (A2063G mutation) from the wild-type strain. All the 51 samples treated with DNA extraction were positive using the LNA probe-based real-time PCR. The results of the real-time PCR with DNA extraction were consistent with the sequence analysis. Of the 51 samples without DNA extraction, on the other hand, 41 (80.4 %) were positive by real-time PCR. Of 29 sputum samples without DNA extraction, 23 (79.3 %) were positive by real-time PCR; of the 22 saliva samples without DNA extraction, 18 (81.8 %) were positive by real-time PCR. There was a statistically significant difference in the amplified DNA levels with extraction between the direct real-time PCR-positive samples (mean ± SD, 7.5 ± 1.6 log copies/ml) and PCR-negative samples (4.2 ± 0.8 log copies/ml) ( P < 0.001). Saliva was useful for a template for PCR as well as sputum. In addition, crude samples were useful for real-time PCR when the samples had medium or high DNA levels. However, samples with low DNA levels sometimes showed false-negative results in direct real-time PCR. [ABSTRACT FROM AUTHOR]

We evaluated Clearline Influenza A/B/(H1N1)2009, a new multi-line immunochromatographic assay for rapid detection of antigens of influenza A (Flu A), B (Flu B), and A(H1N1)2009 viruses. Clearline detected Flu A, Flu B, and A(H1N1)2009 viruses with a detection limit of 4.6 × 10 to 7.5 × 10 pfu/assay. The sensitivity and specificity of detection of influenza virus by Clearline, using RT-PCR as reference standard, were determined for A(H1N1)2009, Flu A, and Flu B, in nasopharyngeal aspirate, nasopharyngeal swab, and self-blown nasal discharge specimens. Sensitivity for nasopharyngeal aspirate specimens was: A(H1N1)2009 = 97.3 %, Flu A = 94.5 %, and Flu B = 96.8 %, and specificity was Flu A = 99.1 % and Flu B = 100 %. Sensitivity for nasopharyngeal swab specimens was: A(H1N1)2009 = 91.9 %, Flu A = 92.8 %, and Flu B = 100 %, and specificity was Flu A = 98.2 % and Flu B = 100 %. Sensitivity for self-blown nasal discharge specimens was: A(H1N1)2009 = 75.7 %, Flu A = 86.5 %, and Flu B = 76.2 %, and specificity was Flu A = 98.4 % and Flu B = 100 %. Sensitivity and specificity of Clearline were sufficient for nasopharyngeal aspirate and swab specimens. For self-blown nasal discharge specimens, sensitivity was lower than for nasopharyngeal aspirates and nasopharyngeal swabs. The sensitivity of Clearline for A(H1N1)2009 was good even 6 h after the onset of symptoms. These findings suggest that Clearline may be useful for early clinical diagnosis of influenza. [ABSTRACT FROM AUTHOR]

Rapid and more sensitive methods for the detection and quantification of viable Legionella cells have been developed. In this paper, a comparative analysis of environmental water samples using the ScanVIT-Legionella™ method and the traditional 'gold standard' method of culturing is realised indicating the usefulness of the ScanVIT method. The ScanVIT-Legionella™ method was performed on environmental water samples from different locations of Huesca region (Spain). Legionella micro-colonies should appear green colour and Legionella pneumophila micro-colonies appear red. Twenty-one environmental water samples analysed by standard culture plus five control samples (Two sterile water samples with Legionella as positive controls and three sterile water samples as negative controls). All of them were used to apply ScanVIT-Legionella™ method. From of 21 environmental samples eleven were positive, six negative with both methods and four samples were negative for culture method and positive for ScanVIT-Legionella™ method. The positive control samples were positive and the negative were negative for both methods. A comparative analysis of the results obtained with two methods showed a strong positive determination coefficient (R = 0.99753). The results demonstrate the usefulness of the ScanVIT-Legionella™ method as a rapid diagnostic tool in order to provide a diagnosis as quick as possible. ScanVIT-Legionella™ method offers a series of advantages such as quickly diagnosis, higher sensitivity and the possibility to identify Legionella spp. and L. pneumophila simultaneously. [ABSTRACT FROM AUTHOR]

A central nervous system (CNS) infection, such as meningitis, is a serious and life-threatening condition. Bacterial meningitis can be severe and may result in brain damage, disability or even death. Rapid diagnosis of CNS infections and identification of the pathogenic microorganisms are needed to improve the patient outcome. Bacterial culture of a patient's cerebrospinal fluid (CSF) is currently considered the 'gold standard' for diagnosing bacterial meningitis. From the CSF cultures researchers can assess the in vitro susceptibility of the causative microorganism to determine the best antibiotic treatment. However, many of the culture assays, such as microscopy and the latex agglutination test are not sensitive. To enhance pathogen detection in CSF samples we developed a multi-target real-time PCR assay that can rapidly identify six different microorganisms: Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus agalactiae, Listeria monocytogenes and Cryptococcus neoformans. In this study we applied this PCR analysis to 296 CSF samples from patients who were suspected of having meningitis. Of the 296 samples that were examined, 59 samples were positive according to the CSF culture and/or molecular assays. Forty-six CSF samples were positive for both the CSF culture and our real-time PCR assay, while 13 samples were positive for the real-time PCR but negative for the traditional assays. This discrepancy may have been caused by the fact that these samples were collected from 23 patients who were treated with antimicrobials before CSF sampling. [ABSTRACT FROM AUTHOR]

Rapid diagnosis of Mycoplasma pneumoniae pneumonia is required for treatment with effective antimicrobial agents without delay; however, this capacity has not yet been established in clinical practice. Recently, a novel nucleic acid amplification method termed loop-mediated isothermal amplification (LAMP) has been used to rapidly diagnose various infectious diseases. In this study, we prospectively evaluated the efficacy of the LAMP assay to rapidly diagnose M. pneumoniae pneumonia in clinical practice. Three hundred sixty-eight children (median age, 3.8 years; range, 0.1-14.3 years) admitted to our hospital between April 2009 and March 2010 for community-acquired pneumonia were enrolled in this study. We obtained throat swabs on admission to detect M. pneumoniae DNA and paired serum samples on admission and at discharge to assay M. pneumoniae antibody titers . M. pneumoniae pneumonia was diagnosed by either a positive LAMP assay or a fourfold or greater increase in antibody titer. Overall, 46 children (12.5% of the patients with pneumonia) were diagnosed with M. pneumoniae pneumonia; of these, 27 (58.7%) were aged less than 6 years. Of the aforementioned 46 children, 38 (82.6%) and 37 (80.4%) were identified by LAMP and serology, respectively. When the results of serology were taken as the standard, the sensitivity and specificity and positive and negative predictive values of the LAMP assay were 78.4%, 97.3%, 76.3%, and 97.6%, respectively. We concluded the LAMP assay may be useful for rapid diagnosis of M. pneumoniae pneumonia. [ABSTRACT FROM AUTHOR]

To clarify the clinical symptoms of the influenza A virus during the 2009 pandemic influenza outbreak, we describe the clinical features of outpatients diagnosed with type A influenza by use of the rapid influenza diagnostic test (RIDT) from September to December 2009. Questionnaires were used to collect prospective data on 1,122 cases with influenza-like illness at our medical institutions. The independent predictors of influenza A virus were identified on the basis of demographic features and the clinical symptoms of the patients who tested positive for influenza A virus in the RIDT test. Of the 1,122 cases tested, 389 (34.7%) were positive for the influenza A virus. The median age of the influenza-positive patients was 14, and 58.9% of the patients were male. The symptoms fever, cough, rhinorrhea, and headache were statistically dominant. A history of recent contact with persons suffering from influenza or influenza-like illness at home, school, or in the workplace was significantly more common in the positive group than in the negative group. Pneumonia was observed in 2 (0.5%) of the positive patients, but the symptoms were only severe enough to require hospitalization in 1 of the 2. No deaths were observed among the 389 RIDT-positive patients. Although the spread of influenza A virus was both rapid and extensive, mainly among children under the age of 18, it seemed to be mild. Appropriate interpretation of the RIDT on the basis of recent clinical information, and early treatment with antiviral drugs might help to prevent severe illness from influenza pandemics in the future. [ABSTRACT FROM AUTHOR]

Background: Spontaneous bacterial peritonitis (SBP) is a common serious complication of cirrhosis, and carries a high morbidity and mortality. Rapid diagnosis and prompt treatment of this condition may improve survival of such patients. Objective: To validate the diagnostic efficacy of a leukocyte esterase reagent (LER) strip test for rapid, bedside diagnosis of SBP. Methods: We prospectively studied 175 patients with liver cirrhosis and ascites [mean age 48 (SD 16.4) years; 146 men] between August 2007 and December 2008. Alcohol was the most common (124 of 175; 70.8%) cause of liver cirrhosis. All patients underwent abdominal paracentesis, and the ascitic fluid was processed for cell count, LER strip (Magistik 10) test and culture. Two different cut-offs for calling the LER strip test positive were tried, namely when the color turned light blue [grade 2: >125 polymorphonuclear leucocytes (PMNL)/μL] or it turned purple (grade 3: >500 PMNL/μL). Sensitivity, specificity, positive predictive value, negative predictive value were calculated, using PMN count by microscopy exceeding 250 PMNL/μL. Results: LER strip using the more stringent purple-color cut off to diagnose SBP had a sensitivity of 92% and specificity of 100%. The corresponding figures using the light-blue color cut-off were 97% and 89%, respectively. Conclusions: LER strip testing of ascitic fluid is a rapid, cheap and sensitive bed side tool for the diagnosis of SBP. [ABSTRACT FROM AUTHOR]

The percentage of individuals receiving influenza vaccine is markedly lower in Japan than in many other economically advanced countries. To increase the rate of coverage, the current practice of giving people two inoculations instead of one needs to be changed. In addition, free vaccination services for the elderly and high-risk patients need to be offered. Amantadine is available for the treatment of influenza type A infection in Japan. Moreover, zanamivir, a neuraminidase inhibitor effective against both influenza type A and B viruses, has been approved in Japan. A rapid diagnosis kit for influenza type A virus is available in Japan. With the current threat of new pandemic influenza viruses emerging, it is necessary to actively confront influenza in Japan by increasing the vaccine coverage rate, by employing amantadine and neuraminidase inhibitors for influenza virus infection, and by providing rapid diagnosis of influenza. [ABSTRACT FROM AUTHOR]

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The authors report their experience with CT scanning in a series of 4 cases of subdural empyema, over a period of 10 months; all the patients were male, aged 16 to 25 years. A review of the literature shows that this disease, in spite of the large variety and efficacy of the available antibiotics, is marked by a significant mortality and morbidity rate if the diagnosis is delayed. Study of the cases reported here, as well as those in the literature, attests that CT scanning of the brain is nowadays the method of choice for early diagnosis of these lesions, and for timing of appropriate intervention according to variations in the development stage of the suppuration. From the study of this series of cases, it is clear that in the future, CT scanning of the brain will certainly allow the hope of a greater survival rate and an appreciable decrease in morbidity in cases of subdural empyema. [ABSTRACT FROM AUTHOR]

Molecular biology allowed considerable improvements in diagnostic procedures by production of new and more specific sonds for the detection of traces of viruses, both on the nucleic acid and protein levels, and by determining the immune response of the host to specific antigens. Improvements in sensitivity and improved correlation to the stage of viral disease are already evident from several applications and strongly suggest a broad application of these approaches. [ABSTRACT FROM AUTHOR]

A 73-year-old woman, who suffered from erythema with bullae and pustules on her abdomen and anterior right thigh, visited our hospital without an awareness of the causative agents. The lesions appeared like first and second degree burns. The small amount of detached roof of bulla was sampled without skin biopsy before the ointment treatment. The sample was sonicated in an ultrasonic bath for 1 min in n-pentane, and then 1 μl of the extract was analyzed by gas chromatography-mass spectrometry (GC–MS). The causative agent was determined to be kerosene. An examination of blood samples collected at the first visit failed to detect kerosene, though traces of trimethylbenzene were detected. A GC–MS analysis of the small sample of lesional epidermis was very useful to identify kerosene as a causative agent. [ABSTRACT FROM AUTHOR]

Dengue virus serotype 1 was isolated from the blood of a patient who had returned to Switzerland from Brazil with fever of unknown origin. After 2 days of culture on Aedes albopictus (C6/36) cell line, the dengue virus was identified as serotype 1 using a type-specific indirect immunofluorescence assay. The diagnosis was confirmed by seroconversion of anti-dengue virus-specific IgM and IgG antibodies and by amplification of a serotype 1-specific region of the dengue virus genome. This is the first description of dengue virus isolation from the blood of a traveler returning to Switzerland. We recommend detection of dengue viremia during the 1st week of illness, before serological. tests usually yield conclusive results, as the most efficient means of early specific diagnosis. [ABSTRACT FROM AUTHOR]

A one-step diagnostic test based on an immunochromatographic (IC) assay for adenovirus was evaluated with purified adenovirus and clinical specimens. According to five clinically common serotypes of purified adenovirus tested, the IC test was more sensitive than two commercially available enzyme immunoassay (EIA) test kits. For tonsilopharyngeal specimens from 63 febrile pediatric patients with suspected adenoviral upper respiratory tract infection, the sensitivity and specificity of the IC test against viral isolation by cell culture was 88.5% (23/26) and 100% (37/37), respectively. The IC test, which is quicker and easier to perform than EIA test kits, is very useful in the rapid diagnosis of adenoviral upper respiratory tract infection of pediatric patients. [ABSTRACT FROM AUTHOR]

A 21-year-old women presented with an acute febrile illness after a two-week holiday in Jamaica. Her symptoms started two days after return, with sudden onset of continuous higher fever (> 39 °C), dizziness and nausea. Three days later she developed a generalized macular rash, which led to the tentative diagnosis “acute dengue fever”. Laboratory confirmation was achieved by demonstrating anti-dengue IgM and IgG antibodies in paired sera; in addition, flavivirus particles were directly visualized by electron microscopy. [ABSTRACT FROM AUTHOR]