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• The high mortality rate (36 %–63 %) of candidemia underlines the urgency of diagnosis. • T2Candida, which allows rapid diagnosis, showed 95.3 % agreement with blood culture. • False negatives on T2Candida should be considered in cases of C. glabrata infection. We evaluated the clinical performance of the T2Candida assay. The overall agreement of the T2Candida assay results with the blood culture results was 95.3 % (121/127). The T2Candida assay detected three Candida albicans/tropicalis -positive specimens and one Candida krusei/glabrata- positive specimen; however, it did not detect two Candida glabrata specimens. [ABSTRACT FROM AUTHOR]

Currently, more and more studies suggested that reductionism was lack of holistic and integrative view of biological processes, leading to limited understanding of complex systems like microbiota and the associated diseases. In fact, microbes are rarely present in individuals but normally live in complex multispecies communities. With the recent development of a variety of metaomics techniques, microbes could be dissected dynamically in both temporal and spatial scales. Therefore, in-depth understanding of human microbiome from different aspects such as genomes, transcriptomes, proteomes, and metabolomes could provide novel insights into their functional roles, which also holds the potential in making them diagnostic biomarkers in many human diseases, though there is still a huge gap to fill for the purpose. In this mini-review, we went through the frontlines of the metaomics techniques and explored their potential applications in clinical diagnoses of human diseases, e.g., infectious diseases, through which we concluded that novel diagnostic methods based on human microbiomes shall be achieved in the near future, while the limitations of these techniques such as standard procedures and computational challenges for rapid and accurate analysis of metaomics data in clinical settings were also examined. [ABSTRACT FROM AUTHOR]

Porcine epidemic diarrhoea virus (PEDV) is a member of the genus Alphacoronavirus in the family Coronaviridae. It causes acute watery diarrhoea and vomiting in piglets with high a mortality rate. Currently, the GII genotype, PEDV, possesses a high separation rate in wild strains and is usually reported in immunity failure cases, which indicates a need for a portable and sensitive detection method. Here, reverse transcription–recombinase aided amplification (RT-RAA) was combined with the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas12a system to establish a multiplexable, rapid and portable detection platform for PEDV. The CRISPR RNA (crRNA) against Spike (S) gene of GII PEDV specifically were added into the protocol. This system is suitable for different experimental conditions, including ultra-sensitive fluorescence, visual, UV light, or flow strip detection. Moreover, it exhibits high sensitivity and specificity and can detect at least 100 copies of the target gene in each reaction. The CRISPR/Cas12a detection platform requires less time and represents a rapid, reliable and practical tool for the rapid diagnosis of GII genotype PEDV. [ABSTRACT FROM AUTHOR]

Frozen section examination could provide pathological diagnosis for surgery of thyroid nodules, which is time‐consuming, skill‐ and experience‐dependent. This study developed a rapid classification method for thyroid nodules and machine learning. Total 69 tissues were collected including 43 nodules and 26 nodule‐adjacent tissues. Intraoperative frozen section was first performed to give accurate diagnosis, and the rest frozen specimen were pretreated for probe electrospray ionization mass measurement. By multivariate analysis of mass scan data, a series compounds were found downregulated in the extraction solution of papillary thyroid carcinoma (PTC), but some were found upregulated by mass spectrometry imaging. m/z 758.5713 ([PC[34:2] + H]+), m/z 772.5845 ([PC[32:0] + K]+), and m/z 786.6037 ([PC[36:2] + H]+) were firstly identified as potential biomarkers for nodular goiter (NG). Machine learning was employed by means of support vector machine (SVM) and random forest (RF) algorithms. For classification of PTC from NG, SVM and RF algorithms exhibited the same performance and the concordance was 94.2% and 94.4% between prediction and pathological diagnosis with positive and negative mass dataset, respectively. For the classification of PTC from PTC adjacent tissues, SVM was better than RF and the concordance was 93.8% and 83.3% with positive and negative mass dataset, respectively. With the identified compounds as training features, the sensitivity and specificity are 87.5% and 88.9% for the test set. The developed method could also correctly predict the malignancy of one medullary thyroid carcinoma and one adenomatous goiter (benign). The diagnosis time is about 10 min for one specimen, and it is very promising for the intraoperative diagnosis of papillary thyroid carcinoma. [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]

Background: Legionella-related community acquired pneumonia (CAP) is a disease with an increasing incidence and a high mortality rate, especially if empirical antibiotic therapy is inadequate. Antibiotic treatment highly relies on clinical symptoms, although proven non-specific, because currently available diagnostic techniques provide insufficient accuracy for detecting Legionella CAP on admission. This study validates a diagnostic scoring system for detection of Legionella-related CAP, based on six items on admission (Legionella prediction score).Methods: We included patients with Legionella-related CAP admitted to five large Dutch hospitals between 2006 and 2016. Controls were non-Legionella-related CAP patients. The following six conditions were rewarded one point if present: fever > 39.4 °C; dry cough; hyponatremia (sodium) < 133 mmol/L; lactate dehydrogenase (LDH) > 225 mmol/L; C-reactive protein (CRP) > 187 mg/L and platelet count < 171 × 109/L. The accuracy of the prediction score was assessed by calculating the area under the curve (AUC) through logistic regression analysis.Results: We included 131 cases and 160 controls. A score of 0 occurred in non-Legionella-related CAP patients only, a score of 5 and 6 in Legionella-related CAP patients only. A cut-off ≥ 4 resulted in a sensitivity of 58.8% and a specificity of 93.1%. The AUC was 0.89 (95% CI 0.86-0.93). The strongest predictors were elevated LDH, elevated CRP and hyponatremia.Conclusions: This multi-centre study validates the Legionella prediction score, an easily applicable diagnostic scoring system, in a large group of patients and finds high diagnostic accuracy. The score shows promise for future prospective validation and could contribute to targeted antibiotic treatment of suspected Legionella CAP. [ABSTRACT FROM AUTHOR]

Biofilm infection has been identified as a crucial factor of the pathogenesis of chronic wound, but wound biofilm diagnosis remains as an unmet clinical need. We previously proposed a modified wound blotting technique using Alcian blue staining for biofilm detection that was characterized as being non-invasive, time-saving, non-expansive, and informative for biofilm distribution. In this study, we adapted a novel Alcian blue grading method as the severity of biofilm infection for the wound blotting technique and compared its biofilm detection efficacy with MolecuLight i:X- a point-of-care florescence imaging device to detect bacteria and biofilm in wounds. Moreover, their predictive value of complete wound healing at 90 days was analyzed. When validated with wound culture results in the 53 enrolled subjects with chronic wounds, the modified wound blotting method showed a strong association with wound culture, while MolecuLight i:X only exhibited a weak association. In predicting 90-day wound outcomes, the modified wound blotting method showed a strong association (Kendall's tau value = 0.563, p < 0.001), and the wound culture showed a moderate association (Spearman's rho = 0.535, p < 0.001), but MolecuLight i:X exhibited no significant association (p = 0.184). In this study, modified wound blotting with the Alcian blue grading method showed superior value to MolecuLight i:X both in biofilm detection and predictive validity in 90-day wound-healing outcomes. [ABSTRACT FROM AUTHOR]

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Systemic fungal infection can disseminate to the skin and require prompt treatment, making early diagnosis very important. This study describes the use of a simple, quick touch biopsy method for the diagnosis of invasive mycoses in patients with AIDS with cutaneous manifestations. We identified fungal infections in 24 of the 29 investigated patients. Histoplasma capsulatum, Cryptococcus neoformans, Talaromyces artroroseus, Aspergillus flavus, Candida tropicalis, and Malassezia sp. were visualized directly in samples obtained from cutaneous lesions and confirmed by culture and molecular examination. The results suggested that touch biopsy is a simple, rapid method for the diagnosis of systemic mycoses with skin dissemination. It can be performed using simple tools and provides quick results, allowing for early intervention with appropriate antifungal therapy.

Abstract Background Bloodstream infection (BSI) is a common urgent condition at the emergency department (ED). However, current guidelines for diagnosis do not specify the juncture at which blood cultures should be taken. The decision whether or not to obtain hemoculture is based solely upon clinical judgment and potential outcomes of inappropriately ordered cultures. This study aimed to find clinical factors present on ED arrival that are predictive of bloodstream infection. Methods This study was conducted retrospectively at the ED of a single tertiary care hospital in Thailand. We included adult patients with suspected infection based on blood culture who were treated with intravenous antibiotics during their ED visit. Independent positive predictors for positive blood culture were calculated by logistic regression analysis. Results A total of 169,578 patients visited the ED during the study period, 12,556 (7.40%) of whom were suspected of infection. Of those, 8177 met the study criteria and were categorized according to blood culture results (741 positive; 9.06%). Six clinical factors, including age over 55 years, moderate to severe CKD, solid organ tumor, liver disease, history of chills, and body temperature of over 38.3 °C, were associated with positive blood culture. Conclusions Clinical factors at ED arrival can be used as predictors of bloodstream infection.

Abstract Background Probe electrospray ionization-mass spectrometry (PESI-MS) can rapidly visualize mass spectra of small, surgically obtained tissue samples, and is a promising novel diagnostic tool when combined with machine learning which discriminates malignant spectrum patterns from others. The present study was performed to evaluate the utility of this device for rapid diagnosis of colorectal liver metastasis (CRLM). Methods A prospectively planned study using retrospectively obtained tissues was performed. In total, 103 CRLM samples and 80 non-cancer liver tissues cut from surgically extracted specimens were analyzed using PESI-MS. Mass spectra obtained by PESI-MS were classified into cancer or non-cancer groups by using logistic regression, a kind of machine learning. Next, to identify the exact molecules responsible for the difference between CRLM and non-cancerous tissues, we performed liquid chromatography-electrospray ionization-MS (LC-ESI-MS), which visualizes sample molecular composition in more detail. Results This diagnostic system distinguished CRLM from non-cancer liver parenchyma with an accuracy rate of 99.5%. The area under the receiver operating characteristic curve reached 0.9999. LC-ESI-MS analysis showed higher ion intensities of phosphatidylcholine and phosphatidylethanolamine in CRLM than in non-cancer liver parenchyma (P

Objective: The aim of this study was to assess the clinical utility of metagenomic next-generation sequencing (mNGS) on smear-negative extrapulmonary specimens collected in China. Methods: Specimens were tested by mNGS and other routine tests for tuberculosis (TB). The diagnostic accuracy of mNGS was calculated and compared with the final clinical diagnosis. Results: The sensitivity of mNGS was found to be significantly higher than the sensitivities of the other routine TB tests. Receiver operating characteristic curve analysis showed that mNGS achieved the highest area under the curve (AUC) value of 0.79. The mNGS positive rate was highest for tuberculous meningitis. All non-tuberculous extrapulmonary pathogens were directly and simultaneously detected. Conclusions: mNGS appeared to be superior to all previous etiological tests for TB on smear-negative extrapulmonary specimens and could identify all possible pathogens at once within 48 h.

Avian leukosis caused by avian leukosis virus (ALV), belonging to the genus Alpharetrovirus of the family Retroviridae, is associated with benign and malignant tumors in hemopoietic cells in poultry. Although several methods have been developed for ALV detection, most of them are not suitable for rapid on-site testing due to instrument limitations, professional operators, or the low sensitivity of the method. Herein, we described the real-time recombinase polymerase amplification (RPA) assay for rapid detection of ALV subgroup J (ALV-J). The major viral structural glycoprotein gp85, highly specific for the subgroup, was used as the molecular target for the real-time RPA assay. The results were obtained at 38°C within 20 min, with the detection sensitivity of 10 copies/μl of standard plasmid pMD18-T-gp85 as the template per reaction. Real-time RPA was capable of ALV-J-specific detection without cross-reaction with other non-targeted avian pathogens. Of the 62 clinical samples tested, the ALV-positive rates of real-time RPA, PCR, and real-time PCR were 66.13% (41/62), 59.68% (37/62), and 67.74% (42/62), respectively. The diagnostic agreement between real-time RPA and real-time PCR was 98.39% (61/62), and the kappa value was 0.9636. The developed real-time ALV-J assay seems promising for rapid and sensitive detection of ALV-J in diagnostic laboratories. It is suitable for on-site detection, especially in a poor resource environment, thus facilitating the prevention and control of ALV-J.

ABSTRACT A rapid and sensitive diagnosis is crucial for the management of tuberculosis (TB). A simple and label-free approach via homobifunctional imidoesters with a microfluidic platform (SLIM) assay showed a higher sensitivity than the Xpert MTB/RIF assay in the diagnosis of pulmonary TB (PTB). Here, we evaluated the efficacy of the SLIM assay for oral swab samples from cases of suspected PTB. Patients with clinically suspected PTB were prospectively enrolled and oral swab samples were processed using the SLIM assay and the attending physicians were blinded to the results of the SLIM assay. TB cases were defined as those treated with anti-TB chemotherapy for at least 6 months at the discretion of the specialists based on their clinical features and conventional laboratory results, including the Xpert assay. A total of 272 patients (with TB, n = 128 [47.1%]; without TB, n = 144 [52.9%]; mean age, 59.8 years) were enrolled. Overall, the sensitivity of the oral swab-based SLIM assay (65.6%) was higher than that of the sputum-based Xpert assay (43.4%; P = 0.001). Specifically, the SLIM oral swab assay showed a notably higher sensitivity in culture-negative TB cases compared with the Xpert assay (69.0% [95% CI: 49.2 to 84.7%] versus 7.4% [95% CI: 0.9 to 24.3%]; P = 0.001). The specificity of the SLIM and the Xpert assays was 86.1% (95% CI: 79.3 to 91.3%) and 100% (95% CI: 97.2 to 100%), respectively. When only culture-confirmed cases were analyzed, the SLIM oral swab was comparable to sputum Xpert in sensitivity (64.7% versus 54.3%, P = 0.26). The oral swab-based SLIM assay showed a superior sensitivity for TB diagnosis over the sputum-based Xpert assay, especially for culture-negative cases. IMPORTANCE The development of a rapid, accessible, and highly sensitive diagnostic tool is a major challenge in the control and management of tuberculosis. Gene-based diagnostics is recommended for the rapid diagnosis of pulmonary tuberculosis (PTB), but its sensitivity, such as Xpert MTB/RIF assay (Xpert), drops in cases with a low bacterial load. It can only be applied to sputum samples, and it is quite difficult for some patients to produce an adequate amount of sputum. We evaluated the clinical validity of an oral swab-based microfluidic system, i.e., the SLIM assay. The SLIM assay showed a significantly higher sensitivity than the Xpert assay, especially in smear-negative TB cases. This non-sputum-based SLIM assay can be a useful diagnostic test by overcoming the limitations of conventional sputum-based tests in pulmonary TB.

Currently, more and more studies suggested that reductionism was lack of holistic and integrative view of biological processes, leading to limited understanding of complex systems like microbiota and the associated diseases. In fact, microbes are rarely present in individuals but normally live in complex multispecies communities. With the recent development of a variety of metaomics techniques, microbes could be dissected dynamically in both temporal and spatial scales. Therefore, in-depth understanding of human microbiome from different aspects such as genomes, transcriptomes, proteomes, and metabolomes could provide novel insights into their functional roles, which also holds the potential in making them diagnostic biomarkers in many human diseases, though there is still a huge gap to fill for the purpose. In this mini-review, we went through the frontlines of the metaomics techniques and explored their potential applications in clinical diagnoses of human diseases, e.g., infectious diseases, through which we concluded that novel diagnostic methods based on human microbiomes shall be achieved in the near future, while the limitations of these techniques such as standard procedures and computational challenges for rapid and accurate analysis of metaomics data in clinical settings were also examined.

Porcine epidemic diarrhoea virus (PEDV) is a member of the genus Alphacoronavirus in the family Coronaviridae. It causes acute watery diarrhoea and vomiting in piglets with high a mortality rate. Currently, the GII genotype, PEDV, possesses a high separation rate in wild strains and is usually reported in immunity failure cases, which indicates a need for a portable and sensitive detection method. Here, reverse transcription–recombinase aided amplification (RT-RAA) was combined with the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas12a system to establish a multiplexable, rapid and portable detection platform for PEDV. The CRISPR RNA (crRNA) against Spike (S) gene of GII PEDV specifically were added into the protocol. This system is suitable for different experimental conditions, including ultra-sensitive fluorescence, visual, UV light, or flow strip detection. Moreover, it exhibits high sensitivity and specificity and can detect at least 100 copies of the target gene in each reaction. The CRISPR/Cas12a detection platform requires less time and represents a rapid, reliable and practical tool for the rapid diagnosis of GII genotype PEDV.

Candida auris has arisen as an important multidrug-resistant fungus because of several nosocomial outbreaks and elevated rates of mortality. Accurate and rapid diagnosis of C. auris is highly desired; nevertheless, current methods often present severe limitations and produce misidentification. Herein a sensitive, selective, and time-competitive biosensor based on oligonucleotide-gated nanomaterials for effective detection of C. auris is presented. In the proposed design, a nanoporous anodic alumina scaffold is filled with the fluorescent indicator rhodamine B and the pores blocked with different oligonucleotides capable of specifically recognize C. auris genomic DNA. Gate opening modulation and cargo delivery is controlled by successful DNA recognition. C. auris is detected at a concentration as low as 6 CFU/mL allowing obtaining a diagnostic result in clinical samples in one hour with no prior DNA extraction or amplification steps.

Background: Typhoid fever is a serious public health problem. It causes severe systemic infection in lesser developed areas of the world. Although blood culture is regarded as the gold standard for diagnosis, it is a time taking procedure. An early and accurate diagnosis is necessary for an effective treatment. Aims: The present study was done to comparatively evaluate rapid card and Widal slide agglutination tests for rapid diagnosis of typhoid cases. Settings and Design: The study design was a cross-sectional descriptive study done over a period of 6 months from January to June 2018. Materials and Methods: A total of 265 patients suspected of typhoid fever who gave their consent were included in the study whose blood samples were tested by both rapid card and Widal slide agglutination tests. Statistical Analysis Used: The collected data were analyzed using SPSS Data Editor Software version 20. Percentage of variables was calculated. Results: Of 265 patients, 97 patients were positive by the Widal slide test, whereas 113 patients were positive by the rapid card test, with 96.9% sensitivity and 88.7% specificity. Of 113 positives, 83 cases were positive for immunoglobulin M (IgM) only, whereas 30 cases were positive for both IgM and IgG. Conclusion: Rapid card test is a simple and easy to perform the diagnostic test for rapid detection of typhoid cases with an additional advantage of separate determination of IgM and IgG antibody, thereby aiding in identification of current infection and previous exposure so that appropriate and timely treatment could be given to the patients.

Context: Early diagnosis is the mainstay in the management of severe cutaneous adverse reactions (SCARs) to drugs. Aims: To study the role of frozen section in the rapid diagnosis of SCARs and the impact on outcome of the affected patients. Settings and Design: A single-blind, hospital-based study was conducted from December 2014-July 2016. Methods and Material: We biopsied 32 adults with SCARs diagnosed by clinical features and standard criteria. The histopathological features seen on frozen sections were compared to that of paraffin blocks. The impact of rapid diagnosis on the clinical outcome was studied in toxic epidermal necrolysis (TEN), Stevens-Johnson syndrome (SJS), drug rash with eosinophilia and systemic symptoms (DRESS) and acute generalized exanthematous pustulosis (AGEP). Statistical Analysis: Z test was used to compare two proportions. Kappa statistic, sensitivity, specificity, positive predictive value, and negative predictive value of the frozen section diagnosis were calculated in TEN/SJS and DRESS using MedCalc software. Results: Frozen and paraffin sections were done in TEN/SJS spectrum (13), DRESS (17), and AGEP (2). The sensitivity, specificity and kappa values for frozen section diagnosis in SJS/TEN and DRESS were 91.7%, 95%, 0.867 and 94.4%, 100%, 0.937 respectively. The concordance between frozen and paraffin section diagnosis was 100% in TEN, SJS, DRESS and AGEP. All the 6 patients with TEN and 2 with AGEP survived. Taking the worst-case scenario, the mortality in SJS was 28.6%. The mortality among patients with DRESS was 11.8%. Conclusions: Frozen section helps in the rapid diagnosis and early treatment of SCARs and differentiates it from diseases that mimic it.

Currently, the standard assay employed to diagnose human orthopneumovirus infection is real-time reverse transcriptase PCR assay (rRT-PCR), a costly and time-consuming procedure that requires the manipulation of infectious viruses. In addition to RT-PCR, serological tests can complement the molecular diagnostic methods and have proven to be important tools in sero-surveillance. In this study, we report the development, optimization, and validation of a novel and rapid in-house diagnostic ELISA kit to detect human orthopneumovirus in clinical samples. We developed three sensitive ELISA formats through the immunization of rats with novel recombinant pPOE-F or pPOE-TF vectors. The two vectors expressed either the full-length (pPOE-F) or the truncated form (pPOE-TF) of the fusion (F) protein. The developed ELISA kits were optimized for coating buffer, capture antibody, blocking buffer, sample antigen, detection antibodies, and peroxidase-conjugated antibody, and validated using 75 rRT-PCR-confirmed nasopharyngeal aspirate (NPA) human orthopneumovirus samples and 25 negative samples collected from hospitalized children during different epidemic seasons between 2014 and 2017. Our results indicate that rats immunized with pPOE-F or pPOE-TF showed significant induction of high levels of MPAs. Validation of the ELISA method was compared to the rRT-PCR and the sensitivity hierarchy of these developed ELISA assays was considered from highest to lowest: indirect competitive inhibition ELISA (93.3%) > indirect antigen-capture ELISA (90.6%) > direct antigen-capture ELISA (86.6%). The development of the rapid in-house diagnostic ELISA kits described in this study demonstrates that a specific, rapid and sensitive test for human orthopneumovirus antigens could be successfully applied to samples collected from hospitalized children during different epidemics and can help in the efficient diagnosis of respiratory syncytial viral infections. [ABSTRACT FROM AUTHOR]

Community-acquired pneumonia requiring hospital admission is a prevalent and potentially serious infection, especially in high-risk patients (e.g., those requiring ICU admission or immunocompromised). International guidelines recommend early aetiological diagnosis to improve prognosis and reduce mortality. Syndromic panels that detect causative pathogens by molecular methods are here to stay. They are highly sensitive and specific for detecting the targets included in the test. A growing number of studies measuring their clinical impact have observed increased treatment appropriateness and decreased turnaround time to aetiological diagnosis, need for admission, length of hospital stay, days of isolation, adverse effects of medication and hospital costs. Its use is recommended a) per a pre-established protocol on making the diagnosis and managing the patient, b) together with an antimicrobial stewardship programme involving both the Microbiology Service and the clinicians responsible for the patient, and c) the final evaluation of the whole process. However, we recall that microbiological diagnosis with traditional methods remains mandatory due to the possibility that the aetiological agent is not included among the molecular targets and to determine the antimicrobial susceptibility of the pathogens detected. [ABSTRACT FROM AUTHOR]

Background: Burkholderia pseudomallei (B. pseudomallei), as a highly pathogenic organism, causes melioidosis, which is a disease of public health importance in many tropical developing countries. Here, we present and validate a novel detection technique, termed multiple cross displacement amplification combined with nanoparticles-based lateral flow biosensor (MCDA-NB), for identifying B. pseudomallei and diagnosing melioidosis. Results: B. pseudomallei-MCDA targets the TTS1 (Type III secretion system gene cluster 1) to specifically design ten MCDA primers. The nanoparticles-based biosensor (NB) can be combined with B. pseudomallei-MCDA for visually, objective, simply and rapidly reporting reaction results. The optimal amplification conditions of B. pseudomallei-MCDA were 66 °C for 30 min. Assay's sensitivity was 100 fg of genomic DNA in the pure cultures, and the analytical specificity was 100% by the examination of 257 strains, including 228 B. pseudomallei and 29 non-B. pseudomallei. As a result, the whole detection procedure was completed within 50 min, including 15 min for genomic DNA preparation, 30 min for l MCDA reaction, and 2 min for the interpretation of the results visually by biosensor. Conclusions: B. pseudomallei-MCDA assay is a rapid, sensitive and specific method for the detection of B. pseudomallei, and can be used as a potential tool for melioidosis diagnose in basic, field and clinical laboratories. [ABSTRACT FROM AUTHOR]

The traditional methods of diagnosing Salmonella which are time-consuming and sometimes problematic, are still used to identify Salmonella serotypes in clinical and food samples, but with the invention of rapid molecular detection methods, these problems have been largely eliminated. The present study aimed to rapidly detect different Salmonella isolates based on invA chromosomal gene search and also to identify acute isolates containing spv operon virulence genes. To this end, 20 human isolates of Salmonella were obtained from hospitals in Tabriz and 20 isolates of this bacterium were isolated from traditional cheese available on Tabriz consumer market. The molecular confirmation of isolates was first evaluated using specific primers of invA gene by simplex PCR method. Then, in order to evaluate the acute strains of the bacterium based on the presence of operon spv, the presence of spvA, B, C and R genes was examined by multiplex PCR using the relevant specific primers. The results showed that firstly, all isolates had molecular confirmation. Secondly, all 40 tested isolates had 3 spvA, C and R genes, but none of them had spvB gene. It seems that due to the limitations and problems in the traditional laboratory examination of Salmonella, PCR can be used as a rapid method to detect Salmonella infection. Also, the presence of 3 out of 4 virulence genes of opron spv in different Salmonella isolates in Tabriz region should be considered an undesirable finding, which emphasizes the need to further observe principles of control and prevention in animal and human communities.

Objective: Metagenomic Next-Generation Sequencing (mNGS) has been applied as a novel method of detection pathogens for infectious diseases, but its value in the rapid diagnosis of tuberculous meningitis(TBM)has not been clarified based on large samples. Methods: A retrospective analysis was conducted on 51 inpatients with suspected TBM who underwent mNGS and four other tests in cerebrospinal fluid (CSF). Results: Among 51 included patients, 45 cases were diagnosed as TBM (38 definite, 5 probable, 2 possible) and 6 cases as non-TBM. Using final diagnosis as reference standard, the sensitivity, specificity, PPV (positive predictive value), and NPV (negative predictive value) of mNGS in CSF for TBM were 84.44%(38/45, 69.94%–93.01%), 100%(6/6, 51.68%–100%), 100%(40/40, 88.57%–100%) and 46.15%(6/13, 20.40%–73.88%). The diagnostic sensitivity of mNGS(84.4%)was significantly higher than that of AFB (0%, P = 0.000), MGIT960 culture(22.2%, P = 0.000), MTB PCR(24.4%, P = 0.000) and Xpert MTB/RIF(40%, P = 0.000). The ROC curve showed that CSF protein quantification and CSF cell count might be valuable in the prediction of NGS positive detection of MTB (Mycobacterium tuberculosis). Conclusion: CSF mNGS had high sensitivity, specificity and PPV in the diagnosis of TBM. Patients with a significant increase in CSF cell number and protein quantification might have a higher likelihood of positive MTB detection of NGS.

Abstract Background Porcine Deltacoronavirus (PDCoV) is a newly emerging Coronavirus that was first identified in 2012 in Hong Kong, China. Since then, PDCoV has subsequently been reported worldwide, causing a high number of neonatal piglet deaths and significant economic losses to the swine industry. Therefore, it is necessary to establish a highly sensitive and specific method for the rapid diagnosis of PDCoV. Results In the present study, a highly sensitive and specific diagnostic method using recombinase polymerase amplification combined with a lateral flow dipstick (LFD-RPA) was developed for rapid and visual detection of PDCoV. The system can be performed under a broad range of temperature conditions from 10 to 37 °C, and the detection of PDCoV can be completed in 10 min at 37 °C. The sensitivity of this assay was 10 times higher than that of conventional PCR with a lower detection limit of 1 × 102 copies/µl of PDCoV. Meanwhile, the LFD-RPA assay specifically amplified PDCoV, while there was no cross-amplification with other swine-associated viruses, including Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Porcine kobuvirus (PKoV), Foot and mouth disease virus (FMDV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine circovirus type 2 (PCV2), Classical swine fever virus (CSFV) and Seneca valley virus (SVV). The repeatability of the test results indicated that this assay had good repeatability. In addition, 68 clinical samples (48 fecal swab specimens and 20 intestinal specimens) were further tested by LFD-RPA and RT-PCR assay. The positive rate of LFD-RPA clinical samples was 26.47% higher than that of conventional PCR (23.53%). Conclusions The LFD-RPA assay successfully detected PDCoV in less than 20 min in this study, providing a potentially valuable tool to improve molecular detection for PDCoV and to monitor the outbreak of PDCoV, especially in low-resource areas and laboratories.

16S rRNA gene-targeted next-generation sequencing (NGS) can detect microorganisms in a comprehensive reference database. To date, NGS has been successfully applied to samples such as urine, blood, and synovial fluid. However, there is no data for continuous ambulatory peritoneal dialysis (CAPD) fluid. The purpose of this study was to evaluate the clinical usefulness of microbiome analysis of CAPD fluids for the diagnosis of CAPD peritonitis.We included 21 patients with high suspicion of CAPD peritonitis. Routine CAPD fluid culture was performed using a pellet of 50 mL CAPD fluid onto the chocolate and blood agar for two days, and thioglycollate broth for one week. 16S rRNA gene-targeted NGS of pellets, stored at -70°C was performed with MiSeq (Illumina, USA). Many colonized or pathogenic bacteria were detected from CAPD fluids using NGS and the microbiomes were composed of 1 to 29 genera with a cut-off 1.0. Compared to the culture results, NGS detected the same pathogens in 6 of 18 valid results (three samples failed with low read count). Additionally, using NGS, anaerobes such as Bacteroides spp. and Prevotella spp. were detected in six patients. In two of five samples in which no bacterial growth was detected, possible pathogens were detected by NGS.To our knowledge, this is the first report about the application of 16S rRNA gene-targeted NGS for diagnosis of CAPD peritonitis. Etiology of culture-negative CAPD peritonitis can be better defined in NGS. Furthermore, it also helped the detection of anaerobic bacteria.

Bordetella pertussis is the most frequent causative agent for pertussis, which is a highly contagious disease. Here, we developed a method based on loop-mediated isothermal amplification (LAMP) and nanoparticle-based lateral flow biosensor (LFB) for the timely diagnosis of B. pertussis infections. A set of six primers was designed for LAMP reactions, and the LAMP results were rapidly and visually indicated using LFB. The recommended condition for the B. pertussis LAMP reactions is 40 min at 66°C. Our results confirmed that the LAMP-LFB assay could specifically detect B. pertussis and did not cross-react with non-B. pertussis isolates. The sensitivity of the B. pertussis LAMP-LFB assay was 50 fg per reaction. In particular, 108 nasopharyngeal swab (NPS) samples were collected to evaluate the B. pertussis LAMP-LFB assay, and the results were compared with those of the quantitative PCR (qPCR) method. The positive rates of B. pertussis LAMP-LFB and qPCR were 40.7% and 38.8%, respectively, and the agreement between the LAMP-LFB and qPCR results was 98%, with a kappa value of 0.96. The whole process of LAMP-LFB can be completed within 1 h, which is much shorter than that of qPCR, including about 15 min of rapid DNA extraction, 40 min of LAMP reaction, and within 2 min of the LFB test. Collectively, the B. pertussis LAMP-LFB assay developed in this report offers a new option for the rapid, reliable, and simple diagnosis of B. pertussis infections.

Orf is an important zoonotic disease caused by the Orf virus (ORFV) which can cause contagious pustular dermatitis in goats and sheep. Orf is widespread in most sheep-raising countries in the world, causing huge economic losses. Although diagnostic methods for ORFV infection already exist, it is still necessary to develop a time-saving, labor-saving, specific, low-cost and visual diagnostic method for rapid detection of ORFV in the field and application in grassroots laboratories. This study establishes a DNA extraction–free, real-time, visual recombinase–aided amplification (RAA) method for the rapid detection of ORFV. This method is specific to ORFV and does not cross-react with other common DNA viruses. The detection limits of the real-time RAA and visual judgment of the RAA assay at 95% probability were 13 and 21 copies per reaction for ORFV, respectively. Compared with qPCR, the sensitivity and specificity of the real-time RAA assay were 100%, and those of the visual RAA assay were 92.31% and 100.0%, respectively. The DNA extraction–free visual detection method of RAA established in this study can meet the needs of rapid onsite detection and grassroots laboratories and has important reference value and significance for the early diagnosis of diseased animals.

The prevalence of Herpes simplex virus 2 (HSV2) is increasing at an alarming rate in the world. Most of the HSV2 cases are not diagnosed properly, although a range of molecular and serological diagnoses exist. Herein, we have reported a very rapid detection method specific for HSV2 using real-time PCR. The primers specific for HSV2 were designed using the Primer-BLAST tool and 120 base pairs of the polymerase gene were amplified using real-time PCR with SYBR Green dye. The designed primer pair was found highly efficient in detecting only HSV2 DNA, but not HSV1. The threshold cycle (Ct) value for HSV2 reactions by designed primers was found to be an average of 22.55 for a standard copy number of viral DNA that may denote the efficiency of the primers. The melting temperature (Tm) of the amplicon using designed primers (82.6 0C) was also higher than that using reference primers (about 78 0C), indicating the high GC content of the amplified template. The designed primer pair will help clinicians to detect the HSV2 DNA specifically and diagnose the associated disease rapidly.

Introduction: Sporotrichosis, a prevalent deep fungal infection in clinical settings, currently lacks rapid and accurate diagnostic methodologies. This study explores a novel rapid diagnosis method for sporotrichosis by combining FTA cards and nested PCR with fungal fluorescence staining., Methods: The study involved skin lesion tissues from 26 patients diagnosed with sporotrichosis (Experimental Group). The Positive Control Group consisted of fungal suspensions from clinical strains of Sporothrix, while the Negative Control Group included fungal solutions of other fungi, namely Trichophyton rubrum, Trichophyton mentagrophyte, and Candida albicans. DNA was extracted from the slurry of skin lesions in the Experimental Group and from fungal suspensions in the Control Group using FTA cards, followed by nested PCR amplification. Subsequently, nested PCR amplification was performed. Histopathological examinations, including HE and fluorescence staining, were conducted on paraffin sections prepared from skin lesion tissues in the Experimental Group., Results: Among the 26 clinical skin lesion tissues in the Experimental Group, 8 cases showed a specific positive band upon nested PCR amplification, resulting in a positive rate of 30.8%. In the Control Group, the fungal solution of the clinical strain of Sporothrix showed a specific positive band upon nested PCR amplification, while all other fungi Negative Control Group tested negative. Histopathological examination revealed granulomatous inflammatory changes in most samples after HE staining. Fluorescence staining detected spores in 17 cases, resulting in a detection rate of 65.4% (17/26)., Conclusion: The combination of FTA cards with nested PCR method proved to be simple and rapid but demonstrated a relatively low positive rate. Fungal fluorescence staining significantly improved the sensitivity of detecting sporotrichosis in histopathological examinations, thereby improving the speed and efficiency of diagnosis., (© 2024 S. Karger AG, Basel.)

Equine piroplasmosis (EP) is a protozoal disease affecting equids, caused by Theileria equi and Babesia caballi . EP is conventionally diagnosed using microscopic, molecular, and/or serological methods, which are time-consuming. Consequently, there is a need for faster testing methods. In this study, we evaluated the application of the Sysmex XN-31 automated hematology analyzer, originally a rapid test for detecting malaria in humans, for the diagnosis of EP. The cultured parasites were measured using the XN-31 that had been customized for horse blood samples (XN-31m). The following parameters were evaluated: limit of detection (LoD), limit of quantification (LoQ), linearity, carryover, precision, and correlation with microscopic examination. The XN-31m detected infected red blood cells (RBCs) in approximately 1 minute. The LoD and LoQ for B. caballi were 4.54 infected RBCs/μL and 14.10 infected RBCs/μL, while those for T. equi were 5.80 infected RBCs/μL and 11.44 infected RBCs/μL, respectively. Linearity showed excellent correlation ( R 2 > 0.99), and carryover never exceeded 0.5%. The coefficient of variation was under 5%. The correlation between the results obtained using XN-31m and microscopic examination was high ( R 2 > 0.98). In conclusion, the XN-31 analyzer detected B. caballi and T. equi parasites in approximately 1 minute with high sensitivity. The results indicate the potential of the XN-31 analyzer as a fast and user-friendly diagnostic method for EP., Importance: In this study, we demonstrated that the automated hematology analyzer, XN-31, can detect red blood cells infected with Babesia caballi and Theileria equi in about 1 minute. We evaluated the diagnostic performance of the XN-31 analyzer for equine piroplasmosis, providing evidence of its potential as a diagnostic tool for this disease., Competing Interests: Yuji Toya, Mikako Sengoku, and Seiichiro Tsuchiya are employees of Sysmex corp.

Primary atopic disorders (PAD) are monogenic disorders caused by pathogenic gene variants encoding proteins that are key for the maintenance of a healthy skin barrier and a well-functioning immune system. Physicians face the challenge to find single, extremely rare PAD patients/families among the millions of individuals with common allergic diseases. We describe case scenarios with signature PAD. We review the literature and deduct specific clinical red flags for PAD detection. They include a positive family history and/or signs of pathological susceptibility to infections, immunodysregulation, or syndromic disease. Results of conventional laboratory and most immunological lab studies are not sufficient to make a definitive diagnosis of PAD. In the past, multistep narrowing of differential diagnoses by various immunological and other laboratory tests led to testing of single genes or gene panel analyses, which was a time-consuming and often unsuccessful approach. The implementation of whole-genomic analyses in the routine diagnostics has led to a paradigm shift. Upfront genome-wide analysis by whole genome sequencing (WGS) will shorten the time to diagnosis, save patients from unnecessary investigations, and reduce morbidity and mortality. We propose a rational, clinical landmark-based approach for deciding which cases pass the filter for carrying out early WGS. WGS result interpretation requires a great deal of caution regarding the causal relationship of variants in PAD phenotypes and absence of proof by adequate functional tests. In case of negative WGS results, a re-iteration attitude with re-analyses of the data (using the latest data base annotation)) may eventually lead to PAD diagnosis. PAD, like many other rare genetic diseases, will only be successfully managed, if physicians from different clinical specialties and geneticists interact regularly in multidisciplinary conferences., Competing Interests: TN: Non-profit organizations (travel expenses, no fees): Jeffrey Modell Foundation (parent organization, donations from the industry); info4pi.org; PENTA Global Pediatric Research Network (with donations from industry); penta-id.org; JIR Cohort (research network with donations from industry); Publishers (royalties): UpToDate, Inc; Springer, Elsevier; Indirect interests (membership) Transparency International; Parents’ associations (children with cancer Krefeld, children’s cancer clinic Düsseldorf); SvH: None. Figure 1.Physiology of the normal skin/mucosa barrier (upper panel) and newborn of 37 + 6 weeks gestation with Netherton Syndrome (pathogenic variants in SPINK5): Ichthyosiform erythroderma with generalized scaling, severe cutaneous inflammation resulting in debilitating pruritus and extremely sensitive skin (lower panel, provided by Dr. Arpe, St. Marien Hospital, Düren, Germany). SPINK5 (serine protease inhibitor Kazal-type 5) encodes the multidomain serine protease inhibitor LEKTI (lymphoepithelial Kazal-type-related inhibitor), which controls the kallikrein network of the epidermis and regulates desmosome turnover. Desmosomes seal the keratinocyte network. Corneodesmosin (CDSN), Desmoglein1 (DSG1), or Desmoplakin (DSP) are key intercellular adhesion molecules. Filaggrin monomers contribute to epidermal water retention through their hygroscopic properties. Activation of Dectin 1 or IL17 receptor (IL17R) leads to activation of CARD14 and NFκB, important for keratinocyte homeostasis (upper panel from left to right).Figure 2A.Physiology of mast cell activation (left panel) and clinical images of an 8-year-old girl with PLCγ2-associated antibody deficiency and immune dysregulation (right panel; PLCG2 variant c.313G>A p.(Val10Ile), Department of Human Genetics, University of Göttingen) and urticaria on the right wrist after exposure to cooling (swimming pool) (above) and vesiculobullous eruptions on colder areas, knees (below). SCF is the primary growth factor for mast cells, KIT, the cognate receptor for SCF. EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2) is encoded by ADGRE2. Attachment to the membrane is mediated by a noncovalently bound subunit of the receptor, which activates EMR2/ADGRE2 in mast cells, when forcefully dissociated by physical shearing forces/mechanical stress (vibration), leading to degranulation. Phosphoinositide-specific phospholipase Cγ2 (PLCγ2) is important for B-cell differentiation and function. In mast cells, PLCγ2 is downstream of the IgE receptor and catalyzes the hydrolysis of phosphatidylinositol 4, 5-bisphosphate to the secondary messengers inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 induces the release of Ca2+ from the ER. PLCγ2 can be activated by cold temperature, which leads to spontaneous calcium flux and degranulation. Tryptase (TPSAB1) is present in mast cell secretory granules. α- and β-tryptases form tetramers stabilized by heparin and are released after degranulation, where they contribute to allergic inflammation, inducing pruritus (adapted from Carlberg and Velleuer. Molecular Immunology: How Science Works, Publisher, Springer International Publishing, 2022 and [6, 7, 8]).Figure 2B.Physiology of granulocyte NLRP3 inflammasome activation (left panel) and a 2-month-old boy with CINCA/NOMID (right panel; GOF variant in NLRP3; Institute for Clinical Chemistry, Ludwig Maximilian University, Munich, Germany) with continuous fever since birth, urticarial rash in changing locations (above), marked eosinophilia (up to 35,000/μL) and fast recovery after institution of IL1-blocking treatment (Anakinra) (below). The NLRP3 inflammasome is a multiprotein complex composed of NLRP3, adapter protein ASC, and protease caspase-1. Upon various stimuli (e.g., microbial ligands, crystals) NLRP3 inflammasome activates caspase‐1 to induce the release of proinflammatory IL1β (adapted from Carlberg and Velleuer. Molecular Immunology: How Science Works, Publisher, Springer International Publishing, 2022). Table 1.Other primary atopic diseases with disrupted epithelial and/or mucosal skin barrier [9, 10, 11]. DiseaseOMIM-PGeneInheritancePhenotypeFilaggrin deficiency#146700FLGAD or AR (LOF)– Ichthyosis vulgaris – Early-onset persistent atopic eczema – Elevated risk of food allergy and eczema herpeticumSevere dermatitis – multiple allergies – metabolic wasting syndrome (SAM)#615508DSG1; DSPAD or AR (LOF)– Life-threatening condition, severe dermatitis, multiple allergies, metabolic wasting – Hypotrichosis, palmoplantar hyperkeratosis, enamel defects, recurrent (skin) infections – In some patients little systemic involvement Peeling skin syndrome type B#270300CDSNAR (LOF)– Lifelong patchy peeling of the skin with chronic pruritus – Frequent food allergy, recurrent skin infections – Usually no failure to thriveCARD14 deficiency#602723CARD14AD (LOF)– Severe atopy, severe pyogenic and viral skin and respiratory tract infections due to impaired NFkB activation and impaired epidermal secretion of antimicrobial peptidesAD = autosomal dominant; AR = autosomal recessive; LOF = loss of function. Table 2.Other primary atopic disorders with dysregulated granulocyte / mast cell function [8, 13, 14]. DiseaseOMIM-PGeneInheritancePhenotypeCryopyrin-associated periodic syndromes (CAPS) composed of 1. Muckle-Wells syndrome 2. Familial cold autoinflammatory syndrome (FCAS1) 3. Neonatal onset multisystem inflammatory disease (NOMID) or chronic infantile neurologic cutaneous and articular syndrome (CINCA)#191900 #607115 #120100NLRP3AD (GOF)– Maculopapular, non-pruritic and most predominantly urticarial rashes – Arthritis, chills, fever and leukocytosis, e.g., after cold exposure (FCAS1) – Recurrent fever, musculoskeletal symptoms, abdominal and thoracic serositis, headache, ophthalmic and auditory nerve inflammation potentially leading to deafness and blindness preventable by IL1-directed treatment – Severity of symptoms variable between and within conditions, and not indicative of a particular diseaseNLRC4 or NLRP12-associated autoinflammatory diseases#616115 #611762NLRC4 NLRP12AD (GOF)Fever, arthritis/arthralgia, rash, abdominal pain, diarrhea, myalgia/fatigue and conjunctivitis triggered by cold exposureAD = autosomal dominant; GOF = gain of function. Figure 3.Central tolerance induction physiology (left panel) and neonate with Omenn syndrome and erythroderma (right panel, above) who was successfully treated by cord blood transplantation (below); (pictures kindly provided by H. Ott, Hannover, published in [16]). The thymic selection of T cells during the first decade of life produces millions of antigenically distinct T cells carrying a very diverse T-cell receptor (TCR) repertoire prepared to encounter millions of variants of microbial and other antigens. Some autoreactive T-cell clones are also generated. This selection process is called central tolerance induction. Figure 4.Physiology of Tregs (regulatory T cells) (left panel) and an infant with IPEX syndrome (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) (right panel, above). The patient presented with severe autoimmune enteropathy and growth charts showing failure to thrive, length and weight < 3rd percentile (right panel, below). Tregs induce peripheral tolerance, preventing development of autoimmune and allergic diseases. Their expression of the high affinity trimeric α/β/γ IL2 receptor (IL2R) helps them to selectively bind IL2 and draw it away from T effector cells (Teff) expressing only the low moderate affinity (β/γ) IL2R. FOXP3 is the lineage defining transcription factor for the development of Tregs. High levels of FOXP3 expression are directly linked to the suppressive capacity of Tregs. FOXP3 expression leads to the upregulation of the high affinity IL2 receptor as well as CTLA-4 on Tregs. CTLA-4 competes with the co-stimulatory molecule, CD28, for binding to CD80 and CD86 on antigen presenting cells (APCs). This is one other mechanism by which Tregs suppress immune responses. IKZF (IKAROS family zinc finger) have been shown to be critical for Treg and hematopoietic development. Table 3.Other primary atopic diseases Tregopathies [17, 18]. DiseaseOMIM-PGeneInheritancePhenotypeDisorders of Treg transcriptional programsIKAROS-associated (IKZF1) disease#616873IKZF1AD (GOF or LOF)– Onset > 1 to up to 40 years of age – Many present with atopic diseases – Autoimmunity/immune dysregulation can be profound: e.g. autoimmune cytopenias (Evans Syndrome), lymphoproliferation, plasma cell expansion (IgG4+), type 1 diabetes, thyroiditis alopecia, vitiligo, celiac disease, colitis) – Sinopulmonary infections – Expressivity of GOF variants is variable, asymptomatic individuals are commonDisorders of IL2 or CTLA4 signalingInterleukin 2 receptor a (IL2RA (CD25)) or interleukin-2 receptor β (IL2RB (CD122))#606367 #618495IL2RA (CD25), IL2RB (CD122)AR (LOF)IPEX-like (see above) and SCID-like presentations CTLA4 haploinsufficiency#152700CTLA4AD (LOF)Onset throughout childhood and adulthood. – Lymphoproliferation, lymphoma, gastric cancer, polyautoimmunity (e.g., autoimmune enteropathies, cytopenias, skin disease, interstitial lung disease, and neurologic manifestations). – Increased severity of infections (e.g., CMV, EBV). – Often initially diagnosed as CVID – Some parents and relatives with pathogenic variants asymptomatic.AD = autosomal dominant; AR = autosomal recessive; IPEX = immune dysregulation, polyendocrinopathy, enteropathy, X-linked; SCID = severe combined immunodeficieny; CMV = cytomegalovirus; EBV = Ebstein-Barr virus; CVID = common variable immunodeficiency. Table 4.Other defects in downstream TCR signalling, cytoskeleton activation and immune synapse formation [3, 19, 21, 22]. DiseaseOMIMGeneInheritancePhenotypeWiskott-Aldrich syndrome#301000WASXL (LOF)Onset: early childhood with the classic triad: – Thrombocytopenia (pathognomonic small mean platelet volume (< 5 fL)) – Recurrent severe infections (impetigo, cellulitis, skin abscesses, molluscum contagiosum; Herpesviruses, including herpes simplex and varicella-zoster virus, Epstein-Barr virus) – Eczema, often unresponsive to conventional treatment. – Early death may result from bleeding. – Autoimmunity and malignancy more common with increasing age. – Milder phenotype: X-linked thrombocytopenia without eczema or immunodeficiency.WIP deficiency#614493WIPF1AR (LOF)– WAS-like presentation, but very early onset of a severe immunodeficiencyNCKAP1L deficiency (HEM-1 hematopoietic protein 1)#618982NCKAP1LAR (LOF)– Atopic disease and hyperinflammation, chronic hepatosplenomegaly, lymphadenopathy – Recurrent fever and upper respiratory tract infections, skin rashes, abscesses, ulcers, autoimmune manifestations and FTTDOCK8 deficiency#243700DOCK8AR (LOF)– Extensive, disfiguring, concurrently occurring cutaneous viral infections, particularly HSV, human papillomavirus, molluscum contagiosum and varicella-zoster virus. – Invasive infections (wide spectrum of Gram-positive and Gram-negative bacteria, viruses and intracellular fungi, e.g., histoplasma capsulatum). – Mucocutaneous candidiasis and recurrent gastro-intestinal tract infections are common. – Severe and extensive food allergies. – High risk of developing malignancies, particularly lymphomas and squamous cell carcinomas. CARMIL2 (RLTPR) deficiency#618131CARMIL2AR (LOF)– Onset early infancy; – Atopic and seborrheic dermatitis and psoriasis-like rashes. – Viral (EBV, CMV, and varicella), bacterial, mycobacterial and fungal infections – Early or very early onset inflammatory bowel disease (VEOIBD) – Autoimmune polyendocrinopathy syndrome (APS) – Characteristic feature is EBV-associated leiomyoma (<20% of cases)STK4 deficiency#614868STK4AR (LOF)– Onset at school age – Infections (cutaneous viral infections, recurrent pneumonia, EBV-associated lymphoproliferation) – Autoimmune or inflammatory diseases and atopic dermatitis/atopyTBX21 deficiency (T-bet, T-box transcription factor 21) #619630TBX21AR (LOF)– Mendelian susceptibility to mycobacterial disease (MSMD) – Persistent upper airway inflammation Moesin-associated immunodeficiency (X-MAID)#300988MSNXLR (LOF) – Skin manifestations, mainly eczema, molluscum contagiosum and increased susceptibility to bacterial and viral infections and VEOIBDXL = X linked; LOF = loss of function; AD = autosomal dominant; AR = autosomal recessive; XLR = X-linked recessive; WAS = Wiscott-Aldrich syndrome; FTT = failure to thrive; HSV = Herpes simplex virus; EBV = Ebstein-Barr virus; CMV = cytomegalovirus; VEOIBD = very early onset inflammatory bowel disease. Figure 5.Physiology of the actin cytoskeleton (left panel) and a 3-year-old boy (right panel, above) with ARPC1B (actin related protein 2/3 complex subunit 1B) deficiency, presenting with ulcerating severe cutaneous viral infections (left shoulder and on the back) and after successful stem cell transplantation at 5 years of age (right panel, below). Rearrangement of actin cytoskeletons is key for immune cell activation, migration and adhesion. Human actin-related protein 2/3 complex (Arp 2/3) has ARPC1 component isoforms (ARPC1B, expressed in blood cells, ARPC1A in non-hematopoietic tissues). WASP (Wiskott Aldrich syndrome protein), WIPF1 (WASP interacting protein family member 1), DOCK8 (dedicator of cytokinesis 8), and NCKAP1 (Nck-associated protein 1 also called HEM1 (hematopoietic protein 1)). The RLTPR or CARMIL-2 protein, the TCR, and CD28 form microclusters that interact with actin and are key for the formation of the immunological synapse. Moesin (MSN) links actin filaments to the plasma membrane which increases cell rigidity and polarity and STK4 controls the translocation of vesicles to the surface and can activate actin (both not shown in Figure).Figure 6.Physiology of STAT3 signaling (left panel) and a 7-year-old boy with coarse facial features (right panel, above), eczema, cold skin abscesses (middle) and delayed dentition at 10 years of age (below, different patient). IL6 signals through the IL6RST (signal transducer of IL6 receptor complex), composed of glycoprotein 130 (GP130) and the IL6R. Other cytokines of the IL6 cytokine family utilize GP130 encoded by IL6ST (IL11, OSM, LIF, CNTF, CT-1, CLCF1, IL27, IL35, and IL39). IL6 then activates through JAK-STAT signal transduction the zinc finger protein 341 (ZNF341) TF, which promotes STAT3 transcription. IL6 and IL23 activate STAT3, which turns on RORC, encoding RORγt the master regulator for a TH1/TH17 T-cell response shift as well as chondrogenesis, bone repair, angiogenesis/vascularization (induction of vascular endothelial factor 3 (VEGF3)) [23]. The phosphoglucomutase 3 (PGM3) (not shown in figure) enzyme catalyzing the isomerization of N-acetyl-glucosamine-6-phosphate to N-acetyl-glucosamine-1-phosphate during the generation of UDP-N-acetyl-glucosamine UDP-GlcNAc. During glycosylation, sugar chains are added to either proteins or lipids, using basic sugar building blocks (UDP-GlcNAcs) to make N-glycans, O-glycans, proteoglycans, and glycosylphosphatidylinositol (GPI)-anchored proteins all participating in cell signaling. Table 5.PAD due to CBMopathies and cytokine signaling dysfunction [4, 7, 26]. DiseaseOMIMGeneInheritancePhenotypeCBMopathiesCARD11-associated atopy with dominant interference of the NFkB signaling (CADINS)#617638CARD11AD (LOF)– Very severe atopic dermatitis (close to 90% of cases) later followed by asthma and food allergy. Cutaneous viral and respiratory tract infections, potentially severe and causing FTT (e.g., due to diarrhea). Some patients display skeletal features as in STAT3 deficiency (broad nose, retained teeth).MALT1 deficiency#615468MALT1AR (LOF)– Left untreated it is thought to be fatal – Recurrent severe bacterial viral and fungal infections of the skin and/or respiratory and gastrointestinal tract infections, FTT – Periodontal disease, aphthous ulcers, cheileitis and gingivitis.TGFβ pathwayTransforming growth factor β receptor 1 and 2 (TGFBR 1 and 2) deficiency (Loeys-Dietz syndrome 1 and 2 )#609192 #610168TGFBR1, TGFBR2AD (LOF)– IgE-mediated food allergy, EGID, allergic asthma, and atopic dermatitis. Additional connective tissue abnormalities overlapping with those seen in STAT3 pathway disorders (hyperextensible joints, scoliosis, etc.)Erbb2-interacting protein (ERBIN) deficiencyNoneERBINAD (LOF)– Significant EGID, connective tissue abnormalities. – Enhanced TGF-β pathway activation leading to increased IL4Rα expression and enhanced Th2 differentiation and IgE productionJAK1/STAT5b pathwayJAK1 GOF#618999JAK1AD (GOF)– Severe atopic dermatitis and allergic asthma. Immune dysregulation such as autoimmunity (e.g., thyroid disease), poor growth, hepatosplenomegaly, eosinophilic enteritis. STAT5B hypereosinophilic syndrome#102578STAT5BSomatic (GOF) in multiple hematopoietic lineages– Neonatal onset dermatitis, urticarial rash and diarrhea, neonatal hypereosinophilia. – Patients with somatic GOF STAT5B variants have presented with leukemia and lymphomas.STAT5B deficiency#618985STAT5BAD (dominant negative) and AR (LOF)– Recurrent viral infections due to poor IL-2-mediated effector functions and chronic pulmonary disease (lymphocytic interstitial pneumonitis). – Extreme short stature due to the roles of STAT5b in growth factor signaling (Growth-hormone insensitive dwarfism, dysmorphic features). – Atopy, e.g., eczema. Prominent autoimmunity similar to IPEX (Modestly reduced Treg number and function). The AD form of STAT5B deficiency has growth-failure, eczema, but no immunodeficiency.IL4/STAT6 pathwaySTAT6-GOF disease (signal transducer and activator of transcription 6)#620532STAT6AD (GOF)– Severe early-onset, multi-system allergic disease, e.g. severe and treatment-resistant dermatitis, marked eosinophilic gastrointestinal disease. – Skin and respiratory infections as well as lymphadenopathy, cobblestone-like appearance of the buccal mucosa, polypoid nodules in the intestinal tract, and notably, B-cell lymphomas. – Non-immunological features: some resembling AD HIES, some are additional: renal fibrosis, short stature and hypotrichosisGOF = gain of function; LOF = loss of function; AD = autosomal dominant; AR = autosomal recessive; FTT = failure to thrive; EGID = eosinophilic gastrointestinal disease; IPEX = immune dysregulation, polyendocrinopathy, enteropathy, X-linked; HIES = hyper IgE syndrome . Table 6.Other primary atopic disorders with STAT3 dysfunction [4, 5, 6, 7, 24, 25]. DiseaseOMIMGeneInheritancePhenotypeZNF341 deficiency#618282ZNF341AR (LOF)– Phenocopy of AD-HIES Partial IL6 signal transducer (IL6ST) (GP130 deficiency)#619752IL6STAD (LOF)– Demonstrates phenotypic overlap with AD HIES but also diarrhea, keratitis, and neurodevelopmental delay. Complete IL6 signal transducer (IL6ST) (GP130 deficiency) #618523IL6STAR (LOF)– Death in utero or in neonatal period in most affected individuals. – Stuve-Wiedemann-like syndrome; skeletal dysplasia, osteoporosis, lung dysfunction, renal abnormalities, thrombocytopenia, eczema. IL6R deficiency#618944IL6RAR (LOF)– Atopic dermatitis, elevated IgE, bacterial sinopulmonary infection, and substantial skin and soft tissue infections – often due to staphylococcus. No connective tissue abnormalitiesPhosphoglucomutase 3 deficiency#615816PGM3AR (LOF) hypomorphic– Syndromic immunodeficiency due to glycosylation defect. Atopic dermatitis, bronchiectasis, and scoliosis but lack of characteristic AD HIES facies, chemotaxis defects, cold abscesses and connective tissue disease. – Developmental delay, primary neurocognitive deficits, hypomyelination and skeletal dysplasiaAD = autosomal dominant; AR = autosomal recessive; HIES = hyper IgE syndrome. Figure 7.A: CBM physiology CBM complexes are critical TCR signaling adapters. Antigen receptor stimulation leads to phosphorylation of CARD 11. Activated CARD 11 is recruited into the CBM complex to the ubiquitin regulatory proteins LUBAC and TRAF6 which ligate unique ubiquitin chains to BCL 10/MALT1 (not shown in Figure) and eventually lead to the activation of NFkB, JNK and ASCT2 regulating glutamine metabolism and activating MTOR. B: JAK1/STAT5/ TGFβ/STAT6 Physiology. TGFβ has roles in immune tolerance, cell cycle arrest, and wound healing. TGFβ signals through TGFBR1 and 2 Receptor. TGFβ increases IL4Rα expression and leads to T helper type 2 (TH2) differentiation and IgE production. ERBIN is a negative regulator of transforming growth factor (TGF)-signaling through interaction with SMAD proteins. STAT3 induces and then forms a complex with ERBIN. STAT5 is activated by signaling through receptors for various cytokines including IL2, hematopoietic growth factors, as well as growth hormone. STAT5B is an important regulator of FOXP3 expression, the essential TF for Tregs. STAT6 signaling induces activation of group 2 innate lymphoid cells (ILC2s), differentiation of TH2 cells from naïve CD4+ T cells and induction of T follicular helper (TFH) cells, which are crucial for B-cell affinity maturation and immunoglobulin class-switch recombination (CSR) in the germinal center. In non-immune cells, IL4/IL13 STAT6 is involved in collagen production of fibroblasts, the development of mucus-secreting goblet cells, eosinophil-attracting chemokine production from epithelial cells and induction of bronchial smooth muscle hyper-responsiveness.Table 7.Screening landmarks F, AD, ID, SY regarding history and physical findings. Presence of Red flags in > 2 landmarks will trigger whole genome analysis. Abnormal findings in lab investigations are only significant in presence of landmarks F, A, ID, SY. Land-markCategoryToolsRed FlagFFamily historyDraw a pedigree§, find inheritance pattern; know geographical distributions of consanguinity$– Presence of family members with IEI; family members fulfill other landmarks (see below)AAtopy disease history Get details: onset, extent, course– Onset: at birth/first 1 – 2 months of life – Extent: multiple simultaneous atopic manifestations such as severe asthma, life-threatening anaphylaxis, allergic rhinoconjunctivitis, multiple food allergies, eosinophilic esophagitis and gastroenteritis, protein-losing enteropathy – Course: very severe, not responsive to standard therapy IDImmunodeficiency, non-atopic past medical historyEvaluate pathological susceptibility to infections, signs of immunodysregulation, past medical history (ELVIS**, GARFIELD,** see next column)General– Failure to thrive+– Malignancy (e.g., lymphomas, leiomyosarcoma) Pathological susceptibility to infections (ELVIS)** – Unusual, opportunistic pathogens: e.g. pneumocystis – Localization: unusual, e.g., organ abscesses – Course: exceptionally long duration – Intensity: exceptionally severe, e.g., admission to intensive care unit. – Sum: Too many, e.g., in adults, >3 or in children > 6 infections per year which require treatment (including antibiotics) and each lasting more than 4 weeks Immunodysregulation (GARFIELD)** – Granuloma – Autoimmune disease – Recurring fever and chronic inflammation – Unusual eczema – Lymphoproliferation – Chronic, inflammatory bowel Disease SYSyndromic diseaseSkilled examination by experienced physician, clinical geneticist; supplementary use of artificial intelligence (next generation phenotyping (e.g., GestaltMatcher)++Key clinical findings such as hypotrichosis, neurodevelopmental delay, skeletal or connective tissue abnormalities (e.g., coarse facies, vascular anomalies, bleeding)! Caveat !: Except newborn SCID screening, results of conventional laboratory studies are not sufficient to make a definitive diagnosis of PAD. High levels of IgE or eosinophilia do occur in many PADs but are also common in non-primary allergic diseases. Abnormal findings in laboratory investigation count only as significant in the presence of landmarks F, A, ID, SY.LILab investigationsSCID newborn screening; full blood count; serum IgG, IgA, IgM, IgE; specific IgE panels to common aeroallergens and food allergens; Skin prick tests; urine glycans, serum tryptase; N- und O-glycosylation in serum transferrin electrophoresis, flow cytometry with immunodeficiency adapted markers; mast cell metabolites (e.g., histamine, leukotriens) in urine– Pathological TREC screening – Abnormal absolute numbers and relative percentages in full blood count (e.g., cytopenia; neutropenia, lymphopenia, thrombocytopenia, anemia) – Low IgG- SD age-adjusted; IgA < 5 mg/dL – Very high TH2 biomarkers: severe eosinophilia (>5,000 cells/mm3); serum total IgE > 2,000 kUIEI = inborn errors of immunity; SCID = severe combined immunodeficiency; PAD = primary atopic disease; TREC = T cell receptor excision circle;SD = standard deviation.&Online resources and instructions on How to Draw a Pedigree - Iowa Institute of Human Genetics (uiowa.edu); (Definition of pedigree - NCI Dictionary of Genetics Terms - NCI (cancer.gov); $Consang.net. **acronyms adapted from the German guideline for Diagnostics in Primary immunodeficiencies (S. Farmand, manuscript in preparation) and [31]; ELVIS = Erreger (pathogens), Lokalisation (localization), Verlauf (course), Intensität (intensity), Summe (sum); GARFIELD = Granulome (granuloma), Autoimmunität (autoimmunity), Rezidivierendes Fieber (recurrent fever), Darmentzündung (chronic inflammatory bowel disease). +Growth hormone evaluation and bone age study if there is short stature; delayed bone age and growth hormone deficiency may be found. ++www.db.gestatmatcher.org.Figure 8Rational, clinical landmark-based approach to identify patients/families with primary atopic disorders by early, upfront whole genome sequencing. If > 2 red flags are fulfilled, genetic analysis is indicated. In the following sections we discuss the different methods of genetic analysis and the cornerstones of genetic result interpretation in individuals with (suspected) primary atopic disorder. Details on red flags are given in Table 7., (© Dustri-Verlag Dr. K. Feistle.)

Mycoplasma pneumoniae is a significant pathogen responsible for community-acquired pneumonia, predominantly affecting children and adolescents. Here, we devised a rapid method for M. pneumoniae that combined multiple cross displacement amplification (MCDA) with real-time fluorescence technology. A set of ten primers, which were specifically designed for M. pneumoniae detection, were employed in a real-time fluorescence MCDA reaction. Of these, one primer incorporated a restriction endonuclease recognition sequence, a fluorophore, and a quencher, facilitating real-time fluorescence detection. The real-time (RT)-MCDA reactions were monitored in a simple real-time fluorescence instrument and conducted under optimised conditions (64°C for 40 min). The detection limit of the M. pneumoniae RT-MCDA assay for genomic DNA extracted from M. pneumoniae culture was down to 43 fg/µl. This assay accurately identified M. pneumoniae strains without cross-reacting with other bacteria. To validate its practical application, we tested the M. pneumoniae RT-MCDA assay using genomic DNA extracted from clinical samples. The assay's detection capability proved comparable with real-time PCR, MCDA-based biosensor detection, and visual inspection under blue light. The entire process, including rapid DNA extraction and real-time MCDA detection, was completed within 1 h. Overall, the M. pneumoniae RT-MCDA assay reported here is a simple and effective diagnostic tool for rapid M. pneumoniae detection, which holds significant potential for point-of-care testing and in resource-limited regions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Xiao, Zhang, Xu, Fu, Huang, Jia, Sun, Xu, Zheng, Zhou, Wang and Meng.)

Background: Cytological detection of early esophageal squamous cell carcinoma (ESCC) remains challenging. Therefore, we introduced a rapid cytological screening method and evaluated its efficacy as a pre-endoscopy screening for early ESCC and precursor lesions. Methods: This method consisted of a sponge sample retrieval, automatic liquid-based cytological treatment and slides preparation, computer-assisted screening and manual diagnosis. Efficacy for detection of early ESCC and precursor lesions was evaluated. Also, diagnostic efficiency was compared with manual diagnosis. Results: Eighty-three patients with early ESCC and precursor lesions and 2,090 asymptomatic participants with high risks of ESCC were enrolled. Whole procedure was accomplished within two working days. Abnormal cells were detected in all 83 patients, and in 272 (13.01%) subjects among 2,090 asymptomatic participants. Early ESCC, high-grade intraepithelial neoplasia, low-grade intraepithelial neoplasia and reflux esophagitis and normal endoscopic findings were detected in 8, 13, 11, 187 and 53 participants with abnormal cells, respectively. The calculated sensitivity, specificity, positive predictive value and negative predictive value for detection of early ESCC and precursor lesions were 100%, 88.34%, 11.76%, and 100%, respectively. Compared with manual diagnosis, this method was accomplished in a shorter time duration (5.4 ± 0.45 min vs 320.2 ± 132.4 min, p < 0.001), a higher diagnostic accuracy (96.7% vs74.4%, p = 0.015) and a better inter-observer agreement (93.3% vs66.7%, K = 0.286, p < 0.001). Conclusions: Our study provides a promising methodology as pre-endoscopy screening for early ESCC and precursor lesions. [ABSTRACT FROM AUTHOR]

Schistosoma mekongi infection is endemic in countries along the Mekong River and certain of its tributaries in the lower Mekong basin, especially in Lao People's Democratic Republic and Cambodia. Diagnosis of schistosomiasis is crucial before treatment and epidemiological surveys before and/or after an intervention, such as a mass drug administration. A newly developed immunochromatographic test (ICT) for the diagnosis of schistosomiasis mekongi, based on antiparasite antibody detection in human sera, was evaluated. The schistosomiasis mekongi-ICT (Smk-ICT) strip was developed using somatic antigen from adult S. mekongi. In total, 209 serum samples were examined, including 14 from parasitologically proven schistosomiasis mekongi patients, 30 from schistosomiasis japonica patients, other parasitosis (n = 135), and healthy volunteers (n = 30) from areas not endemic for S. mekongi. Eleven schistosomiasis mekongi samples were positive according to the Smk-ICT, whereas all healthy control samples were negative. Cross-reactions with paragonimiasis heterotremus, sparganosis, trichinellosis, and taeniasis saginata samples were observed at 2.4% (4/165). The diagnostic sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 78.6% (95% confidence interval [CI] 49.2–95.3), 97.6% (95% CI 93.9–99.3), 73.3% (95% CI 44.9–92.2), 98.2% (95% CI 94.7–99.6), and 96.1% (95% CI 92.1–98.4), respectively. The Smk-ICT kit might be useful to assess the prevalence of disease before establishing transmission control and mass deworming campaigns in countries in the Mekong River subregion. [ABSTRACT FROM AUTHOR]

Cancer remains an intractable medical problem. Rapid diagnosis and identification of cancer are critical to differentiate it from nonmalignant diseases. High‐throughput biofluid metabolic analysis has potential for cancer diagnosis. Nevertheless, the present metabolite analysis method does not meet the demand for high‐throughput screening of diseases. Herein, a high‐throughput, cost‐effective, and noninvasive urine metabolic profiling method based on TiO2/MXene‐assisted laser desorption/ionization mass spectrometry (LDI‐MS) is presented for the efficient screening of bladder cancer (BC) and nonmalignant urinary disease. Combined with machine learning, TiO2/MXene‐assisted LDI‐MS enables high diagnostic accuracy (96.8%) for the classification of patient groups (including 47 BC and 46 ureteral calculus (UC) patients) from healthy controls (113 cases). In addition, BC patients can also be identified from noncancerous UC individuals with an accuracy of 88.3% in the independent test cohort. Furthermore, metabolite variations between BC and UC individuals are investigated based on relative quantification, and related pathways are also discussed. These results suggest that this method, based on urine metabolic patterns, provides a potential tool for rapidly distinguishing urinary diseases and it may pave the way for precision medicine. [ABSTRACT FROM AUTHOR]

Leishmania belongs to a genus of the protozoan parasites that causes leishmaniasis, and includes cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). In this case, Leishmania amastigotes were found on cytomorphology examination of the bone marrow specimen, followed by 1,076 Leishmania donovani reads using metagenomic next generation sequencing (mNGS). Since being definitely diagnosed with VL/HIV coinfection, the patient was treated with liposomal amphotericin B as the parasite-resistant therapy and was discharged after clinical cure. But nearly a year later, on the mNGS follow-up, L. donovani was detected in the patient's blood plasma specimen with 941 reads, suggesting that a relapse of leishmaniasis had occurred. These results indicate that leishmaniasis still exists in China and may represent a public health concern. This case could be helpful in the differential diagnosis of leishmaniasis, and for determining disease progression, prevention, and control of vectors and reservoir hosts. [ABSTRACT FROM AUTHOR]

• The increase of extended-spectrum beta-lactamase (ESBL)-producing pathogens makes it difficult to select antibiotics. • ESBL-producing pathogens were isolated from 22.6% of urine samples. • Cica-beta test had diagnostic accuracy of 94.9% for the rapid detection of ESBLs. The rapid increase of extended-spectrum beta-lactamase (ESBL)-producing pathogens makes it difficult to choose appropriate antimicrobials in patients with Gram-negative bacterial infection. The Cica-beta reagent (Kanto Chemical, Tokyo, Japan) is a chromogenic test that detects ESBLs from bacterial colonies. This study aimed to reveal whether Cica-beta reagent could detect ESBLs directly from urine samples to facilitate rapid diagnosis of antibiotic susceptibility. A prospective study was conducted from July 2019 to November 2019. Patients in whom urine culture tests were performed were eligible. Each urine sample was centrifuged, and the pellet was mixed with Cica-beta reagent. The test was considered positive when the enzymatic reaction turned from yellow to red or orange. In total, 350 urine samples were analysed. Urinary tract infection (UTI) was diagnosed in 214 patients. ESBL-producing Enterobacterales were isolated from 79 samples. The Cica-beta test showed sensitivity of 79.8% and specificity of 99.3% in patients with Gram-negative bacteriuria. Sensitivity and specificity improved to 90.2% and 100%, respectively, in patients with UTI. The Cica-beta test could be an efficient test for the detection of ESBL-producing pathogens in urine. By providing immediate information about ESBLs, it might be a useful point-of-care test to guide appropriate antimicrobial use in patients with UTI. [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]

The purpose of this study was to evaluate the SARS‐CoV‐2 immunoglobulin M/immunoglobulin G (IgM/IgG) rapid antibody test results in symptomatic patients with COVID‐19 and their chest computed tomography (CT) data. A total of 320 patients admitted to our hospital for different durations due to COVID‐19 were included in the study. Serum samples were obtained within 0–7 days from COVID‐19 patients confirmed by reverse‐transcription polymerase chain reaction (RT‐PCR) and chest CT scan. According to the SARS‐CoV‐2 RT‐PCR results, the patients included in the study were divided into two groups: PCR positive group (n = 46) and PCR negative group (n = 274). The relationship between chest CT and rapid antibody test results were compared statistically. Of the 320 COVID‐19 serum samples, IgM, IgG, and IgM/IgG were detected in 8.4%, 0.3%, and 11.6% within 1 week, respectively. IgG/IgM antibodies were not detected in 79.7% of the patients. In the study, 249 (77.8%) of 320 patients had positive chest CT scans. Four (5.6%) of 71 patients with negative chest CT scans had IgM and two (2.8%) were both IgM/IgG positive. IgM was detected in 23 (9.2%), IgG in one (0.4%), and IgM/IgG in 35 (14%) of chest CT scan positive patients. The rate of CT findings in patients with antibody positivity was found to be significantly higher than those with antibody negativity. The results of the present study show the accurate and equivalent performance of serological antibody assays and chest CT in detecting SARS‐CoV‐2 within 0–7 days from the onset of COVID19 symptoms. When RT‐PCR is not available, we believe that the combination of immunochromatographic test and chest CT scan can increase diagnostic sensitivity for COVID‐19. [ABSTRACT FROM AUTHOR]

Background: Current assays for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rely on time consuming, costly and laboratory based methods for virus isolation, purification and removing inhibitors. To address this limitation, we propose a simple method for testing RNA from nasopharyngeal swab samples that bypasses the RNA purification step.Methods: In the current project, we have described two extraction-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of SARS-CoV-2 by using E gene and RdRp gene as the targets.Results: Here, results showed that reverse transcription loop-mediated isothermal amplification assays with 88.4% sensitive (95% CI: 74.9-96.1%) and 67.4% sensitive (95% CI: 51.5-80.9%) for E gene and RdRp gene, respectively.Conclusion: Without the need of RNA purification, our developed RT-LAMP assays for direct detection of SARS-CoV-2 from nasopharyngeal swab samples could be turned into alternatives to qRT-PCR for rapid screening. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is major health concern and reason of deaths from decades to current date. Even though with a lot of advancements, diagnostic techniques, and discovery of standard antibiotics TB remains crucial challenge and can create worst scenario for human health in near future. Nanoparticles play emerging role in diagnosis and treatment of TB. In this study, we developed mesoporous silica nanoparticles containing gold (MSNs@GNPs) for rapid diagnosis and treatment of TB. The physicochemical characterization revealed effective surface morphology and particles diameter, that is applicable for in vitro applications. The in vitro antimicrobial analysis revealed that the designed MSNs@GNPs has retained significantly lower minimal inhibitory concentration (MIC) values and can effectively demolish mycobacterium tuberculosis (Mtb). Furthermore, the diagnosis efficiency of the MSNs@GNPs was evaluated by calorimetric analysis. Which demonstrates that MSNs@GNPs can be used for rapid diagnosis of the tuberculosis when applied on in vitro culture of the Mtb. The current study needs further verification on human's clinical samples from tuberculosis patients. However, MSNs@GNPs can be a versatile clinical approach for the rapid diagnosis and clinical treatment of the tuberculosis. [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]

Hemoglobin Constant Spring (Hb CS) and Hb Pakse' (PS) are the common non-deletional α+-thalassemia found in Thailand. These two variants can cause severe thalassemia syndromes, especially in fetus and neonate. Molecular diagnosis is the only confirmatory method because Hb CS and Hb PS are usually missed by routine screening and Hb analysis. Therefore, we aimed to develop rapid direct PCR for the diagnosis of Hb CS and PS genes. Multiplex direct PCR assays for identifying the Hb CS and PS genes in whole blood (WB) and amniotic fluid (AF) specimens were developed. The assays were firstly validated on 290 unrelated whole blood specimens. Hb CS and PS carriers were identified in 67 (23.1%) and 6 (2.1%) cases, respectively. A 100% concordant result as compared to routine PCR assay was observed. The direct PCR assays have been applied successfully for prenatal diagnosis in two families. The result showed that the fetuses were affected by homozygous Hb CS and compound heterozygous Hb CS/Hb PS. Accurate prenatal diagnosis of these families was observed using the newly developed assays. These assays should be applicable in routine thalassemia diagnostics as well as in the large-scale screening of Hb CS and PS in the region. [ABSTRACT FROM AUTHOR]

Leishmania belongs to a genus of the protozoan parasites that causes leishmaniasis, and includes cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). In this case, Leishmania amastigotes were found on cytomorphology examination of the bone marrow specimen, followed by 1,076 Leishmania donovani reads using metagenomic next generation sequencing (mNGS). Since being definitely diagnosed with VL/HIV coinfection, the patient was treated with liposomal amphotericin B as the parasite-resistant therapy and was discharged after clinical cure. But nearly a year later, on the mNGS follow-up, L. donovani was detected in the patient’s blood plasma specimen with 941 reads, suggesting that a relapse of leishmaniasis had occurred. These results indicate that leishmaniasis still exists in China and may represent a public health concern. This case could be helpful in the differential diagnosis of leishmaniasis, and for determining disease progression, prevention, and control of vectors and reservoir hosts.

The prevalence of Herpes simplex virus 2 (HSV2) is increasing at an alarming rate in the world. Most of the HSV2 cases are not diagnosed properly, although a range of molecular and serological diagnoses exist. Herein, we have reported a very rapid detection method specific for HSV2 using real-time PCR. The primers specific for HSV2 were designed using the Primer-BLAST tool and 120 base pairs of the polymerase gene were amplified using real-time PCR with SYBR Green dye. The designed primer pair was found highly efficient in detecting only HSV2 DNA, but not HSV1. The threshold cycle (Ct) value for HSV2 reactions by designed primers was found to be an average of 22.55 for a standard copy number of viral DNA that may denote the efficiency of the primers. The melting temperature (Tm) of the amplicon using designed primers (82.6 0C) was also higher than that using reference primers (about 78 0C), indicating the high GC content of the amplified template. The designed primer pair will help clinicians to detect the HSV2 DNA specifically and diagnose the associated disease rapidly.

ABSTRACT Emerging SARS-CoV-2 (SC-2) variants with increased infectivity and vaccine resistance are of major concern. Rapid identification of such variants is important for the public health decision making and to provide valuable data for epidemiological and policy decision making. We developed a multiplex reverse transcriptase quantitative PCR (RT-qPCR) assay that can specifically identify and differentiate between the emerging B.1.1.7 and B.1.351 SC-2 variants. In a single assay, we combined four reactions—one that detects SC-2 RNA independently of the strain, one that detects the D3L mutation, which is specific to variant B.1.1.7, one that detects the 242 to 244 deletion, which is specific to variant B.1.351, and the fourth reaction, which identifies the human RNAseP gene, serving as an endogenous control for RNA extraction integrity. We show that the strain-specific reactions target mutations that are strongly associated with the target variants and not with other major known variants. The assay’s specificity was tested against a panel of respiratory pathogens (n = 16), showing high specificity toward SC-2 RNA. The assay’s sensitivity was assessed using both in vitro transcribed RNA and clinical samples and was determined to be between 20 and 40 viral RNA copies per reaction. The assay performance was corroborated with Sanger and whole-genome sequencing, showing complete agreement with the sequencing results. The new assay is currently implemented in the routine diagnostic work at the Central Virology Laboratory, and may be used in other laboratories to facilitate the diagnosis of these major worldwide-circulating SC-2 variants. IMPORTANCE This study describes the design and utilization of a multiplex reverse transcriptase quantitative PCR (RT-qPCR) to identify SARS-COV-2 (SC2) RNA in general and, specifically, to detect whether it is of lineage B.1.1.7 or B.1.351. Implementation of this method in diagnostic and research laboratories worldwide may help the efforts to contain the COVID-19 pandemic. The method can be easily scaled up and be used in high-throughput laboratories, as well as small ones. In addition to immediate help in diagnostic efforts, this method may also help in epidemiological studies focused on the spread of emerging SC-2 lineages.