Diagnostic Assays Research Articles

Non-Invasive Determination of the Paternal Inheritance in Pregnancies at Risk for β-Thalassaemia by Analyzing Cell-Free Fetal DNA Using Targeted Next-Generation Sequencing

Non-invasive prenatal testing (NIPT) has been widely adopted for the screening of chromosomal abnormalities; however, its adoption for monogenic disorders, such as β-thalassaemia, has proven challenging. Haemoglobinopathies are the most common monogenic disorders globally, with β-thalassaemia being particularly prevalent in Cyprus. This study introduces a non-invasive prenatal haplotyping (NIPH) assay for β-thalassaemia, utilizing cell-free DNA (cfDNA) from maternal plasma. The assay determines paternal inheritance by analyzing highly heterozygous single-nucleotide variants (SNVs) in the β-globin gene cluster. To identify highly heterozygous SNVs in the population, 96 randomly selected samples were processed using Illumina DNA-prep NGS chemistry. A custom, high-density NGS genotyping panel, named HAPLONID, was designed with 169 SNVs, including 15 common pathogenic ones. The AmpliSeq for Illumina assay was then applied to cfDNA to evaluate the panel’s efficiency in performing NIPT for β-thalassaemia. Analysis revealed 219 highly polymorphic SNVs, and the sequencing of 17 families confirmed successful paternal allele determination. The NIPH assay demonstrated 100% success in diagnostic interpretation. This study achieved the advancement of an integrated NGS-NIPT assay for β-thalassaemia, bringing it one step closer to being a diagnostic assay and thereby enabling a reduction in the number of risky invasive prenatal sampling procedures in Cyprus and elsewhere.

Clinical evaluation of long-read sequencing-based episignature detection in developmental disorders

BackgroundA subset of developmental disorders (DD) is characterized by disease-specific genome-wide methylation changes. These episignatures inform on the underlying pathogenic mechanisms and can be used to assess the pathogenicity of genomic variants as well as confirm clinical diagnoses. Currently, the detection of these episignature requires the use of indirect methylation profiling methodologies. We hypothesized that long-read whole genome sequencing would not only enable the detection of single nucleotide variants and structural variants but also episignatures.MethodsGenome-wide nanopore sequencing was performed in 40 controls and 20 patients with confirmed or suspected episignature-associated DD, representing 13 distinct diseases. Following genomic variant and methylome calling, hierarchical clustering and dimensional reduction were used to determine the compatibility with microarray-based episignatures. Subsequently, we developed a support vector machine (SVM) for the detection of each DD.ResultsNanopore sequencing-based methylome patterns were concordant with microarray-based episignatures. Our SVM-based classifier identified the episignatures in 17/19 patients with a (likely) pathogenic variant and none of the controls. The remaining patients in which no episignature was identified were also classified as controls by a commercial microarray assay. In addition, we identified all underlying pathogenic single nucleotide and structural variants and showed haplotype-aware skewed X-inactivation evaluation directs clinical interpretation.ConclusionThis proof-of-concept study demonstrates nanopore sequencing enables episignature detection. In addition, concurrent haplotyped genomic and epigenomic analyses leverage simultaneous detection of single nucleotide/structural variants, X-inactivation, and imprinting, consolidating a multi-step sequential process into a single diagnostic assay.

A Serological Assay for the Detection of Antibodies Generated Against California Serogroup Virus Infection.

The California serogroup (CSG) viruses are orthobunyaviruses endemic in North America and responsible for the second most common cause of mosquito-borne viral encephalitis in the United States. As the CSG viruses have been neglected and are poorly studied, there are no commercial diagnostic serological assays or reagents available for detection. Therefore, diagnostic laboratories have had to rely on the development of their own in-house serological assays. To develop serological assays, antigenic materials such as recombinant protein, virus-like particles (VLP), or inactivated virus can be used in the IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) to detect CSG virus-specific IgM antibodies. All positive MAC-ELISA results should be confirmed by a well-defined gold standard test method such as the plaque-reduction neutralization test (PRNT).

Homologous recombination deficiency in ovarian cancer: Global expert consensus on testing and a comparison of companion diagnostics.

Poly (ADP ribose) polymerase inhibitors (PARPis) are a treatment option for patients with advanced high-grade serous or endometrioid ovarian carcinoma (OC). Recent guidelines have clarified how homologous recombination deficiency (HRD) may influence treatment decision-making in this setting. As a result, numerous companion diagnostic assays (CDx) have been developed to identify HRD. However, the optimal HRD testing strategy is an area of debate. Moreover, recently published clinical and translational data may impact how HRD status may be used to identify patients likely to benefit from PARPi use. We aimed to extensively compare available HRD CDx and establish a worldwide expert consensus on HRD testing in primary and recurrent OC. A group of 99 global experts from 31 different countries was formed. Using a modified Delphi process, the experts aimed to establish consensus statements based on a systematic literature search and CDx information sought from investigators, companies and/or publications. Technical information, including analytical and clinical validation, were obtained from 14 of 15 available HRD CDx (7 academic; 7 commercial). Consensus was reached on 36 statements encompassing the following topics: 1) the predictive impact of HRD status on PARPi use in primary and recurrent OC; 2) analytical and clinical validation requirements of HRD CDx; 3) resource-stratified HRD testing; and 4) how future CDx may include additional approaches to help address unmet testing needs. This manuscript provides detailed information on currently available HRD CDx and up-to-date guidance from global experts on HRD testing in patients with primary and recurrent OC.

Development and Clinical Detection of Rapid Molecular Diagnostic System for Pathogenic Dermatophytes of Tinea Capitis of Multiple Centres in China.

Tinea capitis remains a common fungal infection in children worldwide. Species identification is critical for determining the source of infection and reducing transmission. In conventional methods, macro- and microscopic analysis is time-consuming and results in slow fungal growth or low specificity. We propose a rapid real-time diagnostic PCR method that allows species-specific identification of dermatophytes, including the Microsporum canis complex, Trichophyton mentagrophytes complex, Trichophyton rubrum complex and Trichophyton tonsurans, in patients with tinea capitis. Hair and scrapings samples were collected from 231 patients with tinea capitis who were positive for fungal elements via direct microscopy with potassium hydroxide. Each sample was subjected to a two-step real-time PCR (RT-PCR) assay, which was designed on the basis of differences in the DNA fragments of the internal transcribed spacer (ITS) and β-tubulin covering the Microsporum canis complex, T. mentagrophyte complex, T. rubrum complex, T. tonsurans, T. verrucosum, T. schoenleinii and N. gypseum. In total, 186/231 samples (80.52%) were positive for fungal culture. The two-step RT-PCR was positive in 215/231 samples (93.07%), among which 179 were culture positive. The combined efficacy was 96.81%, which was significantly different when the RT-PCR assays were performed in parallel with fungal culture. A total of 126 samples (54.55%) were identified as Microsporum canis by fungal culture, among which the positive rate of M. canis complex RT-PCR was 97.62% (123/126). A total of 45 samples were negative for fungal culture, of which 80.0% (36/45) were positive by RT-PCR, and the percentage of M. canis complex-positive samples was 53.33% (24/45). The RT-PCR assays were negative for 16/231 samples, among which 7 were culture positive, including M. canis (n = 3), T. violaceum (n = 3) and N. gypseum (n = 1). We developed a new diagnostic assay system using a rapid real-time TaqMan PCR assay with specific primers that can be applied in routine laboratory practice for hair and skin samples of tinea capitis to detect dermatophytes and increase diagnostic efficiency.

Designing of immunodiagnostic assay using polyclonal antibodies for detection of Enteropathogenic Escherichia coli strains.

Enteropathogenic Escherichia coli (EPEC) is a significant bacterial pathogen that causes infantile diarrhea, particularly in low- and middle-income countries. The lack of a reliable diagnostic method greatly contributes to the increased occurrence and severity of the disease. This study aimed at developing of a cost-effective, rapid, and efficient immunodiagnostic assay for detecting EPEC infection. Lipopolysaccharide (LPS) was extracted from overnight EPEC cultures and combined with alum adjuvant, and then injected into mice for three rounds of immunizations. Subsequently, sera were collected after each immunization and utilized in agglutination assays conducted on glass slides. Both the LPS and colonies of the EPEC isolate used for LPS preparation were employed in these agglutination assays. To evaluate the assay's performance, a total of 34 bacteria, which comprise pathogenic, non-diarrheic E. coli and non-E. coli pathogenic bacteria were used. The developed assay detected EPEC, which yielded positive reactions within 6 minutes on average for both purified LPS and bacterial isolates. The assay exhibited 100% sensitivity and a 95.83% specificity for the detection of EPEC local isolates. Moreover, the assay also detected a low number of bacteria forming units (104X 104 CFU/ml) in spiked fecal samples. This study conclusively confirms that the developed immunodiagnostic assay possesses multiple favorable characteristics, including user-friendliness, high sensitivity, high specificity, cost-effectiveness, and time-efficiency. Hence, this assay can be used as ideal diagnostic assay, which is highly suitable for the detection and screening of EPEC infection in both humans and cattle in one health perspective of resource-limited laboratories.

Development of reverse transcription recombinase polymerase amplification assay for rapid diagnostics of Peanut mottle virus

Development of reverse transcription recombinase polymerase amplification assay for rapid diagnostics of Peanut mottle virus

Ultrasensitive Protein Aggregate Quantification Assays for Neurodegenerative Diseases on the Simoa Platform.

Nanoscale aggregates play a key role in the pathogenesis of neurodegenerative disorders such as Alzheimer's and Parkinson's disease. However, quantifying these aggregates in complex biological samples, such as biofluids and postmortem brain tissue, has been challenging due to their low concentration and small size, necessitating the development of methods with high sensitivity and specificity. Here, we have developed ultrasensitive assays utilizing the Quanterix Simoa platform to detect α-synuclein, β-amyloid and tau aggregates, including those with common posttranslational modifications such as truncation of α-synuclein and AT8 phosphorylation of tau aggregates. All assays had a detection limit in the low pM range. As a part of this work, we developed silica-nanoparticle calibrators, allowing for the quantification of all aggregates. These assays were validated for aggregate and target specificity through denaturation and cross-reactivity experiments. We then applied these assays to brain homogenate samples from Alzheimer's disease and control samples, demonstrating their applicability to postmortem tissue. Lastly, we explored the potential of these assays for blood-based diagnostics by detecting aggregates in serum samples from early Alzheimer's disease patients.

Technical evaluation of the InBios Strongy Detect IgG ELISA assay for the diagnosis of Strongyloides stercoralis infection

Abstract Background Strongyloidiasis is a neglected tropical disease (NTD) caused by the soil-transmitted helminth Strongyloides stercoralis, recently included in the 2030 targets of the World Health Organization for the control of STHs. Assessment of infection prevalence is fundamental for decision-making about the implementation of control programs, but diagnostic assays to be applied in such context require evaluation. Seroassays based on recombinant antigens, which could be produced in a standardized and scalable manner, are particularly appealing for use in control programs. In this study, we performed a technical evaluation of the InBios Strongy Detect IgG ELISA, based on recombinant antigens NIE and SsIR, which has shown promising for field use. Methods A total of 46 plasma samples from Ethiopian children were used for this technical evaluation. Repeatability was evaluated on duplicate samples per plate, on four plates per day for 3 days using Bland–Altman plots, analysis of residuals, and variance components analysis. Three samples were selected for evaluation of the uniformity of test results within a single plate (border effect) by two-sided t-test. Correlation between samples and internal ELISA positive controls was analyzed using Spearman’s rank correlation coefficient applied on the results of 777 samples analyzed with the assay in a previous field-based study. Results Within and between plate residuals ranged from −0.05 to + 0.05 and −0.1 to + 0.1, respectively. Total variance was estimated at 0.327; 99.6% of variation could be attributed to the samples. There was no systematic border effect and a negligible correlation between positive internal control and samples results (R2 = 0.213; p < 0.001). Conclusion The results obtained in this study, in highly controlled conditions, point toward the InBios Strongy Detect IgG ELISA assay being reproducible, with no systematic border effect. These results encourage further assay’s development and evaluation for use in practice, including determination of preset cutoff values for positivity, which is currently not provided. Graphical Abstract

Comparative evaluation of rapid diagnostic test and PCR-based diagnostic assay for identification of trypanosomes in cattle of Apac and Kiryandongo districts, Uganda: A cross sectional study

BackgroundDiagnostic tests and knowledge of their diagnostic accuracies are important for animal trypanosomosis surveillance and treatment.Methodology: A cross-sectional study was conducted in November 2021 to compare the performance of rapid diagnostic test (RDT) and PCR-based assay for the detection of trypanosome infections. Random sampling and probability proportional to size sampling were used to sample study households and animals from the sampled household respectively. Animals were screened for the presence of trypanosomes using both tests. A total of 100 cattle (52 from Apac and 48 from Kiryandongo districts) were included in the study. The percentage of positive tests, sensitivity, and specificity of the tests using mini PCR as a reference were computed. Cohen’s kappa statistics was computed to assess how well the rapid diagnostic test agrees with the mini PCR. McNemar’s statistic assessed if the proportion of positive tests identified by mini PCR significantly differed from that identified by the rapid diagnostic test.ResultsThe mini PCR test detected 31.2% Trypanosome spp positive samples in Kiryandongo while it detected only 5.7% positive samples in Apac district. The rapid diagnostic test (RDT) analysis detected 91.6% Trypanosome spp positive samples in Kiryandongo district and only 46.1% in Apac district. T. congolense was the most common Trypanosoma species identified in Apac (36.5%) and Kiryandongo (47.9%) by the RDT. The percentage of each of the Trypanosome species (T.vivax, T.congolense, and mixed infection of T.vivax and T. congolense) detected by RDT differed significantly (p < 0.001) between the two districts. The RDT had a high sensitivity of 94.4% (95% Confidence Interval (CI): 72.7–99.9) but a very low specificity of 36.6% (95% CI: 26.2–48.0). The kappa test showed evidence of only a slight agreement (kappa = 0.1547, Accuracy = 0.48 (95% Confidence Interval (CI): 0.379,0.5822) between the two tests. The observed agreement between the tests was 47% while the expected agreement was 37.84%.ConclusionThis study found high sensitivity but low specificity of RDT using mini PCR as a reference.

Genome-wide association studies unveil major genetic loci driving insecticide resistance in Anopheles funestus in four eco-geographical settings across Cameroon

BackgroundInsecticide resistance is jeopardising malaria control efforts in Africa. Deciphering the evolutionary dynamics of mosquito populations country-wide is essential for designing effective and sustainable national and subnational tailored strategies to accelerate malaria elimination efforts. Here, we employed genome-wide association studies through pooled template sequencing to compare four eco-geographically different populations of the major vector, Anopheles funestus, across a South North transect in Cameroon, aiming to identify genomic signatures of adaptive responses to insecticides.ResultsOur analysis revealed limited population structure within Northern and Central regions (FST<0.02), suggesting extensive gene flow, while populations from the Littoral/Coastal region exhibited more distinct genetic patterns (FST>0.049). Greater genetic differentiation was observed at known resistance-associated loci, resistance-to-pyrethroids 1 (rp1) (2R chromosome) and CYP9 (X chromosome), with varying signatures of positive selection across populations. Allelic variation between variants underscores the pervasive impact of selection pressures, with rp1 variants more prevalent in Central and Northern populations (FST>0.3), and the CYP9 associated variants more pronounced in the Littoral/Coastal region (FST =0.29). Evidence of selective sweeps was supported by negative Tajima’s D and reduced genetic diversity in all populations, particularly in Central (Elende) and Northern (Tibati) regions. Genomic variant analysis identified novel missense mutations and signatures of complex genomic alterations such as duplications, deletions, transposable element (TE) insertions, and chromosomal inversions, all associated with selective sweeps. A 4.3 kb TE insertion was fixed in all populations with Njombe Littoral/Coastal population, showing higher frequency of CYP9K1 (G454A), a known resistance allele and TE upstream compared to elsewhere.ConclusionOur study uncovered regional variations in insecticide resistance candidate variants, emphasizing the need for a streamlined DNA-based diagnostic assay for genomic surveillance across Africa. These findings will contribute to the development of tailored resistance management strategies crucial for addressing the dynamic challenges of malaria control in Cameroon.

Development of a LAMP protocol to identify the parasitoid Carcelia iliaca from oak processionary moth (Thaumetopoea processioneae) larval tissue to understand and enhance biocontrol management plans

Abstract Oak processionary moth (Thaumetopoea processionea) (OPM) Linnaeus, 1758 (Lepidoptera: Notodontidae) is a serious forestry pest and risk to public health in the UK. The economic and environmental cost of chemical pesticides in managing OPM has driven the need for sustainable, strategies which fit into integrated pest management frameworks, including the use of novel biocontrol methods such as conservation biocontrol. Carcelia iliaca Ratzeburg, 1840 (Diptera: Tachinidae), a specific parasitoid of OPM, is currently the main biocontrol agent of the UK OPM population. However, basic information on C. iliaca life history and rates of parasitism are currently lacking, partly driven by the risks OPM pose to human health, making both study and incorporation of biocontrol into management plans difficult. Here, we design and validate a molecular diagnostic assay based on loop‐mediated isothermal amplification (LAMP) to detect C. iliaca from OPM larval tissue samples collected in the field, overcoming the challenges of studying problematic invasive species such as these. To assess assay performance, diagnostic sensitivity, which was 91%, and specificity, which was 75%, are used alongside limit of detection (600 pg). We discuss the wider applications for LAMP as a cost‐effective tool for studying the natural enemies of insect pests which can be used to inform conservation biocontrol management strategies.

Single and dual RPA-CRISPR/Cas assays for point-of-need detection of Stewart's wilt pathogen (Pantoea stewartii subsp. stewartii) of corn and Maize dwarf mosaic virus.

Pantoea stewartii subsp. stewartii and Maize dwarf mosaic virus (MDMV) infections severely affect corn productivity worldwide. Rapid point-of-need diagnoses of quarantine pathogens P. stewartii subsp. stewartii and MDMV are required for early detection, timely disease management and ensuring phytosanitary regulations. Recombinase polymerase amplification (RPA) is an isothermal technique suitable for rapid diagnostics using minimally processed samples. Integrating CRISPR/Cas collateral activities with the RPA assays further enhances the specificity and sensitivity of molecular toolkits for diagnostic assays. RPA-CRISPR/Cas12a assay targeting the intergenic spacer region between capsular polysaccharide genes cpsA and cpsB of P. stewartii subsp. stewartii detected 1 × 10-6 ng DNA μL-1 using real-time fluorescence and blue light observation methods, and 1 × 10-4 ng DNA μL-1 using the lateral flow dipstick (LFD). Likewise, RPA-CRISPR/Cas13a assay detected MDMV coat protein (CP) gene with an ultrasensitive detection limit of 3.69 × 10-7 ng μL-1 using the real-time fluorescence and blue light observation methods, and 3.69 × 10-5 ng μL-1 using the LFD. The dual RPA-CRISPR/Cas assays detected both pathogens without compromising the speed and/or detection sensitivity of the single assays. The validated assays provide a useful and sensitive molecular tool for detecting two quarantine pathogens of maize within a minimal resource framework suitable for fast-tracking the containment strategies. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Increased Risk of Myositis-Specific and Myositis-Associated Autoantibodies After COVID-19 Pandemic and Vaccination: A Spanish Multicenter Collaborative Study.

Background: Emerging evidence suggests that SARS-CoV-2 infection and vaccines may trigger autoimmune responses in predisposed individuals. Idiopathic inflammatory myopathies (IIMs) are diseases with diverse clinical manifestations, often associated with myositis autoantibodies (MAs). Diagnosing IIM is challenging due to limitations in classification criteria and diagnostic assays. This study aimed to describe the incidence of IIM following SARS-CoV-2 infection or vaccination and compare rates between exposures. Methods: A multicenter observational study was conducted with 788 patients from 11 Spanish referral centers. A total of 1209 autoantibodies including myositis-specific autoantibodies (MSAs) and myositis-associated autoantibodies (MAAs), were analyzed using line blot immunoassay (LIA). Results: The study identified distinct patterns in aminoacyl-tRNA synthetase (ARS) antibody frequencies compared to pre-pandemic periods. Anti-PL-7 was the most prevalent ARS antibody (14.85%), while anti-Jo-1 was less frequent (7.23%). Anti-MDA5, commonly linked to SARS-CoV-2 infection, was detected in 11.68%. ANA positivity was observed in 60.66%, suggesting an autoimmune background. The most frequent diagnoses were anti-synthetase syndrome (ASSD) or IIM-non-ASSD (21.31%), followed by other systemic autoimmune diseases (SAIDs) (13.57%). Among the cohort, 91.13% received at least one dose of a messenger RNA (mRNA) COVID-19 vaccine, with a median of three doses per patient. Patients with prior SARS-CoV-2 infection or heterologous vaccination showed a higher frequency of multiple autoantibody positivity (p < 0.05), reflecting distinct immune signatures. Conclusions: This study provides valuable insights into the autoimmune risks and phenotypes associated with SARS-CoV-2 infection and vaccination, establishing a basis for further research on IIM and its link to MSAs and MAAs.

Development of a novel test of splenic function for use in a clinical diagnostic laboratory.

The spleen is prone to both physical damage and functional impairment, which can be difficult to detect before catastrophic complications occur. Currently available tests of splenic function are laborious, user-dependent and unreliable, so there is an unmet need for a reliable test offered routinely in diagnostic laboratories. In this study, we have assessed a simple flow cytometry-based method measuring high mannose glycans (HMGs) on erythrocytes, which has previously been proposed as a potential test of splenic function. We developed the test as a diagnostic assay using blood from a range of control and potentially hyposplenic samples, including people with sickle cell disease. HMG expression correlated well with manual pit counting, an established method for assessing splenic function (r = 0.6). A threshold of >36% difference compared to the mean of control samples was used to define hyposplenism. At this threshold, the test is 93% sensitive and 100% specific for detecting splenic dysfunction. The test was highly reproducible and stable in blood samples of up to 4 days old. This test is non-invasive with quantitative data output and requires significantly less operator time than other available techniques, making it a robust new clinical assay for determining splenic function.

Advances in diagnostic assays for Clostridioides difficile infection in adults

Clostridioides difficile (C. difficile) was a gram-positive anaerobic bacterium in the gut, exhibiting clinical manifestations ranging from mild diarrhoea to fatal pseudomembranous colitis. C. difficile infection (CDI) remains a serious public health problem and accounted for an estimated 360,075 cases in the United States in 2021. It has attracted the utmost attention of the world health organization (WHO). Since publication of a review of the diagnosis of CDI in adults, new clinical diagnostic assays have become available and clinical practice guidelines were updated. This paper presents a comprehensive review of contemporary laboratory diagnostic approaches for CDI in adult patients, with a focus on the utilisation and potential advancements of five sophisticated methodologies, CRISPR in conjunction with nucleic acid amplification tests (NAATs), gene sequencing technology, ultra-high performance liquid chromatography-mass spectrometry, Raman spectroscopy, and real-time cell analysis (RTCA). It can provide new perspectives and ideas for the early diagnosis of CDI in clinical settings.

Characterization of Shigella flexneri serotype 6 strains from geographically diverse low- and middle-income countries.

Shigella flexneri serotype 6 (Sf6) is one of the most common serotypes recovered from surveillance studies of moderate to severe diarrhea. Despite the clinical significance of Sf6, this serotype is understudied. In this work, we have performed both serotype-specific genomic and phenotypic comparisons of Sf6 isolates to one another and non-S. flexneri serotypes. Comparative genomic analyses identified significant nucleotide homology between Sf6 strains (n = 325), despite a broad range of collection timeframes and geographic locations. We identified Sf6 specific factors, including a potential novel Shigella virulence factor (type II secretion system). Additionally, we identified established Shigella virulence genes (ospG) and metabolic genes (rutABCDEFGR) that were absent in Sf6 strains while present in the majority of 728 non-Sf6 strains. Complete sequencing of 11 clinical Sf6 strains, demonstrated that the Sf6 virulence plasmid (pINV) is ~38 kb smaller than the average non-Sf6 pINV (~228 kb). Comparisons of S. flexneri species level antibiotic susceptibility highlighted that clinical Sf6 isolates from Africa in the Global Enteric Multicenter Study (GEMS) and Vaccine Impact on Diarrhea in Africa (VIDA) study demonstrated geographic, serotype-specific susceptibility pattern. Phenotypic analyses of Sf6 identified reduced intracellular invasion and cytokine induction from HT-29 cells, as well as reduced Ipa protein effector secretion, compared with S. flexneri serotype 2a strain 2457T. Together these data highlight conserved and unique serotype-specific genotypic and phenotypic features for Sf6. This level of conservation has not been noted for other S. flexneri serotypes and is promising for vaccine and diagnostic assays to provide global Sf6-specific coverage.IMPORTANCEShigellosis is an ongoing global public health crisis with >270 million annual episodes among all age groups; however, the greatest disease burden is among children in low- and middle-income countries (LMIC). The lack of a licensed Shigella vaccine and the observed rise in antimicrobial-resistant Shigella spp. highlights the urgency for effective preventative and interventional strategies. The inclusion of S. flexneri serotype 6 (Sf6) is a necessary component of a multivalent vaccine strategies based on its clinical and epidemiological importance. Given the genomic diversity of Sf6 compared with other S. flexneri serotypes and Sf6 unique O-antigen core structure, serotype-specific characterization of Sf6 is a critical step to inform Shigella-directed vaccine and alternative therapeutic designs. Herein, we identified conserved genomic content among a large collection of temporally and geographically diverse Sf6 clinical isolates and characterized genotypic and phenotypic properties that separate Sf6 from non-Sf6 S. flexneri serotypes.

Plasma proteomics for novel biomarker discovery in childhood tuberculosis.

Failure to rapidly diagnose tuberculosis disease (TB) and initiate treatment is a driving factor of TB as a leading cause of death in children. Current TB diagnostic assays have poor performance in children, and identifying novel non-sputum-based TB biomarkers to improve pediatric TB diagnosis is a global priority. We sought to develop a plasma biosignature for TB by probing the plasma proteome of 511 children stratified by TB diagnostic classification and HIV status from sites in four low- and middle-income countries, using high-throughput data-independent acquisition mass-spectrometry (DIA-PASEF-MS). We identified 47 proteins differentially regulated (BH adjusted p-values < 1%) between children with microbiologically confirmed TB and children with non-TB respiratory diseases (Unlikely TB). We further employed machine learning to derive three parsimonious biosignatures encompassing 4, 5, or 6 proteins that achieved AUCs of 0.86-0.88 all of which exceeded the minimum WHO target product profile accuracy thresholds for a TB screening test (70% specificity at 90% sensitivity, PPV 0.65-0.74, NPV 0.92-0.95). This work provides insights into the unique host response in pediatric TB disease, as well as a non-sputum biosignature that could reduce delays in TB diagnosis and improve detection and management of TB in children worldwide.

Survey of anatomic and clinical pathology services and infrastructure in Nigeria.

The paucity of data regarding the availability and extent of diagnostic medical services across sub-Saharan Africa hinders appropriate allocation of resources to improve health care in these regions. We assessed anatomic pathology (AP) and clinical pathology (CP) services in Nigeria, one of the most populous and fastest-growing countries in the world. Two individual surveys (AP focused and CP focused) were developed by subject matter experts and administered to individuals involved in pathology and laboratory medicine diagnostic services at hospitals and laboratories across Nigeria between June and August 2022 using the American Society for Clinical Pathology email listserv. A total of 75 responses (29 AP and 46 CP) were received from 48 unique laboratories. Twenty-four sites provided AP services and 35 provided CP services. Eleven respondents performed both AP and CP services. Among AP services, basic surgical and cytopathology capabilities were available at most sites; however, the availability of automated technologies (eg, automated sample processing and staining) was more variable. Advanced diagnostic techniques, (eg, immunohistochemistry, human papillomavirus testing, molecular diagnostics) were rarely performed. The most frequently available CP services included hematology, microbiology, and chemistry. Microbiology services appeared to be among the most robust laboratory medicine services, particularly parasitology and bacteriology testing. Similar to AP services, more advanced diagnostic assays, such as flow cytometry, cytogenetics, and molecular testing, were largely unavailable. These findings augment earlier studies and identify gaps that should be prioritized from a policy perspective to improve medical services and the overall health care infrastructure in Nigeria.

Leptin Levels in Acute and Recovered Eating Disorders: An Arm-Based Network Meta-Analysis.

This study aimed to provide a BMI-adjusted meta-analytical calculation of blood leptin levels across different eating disorders (EDs) including anorexia nervosa (AN), bulimia nervosa (BN), binge eating disorder (BED), recovered EDs, and healthy controls (HCs). The goal was to understand BMI-independent leptin alterations and their potential as biomarkers. PubMed and ClinicalTrials.gov were searched for studies reporting serum leptin in AN, BN, BED, or recovered EDs. A multilevel network meta-analysis using a linear mixed-effects meta-regression model, adjusting for BMI, sex, and assay type, was performed on 146 studies (5048 patients, 3525 controls). Significant differences in leptin levels were found across EDs. AN patients exhibited the lowest leptin levels, while BED patients had the highest. BN and recovered AN patients had leptin levels similar to AN, significantly lower than HCs. BMI, sex, and assay type were significant covariates. The model accounted for heterogeneity due to diagnostic criteria, assay types, and study-level differences. Leptin levels in EDs are significantly altered beyond BMI effects, suggesting disease-specific factors. These findings support leptin's potential as a biomarker for ED staging and prognosis. Further research is needed to explore leptin's role in ED pathogenesis and trajectory, to identify subpopulations and improve clinical interventions.