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The accurate detection and quantification of biological species that are rarely present but potentially devastating is of paramount importance for the life sciences, biosecurity, food safety, and environmental monitoring. Consequently, there has been significant interest in the sensitive and accurate detection of nucleic acids, leveraging both chemical and biological methods. Among these, quantitative polymerase chain reaction (qPCR) is regarded as the gold standard due to its sensitivity and precision in identifying specific nucleic acid targets. Despite the widespread adoption of qPCR for nucleic acid detection, the analysis of qPCR data typically depends on the use of calibrated standard curves and a threshold method to interpret signal measurements. In this study, we use a stochastic simulation to show the limitations of the threshold method due to its assumptions on amplification kinetics. We propose a new approach for the absolute quantification of nucleic acids that overcomes these limitations by reconstructing the efficiency profile across amplification cycles and using cumulative amplification folds to build a standard curve, thus avoiding the constant efficiency assumption. Our method, validated through experiments with nucleic acid amplification in the presence of potent inhibitors, demonstrates improved accuracy in quantifying nucleic acids, avoiding the systematic errors of the threshold method. This innovation enhances the reliability of nucleic acid quantification, especially where traditional methods struggle with kinetic variability. [ABSTRACT FROM AUTHOR]

The obligate intracellular bacterium Chlamydia has a unique developmental cycle that alternates between two contrasting cell types. With a hardy envelope and highly condensed genome, the small elementary body (EB) maintains limited metabolic activities yet survives in extracellular environments and is infectious. After entering host cells, EBs differentiate into larger and proliferating reticulate bodies (RBs). Progeny EBs are derived from RBs in late developmental stages and eventually exit host cells. How expression of the chlamydial genome consisting of nearly 1,000 genes governs the chlamydial developmental cycle is unclear. A previous microarray study identified only 29 Chlamydia trachomatis immediate early genes, defined as genes with increased expression during the first hour postinoculation in cultured cells. In this study, we performed more sensitive RNA sequencing (RNA-Seq) analysis for C. trachomatis cultures with high multiplicities of infection. Remarkably, we observed well over 700 C. trachomatis genes that underwent 2- to 900-fold activation within 1 hour postinoculation. Quantitative reverse transcription real-time PCR analysis was further used to validate the activated expression of a large subset of the genes identified by RNA-Seq. Importantly, our results demonstrate that the immediate early transcriptome is over 20 times more extensive than previously realized. Gene ontology analysis indicates that the activated expression spans all functional categories. We conclude that over 70% of C. trachomatis genes are activated in EBs almost immediately upon entry into host cells, thus implicating their importance in initiating rapid differentiation into RBs and establishing an intracellular niche conducive with chlamydial development and growth.

Purpose: To investigate the possible antimicrobial activity of glycine air polishing by comparing peri-implant microbiota before and after treatment. Materials and Methods: A total of 15 patients who received implant-supported full-arch fixed rehabilitations were included. After prosthesis removal (T0), Plaque Index (PI), probing depth (PD), and bleeding on probing (BOP) were recorded. In each hemiarch, the implant with the highest PD score was selected for microbiologic sample collection from the peri-implant sulcus (T0). All patients received two different hygienic protocols (randomly administered, one per each hemiarch): glycine air-polishing (G) and cleansing with cotton pellets soaked in saline (C). At 7 days (T1) and 3 months (T2) after the intervention, PI and BOP were recorded, and new microbiologic samples were taken. Traditional polymerase chain reaction (PCR) and quantitative PCR real-time were employed for microbiologic analysis to investigate how the presence of different bacterial species varied according to the hygienic treatment performed. Results: Treatment G provided a significantly higher PI score reduction around implants compared to treatment C (P = .015). No statistical difference was found in the microbial population around G and C implant sites, with Tannerella forsythia being the most commonly detected bacterial species in both G and C groups. No statistical differences were found between the antimicrobial activity of treatments C and G. Conclusions: Glycine powder air polishing is a valid method for professional hygienic care of implants and was more effective in PI reduction compared to the control treatment. However, its antimicrobial efficacy cannot be confirmed by the outcomes of the present study. [ABSTRACT FROM AUTHOR]

OBJECTIVES: Viral lower respiratory tract infection (vLRTI) contributes to substantial morbidity and mortality in children. Diagnosis is typically confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) of nasopharyngeal specimens in hospitalized patients; however, it is unknown whether nasopharyngeal detection accurately reflects presence of virus in the lower respiratory tract (LRT). This study evaluates agreement between viral detection from nasopharyngeal specimens by RT-PCR compared with metagenomic next-generation RNA sequencing (RNA-Seq) from tracheal aspirates (TAs). DESIGN: This is an analysis of of a seven-center prospective cohort study. SETTING: Seven PICUs within academic childrens hospitals in the United States. PATIENTS: Critically ill children (from 1 mo to 18 yr) who required mechanical ventilation via endotracheal tube for greater than or equal to 72 hours. INTERVENTIONS: We evaluated agreement in viral detection between paired upper and LRT samples. Results of clinical nasopharyngeal RT-PCR were compared with TA RNA-Seq. Positive and negative predictive agreement and Cohens Kappa were used to assess agreement. MEASUREMENTS AND MAIN RESULTS: Of 295 subjects with paired testing available, 200 (68%) and 210 (71%) had positive viral testing by RT-PCR from nasopharyngeal and RNA-Seq from TA samples, respectively; 184 (62%) were positive by both nasopharyngeal RT-PCR and TA RNA-Seq for a virus, and 69 (23%) were negative by both methods. Nasopharyngeal RT-PCR detected the most abundant virus identified by RNA-Seq in 92.4% of subjects. Among the most frequent viruses detected, respiratory syncytial virus demonstrated the highest degree of concordance (κ = 0.89; 95% CI, 0.83-0.94), whereas rhinovirus/enterovirus demonstrated lower concordance (κ = 0.55; 95% CI, 0.44-0.66). Nasopharyngeal PCR was more likely to detect multiple viruses than TA RNA-Seq (54 [18.3%] vs 24 [8.1%], p ≤ 0.001). CONCLUSIONS: Viral nucleic acid detection in the upper versus LRT reveals good overall agreement, but concordance depends on the virus. Further studies are indicated to determine the utility of LRT sampling or the use of RNA-Seq to determine LRTI etiology.

We report a cluster of clade I monkeypox virus infections linked to sexual contact in the Democratic Republic of the Congo. Case investigations resulted in 5 reverse transcription PCR-confirmed infections; genome sequencing suggest they belonged to the same transmission chain. This finding demonstrates that mpox transmission through sexual contact extends beyond clade IIb.

Detection sensitivity of aquatic species using environmental DNA (eDNA) generally decreases in turbid water but is poorly characterized. In this study, eDNA detection targeted delta smelt (Hypomesus transpacificus), a critically endangered estuarine fish associated with turbid water. eDNA sampling in the field was first paired with a trawl survey. Species-specific detection using a Taqman qPCR assay showed concordance between the methods, but a weak eDNA signal. Informed by the results of field sampling, an experiment was designed to assess how turbidity and filtration methods influence detection of a rare target. Water from non-turbid (5 NTU) and turbid (50 NTU) estuarine sites was spiked with small volumes (0.5 and 1 mL) of water from a delta smelt tank to generate low eDNA concentrations. Samples were filtered using four filter types: cartridge filters (pore size 0.45 μm) and 47 mm filters (glass fiber, pore size 1.6 μm and polycarbonate, pore sizes 5 and 10 μm). Prefiltration was also tested as an addition to the filtration protocol for turbid water samples. eDNA copy numbers were analyzed using a censored data method for qPCR data. The assay limits and lack of PCR inhibition indicated an optimized assay. Glass fiber filters yielded the highest detection rates and eDNA copies in non-turbid and turbid water. Prefiltration improved detection in turbid water only when used with cartridge and polycarbonate filters. Statistical analysis identified turbidity as a significant effect on detection probability and eDNA copies detected; filter type and an interaction between filter type and prefilter were significant effects on eDNA copies detected, suggesting that particulate-filter interactions can affect detection sensitivity. Pilot experiments and transparent criteria for positive detection could improve eDNA surveys of rare species in turbid environments.

BACKGROUND: Multiplex PCR amplifies numerous targets in a single tube reaction and is essential in molecular biology and clinical diagnostics. One of its most important applications is in the targeted sequencing of pathogens. Despite this importance, few tools are available for designing multiplex primers. RESULTS: We developed primerJinn, a tool that designs a set of multiplex primers and allows for the in silico PCR evaluation of primer sets against numerous input genomes. We used primerJinn to create a multiplex PCR for the sequencing of drug resistance-conferring gene regions from Mycobacterium tuberculosis, which were then successfully sequenced. CONCLUSIONS: primerJinn provides a user-friendly, efficient, and accurate method for designing multiplex PCR primers for targeted sequencing and performing in silico PCR. It can be used for various applications in molecular biology and bioinformatics research, including the design of assays for amplifying and sequencing drug-resistance-conferring regions in important pathogens.

Newcastle disease (ND) is among the most important poultry diseases worldwide. It is the major threat to poultry production in Africa and causes major economic losses for both local and commercial chickens. To date, half of ND class II genotypes have been reported in Africa (I, IV, V, VI, VII, XI, XIII, XIV, XVII, XVIII, and XXI). The information on the circulating NDV genotypes is still scarce despite the endemic nature of ND in most countries on the African continent.A total of 659 oro-cloacal swabs were collected from local chickens in Mawenzi live bird market located in Morogoro, Tanzania, between June 2020 and May 2021. Newcastle disease virus was detected by using reverse transcription real-time polymerase chain reaction (RT-qPCR) and conventional PCR followed by sequencing of PCR products. The prevalence of NDV in the surveilled live bird markets was 23.5%. Sequencing and phylogenetic analysis revealed the presence of sub-genotype VII.2. The detected sub-genotype VII.2 has phylogenetic links to Zambian NDV strains implying a Southeast dissemination of the virus, considering that it was first detected in Mozambique. This study underscores the need of active NDV surveillance to determine the distribution of this NDV genotype in the country and monitor its spread and contribution to the emergence of new ND viruses.

Microbial infections of the brain can lead to dementia, and for many decades microbial infections have been implicated in Alzheimers disease (AD) pathology. However, a causal role for infection in AD remains contentious, and the lack of standardized detection methodologies has led to inconsistent detection/identification of microbes in AD brains. There is a need for a consensus methodology; the Alzheimers Pathobiome Initiative aims to perform comparative molecular analyses of microbes in post mortem brains versus cerebrospinal fluid, blood, olfactory neuroepithelium, oral/nasopharyngeal tissue, bronchoalveolar, urinary, and gut/stool samples. Diverse extraction methodologies, polymerase chain reaction and sequencing techniques, and bioinformatic tools will be evaluated, in addition to direct microbial culture and metabolomic techniques. The goal is to provide a roadmap for detecting infectious agents in patients with mild cognitive impairment or AD. Positive findings would then prompt tailoring of antimicrobial treatments that might attenuate or remit mounting clinical deficits in a subset of patients.

The aim of this study was to use environmental sampling to determine the frequency of detection of selected equine respiratory viruses and bacteria in horses attending a multi-week equestrian show during the winter months. At four time points during showing, environmental sponge samples were collected from all stalls on the property and tested for the presence of equine herpesvirus-1 (EHV-1), EHV-2, EHV-4, equine influenza virus (EIV), equine rhinitis B virus (ERBV), Streptococcus equi ss. equi (S. equi), and S. equi ss. zooepidemicus (S. zooepidemicus) using real-time PCR (PCR). Environmental sponges were collected from all 53 barns by using one sponge for up to 10 stalls. Further, 2/53 barns were randomly selected for individual stall sampling in order to compare the results between individual and pooled stall samples. A total of 333/948 (35.13%, 95% CI 32.09-38.26%) pooled environmental stall sponges tested PCR-positive for at least one of the selected respiratory pathogens. Streptococcus zooepidemicus was the most commonly detected pathogen in pooled samples (28.69%, 95% CI 25.83-31.69%), followed by EHV-2 (14.45%, 95% CI 12.27-16.85%), EHV-4 (1.37%, 95% CI 0.73-2.33%), and a very small percentage of pooled stall sponges tested PCR-positive for EHV-1, ERBV, EIV, and S. equi. In individual samples, 171/464 (36.85%, 95% CI 32.45-41.42%) environmental stall sponges tested PCR-positive for at least one of the selected pathogens, following a similar frequency of pathogen detection as pooled samples. The detection frequency of true respiratory pathogens from environmental samples was higher during the winter months compared to previous studies performed during spring and summer, and this testing highlights that such pathogens circulate with greater frequency during the colder months of the year. The strategy of monitoring environmental stall samples for respiratory pathogens circumvents the often labor-intensive collection of respiratory secretions from healthy horses and allows for a more efficient assessment of pathogen buildup over time. However, environmental stall testing for respiratory pathogens should not replace proper biosecurity protocols, but it should instead be considered as an additional tool to monitor the silent circulation of respiratory pathogens in at-risk horses.

BACKGROUND AND AIM: Infectious bovine keratoconjunctivitis (IBK) causes a significant economic loss to cattle industries in many countries, including Kazakhstan. Although Moraxella bovis is recognized as an etiologic agent of IBK, other bacterial and viral agents have been suspected to play a role in the pathogenesis of this disease. This study aimed to evaluate samples collected from the eyes of IBK-affected cattle in Eastern Kazakhstan at different stages of IBK for the presence of Mor. bovis, Moraxella bovoculi, Mycoplasma bovis, Mycoplasma bovoculi, and Bovine Herpes Virus Type 1 (BHV-1) and to characterize Mor. bovoculi pilA gene sequence diversity from Mor. bovoculi positive samples. MATERIALS AND METHODS: Individual ocular swabs (n = 168) were collected from cattle that had clinical signs of IBK during the summer of 2022 on farms in the Abay region of Kazakhstan. Eye lesion scores (1, 2, and 3) were assigned depending on the degree of ocular damage. Infectious bovine keratoconjunctivitis-associated organisms were detected using a multiplex real-time polymerase chain reaction assay. The Mor. bovoculi pilA gene was sequenced from Mor. bovoculi positive samples. RESULTS: Mycoplasma bovis and BHV-1 were not detected in any of the collected samples. Mycoplasma bovoculi was identified in the majority of samples overall, usually in mixed infection with Moraxella spp. Moraxella bovoculi was detected in 76.2% of animals and predominated in animals with eye lesion scores 2 and 3. Mycoplasma bovoculi was detected only in association with Mor. bovis and/or Mor. bovoculi in animals with eye lesion scores 2 and 3. Moraxella bovis was found in 57.7% of animals and was always identified in association with another organism. Sequencing of the pilA gene in 96 samples from Mor. bovoculi positive samples identified five PilA groups. The majority belonged to PilA group A. However, three new PilA groups were identified and designated PilA groups N, O, and P. CONCLUSION: The results indicate a high prevalence of Myc. bovoculi and Mor. bovoculi in eyes of cattle with IBK on livestock farms in Eastern Kazakhstan. Additional novel Mor. bovoculi PilA groups were identified.

Limit of detection (LOD), speed, and cost for some of the most important diagnostic tools, i.e., lateral flow assays (LFA), enzyme-linked immunosorbent assays (ELISA), and polymerase chain reaction (PCR), all benefited from both the financial and regulatory support brought about by the pandemic. From those three, PCR has gained the most in overall performance. However, implementing PCR in point of care (POC) settings remains challenging because of its stringent requirements for a low LOD, multiplexing, accuracy, selectivity, robustness, and cost. Moreover, from a clinical point of view, it has become very desirable to attain an overall sample-to-answer time (t) of 10 min or less. Based on those POC requirements, we introduce three parameters to guide the design towards the next generation of PCR reactors: the overall sample-to-answer time (t); lambda (λ), a measure that sets the minimum number of copies required per reactor volume; and gamma (γ), the systems thermal efficiency. These three parameters control the necessary sample volume, the number of reactors that are feasible (for multiplexing), the type of fluidics, the PCR reactor shape, the thermal conductivity, the diffusivity of the materials used, and the type of heating and cooling systems employed. Then, as an illustration, we carry out a numerical simulation of temperature changes in a PCR device, discuss the leading commercial and RT-qPCR contenders under development, and suggest approaches to achieve the PCR reactor for RT-qPCR of the future.

In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist in cells and tissues throughout the body during ART (HIV reservoir). One such technique for HIV RNA quantitation, HIV transcription profiling, developed in the Yukl laboratory, measures a series of HIV RNA species using droplet digital PCR. To take advantage of advances in digital (d)PCR, we adapted the HIV transcription profiling technique to Qiagens dPCR platform (QIAcuity) and compared its performance to droplet digital (dd)PCR (Bio-Rad QX200 system). Using RNA standards, the two technologies were tested in parallel and assessed for multiple parameters including sensitivity, specificity, linearity, and intra- and inter-assay variability. The newly validated dPCR assays were then applied to samples from PLWH to determine HIV transcriptional activity relative to HIV reservoir size. We report that HIV transcriptional profiling was readily adapted to dPCR and assays performed similarly to ddPCR, with no differences in assay characteristics. We applied these assays in a cohort of 23 PLWH and found that HIV reservoir size, based on genetically intact proviral DNA, does not predict HIV transcriptional activity. In contrast, levels of total DNA correlated with levels of most HIV transcripts (initiated, proximally and distally elongated, unspliced, and completed, but not multiply spliced), suggesting that a considerable proportion of HIV transcripts likely originate from defective proviruses. These findings may have implications for measuring and assessing curative strategies and clinical trial outcomes.

A definitive diagnosis of nevus or melanoma is not always possible for histologically ambiguous melanocytic neoplasms. In such cases, ancillary molecular testing can support a diagnosis of melanoma if certain chromosomal aberrations are detected. Current technologies for copy number variation (CNV) detection include chromosomal microarray analysis (CMA) and fluorescence in situ hybridization. Although CMA and fluorescence in situ hybridization are effective, their utilization can be limited by cost, turnaround time, and inaccessibility outside of large reference laboratories. Droplet digital polymerase chain reaction (ddPCR) is a rapid, automated, and relatively inexpensive technology for CNV detection. We investigated the ability of ddPCR to quantify CNV in cyclin-dependent kinase inhibitor 2A ( CDKN2A ), the most commonly deleted tumor suppressor gene in melanoma. CMA data were used as the gold standard. We analyzed 57 skin samples from 52 patients diagnosed with benign nevi, borderline lesions, primary melanomas, and metastatic melanomas. In a training cohort comprising 29 randomly selected samples, receiver operator characteristic curve analysis revealed an optimal ddPCR cutoff value of 1.73 for calling CDKN2A loss. In a validation cohort comprising the remaining 28 samples, ddPCR detected CDKN2A loss with a sensitivity and specificity of 94% and 90%, respectively. Significantly, ddPCR could also identify whether CDKN2A losses were monoallelic or biallelic. These pilot data suggest that ddPCR can detect CDKN2A deletions in melanocytic tumors with accuracy comparable with CMA. With further validation, ddPCR could provide an additional CNV assay to aid in the diagnosis of challenging melanocytic neoplasms.

We report a case of a 53-year-old HIV-negative patient in San Francisco, California, USA, with no classic mpox prodromal symptoms or skin lesions who experienced fulminant, vision-threatening scleritis, keratitis, and uveitis. Deep sequence analysis identified monkeypox virus RNA in the aqueous humor. We confirmed the virus on the cornea and sclera by PCR.

Abstract Introduction The diagnosis of intestinal tuberculosis is challenging even nowadays. This study aims to report the positivity rates of new diagnostic methods such as immunohistochemistry and Real-Time Polymerase Chain Reaction in patients with intestinal tuberculosis, as well as describe the pathological and endoscopic features of intestinal tuberculosis in our population. Methods This was a retrospective observational study conducted in patients diagnosed with intestinal tuberculosis, between 2010 to 2023 from the Hospital Nacional Daniel Alcides Carrion and a Private Pathology Center, both located in Peru. Clinical data was obtained, histologic features were independently re-evaluated by three pathologists; and immunohistochemistry and real-time Polymerase Chain Reaction evaluation were performed. The 33 patients with intestinal tuberculosis who fulfilled the inclusion criteria were recruited. Results Immunohistochemistry was positive in 90.9% of cases, while real-time Polymerase Chain Reaction was positive in 38.7%. The ileocecal region was the most affected area (33.3%), and the most frequent endoscopic appearance was an ulcer (63.6%). Most of the granulomas were composed solely of epithelioid histiocytes (75.8%). Crypt architectural disarray was the second most frequent histologic finding (78.8%) after granulomas, but most of them were mild. Conclusion Since immunohistochemistry does not require an intact cell wall, it demonstrates higher sensitivity compared to Ziehl–Neelsen staining. Therefore, it could be helpful for the diagnosis of paucibacillary tuberculosis.

This study developed a real-time polymerase chain reaction (real-time PCR) method for the detection of Lactobacillus kefiranofaciens subsp. kefiranofaciens. Based on the 16S rDNA gene sequence and whole genome sequence of the type strain of L. kefiranofaciens subsp. kefiranofaciens ZW3, specific primers were designed and screened. The fluorescent dye SYBR Green I was used in the real-time PCR method. Its specificity, sensitivity and repeatability were evaluated, and this method was applied to detect several strains of this species and its mixtures with other lactic acid bacteria. The results showed that the proposed method was highly specific, sensitive and repeatable. The standard curve was linear with a determination coefficient (R2) of 0.965. Moreover, this method could specifically detect L. kefiranofaciens subsp. kefiranofaciens from its mixture with other lactic acid bacteria. In summary, the real-time PCR method could quickly and accurately detect L. kefiranofaciens subsp. kefiranofaciens, providing a new method for the specific qualitative and quantitative detection of L. kefiranofaciens subsp. kefiranofaciens.

Background and Aim: Feline coronavirus (FCoV), feline panleukopenia virus (FPV), and feline leukemia virus (FeLV) are prevalent throughout China and significantly threaten cat health. These viruses cause similar manifestations and pathological damage. Rapid and accurate diagnosis depends on detection in the laboratory. This study aimed to establish a reliable and rapid method for accurate detection of FCoV, FPV, and FeLV so that a definite diagnosis can be made and effective measures can be taken to prevent and control viral infection. Materials and Methods: We designed three pairs of specific primers and probes for the detection of FCoV 5′ untranslated region, FPV viral protein 2, and FeLV pol genes. Recombinant plasmid constructs were generated for use as standard plasmid constructs. Optimal reaction conditions, including primer and probe concentrations, reaction cycles, and annealing temperatures, were obtained on the basis of optimization tests. One-step triplex real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was successfully established to simultaneously detect FCoV, FPV, and FeLV. The specificity, sensitivity, and repeatability of the assay were analyzed, and its applicability was validated by testing 1175 clinical samples. Results: One-step triplex RT-qPCR had a high degree of specificity only for the detection of FCoV, FPV, and FeLV; it had high sensitivity with limits of detection of 139.904, 143.099, and 152.079 copies/reaction for p-FCoV, p-FPV, and p-FeLV standard plasmid constructs, respectively, and it had reliable repeatability with 0.06%–0.87% intra-assay coefficients of variations. A total of 1175 clinical samples were examined for FCoV, FPV, and FeLV using triplex RT-qPCR, and the FCoV, FPV, and FeLV positivity rates were 18.47%, 19.91%, and 47.57%, respectively. The clinical sensitivity and specificity of one-step triplex RT-qPCR were 93.07% and 97.99%, respectively. Conclusion: We developed a rapid and reliable one-step triplex RT-qPCR method for the detection of FCoV, FPV, and FeLV, which could be used as a diagnostic tool for clinical monitoring and diagnosis.

Background and Aim: Leptospirosis is a re-emerging zoonosis that is under-reported in tropical countries, and canines can be a potential reservoir of the disease. The objective of this study was to diagnose Leptospira spp. that is actively infected and re-infected in stray dogs and cats from Bogota, D.C., Colombia. Materials and Methods: A sample of 200 animals, including dogs and cats from the animal protection programs of Bogota, Colombia, were used in this study. Blood was collected from these animals for serum and DNA analysis. Conventional polymerase chain reaction (PCR) was performed using the 16s rRNA primer set, and higher-quality amplification products were sequenced by Sanger. For serodiagnosis, a group of PCR-positive samples was tested using the microagglutination test (MAT). Results: The overall PCR positivity of stray dogs and cats was 56%, 52.9%, and 65.3% in dogs and cats, respectively. The MAT seropositivity was 77.3%, and only dogs showed titers higher than 1:400. Canicola, Icterohaemorrhagiae, Pomona, Hardjo Prajitno, and Canicola and Hardjo prajitno were the serogroups associated with dogs and cats, respectively. Phylogenetic analysis revealed that the strains belonging to Leptospira interrogans serovars related to isolated samples of American, European, and Asian bats (Myotis myotis), dogs, and bovines of American origin. Conclusion: These results showed that stray dogs and cats were previously exposed to different serovars of Leptospira spp. and re-infected with other serovars that actively participated in the transmission cycle. These findings highlight the importance of actively diagnosing infectious animals to design effective intervention strategies.

AIM: To investigate the stability of the seven housekeeping genes: beta-actin (ActB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18s ribosomal unit 5 (18s), cyclophilin A (CycA), hypoxanthine-guanine phosphoribosyl transferase (HPRT), ribosomal protein large P0 (36B4) and terminal uridylyl transferase 1 (U6) in the diabetic retinal tissue of rat model. METHODS: The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) in two groups; normal control rats and streptozotocin-induced diabetic rats. The stability analysis of gene expression was investigated using geNorm, NormFinder, BestKeeper, and comparative delta-Ct (ΔCt) algorithms. RESULTS: The 36B4 gene was stably expressed in the retinal tissues of normal control animals; however, it was less stable in diabetic retinas. The 18s gene was expressed consistently in both normal control and diabetic rats' retinal tissue. That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats. Furthermore, there was no ideal gene stably expressed for use in all experimental settings. CONCLUSION: Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

Abstract Purpose The clinical significance of negative toxin enzyme immunoassays (EIA) for Clostridioides difficile infections (CDIs) is unclear. Our study aimed to investigate the significance of toxin EIA-negative in the diagnosis and prognosis of CDI. Methods All stool specimens submitted for C. difficile toxin EIA testing were cultured to isolate C. difficile. In-house PCR for tcdA, tcdB, cdtA, and cdtB genes were performed using C. difficile isolates. Stool specimens were tested with C. difficile toxins A and B using EIA kit (RIDASCREEN Clostridium difficile toxin A/B, R-Biopharm AG, Darmstadt, Germany). Characteristics and subsequent CDI episodes of toxin EIA-negative and -positive patients were compared. Results Among 190 C. difficile PCR-positive patients, 83 (43.7%) were toxin EIA-negative. Multivariate analysis revealed independent associations toxin EIA-negative results and shorter hospital stays (OR = 0.98, 95% CI 0.96–0.99, p = 0.013) and less high-risk antibiotic exposure in the preceding month (OR = 0.38, 95% CI 0.16–0.94, p = 0.035). Toxin EIA-negative patients displayed a significantly lower white blood cell count rate (11.0 vs. 35.4%, p

Tung Nguyen Thanh,1 Sang Trieu Tien,2 Phong Nguyen Van,2 Son Dang Thai,3 Thuc Luong Cong,4 Tuan Dinh Le,5 Son Tien Nguyen,5 Tuan Tran Van,1 Hoang Huy Duong,6 Tien Minh Bui,7 Kien Trung Nguyen7 1Military Institute of Clinical Embryology and Histology, Vietnam Military Medical University, Hanoi, 100000, Vietnam; 2Department of Biology and Medical Genetics, Vietnam Military Medical University, Hanoi, 100000, Vietnam; 3Institute of Biological and Food Technology, Hanoi Open University, Hanoi, 100000, Vietnam; 4Cardiovascular Center, Military Hospital 103, Vietnam Military Medical University, Hanoi, 100000, Vietnam; 5Department of Rheumatology and Endocrinology, Military Hospital 103, Vietnam Military Medical University, Hanoi, 100000, Vietnam; 6Department of Neurology, Thai Binh University of Medicine and Pharmacy, Thai Binh, 410000, Vietnam; 7Department of Obstetrics and Gynecology, Thai Binh University of Medicine and Pharmacy, Thai Binh, 410000, VietnamCorrespondence: Sang Trieu Tien, Department of Biology and Medical Genetics, Vietnam Military Medical University, Hanoi, 100000, Vietnam, Email trieusangk83@yahoo.com.vnBackground: To optimize the multiplex polymerase chain reaction (M-PCR) technique to diagnose microdeletions of azoospermia factors (AZF) on the Y chromosome and initially apply the technique to diagnose male patients with sperm density less than 5× 106 million sperm/mL was assigned to do a test to check for AZF microdeletions on the Y chromosome.Methods: Based on the positive control samples which belong to male subjects who have had 2 healthy children without any assisted reproductive technologies, the M-PCR method was developed to detect simultaneously and accurately AZF microdeletions on 32 male patients with sperm densities below 5× 106 million sperm/mL of semen at the Department of Biology and Medical Genetics – Vietnam Military Medical University.Results: Successful optimization of the M-PCR technique including 7 reactions arranged according to each AZFabc region using 24 STS/gene on the Y chromosome. Initial application to diagnose AZF deletion on 32 azoospermic and oligospermic men reveals that AZFa deletion accounts for 6.25% (2/32); deletion of all 3 regions AZFa,b,c with 18.75% (6/32 cases); The combined deletion rate of AZFb,c is highest, accounting for 56.24% (18/32 patients).Conclusion: Successfully optimized the M-PCR technique in identifying AZF microdeletions using 24 sequence tagged sites (STS)/gene for azoospermic and oligozoospermic men. The M-PCR technique has great potential in the application of AZF deletion diagnosis.Keywords: male infertility, azoospermia factors, AZF, multiplex polymerase chain reaction, M-PCR, sequence tagged sites, STS

Background: Even though a few years have passed since the coronavirus disease 2019 (COVID-19) outbreak, information regarding certain aspects of the disease, such as post-infection immunity, is still quite limited. This study aimed to evaluate post-infection protection and COVID-19 features among healthcare workers (HCWs), during three successive surges, as well as the rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection, reactivation, re-positivity, and severity.Methods: This cross-sectional population-level observational study was conducted from 20 April 2020 to 18 February 2021. The study population included all HCWs in public or private hospitals in Fars Province, Southern Iran. The infection rate was computed as the number of individuals with positive polymerase chain reaction (PCR) tests divided by the total number of person-days at risk. The re-infection was evaluated after 90 days. Results: A total of 30,546 PCR tests were performed among HCWs, of which 13,749 (61.94% of total HCWs) were positive. Considering the applied 90-day threshold, there were 44 (31.2%) cases of reactivation and relapse, and 97 (68.8% of infected and 1.81% of total HCWs) cases of reinfection among 141 (2.64%) diagnosed cases who experienced a second episode of COVID-19. There was no significant difference in symptoms (P=0.65) or the necessity for ICU admission (P=0.25). The estimated protection against repeated infection after a previous SARS-CoV-2 infection was 94.8% (95% CI=93.6-95.7).Conclusion: SARS-CoV-2 re-positivity, relapse, and reinfection were rare in the HCW population. After the first episode of infection, an estimated 94.8% protection against recurring infections was achieved.

Background and Aim: Filarial nematode typically produces a larval stage (microfilariae) in the bloodstream of vertebrate hosts, where microfilariae reside in the blood or subcutaneous tissues. Filarial nematodes cause human diseases, such as river blindness and elephantiasis, which are widely studied. However, in avian species, they are overlooked because they are nonpathogenic. In Thailand, microfilaria can be found in wild birds and domestic chickens. Recently, an increase in the number of blood samples submitted to veterinary diagnostic laboratories may have increased the number of microfilariae. Therefore, knowledge about filarial species and reliable detection methods are important. Therefore, this study aimed to investigate the efficacy of buffy coat smear and polymerase chain reaction (PCR)-based methods for the detection of microfilaria in domestic chickens. In addition, parasites were identified using the sequence of the cytochrome c oxidase subunit 1 (COX1) gene. Materials and Methods: Giemsa-stained buffy coat smears from a previous study were reanalyzed. These available buffy coat smears were prepared from 55 domestic chickens raised as backyard free-ranging in Southern Thailand. Fifty-seven frozen genomic DNA extracted from chicken blood were used to detect the presence of the COX1 gene in Onchocercidae nematodes. The nested PCR protocol for amplification of the OnchoCOI_ R2-OnchoCOI_ R2 fragment of the COX1 gene was applied from a previous report. Sequences of COX1 were analyzed to identify Onchocercidae nematodes and if they were single or mixed infections. We constructed Bayesian phylogenetics to identify parasites and assessment of the relationship between filarial nematodes in avian species and other vertebrate hosts. Results: Buffy coat smears from 15 samples revealed microfilaria. Of these 15 samples, only eight were positive for COX1 nested-PCR amplification. The other two buffy coat-negative samples were also positive for nested-PCR. Sequencing of these 11 nested PCR-positive samples revealed that almost all of them were Onchocercidae nematodes. Bayesian phylogenetic analysis showed that chicken Onchocercidae spp. were grouped with other avian filarial nematodes. However, all chickens Onchocercidae spp. showed a double peak in the sequencing chromatogram, indicating mixed filarial infection (species or haplotypes). Therefore, no chicken Onchocercidae sequence was deposited on National Center for Biotechnology Information, GenBank. Conclusion: Giemsa-stained buffy coat smear was a reliable method for the detection of chicken microfilaria in routine veterinary diagnostic laboratories. Development of a new PCR-based method is necessary. This method may provide greater sensitivity and specificity of detection. In addition, the PCR method allowed us to access the genetic characteristics of nematodes, which helped us maximize our knowledge of nematodes. Further investigations, such as the pathogenicity of filarial nematodes in chickens and their potential vectors, are required.

Shuai Li, Xue Han, Jing Ma, Guo-Hong Huang, Shu-Ting Yang, Chang-Min Wang Clinical Laboratory Center, People’s Hospital of Xinjiang Uygur Autonomous Region, Urumqi, Xinjiang, 830001, People’s Republic of ChinaCorrespondence: Chang-Min Wang, Department of Clinical Laboratory Center, People’s Hospital of Xinjiang Uygur Autonomous Region, No. 91 Tianchi Street, Tianshan District, Urumqi, Xinjiang, 830001, People’s Republic of China, Tel +86 13609992616, Fax +86− 9918564168, Email wang_c3223min@yeah.netObjective: To investigate the value of metagenomic Next-Generation Sequencing (mNGS) in diagnosing Pneumocystis jirovecii pneumonia (PJP) in non-human immunodeficiency virus (HIV)-infected patients.Methods: In this retrospective study, non-HIV-infected patients with PJP and those diagnosed with non-PJP from August 2022 to December 2024 were selected as subjects. The presence of Pneumocystis jirovecii (PJ) and other co-pathogens in bronchoalveolar lavage fluid (BALF) was analyzed, and the diagnostic efficacy of NGS, polymerase chain reaction (PCR) and serum 1,3-β-D-glucan (BDG) in PJP was compared with the reference standard of clinical compound diagnosis.Results: Eighty-nine non-HIV-infected patients were recruited, with dyspnea as the primary symptom (69.66%) and solid malignant tumor as the most common underlying disease (20.22%). Taking clinical compound diagnosis as the reference standard, the sensitivity, specificity, negative predictive value and positive predictive value of mNGS were higher than those detected by PCR and serum BDG. Among 42 non-HIV-infected patients with PJP who underwent mNGS and conventional pathogen detection of BALF, 6 had simple PJ infection and 36 had combined PJ infection. The detection rate of mNGS in mixed infections was significantly higher than that of conventional pathogen detection (85.71 vs 61.70%, P = 0.012). A total of 127 pathogens were detected in BALF using mNGS, among which fungi had the highest detection rate (46.46%). The fungi, viruses and bacteria detected were mainly Pneumocystis jirovecii, human gammaherpesvirus 4 and Acinetobacter baumannii.Conclusion: mNGS is highly effective in diagnosing non-HIV-infected patients with PJP and exhibits ideal performance in the detection of co-pathogens. In addition, it has certain value for clinical diagnosis and guidance of targeted anti-infective drug treatment.Keywords: metagenomic next-generation sequencing, Pneumocystis jirovecii pneumonia, polymerase chain reaction, non-HIV infection

Introduction: Duchenne Muscular Dystrophy (DMD) is an X-linked recessive neuromuscular disorder characterised by progressive, irreversible muscle weakness. It is caused by a mutation in the dystrophin or DMD gene, leading to the absence of the essential muscle protein Dystrophin in DMD. Muscle strength continually diminishes, and death usually occurs from chronic respiratory insufficiency and/or cardiac failure. Due to the high mortality rate, early diagnosis is crucial to allow appropriate planning for patient care and treatment. In India, the traditional multiplex Polymerase Chain Reaction (PCR) assay is the most common method to detect Dystrophin gene deletion mutations. Aim: To describe the spectrum of deletion patterns in the dystrophin gene in muscular dystrophy patients attending a tertiary care hospital in Northeast India. Materials and Methods: A hospital-based cross-sectional study was conducted on a total of 53 suspected DMD patients attending the Department of Neurology and Department of Paediatrics at Assam Medical College, Dibrugarh, Assam, India from January 2016 to December 2022. Multiplex PCR was performed to study the deletion patterns for the 25 most common exons of the DMD gene for all patients at the Genetic lab, Department of Anatomy. Deletion mutations at different multiple exons were found, and the results were statistically analysed using Statistical Package for Social Sciences (SPSS) to calculate the mean and standard deviation. The results were presented in tabular form as percentages. R esults: Out of the 53 cases suspected to have DMD on the basis of clinical presentation and high serum Creatine Kinase (CK) levels, 34 patients (64.2%) showing deletion mutations in the 25 most common exons of the DMD gene were included in the study. Deletions were most common in 15 (44.1%) patients in the distal hotspot region of exons 44-55. The most common gene deleted was exon 50 in 11 (32.4%) patients. The age at which symptoms were noticed was 4.7±2.01 years. The mean age at diagnosis was 8.4±2.4 years. C onclusion: In the present study, most patients suspected of DMD based on clinical and laboratory findings had deletions in the DMD gene, with the most common region for deletion in the dystrophin gene being the distal hotspot region, and exon 50 being the most commonly deleted.

Abstract Given the limitation of current routine approaches for pancreatic cancer screening and detection, the mortality rate of pancreatic cancer cases is still critical. The development of blood-based molecular biomarkers for pancreatic cancer screening and early detection which provide less-invasive, high-sensitivity, and cost-effective, is urgently needed. The goal of this study is to identify and validate the potential molecular biomarkers in white blood cells (WBCs) of pancreatic cancer patients. Gene expression profiles of pancreatic cancer patients from NCBI GEO database were analyzed by CU-DREAM. Then, mRNA expression levels of three candidate genes were determined by quantitative RT-PCR in WBCs of pancreatic cancer patients (N = 27) and healthy controls (N = 51). ROC analysis was performed to assess the performance of each candidate gene. A total of 29 upregulated genes were identified and three selected genes were performed gene expression analysis. Our results revealed high mRNA expression levels in WBCs of pancreatic cancer patients in all selected genes, including FKBP1A (p

Abstract Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus, enable one-pot exponential DNA amplification known as polymerase chain reaction (PCR). However, properties other than thermostability - such as fidelity, processivity, and compatibility with modified nucleotides - are important in contemporary molecular biology applications. Here, we describe the engineering and characterization of a fusion between a DNA polymerase identified in the marine archaea Nanoarchaeum equitans and a DNA binding domain from the thermophile Sulfolobus solfataricus. The fusion creates a highly active enzyme, Neq2X7, capable of amplifying long and GC-rich DNA, unaffected by replacing dTTP with dUTP in PCR, and tolerant to various known PCR inhibitors. This makes it an attractive DNA polymerase for use, e.g., with uracil excision (USER) DNA assembly and for contamination-free diagnostics. Using a magnification via nucleotide imbalance fidelity assay, Neq2X7 was estimated to have an error rate lower than 2 ∙ 10−5 bp−1 and an approximately 100x lower fidelity than the parental variant Neq2X, indicating a trade-off between fidelity and processivity – an observation that may be of importance for similarly engineered DNA polymerases. Neq2X7 is easy to produce for routine application in any molecular biology laboratory, and the expression plasmid is made freely available.

Trypanosoma evansi is a widespread hemoprotozoa that causes trypanosomiasis in both livestock and wildlife. One of the susceptible animals is the Sumatran elephant, which is included in the endangered species category. The infection of this parasite often develops into chronic and sub-clinical forms in elephants, therefore it may become unnoticed and hard to diagnose. This study aimed to analyze and evaluate the infection of T. evansi in semi-captive Sumatran elephants in Way Kambas National Park, Indonesia, using various diagnostic tests. The prevalence of T. evansi in a total of 53 Sumatran elephants was estimated using a card agglutination test for trypanosomiasis (CATT) in 2016. A longitudinal study was later conducted in 2019 using Giemsa stained blood smear (GSBS) and polymerase chain reaction (PCR). The results showed that 26.4% of samples (14/53) were positively detected by both CATT in 2016 and PCR in 2019, while GSBS was unable to detect the parasites in all samples. Furthermore, four individuals were confirmed to have persistent infections. This study concluded that the ability of CATT and PCR were more convincing over GSBS for the diagnosis of sub-clinical trypanosomiasis in Sumatran elephants. However, it is recommended to use a combination of CATT as a screening tool and PCR as a confirmatory test for reliable results.

Aim: The fight against the coronavirus disease-2019 (COVID-19) pandemic has proven crucial, necessitating the need for faster, cheaper, and more reliable detection methods. This study evaluated the performance of a rapid antigen test for the diagnosis of COVID-19 compared with reverse transcription-polymerase chain reaction (RT-PCR) results. Methods: This prospective study included 169 participants. Two simultaneous nasopharyngeal swabs were collected from the participants. Samples were tested for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) using the Panbio™ COVID-19 Ag rapid test (Abbott Rapid Diagnostics, Jena, Germany) and the Bio-Speedy® SARS-CoV-2 RT-PCR kit (Bioeksen, Istanbul, Turkey). Results: Reverse transcription-polymerase chain reaction results were positive in 90 (53.2%) of 169 patients. The antigen rapid diagnostic test (Ag-RDT) was able to find 66 (73.3%) of the 90 RT-PCR positive samples as positive (p

OBJECTIVE: To determine the Frequency of Germinal Center B (GCB) type Diffuse Large B Cell Lymphoma (DLBCL) in Karachi, Pakistan, and to screen the DLBCL patients for EZH2 mutation. METHODOLOGY: A cross-sectional study was conducted on DLBCL patients, and IHC analysis, PCR, and sequencing of the EZH2 gene were used. This study was conducted from August 2016 to July 2020. Surgical specimens received in the histopathology section of DDRRL centrally and from different collection units throughout Sindh having been diagnosed with DLBCL were collected. Three markers (CD10, BCL6, and MUM1) were used for subtyping. DNA was manually extracted from the FFPE Blocks of DLBCL cases using phenol: chloroform: isopropanol alcohol. Polymerase chain reaction (PCR) was performed, and samples were sent from Dow University to Korea (Macrogen) for sequencing. The amplified product was 189bp. Primers were used for EZH2 to flank the region containing the tyrosine domain on exon 16 and exon 18. RESULTS: Out of 116 cases, 60.3% and 39.7% were male and female, respectively. The mean age was 49.73±16.38 years. The mean survival duration of cases diagnosed with GCB was 8.21±7.39 months. Polymorphism of EZH2 mutation (Tyr 701 Asn) was found to be positive in GCB-DLBCL for three samples (16.6%). CONCLUSION: In the current study, Sanger sequencing was used to detect any mutation in the coding region of EZH2, and a unique mutation was reported (Tyr701Asn).

The contamination status of cold resistant Morganella psychrotolerans in commercially available aquatic products was investigated, and the generation of histamine by its isolates was explored. A total of 100 samples of commercially available fish from Guangdong province were surveyed. The results revealed a widespread distribution of M. psychrotolerans with a contamination rate of 34%. The contamination rates for red-fleshed and white-fleshed fish were 35.9% and 27.3%, respectively. Specifically, the mackerel samples had a contamination rate of 38.24%, and the tuna samples had a contamination rate of 29.41% among all positive samples. Sixteen strains of M. psychrotolerans were isolated from the positive samples, and all isolates produced histamine levels exceeding 1 000 mg/L after 48 h incubation at 20 ℃. Notably, isolate 1 showed higher histamine-producing capacity than the type strain. Although the two strains exhibited no significant differences in growth curves at varying temperatures (4 and 20 ℃), their histamine-producing capacities were different. After 10 days of culture at 4 ℃, histamine production was higher by isolate 1 than the type strain, while the opposite result was observed after 60 h of culture at 20 ℃. These findings offer crucial theoretical support for addressing the problem of histamine accumulation in cold chain aquatic products.

Background: High-risk Human Papillomavirus (HPV) genotypes are found in malignant oral epithelial lesions, and HPV infection is proposed as a risk factor for initiating Squamous cell carcinoma (SCC) in the head and neck region. This study suggests a practical approach to detect HPV in HPV-associated oral epithelial dysplasia (HAOED).Methods: Fifty-four oral epithelial dysplasia specimens were examined, comprising twenty-seven cases diagnosed with high-grade dysplasia and twenty-seven cases diagnosed with low-grade dysplasia using a binary grading system. To assess the cases for HPV, the specimens were examined for p16 protein using an immunohistochemical (IHC) study, and then, the Chromatin In Situ Hybridization (CISH) test was performed for all positive cases. Chromatin Immunoprecipitation-Polymerase Chain Reaction (ChIP-PCR) was performed on CISH-positive specimens to assess the outcome. This cross-sectional study was conducted in 2020 at Tehran University of Medical Science. SPSS software version 22.0 was used to perform the Chi square or Fisher’s exact test to examine the relationship between variables (statistically significant level P0.99), and in the nine cases, undergone the ChIP-PCR study, two cases (22.2%) showed positivity for HPV-16, while one case (11.1%) demonstrated positivity for HPV-51.Conclusion: Regarding HAOED, here, we proposed a step-by-step combination approach using different diagnostic methods, including IHC for p16 protein, CISH, and ChIP-PCR based on a complementary algorithm.

Detecting Ganoderma boninense in Indonesia is crucial for effectively controlling and mitigating the spread of basal stem disease in oil palm fields. While there is ongoing development of tolerant plants, no such plant has been successfully created yet. Consequently, researchers are actively studying detection methods for Ganoderma boninense. One established and highly accurate approach is the use of polymerase chain reaction (PCR) techniques for molecular detection. However, this method requires time‐consuming sample preparation, which can pose challenges in plantation settings. To address this problem, a portable lab‐on‐chip device has been introduced. This technology enables easy and automatic DNA retrieval from liquid samples by absorbing lysed DNA using magnetic beads. An efficient mechanism for manipulating the magnetic bead within the semiconductor has been successfully implemented. The extraction process typically takes around 15 minutes using a modified methodology on the chip device approach. The chip facilitates the retrieval of two samples with a capacity of 120 µL for each sample. The PCR method was utilized to validate the equivalence of the lab‐on‐chip device extraction to the standard extraction method. This represents a promising alternative for expedited and simplified detection of Ganoderma boninense in field conditions.

Aims: Continuous local antibiotic perfusion (CLAP) has recently attracted attention as a new drug delivery system for orthopaedic infections. CLAP is a direct continuous infusion of high-concentration gentamicin (1,200 μg/ml) into the bone marrow. As it is a new system, its influence on the bone marrow is unknown. This study aimed to examine the effects of high-concentration antibiotics on human bone tissue-derived cells. Methods: Cells were isolated from the bone tissue grafts collected from six patients using the Reamer-Irrigator-Aspirator system, and exposed to different gentamicin concentrations. Live cells rate, apoptosis rate, alkaline phosphatase (ALP) activity, expression of osteoblast-related genes, mineralization potential, and restoration of cell viability and ALP activity were examined by in vitro studies. Results: The live cells rate (the ratio of total number of cells in the well plate to the absorbance-measured number of live cells) was significantly decreased at ≥ 500 μg/ml of gentamicin on day 14; apoptosis rate was significantly increased at ≥ 750 μg/ml, and ALP activity was significantly decreased at ≥ 750 μg/ml. Real-time reverse transcription-polymerase chain reaction results showed no significant decrease in the ALP and activating transcription factor 4 transcript levels at ≥ 1,000 μg/ml on day 7. Mineralization potential was significantly decreased at all concentrations. Restoration of cell viability was significantly decreased at 750 and 1,000 μg/ml on day 21 and at 500 μg/ml on day 28, and ALP activity was significantly decreased at 500 μg/ml on day 28. Conclusion: Our findings suggest that the exposure concentration and duration of antibiotic administration during CLAP could affect cell functions. However, further in vivo studies are needed to determine the optimal dose in a clinical setting. Cite this article: Bone Joint Res 2024;13(3):92–101.

Anbing Zhang,1,* Jinguang Liang,2,* Xiaoli Lao,1,3 Xiuqiong Xia,1 Jianping Liang1,3 1Department of Respiratory and Critical Care Medicine, Zhongshan People’s Hospital, Zhongshan, Guangdong Province, People’s Republic of China; 2Department of Respiratory and Critical Care Medicine, Zhongshan Huangpu People’s Hospital, Zhongshan, Guangdong Province, People’s Republic of China; 3Graduate School, Guangdong Medical University, Zhanjiang, Guangdong Province, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jianping Liang, Department of Respiratory and Critical Care Medicine, Zhongshan People’s Hospital, 2 Sunwen East Road, Zhongshan, 528400, People’s Republic of China, Tel +86-159-8910-7884, Fax +86-760-8988-0256, Email ljpzhongshan@163.comAbstract: We report a case of pneumonia caused by coinfection with Chlamydia psittaci and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron XBB.1 variant, confirmed using metagenomic next-generation sequencing (mNGS) and quantitative polymerase chain reaction (qPCR). C. psittaci and SARS-CoV-2 were detected in bronchoalveolar lavage fluid using mNGS. Additionally, mNGS detected C. psittaci in blood and nasopharyngeal specimens and was more sensitive than qPCR. The patient recovered after treatment with moxifloxacin. This report highlights the use of coinfections of C. psittaci and SARS-CoV-2, as mNGS has already been recognized to be a diagnostic tool for identifying coinfections.Keywords: coinfection, quantitative reverse-transcription polymerase chain reaction, bronchoalveolar lavage fluid, zoonoses

Sheng Yin,1 YingRui Lin,1 Bingqi Wang,1 Yizhi Peng,2 Zeyou Wang,1 Xiaolin Zhu,1 Hao Liang,1 Xianping Li,1 Min Wang1 1Department of Laboratory Medicine, The Second Xiangya Hospital, Central South University, Changsha, Hunan, 410011, People’s Republic of China; 2Department of Laboratory Medicine, Hunan Cancer Hospital, Central South University, Changsha, Hunan, 410031, People’s Republic of ChinaCorrespondence: Min Wang, Department of Laboratory Medicine, The Second Xiangya Hospital, Central South University, Changsha, Hunan, 410011, People’s Republic of China, Tel +86 13298697558, Email wangmin0000@csu.edu.cnPurpose: Bloodstream infection(BSI) is linked with high mortality, underscoring the significance of prompt etiological diagnosis for timely and precise treatment. This study aims to investigate the diagnostic value of droplet digital polymerase chain reaction(ddPCR) in combination with conventional inflammatory markers [interleukin-6(IL-6) and procalcitonin(PCT)] concerning disease progression and treatment prognosis in BSI patients. Furthermore, the study aims to explore a more efficient clinical application strategy.Patients and Methods: This prospective case seried study centers on 176 patients suspected of or confirmed with BSI. Blood samples were collected to extract nucleic acids for identifying pathogens (bacteria, fungi, and viruses) and determining copy loads via ddPCR.Results: The sensitivity of ddPCR was markedly higher compared to the culture method (74.71% vs 31.03%). A positive correlation existed between bacterial load and levels of inflammatory markers [IL-6 (P= 0.0182), PCT (P= 0.0029), and CRP (P= 0.0005)]. In suspected BSI cases, the combination of ddPCR and inflammatory markers could predict sepsis risk [ROC: Area under the curve(AUC)=0.6071, P= 0.0383]. Within confirmed BSI patients, the ddPCR bacterial load of those with SOFA< 7 was lower than that of the SOFA≥ 7 (P= 0.0334). ddPCR (OR: 1.789, P= 0.035) monitoring combined with PCT (OR: 1.787, P= 0.035) holded predictive value for SOFA progression (AUC=0.7913, P= 0.0003). Similarly, BSI survivors displayed a lower burden than non-survivors (P= 0.0170). Additionally, ddPCR combinated with IL-6 provided a more accurate and expedited insight into clinical outcomes prediction for BSI confirmed patients (AUC=0.7352, P= 0.0030). Serial monitoring of bacterial load by ddPCR effectively mirrored the clinical course of BSI in patients. Notably, patients with positive ddPCR virus infection exhibited significantly reduced lymphocyte counts (P= 0.0003).Conclusion: In a clinical context, qualitative ddPCR results and quantitative continuous monitoring can more precisely assess sepsis progression and treatment prognosis in BSI patients. Furthermore, ddPCR results offer quicker and more accurate reference points for clinical antibacterial and antiviral interventions.Keywords: bloodstream infection, droplet digital polymerase chain reaction, interleukin-6, procalcitonin, prognosis

Objective: Malaria has been eradicated in Türkiye as of 2010, but there are imported cases. In this study, we aimed to compare the diagnostic value of two rapid tests; SD Bioline Malaria Ag Pf/Pan (SD-Pf/Pan) and SD Bioline Malaria Ag Pf/Pv (SD-Pf/Pv) with microscopy and real time-polymerase chain reaction (RT-PCR). Methods: Blood samples were taken from all participants. Thick drop smears were prepared. Thick drop smears were examined for malaria positive/negative distinction under the light microscopy. Then, two rapid diagnostic tests (SD-Pf/Pan and SD-Pf/Pv) were performed. After DNA extraction from blood samples, RT-PCR was typed. The data were evaluated with SPSS 21 program of statistics. Results: A total of 30 cases out of 66 suspected malaria cases were detected as positive with microscopy and RT-PCR. Twenty-seven patients were found positive with both SD-Pf/Pan and SD-Pf/Pv tests. Based on the microscopic results as a reference method, SD-Pf/Pan and SD-Pf/Pv rapid diagnostic tests had a 90% sensitivity, 100% specificity, 100% positive predictive value (PPV), and 92.86% negative predictive value (NPV). Based on the RT-PCR results as a reference method, for detection of P. falciparum, both tests had a 95.65% sensitivity, 100% specificity, 100% PPV, and 88.89% NPV. Moreover, while SD-Pf/Pv had a sensitivity, specificity, PPV, and NPV of 100% in detection of P. vivax; SD-Pf/Pan has a 77.78% sensitivity of, 61.90% specificity of, 46.67% PPV, and 86.67% NPV SD-Pf/Pan for detection of PAN. Conclusion: As a result, high sensitivity and specificity were detected in both kits in the diagnosis of malaria infections caused by P. falciparum and P. vivax. Rapid diagnostic tests can be used safely in diagnosis however the diagnosis should be supported by microscopy and RT-PCR methods when they are applicable.

Background and Aim: Bovine tuberculosis (TB) is a zoonotic disease of great public health importance, particularly in Indonesia, where control measures are limited or are not implemented. This study aimed to detect the presence of Mycobacterium pathogens in milk samples from dairy cattle in Pasuruan regency and Surabaya City, East Java, using Ziehl–Neelsen acid-fast staining and polymerase chain reaction (PCR). Materials and Methods: Milk samples were aseptically collected from 50 cattle in the Lekok Subdistrict, Pasuruan Regency, and 44 from dairy farms in the Lakarsantri Subdistrict, Wonocolo Subdistrict, Mulyorejo Subdistrict, and Kenjeran Subdistrict, Surabaya, East Java. To detect Mycobacteria at the species level, each sample was assessed by Ziehl–Neelsen staining and PCR using the RD1 and RD4 genes. Results: The results of PCR assay from 50 samples in Lekok Subdistrict, Pasuruan Regency showed that 30 samples (60%) were positive for Mycobacterium tuberculosis and two samples (4%) were positive for Mycobacterium bovis, although Ziehl–Neelsen staining did not show the presence of Mycobacterium spp. In the Surabaya region, 31 samples (70.45%) were positive for M. tuberculosis and three samples (6.8%) were positive for M. bovis. Six samples (13.63%) from all PCR-positive samples could be detected microscopically with Ziehl–Neelsen. Conclusion: The presence of bovine TB in this study supports the importance of using a molecular tool alongside routine surveillance for a better understanding of the epidemiology of bovine TB in East Java.

Background: A novel coronavirus severe acute respiratory syndrome coronavirus-2 (SARSCoV-2) that emerged in China in December 2019 has spread rapidly globally to many countries including India and World Health Organization declared it as a pandemic on March 11th, 2020. Aims and Objectives: The current study endeavors to determine the SARS-CoV-2 positivity rate as well as epidemiological, clinical, and diagnostic profiles from the second wave. Materials and Methods: We performed a retrospective analysis of all suspected COVID-19 cases from January 2021 to October 2021 presenting at a large testing center for SARS-CoV-2 infection by real-time polymerase chain reaction (RT-PCR). Descriptive analysis has been performed for profiling of clinical-epidemiological aspects of suspected cases. Results: A total of 694427 participants were enrolled during the study from January 2021 to October 2021. Overall RT-PCR positivity rate was found to be 1.7% in the year 2021 and the positivity was maximum in April 2021 which represents the second wave of COVID-19 infection in India. In the study population, more than half (57.07%) of the persons screened for COVID-19 infection were between 21 and 40 years of age, and about two-thirds (65.20%) of the persons screened were male followed by 34.79% were female. Conclusions: SARS-CoV-2 poses a high burden of infections in the community. Males had a higher RT-PCR detection rate as compared to females. The younger age group (80 years) expressed the highest positivity rate.

In a span of 4 months, there were 7 cases of meningococcal meningitis who presented with diagnostic dilemma and also a few atypical manifestations. A retrospective data analysis was carried out in children diagnosed with meningococcal meningitis who were admitted from October 2022 to January 2023. There were 7 confirmed cases of Neisseria meningitidis infections during the study period. The median age of presentation was 6 years (range: 4.5 months–9 years) with four males and three females. All the children presented with fever, but none had signs of meningeal irritation as such. Routine blood and cerebrospinal fluid (CSF) analysis were also near normal. Reverse transcriptase polymerase chain reaction (RT-PCR) for N. meningitidis was positive in the CSF samples of all the seven cases. All of the patients recovered completely, except that there were residues of neurodeficit in one patient. In endemic areas, pediatricians should have a low threshold of suspicion for N. meningitidis in children presenting with signs and symptoms involving the central nervous system and consider an RT-PCR of the CSF sample even if routine blood and CSF studies are near normal.

A targeted metabologenomic method was developed to selectively discover terminal oxazole‐bearing natural products from bacteria. For this, genes encoding oxazole cyclase, a key enzyme in terminal oxazole biosynthesis, were chosen as the genomic signature to screen bacterial strains that may produce oxazole‐bearing compounds. Sixteen strains were identified from the screening of a bacterial DNA library (1,000 strains) using oxazole cyclase gene‐targeting polymerase chain reaction (PCR) primers. The PCR amplicon sequences were subjected to phylogenetic analysis and classified into nine clades. 1H−13C coupled‐HSQC NMR spectra obtained from the culture extracts of the hit strains enabled the unequivocal detection of the target compounds, including five new oxazole compounds, based on the unique 1JCH values and chemical shifts of oxazole: lenzioxazole (1) possessing an unprecedented cyclopentane, permafroxazole (2) bearing a tetraene conjugated with carboxylic acid, tenebriazine (3) incorporating two modified amino acids, and methyl‐oxazolomycins A and B (4 and 5). Tenebriazine displayed inhibitory activity against pathogenic fungi, whereas methyl‐oxazolomycins A and B (4 and 5) selectively showed anti‐proliferative activity against estrogen receptor‐positive breast cancer cells. This metabologenomic method enables the logical and efficient discovery of new microbial natural products with a target structural motif without the need for isotopic labeling. [ABSTRACT FROM AUTHOR]

Emerging technologies focused on the detection and quantification of circulating tumor DNA (ctDNA) in blood show extensive potential for managing patient treatment decisions, informing risk of recurrence, and predicting response to therapy. Currently available tissue-informed approaches are often limited by the need for additional sequencing of normal tissue or peripheral mononuclear cells to identify non-tumor-derived alterations while tissue-naïve approaches are often limited in sensitivity. Here we present the analytical validation for a novel ctDNA monitoring assay, FoundationOne®Tracker. The assay utilizes somatic alterations from comprehensive genomic profiling (CGP) of tumor tissue. A novel algorithm identifies monitorable alterations with a high probability of being somatic and computationally filters non-tumor-derived alterations such as germline or clonal hematopoiesis variants without the need for sequencing of additional samples. Monitorable alterations identified from tissue CGP are then quantified in blood using a multiplex polymerase chain reaction assay based on the validated SignateraTM assay. The analytical specificity of the plasma workflow is shown to be 99.6% at the sample level. Analytical sensitivity is shown to be >97.3% at ≥5 mean tumor molecules per mL of plasma (MTM/mL) when tested with the most conservative configuration using only two monitorable alterations. The assay also demonstrates high analytical accuracy when compared to liquid biopsy-based CGP as well as high qualitative (measured 100% PPA) and quantitative precision (<11.2% coefficient of variation). [ABSTRACT FROM AUTHOR]

Background: Chronic rhinosinusitis (CRS) is an inflammatory disease affecting the sinuses or nose. Persistent inflammatory responses can lead to tissue remodeling, which is a pathological characteristics of CRS. Activation of fibroblasts in the nasal mucosal stroma, differentiation and collagen deposition, and subepithelial fibrosis have been associated with CRS. Objectives: We aimed to assess the inhibitory effects of doxycycline and deoxycholic acid-polyethyleneimine conjugate (DA3-Doxy) on myofibroblast differentiation and extracellular matrix (ECM) production in nasal fibroblasts stimulated with TGF-β1. Methods: To enhance efficacy, we prepared DA3-Doxy using a conjugate of low-molecular-weight polyethyleneimine (PEI) (MW 1800) and deoxycholic acid (DA) and Doxy. The synthesis of the DA3-Doxy polymer was confirmed using nuclear magnetic resonance, and the critical micelle concentration required for cationic micelle formation through self-assembly was determined. Subsequently, the Doxy loading efficiency of DA3 was assessed. The cytotoxicity of Doxy, DA3, PEI, and DA-Doxy in nasal fibroblasts was evaluated using the WST-1 assay. The anti-tissue remodeling and anti-inflammatory effects of DA3-Doxy and DA3 were examined using real-time polymerase chain reaction (Real-time PCR), immunocytochemistry, western blot, and Sircol assay. Results: Both DA3 and DA3-Doxy exhibited cytotoxicity at 10 μg/ml in nasal fibroblasts. Doxy partially inhibited α-smooth muscle actin, collagen types I and III, and fibronectin. However, DA3-Doxy significantly inhibited α-SMA, collagen types I and III, and fibronectin at 5 μg/ml. DA3-Doxy also modulated TGF-β1-induced changes in the expression of MMP 1, 2, and 9. Nonetheless, TGF-β1-induced expression of MMP3 was further increased by DA3-Doxy. The expression of TIMP 1 and 2 was partially reduced with 5 μg/ml DA3-Doxy. Conclusions: Although initially developed for the delivery of genetic materials or drugs, DA3 exhibits inhibitory effects on myofibroblast differentiation and ECM production. Therefore, it holds therapeutic potential for CRS, and a synergistic effect can be expected when loaded with CRS treatment drugs. [ABSTRACT FROM AUTHOR]

Background: Osteonecrosis of the femoral head caused by glucocorticoids (GIONFH) is a significant issue resulting from prolonged or excessive clinical glucocorticoid use. Astaxanthin, an orange-red carotenoid present in marine organisms, has been the focus of this study to explore its impact and mechanism on osteoblast apoptosis induced by dexamethasone (Dex) and GIONFH. Methods: In this experiment, bioinformatic prediction, molecular docking and dynamics simulation, cytotoxicity assay, osteogenic differentiation, qRT-PCR analysis, terminal uridine nickend labeling (TUNEL) assay, determination of intracellular ROS, mitochondrial function assay, immunofluorescence, GIONFH rat model construction, micro-computed tomography (micro-CT) scans were performed. Results: Our research demonstrated that a low dose of astaxanthin was non-toxic to healthy osteoblasts and restored the osteogenic function of Dex-treated osteoblasts by reducing oxidative stress, mitochondrial dysfunction, and apoptosis. Furthermore, astaxanthin rescued the dysfunction in poor bone quality, bone metabolism and angiogenesis of GIONFH rats. The mechanism behind this involves astaxanthin counteracting Dex-induced osteogenic damage by activating the Nrf2 pathway. Conclusion: Astaxanthin shields osteoblasts from glucocorticoid-induced oxidative stress and mitochondrial dysfunction via Nrf2 pathway activation, making it a potential therapeutic agent for GIONFH treatment. [ABSTRACT FROM AUTHOR]