A novel molecular assay conducted on the BD Max system to facilitate reflex testing for vanA and vanB in clinical isolates of enterococci.

Infections caused by vancomycin-resistant enterococci (VRE) are common. Real-time PCR assays targeting vanA and vanB facilitate screening of patients in healthcare settings to limit the risk of dissemination, especially amongst those at high-risk of infection or with limited treatment options. Such assays are commonly performed as reflex testing procedures where they augment phenotypic techniques and shorten turnaround time to benefit timely clinical management. 'Random access' and 'sample-to-result' real-time PCR platforms are suited for this application as they are of low complexity and less technically demanding. Modelled on these attributes, we configured a real-time PCR assay (VRE BD) for detection of vanA/B in clinical isolates of enterococci, adapted for the BD Max System (Becton Dickinson). We applied an unconventional approach by testing suspensions of microorganisms in water to circumvent the traditional pre-analytical genomic extraction process. Our objective of this study was to assess the performance of this assay for detection of VRE in cultures by validating against a traditional real-time PCR assay based on the LightCycler 2.0 platform (Roche, VRE RO). A high level of analytical sensitivity and specificity (≥99.0%) for both genes was obtained when testing suspensions derived from blood agar. Results for suspensions obtained from chromID VRE (Edwards Group) showed a similar level of performance for vanA detection (100%), but not for the vanB target (≥90.9%) where a lesser number of isolates were available for testing. However, our results for VRE detection in isolates from these media were repeatable and reproducible, and equated to positive and negative predictive values of ≥95.2% and ≥97.8%, respectively. Furthermore, the VRE BD assay was also able to accurately detect VRE in clinical and spiked BacT/ALERT (bioMérieux) blood cultures. Thus, the technical simplicity, short turnaround time and robustness of this high performing assay for VRE is suitable for reflex testing. In addition, the format developed for the BD Max platform has potential application for reflex testing other molecular targets of clinical importance.

An improved Trizol method for extracting total RNA from Eleutherococcus senticosus (Rupr. & Maxim.) Maxim leaves.

INTRODUCTION: High-quality nucleic acids are the basis for molecular biology experiments. Traditional RNA extraction methods are not suitable for Eleutherococcus senticosus Maxim. OBJECTIVE: To find a suitable method to improve the quality of RNA extracted, we modified the RNA extraction methods of Trizol. METHODOLOGY: Based on the conventional Trizol method, the modified Trizol method 1 and modified Trizol method 2 were used as the control for extraction of RNA from E. senticosus Maxim leaves. The modified Trizol method 1 added ß-mercaptoethanol on the conventional Trizol method. After RNA was dissolved, a mixed solution of phenol, chloroform, and isoamyl alcohol was added to denature protein and inhibit the degradation of RNA. The modified Trizol method 2 adds PVPP to grind on the basis of modified Trizol method 1, so as to better remove phenols from leaves, and eliminates the step of incubation at -20°C to reduce extraction time and RNA degradation. Chloroform, CTAB, and CH3COONa were used instead of a phenol, chloroform, and isoamyl alcohol mixed solution to ensure complete separation of nucleic acid from plant tissues and to obtain high-purity RNA. RESULTS: The research results showed that the quality of RNA extracted by conventional Trizol method, modified Trizol method 1, was incomplete, accompanied with different degrees of contamination of polysaccharides, polyphenols, and DNA. The modified Trizol method 2 could better extract RNA from E. senticosus Maxim leaves. The ratio of A260/A280 was in the range of 1.8-2.0, and the yield of RNA was the highest, which was 1.68 and 1.15 times compared with that by conventional Trizol method and modified Trizol method 1 extraction, respectively. The reverse transcription cDNA was further tested through PCR with the specific primers. The amplified fragments are displayed in clear and bright bands in accordance with the expected size. CONCLUSION: The modified Trizol method 2 could better extract RNA from E. senticosus Maxim leaves. High-quality RNA has more advantages in molecular biology study of E. senticosus Maxim.

Comparison of serological and molecular methods for differentiation between genotype A and genotype B strains of small ruminant lentiviruses.

Introduction: Small ruminant lentiviruses (SRLV) cause multisystemic, degenerative and chronic disease in sheep and goats. There are five genotypes (A, B, C, D and E), of which A and B are the most widespread. The purpose of this study was to evaluate the serotyping efficiency of the Eradikit SRLV Genotyping ELISA and the molecular typing efficiency of a newly developed nested real-time PCR targeting the long terminal repeat-gag (LTR-gag) region using samples from animals infected with subtypes of SRLV known to circulate in Poland. Material and Methods: A total of 97 sera samples taken from 34 sheep and 63 goats were immunoassayed, and 86 DNA samples from 31 sheep and 55 goats were tested with the PCR. All ruminants were infected with known SRLV strains of the A1, A5, A12, A13, A16, A17, A18, A23, A24, A27, B1 and B2 subtypes. Results: A total of 69 (80.2%, 95% confidence interval 71.6%-88.8%) out of 86 tested samples gave positive results in the PCR. In 17 out of the 86 (19.8%) samples, no proviral DNA of SRLV was detected. The differentiation between MVV (genotype A) and CAEV (genotype B) by PCR matched the predating phylogenetic analysis invariably. No cross-reactivity was observed. On the other hand, the proportion of samples genotyped the same by the older phylogenetic analysis and the Eradikit SRLV Genotyping ELISA was 42.3%. The test was unable to classify 40.2% of samples, and 17.5% of sera were incorrectly classified. Conclusion: Our results showed that the Eradikit SRLV genotyping kit is not a reliable method for predicting SRLV genotype, while the nested real-time PCR based on the LTR-gag region did prove to be, at least for genotypes A and B.

Quadruplex qPCR for detection and discrimination of C. Coli,C. fetus, and C. Jejuni from other Campylobacter species in chicken and sheep meat.

Campylobacter is gram-negative bacteria considered the predominant genera isolated from poultry samples and associated with gastroenteritis. Due to the problems in conventional cultural methods of time-consuming and technically demanding requirements, a rapid and feasible method for their identification and discrimination of the closely related spp. Including Campylobacter coli, Campylobacter fetus, and Campylobacter jejuni is needed. This study analyzes the chicken and sheep meats samples (n = 125) using culture and pre-enrichment-based Quadraplex real-time PCR by targeting OrfA, CstA, HipO, and 16 S rRNA genes of C. coli, C. fetus, C. jejuni and Campylobacter spp. Respectively. The analysis of 125 chicken and sheep meat samples by culture and real-time PCR showed high concordance between the results of the two methods. The present study show high prevalence of Campylobacter species (35% and 32% from chicken and meat respectively) of which C. jejuni were the most abundant. Reaction efficiencies were between 90 and 110%, and detect as low as 8.9 fg in C. jejuni. The need for quick detection and discrimination methods in sheep and chicken meat can be met using the described Quadraplex real-time PCR methodology.

Molecular and serological prevalence of Leptospira spp. among slaughtered cattle and associated risk factors in the Bahr El Ghazal region of South Sudan.

INTRODUCTION: Leptospirosis is a neglected emerging and zoonotic disease reported worldwide. This study sought to determine the molecular and serological prevalence of Leptospira spp. and the associated risk factors in slaughtered cattle from the Bahr El Ghazal region of South Sudan. MATERIALS AND METHODS: Between January 16th and February 25th, 2023, blood and urine samples were collected from 402 cattle at the Lokoloko Municipal Slaughterhouse in Western Bahr El-Ghazal State. Serum samples were tested using the microscopic agglutination test (MAT), with a panel of 12 serovars (sv) from 12 serogroups (sg) and 4 species (spp) of Leptospira spp. These serovars had been previously identified in Sudan and the East African region. Simultaneously, 400 corresponding urine samples were screened using qualitative real-time polymerase chain reaction (PCR) to detect the shedding of Leptospira spp. in urine. To identify the associated risk factors, the age, sex, breed and body condition score of each sampled cattle was noted at the time of sampling and subsequently analysed using logistic regression models. RESULTS: Among the 402 serum samples screened, a substantial 81.8% (329/402, 95% CI 77.9-85.3) displayed seropositivity for Leptospira spp. with a MAT titre ≥ 100. The prevalence of urine shedding determined by PCR was 6% (23/400, 95% CI 3.8-8.4), while probable recent leptospirosis with a MAT ≥ 1:800 was observed in 33.1% (133/402, 95% CI 28.6-37.8) of the cattle. Multiple reactions were detected in 34.8% (140/402, 95% CI 30.6-39.5) serum samples. The seropositivity was against L. borgpetersenii sg. Tarassovi (78.6%; 316/402, 95% CI 74.4-82.3), followed by L. borgpetersenii sg. Ballum at 20.4% (82/402, 95% CI, 16.7-24.4%), L. kirschneri sg. Autumnalis At 8.7% (35/402, 95% CI 5.7-11.7), L. interrogans sg. of Pomona at 7.0% (28/402, 95% CI 4.5-9.5), and L. interrogans sg. Hebdomadis was 5.0% (20/402, 95% CI 2.8-7.2). Several risk factors are associated with seropositivity. Older animals (≥ 2 years) had 2.0 times greater odds (95% CI 1.14-3.5) of being seropositive than younger animals (< 2 years), P-value = 0.016. Female animals demonstrated 2.1 times greater odds (95% CI 1.2-3.6) of seropositivity than males did (P-value = 0.008). Additionally, Felata/Mbororo cattle exhibited 2.4 times greater odds (95% CI 1.3-4.5) of being seropositive than did local Nilotic cattle (P-value = 0.005). The agreement between the MAT and PCR results was poor, as indicated by a kappa statistic value of 0.001 and a P-value of 0.913. But there was a moderate agreement between MAT high titres ≥ 800 and PCR positivity with a kappa statistic value = 0.501 and a P-value < 0.001. CONCLUSION: In addition to the high seroprevalence, Leptospira spp. were found in the urine of slaughtered cattle, suggesting that leptospirosis is endemic to the study area. This finding underscores the significance of cattle as potential sources of infection for slaughterhouse workers, the general public, and other animal species. To address this issue effectively in the Bahr El Ghazal Region and South Sudan, a comprehensive strategy involving a multidisciplinary approach is essential to minimize disease among animals, hence reducing potential zoonotic risks to humans.

Microplastics in granular sequencing batch reactors: Effects on pollutant removal dynamics and the microbial community.

This study investigated the relationship between microplastic (MP) presence and pollutant removal in granular sludge sequencing batch reactors (GSBRs). Two types of MPs, polyethylene (PE) and polyethylene terephthalate (PET), were introduced in varying concentrations to assess their effects on microbial community dynamics and rates of nitrogen, phosphorus, and organic compound removal. The study revealed type-dependent variations in the deposition of MPs within the biomass, with PET-MPs exhibiting a stronger affinity for accumulation in biomass. A 50 mg/L dose of PET-MP decreased COD removal efficiency by approximately 4 % while increasing P-PO4 removal efficiency by around 7 % compared to the control reactor. The rate of nitrogen compounds removal decreased with higher PET-MP dosages but increased with higher PE-MP dosages. An analysis of microbial activity and gene abundance highlighted the influence of MPs on the expression of the nosZ and ppk1 genes, which code enzymes responsible for nitrogen and phosphorus transformations. The study also explored shifts in microbial community structure, revealing alterations with changes in MP dose and type. This research contributes valuable insights into the complex interactions between MP, microbial communities, and pollutant removal processes in GSBR systems, with implications for the sustainable management of wastewater treatment in the presence of MP.

Comparison of Bordetella species identification among differing rt-PCR assays in the United States.

In the United States, the general laboratory method for diagnosing pertussis, caused by Bordetella pertussis, is real-time PCR (rt-PCR) targeting insertion sequence 481 (IS481). Other Bordetella species (parapertussis, holmesii, and bronchiseptica) can also cause a pertussis-like syndrome, and some commercial laboratory assays include the insertion sequence 1001 (pIS1001) that can detect B. parapertussis/B. bronchiseptica (BppBb). Because IS481 exists in B. pertussis and B. holmesii, current commercial assays cannot differentiate these two species. We used a multiplex rt-PCR assay containing species-specific targets to Bordetella to evaluate clinical specimens detected as B. pertussis/B. holmesii (BpBh) or BppBb by commercial laboratories. A sample of 3,984 clinical specimens positive for IS481 or pIS1001 from two commercial laboratories during 2012-2019 were re-tested at CDC. Agreement of Bordetella species between the CDC and commercial laboratory assays, and the proportion of commercial laboratory specimens that were non-B. pertussis by CDC's assay was assessed. Overall agreement in Bordetella species detection and identification between the CDC and commercial lab assays was 85%. Agreement for identifying B. pertussis was 87% for 3,663 BpBh specimens and 98% for identifying B. parapertussis in 310 BppBb specimens. CDC's assay detected B. holmesii in 55/3,984 (1.4%) specimens. Most discrepant results (410/490, 82%) were BpBh specimens interpreted as indeterminate B. pertussis at CDC. We found a small portion of B. holmesii in a sample of IS481-positive clinical specimens originally identified by commercial laboratory rt-PCR assays, suggesting that commercial PCR assays are a reliable diagnostic tool for correctly identifying Bordetella species in most patients with suspected pertussis. IMPORTANCE: When testing specimens collected from patients with suspected pertussis, large-scale commercial laboratories in the United States employ an IS481-based assay that cannot differentiate between Bordetella pertussis and Bordetella holmseii. The level of B. holmesii causing pertussis-like illness in the United States is not well-understood given that only B. pertussis is nationally notifiable. After re-testing with a multiplex, species-specific rt-PCR assay, our data show low levels of B. holmesii identified in a sample of IS481-positive clinical specimens originally identified by commercial laboratory rt-PCR assays. These results reinforce the validity of large-scale commercial rt-PCR testing as a reliable diagnostic tool for pertussis in the United States.

Sample-to-answer direct real-time PCR detection of Anaplasma phagocytophilum, Ehrlichia spp., and Babesia spp. infections in whole-blood specimens.

Emerging tick-borne illnesses, such as anaplasmosis, babesiosis, or ehrlichiosis, are caused by obligate intracellular pathogens that have clinically comparable presentations. Diagnostics used in laboratories today are serologic assays and blood smear analyses, which have known diagnostic limits. This study evaluated the performance of a sample-to-answer direct real-time PCR laboratory-developed test for the multiplex qualitative detection of Anaplasma, Babesia, and Ehrlichia DNA in whole-blood specimens. Compared to two standard-of-care (SOC) methods, the DiaSorin tick-borne laboratory-developed test for Anaplasma detection demonstrated a positive percent agreement (PPA) and negative percent agreement (NPA) of 100% (95% CI, 0.80 to 1.0) and 89% (95% CI, 0.74 to 0.97), respectively with a discordant rate of 9.3% against microscopy. After discordant resolution, the NPA increased to 100%. For Babesia, the test demonstrated a PPA of 100% (95% CI, 0.90 to 1.0) and NPA of 100% (95% CI, 0.90 to 1.0). Compared to a SOC PCR method Anaplasma samples showed a PPA of 100% (95% CI, 0.66 to 1.0) and NPA of 100% (95% CI, 0.90 to 1.0). Ehrlichia results showed a PPA of 100% (95% CI, 0.69 to 1.0) and NPA of 100% (95% CI, 0.90 to 1.0). The total percent agreement was 98% (95% CI, 0.95 to 0.99) with a κ statistic of 0.95 (95% CI, 0.90 to 0.99) or almost perfect agreement compared to SOC methods. This laboratory-developed test for detecting Anaplasma, Babesia, and Ehrlichia DNA provides rapid and reliable detection of tick-borne infections without nucleic acid extraction. IMPORTANCE: This work demonstrates that detection of tick-borne illnesses, such as anaplasmosis, babesiosis, or ehrlichiosis, can be performed directly from whole blood with no extraction. The assay described here has a high positive and negative percent agreement with existing methods and is used as the standard of care. An increasing incidence of tick-borne illness combined with shortage of well-trained technologists to perform traditional manual testing, testing options that can be adapted to various lab settings, are of the utmost importance.

Evaluation of the sensitivity of a measles diagnostic real-time RT-PCR assay incorporating recently observed priming mismatch variants, 2024.

We investigated a variant of measles virus that encodes three mismatches to the reverse priming site for a widely used diagnostic real-time RT-PCR assay; reduction of sensitivity was hypothesised. We examined performance of the assay in context of the variant using in silico data, synthetic RNA templates and clinical specimens. Sensitivity was reduced observed at low copy numbers for templates encoding the variant sequence. We designed and tested an alternate priming strategy, rescuing the sensitivity of the assay.

Detection of Bordetella spp. in children with pertussis-like illness from 2018 to 2024 in China.

OBJECTIVE: To evaluate the role of of Bordetella pertussis (B. pertussis), B. parapertussis, B. holmesii, and B. bronchiseptica on pertussis resurgence in China, particularly the sharp rise since the latest winter. METHODS: Nasopharyngeal swabs collected from children with pertussis-like illness from January 2018 to March 2024 were cultured to detect B. pertussis, B. parapertussis, B. holmesii, and B. bronchiseptica, and tested for all of these except for B. bronchiseptica using a pooled real-time polymerase chain reaction (PCR) kit targeting insertion sequences ptxS1, IS481, IS1001, and hIS1001. RESULTS: Out of the collected 7732 nasopharyngeal swabs, 1531 cases tested positive for B. pertussis (19.8%, 1531/7732), and 10 cases were positive for B. parapertussis (0.1%, 10/7732). B. holmesii and B.bronchiseptica was not detected. The number of specimens and the detection rate of B. pertussis were 1709 and 26.9% (459/1709) in 2018, 1936 and 20.7% (400/1936) in 2019, which sharply declined to 308 and 11.4% (35/308) in 2020, 306 and 4.2% (13/306) in 2021, and then notably increased to 754 and 17.6% (133/754) in 2022, 1842 and 16.0% (295/1842) in 2023, 196 and 22.4% (196/877) in the first quarter of 2024. The proportion of children aged 3 to less than 6 years (preschool age) and 6 to 16 years (school age) in pertussis cases increased significantly during the study period, especially the proportion of school-aged children increased from 2.0% (9/459) in 2018 to 40.8% (80/196) in 2024. CONCLUSIONS: B. pertussis was the predominant pathogen among children with pertussis-like illness in China, with sporadic detection of B. parapertussis and no detection of B. holmesii or B.bronchiseptica. The preschool and school-age children are increasingly prevalent in B. pertussis infection cases, which may be associated with the latest rapid escalation of pertussis outbreak.

Molecular and Serological Surveillance for Mycobacterium leprae and Mycobacterium lepromatosis in Wild Red Squirrels (Sciurus vulgaris) from Scotland and Northern England.

Leprosy is a poverty-associated infectious disease in humans caused by Mycobacterium leprae or M. lepromatosis, often resulting in skin and peripheral nerve damage, which remains a significant public health concern in isolated areas of low- and middle-income countries. Previous studies reported leprosy in red squirrels in the British Isles, despite the fact that autochthonous human cases have been absent for centuries in this region. To investigate the extent of M. leprae and M. lepromatosis presence in wild red squirrels in the northern UK, we analyzed 220 blood/body cavity fluid samples from opportunistically sampled red squirrels (2004-2023) for specific antibodies against phenolic glycolipid-I, a cell wall component specific for these leprosy bacilli. Additionally, we assessed bacillus-derived DNA by real-time PCR (qPCR) in 250 pinnae from the same cohort. M. lepromatosis and M. leprae DNA were detected by qPCR in 20.4% and 0.8% of the squirrels, respectively. No cases of co-detection were observed. Detectable levels of anti-PGL-I antibodies by UCP-LFA were observed in 52.9% of animals with the presence of M. lepromatosis determined by qPCR, and overall in 15.5% of all animals. In total, 22.6% (n = 296) of this UK cohort had at least some exposure to leprosy bacilli. Our study shows that leprosy bacilli persist in red squirrels in the northern UK, emphasizing the necessity for ongoing molecular and serological monitoring to study leprosy ecology in red squirrels, gain insight into potential zoonotic transmission, and to determine whether the disease has a conservation impact on this endangered species.

Screening of Reference Genes for Quantitative Real-Time PCR Analysis in Tissues and during Testis Development, and Application to Analyze the Expression of kifc1 in Hemibarbus labeo (Teleostei, Cypriniformes, Cyprinidae).

The selection of proper reference genes is vital for ensuring precise quantitative real-time PCR (qPCR) assays. This study evaluates the stability of the expression of nine candidate reference genes in different tissues and during testicular development in H. labeo. The results show that eef1a is recommended as a reference gene for qPCR analysis in tissues and during testicular development. Furthermore, we evaluated the optimal number of reference genes needed when calculating gene expression levels using the geomean method, revealing that two reference genes are sufficient. Specifically, eef1a and rps27 are recommended for analysis of gene expression in tissues, whereas eef1a and actb are advised for evaluating gene expression during testicular development. In addition, we examined the expression pattern of kifc1, a kinesin involved in the reshaping of spermatids. We detected peak expression levels of kifc1 in testes, with its expression initially increasing before decreasing throughout testicular development. The highest expression of kifc1 was observed in stage IV testes, the active period of spermiogenesis, suggesting a possible role for kifc1 in the regulation of the reshaping of spermatids and hence testicular development. This study represents the first investigation of reference genes for H. labeo, providing a foundation for studying gene expression patterns and investigating gene expression regulation during testicular development.

"GLI1 Subcellular Localization and Overexpression as Prognostic Factors for Disease-Free Survival in Colorectal Carcinoma".

PURPOSE: Glioma-associated oncogene homolog-1 (GLI1) is amplified in human glioblastoma, and there is growing evidence suggesting its significant role in tumor development and metastasis. Our aim was to investigate the role of the GLI-1 gene in the progression of colorectal cancer (CRC) and its correlation with various clinicopathological features. Additionally, we examined the impact of the GLI-1 gene and other factors on the prognosis of CRC. METHODS: We analyzed a total of 98 confirmed CRC cases and adjacent normal tissue controls. Patients suspected of having colon cancer underwent a colonoscopy and targeted biopsy, while those with rectal cancer underwent CT scans and MRI. GLI1 expression was detected using real-time PCR assay, Western blotting, and immunohistochemistry. RESULTS: The GLI1 gene was observed to be overexpressed in tumor tissues at both the protein and mRNA levels (p < 0.05). In addition, GLI1 overexpression was significantly associated with various factors such as tumor invasion (T3/T4), presence of lymph nodes, lymph node metastasis (LNM), stage (III/IV), tumor site (colon), tumor size (≥ 3 cm), localization (nucleocytoplasmic), strong staining intensity and recurrence (p < 0.05). The results of survival analysis showed that the patients with overexpression of GLI1 had a significantly lower DFS rate which was 21 months compared to those with normal expression who had 31 months (p < 0.05). Moreover, individuals with early onset disease (15 months) were more likely to have cytoplasmic localization of the GLI1 gene as opposed to nucleo-cytoplasmic localization of GLI1 which presented late-onset disease( 23 months) (p < 0.05). Finally, Stage and PNI (p < 0.05) were found to independently affect outcomes of CRC according to Cox regression analysis. CONCLUSION: High expression of GLI-1 in CRC is associated with adverse pathology and poor prognosis for patients. The correlation between cytoplasmic localization of GLI-1 and reduced disease-free survival holds potential for guiding prognosis and treatment. Further research is needed to develop strategies targeting GLI-1 for improved outcomes.

Real-time polymerase chain reaction detection and surgical treatment of thoracic and lumbar spondylitis due to Brucella infection: two typical case reports.

Background: Spondylitis caused by Brucella infection is a rare but challenging condition, and its successful management depends on timely diagnosis and appropriate treatment. This study reports two typical cases of thoracic and lumbar brucellosis spondylitis, highlighting the pivotal roles of real-time polymerase chain reaction (real-time PCR) detection and surgical intervention. Case presentation: Case 1 involved a 49-year-old male shepherd who presented with a 6-month history of fever (40°C), severe chest and back pain, and 2-week limited lower limb movement with night-time exacerbation. Physical examination revealed tenderness and percussion pain over the T9 and T10 spinous processes, with grade 2 muscle strength in the lower limbs. CT showed bone destruction of the T9 and T10 vertebrae with narrowing of the intervertebral space, whereas MRI demonstrated abnormal signals in the T9-T10 vertebrae, a spinal canal abscess, and spinal cord compression. The Rose Bengal plate agglutination test was positive. Case 2 was a 59-year-old man who complained of severe thoracolumbar back pain with fever (39.0°C) and limited walking for 2 months. He had a 2.5 kg weight loss and a history of close contact with sheep. The Rose Bengal test was positive, and the MRI showed inflammatory changes in the L1 and L2 vertebrae. Diagnosis and treatment: real-time PCR confirmed Brucella infection in both cases. Preoperative antimicrobial therapy with doxycycline, rifampicin, and ceftazidime-sulbactam was administered for at least 2 weeks. Surgical management involved intervertebral foraminotomy-assisted debridement, decompression, internal fixation, and bone grafting under general anesthesia. Postoperative histopathological examination with HE and Gram staining further substantiated the diagnosis. Outcomes: both patients experienced significant pain relief and restored normal lower limb movement at the last follow-up (4-12 weeks) after the intervention. Conclusion: Real-time PCR detection offers valuable diagnostic insights for suspected cases of brucellosis spondylitis. Surgical treatment helps in infection control, decompression of the spinal cord, and restoration of stability, constituting a necessary and effective therapeutic approach. Prompt diagnosis and comprehensive management are crucial for favorable outcomes in such cases.

Epigenetic alterations in preeclampsia: a focus on microRNA149 and tetrahydrofolate reductase gene polymorphisms in Egyptian women.

BACKGROUND: Preeclampsia (PE) poses a substantial risk to prenatal and maternal health. Folic acid (FA) and methylenetetrahydrofolate reductase (MTHFR) play roles in DNA methylation and genomic integrity maintenance, with MTHFR polymorphisms potentially impacting PE occurrence. Human microRNA 149 (miR-149) remains underexplored in PE despite its involvement in folate metabolism. This study seeks to evaluate serum miR-149 levels with the MTHFR C677T polymorphism for diagnosing PE. METHODS: Seventy females aged 28-40 gestational weeks were divided into control and Preeclampsia groups. Serum miR-149 and MTHFR gene levels were evaluated using real-time PCR. RESULTS: Preeclamptic patients showed significantly lower serum miR-149 levels than healthy controls (P ≤ 0.01). PE cases showed a higher frequency of the TT genotype and T allele of the C677T polymorphism (OR = 0.181, 2.882, respectively), implicating them as genetic risk factors. The CT genotype also increased PE risk (OR = 0.26), while no significant difference was observed in the CC genotype. CONCLUSION: Merging miR-149 and MTHFR polymorphism assessment improves discrimination between healthy and PE groups, offering valuable insights into PE pathogenesis and potential diagnostic strategies.

Impact of Interleukin-17 Receptor A Gene Variants on Asthma Susceptibility and Clinical Manifestations in Children and Adolescents.

Several single nucleotide polymorphisms (SNPs) in multiple interleukin receptor genes could be associated with asthma risk and/or phenotype. Interleukin-17 (IL-17) has been implicated in tissue inflammation and autoimmune diseases. As no previous studies have uncovered the potential role of IL17 receptor A (RA) gene variants in asthma risk, we aimed to explore the association of four IL17RA SNPs (i.e., rs4819554A/G, rs879577C/T, rs41323645G/A, and rs4819555C/T) with asthma susceptibility/phenotype in our region. TaqMan allelic discrimination analysis was used to genotype 192 individuals. We found that the rs4819554 G/G genotype significantly reduced disease risk in the codominant (OR = 0.15, 95%CI = 0.05-0.45, p < 0.001), dominant (OR = 0.49, 95%CI = 0.26-0.93, p = 0.028), and recessive (OR = 0.18, 95%CI = 0.07-0.52, p < 0.001) models. Similarly, rs879577 showed reduced disease risk associated with the T allele across all genetic models. However, the A allele of rs41323645 was associated with increased disease risk in all models. The G/A and A/A genotypes have higher ORs of 2.47 (95%CI = 1.19-5.14) and 3.86 (95%CI = 1.62-9.18), respectively. Similar trends are observed in the dominant 2.89 (95%CI = 1.47-5.68, p = 0.002) and recessive 2.34 (95%CI = 1.10-4.98, p = 0.025) models. For the rs4819555 variant, although there was no significant association identified under any models, carriers of the rs4819554*A demonstrated an association with a positive family history of asthma (71.4% in carriers vs. 27% in non-carriers; p = 0.025) and the use of relievers for >2 weeks (52.2% of carriers vs. 28.8% of non-carriers; p = 0.047). Meanwhile, the rs4819555*C carriers displayed a significant divergence in the asthma phenotype, specifically atopic asthma (83.3% vs. 61.1%; p = 0.007), showed a higher prevalence of chest tightness (88.9% vs. 61.5%; p = 0.029), and were more likely to report comorbidities (57.7% vs. 16.7%, p = 0.003). The most frequent haplotype in the asthma group was ACAC, with a frequency of 22.87% vs. 1.36% in the controls (p < 0.001). In conclusion, the studied IL17RA variants could be essential in asthma susceptibility and phenotype in children and adolescents.

Establishment and evaluation of a qPCR method for the detection of pfmdr1 mutations in Plasmodium falciparum, the causal agent of fatal malaria.

Drug resistance surveillance is a major integral part of malaria control programs. Molecular methods play a pivotal role in drug resistance detection and related molecular research. This study aimed to develop a rapid and accurate detection method for drug resistance of Plasmodium falciparum (P. falciparum). A quantitative real-time PCR (qPCR) assay has been developed that identifies the mutation at locus A256T in the P.falciparum multi-drug resistance(pfmdr1) gene producing amino acid change at position 86. The results of 198 samples detected by qPCR were consistent with nested PCR and sequencing, giving an accuracy of 94.3%. The sensitivity, specificity, positive and negative predictive value of qPCR were 85.7%, 97.6%, 90.0% and 96.4%, respectively. The results of qPCR are basically consistent with the nested PCR, which is expected to replace the nested PCR as a new molecular biological method for drug resistance detection, providing reliable technical support for global malaria prevention and control.

Torquetenovirus Viremia Quantification Using Real-Time PCR Developed on a Fully Automated, Random-Access Platform.

Quantification of Torquetenovirus (TTV) viremia is becoming important for evaluating the status of the immune system in solid organ transplant recipients, monitoring the appearance of post-transplant complications, and controlling the efficacy of maintenance immunosuppressive therapy. Thus, diagnostic approaches able to scale up TTV quantification are needed. Here, we report on the development and validation of a real-time PCR assay for TTV quantification on the Hologic Panther Fusion® System by utilizing its open-access channel. The manual real-time PCR previously developed in our laboratories was optimized to detect TTV DNA on the Hologic Panther Fusion® System. The assay was validated using clinical samples. The automated TTV assay has a limit of detection of 1.6 log copies per ml of serum. Using 112 samples previously tested via manual real-time PCR, the concordance in TTV detection was 93% between the assays. When the TTV levels were compared, the overall agreement between the methods, as assessed using Passing-Bablok linear regression and Bland-Altman analyses, was excellent. In summary, we validated a highly sensitive and accurate method for the diagnostic use of TTV quantification on a fully automated Hologic Panther Fusion® System. This will greatly improve the turnaround time for TTV testing and better support the laboratory diagnosis of this new viral biomarker.

PROMER technology: A new real-time PCR tool enabling multiplex detection of point mutation with high specificity and sensitivity.

Real-time polymerase chain reaction (real-time PCR) is a powerful tool for the precise quantification of nucleic acids in various applications. In cancer management, the monitoring of circulating tumor DNA (ctDNA) from liquid biopsies can provide valuable information for precision care, including treatment selection and monitoring, prognosis, and early detection. However, the rare and heterogeneous nature of ctDNA has made its precise detection and quantification challenging, particularly for ctDNA containing hotspot mutations. We have developed a new real-time PCR tool, PROMER technology, which enables the precise and sensitive detection of ctDNA containing cancer-driven single-point mutations. The PROMER functions as both a PRObe and priMER, providing enhanced detection specificity. We validated PROMER technology using synthetic templates with known KRAS point mutations and demonstrated its sensitivity and linearity of quantification. Using genomic DNA from human cancer cells with mutant and wild-type KRAS, we confirmed that PROMER PCR can detect mutant DNA. Furthermore, we demonstrated the ability of PROMER technology to efficiently detect mutation-carrying ctDNA from the plasma of mice with human cancers. Our results suggest that PROMER technology represents a promising new tool for the precise detection and quantification of DNA containing point mutations in the presence of a large excess of wild-type counterpart.

Comparative Evaluation of Mogibacterium timidum in the Subgingival Plaque of Periodontally Healthy and Chronic Periodontitis Patients: A Real-Time Polymerase Chain Reaction (PCR) Study.

BACKGROUND: Mogibacterium timidum is a new genus of anaerobic bacteria discovered in the year 2000. It is one of the most common bacteria present in the host microbial flora of dental plaque. The levels of M. timidum are supposedly higher in inflammatory conditions. AIMS AND OBJECTIVES: This study aimed to quantify the levels of M. timidum species in the subgingival plaque samples of healthy patients and patients with chronic periodontitis. MATERIALS AND METHODS: A total of 24 samples of the subgingival plaque, 12 healthy samples and 12 samples of chronic periodontitis patients, were collected in a buffer solution using a sterile Gracey curette. These samples were then sent to a laboratory for real-time polymerase chain reaction (PCR) testing. RESULTS: M. timidum was found in higher quantities in plaque samples taken from chronic periodontitis patients when compared to healthy patients. CONCLUSION: M. timidum can be said to be associated with chronic periodontitis condition. Further studies are required to know the exact nature of the pathogen.