Comprehensive investigation of malignant epithelial cell-related genes in clear cell renal cell carcinoma: development of a prognostic signature and exploration of tumor microenvironment interactions.

Clear cell renal cell carcinoma (ccRCC) is a prevalent malignancy with complex heterogeneity within epithelial cells, which plays a crucial role in tumor progression and immune regulation. Yet, the clinical importance of the malignant epithelial cell-related genes (MECRGs) in ccRCC remains insufficiently understood. This research aims to undertake a comprehensive investigation into the functions and clinical relevance of malignant epithelial cell-related genes in ccRCC, providing valuable understanding of the molecular mechanisms and offering potential targets for treatment strategies. Using data from single-cell sequencing, we successfully identified 219 MECRGs and established a prognostic model MECRGS (MECRGs' signature) by synergistically analyzing 101 machine-learning models using 10 different algorithms. Remarkably, the MECRGS demonstrated superior predictive performance compared to traditional clinical features and 92 previously published signatures across six cohorts, showcasing its independence and accuracy. Upon stratifying patients into high- and low-MECRGS subgroups using the specified cut-off threshold, we noted that patients with elevated MECRGS scores displayed characteristics of an immune suppressive tumor microenvironment (TME) and showed worse outcomes after immunotherapy. Additionally, we discovered a distinct ccRCC tumor cell subtype characterized by the high expressions of PLOD2 (procollagen-lysine,2-oxoglutarate 5-dioxygenase 2) and SAA1 (Serum Amyloid A1), which we further validated in the Renji tissue microarray (TMA) cohort. Lastly, 'Cellchat' revealed potential crosstalk patterns between these cells and other cell types, indicating their potential role in recruiting CD163 + macrophages and regulatory T cells (Tregs), thereby establishing an immunosuppressive TME. PLOD2 + SAA1 + cancer cells with intricate crosstalk patterns indeed show promise for potential therapeutic interventions.

Comparison of the short- and long-term prognosis of early-onset colorectal cancer compared with later-onset colorectal cancer: A systematic review and meta-analysis.

Background and Aims: The annual incidence of early-onset colorectal cancer (EOCRC) is increasing at an alarming rate. The prognosis of EOCRC remains controversial, and whether the early onset is a risk factor for colorectal cancer remains unclear. Methods: We searched four electronic bibliographic databases from database inception to April 25, 2022 for studies that included both early- and later-onset patients and performed a prognostic analysis. Random-effects models were used to summarize the prognostic information extracted by the investigators, including overall survival (OS), cancer-special survival (CSS), and disease-free survival (DFS). Network meta-analysis (NMA) was used to compare patients' long-term prognoses in different age subgroups. Results: After 694 reports were screened, 13 studies were included in the final analysis, with a total of 448,781 CRC cases. In the meta-analysis of the 5-year OS, EOCRC had a better prognosis compared to LOCRC (hazard ratio [HR] 0.87, 95% confidence interval [CI], 0.74-0.99; relative risk [RR] 0.83, 95% CI, 0.78-0.89). No difference in prognosis was found between the two groups in terms of 5-year CSS (RR 0.99, 95% CI, 0.93-1.05), 5-year DFS (RR 0.90, 95% CI, 0.74-1.09), and short-term OS. In the NMA, patients aged <30 years had the worst outcome (surface under the cumulative ranking curve [SUCRA], 15.8%) in 5-year OS; consistent results were observed in the analysis of 5-year CSS (<30 years, SUCRA 4.5%), but the difference was not statistically significant. Conclusion: Although patients with early-onset CRC had better OS than those with later-onset CRC, there was no difference in the CSS. Meanwhile, the trend for survival was worse in younger patients, especially in those ages 18-29 years. Thus, more attention should be paid to early diagnosis and treatment of EOCRC. Systematic Review and Meta­Analysis Registration: The systematic review and Meta-analysis protocol was registered with PROSPERO (registration number CRD42022334697).

TLR7 modulates extramedullary splenic erythropoiesis in P. yoelii NSM-infected mice through the regulation of iron metabolism of macrophages with IFN-γ.

Splenomegaly is a prominent clinical manifestation of malaria and the causes remain incompletely clear. Anemia is induced in malaria and extramedullary splenic erythropoiesis is compensation for the loss of erythrocytes. However, the regulation of extramedullary splenic erythropoiesis in malaria is unknown. An inflammatory response could facilitate extramedullary splenic erythropoiesis in the settings of infection and inflammation. Here, when mice were infected with rodent parasites, Plasmodium yoelii NSM, TLR7 expression in splenocytes was increased. To explore the roles of TLR7 in splenic erythropoiesis, we infected wild-type and TLR7 -/- C57BL/6 mice with P. yoelii NSM and found that the development of splenic erythroid progenitor cells was impeded in TLR7 -/- mice. Contrarily, the treatment of the TLR7 agonist, R848, promoted extramedullary splenic erythropoiesis in wild-type infected mice, which highlights the implication of TLR7 on splenic erythropoiesis. Then, we found that TLR7 promoted the production of IFN-γ that could enhance phagocytosis of infected erythrocytes by RAW264.7. After phagocytosis of infected erythrocytes, the iron metabolism of RAW264.7 was upregulated, evidenced by higher iron content and expression of Hmox1 and Slc40a1. Additionally, the neutralization of IFN-γ impeded the extramedullary splenic erythropoiesis modestly and reduced the iron accumulation in the spleen of infected mice. In conclusion, TLR7 promoted extramedullary splenic erythropoiesis in P. yoelii NSM-infected mice. TLR7 enhanced the production of IFN-γ, and IFN-γ promoted phagocytosis of infected erythrocytes and the iron metabolism of macrophages in vitro, which may be related to the regulation of extramedullary splenic erythropoiesis by TLR7.

Development of a novel gene signature to predict prognosis and response to PD-1 blockade in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common kidney malignancy characterized by a poor prognosis. The treatment efficacy of immune checkpoint inhibitors (ICIs) also varies widely in advanced ccRCC. We aim to construct a robust gene signature to improve the prognostic discrimination and prediction of ICIs for ccRCC patients. In this study, adopting differentially expressed genes from seven ccRCC datasets in GEO (Gene Expression Omnibus), a novel signature (FOXM1&TOP2A) was constructed in TCGA (The Cancer Genome Atlas) database by LASSO and Cox regression. Survival and time-dependent ROC analysis revealed the strong predictive ability of our signature in discovery set, two online validation sets and one tissue microarray (TMA) from our institution. High-risk group based on the signature comprises more high-grade (G3&G4) and advanced pathologic stage (stageIII/IV) tumors and presents hyperactivation of cell cycle process according to the functional analysis. Meanwhile, high-risk tumors demonstrate an immunosuppressive phenotype with more infiltrations of regulatory T cells (Tregs), macrophages and high expressions of genes negatively regulating anti-tumor immunity. Low-risk tumors have an improved response to anti-PD-1 therapy and the predictive ability of our signature is better than other recognized biomarkers in ccRCC. A nomogram containing this signature showed a high predictive accuracy with AUCs of 0.90 and 0.84 at 3 and 5 years. Overall, this robust signature could predict prognosis, evaluate immune microenvironment and response to anti-PD-1 therapy in ccRCC, which is very promising in clinical promotion.

PRMT1 is a novel molecular therapeutic target for clear cell renal cell carcinoma.

Background and Objective: Epigenetic alterations are common events in clear cell renal cell carcinoma (ccRCC), and protein arginine methyltransferase 1 (PRMT1) is an important epigenetic regulator in cancers. However, its role in ccRCC remains unclear. Methods: We investigated PRMT1 expression level and its correlations to clinicopathological factors and prognosis in ccRCC patients based on ccRCC tissue microarrays (TMAs). Genetic knockdown and pharmacological inhibition using a novel PRMT1 inhibitor DCPT1061 were performed to investigate the functional role of PRMT1 in ccRCC proliferation. Besides, we confirmed the antitumor effect of PRMT1 inhibitor DCPT1061 in ccRCC cell-derived tumor xenograft (CDX) models as well as patient-derived tumor xenograft (PDX) models. Results: We found PRMT1 expression was remarkably upregulated in tumor tissues and associated with poor pathologic characters and outcomes of ccRCC patients. Furthermore, genetic knockdown and pharmacological inhibition of PRMT1 by a novel potent inhibitor DCPT1061 dramatically induced G1 cell cycle arrest and suppressed ccRCC cell growth. Mechanistically, RNA sequencing and further validation identified Lipocalin2 (LCN2), a secreted glycoprotein implicated in tumorigenesis, as a crucial regulator of ccRCC growth and functional downstream effector of PRMT1. Epigenetic silencing of LCN2 autocrine secretion by PRMT1 deficiency decreased downstream p-AKT, leading to reduced p-RB and cell growth arrest through the neutrophil gelatinase associated lipocalin receptor (NGALR). Moreover, PRMT1 inhibition by DCPT1061 not only inhibited tumor growth but also sensitized ccRCC to sunitinib treatment in vivo by attenuating sunitinib-induced upregulation of LCN2-AKT-RB signaling. Conclusion: Taken together, our study revealed a PRMT1-dependent epigenetic mechanism in the control of ccRCC tumor growth and drug resistance, indicating PRMT1 may serve as a promising target for therapeutic intervention in ccRCC patients.

SUV39H1 deficiency suppresses clear cell renal cell carcinoma growth by inducing ferroptosis.

Clear cell renal cell carcinoma (ccRCC) is a common kidney malignancy characterized by a poor prognosis. Suppressor of variegation 3-9 homolog 1 (SUV39H1), which encodes a histone H3 lysine 9 methyltransferase, has been reported to act as an oncogene in many cancers. However, it is unclear whether SUV39H1 is involved in ccRCC. Here, we report that SUV39H1 expression is frequently upregulated in ccRCC tumors and is significantly correlated with ccRCC progression. SUV39H1 expression level is an independent risk factor for cancer prognosis, and integration with several known prognostic factors predicted ccRCC patient prognosis with improved accuracy than the conventional SSIGN (stage, size, grade and necrosis) prognostic model. Mechanistically, we discovered that siRNA knockdown or pharmacological inhibition of SUV39H1 induced iron accumulation and lipid peroxidation, leading to ferroptosis that disrupted ccRCC cell growth in vitro and in vivo. We also show that SUV39H1 deficiency modulated the H3K9me3 status of the DPP4 (dipeptidyl-peptidase-4) gene promoter, resulting in upregulation of its expression that contributes to ferroptosis. Taken together, our findings provide the mechanistic insight into SUV39H1-dependent epigenetic control of ccRCC tumor growth and indicate that SUV39H1 may serve as a potential therapeutic target for ccRCC treatment.

An Observational Study: Is N-Acetylcysteine Helpful in Performance Improvement of Mycoplasma IST2 Testing through Sample Homogenization?

BACKGROUND: Culture is still the gold standard for the detection of genital mycoplasma which could cause urogenital infections in humans. Mycoplasma IST2 is a commercial kit widely used for the detection of M. hominis and Ureaplasma species. Its accuracy was partially impaired because clinical specimens are usually mixed with purulent or transparent mucus. We aimed to solve this problem through sample homogenization by N-acetylcysteine (NAC) treatment. METHODS: Twenty-two endocervical swab samples were collected from 22 female patients with suspected mycoplasma infection, while 11 of these specimens were with purulent or transparent mucus. Mycoplasma IST2 testing kit was used for mycoplasma culture and AST for the control group and NAC-treated group. RESULTS: Genital mycoplasma was detected in 15 of 22 samples for both groups. The colony number in 6 out of 11 purulent specimens (54.5%) was more than 104 CFU/ml of genital mycoplasma for the NAC-treated group, while only one of 11 (9.1%) for the control group. For the nonpurulent specimens, no significant difference had been found in colony counting of genital mycoplasma between the control group and NAC-treated group (P > 0.05). The results of antimicrobial susceptibility testing for the NAC-treated group were highly similar to those for the control group. CONCLUSIONS: Our results demonstrate that NAC is helpful in sample homogenization and NAC treatment can improve the detection efficiency of mycoplasma with Mycoplasma IST2 testing.

Identification of biomarkers of chromophobe renal cell carcinoma by weighted gene co-expression network analysis.

BACKGROUND: Chromophobe renal cell carcinoma (ChRCC) is the second common subtype of non-clear cell renal cell carcinoma (nccRCC), which accounting for 4-5% of renal cell carcinoma (RCC). However, there is no effective bio-marker to predict clinical outcomes of this malignant disease. Bioinformatic methods may provide a feasible potential to solve this problem. METHODS: In this study, differentially expressed genes (DEGs) of ChRCC samples on The Cancer Genome Atlas database were filtered out to construct co-expression modules by weighted gene co-expression network analysis and the key module were identified by calculating module-trait correlations. Functional analysis was performed on the key module and candidate hub genes were screened out by co-expression and MCODE analysis. Afterwards, real hub genes were filter out in an independent dataset GSE15641 and validated by survival analysis. RESULTS: Overall 2215 DEGs were screened out to construct eight co-expression modules. Brown module was identified as the key module for the highest correlations with pathologic stage, neoplasm status and survival status. 29 candidate hub genes were identified. GO and KEGG analysis demonstrated most candidate genes were enriched in mitotic cell cycle. Three real hub genes (SKA1, ERCC6L, GTSE-1) were selected out after mapping candidate genes to GSE15641 and two of them (SKA1, ERCC6L) were significantly related to overall survivals of ChRCC patients. CONCLUSIONS: In summary, our findings identified molecular markers correlated with progression and prognosis of ChRCC, which might provide new implications for improving risk evaluation, therapeutic intervention, and prognosis prediction in ChRCC patients.

Electrochemical determination of microRNAs based on isothermal strand-displacement polymerase reaction coupled with multienzyme functionalized magnetic micro-carriers.

MicroRNAs (miRNAs) show great potential for disease diagnostics due to their specific molecular profiles. Detection of miRNAs remains challenging and often requires sophisticated platforms. Here we report a multienzyme-functionalized magnetic microcarriers-assisted isothermal strand-displacement polymerase reaction (ISDPR) for quantitative detection of miRNAs. Magnetic micro-carriers (MMCs) were functionalized with molecular beacons to enable miRNAs recognition and magnetic separation. The target miRNAs triggered a phi29-mediated ISDPR, which can produce biotin-modified sequences on the MMCs. Streptavidin-alkaline phosphatase was then conjugated to the MMC surface through biotin-streptavidin interactions. In the presence of 2-phospho-L-ascorbic acid, miRNAs were quantitatively determined on a screen-printed carbon electrode from the anodic current of the enzymatic product. We show that this method enables detection of miRNAs as low as 9 fM and allows the discrimination of one base mismatched sequence. The proposed method was also successfully applied to analyze miRNAs in clinical tumor samples. This paper reports a new strategy for miRNAs analysis with high sensitivity, simplicity, and low cost. It would be particularly useful for rapid point-of-care testing of miRNAs in clinical laboratory.

Elevation of miR-27b by HPV16 E7 inhibits PPARγ expression and promotes proliferation and invasion in cervical carcinoma cells.

MicroRNAs (miRNAs) have been reported to be involved in multiple biological pathways that can influence tumor progression and metastasis. High-risk human papillomavirus (HR-HPVs) is aetiologically correlated to cervical cancer. Recently, miRNAs were reported to be regulated by virus and play pivotal roles in HPV-related tumor progression. However, the underlying mechanism remains poorly understood. In the present study, we report that HPV16 E7 upregulated miR-27b to promote proliferation and invasion in cervical cancer. The results showed that PPARγ, as a target of miR-27b, played a significant role in suppressing cervical cancer progression by downregulating the sodium-hydrogen exchanger isoform 1 (NHE1). It was also shown that the inhibition of miR-27b diminished the ability of HPV16 E7 to suppress PPARγ or activate NHE1 expression. In addition, we observed high expression of miR-27b and NHE1, but low expression of PPARγ in HPV16-positive cervical cancer tissues. In summary, the present study revealed that miR-27b is upregulated by HPV16 E7 to inhibit PPARγ expression and promotes proliferation and invasion in cervical carcinoma cells.

Practical Prediction of Ten Common Streptococcus pneumoniae Serotypes/Serogroups in One PCR Reaction by Multiplex Ligation-Dependent Probe Amplification and Melting Curve (MLPA-MC) Assay in Shenzhen, China.

BACKGROUND: Streptococcus pneumoniae has more than 95 distinct serotypes described to date. However, only certain serotypes are more likely to cause pneumococcal diseases. Thus serotype surveillance is important for vaccine formula design as well as in post-vaccine serotype shift monitor. The goal of this study was to develop a practical screening assay for ten Shenzhen China common pneumococcal serotypes/serogroups in one molecular reaction. METHODS: A molecular assay, based on multiplex ligation-dependent probe amplification (MLPA) and melting curve (MC) analysis, was developed in an integrated approach (MLPA-MC) for the detection of ten capsular serotypes/serogroups 4, 6 (6A/6B/6C/6D), 9V/9A, 14, 15F/15A, 15B/15C, 18 (18F/18A/18B/18C), 19F, 19A and 23F. We designed serotype/serogroup-specific MLPA probes and fluorescent detection probes to discriminate the different serotypes/serogroups in one molecular reaction. The three steps of MLPA-MC assay are continuous reactions in one well detected by LightCycler 480. A total of 210 S. pneumoniae isolates from our local Maternity and Child Health Hospital were randomly chosen to evaluate the assay against published multiplex PCR assays. RESULTS: Our results showed that 198 (94.3%) of S. pneumoniae isolates were type-able by our assays and the results were in complete concordance with the published multiplex PCRs. Using the MLPA-MC assay, 96 S. pneumoniae isolates could be typed within 3 hours with limited hands-on time. This serotype/serogroup-screening assay can be easily modified or extended by modification of the serotype/serogroup-specific MLPA probes combinations according to the needs of different laboratories. CONCLUSIONS: We recommend use of this assay as a starting point for screening serotype/serogroup frequencies. There is a need for this assay to be combined with other molecular typing assays, like published serotype specific PCRs, or even the Quellung reaction for serotype confirmation.

Optimization of the Original TRIzol-Based Technique Improves the Extraction of Circulating MicroRNA from Serum Samples.

BACKGROUND: MicroRNAs (miRNAs) are a group of small endogenous, non-coding RNA molecules that have been demonstrated to be essential regulators of many critical biological and pathological processes. Because of their high stability in blood and the strong implication of miRNA expression profiles for human diseases, miRNAs are currently emerging as promising circulating biomarkers for the diagnosis or prognosis of a variety of human diseases. The TRIzol-based technique has been widely used for cell or tissue RNA extraction because of its economy and reliability. However, the original TRIzol-based RNA isolation protocol was not specifically designed for microRNA extraction from serum samples. When it was used to extract serum microRNAs, due to the short sequence and low level of microRNAs, the isolation efficiency in most cases could not meet the requirement. To address this issue, in this study, an improved TRIzol-based protocol was established by modifying the extraction procedure of the original TRIzol-based protocol. The performance of the improved TRIzol-based protocol was evaluated by comparison with other methods. METHODS: Total RNA was isolated by the improved TRIzol-based method, the original method, and two other commonly used methods. RT-qPCR and spectrophotometry were used to examine the quality and yield of total RNA. RESULTS: The improved method was more efficient than the original protocol and more suitable for real-time PCR-based profiling experiments. CONCLUSIONS: The optimized TRIzol-based method described in this report is suitable for the extraction of serum microRNAs and useful for the development of microRNAs as diagnostic biomarkers.

microRNA-141 inhibits cell proliferation and invasion and promotes apoptosis by targeting hepatocyte nuclear factor-3ß in hepatocellular carcinoma cells.

BACKGROUND: Hepatocyte nuclear factor-3ß (HNF-3ß) plays a critical role in hepatocyte differentiation and controls liver-specific gene expression during the development of hepatocellular carcinoma (HCC), but the molecular basis of this process has not been fully elucidated. microRNAs (miRNAs) are powerful, post-transcriptional regulators of gene expression. Whether miRNAs can impact the effects of HNF-3ß in HCC is still unknown. METHODS: HNF-3ß and miR-141 expression levels were detected in HepG2 cells, using real-time quantitative RT-PCR (qRT-PCR). Luciferase reporter assays and Western blots were used to validate HNF-3ß as a direct target gene of miR-141. Cell proliferation, invasion, and apoptosis were also examined to confirm whether miR-141 could impact on HNF-3ß in HCC. RESULTS: In this study, we found that HNF-3ß protein levels were consistently upregulated in HCC clinical tissues compared with matched normal adjacent tissues. However, the mRNA levels of HNF-3ß varied in random tissues, suggesting that a post-transcriptional mechanism was involved in its regulation. We used bioinformatic analyses to search for miRNAs that could potentially target HNF-3ß, and identified specific targeting sites for miR-141 in the 3'-untranslated region (3'-UTR) of the HNF-3ß gene. By overexpressing miR-141 in HepG2 cells, we experimentally validated that miR-141 directly regulated HNF-3ß expression. Furthermore, the biological consequences of targeting HNF-3ß by miR-141 were examined using cell proliferation, invasion and apoptosis assays in vitro. We demonstrated that the repression of HNF-3ß by miR-141 suppressed the proliferation and invasion and promoted the apoptosis of HepG2 cells. CONCLUSIONS: miR-141 functions as a tumor suppressor in HCC cells through the inhibition of HNF-3ß translation.

The impact of hepatitis B virus x protein and microRNAs in hepatocellular carcinoma: a comprehensive analysis.

microRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs, approximately 22 nucleotides (nts) in length, widely found in animals, plants, and viruses. Mature miRNAs control gene expression at a post-transcriptional level through blocking protein translation or inducing mRNA degradation. Many recent studies have shown that hepatitis B virus x protein (HBx), a viral protein with a crucial role in hepatogenesis, is associated with the regulation of miRNAs. This interaction impacts fundamental tumor processes, such as cell proliferation, differentiation, and apoptosis. In this review, we summarized the recent literature on the roles of HBx-regulated miRNAs in the pathogenesis of hepatocellular carcinoma.

Electrochemical enzyme-linked immunosorbent assay (ELISA) for α-fetoprotein based on glucose detection with multienzyme-nanoparticle amplification.

Since glucose biosensors are one of the most popular and widely used point-of-care testing devices, a novel electrochemical enzyme-linked immunosorbent assay (ELISA) for protein biomarkers has been developed based on a glucose detection strategy. In this study, α-fetoprotein (AFP) was used as the target protein. An electrochemical ELISA system was constructed using anti-AFP antibodies immobilized on microwell plates as the capture antibody (Ab1) and multi-label bioconjugates as signal tracer. The bioconjugates were synthesized by attaching glucoamylase and the secondary anti-AFP antibodies (Ab2) to gold nanoparticles (AuNPs). After formation of the sandwich complex, the Ab2-glucoamylase-AuNPs conjugates converted starch into glucose in the presence of AFP. The concentration of AFP can be calculated based on the linear relation between AFP and glucose, the concentration of which can be detected by the glucose biosensor. When the AFP concentration ranged from 0.05 to 100 ng/mL, a linear calibration plot (i (µA) = 13.62033 - 2.86252 logCAFP (ng/mL), r = 0.99886) with a detection limit of 0.02 ng/mL was obtained under optimal conditions. The electrochemical ELISA developed in this work shows acceptable stability and reproducibility, and the assay for AFP spiked in human serum also shows good recovery (97.0%-104%). This new method could be applied for detecting any protein biomarker with the corresponding antibodies.

High-resolution melting curve analysis for rapid detection of rifampin resistance in Mycobacterium tuberculosis: a meta-analysis.

A rapid, simple, accurate, and affordable method for the detection of drug-resistant tuberculosis is very critical for the selection of antimicrobial therapy and management of patient treatment. High-resolution melting curve analysis has been used for the detection of rifampin resistance in Mycobacterium tuberculosis and has shown promise. We did a systematic review and meta-analysis of published studies to evaluate the accuracy of high-resolution melting curve analysis for the detection of rifampin resistance in clinical M. tuberculosis isolates. We searched the PubMed, BIOSIS Previews, and Web of Science databases to identify studies and included them according to predetermined criteria. We used the DerSimonian-Laird random-effects model to calculate pooled measures and applied Moses' constant for linear models to fit the summary receiver operating characteristic curve. According to the selection criteria, most of the identified studies were excluded, and only seven studies were included in the final analysis. The overall sensitivity of the high-resolution melting curve analysis was 94% (95% confidence interval [CI], 92% to 96%), and the overall specificity was very high at 99% (95% CI, 98% to 100%). The values for the pooled positive likelihood ratio, negative likelihood ratio, and diagnostic odds ratio were 63.39 (95% CI, 30.21 to 133.00), 0.06 (95% CI, 0.04 to 0.09), and 892.70 (95% CI, 385.50 to 2,067.24), respectively. There was no significant heterogeneity across all included studies for the measurements we evaluated. The summary receiver operating characteristic curve for the same data shows an area of 0.99 and a Q* value of 0.97. High-resolution melting curve analysis has high sensitivity and specificity for the detection of rifampin resistance in clinical M. tuberculosis isolates. This method might be a good alternative to conventional drug susceptibility tests in clinical practice.

Commercial MPT64-based tests for rapid identification of Mycobacterium tuberculosis complex: a meta-analysis.

OBJECTIVE: We did a systematic review and meta-analysis of published studies to evaluate the accuracy of commercial MPT64-based immunochromatographic tests for rapid identification of Mycobacterium tuberculosis complex. METHODS: We identified studies by searching Pubmed, BIOSIS Previews and Web of Science, and included studies using predetermined inclusion criteria. The data were pooled using the DerSimonian-Laird random effects model. RESULTS: A total of 28 studies were included in the final analysis. Pooled estimates were 97% (confidence interval [CI] 96-97%) for sensitivity and 98% (CI 98-99%) for specificity. The summary receiver operating characteristic curve showed an area of 0.9968 and a Q* of 0.98. Subgroup analysis showed that test accuracy did not depend on commercial kit, reference test and medium. CONCLUSIONS: Commercial MPT64-based immunochromatographic tests are highly sensitive and specific for rapid identification of M. tuberculosis complex. They are good alternatives to biochemical test and molecular assays. Nevertheless, additional studies are required in setting with high prevalence of mpt64 mutations or high contamination of cultures.

Comparative evaluation of two rapid methods for differentiating mycobacteria.

The real-time PCR with duplex primer sets and the MPB64-based immunochromatographic assay are newly developed methods for rapid differentiation of mycobacteria. The aim of this study is to evaluate the two methods for differentiation between Mycobacterium tuberculosis complex and nontuberculous mycobacteria. A total of 95 clinical mycobacterial isolates belonging to 22 different species and 16 reference strains of 16 different species were differentiated by duplex real-time PCR method and MPB64-based immunochromatographic assay method. The two methods were evaluated by comparison with conventional biochemical technique as the gold standard method. The duplex real-time PCR method correctly differentiated all reference strains as well as the MPB64-based immunochromatographic assay method. For clinical isolates, the accuracy of the duplex real-time PCR method (100%) was slightly higher than the MPB64-based immunochromatographic assay method (97.9%), but there was no statistical significance between the two methods (P > 0.05), and there was an excellent agreement between them (Kappa = 0.957). The duplex real-time PCR method possesses greater potential for differentiation of mycobacteria in the clinical laboratory than the MPB64-based immunochromatographic assay method. However, the MPB64-based immunochromatographic assay method is more convenient than the duplex real-time PCR method when the number of sample is small.

Mutation characterization of gyrA and gyrB genes in levofloxacin-resistant Mycobacterium tuberculosis clinical isolates from Guangdong Province in China.

OBJECTIVE: Fluoroquinolone (FQ)-resistant Mycobacterium tuberculosis (MTB) clinical isolates have emerged in many areas of the world. The aim of this study was to observe the molecular characterization of gyrA and gyrB genes in FQ-resistant MTB clinical isolates from Guangdong Province in China. MATERIALS AND METHODS: A total of 62 MTB clinical strains were originally isolated from patients with pulmonary tuberculosis. The phenotype of susceptibility of each strain was determined by the absolute concentration method and the sequences of the QRDR in gyrA and gyrB genes were detected with DNA direct sequencing technique. RESULTS: 44 of 60 (73.3%) levofloxacin-resistant MTB clinical isolates, including 17 of 18 (94.4%) high-level resistant strains and 27 of 42 (64.3%) low-level resistant strains, had mutation in QRDR of gyrA gene. The mutation patterns involved six patterns of single codon mutation (H70R, A90V, S91A, D94G, D94A or D94N) and one pattern of double codons mutation (A90V with D94A). Of 60 levofloxacin-resistant MTB clinical isolates, only one (1.6%) mutated in gyrB gene (T511N). CONCLUSIONS: These findings confirm that mutations of gyrA codons 90, 91 and 94 constitute the primary mechanism of FQ resistance in MTB. Furthermore, our findings indicate that the regional investigations are necessary for the comprehensive understanding of FQ resistance of MTB.