Formosanin C inhibits pulmonary metastasis by targeting stearyl CoA desaturase-1.

BACKGROUND: Cisplatin (DDP) as the first-line drug has been used in cancer therapy. However, side effects and drug resistance are the challenges of DDP. Disordered lipid metabolism is related to DDP resistance. STUDY DESIGN: In this study, formosanin C (FC) as the main compound of Rhizoma Paridis saponins (RPS) inhibits pulmonary metastasis by targeting stearyl CoA desaturase-1. METHODS AND RESULTS: RPS prolonged the survival period of mice, reduced pulmonary metastases and alleviated colon toxicity caused by DDP. FC as the main compound of RPS enhanced the anti-tumor and anti-metastatic effects of DDP. FC decreased the mRNA level of SCD1 and the content of lipid droplets (LDs) in lung cancer cells. Molecular dynamics and isothermal titration calorimetry verified the binding stability and spontaneously between FC and SCD1. SiSCD1 reduced the content of LDs in cell lines and increased mitochondria (mtROS), which was consistent with the results of FC treatment. The combination group decreased DNA repair associated protein as well as DDP resistance markers such as ERCC1 and 53bp1, and increased DNA damage marker like γH2AX, which indirectly confirmed the occurrence of mtROS. In addition, FC combination with DDP also affected epithelial-mesenchymal transition-related protein like VIM and CDH1 in vivo experiments, and thereby inhibited pulmonary metastasis. CONCLUSION: Our research indicated that the FC as the main compound of RPS targeted the CY2 domain of SCD1, inhibited lipid metabolism in mice, and thereby suppressed cancer metastases. This provided support for use of FC to treat cancer based on lipid metabolism pathway.

Cogan's Syndrome Combined with Hypertrophic Pachymeningitis: A Case Report.

Cogan's syndrome (CS) is a rare chronic inflammatory disease, characterized by interstitial keratitis and vestibular auditory dysfunction. Hypertrophic pachymeningitis (HP) is a rare chronic aseptic inflammatory disease of the central nervous system. This article reports a patient with CS coexisting with HP. The patient was a 66-year-old male with fever, headache, red eyes, hearing loss, and significantly elevated inflammatory markers. Cerebrospinal fluid examination, blood culture, and tests for autoantibodies such as antinuclear antibodies were negative. Pure tone audiology (PTA) indicated bilateral sensorineural deafness. Both Positron emission tomography-computed tomography (PET/CT) and vascular color Doppler ultrasound suggest the presence of vasculitis. Considering Cogan's syndrome, the patient received 40 mg of methylprednisolone intravenously once daily. The brain's magnetic resonance imaging (MRI) revealed slightly thickened and enhanced dura mater, suggesting HP. The dose of methylprednisolone was increased to 40 mg intravenously every 8 hours, leading to the patient's improved symptoms and decreased inflammatory markers. Both CS and HP are rare chronic inflammatory diseases, and their coexistence is even rarer, with only two reported cases in literature up to date. The coexistence of CS and HP should be considered when the CS patients with headaches do not respond well to treatment.

Synthesis of Core M3 Matriglycan Constituents via an Additive-Controlled 1,2-cis-Xylopyranosylation with O-Xylosyl Imidates as Donors.

A highly stereoselective strategy for 1,2-cis-xylopyranoside bond formation was established via a preactivation-based, additive-modulated trichloroacetimidate glycosidation strategy. The current protocol is mild, practical, and successful with various xylopyranosyl donors and glycosyl acceptors, including acceptors that are reported to be less reactive due to steric hindrance. The utility of this method was demonstrated with the facile assembly of matriglycan constituent tetra- and hexasaccharides.

Immunoglobulin G4-related Disease with Multiple Organs Involvement Depicted on FDG PET/CT: A Case Report and Literature Review.

INTRODUCTION: Immunoglobulin G4-related disease (IgG4-RD) is a relatively rare immune-mediated chronic inflammatory disease with fibrosis newly defined in recent years. It can involve multiple systems and organs with complex clinical manifestations. Due to mass-like lesions, it is easily misdiagnosed as tumors. CASE REPORT: Herein, we report a 57-year-old woman treated for submandibular mass and anosmia. The serum IgG4 level was increased. The biopsy of the submandibular gland indicated salivary gland tissue and hyperplasia of fibrous tissue and lymphoid tissue. Immunohistochemical examination showed a large number of IgG4-positive plasma cells. M protein was found in the patient's serum by immunofixation electrophoresis, and plasma cell diseases were excluded by bone marrow puncture. PET/CT examination showed that besides the submandibular glands, the parotid gland, common bile duct, the transitional part of the left renal pelvis and ureter, retroperitoneum in the lower abdomen, and multiple lymph nodes were also involved. The patient was diagnosed with IgG4-RD, and after treatment with glucocorticoid, the enlargement of submandibular glands and decreased olfactory function improved. After 14 weeks of treatment, the serological examinations, PET/CT, and ultrasound re-examination results showed significant improvement. So far, the patient has been followed up for 27 months and is in continuous remission. CONCLUSION: This case report aims to raise awareness of IgG4-RD and explore the value of PET/CT in the diagnosis and efficacy monitoring of the disease.

SARS-CoV-2 E protein: Pathogenesis and potential therapeutic development.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic, which has seriously affected human health worldwide. The discovery of therapeutic agents is extremely urgent, and the viral structural proteins are particularly important as potential drug targets. SARS-CoV-2 envelope (E) protein is one of the main structural proteins of the virus, which is involved in multiple processes of the virus life cycle and is directly related to pathogenesis process. In this review, we present the amino acid sequence of the E protein and compare it with other two human coronaviruses. We then explored the role of E protein in the viral life cycle and discussed the pathogenic mechanisms that E protein may be involved in. Next, we summarize the potential drugs against E protein discovered in the current studies. Finally, we described the possible effects of E protein mutation on virus and host. This established a knowledge system of E protein to date, aiming to provide theoretical insights for mitigating the current COVID-19 pandemic and potential future coronavirus outbreaks.

A facile turn-on chemiluminescence probe for sensitive imaging on aminopeptidase N activity.

Aminopeptidase N, as a target for drug discovery, shows marked relationships with many diseases, especially liver injury and cancer. Here, we explored a chemiluminescence (CL) probe for sensing APN by tethering the APN-specific substrate group to the ortho-acrylated phenoxy-dioxetane scaffold. In this way, two CL probes (APN-CL and BAPN-CL) were designed with noncapped leucine and butoxy-carbonyl capped leucine as the protecting group to preserve the chemiexcitation energy. The uncovered leucine was demonstrated to be essential for detection of APN activity by comparing the CL intensity of two CL probes. Probe APN-CL was turned on upon APN cleavage, resulting in a high chemiluminescent emission, whereas the chemiexcitation energy of probe BAPN-CL was still restrained even with the high-level APN. The result was further elucidated by molecular docking simulations. Probe APN-CL exhibited a fast response and high sensitivity with a detection limit of 0.068 U/L, and an excellent specificity for the discrimination of APN from biological ions, small molecules, and other proteases commonly found in living system. By virtue of good stability and cell viability, probe APN-CL imaged abnormal levels of APN in tumour cells and tumour-bearing mice. Moreover, this probe APN-CL could be easily used to evaluate APN inhibitors and APN levels in plasma samples from 20 patients. Overall, as a facile and cost-effective probe, APN-CL will be a promising alternative in the early diagnosis of pathologies and for cost-effective screening of inhibitors.

Efficacy of different sequential patterns after crizotinib progression in advanced anaplastic lymphoma kinase-positive non-small cell lung cancer.

BACKGROUND: The efficacy difference between the second- and third-generation of anaplastic lymphoma kinase-tyrosine kinase inhibitors (ALK-TKIs) after crizotinib failure in advanced ALK-positive non-small cell lung cancer (NSCLC) has not been clarified. This study evaluates the efficacy of different sequential patterns after crizotinib progression. METHODS: Data of patients who met the study criteria were retrospectively analyzed. The Kaplan-Meier method was used to draw survival curves, log-rank method was used to compare the differences between groups, and Cox multivariate analysis was used to evaluate the significance of influencing factors. RESULTS: A total of 128 patients developed disease progression after crizotinib. The overall survival (OS) of 57 patients in the sequential second-generation ALK-TKIs group was significantly longer than that of 65 patients with other systemic treatment (58.5 months vs. 33.0 months, p < 0.001); The OS of the direct sequential lorlatinib group was significantly longer than the second-generation ALK-TKIs group (114.0 months vs. 58.5 months, p = 0.020). Similarly, of the 48 patients who developed disease progression after first- and second-generation ALK-TKIs treatment, 16 patients with sequential lorlatinib had significantly longer OS than the others (62.0 months vs. 43.0 months, p = 0.014). The progression-free survival (PFS) of second-line and third- or later-line lorlatinib were statistically different (20.0 months vs. 5.5 months, p = 0.011). CONCLUSIONS: The application of next-generation ALK-TKIs after crizotinib progression significantly prolonged survival, whereas direct sequencing lorlatinib seemed advantageous. Similarly, lorlatinib also prolonged survival in patients with first- and second-generation ALK-TKIs failure.

Q-marker identification of Paris polyphylla var. yunnanensis (Franch.) Hand.-Mazz. in pulmonary metastasis of liver cancer mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhizoma Paridis saponins (RPS) as the mainly active components of Paris polyphylla var. yunnanensis (Franch.) Hand.-Mazz., possess tumor therapeutic potential. However, the anti-tumor material basis of RPS in liver cancer pulmonary metastasis remains poorly understood. The objective of this study was to identify the distribution and anti-cancer effects of RPS in liver cancer pulmonary metastatic model. MATERIALS AND METHODS: In this study, a mouse liver cancer pulmonary metastasis model was established to determine the distribution of different saponins in the tissues by UPLC-MS and plasma protein binding rate. RESULTS: As a result, RPS prolonged the survival time and inhibited the pulmonary metastasis in H22 injected mice through its underlying mechanism. UPLC-MS identified saponins from RPS such as PVII, PH, PVI, PII, gracillin and PI in tissues, which may be regarded as the Q-markers in RPS. Surprisingly, the concentration of PI, PII and gracillin as diosgenyl saponins was higher than that of pennogenyl saponins in the liver and lung. Besides, plasma protein binding rate of PII was higher than that of PVII. CONCLUSION: These findings suggested that PVII, PH, PVI, PI, PII and gracillin are regarded as the Q-markers of RPS in liver cancer pulmonary metastasis. The concentration of PI, PII and gracillin as diosgenyl saponins was higher than that of pennogenyl saponins in the liver and lung. It would be helpful for understanding the importance of RPS with anticancer activities in the future.

A Highly Efficient Fluorescent Sensor Based on AIEgen for Detection of Nitrophenolic Explosives.

The detection of nitrophenolic explosives is important in counterterrorism and environmental protection, but it is still a challenge to identify the nitroaromatic compounds among those with a similar structure. Herein, a simple tetraphenylethene (TPE) derivative with aggregation-induced emission (AIE) characteristics was synthesized and used as a fluorescent sensor for the detection of nitrophenolic explosives (2, 4, 6-trinitrophenol, TNP and 2, 4-dinitrophenol, DNP) in water solution and in a solid state with a high selectivity. Meanwhile, it was found that only hydroxyl containing nitrophenolic explosives caused obvious fluorescence quenching. The sensing mechanism was investigated by using fluorescence titration and 1H NMR spectra. This simple AIE-active probe can potentially be applied to the construction of portable detection devices for explosives.

Knockdown of FBI-1 Inhibits the Warburg Effect and Enhances the Sensitivity of Hepatocellular Carcinoma Cells to Molecular Targeted Agents via miR-3692/HIF-1α.

The transcription suppressor factor FBI-1 (the factor that binds to inducer of short transcripts-1) is an important regulator of hepatocellular carcinoma (HCC). In this work, the results showed that FBI-1 promoted the Warburg effect and enhances the resistance of hepatocellular carcinoma cells to molecular targeted agents. Knockdown of FBI-1 via its small-interfering RNA (siRNA) inhibited the ATP level, lactate productions, glucose uptake or lactate dehydrogenase (LDH) activation of HCC cells. Transfection of siFBI-1 also decreased the expression of the Warburg-effect-related factors: hypoxia-inducible factor-1 alpha (HIF-1α), lactate dehydrogenase A (LDHA), or GLUT1, and the epithelial-mesenchymal transition-related factors, Vimentin or N-cadherin. The positive correlation between the expression of FBI-1 with HIF-1α, LDHA, or GLUT1 was confirmed in HCC tissues. Mechanistically, the miR-30c repressed the expression of HIF-1α by binding to the 3'-untranslated region (3'-UTR) of HIF-1α in a sequence-specific manner, and FBI-1 enhanced the expression of HIF-1α and HIF-1α pathway's activation by repressing the expression of miR. By modulating the miR-30c/HIF-1α, FBI-1 promoted the Warburg effect or the epithelial-mesenchymal transition of HCC cells and promoted the resistance of HCC cells to molecular targeted agents.

Enantioselective recognition of chiral acids by supramolecular interactions with chiral AIEgens.

Novel chiral AIEgens bearing optically pure amino groups were synthesized and showed excellent discrimination for a series of chiral acidic compounds and amino acids. Interestingly, after supramolecular assembly with 4-sulfocalix[4]arene, the obtained complexes showed enhanced enantioselectivity for chiral acids.

Droplet digital PCR (ddPCR) for the detection and quantification of Ureaplasma spp.

BACKGROUND: Ureaplasma spp. are associated with various infectious diseases in females, but there is still limited evidence regarding whether they are related to nonspecific cervicitis. The aim of this study was to develop and evaluate a digital droplet PCR (ddPCR) assay for the detection and quantification of Ureaplasma spp. in cervical swabs. METHODS: A total of 267 non-specific cervicitis (NSC) patients and 195 asymptomatic females were included in this study. We produced standard curves for Ureaplasma spp. to evaluate the analytical performance of the ddPCR assay. Then, we detected and quantified the bacterial load of Ureaplasma spp. in cervical swabs. RESULTS: The prevalences of U. parvum were 37.8% (101/267) and 29.7% (58/195),  U. urealyticum were 9.0% (24/267) and 8.7% (17/195) in the NSC group and control group, respectively. In addition, the median copy number of U. parvum was 2.5 × 104 copies/ml (n = 101) in the NSC group and 9.2 × 103 copies/ml (n = 58) in the control group. The U. parvum load in the NSC group was significantly higher than that in the asymptomatic individuals (P < 0.001). whereas the median load of U. urealyticum was 8.4 × 103 copies/ml (n = 24) and 1.4 × 103 (n = 17) copies/ml in the two groups, respectively, , the difference was not statistically significant (P = 0.450). CONCLUSIONS: Our study is the first to develop a droplet digital PCR (ddPCR) method for the detection and quantification of Ureaplasma spp. in clinical samples, and the method has excellent analytical performance and a wide range of clinical application prospects.

Pathogenesis and therapeutic strategy in platinum resistance lung cancer.

Platinum compounds (cisplatin and carboplatin) represent the most active anticancer agents in clinical use both of lung cancer in mono-and combination therapies. However, platinum resistance limits its clinical application. It is necessary to understand the molecular mechanism of platinum resistance, identify predictive markers, and develop newer, more effective and less toxic agents to treat platinum resistance in lung cancer. Here, it summarizes the main molecular mechanisms associated with platinum resistance in lung cancer and the development of new approaches to tackle this clinically relevant problem. Moreover, it could lead to the development of more effective treatment for refractory lung cancer in future.

The role of a two-assay serological testing strategy for anti-HCV screening in low-prevalence populations.

HCV screening depends mainly on a one-assay anti-HCV testing strategy that is subject to an increased false-positive rate in low-prevalence populations. In this study, a two-assay anti-HCV testing strategy was applied to screen HCV infection in two groups, labelled group one (76,442 people) and group two (18,415 people), using Elecsys electrochemiluminescence (ECL) and an Architect chemiluminescent microparticle immunoassay (CMIA), respectively. Each anti-HCV-reactive serum was retested with the other assay. A recombinant immunoblot assay (RIBA) and HCV RNA testing were performed to confirm anti-HCV positivity or active HCV infection. In group one, 516 specimens were reactive in the ECL screening, of which CMIA retesting showed that 363 (70.3%) were anti-HCV reactive (327 positive, 30 indeterminate, 6 negative by RIBA; 191 HCV RNA positive), but 153 (29.7%) were not anti-HCV reactive (4 positive, 29 indeterminate, 120 negative by RIBA; none HCV RNA positive). The two-assay strategy significantly improved the positive predictive value (PPV, 64.1% & 90.1%, P < 0.05). In group two, 87 serum specimens were reactive according to CMIA screening. ECL showed that 56 (70.3%) were anti-HCV reactive (47 positive, 8 indeterminate, 1 negative by RIBA; 29 HCV RNA positive) and 31 (29.7%) were anti-HCV non-reactive (25 negative, 5 indeterminate, 1 positive by RIBA; none HCV RNA positive). Again, the PPV was significantly increased (55.2% & 83.9%, P < 0.05). Compared with a one-assay testing strategy, the two-assay testing strategy may significantly reduce false positives in anti-HCV testing and identify inactive HCV infection in low-seroprevalence populations.

Efficacy and Safety of Banxia XieXin Decoction, a Blended Traditional Chinese Medicine, as Monotherapy for Patients With Advanced Hepatocellular Carcinoma.

PURPOSE: To explore a new therapeutic option for patients with hepatocellular carcinoma (HCC), the efficacy and safety of a group of traditional Chinese medicines (Banxia XieXin recipe) as monotherapy for patients with advanced HCC was studied. MATERIALS AND METHODS: The study included 68 patients with advanced HCC from August 16,2016 to August 15,2019 for analysis. These eligible patients received treatment with Banxia XieXin recipe for at least 1 month. The primary endpoints were progression-free survival (PFS) and overall survival (OS). The secondary efficacy endpoints included objective response rate (ORR) and disease control rate (DCR). In addition, safety was also assessed. RESULTS: The median treatment duration of these 68 patients was 10.3 months (range = 1.6-33.5 months), and follow-up is still ongoing. The median PFS was 6.07 months (95% confidence interval [CI] = 3.748-8.392 months), and the median OS was 12.60 months (95% CI = 8.019-17.181 months). The ORR was 10.3% and the DCR was 41.2%. In the subgroup analysis, the median OS in the transcatheter arterial chemoembolization (TACE) group was not reached, and the median OS in the NO TACE group was 11.30 months (95% CI = 3.219-19.381 months). In addition, no drug-related serious adverse events were observed during the study. CONCLUSION: This is the first clinical analysis of traditional Chinese medicine as a single treatment for advanced HCC. The obtained results are encouraging as they suggest that this panel of Chinese herbs is safe and it may be effective for patients with advanced HCC in a real-world clinical setting.

Paris saponin II-induced paraptosis-associated cell death increased the sensitivity of cisplatin.

Paris Saponin II (PSII) has been regarded as an effective and imperative component isolated from Rhizoma Paridis saponins (RPS) and exhibited strong anti-tumor effects on a variety of cancer. Our results revealed that human non-small lung cancer cell lines NCI-H460 and NCI-H520 were exposed to 1 µM of PSII, which inhibited the proliferation of lung cancer cells and activated apoptosis, autophagy and paraptosis. PSII induced paraptosis-associated cell death prior to apoptosis and autophagy. It induced paraptosis based on ER stress through activation of the JNK pathway. Meanwhile, PSII increased the cytotoxicity of cisplatin through paraptosis-associated pathway. All in all, PSII induced paraptosis based on induction of non-apoptotic cell death, which would be a possible approach to suppress the multi-drug resistant to apoptosis.

Curcumin-enhanced antitumor effects of sorafenib via regulating the metabolism and tumor microenvironment.

Curcumin, the main active ingredient of turmeric, is widely used as a kind of food additive and also displays a range of pharmacological activities, such as anti-inflammation, anti-tumor, liver and kidney protection, and so forth. Sorafenib was the first targeted agent against hepatocellular carcinoma (HCC), whose intolerance is related to the promotion of lipid synthesis and epithelial-to-mesenchymal transition (EMT) formation. In this study, biochemical analysis, immune cells composition, the tumor microenvironment, metabolomics, and relative metabolic enzymes and transporters were detected in H22-bearing mice treated with curcumin combined with sorafenib vs. control groups. It was found that curcumin protected against liver cancer progression through reducing the level of alpha fetoprotein in liver tissues, increasing the number of immune cells, like NK cells, inhibiting EMT via the regulation of IL-6/JAK/STAT3 and IL-1ß/NF-κB pathways, suppressing anaerobic glycolysis through the inhibition of LDH and HIF-1α, and decreasing the lipid synthesis via the downregulation of FASN, and upregulated the serum HDL-C and mRNA levels of apoA1 in the sorafenib-treated mice. Furthermore, curcumin regulation of the disorder of glycolipid metabolism and EMT was also based on the PI3K/AKT pathway. A docking study was performed and proved the strong affinity between curcumin and the proteins of STAT3, FASN, and AKT. All in all, this experiment provided evidence for the addition of curcumin in the diet to enhance the antitumor efficacy of sorafenib through activating immune function, downregulating EMT, and reversing disorders of the metabolism.

Experimental Study of Shenfu Injection on the Prevention and Treatment of Paclitaxel Chemotherapy DRG Neuron Injury.

PURPOSE: The purpose of this paper is investigating the effect and mechanism of Shenfu injection (a Traditional Chinese Medicine injection form) on prevention and treatment of paclitaxel chemotherapy in peripheral nerve injury. METHODS: Wistar rat dorsal root ganglion cells were cultured in vitro and divided into groups of MOCK, PT, PT + LD, and PT + HD. Each group was cultured at a total serum concentration of 10%, including 10% blank serum in the MOCK group, 0.73 (IC30) µmol/L paclitaxel + 10% blank serum in the PT group, and 10% and 5% drug-containing serum and equal amount of paclitaxel were added into the high- and low-dosage groups, respectively. After culturing for 24 hours, the following tests were performed: (1) cell proliferation detected by using CCK-8 and a microplate reader; (2) axon length detected by cellular immunostaining and detection analysis on antibody ß-tubulin III; and (3) changes in mitochondrial membrane potential by analyzing immunofluorescence staining with JC-1 probe. RESULTS: (1) Cell proliferation: OD values of the MOCK group and PT group were 0.43 ± 0.02 and 0.25 ± 0.03, respectively (P < 0.05), while OD values of groups PT + LD and PT + HD were 0.41 ± 0.05 and 0.46 ± 0.03, respectively, higher than group PT (P < 0.05), while OD values of groups PT + LD and PT + HD were 0.41 ± 0.05 and 0.46 ± 0.03, respectively, higher than group PT (µmol/L paclitaxel + 10% blank serum in the PT group, and 10% and 5% drug-containing serum and equal amount of paclitaxel were added into the high- and low-dosage groups, respectively. After culturing for 24 hours, the following tests were performed: (1) cell proliferation detected by using CCK-8 and a microplate reader; (2) axon length detected by cellular immunostaining and detection analysis on antibody µmol/L paclitaxel + 10% blank serum in the PT group, and 10% and 5% drug-containing serum and equal amount of paclitaxel were added into the high- and low-dosage groups, respectively. After culturing for 24 hours, the following tests were performed: (1) cell proliferation detected by using CCK-8 and a microplate reader; (2) axon length detected by cellular immunostaining and detection analysis on antibody P < 0.05), while OD values of groups PT + LD and PT + HD were 0.41 ± 0.05 and 0.46 ± 0.03, respectively, higher than group PT (P < 0.05), while OD values of groups PT + LD and PT + HD were 0.41 ± 0.05 and 0.46 ± 0.03, respectively, higher than group PT (. CONCLUSION: Shenfu injection can prevent the toxicity of DRG neurons induced by paclitaxel, and its mechanism may be related to the alleviation of mitochondrial dysfunction.

Real-time PCR assay may be used to verify suspicious test results of Ureaplasmas spp. from the liquid culture method.

Ureaplasma spp. are associated with female genital tract infections and are mainly tested by liquid culture in developing countries. To evaluate the accuracy of liquid culture, 686 vaginal swabs were collected and tested by using the Mycoplasma Culturing, Identification, Enumeration, and Susceptibility (IES) Kit. Then these culture broths were verified using real-time PCR. Among 368 Ureaplasma positive broths, 263 contained Ureaplasma parvum, 30 contained Ureaplasma urealyticum, 57 contained both, and 18 were negative by real-time PCR. In 318 Ureaplasmas negative broths, 78 were found to be Ureaplasma positive by real-time PCR. Using real-time PCR as the reference, the false positive rate of the liquid culture was 7.0%. It has been suggested that the liquid culture positive broth should be inoculated onto solid agar to eliminate false-positives. However, solid culture is rarely used due to low sensitivity and being time consuming. Real-time PCR may be performed to replace solid culture to verify suspicious liquid culture results.