结核分枝杆菌异烟肼耐药性的噬菌体测定技术研究

目的建立噬菌体生物扩增法快速测定异烟肼（INH）耐药性的方法．并探讨其在结核分枝杆菌（MTB）INH耐药性测定中的应用价值。方法用PhaB法检测167株MTB临床分离株INH耐药性，并与Bactec-960测定结果比较，分析PhaB法的敏感性、特异性和准确性。结果0．2μg／ml INH与菌液37℃作用48h后，通过噬菌体检测．以噬菌斑减少95％为最适判断标准．噬菌斑减少高于95％的判为敏感株，噬菌斑减少低于95％的为耐药株。以Bactec-960测定结果为判断标准，则PhaB的敏感性、特异性、阳性和阴性预测值及符合率分别为96．4％、96、4彤、93．1％、98．2％和96．4％。结论PhaB检测INH耐药性具有很高的敏感性和特异性．与Bactec-960的符合率达96．4％，只需3d时间．且操作简便、不需特殊仪器设备．可作为MTB的INH耐药性快速筛选方法。     

延迟放瓶对两种血培养系统BacT/AlERT3D与BACTEC 9120阳性检出率的影响

目的 比较延迟放瓶对两种血培养系统BacT/AlERT 3D与BACTEC 9120阳性检出率的影响.方法 将4种常见临床菌种制成不同浓度菌液分别接种到BacT/AlERT FA与Bactec Plus(Aerobic)培养瓶中,将培养瓶分为立即放入BacT/AlERT 3D和BACTEC 9120培养仪中和分别在室温(22℃)和35℃孵育条件下延迟8、16、24 h后放入上述培养仪中等各组,记录报阳时间和计算阳性检出率.结果 无论是室温(22℃)还是35℃孵育,BacT/AlERT 3D中的培养瓶在分别延迟0、8、16、24 h放入时.阳性检出率差异无统汁学意义;而BACTEC 9120中的培养瓶分别延迟0、8、16 h时其阳性榆出率差异无统计学意义;而延时24 h放瓶时其阳性检出率则显著下降;BacT/AlERT 3D和BACTEC 9120在延时放瓶0、8、16 h时两者的阳性检出率差异均无统计学意义;而延迟24 h放瓶时前者的阳性检出率明显高于后者;两种血培养仪在22℃孵育的阳性检出率和35℃孵育时的检出率差异无统计学意义.结论 临床上血培养瓶应尽可能早的放人培养仪,BacT/AlERT 3D的延迟放瓶时间应在24 h以内,BACTEC 9120则应在16 h以内,延时孵育温度应为室温.     

Significance of Nuclear Glutathione S-Transferase π in Resistance to Anti-cancer Drugs

Recent study has shown that nuclear glutathione S-transferase (GST) π accumulates in cancer cells resistant to doxorubicin hydrochloride (DOX) and may function to prevent nuclear DNA damage caused by DOX (Goto et al., FASEB J. , 15, 2702–2714 (2001)). It is not clear if the amount of nuclear GSTπ increases in response to other anti-cancer drugs and if so, what is the physiological significance of the nuclear transfer of GSTπ in the acquisition of drug-resistance in cancer cells. In the present study, we employed three cancer cell lines, HCT8 human colonic cancer cells, A549 human lung adenocarcinoma cells, and T98G human glioblastoma cells. We estimated the nuclear transfer of GSTπ induced by the anti-cancer drugs cisplatin (CDDP), irinotecan hydrochloride (CPT-11), etoposide (VP-16) and 5-fluorouracil (5-FU). It was found that: (1) Nuclear GSTjt accumulated in these cancer cells in response to CDDP, DOX, CPT-11, VP-16 and 5-FU. (2) An inhibitor of the nuclear transport of GSTπ, edible mushroom lectin ( Agaricus bisporus lectin, ABL), increased the sensitivity of the cancer cells to DOX and CDDP, and partially to CPT-11. Treatment with ABL had no apparent effect on the cytotoxicity of VP-16 and 5-FU. These results suggest that inhibitors of the nuclear transfer of GSTπ have practical value in producing an increase of sensitivity to DOX, CDDP and CPT-11.     

Expression of glutathione S-transferase π and cisplatin resistance in human ovarian cancer

Glutathione S-transferase π (GSTπ) expression in tumor cells is thought to relate to cisplatin resistance. We attempted to clarify immunohistochemically the correlation between GSTπ expression and clinical response in ovarian cancer. Fifty-nine patients with ovarian cancer underwent initial debulking surgery and received from three to five courses of cisplatin-based chemotherapy after surgery. Immunostaining for GSTπ was performed on formalin fixed sections of the patients' tumor by the streptoavidin-biotin-peroxidase complex method. Positive staining of GSTπ was observed diffusely in nuclei and cytoplasm of cancer cells. Of 59 tumors, 38 (64.4%) were GSTπ positive. Twenty-three of 25 patients (92.0%) who showed no response to chemotherapy had GSTπ positive tumor cells. The predictive value of positive GSTπ staining for drug resistance was 79.3% (23/29). The 5-year survival rate of patients with GSTπ positive tumors was significantly lower than that of those with GSTπ negative tumors by the Kaplan-Meier method ( P < 0.01). The results suggest that overexpression of GSTπ is related to resistance to cisplatin and to prognosis in patients with ovarian cancer.     

FK960, a potential anti-dementia drug,increases synaptic density in the hippocampal CA3 region of aged rats

There is accumulating evidence suggesting that synapse formation in the adult brain is dynamically regulated, and that this regulation plays a role in cognitive function. A decrease in synaptic density is reportedly related to memory deficits in aged animals as well as in Alzheimer's patients. FK960 [N-(4-acetyl-1-piperazinyl)-p-fluorobenzamide monohydrate], a novel anti-dementia drug, has been shown to ameliorate experimental amnesia in rats and monkeys through activation of the somatostatinergic nervous system in the hippocampus. Furthermore, FK960 has been shown to be considerably more effective in a model of spontaneous amnesia in aged rats than cholinesterase inhibitors. In the present electron microscopy study, we demonstrated that the density of axodendritic and axosomatic synapses in the hippocampal CA3 region of aged rats was reduced compared to young rats, and that repeated treatment with FK960 for either 3 or 21 days dose-dependently reversed these deficits in aged rats. This FK960-induced increase in synaptic density was transient and density returned to basal levels at 8 days after the final dose. In contrast, FK960 did not alter synaptic density in the cingulate cortex or hippocampal CA1 region in aged rats, nor the CA3 region of young rats. Collectively, these results suggest that FK960 can selectively and reversibly increase synaptic density in the hippocampal CA3 region of aged rats, and that this activity may play a role in its cognitive-enhancing action.     

Comparison of the conventional diagnostic modalities, bactec culture and polymerase chain reaction test for diagnosis of tuberculosis

Purpose: To evaluate the performance of 65 kDa antigen based PCR assay in clinical samples obtained from pulmonary and extrapulmonary cases of tuberculosis. Methods: One hundred and fifty six samples were processed for detection of Mycobacterium tuberculosis by ZN smear examination, LJ medium culture, BACTEC radiometric culture and PCR tests. Results: A significant difference was seen in the sensitivities of different tests, the figures being 74.4% for PCR test, 33.79% for ZN smear examination, 48.9% for LJ culture and 55.8% for BACTEC culture (P<0.05). However, there was no significant difference (P>0.05) as far as specificity of different tests was concerned. PCR test sensitivity in pulmonary and extrapulmonary clinical samples were 72.7% and 75.9% respectively and found to be significantly higher (P<0.05) when compared with those of other tests. The mean detection time for M.tuberculosis was 24.03 days by LJ medium culture, 12.89 days by BACTEC culture and less than one day by PCR test. Conclusions: PCR is a rapid and sensitive method for the early diagnosis of pulmonary and extrapulmonary tuberculosis.     

Performance of the GenoType MTBDRsl V 2.0 for detecting second-line drugs resistance of Mycobacterium tuberculosis isolates in Tunisia

Rapid detection of the second-line drug (SLD) resistant tuberculosis (TB) strains is challenging to prescribe an immediate adequate treatment and limit the transmission of SLD resistant strains.The study aimed to evaluate the performance of GenoType MTBDRsl V2.0 compared to phenotypic drug susceptibility testing (pDST:MGIT960) to detect resistance to SLD of Mycobacterium tuberculosis (MTB) isolates in Tunisia, between May 2015 and December 2019.As a matter of fact, 103 rifampicin-resistant and multidrug-resistant MTB strains were included. Discrepancies between pDST and MTBDRsl were solved by whole genome sequencing.Compared to pDST, MTBDRsl V2.0 showed a sensitivity of 92.8% (68.5%–98.7%) in detecting resistance to fluoroquinolones. As for second-line injectable drugs, it presented a sensitivity of 80.0% (49.0%–94.3%). MTBDRsl had sensitivities of 100.0% (67.5%–100.0%), 75.0% (40.9%–92.8%) and 100.0% (60.9%–100.0%) respectively for kanamycin, capreomycin and amikacin. The specificity was 100.0% for all the drugs evaluated.As for diagnosing XDR-TB, it had a sensitivity of 57.1% (25.0%–84.1%) and a specificity of 100.0% (96.1%–100.0%).MTBDRsl V2.0 showed a high performance in detecting SLD resistance with a short turnaround time compared with pDST, which made it possible to start an early treatment and to maintain a low prevalence of SLD resistance and XDR-TB in Tunisia.     

Identification and susceptibility testing of Staphylococcus aureus by direct inoculation from positive BACTEC blood culture bottles

This study explored the possibility of combining direct inoculation of tube coagulase and DNase tests, and the VITEK2 system, from BACTEC blood culture bottles in order to achieve rapid identification and susceptibility testing of Staphylococcus aureus. All isolates were identified correctly as S. aureus or coagulase-negative staphylococci (CNS). Antimicrobial susceptibility testing with the VITEK2 system gave 99.6% correct category agreement, with 0.1% very major errors and 0.3% minor errors among S. aureus isolates, and 97.4% correct category agreement, with 0.9% very major errors and 1.7% minor errors among CNS isolates. The results suggested that direct identification and susceptibility testing is sufficiently accurate for immediate reporting.     

Comparison of various microbiological tests including polymerase chain reaction for the diagnosis of osteoarticular tuberculosis

Purpose: To evaluate the utility of the polymerase chain reaction(PCR) test for diagnosing osteoarticular tuberculosis (TB). Methods: Clinical samples (synovial tissue and synovial fluid) obtained from 23 cases of suspected osteoarticular tuberculosis were subjected to Ziehl Neelsen (ZN) smear examination, radiometric BACTEC culture and PCR test for tuberculosis by amplifying 65 kDa antigen coding region of Mycobacterium tuberculosis (M.tb) genome. Results: PCR test was found to be much more sensitive than the ZN smear examination and BACTEC culture(p < 0.05) in the diagnosis of osteoarticular TB. In synovial fluid samples, PCR was positive in 73.9%, ZN smear examination in 17.39% and BACTEC culture in 39.13% of cases.The positivities were relatively lower with synovial tissue samples, the corresponding figures being 60.8, 8.6 and 26.08% respectively. Moreover, on combining the results of synovial fluid and tissues, the corresponding figures further increased to 78.2, 21.7 and 43.3% respectively. Further, sensitivity and specificity for PCR employing BACTEC culture as the “gold standard” was 100% respectively. Using BACTEC culture, the earliest positivity was seen in three days using synovial tissue specimen and 13 days with synovial fluid, the average detection times being 23.2 days and 32.6days respectively. On the other hand, PCR test gave a positive result within 24 hours.Conclusions: PCR test was shown to be much more sensitive than ZN smear examination and BACTEC culture test for diagnosing osteoarticular tuberculosis.