Indicators Of Drug Resistance Research Articles

Эпидемиологические аспекты формирования новых рисков заболеваемости и лечения туберкулеза среди населения.

In accordance with the instruction of the Minister of Health of the Russian Federation No. 19 dated 02/07/2023, specialists currently face several important tasks: from 2015 to 2025, to reduce the incidence of tuberculosis, as well as the number of deaths from tuberculosis by 75%. To do this, it is necessary to identify all the risk factors that may create difficulties in achieving these goals. Purpose of the study is to characterize the epidemiological aspects of the formation of new risks of morbidity and treatment of tuberculosis among the population. Materials and methods. An analysis was carried out among newly identified patients with tuberculosis by molecular genetic (n=1682) and bacterial (n=1630) research methods in the period from 2017 to 2020 according to drug resistance indicators. Results. It was found that in the period from 2015 to 2017, the indicators of drug resistance in tuberculosis patients were almost at the same level. The leading antimicrobial drugs in demand among the population were ceftriaxone, azithromycin, levofloxacin, moxifloxacin. The maximum increase in sales in 2020 relative to 2019 data to 150% was determined among quinolone derivatives of moxifloxacin and levofloxacin, which began to be used in the treatment of coronavirus infection. At the same time, these drugs are traditionally used to treat tuberculosis, but in 2020, an alarming trend was revealed, which was that drug resistance was detected in patients with tuberculosis to these drugs. Among newly diagnosed tuberculosis patients, from 2018 to 2020, an increase in the proportion of patients with primary drug resistance to levofloxacin and moxifloxacin was established by 189-480%; whereas for other antibiotics, the increase averaged up to 60.8%. Findings. The findings suggest an alarming scenario in which drug resistance develops towards highly virulent and highly resistant to antibacterial drugs genotypes.

Long-Term Prophylactic Transcranial Direct Current Stimulation Ameliorates Allodynia and Improves Clinical Outcomes in Individuals With Migraine

ObjectivesMigraine is a common and substantially debilitating disorder that may associate with allodynia, a marker of central sensitization in the pain circuits. Several unmet needs, like limited adherence to drugs due to adverse events and cost-effectivity, still occur in the prophylactic treatment of migraine. Transcranial direct current stimulation (tDCS) has recently been indicated to be beneficial in individuals with migraine with and without allodynia. However, to our knowledge, there are no studies evaluating the efficacy of six-month tDCS in migraine. Materials and MethodsThis study was a randomized double-blind parallel-group sham-controlled five-month extension study after a one-month lead-in trial of tDCS in individuals with migraine. A total of 23 individuals with migraine with allodynia who completed the lead-in trial were recruited after their consent and were administered three consecutive sessions of 2-mA anodal 20-minute tDCS over the left primary motor cortex every month for an additional five months. Pain-related outcomes were determined using monthly headache diaries. Allodynia, depression, anxiety, and disability because of migraine also were assessed throughout the study. ResultsImprovements in allodynia levels, attack frequency, number of rescue medications, and attack duration were higher, and mostly gradual during the trial, in the active group. Migraine Disability Scale grades also were lower in the active group, whereas no between-group differences were found in depression and anxiety scores. Higher responder rates of migraine attack frequency (56.8% vs 25%), number of headache days (56% vs 16.7%), and migraine attack duration (90.9% vs 8.3%) were observed after six-month tDCS in the active group than in the sham group. ConclusionsLong-term extended tDCS is shown to be a safe, efficacious, and plausible modality for prophylactic treatment in individuals with migraine with allodynia. SignificanceLong-term extended tDCS can alleviate allodynia, which is an indicator of drug resistance and chronicity, and meet the goals of prophylactic treatment in individuals with migraine with allodynia.

Up-regulation of GSTT1 in serous ovarian cancer associated with resistance to TAXOL / carboplatin

Serous ovarian cancer (SOC) is the most common women cancer and the leading cause of cancer-related mortality among the gynaecological malignancies. Although effective chemotherapeutics combined with surgery are developed for the treatment, the five-year survival rate is unsatisfactory due to chemoresistance. To overcome this shortcoming of chemotherapy, we established taxol and carboplatin resistant SOC cell lines for the understandings of the molecular and cellular mechanisms of chemoresistance. Here, we found that these chemoresistant cell lines showed less viability and proliferation, due to more cells arrested at G0/G1 phase. Glutathione-S-transferases-theta1 (GSTT1) was significantly upregulated in these chemoresistant cells, along with other chemoresistant genes. Meanwhile, GSTT1 expression was also significantly upregulated in the SOC patient tissues after taxol treatment, indicating this upregulation was physiologically relevant to chemotherapy. Further, suppression of GSTT1 expression by shRNA in SOC cell lines led to more sensitivity to drug treatment, through increasing divided cells and promoting cell death. Moreover, the expression of DNA topoisomerase 1 (Topo I) was in synergy with that of GSTT1 in the chemoresistant cells, and GSTT1 can bind to Topo I in vitro, which suggested GSTT1 could function through DNA repair mechanism during chemoresistance. In summary, our data imply that GSTT1 may be a potential biomarker or indicator of drug resistance in serous ovarian cancer.

Decreased classical monocytes and CD163 expression in TB patients: an indicator of drug resistance.

Tuberculosis (TB) is a disease instigated by Mycobacterium tuberculosis. Peripheral blood monocytes represent highly efficient effector cells of innate immunity against TB. Little is known about monocyte subsets and their potential involvement in the development of M. tuberculosis drug resistance in patients with TB. This study was conducted to investigate alterations in monocyte subsets, CD163 expression on monocytes, and its serum level in patients without and with rifampicin resistance TB (RR-TB) and healthy controls. A total of 164 patients with TB (84 without RR-TB and 80 patients with RR-TB) and 85 healthy controls were enrolled in this study. The percentages of various monocyte subsets and surface expression of CD163 on monocytes were quantitatively determined using flow cytometry. The serum level of CD163 was determined by commercially available ELISA kits. Decreased frequency of classical monocytes was detected in patients with RR-TB. Non-classical monocytes were decreased in patients without RR-TB; however, intermediate monocytes were raised in patients with RR-TB. The serum level of CD163 was decreased in patients of RR-TB that showsed a positive correlation with the frequency of CD14++CD16-CD163+ and CD14++CD16+CD163+ monocytes. It is concluded that decreased classical monocytes and sCD163 in patients with RR-TB could be an indicator of drug resistance.

HIV Reverse Transcriptase and Protease Genes Variability Can Be a Biomarker Associated with HIV and Hepatitis B or C Coinfection

Variability of the HIV reverse transcriptase (RT) and protease (PR) genes has been used as indicators of drug resistance and as a mean to evaluate phylogenetic relationships among circulating virus. However, these studies have been carried in HIV mono-infected populations. The goal of this study was to evaluate, for the first time, the HIV PR and RT sequences from HIV/HBV and HIV/HCV co-infected patients. HIV PR and RT genes were amplificated and sequenced to resistance analysis. The bioinformatics analysis was performed to infer about sequences clustering and molecular evolution. The results showed that the most frequent amino acid substitutions in RT were L214F (67.6%), I135T (55.9%), and in PR was V15I (41.2%). The molecular clock analysis showed that the HIV circulating in co-infected patients were separated in two clusters in the years 1999–2000. Some patients included as HIV mono-infected according patients’ medical records and inside the co-infected cluster were, in fact, co-infected by PCR analysis. Analysis of the decision trees showed susceptibility to lamivudine and emtricitabine were important attribute to characterize co-infected patients. In conclusion, the results obtained in this study suggest, for the first time, that HIV RT and PR genes variability could be a genetic biomarker to coinfection.

Treatment against coccidiosis in Norwegian lambs and potential risk factors for development of anticoccidial resistance\u2014a questionnaire-based study

The objectives of this study were to investigate the use of anticoccidials in Norwegian sheep flocks and identify farms with management procedures likely to select for drug resistance. Data were obtained by a questionnaire sent to all members of the Norwegian Sheep Recording System in October 2015. The data set consisted of 1215 answers, corresponding to 8.5% of Norwegian sheep flocks. Anticoccidials were used in 82.7% of flocks. Main treatment was at turnout (38.6% of treated flocks) or 1 week after turnout (32.4%). Interestingly, clinical signs possibly related to coccidiosis were observed by almost 40% of the farmers after treatment, which might be an indication of drug resistance. Correlations between the apparently reduced anticoccidial efficacy and management conditions, such as the size of the farms, were found. From the farmers’ perspective, metaphylactic treatment was used in 88.5% of treated flocks, of which approximately one third had no history of clinical coccidiosis. Even though farmers seem aware of the importance of good drenching routines based on reliable estimates of weights and calibration of drench guns, drench gun used for anticoccidial administration was never calibrated in 12.1% of the flocks. Finally, dose estimation was made by visual appraisal in 27.5% of the flocks, which can lead to incorrect dosing. Based on the present study, it cannot be determined whether the apparent treatment failure was related to management practises, incorrect administration of the drug, other infections or actual anticoccidial drug resistance.

Effect of Verbascum thapsus ethanol extract on induction of apoptosis in Trichomonas vaginalis in vitro.

Trichomoniasis is a sexually transmitted disease (STD) caused by a tiny parasite called Trichomonas vaginalis. Metronidazole is used as routine treatment of disease. Some reports have confirmed the potential carcinogenic and teratogenic effects of this drug on fetus and indication of drug resistance. Verbascum thapsus belongs to the family of Scorphulariaceae. Its antiinflammatory properties, disinfectant and skin healing effects are well known. This plant has been used to treat diarrhea and genitourinary infection in traditional medicine. Effects of different concentrations of the Verbascum thapsus extract were tested on the growth and motility of T. vaginalis trophozoites. To evaluate the toxicity of extract, their effects on mice macrophages were measured by MTT([3-(4,5-dimethyl thiazolyl-2)- 2,5-diphenyle tetrazolium bromide ])assay. In this experimental study the effect of Verbascum thapsus ethanol extract on induction apoptosis in T. vaginalis was determined by Flow Cytometry. Results were analyzed by Flow Jo software and the degree of apoptosis was determined. Toxicity percentage of 25-800 μg/ml concentrations of Verbascum thapsus alcoholic extract for mice macrophages was observed between 0.17-0.25 after 12 hours and they were between 0.25-0.42 and0.45-0.95 after 24 and 48h respectively. IC50 (inhibitory concentration, 50%) of Verbascum thapsus ethanol extract and metronidazole after 24h was 39.17 and 0.0326 μg/ml respectively. Flow cytometry results showed the percent of apoptosis following treatment of trophozoites with different concentrations of Verbascum thapsus ethanol extract (25, 50,100,200,400 μg/ml), were 20.7, 37.04, 47.5, 62.72 and 86.35 respectively, while in control group was 2.9. According to this study, Verbascum thapsus extract induces programmed death in T. vaginalis. It is recommended that Verbascum thapsus extract can be considered as a suitable choice for Medical Studies.

MiR-218 targets survivin and regulates resistance to chemotherapeutics in breast cancer

Multidrug resistance (MDR) remains one of the most significant obstacles in breast cancer treatment, and this process often involves dysregulation of a great number of microRNAs (miRNAs). Some miRNAs are indicators of drug resistance and confer resistance to chemotherapeutic drugs, although our understanding of this complex process is still incomplete. We have used a combination of miRNA profiling and real-time PCR in two drug-resistant derivatives of MCF-7 and Cal51 cells. Experimental modulation of miR expression has been obtained by retroviral transfection. Taxol and doxorubicin IC50 values were obtained by short-term drug sensitivity assays. Apoptosis was determined by flow cytometry after annexin V staining, by caspase 3/7 and caspase 9 activity assays and the levels of apoptosis-related proteins bcl-2 and bax by real-time PCR and Western blot. miR target was studied using transient transfection of luciferase constructs with the 3' untranslated regions (UTR) of target mRNAs. Small interfering RNA-mediated genetic knock-down was performed in MDR cells and its modulatory effect on apoptosis examined. The effect of miRNA on tumorigenicity and tumor drug response was studied in mouse xenografts. miRNA profiling of two drug-resistant breast cancer cell models indicated that miR-218 was down-regulated in both MCF-7/A02 and CALDOX cells. Ectopic expression of miR-218 resensitized both drug-resistant cell lines to doxorubicin and taxol due to an increase in apoptosis. miR-218 binds survivin (BIRC5) mRNA 3'-UTR and down-regulated reporter luciferase activity. Experimental down-regulation of survivin by RNA interference in drug-resistant cells did mimic the sensitization observed when miRNA-218 was up-regulated. In addition, resensitization to taxol was also observed in mouse tumor xenografts from cells over-expressing miR-218. miR-218 is involved in the development of MDR in breast cancer cells via targeting survivin and leading to evasion of apoptosis. Targeting miR-218 and survivin may thus provide a potential strategy for reversing drug resistance in breast cancer.

Abstract 911: Vemurafenib FDG-PET preclinical imaging outcomes across BRAF V600E mutant and resistant tumors

Abstract The BRAF inhibitor vemurafenib (marketed as zelboraf) has recently been FDA approved for the treatment of metastatic melanoma in patients harboring BRAF V600 mutations. PET imaging will be essential for guiding therapy in terms of gauging initial drug response, monitoring for disease relapse, and evaluating combination treatments in clinical trials. Herein, we assess 18F-FDG uptake across V600E mutant and vemurafenib resistant cells. We find that vemurafenib is equally effective at reducing FDG uptake in cell lines harboring either heterozygous or homozygous V600E BRAF but is ineffective in cells with acquired resistance or BRAF WT cells. Combination treatment with MEK inhibitor GDC-0973 results in a highly significant enhancement of efficacy across all twenty lines tested. We show that FDG uptake is driven by Glut-1, and that the membrane localization of the transporter paralleled with drug efficacy. Interestingly, we find that vemurafenib induced increased FDG uptake in cells harboring RAS mutations, as well as in tumors that have acquired vemurafenib drug resistance. Hexokinase II, CRAF, KSR and p-MEK protein levels in sensitive and drug resistant tumors were all associated with decreased measurements of FDG uptake in vivo. Overall, we have demonstrated that FDG PET imaging should be effective at monitoring the efficacy of vemurafenib in V600E melanoma mutants and that any detected increases in tumor uptake will likely be an indication of drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 911. doi:1538-7445.AM2012-911

Microcolonies in fluoroquinolone agar proportion susceptibility testing of Mycobacterium tuberculosis: an indicator of drug resistance

Microcolony growth of Mycobacterium tuberculosis on agar proportion susceptibility testing is neither well-defined nor previously reported with fluoroquinolone susceptibility testing. We describe here M. tuberculosis microcolony growth with fluoroquinolones, and assess its clinical significance. We screened 797 M. tuberculosis isolates for ofloxacin resistance (2.0μg/mL) by agar proportion; 19 ofloxacin-resistant and 38 ofloxacin-susceptible isolates were selected for more detailed susceptibility testing with ofloxacin, ciprofloxacin, levofloxacin (all at 2.0μg/mL) and moxifloxacin (0.5μg/mL). The 57 isolates were also tested at two concentrations both above and below the critical concentrations. Microcolonies were defined as colonies 0.2-0.4mm in diameter; confirmed microcolonies were present on repeat testing. Of the 57 isolates tested in detail, 7 grew microcolonies, of which 2 (0.3% of all isolates tested) had confirmed microcolonies on repeat testing (6 tests performed, and microcolonies were present on at least 4). Both M. tuberculosis isolates were ofloxacin-resistant on screening, and had ofloxacin minimum inhibitory concentration (MIC) >8μg/mL. The five other isolates were ofloxacin-susceptible on screening, but had regular colony growth (i.e., resistance) at the drug concentration that initially resulted in microcolonies (ofloxacin 0.5 or 1.0μg/mL). Microcolonies were observed infrequently with fluoroquinolone susceptibility testing, but when confirmed, they were associated with drug resistance.

Promoter methylation‐mediated inactivation of PCDH10 in acute lymphoblastic leukemia contributes to chemotherapy resistance

PCDH10 has been implicated as a tumor suppressor, since epigenetic alterations of this gene have been noted in multiple tumor types. However, to date, studies regarding its role in acute and chronic leukemias are lacking. Here, we have investigated the presence of promoter hypermethylation of two CpG islands of the PCDH10 gene by methylation-specific PCR in 215 cases of various subsets of myeloid- and lymphoid-lineage leukemias. We found that PCDH10 promoter hypermethylation was frequent in both B-cell (81.9%) and T-cell (80%) acute lymphoblastic leukemia (ALL), while it was present in low frequency in most subtypes of myeloid leukemias (25.9%) and rare in chronic myeloid leukemia (2.2%). PCDH10 expression was downregulated via promoter hypermethylation in primary ALL samples (N = 4) and leukemia cell lines (N = 11). The transcriptional repression caused by PCDH10 methylation could be restored by pharmacologic inhibition of DNA methyltransferases. ALL cell lines harboring methylation-mediated inactivation of PCDH10 were less sensitive to commonly used leukemia-specific drugs suggesting that PCDH10 methylation might serve as a biomarker of chemotherapy response. Our results demonstrate that PCDH10 is a target of epigenetic silencing in ALL, a phenomenon that may impact lymphoid-lineage leukemogenesis, serve as an indicator of drug resistance and may also have potential implications for targeted epigenetic therapy.

Abstract 50: Comparison of NMR lipid profiles in mitotic arrest and apoptosis as indicators of drug resistance

Abstract Aim: To compare NMR-visible lipids in paclitaxel exposed cells undergoing apoptosis or mitotic arrest in order to explore their utility as a biomarker of drug resistance. Methods: Cultured cervical cell lines (HeLa, C33A and Me180, ATCC, USA) were exposed to 1 µM paclitaxel (Sigma, UK) for 8, 16, 24, and 48 hours (n=3). Diffusion-weighted (DW) spectra were acquired using a 11.74T spectrometer (Avance Bruker BioSpin, Germany) using a stimulated echo sequence with bipolar gradients. Cellular morphology was assessed with transmission electron microscopy (TEM) using uranyl acetate followed by lead citrate staining. Following TOPRO-3 and nile red co-staining, flow cytometry and confocal microscopy verified cell size and viability, visualized cell cycle phase distribution, apoptotic features, and accumulation of cytoplasmic lipid droplets. Western blots were used to assess activation (by phosphorylation) of cytoplasmic phospholipase A2 (P-cPLA2) and expression of fatty acid synthase (FAS) at each stage. Results: 24 h after exposure to paclitaxel, all lines showed >65% mitotic arrest, and HeLa showed apoptosis as well. At 48 h HeLa cells progressed to apoptosis (due to mitotic catastrophe) while C33A and Me180 cells progressed beyond mitotic arrest to normal morphology or multinucleation indicating resistance. There was significant increase in saturated and unsaturated lipids at 24 h (mitotic arrest) followed by a further striking increase at 48 h in all lines, especially in the 5.3ppm peak (unsaturated) in HeLa at 48 h. Unsaturated lipids increased more than saturated lipids in all lines, triglycerides increase the most in HeLa cells. Resistant lines showed lower increased methylene / methyl (1.3 ppm / 0.9 ppm) ratio (from 1.3 to 2.8, 1.7 to 2.8 compared with 1.5 to 3.8 in HeLa cells at 48 h). The levels of P-cPLA2 and FAS were unchanged at 24 h followed by a drop at 48 h in HeLa cells. The percentage of cells displaying lipid droplets increased significantly at 24 and 48 h in all lines with increase in droplet size only in HeLa cells at 24 and 48 h. This implies that the larger droplets are associated with apoptosis but that smaller ones are less specific. Apoptotic cells showed 3-4 x the number of droplets compared to cells arrested in mitosis. Conclusion: After exposure of cells to paclitaxel, increase in lipids on NMR results from increased visibility not synthesis (more droplets, FAS not increased). In apoptosis predominant increases are in unsaturated fatty acids in larger droplets, whereas paclitaxel resistant lines accumulate smaller droplets with significantly less triglycerides. Acknowledgements: This work was funded by the EC FP6 Marie Curie Action: Early Stage Training (contact No. 020718). We also acknowledge the support received for the CRUK and EPSRC Cancer Imaging Centre in association with the MRC and Department of Health (England) (grant C1060/ A10334) and NHS Funding to the NIHR Biomedical Research Centre. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 50.

Abstract B63: Apaf-1 mislocalization is a strong indicator of drug resistance in B cell lymphomas

Abstract Resistance to chemotherapy remains a challenge in the clinical management of diffuse B cell lymphomas despite aggressive chemotherapy such as CHOP and monoclonal CD20. We previously reported that sequestration of Apaf-1 to membrane lipid rafts was responsible for apoptosis resistance in B-cell lymphoma cell lines1. Here, we extended our studies to 60 clinical biopsies from patients with B-cell lymphomas, T-cell lymphomas and reactive lymphadenopathy, to investigate if the resistance to drug-induced apoptosis was, indeed, a function of Apaf-1 mislocalization. Firstly, cells were separated from these biopsies and their sensitivity to a variety of apoptosis inducing agents was assessed. Whereas, most T cell lymphomas as well as reactive lymphadenopathy cells were sensitive to apoptotic stimuli, B cell lymphomas exhibited strong resistance. We then investigated the expression of Apaf-1 and its intracellular localization in these clinical biopsies. To do so, cell fraction was performed to separate cytosol from membrane-enriched fractions. The latter were further subjected to density gradient centrifugation to obtain lipid raft fractions. We show that Apaf-1 was expressed in total cell lysates from B and T cell lymphomas, however upon fractionation the localization was strikingly different. In T cell lymphoma samples as well as in cells derived from reactive lymphadenopathy biopsies, Apaf-1 expression was prominently detected in the cytosol, which correlated with the sensitivity of the cells to apoptotic stimuli. In contrast, whereas cytosolic Apaf-1 expression was significantly lower or absent in almost all B cell lymphomas analyzed, increased localization of the protien was detected in membrane lipid rafts. The latter was confirmed by immunohistochemical analysis of tissues from the same biopsy specimens. Interestingly, the resistance of B cell lymphomas to apoptotic execution (drug-induced or death receptor-mediated) was significantly bypassed upon incubation of cells with pharmacological agents that facilitated the dissociation of Apaf-1 from the lipid rafts to the cytosol. Taken together, our results implicate Apaf-1 mislocalization as a potential diagnostic marker for B-cell lymphomas as well as a predictor of response to therapeutic management. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B63.

Viral dynamics in hepatitis B virus infection.

Treatment of chronic hepatitis B virus (HBV) infections with the reverse transcriptase inhibitor lamivudine leads to a rapid decline in plasma viremia and provides estimates for crucial kinetic constants of HBV replication. We find that in persistently infected patients, HBV particles are cleared from the plasma with a half-life of approximately 1.0 day, which implies a 50% daily turnover of the free virus population. Total viral release into the periphery is approximately 10(11) virus particles per day. Although we have no direct measurement of the infected cell mass, we can estimate the turnover rate of these cells in two ways: (i) by comparing the rate of viral production before and after therapy or (ii) from the decline of hepatitis B antigen during treatment. These two independent methods give equivalent results: we find a wide distribution of half-lives for virus-producing cells, ranging from 10 to 100 days in different patients, which may reflect differences in rates of lysis of infected cells by immune responses. Our analysis provides a quantitative understanding of HBV replication dynamics in vivo and has implications for the optimal timing of drug treatment and immunotherapy in chronic HBV infection. This study also represents a comparison for recent findings on the dynamics of human immunodeficiency virus (HIV) infection. The total daily production of plasma virus is, on average, higher in chronic HBV carriers than in HIV-infected patients, but the half-life of virus-producing cells is much shorter in HIV. Most strikingly, there is no indication of drug resistance in HBV-infected patients treated for up to 24 weeks.

Results of directly observed short-course chemotherapy in 112 842 Chinese patients with smear-positive tuberculosis

SummaryBackground Tuberculosis remains a substantial public health burden in China. The cure rate in treatment programmes has been about 50%. In 1991, a new project based on directly observed short-course chemotherapy was introduced. We report the results of the first 112 842 patients treated.Methods Patients with symptoms suggesting tuberculosis are referred by general health services to the tuberculosis county dispensary for physical examination and fluoroscopy. Those with suspicious findings submit three sputum samples for smear examination. Village doctors deliver free, directly observed therapy to all smear-positive patients on an outpatient basis. New cases are treated with streptomycin (750 mg), isoniazid (600 mg), pyrazinamide (2000 mg), and rifampicin (600 mg) every other day for 2 months, followed by 4 months of alternate-day isoniazid plus rifampicin. Previously treated patients receive streptomycin, isoniazid, pyrazinamide, rifampicin, and ethambutol (1250 mg) every other day for 2 months, followed by 6 months of isoniazid, rifampicin, and ethambutol. Patients are re-examined and smears are repeated at 2 months (then 3 months if still positive) and at 5 and 6 months (new cases) or 5 and 8 months (previously treated cases).Findings Nearly 1·6 million patients with suspected tuberculosis were referred for fluoroscopy between 1991 and 1994. 48% had suspicious findings on fluoroscopy and underwent sputum examination. 104444 new smear-positive cases and 95 616 previously treated smear-positive cases have been diagnosed. Results of treatment for those enrolled up to the first quarter of 1994 show cure rates of 89·7% among new cases (55213) and 81·1% among those previously treated (57 629). The failure rate in previously treated cases, an indicator of drug resistance, fell from 17·6% among 437 patients enrolled in 1991 to 6·2% among 7822 patients enrolled in the first quarter of 1994.Interpretation Our experience shows that high rates of compliance and cure can be achieved on a mass scale and in a short time by means of directly observed short-course chemotherapy and the WHO technical strategy for effective tuberculosis control.

Parallel assessment of glutathione-based detoxifying enzymes, O 6-alkylguanine-DNA alkyltransferase and P-glycoprotein as indicators of drug resistance in tumor and normal lung of patients with lung cancer : Oberli-Schrammle AE, Joncourt F, Stadler M, Altermatt HJ, Buser K, Ris HB et al. University of Berne, Dept. for Clinical-Experimen. Res., Tiefenaustrasse 120, CH-3004 Berne. Int J Cancer 1994;59:629–636

Parallel assessment of glutathione-based detoxifying enzymes, O 6-alkylguanine-DNA alkyltransferase and P-glycoprotein as indicators of drug resistance in tumor and normal lung of patients with lung cancer : Oberli-Schrammle AE, Joncourt F, Stadler M, Altermatt HJ, Buser K, Ris HB et al. University of Berne, Dept. for Clinical-Experimen. Res., Tiefenaustrasse 120, CH-3004 Berne. Int J Cancer 1994;59:629–636

Glutathione content of human malignant melanoma cell lines

We measured the glutathione content of a panel of human malignant melanoma cell lines by flow cytometry after staining with mercury orange, using visible light (488 nm) excitation, and compared the values obtained with those measured biochemically using a modified Tietze assay. Glutathione levels were depleted by culturing cells with various concentrations of buthionine sulphoximine to provide a suitable spread of glutathione concentrations. The two assays showed good correlation (r = 0.93). We found a number of technical factors to be critically important. In particular, the conditions of staining, cell number, and method of mixing media, cells and stain were responsible for wide variations in fluorescence intensity. We applied the flow cytometric technique to cell suspensions obtained by fine needle aspiration biopsy of human malignant melanoma deposits, and observed a proportion of cells with high glutathione levels in many samples. The results confirm the feasibility of measuring glutathione content by visible light flow cytometry, and raise the possibility of monitoring glutathione content as an indicator of drug resistance in clinical therapy of human malignant melanoma.

In vivo and in vitro assessment of the sensitivity of Plasmodium falciparum to chloroquine in four districts of Tanga region, Tanzania

The sensitivity of Plasmodium falciparum to chloroquine was tested in Muheza, Pangani, Tanga and Korogwe districts in north-eastern Tanzania by applying both in vivo and in vitro tests in schoolchildren. A total dose of 25 mg chloroquine base/kg body-weight given over a period of three days (10 mg/kg on days 0 and one; and 5 mg/kg on day 2) failed to clear asexual parasites from the peripheral blood by day 7 in 12·5% of the children tested at Muheza, 5·9% at Pangani, 31·8% at Tanga, and 39·5% at Korogwe. In vitro micro tests were successfully carried out on 44 isolates at Muheza, 29 isolates at Pangani, 45 isolates at Tanga and 44 isolates at Korogwe. Schizont maturation at chloroquine concentrations of 1·14 μ mol/litre of blood and above, an indication of drug resistance, was observed in 20·5% of the isolates at Muheza, 41·4% at Pangani, 51·1% at Tanga and 45·5% at Korogwe. In vivo and in vitro results of the tests for resistance have been compared.

The response of blood-inoculated and sporozoite-induced infections of Plasmodium relictum to drugs

1. When sporozoite-induced infections of Plasmodium relictum are treated with atebrin (mepacrine) in doses suppressive to blood-inoculated infections, for periods exceeding that of normal incubation, parasites devoid of pigment appear in the erythrocytes of the peripheral blood. A small proportion of these may develop into schizonts, but the number of merozoites produced is fewer than normal.2. A similar phenomenon occurs when quinine is substituted for atebrin, but the drug appears to be excreted more rapidly so that the inhibitive effect upon growth is less marked.3. When plasmoquine is given in the maximum tolerated dose to birds infected with sporozoites, parasites are not seen in the peripheral blood during treatment, though the infection is not sterilized. With smaller doses parasites, including a few small schizonts, are found in the erythrocytes during treatment.4. Merozoites produced in the erythrocytes during drug treatment are not capable of further multiplication when inoculated into uninfected birds having a similar blood-concentration of drug, so long as treatment continues, though they develop normally when transferred to untreated birds.5. No indication of drug resistance has been observed.6. The possible mechanisms by which blood-inoculated infections are eradicated, or resist eradication, are discussed.We wish to thank Miss Vincent (Mrs Thorpe) and Dr Parr Tate for access to their preliminary unpublished experiments, and the latter for helpful criticism.