An analytical method for assessing stage-specific drug activity in Plasmodium vivax malaria : implications for ex vivo drug susceptibility testing

The emergence of highly chloroquine (CQ) resistant P. vivax in Southeast Asia has created an urgent need for an improved understanding of the mechanisms of drug resistance in these parasites, the development of robust tools for defining the spread of resistance, and the discovery of new antimalarial agents. The ex vivo Schizont Maturation Test (SMT), originally developed for the study of P. falciparum, has been modified for P. vivax. We retrospectively analysed the results from 760 parasite isolates assessed by the modified SMT to investigate the relationship between parasite growth dynamics and parasite susceptibility to antimalarial drugs. Previous observations of the stage-specific activity of CQ against P. vivax were confirmed, and shown to have profound consequences for interpretation of the assay. Using a nonlinear model we show increased duration of the assay and a higher proportion of ring stages in the initial blood sample were associated with decreased effective concentration (EC50) values of CQ, and identify a threshold where these associations no longer hold. Thus, starting composition of parasites in the SMT and duration of the assay can have a profound effect on the calculated EC50 for CQ. Our findings indicate that EC50 values from assays with a duration less than 34 hours do not truly reflect the sensitivity of the parasite to CQ, nor an assay where the proportion of ring stage parasites at the start of the assay does not exceed 66%. Application of this threshold modelling approach suggests that similar issues may occur for susceptibility testing of amodiaquine and mefloquine. The statistical methodology which has been developed also provides a novel means of detecting stage-specific drug activity for new antimalarials.

Effect of the metabolic inhibitor, methimazole on the drug susceptibility of a triclabendazole-resistant isolate of Fasciola hepatica

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) is altered in the presence of a metabolic inhibitor. The flavin monooxygenase system (FMO) was inhibited using methimazole (MTZ) to see whether a TCBZ-resistant isolate could be made more sensitive to TCBZ action. The Oberon TCBZ-resistant and Cullompton TCBZ-sensitive isolates Were used for these experiments. The FMO system was inhibited by a 2-h pre-incubation in methimazole (100 mu M). Flukes were then incubated for I further 22 h in NCTC medium containing either MTZ; MTZ+nicotinamide adenine dinucleotide phosphate (NADPH) (1 nm); MTZ+NADPH+TCBZ (15 mu g/ml); or MTZ+NADPH+triclabendazole sulphoxide (TCBZ.SO) (15 mu g/ml). Morphological changes resulting from drug treatment and following metabolic inhibition were assessed Using scanning electron microscopy'. After treatment with either TCBZ or TCBZ.SO alone, there was greater surface disruption to the triclabendazole-susceptible than -resistant isolate. However, co-incubation with MTZ and TCBZ/TCBZ.SO lead to more severe surface changes to the TCBZ-resistant isolate than with each drug oil its own; this was not seen for the TCBZ-susceptible Cullompton isolate. Results of this study support the concept of altered drug metabolism in TCBZ-Resistant flukes and this process may play a role in the development of drug resistance.

Effect of the P-glycoprotein inhibitor, R(+)-verapamil on the drug susceptibility of a triclabendazole-resistant isolate of Fasciola hepatica

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by the inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Sligo TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for these experiments and the Pgp inhibitor selected was R(+)-verapamil [R-VPL]. In the first experiment, flukes were initially incubated for 2 h in R-VPL (100 µM), then incubated for a further 22 h in R-VPL+triclabendazole sulphoxide (TCBZ.SO) (50 µg/ml, or 0.1327 µM). For controls, flukes were incubated for 24 h in R-VPL and TCBZ.SO on their own. In a second experiment, flukes were removed from the incubation media following cessation of movement. In the third experiment, Sligo flukes were incubated in lower concentrations of R-VPL (10 µM) and TCBZ.SO (15 µg/ml, or 0.0398 µM). Morphological changes resulting from drug treatment and following Pgp inhibition were assessed by means of scanning electron microscopy. Incubation in R-VPL alone had minimal effect on either isolate. After treatment with TCBZ.SO alone, there was greater surface disruption to the Cullompton than Sligo isolate. However, combined treatment of R-VPL+TCBZ.SO led to more severe surface changes to the Sligo isolate than with TCBZ.SO on its own; this potentiation of drug activity was not seen with the Cullompton isolate. The phenomenon was evident at both concentrations of TCBZ.SO. Inclusion of R-VPL in the incubation medium also reduced the time taken for the flukes to become inactive; again, this effect was more distinct with the Sligo isolate. The results of this study support the concept of altered drug efflux in TCBZ-resistant flukes and indicate that drug transporters may play a role in the development of drug resistance.

Correlation of the in vitro antifungal drug susceptibility with the in vivo activity of fluconazole in a murine model of cerebral infection caused by Cryptococcus gattii

Forty Cryptococcus gattii strains were submitted to antifungal susceptibility testing with fluconazole, itraconazole, amphotericin B and terbinafine. The minimum inhibitory concentration (MIC) ranges were 0.5-64.0 for fluconazole, < 0.015-0.25 for itraconazole, 0.015-0.5 for amphotericin B and 0.062-2.0 for terbinafine. A bioassay for the quantitation of fluconazole in murine brain tissue was developed. Swiss mice received daily injections of the antifungal, and their brains were withdrawn at different times over the 14-day study period. The drug concentrations varied from 12.98 to 44.60 mu g/mL. This assay was used to evaluate the therapy with fluconazole in a model of infection caused by C. gattii. Swiss mice were infected intracranially and treated with fluconazole for 7, 10 or 14 days. The treatment reduced the fungal burden, but an increase in fungal growth was observed on day 14. The MIC for fluconazole against sequential isolates was 16 mu g/mL, except for the isolates obtained from animals treated for 14 days (MIC = 64 mu g/mL). The quantitation of cytokines revealed a predominance of IFN-gamma and IL-12 in the non-treated group and elevation of IL-4 and IL-10 in the treated group. Our data revealed the possibility of acquired resistance during the antifungal drug therapy.

Evaluation of the microscopic observation drug susceptibility assay for detection of Mycobacterium tuberculosis resistance to pyrazinamide

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Environmental isolation, biochemical identification, and antifungal drug susceptibility of Cryptococcus species

Introduction The incidence of opportunistic fungal infections has increased in recent years and is considered an important public health problem. Among systemic and opportunistic mycoses, cryptococcosis is distinguished by its clinical importance due to the increased risk of infection in individuals infected by human immunodeficiency virus. Methods To determine the occurrence of pathogenic Cryptococcus in pigeon excrement in the City of Araraquara, samples were collected from nine environments, including state and municipal schools, abandoned buildings, parks, and a hospital. The isolates were identified using classical tests, and susceptibility testing for the antifungal drugs (fluconazole, itraconazole, voriconazole, and amphotericin B) independently was also performed. After collection, the excrement samples were plated on Niger agar and incubated at room temperature. Results A total of 87 bird dropping samples were collected, and 66.6% were positive for the genus Cryptococcus. The following species were identified: Cryptococcus neoformans (17.2%), Cryptococcus gattii (5.2%), Cryptococcus ater (3.5%), Cryptococcus laurentti (1.7%), and Cryptococcus luteolus (1.7%). A total of 70.7% of the isolates were not identified to the species level and are referred to as Cryptococcus spp. throughout the manuscript. Conclusions Although none of the isolates demonstrated resistance to antifungal drugs, the identification of infested areas, the proper control of birds, and the disinfection of these environments are essential for the epidemiological control of cryptococcosis.

Stable expression of Escherichia coli beta-glucuronidase A (GusA) in Giardia lamblia: application to high-throughput drug susceptibility testing

OBJECTIVES: In order to create a suitable model for high-throughput drug screening, a Giardia lamblia WB C6 strain expressing Escherichia coli glucuronidase A (GusA) was created and tested with respect to susceptibility to the anti-giardial drugs nitazoxanide and metronidazole. METHODS: GusA, a well-established reporter gene in other systems, was cloned into the vector pPacVInteg allowing stable expression in G. lamblia under control of the promoter from the glutamate dehydrogenase (gdh) gene. The resulting transgenic strain was compared with the wild-type strain in a vitality assay, characterized with respect to susceptibility to nitazoxanide, metronidazole and -- as assessed in a 96-well plate format -- to a panel of 15 other compounds to be tested for anti-giardial activity. RESULTS: GusA was stably expressed in G. lamblia. Using a simple glucuronidase assay protocol, drug efficacy tests yielded results similar to those from cell counting. CONCLUSIONS: G. lamblia WB C6 GusA is a suitable tool for high-throughput anti-giardial drug screening.

Comparison of resazurin microtiter assay performance and BACTEC MGIT 960 in the susceptibility testing of Brazilian clinical isolates of Mycobacterium tuberculosis to four first-line drugs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Clinical and epidemiological profiles of individuals with drug-resistant tuberculosis

Drug-resistant tuberculosis (TB) is a growing global threat. Approximately 450,000 people developed multidrugresistant TB worldwide in 2012 and an estimated 170,000 people died from the disease. This paper describes the sociodemographic, clinical-epidemiological and bacteriological aspects of TB and correlates these features with the distribution of anti-TB drug resistance. Mycobacterium tuberculosis (MT) cultures and drug susceptibility testing were performed according to the BACTEC MGIT 960 method. The results demonstrated that MT strains from individuals who received treatment for TB and people who were infected with human immunodeficiency virus were more resistant to TB drugs compared to other individuals (p < 0.05). Approximately half of the individuals received supervised treatment, but most drug-resistant cases were positive for pulmonary TB and exhibited positive acid-fast bacilli smears, which are complicating factors for TB control programs. Primary healthcare is the ideal level for early disease detection, but tertiary healthcare is the most common entry point for patients into the system. These factors require special attention from healthcare managers and professionals to effectively control and monitor the spread of TB drug-resistant cases.

Multidrug- and extensively drug-resistant tuberculosis

We evaluated risk factors and treatment outcomes associated with multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) in Germany in 2004–2006. In 177 (4%) of 4,557 culture-positive TB cases, Mycobacterium tuberculosis isolates were identified as MDR TB; an additional 7 (0.15%) met criteria for XDR TB. Of these 184 patients, 148 (80%) were born in countries of the former Soviet Union. In patients with XDR TB, hospitalization was longer (mean ± SD 202 ± 130 vs. 123 ± 81 days; p = 0.015) and resistance to all first-line drugs was more frequent (36% vs. 86%; p = 0.013) than in patients with MDR TB. Seventy-four (40%) of these 184 patients received treatment with linezolid. Treatment success rates ranged from 59% for the entire cohort (59% for MDR TB and 57% for XDR TB) to 87% for those with a definitive outcome (n = 125; 89% for MDR TB and 80% for XDR TB). Extensive drug susceptibility testing and availability of second- and third-line drugs under inpatient management conditions permit relatively high treatment success rates in MDR- and XDR TB.

Detection and management of drug-resistant tuberculosis in HIV-infected patients in lower-income countries.

SETTING Drug resistance threatens tuberculosis (TB) control, particularly among human immunodeficiency virus (HIV) infected persons. OBJECTIVE To describe practices in the prevention and management of drug-resistant TB under antiretroviral therapy (ART) programs in lower-income countries. DESIGN We used online questionnaires to collect program-level data on 47 ART programs in Southern Africa (n = 14), East Africa (n = 8), West Africa (n = 7), Central Africa (n = 5), Latin America (n = 7) and the Asia-Pacific (n = 6 programs) in 2012. Patient-level data were collected on 1002 adult TB patients seen at 40 of the participating ART programs. RESULTS Phenotypic drug susceptibility testing (DST) was available in 36 (77%) ART programs, but was only used for 22% of all TB patients. Molecular DST was available in 33 (70%) programs and was used in 23% of all TB patients. Twenty ART programs (43%) provided directly observed therapy (DOT) during the entire course of treatment, 16 (34%) during the intensive phase only, and 11 (23%) did not follow DOT. Fourteen (30%) ART programs reported no access to second-line anti-tuberculosis regimens; 18 (38%) reported TB drug shortages. CONCLUSIONS Capacity to diagnose and treat drug-resistant TB was limited across ART programs in lower-income countries. DOT was not always implemented and drug supplies were regularly interrupted, which may contribute to the global emergence of drug resistance.

Trends in anti-tuberculosis drug resistance from 2003--2007 at Pham Ngoc Thach tuberculosis and lung disease hospital, Ho Chi Minh City, Vietnam

Vietnam is one of the countries with the highest prevalence and incidence of tuberculosis (TB) in the world (1). Although Vietnam has had many successes in TB control, it still faces the challenge of drug resistant and multidrug-resistant tuberculosis (MDR-TB). MDR-TB appears to be relatively stable, but data on MDR-TB continues to be scarce and routine testing of all isolates for drug susceptibility is not performed under Vietnam's National Tuberculosis Program (6). Pham Ngoc Thach Hospital (PNT), the leading tuberculosis and lung disease hospital in Ho Chi Minh City, serves as a reference hospital and laboratory for both Ho Chi Minh City and the Southern Vietnam region. This study is an unmatched, nested case-control study consisting of a secondary analysis of a previously created dataset composed of drug susceptibility and basic demographic data from a cohort of patients diagnosed with tuberculosis at PNT from 2003 through 2007 in order to calculate the prevalence of resistance among acid-fast bacilli smear-positive patients. The susceptibility records for the years 2003-2004 were not representative of the entire population, but over the years 2005-2007 the investigator found a decrease in resistance to all primary TB drugs on which records were available, as well as MDR-TB. Overall, females showed a higher proportion of resistance to TB drugs than males, and females had a greater likelihood of presenting with MDR-TB than males (OR=1.77). Persons 35-54 had greater likelihood of having MDR-TB than younger and older age groups. Among the population with HIV data, HIV-positivity was associated with greater likelihood of MDR-TB (OR=1.70, 95% CI=0.97-3.11). This study shows that rates of TB drug resistance are high, but declining, in one of Vietnam's largest TB hospitals, and that females and HIV-positive individuals are possible high-risk groups in this population.^