In vitro assessment of drug resistance in Plasmodium falciparum in five States of India.

Background & objectives: In vitro assays are an important tool to assess baseline sensitivity and monitor the drug response of Plasmodium falciparum over time and place and, therefore, can provide background information for the development and evaluation of drug policies. This study was aimed at determining the in vitro sensitivity of P. falciparum isolates to antimalarials. Methods: The in vitro activity of 108 P. falciparum isolates obtained from five States of India was evaluated using WHO microtest (Mark III) to chloroquine, monodesethylamodiaquine, dihydroartesunate and mefloquine. Samples were collected from the States of Orissa, Jharkhand, Karnataka, Goa and Chhattisgarh from September 2007 to August 2009. In addition, representative samples from different States of India cryopreserved and culture adapted in the Malaria Parasite Bank of National Institute of Malaria Research, New Delhi, were also evaluated. Results: The proportion of isolates resistant to chloroquine and monodesethylamodiaquine was 44.4 and 25 per cent, respectively. Of the 27 isolates resistant to monodesethylamodiaquine, 16 (59.3%) were cross-resistant to chloroquine. No isolate showed resistance to dihydroartesunate and mefloquine. Isolates from Orissa showed the highest degree of resistance to chloroquine and amodiaquine followed by Jharkhand. Forty two isolates were genotyped for pfcrt T76K chloroquine resistant mutation; mutations were seen in 38 (90.47%) isolates. Interpretation & conclusions: The Indian P. falciparum isolates showed a high degree of resistance to chloroquine followed by monodesethylamodiaquine. No resistance was recorded to mefloquine and dihydroartesunate.

Dermatomicosis en ancianos institucionalizados y estudios de sensibilidad in vitro a los antifúngicos sistémicos / Dermatomycosis in institutionalized elderly patients and in vitro sensitivity study to systemic antifungus

Se determinó la prevalencia de dermatomicosis en ancianos institucionalizados de Ciudad Bilívar, Estado Bolívar, Venezuela, y se evaluó la sensibilidad in vitro de los aislamientos clínicos a los antifúngicos itraconazol, fluconazol y terbinafina mediante el método de microdilución en medio líquido, recomendado por el Comité Internacional de Laboratorios Clínicos (M38-P), con algunas modificicaciones. Los hongos fueron identificados mediante métodos tradicionales. Las levaduras se identificaron mediante pruebas bioquímicas, sistema Api 20 C AUX (Biomérieux SA®, France) y crecimiento en medio de Staib. Se estudiaron 74 ancianos, todos recluidos en el Asilo "San Vicente de Paúl" y el Geriátrico "Carlos Fragachán" quienes dieron consentimiento por escrito para participar en el estudio. La edad de los pacientes estuvo comprendida entre 63 y 98 años (80 ± 8,4 años), la mayoría eran hombres (73%). Todos los pacientes tenían lesiones sugestivas de onicomicosis en los pies. El único dermatofito aislado fue Trichophyton rubrum (n=2) el cual resultó sensible al Itraconazol, terbinafina y sensibilidad variable a flucozazol. Asimismo se logró aislar Aspergillus niger (n=5; 6,7%) demostrándose sensible a terbinafina y fluconazol con sensibilidad variable a itraconazol. Candida albicans (n=3; 4,1%) fue sensible a fluconazol, resistentes a itraconazol y variable a la terbinafina. Aspergillus flavus fue aislado en dos casos (2,7%). Además de Geomyces sp, Fusarium oxysporum y Pseudeurotium ovale. Se concluye que existe una prevalencia baja de dermatomicosis en los ancianos institucionalizados de Ciudad Bolívar y que las lesiones clinicamente observadas son debidas a los cambios degenerativos propios de la edad.

A study determine prevalence of dermatomycosis in 74 institutionalized elderly patients was conductted in Ciudad Bolivar, state of Bolivar, Venezuela. Clinical isolates were assayed for in vitro sensitivity to itraconazole, fluconazole, and terbinafine using a slightly modified version of the microdilution method in liquid medium recommended by the International Committee of Clinical Laboratory (M38-P). Traditional methods were used to identify the fungi. The yeasts were identified by Api 20C AUX biochemical testing (bioMérieux SA®, France) and growth on Staib media. The elders, mostly men (73%), from the "San Vicente de Paúl" Nursing Home and the "Carlos Fragachan" Geriatric Hospital, were aged between 63 and 98 (80 ± 8.4 years). All the patients, whose written consent was secured, had lesions suggestive of onychomycosis. Trichophyton rubrum was the only isolated dermatophyte (n=2), which resulted sensitive to itraconazole and terbinafine, with variable sensitivity to fluconazole. Aspergillus niger (n=5;6.7%) was sensitive to terbinafine and fluconazole with variable itraconazole sensitivity. Candida albicans (n=3; 4.1%) was fluconazole sensitive, resistant to itraconazole, and variable to terbinafine. Aspergillus flavus was isolated in two cases 2.7%). Geomyces sp., Fusarium oxysporum, and Pseudeurotium ovale were also isolated. It is concluded that there is a low prevalence of dermatomycosis among institutionalized elders in Ciudad Bolivar, and that the lesions clinically observed were due to degenerative changes naturally occurring with aging.

Assessment of in vitro sensitivity of Plasmodium vivax fresh isolates

<p><b>OBJECTIVE</b>To compare the applicability of the SYBR Green-I assay with the standard schizont maturation assay, for determination of sensitivity of Plasmodium vivax (P. vivax) to chloroquine and a new antifolate WR 99210.</p><p><b>METHODS</b>The study was conducted at Mae Tao Clinic for migrant workers, Tak Province during April 2009 to July 2010. A total of 64 blood samples (1 mL blood collected into sodium heparinized plastic tube) were collected from patients with mono-infection with P. vivax malaria prior to treatment with standard regimen of a 3-day chloroquine. In vitro sensitivity of P. vivax isolates was evaluated by schizont maturation inhibition and SYBR Green-I assays.</p><p><b>RESULTS</b>A total of 30 out of 64 blood samples collected from patients with P. vivax malaria were successfully analyzed using both the microscopic schizont maturation inhibition and SYBR Green-I assays. The failure rates of the schizont maturation inhibition assay (50%) and the SYBR Green-I assay (54%) were similar (P=0.51). The median IC10s, IC50s and IC90s of both chloroquine and WR99210 were not significantly different from the clinical isolates of P. vivax tested. Based on the cut-off of 100 nM, the prevalences of chloroquine resistance determined by schizont maturation inhibition and SYBR Green-I assays were 19 and 11 isolates, respectively. The strength of agreement between the two methods was very poor for both chloroquine and WR99210.</p><p><b>CONCLUSIONS</b>On the basis of this condition and its superior sensitivity, the microscopic method appears better than the SYBR Green-I Green assay for assessing in vitro sensitivity of fresh P. vivax isolates to antimalarial drugs.</p>

Assessment of in vitro sensitivity of Plasmodium vivax fresh isolates

Objective: To compare the applicability of the SYBR Green-I assay with the standard schizont maturation assay, for determination of sensitivity of Plasmodium vivax (P. vivax) to chloroquine and a new antifolate WR 99210. Methods: The study was conducted at Mae Tao Clinic for migrant workers, Tak Province during April 2009 to July 2010. A total of 64 blood samples (1 mL blood collected into sodium heparinized plastic tube) were collected from patients with mono-infection with P. vivax malaria prior to treatment with standard regimen of a 3-day chloroquine.In vitro sensitivity of P. vivax isolates was evaluated by schizont maturation inhibition and SYBR Green-I assays. Results: A total of 30 out of 64 blood samples collected from patients withP. vivax malaria were successfully analyzed using both the microscopic schizont maturation inhibition and SYBR Green-I assays. The failure rates of the schizont maturation inhibition assay (50%) and the SYBR Green-I assay (54%) were similar (P=0.51). The median IC10s, IC50s and IC90s of both chloroquine and WR99210 were not significantly different from the clinical isolates of P. vivax tested. Based on the cut-off of 100 nM, the prevalences of chloroquine resistance determined by schizont maturation inhibition and SYBR Green-I assays were 19 and 11 isolates, respectively. The strength of agreement between the two methods was very poor for both chloroquine and WR99210. Conclusions: On the basis of this condition and its superior sensitivity, the microscopic method appears better than the SYBR Green-I Green assay for assessing in vitro sensitivity of fresh P. vivax isolates to antimalarial drugs.

A study of chloroquine resistance of plasmodium falciparum using the in-vitro sensitivity test and polymerase chain reaction (PCR)

The widespread and increasing resistance of Plasmodium falciparum to antimalarial drugs is one of several factors that contributed to the persistence and even worsening of the malaria problem. Resistance to Chloroquine is of utmost concern, considering that it had been the most reliable antimalarial until the emergence and spread of Chloroquineresistant P. falciparum. Until recently, Chloroquine and Sulfadoxine-Pyrimethamine were the first and second line antimalarials in use in the Philippines. However, this has been changed to a combination of Chloroquine and Sulfadoxine-Pyrimethamine, because of the high percentage of treatment failures in therapeutic efficacy studies done in northern and southern Philippines. The objective of this study is to apply PCR in determining the occurence of Chloroquine resistance in southeastern Mindanao using in-vitro test and polymerase chain reaction (PCR)In the first study, the in-vitro susceptibility of P. falciparum to Chloroquine was tested in 33 isolates using the World Health Organization (WHO) Semi-Microtest Method. These isolates were collected from patients who consulted or were admitted at the regional hospital of Davao del Norte. The results showed that 10 (30.3 percent) were susceptible with IC50 80 nM, 12 (36.4 percent) isolates had decreased sensitivity with 80 nM /- IC50 114 nM, and 11 (33.3 percent) were resistant with IC 114 nM. Ten of these 11 (91 percent) were from Davao del Norte. A closer look at the municipalities of this province showed that the geometric mean (SD) of IC50 of Asuncion was 133 (41) nM and was significantly higher (p=0.025) than nearby Kapalong at 82 (25) nMThe PCR, Apo1 restriction revealed that 30 (90.9 percent) of the isolates manifested the PfCRT (K76T) mutation. These findings are indicative of the presence of Chloroquine resistance among the isolates. Comparison with the results of the invitro test (33.3 percent resistance) showed that the frequency of the PfCRT gene (90.9 percent) was very high. This finding suggests that the mere presence of the PfCRT gene does not mean the expression of Chloroquine resistance. It is possible that other genes such as the Pfmdr and cg2 are also involved in the expression of Chloroquine resistance. The study also shows that PCR and Apo1 restriction may be limited in generating results that can be used to correlate with those of the in-vitro or even in-vivo tests