Effect of cattle and horse feces storage methods on Nematode egg viability and sensitivity for egg hatch test.

The aim of the present study was to validate methods of stool sample conservation for the egg hatch test (EHT). This study involved the use of a bovine naturally infected predominantly by Cooperia spp. and one equine naturally infected predominantly by cyathostomins characterized as susceptible to benzimidazoles in the EHT. Fecal samples were submitted to three treatments: aerobic methods (anaerobic storage in plastic bottles, anaerobic storage in vacuum-sealed bags or aerobic storage in plastic bags), under two temperature conditions (room temperature and refrigeration) analyzed at four different assessment times (48, 72, 96 and 120 h). As the standard test, an assay was also performed within 3 h. The tests were performed in triplicate for each drug concentration and with three experimental repetitions at one-week intervals. Two criteria were used for the storage methods: hatchability in the negative control group and sensitivity of the eggs to thiabendazole, comparing the EC50 and 95% confidence interval for each treatment to those of the standard test and the other repetitions. Bovine samples can be stored for up to 96 h and refrigerated vacuum storage can be used, ensuring hatchability of the negative control and sensitivity of the eggs to thiabendazole. For equine samples, no forms of storage were indicated due to the variation among the repetitions and the reduction in the sensitivity of the eggs to thiabendazole, which could result in a false positive detection of resistance.

In Vitro Activity of Essential Oils against Saprolegnia parasitica.

Saprolegnia spp. water molds severely impact fish health in aquaculture, fish farms and hobby fish tanks colonizing mature and immature stages of fishes, as well as eggs. Considering that there are no drugs licensed for treating and/or control the organism, efficient and environmental low-impact methods to control these oomycetes in aquaculture are needed. The aim of the present report was to evaluate the in vitro sensitivity of Saprolegnia parasitica to essential oils (EOs) from Citrus aurantium L., Citrus bergamia Risso et Poiteau, Citrus limon Burm. f., Citrus paradisi Macfad, Citrussinensis Osbeck, Cinnamomum zeylanicum Blume, Cymbopogon flexuosum (Nees ex Steud.) Watson, Foeniculum vulgare Mill., Illicium verum Hook.f., Litsea cubeba (Lour.) Pers., Origanum majorana L., Origanum vulgare L., Pelargonium graveolens L'Hér., Syzygium aromaticum Merr. & L.M.Perry, and Thymus vulgaris L., by microdilution test. The most effective EOs assayed were T. vulgaris and O. vulgare, followed by C. flexuosum, L. cubeba and C. bergamia. These EOs could be of interest for controlling Saprolegnia infections. Nevertheless, further safety studies are necessary to evaluate if these products could be dispersed in tank waters, or if their use should be limited to aquaculture supplies.

Phenotypic and genotypic characterization of Thai isolates of Plasmodium falciparum after an artemisinin resistance containment project.

BACKGROUND: In Thailand, artemisinin-based combination therapy (ACT) has been used to treat uncomplicated falciparum malaria since 1995. Unfortunately, artemisinin resistance has been reported from Thailand and other Southeast Asian countries since 2003. Malarone®, a combination of atovaquone-proguanil (ATQ-PG), has been used to cease artemisinin pressure in some areas along Thai-Cambodia border, as part of an artemisinin resistance containment project since 2009. This study aimed to determine genotypes and phenotypes of Plasmodium falciparum isolates collected from the Thai-Cambodia border after the artemisinin resistance containment project compared with those collected before. RESULTS: One hundred and nine of P. falciparum isolates collected from Thai-Cambodia border from Chanthaburi and Trat provinces during 1988-2016 were used in this study. Of these, 58 isolates were collected after the containment. These parasite isolates were characterized for in vitro antimalarial sensitivities including chloroquine (CQ), quinine (QN), mefloquine (MQ), piperaquine (PPQ), artesunate (AS), dihydroartemisinin (DHA), ATQ and PG and genetic markers for drug resistance including the Kelch13 (k13), Plasmodium falciparum chloroquine resistance transporter (pfcrt), P. falciparum multidrug resistance 1 (pfmdr1) and cytochrome b (cytb) genes. Mean CQ, QN, MQ, PPQ and AS IC50s of the parasite isolates collected from 2009 to 2016 exhibited significantly higher than those of parasites collected before 2009. Approximately 57% exhibited in vitro MQ resistance. Approximately 94% of the isolates collected from 2009 to 2016 contained the pfmdr1 184F allele. Mutations of the k13 gene were detected in approximately 90% of the parasites collected from 2009 to 2016 which were significantly higher than the parasite isolates collected before. No ATQ-resistant genotype and phenotype of P. falciparum were found among the isolates collected after the containment project. CONCLUSIONS: Although the containment project had been implemented in this area, the expansion of artemisinin-resistant parasites did not decline. In addition, reduced sensitivity of the partner drugs of ACT including MQ and PPQ were identified.

Clinical Impact of Antifungal Susceptibility, Biofilm Formation and Mannoside Expression of Candida Yeasts on the Outcome of Invasive Candidiasis in ICU: An Ancillary Study on the Prospective AmarCAND2 Cohort.

Background: The link between Candida phenotypical characteristics and invasive candidiasis (IC) prognosis is still partially unknown. Methods: Candida strains isolated during the AmarCAND2 study were centrally analyzed for species identification, antifungal susceptibility, biofilm formation, and expression of surface and glycoconjugate mannosides. Correlation between these phenotypical features and patient outcome was sought using a multivariable Cox survival model. Results: Candida albicans was predominant (65.4%, n = 285), with a mortality rate significantly lower than that in patients with non-albicans strains [HR 0.67 (0.46-1.00), p = 0.048]. The rate of fluconazole-resistant strains was low (C. albicans and Candida glabrata: 3.5 and 6.2%, respectively) as well as caspofungin-resistant ones (1 and 3.1%, respectively). Early biofilm formation was less frequent among C. albicans (45.4%) than among non-albicans (81.2%). While the strains of C. albicans showed variable levels of surface mannosides expression, strains isolated from candidemia exhibited a high expression of ß-man, which was correlated with an increased mortality (p = 0.02). Conclusion: Candida albicans IC were associated with lower mortality, and with strains that exhibited less frequently early biofilm formation than non-albicans strains. A high expression of ß-man was associated with increased IC mortality. Further studies are warranted to confirm this data and to evaluate other virulence factors in yeasts.

DNA ploidy and S-phase fraction analysis in peritoneal carcinomatosis from ovarian cancer: correlation with clinical pathological factors and response to chemotherapy.

OBJECTIVE: We investigated the correlation between ploidy or S-phase fraction (SPF) and the clinical pathological characteristics of patients with peritoneal carcinomatosis from ovarian cancer. We also assessed their relation with the in vivo and in vitro response to several chemotherapeutic agents. PATIENTS AND METHODS: Fifty-three patients with peritoneal carcinomatosis from ovarian cancer were enrolled. Frozen tumor tissue was dissociated by a detergent-trypsin method, and the resulting cell suspension was stained with RNase A and propidium iodide. Samples were then analyzed for ploidy and SPF by flow cytometry. Fresh tumor tissue was dissociated by enzymatic digestion, and cells were exposed to different concentrations of cisplatin, adriamycin, carboplatin, gemcitabine and taxol for 72 hours. In vitro drug sensitivity was then measured using the sulforhodamine B assay. RESULTS: No significant correlation was found between ploidy or SPF and patient characteristics, even though primary carcinomas were mainly hyperdiploid and more proliferative than recurrent tumors. SPF differed significantly among ploidy categories (P=0.01), and high SPF was associated with short-term survival (P=0.48). Patients with multiploid tumors were the most resistant to platinum-based chemotherapy, whereas those with hyperdiploid tumors were the most responsive. In vitro multiploid tumors were the least sensitive, while hypodiploid samples showed the highest sensitivity to the tested drugs. Sensitivity to adriamycin was significantly correlated with ploidy (P=0.03), whereas sensitivity to taxol was correlated with SPF (P=0.04). CONCLUSION: Our results indicate that ploidy and SPF could facilitate the choice of therapy for patients with peritoneal carcinomatosis.

In vitro sensitivity of antimalarial drugs and correlation with clinico-parasitological response following treatment with a 3-day artesunate-mefloquine combination in patients with falciparum malaria along the Thai-Myanmar border.

A 3-day artesunate-mefloquine combination therapy has been using as first-line treatment for acute uncomplicated Plasmodium falciparum malaria in Thailand since 1995 on the background of mefloquine resistance. The aim of the present study was to assess sensitivity of P. falciparum isolates (n=44) in an area along the Thai-Myanmar border (year 2009) to artesunate, mefloquine, chloroquine and quinine, including their correlation with clinico-parasitological response. Twenty, 19, and 5 isolates were collected from patients with 'Adequate Clinical and Parasitological Response (ACPR)', 'Late Parasitological Failure (LPF)' and 're-infection', respectively. The IC50 of artesunate and mefloquine were significantly higher in patients with LPF compared with ACPR and re-infection. The proportion of isolates with declined artesunate or mefloquine sensitivity in the LPF group (47.4%) was significantly higher than the ACPR group (5.0%). A weak but statistical significant correlation (r=0.384, p=0.01) was observed between IC50 values of artesunate and parasite clearance time (PCT). There was no significant relationship between in vitro sensitivity of parasite isolates to chloroquine or quinine and clinical response. In vitro susceptibility of P. falciparum isolates to artesunate and mefloquine may be used as a useful reliable tool to predict clinico-pathological response following a 3-day artesunate-mefloquine combination therapy.

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.

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 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. 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.

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.

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>

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