Characterization of antimalarial activity of artemisinin-based hybrid drugs.

In response to the spread of artemisinin (ART) resistance, ART-based hybrid drugs were developed, and their activity profile was characterized against drug-sensitive and drug-resistant Plasmodium falciparum parasites. Two hybrids were found to display parasite growth reduction, stage-specificity, speed of activity, additivity of activity in drug combinations, and stability in hepatic microsomes of similar levels to those displayed by dihydroartemisinin (DHA). Conversely, the rate of chemical homolysis of the peroxide bonds is slower in hybrids than in DHA. From a mechanistic perspective, heme plays a central role in the chemical homolysis of peroxide, inhibiting heme detoxification and disrupting parasite heme redox homeostasis. The hybrid exhibiting slow homolysis of peroxide bonds was more potent in reducing the viability of ART-resistant parasites in a ring-stage survival assay than the hybrid exhibiting fast homolysis. However, both hybrids showed limited activity against ART-induced quiescent parasites in the quiescent-stage survival assay. Our findings are consistent with previous results showing that slow homolysis of peroxide-containing drugs may retain activity against proliferating ART-resistant parasites. However, our data suggest that this property does not overcome the limited activity of peroxides in killing non-proliferating parasites in a quiescent state.

Mecanismos de expressão de resistência aos antibióticos e saúde pública / Mechanisms of expression of resistance to antibiotics and public health / Mecanismos de expresión de la resistencia a los antibióticos y salud pública

Com o avanço da medicina e o aumento do uso de antimicrobianos, a resistência microbiana vem se tornando um problema sério na saúde pública. Para que uma bactéria se torne resistente, são necessários vários fatores, entre eles, o uso indiscriminado e prolongado de antimicrobianos e as resistências intrínsecas e adquiridas. Nesse contexto, o objetivo do trabalho foi explorar os mecanismos de ação dos antimicrobianos, de resistência e a sua importância na saúde pública. Foram utilizadas para a presente pesquisa, as bases de dados Pubmed, Google acadêmico e Scielo. Segundo a Organização Mundial da Saúde define-se resistência ao antibiótico quando o mesmo não produz mais efeito. A inserção cada vez mais frequente de antimicrobianos favorece a resistência, onde provocam uma pressão seletiva sobre os microrganismos, tornando-os resistentes a diversas drogas. O uso indiscriminado de antimicrobianos é o principal fator de resistência microbiana, assim como o uso de antimicrobianos sem exame de cultura e teste de sensibilidade. Neste sentido, conclui-se que é de suma importância a atualização de protocolos que contenham os mecanismos de resistência bacteriana a fim de minimizar o uso indiscriminado de antimicrobianos, assim como capacitar os profissionais da saúde para este problema na saúde pública.

With the advance of medicine and the increase in the use of antimicrobials, microbial resistance has become a serious problem in public health. For a bacterium to become resistant, several factors are necessary, among them, the indiscriminate and prolonged use of antimicrobials and the intrinsic and acquired resistance. In this context, the objective of the work was to explore the mechanisms of action of antimicrobials, resistance and their importance in public health. Pubmed, Google academic and Scielo databases were used for this research. According to the World Health Organization, resistance to antibiotics is defined when it no longer has an effect. The increasingly frequent insertion of antimicrobials favors resistance, where they put selective pressure on microorganisms, making them resistant to various drugs. The indiscriminate use of antimicrobials is the main factor of microbial resistance, as well as the use of antimicrobials without culture examination and sensitivity test. In this sense, it is concluded that it is extremely important to update protocols that contain the mechanisms of bacterial resistance in order to minimize the indiscriminate use of antimicrobials, as well as to train health professionals for this problem in public health.

Con los avances de la medicina y el mayor uso de antimicrobianos, la resistencia microbiana se ha convertido en un grave problema de salud pública. Para que una bacteria se vuelva resistente son necesarios varios factores, entre ellos, el uso indiscriminado y prolongado de antimicrobianos y la resistencia intrínseca y adquirida. En este contexto, el objetivo de este trabajo fue explorar los mecanismos de acción de los antimicrobianos, la resistencia y su importancia en la salud pública. Para esta investigación se utilizaron las bases de datos Pubmed, Google Scholar y Scielo. Según la Organización Mundial de la Salud, la resistencia a un antibiótico se define cuando deja de producir efecto. El uso cada vez más frecuente de antimicrobianos favorece la resistencia, ya que provocan una presión selectiva sobre los microorganismos, haciéndolos resistentes a varios fármacos. El uso indiscriminado de antimicrobianos es el principal factor de resistencia microbiana, así como el uso de antimicrobianos sin pruebas de cultivo y sensibilidad. En este sentido, se concluye que es de suma importancia actualizar los protocolos que contienen los mecanismos de resistencia bacteriana para minimizar el uso indiscriminado de antimicrobianos, así como capacitar a los profesionales de la salud para este problema en la salud pública.

Modulação farmacológica da melatonina na resistência a drogas em neoplasia mamária canina / Modulación farmacológica de melatonina sobre la resistencia a drogas en la neoplasia mamaria canina / Pharmacological modulation of melatonin on drug resistance in canine mammary neoplasia

El cáncer mamario es una enfermedad de frecuente presentación en perras. Dentro de la masa tumoral, hay una subpoblación de células neoplásicas con características de troncalidad (CSCs), que forman estructuras esferoides que crecen libres de anclaje (esferas), y resisten quimioterapia, explicando en parte la recurrencia de algunos cánceres. Melatonina ha mostrado efectos antitumorales sobre células tumorales mamarias; sin embargo, sus efectos han sido poco estudiados en CSCs mamarias caninas. Células CF41.Mg y REM 134 se cultivaron bajo condiciones standard. Esferas derivadas de estas células se cultivaron en placas de ultra baja adherencia, en ausencia de suero fetal bovino y conteniendo diferentes factores de crecimiento. Esferas de ambas líneas celulares mostraron quimioresistencia. Melatonina indujo un efecto antiproliferativo solo en CSC-CF41.Mg, siendo este efecto de mayor magnitud que en células parentales no troncales, sin embargo, no indujo un efecto aditivo con doxorrubicina y mitoxantrona. La invasión celular se redujo en respuesta a concentraciones no citotóxicas de la hormona. Además, resultados preliminares muestran que melatonina no modula la expresión génica de MDR1, molécula clave en el fenómeno de resistencia a drogas. Estos resultados indican que melatonina modula la actividad proliferativa e invasiva de CSCs, efecto que es dependiente del tipo celular.(AU)

O câncer de mama é uma doença comum em cadelas. Dentro da massa tumoral existe uma subpopulação de células neoplásicas com características de tronco (CSCs), que formam estruturas esferóides que crescem livres de ancoragem (esferas) e resistem à quimioterapia, explicando em parte a recorrência de alguns cânceres. A melatonina mostrou efeitos antitumorais em células tumorais mamárias; entretanto, seus efeitos têm sido pouco estudados em CSCs mamárias caninas. As células CF41.Mg e REM 134 foram cultivadas sob condições padrão. Esferas derivadas dessas células foram cultivadas em placas de ultra baixa aderência, na ausência de soro fetal bovino e contendo diferentes fatores de crescimento. As pérolas de ambas as linhas celulares mostraram quimiorresistência. A melatonina induziu um efeito antiproliferativo apenas em CSC-CF41.Mg, sendo este efeito maior do que em células não-tronco parentais, no entanto, não induziu efeito aditivo com doxorrubicina e mitoxantrona. A invasão celular foi reduzida em resposta às concentrações não citotóxicas do hormônio. Além disso, resultados preliminares mostram que a melatonina não modula a expressão gênica de MDR1, uma molécula chave no fenômeno de resistência a drogas. Esses resultados indicam que a melatonina modula a atividade proliferativa e invasiva das CSCs, um efeito que é dependente do tipo celular.(AU)

Comprehensive Transcriptome Analyses in Sea Louse Reveal Novel Delousing Drug Responses Through MicroRNA regulation.

The role of miRNAs in pharmacological responses through gene regulation related to drug metabolism and the detoxification system has recently been determined for terrestrial species. However, studies on marine ectoparasites have scarcely been conducted to investigate the molecular mechanisms of pesticide resistance. Herein, we explored the sea louse Caligus rogercresseyi miRNome responses exposed to delousing drugs and the interplaying with coding/non-coding RNAs. Drug sensitivity in sea lice was tested by in vitro bioassays for the pesticides azamethiphos, deltamethrin, and cypermethrin. Ectoparasites strains with contrasting susceptibility to these compounds were used. Small-RNA sequencing was conducted, identifying 2776 novel annotated miRNAs, where 163 mature miRNAs were differentially expressed in response to the drug testing. Notably, putative binding sites for miRNAs were found in the ADME genes associated with the drugs' absorption, distribution, metabolism, and excretion. Interactions between the miRNAs and long non-coding RNAs (lncRNAs) were also found, suggesting putative molecular gene regulation mechanisms. This study reports putative miRNAs correlated to the coding/non-coding RNAs modulation, revealing novel pharmacological mechanisms associated with drug resistance in sea lice species.

Resistencia antirretroviral en pacientes con virus de inmuno- deficiencia humana en Hospital Nacional Mario Catarino Rivas / Resistance to antiretroviral drugs in patients with human immunodeficiency virus in Mario Catarino Rivas National Hospital

Introducción: La terapia antirretroviral de primera línea utilizada en el país desde hace varios años, consiste en un inhibidor de la transcriptasa reversa no nucleósido con dos inhibidores de la transcriptasa reversa nucleósidos, está asociada al fallo terapéutico y se ha reportado resis- tencia a la misma. Objetivo: conocer la resistencia a la terapia antirretroviral de primera línea en pacientes tratados por Virus de Inmunodeficiencia Humana en Hospital Nacional Mario Catarino Rivas desde octubre 2016 al 2017. Pacientes y métodos: Investigación observacio- nal, descriptiva; tipo cohorte transversal, retrospectiva. Población de 313 pacientes a quienes se le realizó prueba de genotipo, de los cuales se tomaron como muestra 291 pacientes distri- buidos por grupos: sin previa exposición a antirretrovirales, pediátricos, con 12 meses de trata- miento y 48 o más meses de tratamiento a quienes se les realizó prueba de genotipo. Resulta- dos: Hubo amplificación en el 52% (152) de los pacientes, de los cuales el 56% (85) presentó resistencia a tratamiento antirretroviral, con prevalencia de resistencia de inhibidores de trans- criptasa reversa análogo de nucleósidos del 75% (64), los inhibidores de transcriptasa reversa no nucleósidos (ITRNN) con 89% (76) y los inhibidores de proteasa del 15% (13). Se encontró una prevalencia de resistencia primaria del 19% en pacientes de diagnóstico nuevo. Conclu- sión: Se recomienda el cambio de primera línea de terapia antirretroviral ya que se identifica- ron mutaciones de resistencia a los ITRNN en un 91% en los pacientes con diagnóstico recien- te y sin exposición a ARV. La OMS recomienda retirar los ITRNN como primera línea e incluir fármacos con mejor barrera genética, cuando los niveles de resistencia para los ITRNN sean >10%...(AU)

Mycobacterium leprae: aspectos da resistência aos fármacos na poliquimioterapia / Drug resistance in multidrug therapy for Mycobacterium leprae

Introdução: O diagnóstico da hanseníase possui números significativos que causam preocupação à saúde pública. Os casos de resistência medicamentosa nessa doença se iniciaram em meados dos anos 60 e diante do problema, a Organização Mundial da Saúde instituiu em 1981 a poliquimioterapia, associação dos antibióticos rifampicina, dapsona e clofazimina, tratamento atual de escolha. A resistência aos fármacos na hanseníase é reportada pela literatura, desvelando um obstáculo à sua eliminação. Apresentamos nessa revisão os principais aspectos da resistência medicamentosa no tratamento para hanseníase e seus impactos. Metodologia: Revisão sistemática sobre os aspectos da resistência medicamentosa utilizando a pesquisa exploratória como metodologia de abordagem. Foram pesquisados os termos resistência medicamentosa, hanseníase, recidiva, alterações genéticas e os operadores booleanos "and" e "or" na busca. Resultados e discussão: A dificuldade de tomar a medicação corretamente foi um dos principais fatores que acarretaram resistência do bacilo Mycobacterium leprae aos fármacos. Homens de países norte e sul-americanos e asiáticos foram os mais atingidos por episódios de resistência. A resistência medicamentosa é uma das principais causas de recidivas em hanseníase. O principal fármaco causador de resistência medicamentosa descrito nos trabalhos foi a dapsona (46,6%) e a maioria das alterações genéticas encontradas estão no gene rpoB; 23,2% dos registros relatados foram de resistência secundária aos fármacos e, também, sete casos de resistência múltipla a esses medicamentos. Conclusão: Os principais aspectos da resistência medicamentosa na hanseníase são os equívocos ao ingerir os medicamentos e as alterações genéticas na bactéria. Os impactos causados estão na dificuldade de refazer o tratamento, a possibilidade de nova transmissão e o aparecimento de sintomas mais graves.

Introduction: The diagnosis of leprosy has significant numbers causing public health concern. Reports of drug resistance in this disease begun in the mid-1960s and due to this problem, the World Health Organization instituted a multidrug therapy with rifampicin, dapsone, and clofazimine antibiotic association in 1981, which is currently the first-choice treatment for leprosy. Cases of drug resistance have been reported in literature, revealing an obstacle to the eradication of the disease. This paper has the purpose of presenting the key aspects and impacts of drug resistance in the treatment for leprosy. Methods: Systematic review of the drug resistance aspects using exploratory research as an approach methodology. The authors searched the terms drug resistance, leprosy, recurrence, genetic alterations, and the Boolean operators "and" and "or" between them. Results and discussion: The difficulty in taking the medication correctly was one of the key factors that led to drug resistance for Mycobacterium leprae. Men from North and South American, as well as from Asian countries, were the most affected by episodes of resistance. Drug resistance is one of the main causes of leprosy recurrences. Dapsone was the most frequently identified drug resistance in the studies (46.6%), while most of the genetic alterations were found in the rpoB gene; 23.2% of the cases were from secondary resistance episodes, and seven cases of multiple resistance were reported. Conclusion: The misconceptions when taking the treatment and the Mycobacterium leprae genetic alterations have been described as the key aspects of drugs resistance in leprosy and the impacts caused are the difficulty in redoing the treatment, the possibility of new transmission, and the appearance of more severe symptoms.

Ursolic and betulinic semisynthetic derivatives show activity against CQ-resistant Plasmodium falciparum isolated from Amazonia.

ACT's low levels of Plasmodium parasitemia clearance are worrisome since it is the last treatment option against P. falciparum. This scenario has led to investigations of compounds with different mechanisms of action for malaria treatment. Natural compounds like ursolic acid (UA) and betulinic acid (BA), distinguished by their activity against numerous microorganisms, including P. falciparum, have become relevant. This study evaluated the antiplasmodial activity of imidazole derivatives of UA and BA against P. falciparum in vitro. Eight molecules were obtained by semisynthesis and tested against P. falciparum strains (NF54 and CQ-resistant 106/cand isolated in Porto Velho, Brazil); 2a and 2b showed activity against NF54 and 106/cand strains with IC50  < 10 µM. They presented high selectivity indexes (SI > 25) and showed synergism when combined with artemisinin. 2b inhibited the parasite's ring and schizont forms regardless of when the treatment began. In silico analysis presented a tight bind of 2b in the topoisomerase II-DNA complex. This study demonstrates the importance of natural derivate compounds as new candidates for malarial treatment with new mechanisms of action. Semisynthesis led to new triterpenes that are active against P. falciparum and may represent new alternatives for malaria drug development.

Modulation of the Drug Resistance by Platonia insignis Mart. Extract, Ethyl Acetate Fraction and Morelloflavone/Volkensiflavone (Biflavonoids) in Staphylococcus aureus Strains Overexpressing Efflux Pump Genes.

BACKGROUND: Microbial resistance to antibiotics is a global public health problem, which requires urgent attention. Platonia insignis is a native species from the eastern Brazilian Amazon, used in the treatment of burns and wounds. OBJECTIVES: To evaluate the antimicrobial activity of the hydroalcoholic extract of P. insignis (PIHA), the ethyl acetate fraction (PIAE), and its subfraction containing a mixture of biflavonoids (BF). Moreover, the effect of these natural products on the antibiotic activity against S. aureus strains overexpressing efflux pump genes was also evaluated. METHODS: Minimal inhibitory concentrations were determined against different species of microorganisms. To evaluate the modulatory effect on the Norfloxacin-resistance, the MIC of this antibiotic was determined in the absence and presence of the natural products at subinhibitory concentrations. Inhibition of the EtBr efflux assays were conducted in the absence or presence of natural products. RESULTS: PIHA showed a microbicidal effect against S. aureus and C. albicans, while PIAE was bacteriostatic for S. aureus. PIAE and BF at subinhibitory concentrations were able to reduce the MIC of Norfloxacin acting as modulating agents. BF was able to inhibit the efflux of EtBr efflux in S. aureus strains overexpressing specific efflux pump genes. CONCLUSION: P. inignisis, a source of efflux pump inhibitors, including volkensiflavone and morelloflavone, which were able to potentiate the Norfloxacin activity by NorA inhibition, being also able to inhibit QacA/B, TetK and MsrA. Volkensiflavone and morelloflavone could be used as an adjuvant in the antibiotic therapy of multidrug resistant S. aureus strains overexpressing efflux pumps.

Antiplasmodial activity of sulfonylhydrazones: in vitro and in silico approaches.

Malaria is still a life-threatening public health issue, and the upsurge of resistant strains requires continuous generation of active molecules. In this work, 35 sulfonylhydrazone derivatives were synthesized and evaluated against Plasmodium falciparum chloroquine-sensitive (3D7) and resistant (W2) strains. The most promising compound, 5b, had an IC50 of 0.22 µM against W2 and was less cytotoxic and 26-fold more selective than chloroquine. The structure-activity relationship model, statistical analysis and molecular modeling studies suggested that antiplasmodial activity was related to hydrogen bond acceptor count, molecular weight and partition coefficient of octanol/water and displacement of frontier orbitals to the heteroaromatic ring beside the imine bond. This study demonstrates that the synthesized molecules with a simple scaffold allow the hit-to-lead process for new antimalarials to commence.

Quinolinotriazole antiplasmodials via click chemistry: synthesis and in vitro studies of 7-Chloroquinoline-based compounds

Malaria is nowadays one of the most serious health concerns in a global scale and, although there is an evident increase in research studies in this area, pointed by the vast number of hits and leads, it still appears as a recurrent topic every year due to the drug resistance shown by the parasite exposing the urgent need to develop new antimalarial medications. In this work, 38 molecules were synthesized via copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) or "click" chemistry, following different routes to produce 2 different organic azides, obtained from a 4,7 dicholoquinoline, reacted with 19 different commercially available terminal alkynes. All those new compounds were evaluated for their in vitro activity against the chloroquine resistant malaria parasite Plasmodium falciparum (W2). The cytotoxicity evaluation was accomplished using Hep G2 cells and SI index was calculated for every molecule. Some of the quinoline derivatives have shown high antimalarial activity, with IC50 values in the range of 1.72-8.66 µM, low cytotoxicity, with CC50>1000 µM and selectivity index (SI) in the range of 20-100, with some compounds showing SI>800. Therefore, the quinolinotriazole hybrids could be considered a very important step on the development of new antimalarial drugs

Is breaking of a hydrogen bond enough to lead to drug resistance?

Drug resistance is a serious problem in cancer, viral, bacterial, fungal and parasitic diseases. Examination of crystal structures of protein-drug complexes is often not enough to explain why a certain mutation leads to drug resistance. As an example, the crystal structure of the kinase inhibitor dasatinib bound to the Abl1 kinase shows a hydrogen bond between the drug and residue Thr315 and very few contacts between the drug and residues Val299 and Phe317, yet mutations in those residues lead to drug resistance. In the first case, it is tempting to suggest that the loss of a hydrogen bond leads to drug resistance, whereas in the other two cases it is not known why mutations lead to drug resistance in the first place. We carried out extensive molecular dynamics (MD) simulations and free energy calculations to explain drug resistance to dasatinib from a molecular point of view and show that resistance is due to a multitude of subtle effects. Importantly, our calculations could reproduce the experimental values for the binding energies upon mutations in all three cases and shed light on their origin.

Combination of bioactive phytochemicals and synthetic anthelmintics: In vivo and in vitro assessment of the albendazole-thymol association.

The search of novel strategies for anthelmintic control is a crucial need considering the widespread increase in resistant parasitic populations in livestock. Bioactive phytochemicals may contribute to improve parasite control by enhancing the effect of existing anthelmintic drugs. The aim of the current work was to evaluate the in vivo and in vitro pharmaco-chemical interaction and the in vivo efficacy of the combination of albendazole (ABZ) with thymol (TML) in lambs naturally infected with resistant gastrointestinal nematodes. Thirty (30) lambs were allocated into three experimental groups. Each group was treated orally with either ABZ (5 mg/kg), TML (150 mg/kg, twice every 24 h) or the co-administration of both compounds. Blood samples were collected between 0 and 51 h post-treatment and TML, ABZ and its metabolites were measured by HPLC. Individual faecal samples were collected at days -1 and 14 post-treatment to perform the faecal egg count reduction test. Additionally, the effect of TML on the sulphoreduction and sulphonation of ABZ sulphoxide was assessed in vitro using ruminal content and liver microsomes, respectively. The metabolism of TML in the ruminal content was very low and the monoterpene exhibited a low degree of association with the particulate phase of the ruminal content. No changes in the pharmacokinetic behavior of ABZ sulphoxide were observed in the presence of the natural product (TML). In contrast, the ABZ sulphone Cmax and AUC were lower (P 0.002 and 0.001 respectively) in the co-administered animals (0.16 ±â€¯0.07 µg/mL and 3.63 ±â€¯1.21 µg.h/mL) compared with those that received ABZ alone (0.45 ±â€¯0.15 µg/mL and 9.50 ±â€¯2.84 µg.h/mL). TML was detected in the bloodstream between 1 and 48 h post-treatment, which indicates the time of target nematodes being exposed to the bioactive monoterpene. However, the in vivo efficacy of TML was 0% and the presence of this terpene did not increase the efficacy of ABZ. The presence of TML significantly inhibited the ruminal sulphoreduction (P 0.001) and the hepatic sulphonation (P 0.001) of ABZ sulphoxide. These observations point out that in vivo pharmaco-parasitological studies are relevant to corroborate the adverse kinetic/metabolic interactions and the efficacy of bioactive natural products combined with synthetic anthelmintics.

Antifungal and anti-biofilm activity of designed derivatives from kyotorphin.

Kyotorphin (KTP, l-tyrosyl-l-arginine) is an endogenous analgesic neuropeptide first isolated from bovine brain in 1979. Previous studies have shown that kyotorphins possess anti-inflammatory and antimicrobial activity. Six kyotorphins-KTP-NH2, KTP-NH2-DL, ibuprofen-conjugated KTP (IbKTP), IbKTP-NH2, N-methyl-D-Tyr-L-Arg, and N-methyl-L-Tyr-D-Arg-were designed and synthesized to improve lipophilicity and resistance to enzymatic degradation. This study assessed the antimicrobial and antibiofilm activity of these peptides. The antifungal activity of kyotorphins was determined in representative strains of Candida species, including Candida albicans ATCC 10231, Candida krusei ATCC 6258, and six clinical isolates-Candida dubliniensis 19-S, Candida glabrata 217-S, Candida lusitaniae 14-S, Candida novergensis 51-S, Candida parapsilosis 63, and Candida tropicalis 140-S-obtained from the oral cavity of HIV-positive patients. The peptides were synthesized by standard solution or solid-phase synthesis, purified by RP-HPLC (purity >95 %), and characterized by nuclear magnetic resonance. The results of the broth microdilution assay and scanning electron microscopy showed that IbKTP-NH2 presented significant antifungal activity against Candida strains and antibiofilm activity against the clinical isolates. The absence of toxic activity and survival after infection was assessed after injecting the peptide in larvae of Galleria mellonella as experimental infection model. Furthermore, IbKTP-NH2 had strong antimicrobial activity against multidrug-resistant bacteria and fungi and was not toxic to G. mellonella larvae up to a concentration of 500 mM. These results suggest that IbKTP-NH2, in addition to its known effect on cell membranes, can elicit a cellular immune response and, therefore, is promising for biomedical application.

Testing Albendazole Resistance in Fasciola hepatica.

The egg development test is a useful in vitro tool to detect albendazole (ABZ) resistance in Fasciola hepatica. ABZ is the only flukicidal compound with ovicidal activity. The described test is based on the ABZ capacity to affect parasite egg development and hatching in susceptible parasites, while this effect is lost in ABZ-resistant liver fluke isolates. Among many advantages, it is noted that the diagnostic test can be performed on eggs isolated from fecal samples (sheep and cattle), avoiding the sacrifice of animals necessary in controlled efficacy trials. The egg development test described here is a simple, inexpensive, and accessible method, previously employed for diagnosis of ABZ resistance in F. hepatica.

Tocilizumab in glucocorticoid-resistant graves orbitopathy. A case series report of a mexican population.

PURPOSE: To describe pre- to post-treatment changes in clinical activity score (CAS) and exophthalmometry in patients with Graves orbitopathy treated with tocilizumab (TCZ). MATERIAL AND METHODS: Eight Mexican patients presenting with active Graves orbitopathy (CAS>3/7) previously treated with glucocorticoids received 1 monthly dose of TCZ for 6 months. CAS, EUGOGO severity assessment and exophthalmometry were used to evaluate clinical status, with serum measurement of thyroid-stimulating hormone receptor antibodies (TR-Ab) for biochemical evaluation before and after application of TCZ. RESULTS: Eight patients were analyzed: 6 male (75%), 2 female (25%): mean age, 45.9±11.2 years; mean weight, 85±18.3 kg. Mean TR-Ab level at treatment outset was 291.9±96.4%, mean CAS 4.1±0.3 and mean exophthalmometry 21.2±3.2 mm. After TCZ treatment, mean TR-Ab level fell to 172.7±54% (P=0.001), mean CAS to 1.1±0.6 (P=0.001) and mean exophthalmometry to 19.3±2 mm (P=0.02). CONCLUSIONS: TCZ is a therapeutic option for glucocorticoid-resistant orbitopathy, and should be considered in second line due to the cost of treatment or in first line in patients with contraindications to intravenous GC pulse therapy.

5-Nitroimidazole refractory giardiasis is common in Matanzas, Cuba and effectively treated by secnidazole plus high-dose mebendazole or quinacrine: a prospective observational cohort study.

OBJECTIVE: To evaluate the effectiveness and tolerability of secnidazole combined with high-dose mebendazole for treatment of 5-nitroimidazole-resistant giardiasis. METHOD: Adults with microscopically verified Giardia intestinalis monoinfection attending a secondary level hospital in Matanzas City, Cuba were prospectively included in a cohort. A recently introduced treatment ladder consisting of metronidazole as first-line treatment, followed by secnidazole, tinidazole, secnidazole plus mebendazole and quinacrine as second-to fifth-line treatments, respectively, was used. Adverse events and treatment success were determined by questioning and microscopy on concentrated stool samples, respectively on days 3, 5 and 7 after the end of treatment. If G. intestinalis was detected on day 3, 5 or 7, then the infection was classified as refractory and no further microscopy was performed. RESULTS: A total of 456 individuals were included. Metronidazole, 500 mg three times daily for 5 days, cured 248/456 (54%) patients. A single 2-g secnidazole dose as second-line treatment cured 50/208 (24%) patients. A single 2-g tinidazole dose as third-line treatment cured 43/158 (27%) patients. Three rounds of 5-nitroimidazole therapy therefore cured 341/456 (75%) patients. Secnidazole plus mebendazole (200 mg every 8 hours for 3 days) cured 100/115 (87%) of nitroimidazole refractory infections. Quinacrine cured the remaining 15 patients. All treatments were well tolerated. CONCLUSIONS: 5-Nitroimidazole refractory giardiasis was common, indicating that an alternative first-line treatment may be needed. Retreatment of metronidazole refractory giardiasis with an alternative 5-nitroimidazole was suboptimal, indicating cross-resistance. Mebendazole plus secnidazole were well tolerated and effective for the treatment of 5-nitroimidazole refractory G. intestinalis infection in this setting.

Novel Quinolinyl-pyrrolo[3,4-d]pyrimidine-2,5-dione Derivatives Against Chloroquine-resistant Plasmodium falciparum.

INTRODUCTION: In this work DHPMs were combined with the quinoline nucleus to obtain new quinolinyl-pyrrolo[3,4-d]pyrimidine-2,5-dione compounds with improved antiplasmodial activity as well as decreased cytotoxicity. Nineteen quinolinyl-pyrrolo[3,4-d]pyrimidine-2,5-dione derivatives connected by a linker group to quinolone ring moieties with different substituents were synthesized and assayed against P. falciparum. MATERIALS AND METHODS: Nineteen quinolinyl-pyrrolo[3,4-d]pyrimidine-2,5-dione derivatives connected by a linker group to quinoline ring moieties with different substituents were synthesized and assayed against chloroquine-resistant Plasmodium falciparum, along with the reference drug chloroquine. Among these compounds, the derivatives with two methylene carbon spacers showed the best activity accompanied by low cytotoxicity. RESULTS: The derivative without substituents on the aromatic ring (2a) and the derivative with a chlorine group at position 4 (2d) provided the best results, with IC50 = 1.15 µM and 1.5 µM, respectively. CONCLUSION: Compared to the parent drugs, these compounds presented marked decreases in cytotoxicity, with MDL50 values over 1,000 µM and selectivity indexes of >869.5 and >666.6, respectively. The quinolinyl-pyrrolo[3,4-d]pyrimidine-2,5-dione framework appears to be promising for further studies as an antimalarial for overcoming the burden of resistance in P. falciparum.

Intravenous cyclophosphamide monthly pulses in refractory myasthenia gravis.

INTRODUCTION: Refractory myasthenia gravis (MG) is defined as a failure to respond adequately to conventional therapies, the inability to reduce immunosuppressive therapy without clinical relapse or the need for ongoing rescue therapy, severe adverse effects from immunosuppressive therapy (treatment intolerant) or frequent myasthenic crisis even on therapy. Cyclophosphamide (CYC) is a DNA alkylating agent that causes important interference in transcription processes and DNA replication, it has been used in refractory MG with controversial results. We aim to determine the efficacy of CYC in refractory MG in the Mexican population. METHODS: In an observational, longitudinal retrospective study, we identified eight refractory MG patients treated with 30-50 mg/kg monthly CYC for at least 6 months. The efficacy was assessed by Osserman scale considering significant improvement a ≥ 1 point reduction and Myasthenia Gravis Composite Scale. The relapse-free and remission-free period were also calculated using the Kaplan-Meier statistic. RESULTS: Clinical improvement was achieved in 75% of the patients. According to the Kaplan-Meier analysis, the median progression-free survival (PFS) was 9 (6.2-11.5) months and the median time to progression (TTP) was 4 (1-8) months. Response was independent of patient's characteristics, except for the MG age of onset (p = 0.0025). CONCLUSIONS: CYC was effective in all patients with refractory MG for a mean of 9 months, with worsening thereafter, which could be associated with low cumulative dose. The symptomatic improvement with CYC was noted within the 1st month. We conclude that CYC is effective as an induction to remission therapy, although our data suggest it is not effective as a long-term therapy.