Molecular variation of Plasmodium vivax dehydrofolate reductase in Mexico and Nicaragua contrasts with that occurring in South America / Variación molecular de la dehidrofolato reductasa de Plasmodium vivax en México y Nicaragua contrasta con la que ocurre en Sudamérica

Abstract: Objective: To research mutations associated to pyrimethamine resistance in dihydrofolate reductase (pvdhfr) of Plasmodium vivax from Mexico and Nicaragua and compare it to that reported in the rest of America. Materials and methods: Genomic DNA was obtained from P. vivax-infected blood samples. A pvdhfr gene fragment was amplified and sequenced. The identified gene variations were compared to those observed in other affected sites of America. Results: No mutations in pvdhfr were detected in P. vivax from Mexico and Nicaragua. One synonymous change and variation in the repeat domain was detected in Nicaraguan parasites. In South America, a high frequency of variant residues 58R and 117N associated to pyrimethamine resistance was reported. Conclusions: The lack of polymorphisms associated with pyrimethamine resistance suggests that drug-resistant P. vivax has not penetrated Mesoamerica, nor have local parasites been under selective pressure. These data contribute to establish the basis for the epidemiological surveillance of drug resistance.

Resumen: Objetivo: Determinar mutaciones en la dihydrofolato reductasa deP. vivax (Pvdhfr) en parásitos de México y Nicaragua, y comparar con lo reportado en América. Material y métodos: Del ADN de sangres infectadas con P. vivax de pacientes, el gen pvdhfr se amplifico y secuenció, y se contrastócon lo observado en América. Resultados: No se detectaron mutaciones asociadas con la resistencia debida a pirimetamina. Los parásitos de Nicaragua tuvieron una mutación sinónima y variación en la región repetida. Se reportaron frecuentes mutaciones asociadas con la resistencia a la pirimetamina en Sudamérica. Conclusiones: La ausencia de polimorfismos en Pvdhfr sugiere que no se han seleccionado ni introducido parásitos resistentes en la zona de estudio, lo que resulta muy útil para la vigilancia epidemiológica.

Alelos mutantes asociados a la resistencia a cloroquina y sulfadoxina-pirimetamina en Plasmodium falciparum de las fronteras Ecuador-Perú y Ecuador-Colombia / Mutant alleles associated to chloroquine and sulfadoxine-pyrimethanime resistance in Plasmodium falciparum of the Ecuador-Peru and Ecuador-Colombia borders

Se evaluó la frecuencia de mutaciones en los genes pfCRT y DHFR/DHPS del Plasmodium falciparum asociados a la resistencia a cloroquina y sulfadoxina-pirimetamina en 83 cepas provenientes de los distritos Esmeralda y Machala ubicados en las fronteras entre Ecuador-Perú y Ecuador-Colombia durante el año 2002. Se empleó la reacción en cadena de polimerasa (PCR) convencional y sus variantes. El gen pfCRT presentó más de 90% de muestras mutantes en Esmeralda y Machala. Para el gen DHFR, el 90% de las cepas fueron muestras mutantes en Esmeralda, tres fueron mutaciones dobles y una triple; en Machala se encontró 25% de formas mutantes simples y 75% de formas mixtas (formas silvestres/mutantes). En conclusión, la resistencia a cloroquina se ha fijado en las cepas portadoras de la mutación K76T pfCRT, mientras que la impronta genética a la resistencia a pirimetamina está en evolución, principalmente en el distrito de Esmeralda.

The frequency of mutations in pfCRT and DHFR/DHPS genes of Plasmodium falciparum associated with resistance to chloroquine and sulfadoxine-pyrimethamine was evaluated in 83 strains from the districts of Esmeralda and Machala, located on the borders of Ecuador-Peru and Ecuador-Colombia in 2002. Polymerase chain reaction (PCR), conventional and its variants, was used. Mutations in the pfCRT gene were found in more than 90% of the samples from Esmeralda and Machala. For the DHFR gene, 90% of the strains were mutant samples from Esmeralda, 3 were double mutations and 1 was a triple mutation. In Machala, 25% were simple mutant forms and 75% mixed mutant forms (wild forms/mutant). In conclusion, resistance to chloroquine has been fixed in strains carrying K76T pfCRT mutation, whereas genetic imprinting for resistance to pyrimethamine is evolving, particularly in the district of Esmeralda.

Plasmodium vivax dhfr Mutations among Isolates from Malarious Areas of Iran

The use of sulfadoxine and pyrimethamine (SP) for treatment of vivax malaria is uncommon in most malarious areas, but Plasmodium vivax isolates are exposed to SP because of mixed infections with other Plasmodium species. As P. vivax is the most prevalent species of human malaria parasites in Iran, monitoring of resistance of the parasite against the drug is necessary. In the present study, 50 blood samples of symptomatic patients were collected from 4 separated geographical regions of south-east Iran. Point mutations at residues 57, 58, 61, and 117 were detected by the PCR-RFLP method. Polymorphism at positions 58R, 117N, and 117T of P. vivax dihydrofolate reductase (Pvdhfr) gene has been found in 12%, 34%, and 2% of isolates, respectively. Mutation at residues F57 and T61 was not detected. Five distinct haplotypes of the Pvdhfr gene were demonstrated. The 2 most prevalent haplotypes were F57S58T61S117 (62%) and F57S58T61N117 (24%). Haplotypes with 3 and 4 point mutations were not found. The present study suggested that P. vivax in Iran is under the pressure of SP and the sensitivity level of the parasite to SP is diminishing and this fact must be considered in development of malaria control programs.

Prevalence of polymorphisms in DHFR, DHPS, PFMDR1 and PFCRT genes of Plasmodium falciparum isolates in Quang Tri Province, Vietnam.

In 2002 an antimalarial drug resistance survey was carried out in a seasonally endemic area of Vietnam. Sulfadoxine/pyrimethamine (S/P) was the standard treatment recommended for uncomplicated Plasmodium falciparum malaria in that area at the time. Early or late treatment failure as defined by WHO was observed in 14.9% (7/47) of patients. Molecular analysis of treatment failure isolates identified that 5/6 carried two or more dhfr and dhps polymorphisms associated with S/P resistance. Chloroquine resistance-associated polymorphisms occurred in 38.5% (15/39) of the isolates. These results support the move to artemisinin-based combination therapy for malaria in Vietnam.

In vitro study assessing the response of plasmodium falciparum malaria to chloroquine, sulfadoxine/pyrimethamine, quinine and mefloquine in Wad Medani district, Sudan

To assess the in vitro response of Plasmodium falciparum malaria to chloroquine [CQ], sulfadoxine/pyrimethamine [SDX/PYR], Quinine [QU] and Mefloquine [MQ] and monitoring their resistance. In 1999 to 2000, an in vitro study was carried out in Wad Medani district in Sudan. The standard protocol of the WHO in vitro micro-test Mark II was used for the selection of the subjects, the collection of blood samples, the culture techniques, the examination of the post-culture blood slides and the interpretation of the results. In vitro micro-test Mark II were performed on 62 Plasmodium falciparum isolates. Of these isolates, 42 produced successful schizonts growth. The data obtained showed that 29 of 42 isolates [69%] were CQ resistant with an effective concentrations [EC]; EC50 = 399.621 nM, EC90 = 2754.145 nM and EC99 = 13284.967 nM to inhibit the schizont maturation, the values of SDX/PYR showed a flat regression line as an indication of in vitro reduced response with an EC50 = 0.262 nM, EC90 = 147.390 nM and EC99 = 25722.296 nM, and the response to the QU indicated only one of the 42 isolates [2.4%] was resistant with an EC50 = 150.085 nM, EC90 = 822.825 nM and EC99 = 3293.667 nM, while all the 42 isolates were sensitive to MQ with an EC50 = 190.763 nM, EC90 = 615.125 nM and EC99 = 1597.504 nM. The results of this study revealed a high degree of in vitro resistance to CQ and reduced in vitro response to SDX/PYR and QU, while MQ was fully sensitive. The effective concentrations to inhibit 50, 90 and 99% of the parasite maturation were determined for antimalarial drugs efficacy monitoring surveillance in Sudan

A molecular epidemiologic study of point mutations for pyrimethamine-sulfadoxine resistance of Plasmodium falciparum isolates from Lao PDR.

To understand the current condition of pyrimethamine-sulfadoxine (PS) resistant falciparum malaria in Lao PDR, the frequency of point mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum were examined in 50 blood samples collected from the patients with P. falciparum infection in Southern Lao PDR. Point mutations in 5 codons of the DHFR gene, which is known to be related to pyrimethamine resistance, were detected in 15 out of the 50 samples (30%). Among the 15 samples, 10 samples showed a double mutation of codons 59 and 108 (Cys59Arg with Ser108Asn). In the remaining 5 samples, an additional mutation was observed in codon 51 (Asn51 lle), providing a triple mutation of codons 51, 59 and 108. On the other hand, point mutations in the 4 codons of DHPS gene related to sulfadoxine resistance were observed only in 2 samples (4.0%), namely in codon 437 (Ala437Gly). Only one sample showed mutations in both DHFR and DHPS genes. From the results, it should be considered that the frequency of PS resistant malaria is still low in Lao PDR. Continuous monitoring for the PS resistant malaria, however, is necessary because of the increasing use of PS in this country.

Mefloquine--its 20 years in the Thai Malaria Control Program.

Due to the deteriorating efficacy of sulfadoxine-pyrimethamine (SP or Fansidar), from the mid-1970s the Thai Malaria Control Program was actively involved in testing potential replacement drugs to be used as the primary therapy for falciparum malaria in Thailand. In 1983, a large-scale field trial of mefloquine, a long-acting antimalarial drug known for its efficacy against chloroquine- and SP-resistant Plasmodium falciparum, was initiated on the Thai-Cambodian border. The study enrolled over 60,000 patients and eventually led to the formal establishment of mefloquine as the first line drug for the treatment of uncomplicated falciparum malaria in the country. Mefloquine has played a significant role in the control of malaria in Thailand for the past two decades, initially in combination with SP, then by itself, and currently in selected areas as a partner drug in the combination therapy with artesunate. Thailand is the country with the most experience in the use of this drug in a malaria control program. We present here a review of mefloquine's pharmacology and usage in Thailand.

Genome comparison of progressively drug resistant Plasmodium falciparum lines derived from drug sensitive clone

Chloroquine has been the mainstay of malaria chemotherapy for the past five decades, but resistance is now widespread. Pyrimethamine or proguanil form an important component of some alternate drug combinations being used for treatment of uncomplicated Plasmodium falciparum infections in areas of chloroquine resistance. Both pyrimethamine and proguanil are dihydrofolate reductase (DHFR) inhibitors, the proguanil acting primarily through its major metabolite cycloguanil. Resistance to these drugs arises due to specific point mutations in the dhfr gene. Cross resistance between cycloguanil and pyrimethamine is not absolute. It is, therefore, important to investigate mutation rates in P. falciparum for pyrimethamine and proguanil so that DHFR inhibitor with less mutation rate is favored in drug combinations. Hence, we have compared mutation rates in P. falciparum genome for pyrimethamine and cycloguanil. Using erythrocytic stages of P. falciparum cultures, progressively drug resistant lines were selected in vitro and comparing their RFLP profile with a repeat sequence. Our finding suggests that pyrimethamine has higher mutation rate compared to cycloguanil. It enhances the degree of genomic polymorphism leading to diversity of natural parasite population which in turn is predisposes the parasites for faster selection of resistance to some other antimalarial drugs

Insuficiência renal aguda por sulfadiazina: relato de caso / Acute renal failutre due to sulfadiazine: a case review

Sulfadiazina e pirimetamina são drogas importantes para o tratamento da toxoplasmose cerebral. Estamos descrevendo o caso de um paciente que desenvolveu insuficiência renal aguda por precipitação de cristais de sulfadiazina e formação de cálculos renais. O diagnóstico do quadro de insuficiência renal aguda foi clínico e laboratorial, sendo importante a presença de inúmeros cristais de sulfadiazina no exame comum de urina e de cálculos renais observados na ecografia abdominal...

Azitromicina en el tratamiento de la toxoplasmosis aguda experimental / Azithromycin in the treatment of acute experimental toxoplasmosis

La toxoplasmosis es la zoonosis parasitaria más difundida en la naturaleza. Constituye una de las principales infecciones oportunistas en pacientes con SIDA. El tratamiento de primera elección es la asociación de pirimetamina y sulfadiazina, que se ve seriamente limitada por su alta frecuencia de reacciones adversas, lo que ha motivado la búsqueda de esquemas quimioterapéuticos alternativos. Se compara la eficacia de la azitromicina en el tratamiento de toxoplasmosis aguda, en relación a la asociación de pirimetamina y sulfadiazina, en un modelo de infección experimental en ratones, midiéndose la respuesta al tratamiento en días de sobrevida. La asociación de una mayor eficacia en nuestro estudio, por lo que seguiría siendo el esquema de elección en la toxoplasmosis aguda. Sin embargo, la azitromicina prolonga significativamente la sobreida de los ratones infectados con respecto al grupo de ratones no tratados

A simplified in vivo drug sensitivity test for malaria in the field.

The study was intended to develop a simple and reliable in vivo field test for monitoring of sensitivity of P.falciparum to antimalarials. The test is to be used as a built in sustainable monitoring system and applied at regular frequencies to provide guidance in developing a country-wide antimalarial drug policy. The study was conducted as a hospital based study in Mon State in Mudon, Kamawet and Pa-auk hospitals. The criteria matched malaria patients were treated with standard dosages of chloroquine, sulfadoxine-pyrimethamine and mefloquine and blood films were taken on days 0, 2, 3, 4, 7, 14 and 28. The assessment of the in vivo drug response of P.falciparum on days 2, 3 and 4 were compared with WHO standard 28 days and 7 day tests. The following successful tests were carried out for 7 days with different antimalarials: 171 tests with chloroquine and sulfadoxine-pyrimethamine and 167 tests with mefloquine. Tests were also carried out for 28 days: 59 tests with chloroquine, 77 tests with sulfadoxine-pyrimethamine and 78 tests with mefloquine. The results found that 3 day tests, taking blood films on days 0 and 3, can be reliably used as an adjunct to 28-day tests. Since the test is simple and can be used extensively and sustainably throughout the country and the results are applicable to be used for epidemiological purposes, the method is suggested for use as a built-in monitoring method for the malaria control program.

Efficacy of sulfadoxine-pyrimethamine in chloroquine resistant falciparum malaria in Bombay.

Several reports have confirmed the existence of chloroquine resistant Plasmodium falciparum malaria in Bombay. In the management of these cases, of the therapeutic options available, sulfadoxine-pyrimethamine (SP) (administered as a single dose) is preferred, since quinine has to be administered over a period of 7 days while mefloquine is yet to be marketed in India. With increasing reports of falciparum malaria resistant to SP from Thailand and Africa, the present study was conducted to determine the efficacy of SP in the treatment of chloroquine resistant falciparum malaria cases in Bombay. 17 uncomplicated falciparum malaria patients documented to be resistant to chloroquine (10 RI, 5 RII and 2 RIII) by the WHO (1973) in-vivo extended field test criteria and by estimation of plasma chloroquine levels by High Performance Liquid Chromatography were included in the study. These adult patients were then treated with a supervised single dose of 3 tablets of SP and peripheral blood smears were then repeated on days 3, 4, 5, 7, 14, 21, 28, 35 and 42 after treatment. 14 patients responded and were sensitive while 3 patients showed RII grade resistance to SP. These 3 patients then responded to a 7 day course of quinine (30 mg/kg/day)+doxycycline (100 mg/day). These results thus document that SP can be used as an effective second-line antimalarial drug. However one should monitor the patient for plasmodial resistance to sulfadoxine-pyrimethamine.

In vivo study of the response of Plasmodium falciparum to standard mefloquine/sulfadoxine/pyrimethamine (MSP) treatment among gem miners returning from Cambodia.

An in vivo study of the response of P. falciparum to the combination drug, MSP, was conducted among gem miners who contracted malaria from Cambodia in 1991-1992. High level resistance (RII, RIII responses) was observed in 22.5% of the 40 cases attending Mae Sot malaria clinic, west Thailand border, and in 28.1% of the 96 cases attending Bo Rai malaria clinic, east Thailand border. The observations on in vitro studies conducted prior to the MSP treatment and after recrudescence, together with the findings on adequate mefloquine blood levels strongly indicated the serious deterioration of mefloquine efficacy. The first line treatment for the malaria control program needs to be revised and the use of qinghaosu derivatives considered. Intensive measures to combat spreading of the highly resistant strains to other parts of the country should be taken into account.

El uso de la mefloquina en un grupo de pacientes resistentes al tratamiento con fansidar / The use of mefloquine in a group of patients with falciparum malaria resistant to treatment with Fansider

Se trataron con mefloquina, a una dosis de 25 mg/kg de peso repartida en 3 dosis, 7 pacientes cubanos procedentes de la República de Angola (RA) con el diagnóstico de paludismo por Plasmodium falciparum. La parasitemia detectada varió entre 40 y 15 000 parásitos x mm3 de sangre (media 3 453 parásitos x mm3), lo indico una infecciópn ligera. Todos los pacientes tenían como antecedentes proceder de zonas donde se había reportado la resistencia del Plasmodium falciparum a la cloroquina y habían sido tratados con 3 tabletas de fansidar (sulfadoxina + pirimetamina) al salir del área endémica y al llegar a Cuba. En todos los pacientes los análisis de gota gruesa al momento de su llegada a Cuba fueron negativos. Entre 7 y 28 días después de su arribo fueron detectados como positivos al paludismo por Plasmodium falciparum en los análisis de gota gruesa realizados. Después del tratamiento con mefloquina la parasitemia desapareció antes del quinto día sin recrudecimiento subsiguiente durante 2 años de seguimiento. Se consideró la mefloquina como un medicamento eficaz en el tratamiento del paludismo por Plasmodium falciparum

A 5-year surveillance of sensitivity in vivo of Plasmodium falciparum to pyronaridine/sulfadoxine/pyrimethamine in Diaoluo area, Hainan Province.

The surveillance of sensitivity of P. falciparum to pyronaridine/sulfadoxine/pyrimethamine has been carried out in Diaoluo area in Hainan Province where chloroquine-resistant falciparum malaria is endemic, covering an area of 406 square kilometers, with a population of 3745 in 1986. From 1986 all outpatients diagnosed as falciparum malaria were administered with PND/S/P as the only antimalarial. In vivo sensitivity of P. falciparum was measured in some patients who were treated in hospital. It was demonstrated that P. falciparum in the Diaoluo area has retained its sensitivity to a single oral dose of PND/S/P of 500/1,000/50 mg with 100% cure rate for at least 5 years.

In vitro sensitivity of southern African reference isolates of Plasmodium falciparum to chloroquine and pyrimethamine

The in vitro sensitivity to chloroquine and pyrimethamine of 19 culture-adapted southern African reference isolates of Plasmodium falciparum was determined using a 48-hour assay. Four isolates collected in KwaZulu, Natal, were sensitive to chloroquine, and one of these was sensitive to the drug in vivo. Eight isolates from KwaZulu or Mozambique were resistant to chloroquine in vitro. Six of these isolates were chloroquine-resistant in varying degrees in vivo. Four of five isolates from north-eastern Transvaal and two clinically chloroquine-resistant Malawian isolates were resistant to chloroquine in vitro. A wide range of pyrimethamine susceptibilities was detected (0.01 mumol/l to greater than 3.0 mumol/l), although most isolates were inhibited at 0.1 mumol/l, indicating a low level of resistance. These results confirm the presence of both chloroquine and pyrimethamine resistance in the endemic areas of South Africa. This has serious implications for the prophylaxis and treatment of P. falciparum malaria in South Africa

Sensitivity of Plasmodium falciparum to chloroquine and sulfadoxine/pyrimethamine in Nigerian children

The in vivo sensitivity of Plasmodium falciparum to chloroquine and sulfadoxine/pyrimethamine was evaluated in children under 5 years of age in two areas of southern Nigeria in 1987. A modification of the WHO Standard Field and Extended Tests (in vivo) was used, with follow-up on days, 2, 3, 7, and 14 after treatment with 25 mg chloroquine per kg body weight given over 3 days, or with standard doses of sulfadoxine/pyrimethamine. Clinical and parasitological evaluations were performed. At Igbo Ora, in Oyo State, where by day 7 chloroquine was clinically successful in 94.4% of 36 children and sulfadoxine/pyrimethamine in 91.7% of 36 children, there were no parasitological failures in either treatment group. Fever regressed significantly more rapidly with chloroquine than with sulfadoxine/pyrimethamine. At Oban, in Cross River State, initial parasite densities decreased markedly with the chloroquine regimen but 63.6% of 44 children were parasitological failures on days 3, 7, or 14; and all of the 26 children who failed parasitologically and completed follow-up were successfully treated with sulfadoxine/pyrimethamine. By day 7, clinical success was demonstrated for 77.3% of the children treated with chloroquine. The in vitro sensitivity to chloroquine, quinine, and mefloquine at Igbo Ora indicated that isolates of P. falciparum were sensitive to chloroquine and quinine, but had reduced sensitivity to mefloquine. Because of its continued clinical efficacy, chloroquine remains the recommended treatment for children with uncomplicated malaria in Nigeria. Health providers are, however, encouraged to maintain supplies of sulfadoxine/pyrimethamine as an alternative and to refer patients promptly if necessary

Epidemiology of emergence and spread of drug-resistant falciparum malaria in Southeast Asia.

In Southeast Asia the medicated salt project of Pailin, on the Kampuchea-Thai border, demonstrated that drug resistance, especially chloroquine resistance, can develop when a large population of P. falciparum parasites is exposed to intense transmission under intense drug pressure. The selection of resistant parasites being activated by the introduction of non-immune groups. Emergence of drug resistance was the result of continuous and prolonged mass exposure of P. falciparum to pyrimethamine and chloroquine resulting in the selection of resistant mutants. This selection was associated with multiple exposures of the parasites to much higher drug doses, during repeated passages through the non-immune hosts, increasing the degree of resistance. Resistances spread to the receptive areas of Kampuchea and other neighbouring countries through the movements of the temporary migrants who, by then, had become carriers infected with drug resistant falciparum parasites. The rapid and early spread of chloroquine resistance in A. balabacensis areas was not a coincidence but the result of the biological advantages of this species complex in relation to malaria transmission. In Australasia the medicated salt project carried out in Irian Jaya, on the border with Papua New Guinea, also resulted in the development of drug resistance in P. falciparum.

Effects of mycobacterium bovis BCG, bacterial lipopolysaccharide and hydrocortisone on the development of immunity to Plasmodium berghei

Mycobacterium bovis (BCG) aumenta significantemente o desenvolvimento da imunidade nos camundongos CFW, C57BL/6, C57BL/10ScN e BALB/c (Nu/+) para os estágios eritrocitos do Plasmodium berghei. Camundongos tratados com BCG requerem menos ciclos de infecçäo com P. berghei e cura pelo Fansidar (pirimetamina + sulfadoxina) para desenvolverem imunidade sólida a este parasita do que os controles. Contudo, os animais que receberam BCG 30 dias antes do início da imunizaçäo evidenciaram uma perda precoce da imunidade adquirida para o P. berghei, quando comparado com os animais que receberam BCG 14 dias antes ou que näo receberam BCG. Assim, sendo, o BCG aumenta a induçäo na resposta imune do hospedeiro ao P. berghei no curso de infecçöes subseqüentes. O tratamento de camundongos CFW, BALB/c e C57BL/6 com lipopolissacarídeo bacteriano ou hidrocortisona faz com que os animais requeiram um número maior de ciclos de infecçäo e cura para tornarem-se imunes ao P. berghei que os controles. O tratamento dos camundongos C57BL/10ScN com hidrocortisona aboliu completamente a sua habilidade de sobrevida subseqüente a ciclos de infecçäo com P. berghei e cura pelo Fansidar