Molecular prevalence of Merkel cell polyomavirus in nonmelanoma skin cancer in a Brazilian population.

BACKGROUND: Merkel cell polyomavirus (MCPyV), a newly described oncogenic virus, has been found in association with tumours other than Merkel cell carcinoma (MCC). As yet, little is known about the involvement or influence of MCPyV on the development of these tumours and its prevalence in various populations. AIM: To assess the prevalence of MCPyV DNA in cases of nonmelanoma skin cancer (NMSC). METHODS: The prevalence of MCPyV DNA was assessed in 96 cases of NMSC in a Brazilian population comprising 76 subjects, and these results were correlated with epidemiological and demographical data. RESULTS: MCPyV DNA was detected in 23 of 69 (33.3%) basal cell carcinomas, in 2 of 11 (18%) squamous cell carcinomas, 2 of 4 Bowen disease case, 0 of 1 MCC and 4 of 11 other skin disorders. CONCLUSION: Despite the frequent detection of MCPyV DNA in NMSC, its possible role in the development of NMSC still needs further investigation.

Plasmodium falciparum: limited genetic diversity of MSP-2 in isolates circulating in Brazilian endemic areas.

The genetic polymorphism of the surface merozoite protein 2 (MSP-2) was evaluated in Plasmodium falciparum isolates from individuals with uncomplicated malaria living in a Brazilian endemic area of Peixoto de Azevedo. The frequency of MSP-2 alleles and the survival of genetically different populations clones in 104 isolates were verified by Southern blot and SSCP-PCR. Single and mixed infections were observed in similar frequencies and the rate of detection of FC27 and 3D7 allelic families was equivalent. Eight alleles were identified and among them, the sequence polymorphism was mainly attributed to variations in the repetitive region. Interestingly, in three alleles nucleotide polymorphism was identical to that detected in a previous study, conducted in 1992, in a near Brazilian endemic area. This finding demonstrated the genetic similarity between two isolate groups, besides the certain temporal stability in the allelic patterns. The implications of these data for studies on the genetic diversity are also discussed.

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase of isolates from the Amazon Region of Brazil

Since the late 1970s pyrimethamine-sulfadoxine (PS; FansidarTM Hoffman-LaRoche, Basel) has been used as first line therapy for uncomplicated malaria in the Amazon basin. Unfortunately, resistance has developed over the last ten years in many regions of the Amazon and PS is no longer recommended for use in Brazil. In vitro resistance to pyrimethamine and cycloguanil (the active metabolite of proguanil) is caused by specific point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR), and in vitro resistance to sulfadoxine has been associated with mutations in dihydropteroate synthase (DHPS). In association with a proguanil-sulfamethoxazole clinical trial in Brazil, we performed a nested mutation-specific polymerase chain reaction to measure the prevalence of DHFR mutations at codons 50, 51, 59, 108 and 164 and DHPS mutations at codons 436, 437, 540, 581 and 613 at three sites in the Brazilian Amazon. Samples from two isolated towns showed a high degree of homogeneity, with the DHFR Arg-50/Ile-51/Asn-108 and DHPS Gly-437/Glu-540/Gly-581 mutant genotype accounting for all infections in Peixoto de Azevedo (n = 15) and 60 percent of infections in Apiacás (n = 10), State of Mato Grosso. The remaining infections in Apiacás differed from this predominant genotype only by the addition of the Bolivia repeat at codon 30 and the Leu-164 mutation in DHFR. By contrast, 17 samples from Porto Velho, capital city of the State of Rondônia, with much in- and out-migration, showed a wide variety of DHFR and DHPS genotypes.

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase of isolates from the Amazon region of Brazil.

Since the late 1970s pyrimethamine-sulfadoxine (PS; FansidarTM Hoffman-LaRoche, Basel) has been used as first line therapy for uncomplicated malaria in the Amazon basin. Unfortunately, resistance has developed over the last ten years in many regions of the Amazon and PS is no longer recommended for use in Brazil. In vitro resistance to pyrimethamine and cycloguanil (the active metabolite of proguanil) is caused by specific point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR), and in vitro resistance to sulfadoxine has been associated with mutations in dihydropteroate synthase (DHPS). In association with a proguanil-sulfamethoxazole clinical trial in Brazil, we performed a nested mutation-specific polymerase chain reaction to measure the prevalence of DHFR mutations at codons 50, 51, 59, 108 and 164 and DHPS mutations at codons 436, 437, 540, 581 and 613 at three sites in the Brazilian Amazon. Samples from two isolated towns showed a high degree of homogeneity, with the DHFR Arg-50/Ile-51/Asn-108 and DHPS Gly-437/Glu-540/Gly-581 mutant genotype accounting for all infections in Peixoto de Azevedo (n = 15) and 60% of infections in Apiacás (n = 10), State of Mato Grosso. The remaining infections in Apiacás differed from this predominant genotype only by the addition of the Bolivia repeat at codon 30 and the Leu-164 mutation in DHFR. By contrast, 17 samples from Porto Velho, capital city of the State of Rondônia, with much in- and out-migration, showed a wide variety of DHFR and DHPS genotypes.

Antimalarial drug susceptibility testing of Plasmodium falciparum in Brazil using a radioisotope method.

From March 1996 to August 1997, a study was carried out in a malaria endemic area of the Brazilian Amazon region. In vivo sensitivity evaluation to antimalarial drugs was performed in 129 patients. Blood samples (0.5 ml) were drawn from each patient and cryopreserved to proceed to in vitro studies. In vitro sensitivity evaluation performed using a radioisotope method was carried out with the cryopreserved samples from September to December 1997. Thirty-one samples were tested for chloroquine, mefloquine, halofantrine, quinine, arteether and atovaquone. Resistance was evidenced in 96.6% (29/30) of the samples tested for chloroquine, 3. 3% (1/30) for quinine, none (0/30) for mefloquine and none for halofantrine (0/30). Overall low sensitivity was evidenced in 10% of the samples tested for quinine, 22.5% tested for halofantrine and in 20% tested for mefloquine. Means of IC 50 values were 132.2 (SD: 46. 5) ng/ml for chloroquine, 130.6 (SD: 49.6) ng/ml for quinine, 3.4 (SD: 1.3) ng/ml for mefloquine, 0.7 (SD: 0.3) ng/ml for halofantrine, 1 (SD: 0.6) ng/ml for arteether and 0.4 (SD: 0.2) ng/ml for atovaquone. Means of chloroquine IC 50 of the tested samples were comparable to that of the chloroquine-resistant strain W2 (137.57 ng/ml) and nearly nine times higher than that of the chloroquine-sensitive strain D6 (15.09 ng/ml). Means of quinine IC 50 of the tested samples were 1.7 times higher than that of the low sensitivity strain W2 (74.84 ng/ml) and nearly five times higher than that of the quinine-sensitive strain D6 (27.53 ng/ml). These results disclose in vitro high resistance levels to chloroquine, low sensitivity to quinine and evidence of decreasing sensitivity to mefloquine and halofantrine in the area under evaluation.

Characterization of Plasmodium falciparum isolated from the Amazon region of Brazil: evidence for quinine resistance.

The prevalence and severity of drug-resistant malaria is emerging rapidly in the Amazon basin of Brazil. In support of clinical trials using the new antimalarial drug combination of atovaquone and proguanil, we performed in vitro drug sensitivities, molecular characterization of parasite populations using the circumsporozoite protein, merozoite surface antigen-1 (MSA-1), and MSA-2 markers, and an analysis of the Plasmodium falciparum multidrug resistance (pfmdr1) gene sequence and copy number in 26 isolates of P. falciparum obtained in a gold-mining endemic area in Peixoto de Azevedo, Mato Grosso State. All 26 isolates were found to be resistant to chloroquine (50% inhibitory concentration [IC50] = 100-620 nM) and sensitive to mefloquine (IC50 < 23 nM) and halofantrine (IC50 < 6 nM). The isolates also show reduced susceptibility to quinine (IC50 = 48-280 nM). Sequence analysis of the pfmdr1 gene revealed Asn, Phe, Cys, Asp, and Tyr in positions 86, 184, 1034, 1042, and 1246, respectively. These point mutations were similar to that previously described in other Brazilian isolates. Southern blot analysis revealed no amplification of the pfmdr1 gene. These results suggest that three different mechanisms for drug resistance exist for chloroquine, mefloquine, and quinine.

Malaria diagnosis: standardization of a polymerase chain reaction for the detection of Plasmodium falciparum parasites in individuals with low-grade parasitemia.

In Brazil, no study has been done concerning the detection of malaria parasites by polymerase chain reaction (PCR) related to the diagnosis of Plasmodium falciparum malaria. In the present report we describe a highly sensitive methodology for malaria diagnosis using a nested PCR method based on amplification of the p126 P. falciparum gene detected by simple ethidium bromide staining. The P. falciparum Palo Alto strain (culture samples) was serially diluted in blood from an uninfected donor to a final level of parasitemia corresponding to 10(-8)% and was processed for PCR amplification. In each of these dilutions a parasitological examination was performed to compare the sensitivity with that of PCR amplification. Blood samples (field samples) were obtained from 51 malarious patients with positive thick blood smears (TBS) who were living in endemic regions of the Brazilian Amazon. They corresponded to 42 P. falciparum and 9 P. vivax cases, with parasitemia levels ranging from only 16 to 20,200 parasites/microliter for P. falciparum disease and from 114 to 11,000 parasites/microliter for P. vivax malaria. We demonstrate that the use of nested PCR allows the detection of 0.005 parasites/microliter without the use of radioactive material. The use of a 1-ml sample volume and the organic DNA extraction method should be suitable in blood banks and for the evaluation of patients during and after drug treatment.

The art of parasite survival.

Parasites develop and survive in an environment which is often hostile to them. When facing aggressive conditions parasites are able to use various and complex strategies. Echinococcus granulosus, Toxocara canis, Pneumocystis carinii, Entamoeba or Toxoplasma gondii are able to seclude from the environment when stressed by surrounding (immunologic or non-immunologic) aggressive factors. Specific antigens which exert a functional activity during a short period of time appear to be concealed from the immune attack at this crucial moment. This is the case for rhoptry or dense granule antigens of Plasmodium or Toxoplasma sporozoa involved in the formation of the parasitophorous vacuole which are released in a space perfectly isolated from the outside and therefore from antibodies. Some parasites like Schistosoma mansoni or Trypanosoma brucei reveal an amazing opportunistic behavior when they use cytokines of host origin induced by the infectious process for their own development. Leishmania, Toxoplasma and Trypanosoma cruzi are able to invade immunologically competent macrophages and to avoid the triggering of killing mechanisms of these cells. Parasites also take advantage of the genetic restriction of the immune response and it has been observed for Plasmodia that some high molecular weight antigens are unable to induce an immune response in particular strains of mice. Parasite receptors involved in the invasion of host cells by parasites can function in the presence of antibodies which can explain the failure of vaccination attempts targeting this type of molecules. Among the mechanisms developed by parasites to resist to drugs it appears that transmembrane transporters described in many protozoa or helminth parasites could play a role. Moreover, the description of parasite-specific enzymes able to protect them against the damaging effects of oxygen radicals suggests that parasites are potentially able to develop a resistance phenomenon against drugs acting via an oxidative burst.

The art of parasite survival

Parasites develop and survive in an environment which is often hostile to them. When facing aggressive conditions parasites are able to use various and complex strategies. Echinococcus granulosus, Toxocara canis, Pneumocystis carinii, Entamoeba or Toxoplasma gandii are able to seclude from the environment when stressed by surrounding (immunologic or non-immunologic) agressive factors. Specific antigens which exert a functional activity during a short period of time appear to be concealed from the immune attack at this crucial moment. This is the case for rhoptry or dense granule antigens of Plasmodium or Toxoplasma sporozoa involved in the formation of the parasitophorous vacuole which are released in a space perfectly isolated from the outside and therefore from antibodies. Some parasites like Schistosoma mansoni or Trypanosoma brucei reveal an amazing opportunistic behavior when they use cytokines of host origin induced by the infectious process for their own development. Leishmania, Toxoplasma and Trypanosoma cruzi are able to invade immunologically competent macrophages and to avoid the triggering of killing mechanisms of these cells. Parasites also take advantage of the genetic restriction of the immune response and it has been observed for Plasmodia that some high molecular weight antigens are unable to induce an immune response in particular strains of mice. Parasite receptors involved in the invasion of host cells by parasites can function in the presence of antibodies which can explain the failure of vaccination attempts targeting this type of molecules. Among the mechanisms developed by parasites to resist to drugs it appears that transmembrane transporters described in many protozoa or helminth parasites could play a role. Moreover, the description of parasite-specific enzymes able to protect them against the damaging effects of oxygen radicals suggests that parasites are potentially able to develop a resistance phenomenon against drugs acting via an oxidative burst