Mixed Plasmodium falciparum infections and its clinical implications in four areas of the Brazilian Amazon region.

The aim of this study was to assess the prevalence pattern of mixed-Plasmodium falciparum malaria infections in Brazil by molecular diagnosis and to address its clinically important features. DNA was extracted from 115 thick blood film P. falciparum human blood positive samples using the phenol-chloroform method, followed by a semi-nested PCR protocol with species-specific primers. Seventy-three percent of P. falciparum single infections and 26.95% of mixed infections were found. Amongst mixed infections, the majority was double infection (96.77%). Our results suggest that the prevalence of one species over the other can be important on weakening P. falciparum malaria clinical symptoms. We confirm that P. falciparum co-infections frequently occur in Brazilian malaria endemic areas, with underestimated diagnosis. The results point to the need of improving microscopy or changing for another accurate diagnosis technique to differentiate among human malaria species, as this is essential to choose the best treatment and control measure for malaria. More investigations are necessary in order to clarify the role of mixed-infections in the severity of P. falciparum disease.

A new polymerase chain reaction/restriction fragment length polymorphism protocol for Plasmodium vivax circumsporozoite protein genotype (VK210, VK247, and P. vivax-like) determination.

For the molecular diagnosis of Plasmodium vivax variants (VK210, VK247, and P. vivax-like) using DNA amplification procedures in the laboratory, the choice of rapid and inexpensive identification products of the 3 different genotypes is an important prerequisite. We report here the standardization of a new polymerase chain reaction/restriction fragment length polymorphism technique to identify the 3 described P. vivax circumsporozoite protein (CSP) variants using amplification of the central immunodominant region of the CSP gene of this protozoan. The simplicity, specificity, and sensitivity of the system described here is important to determine the prevalence and the distribution of infection with these P. vivax genotypes in endemic and nonendemic malaria areas, enabling a better understanding of their phylogeny.

Bacterial, yeast, parasitic, and viral enteropathogens in HIV-infected children from São Paulo State, Southeastern Brazil.

We present here the frequency of enteropathogens in an HIV-infected children group and investigate their correlation with clinical and sociodemographic characteristics by collecting 100 stool samples from 55 HIV-seropositive Brazilian children. All specimens were processed according to standard methods for bacterial and yeast detection. A commercially available enzyme-linked immunosorbent assay was used to detect protozoan, and to perform virus detection, molecular tests were applied. Consumption of raw vegetables and fruits and severe immunosuppression were significantly associated with diarrhea. Cryptosporidium parvum was the commonest enteropathogen, followed by Candida albicans, enteropathogenic Escherichia coli, and astrovirus. The number of potential pathogenic agents identified in fecal specimens in asymptomatic HIV-seropositive infants is high, which raises the need for additional investigation in this area as well as in other Brazilian regions.

Plasmodium vivax infection among Duffy antigen-negative individuals from the Brazilian Amazon region: an exception?

We present evidence for Plasmodium vivax infection among Duffy blood group-negative inhabitants of Brazil. The P. vivax identification was determined by both genotypic and non-genotypic screening tests. The Duffy blood group was genotyped by PCR/RFLP and phenotyped using a microtyping kit. We detected two homozygous FY*B-33 carriers infected by P. vivax, whose circumsporozoite protein genotypes were VK210 and/or P. vivax-like. Additional efforts are necessary in order to clarify the evidence that P. vivax is being transmitted among Duffy blood group-negative patients from the Brazilian Amazon region.

Molecular screening of Plasmodium sp. asymptomatic carriers among transfusion centers from Brazilian Amazon region.

The transmission of malaria in Brazil is heterogeneous throughout endemic areas and the presence of asymptomatic Plasmodium sp. carriers (APCs) in the Brazilian Amazon has already been demonstrated. Malaria screening in blood banks is based on the selection of donors in respect to possible risks associated with travel or residence, clinical evidence and/or inaccurate diagnostic methods thereby increasing the probability of transfusion-transmitted infection. We evaluated the frequency of APCs in four blood services in distinct areas of the Brazilian Amazon region. DNA was obtained from 400 human blood samples for testing using the phenol-chloroform method followed by a nested-PCR protocol with species-specific primers. The positivity rate varied from 1 to 3% of blood donors from the four areas with an average of 2.3%. All positive individuals had mixed infections for Plasmodium vivax and Plasmodium falciparum. No significant differences in the results were detected among these areas; the majority of cases originated from the transfusion centres of Porto Velho, Rondônia State and Macapá, Amapá State. Although it is still unclear whether APC individuals may act as reservoirs of the parasite, efficient screening of APCs and malaria patients in Brazilian blood services from endemic areas needs to be improved.

[Evaluation of the influence of GSTT1 and GSTM1 null genotypes in head and neck carcinogenesis]. / Avaliação da influência da nulidade dos genótipos GSTT1 e GSTM1 na carcinogênese em cabeça e pescoço.

BACKGROUND: To evaluate the influence of GSTM1 and GSTT1 null genotypes in head and neck carcinogenesis. METHODS: The frequencies of GSTM1 and GSTT1 null genotypes were evaluated by multiplex polymerase chain reaction (PCR) in 45 patients with head and neck squamous cell carcinoma and in 45 control group individuals. Both groups were composed of smokers paired by gender, age and race. RESULTS: The GSTT1 null genotype was found in 24.4% of the patients and 17.7% of the control group (P= 0.606), while 44.4% of the patients and 48.8% of the control group were bearers of the GSTM1 null genotype (P=0.832). No associations between GSTT1 and GSTMI null genotypes and primary tumor sites were found. CONCLUSION: In our study, it was impossible to establish the influence of the GSTT1 and GSTM1 null genotypes in head and neck carcinogenesis.

GSTT1 and GSTM1 polymorphism in cigarette smokers with head and neck squamous cell carcinoma.

UNLABELLED: Gene variability related to carcinogen activation and detoxification may interfere with susceptibility to head and neck cancer. AIM: To investigate the relation between GSTT1 and GSTM1 null polymorphisms and the risk of head and neck squamous cell carcinoma in cigarette smokers. MATERIAL AND METHOD: A case-control study conducted at the Sao Jose do Rio Preto Medical School, Brazil. GSTM1 and GSTT1 null genotype frequencies were evaluated by multiplex PCR in 45 cigarette smokers with head and neck squamous cell carcinomas and 45 cigarette smokers without this disease. RESULTS: The oral cavity was the most prevalent tumor site for squamous cell carcinoma. The GSTT1 null genotype was found in 33.3% of the Experimental Group and 23.3% of the Control Group (p= 0.311). Experimental and Control Groups had GSTM1 null genotype frequencies of 35% and 48.3% (p=0.582). No association between alcohol consumption and GSTT1 and GSTMI null genotypes was found in these groups (p-values>0.05). There were more men, and alcohol consumption was prevalent in both groups. CONCLUSION: In this study we were unable to show a correlation between GSTM1 and GSTT1 genotypes and the development of head and neck squamous cell carcinomas in cigarette smokers.

Polymorphisms of DNA repair genes XRCC1 and XRCC3, interaction with environmental exposure and risk of chronic gastritis and gastric cancer.

AIM: To evaluate the association between polymorphisms XRCC1 Arg194Trp and Arg399Gln and XRCC3 Thr241Met and the risk for chronic gastritis and gastric cancer, in a Southeastern Brazilian population. METHODS: Genotyping by PCR-RFLP was carried out on 202 patients with chronic gastritis (CG) and 160 patients with gastric cancer (GC), matched to 202 (C1) and 150 (C2) controls, respectively. RESULTS: No differences were observed among the studied groups with regard to the genotype distribution of XRCC1 codons 194 and 399 and of XRCC3 codon 241. However, the combined analyses of the three variant alleles (194Trp, 399Gln and 241Met) showed an increased risk for chronic gastritis when compared to the GC group. Moreover, an interaction between the polymorphic alleles and demographic and environmental factors was observed in the CG and GC groups. XRCC1 194Trp was associated with smoking in the CG group, while the variant alleles XRCC1 399Gln and XRCC3 241Met were related with gender, smoking, drinking and H pylori infection in the CG and GC groups. CONCLUSION: Our results showed no evidence of a relationship between the polymorphisms XRCC1 Arg194Trp and Arg399Gln and XRCC3 Thr241Met and the risk of chronic gastritis and gastric cancer in the Brazilian population, but the combined effect of these variants may interact to increase the risk for chronic gastritis, considered a premalignant lesion. Our data also indicate a gene-environment interaction in the susceptibility to chronic gastritis and gastric cancer.

Performance of an immunoenzymatic assay for Cryptosporidium diagnosis of fecal samples.

We evaluated the diagnostic performance of a Cryptosporidium immunoenzymatic assay (ELISA). Fecal samples were collected from 94 HIV-seropositive patients. All specimens were processed with a commercially-available ELISA to detect C. parvum specific coproantigen and with a modified Ziehl-Neelsen stain (ZNm) microscope exam. Overall, sensitivity of the immunoenzymatic test was 100%, with a specificity of 96%; positive and negative predictive values were 89% and 100%, respectively. The commercial ELISA and ZNm proved to be valuable diagnostic tools for Cryptosporidium infection.

Evaluation of an immunochromatography test for malaria diagnosis under different storage conditions.

This study aimed to evaluate the second-generation OptiMal test for malaria diagnosis under various storage conditions. It detected all the positive samples, except for two Plasmodium malariae samples. Further research evaluating diverse environmental conditions are important for ICT test applicability in Brazilian malaria areas.

Enteropathogens detected in a daycare center, Southeastern Brazil: bacteria, virus, and parasite research.

INTRODUCTION: The objective of this study was to determine the prevalence and etiological profile of enteropathogens in children from a daycare center. METHODS: From October 2010 to February 2011 stool samples from 100 children enrolled in a government daycare center in the municipality of São José do Rio Preto, in the state of São Paulo, were collected and analyzed. RESULTS: A total of 246 bacteria were isolated in 99% of the fecal samples; 129 were in the diarrheal group and 117 in the non-diarrheal group. Seventy-three strains of Escherichia coli were isolated, 19 of Enterobacter, one of Alcaligenes and one of Proteus. There were 14 cases of mixed colonization with Enterobacter and E. coli. Norovirus and Astrovirus were detected in children with clinical signs suggestive of diarrhea. These viruses were detected exclusively among children residing in urban areas. All fecal samples were negative for the presence of the rotavirus species A and C. The presence of Giardia lamblia, Entamoeba coli, Endolimax nana and hookworm was observed. A significant association was found between food consumption outside home and daycare center and the presence of intestinal parasites. CONCLUSIONS: For children of this daycare center, intestinal infection due to pathogens does not seem to have contributed to the occurrence of diarrhea or other intestinal symptoms. The observed differences may be due to the wide diversity of geographical, social and economic characteristics and the climate of Brazil, all of which have been reported as critical factors in the modulation of the frequency of different enteropathogens.

GSTT1, GSTM1 and CYP2E1 genetic polymorphisms in gastric cancer and chronic gastritis in a Brazilian population.

AIM: To test the hypothesis that, in the Southeastern Brazilian population, the GSTT1, GSTM1 and CYP2E1 polymorphisms and putative risk factors are associated with an increased risk for gastric cancer. METHODS: We conducted a study on 100 cases of gastric cancer (GC), 100 cases of chronic gastritis (CG), and 150 controls (C). Deletion of the GSTT1 and GSTM1 genes was assessed by multiplex PCR. CYP2E1/PstI genotyping was performed using a PCR-RFLP assay. RESULTS: No relationship between GSTT1/GSTM1 deletion and the c1/c2 genotype of CYP2E1 was observed among the three groups. However, a significant difference between CG and C was observed, due to a greater number of GSTT1/GSTM1 positive genotypes in the CG group. The GSTT1 null genotype occurred more frequently in Negroid subjects, and the GSTM1 null genotype in Caucasians, while the GSTM1 positive genotype was observed mainly in individuals with chronic gastritis infected with H pylori. CONCLUSION: Our findings indicate that there is no obvious relationship between the GSTT1, GSTM1 and CYP2E1 polymorphisms and gastric cancer.

Detection of Mycobacterium tuberculosis complex by nested polymerase chain reaction in pulmonary and extrapulmonary specimens.

OBJECTIVE: To compare the performance of nested polymerase chain reaction (NPCR) with that of cultures in the detection of the Mycobacterium tuberculosis complex in pulmonary and extrapulmonary specimens. METHODS: We analyzed 20 and 78 pulmonary and extrapulmonary specimens, respectively, of 67 hospitalized patients suspected of having tuberculosis. An automated microbial system was used for the identification of Mycobacterium spp. cultures, and M. tuberculosis IS6110 was used as the target sequence in the NPCR. The kappa statistic was used in order to assess the level of agreement among the results. RESULTS: Among the 67 patients, 6 and 5, respectively, were diagnosed with pulmonary and extrapulmonary tuberculosis, and the NPCR was positive in all of the cases. Among the 98 clinical specimens, smear microscopy, culture, and NPCR were positive in 6.00%, 8.16%, and 13.26%, respectively. Comparing the results of NPCR with those of cultures (the gold standard), we found that NPCR had a sensitivity and specificity of 100% and 83%, respectively, in pulmonary specimens, compared with 83% and 96%, respectively, in extrapulmonary specimens, with good concordance between the tests (kappa, 0.50 and 0.6867, respectively). CONCLUSIONS: Although NPCR proved to be a very useful tool for the detection of M. tuberculosis complex, clinical, epidemiological, and other laboratory data should also be considered in the diagnosis and treatment of pulmonary and extrapulmonary tuberculosis.

Influence of HLA-DRB-1 alleles on the production of antibody against CSP, MSP-1, AMA-1, and DBP in Brazilian individuals naturally infected with Plasmodium vivax.

We evaluated the influence of allelic frequency of the human leukocyte antigen (HLA) -DRB1 on the acquisition of antibody response against malaria sporozoite and merozoite peptides in patients with Plasmodium vivax malaria acquired in endemic areas of Brazil. IgG antibodies were detected by enzyme-linked immunosorbent assay against four peptides of circumsporozoite protein (CSP) (amino, carboxyl, and VK210 and VK247 repeats) and peptides of merozoite surface protein 1 (MSP-1), apical membrane antigen 1 (AMA-1), and Duffy-binding protein (DBP). We found an association between HLA-DR3 and HLA-DR5 alleles and lack of antibody response to CSP amino terminal, as well as an association between HLA-DR3 and the highest antibody response to MSP1 (Pv200L). In conclusion, we suggest a potential regulatory role of the HLA-DRB1 alleles in the production of antibodies to a conserved region of P. vivax CSP and MSP1 in Brazilian population exposed to malaria.

Development of PCR-RFLP assay for the discrimination of Plasmodium species and variants of P. vivax (VK210, VK247 and P. vivax-like) in Anopheles mosquitoes.

The identification of Plasmodium species in Anopheles mosquitoes is an integral component of malaria control programs. We developed a new assay to identify Plasmodium falciparum, Plasmodium malariae, and Plasmodium vivax variants. Specific primers were designed to hybridize to CS gene-specific regions. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used to distinguish the P. vivax variants VK210, VK247, and P. vivax-like. The new PCR-RFLP assay revealed good agreement when compared with a nested PCR using artificially infected Anopheles mosquitoes. This sensitive PCR-RFLP method can be useful when detection of Plasmodium species and P. vivax variants is required and may be employed to improve the understanding of malaria transmission dynamics by Anopheles species.

Giardiasis as zoonosis: between proof of principle and paradigm in the Northwestern region of São Paulo State, Brazil.

OBJECTIVE: In order to evaluate the potential zoonotic transmission of Giardia duodenalis, isolates from humans and dogs in the Northwestern region of the São Paulo State, Brazil were characterized based on the ß-giardin gene. METHODS: The samples were analyzed by sequencing of the Nested-PCR products. RESULTS: The A1 and A2 subgenotypes were detected in human and dogs. Cysts of assemblage B, C and D have not been found in any isolates studied. CONCLUSIONS: These results are consistent with the view that giardiasis in the largest endemic region of the Brazil should not be seen as a single entity.

Enteric parasites in HIV-1/AIDS-infected patients from a Northwestern São Paulo reference unit in the highly active antiretroviral therapy era.

INTRODUCTION: We describe the epidemiology of intestinal parasites in patients from an AIDS reference service in Northeastern São Paulo, Brazil. METHODS: Retrospective evaluation was done for all HIV-1/AIDS-positive patients whose Hospital de Base/São José do Rio Preto laboratorial analysis was positive for enteroparasites after diagnosis of HIV-1 infection, from January 1998 to December 2008. Statistical analysis was performed using the R statistical software version 2.4.1. The level of significance adopted was 5%. RESULTS: The most frequent protozoan was Isospora belli (4.2%), followed by Giardia lamblia (3.5%), Entamoeba coli (2.8%), and Cryptosporidium parvum (0.3%). Ancylostoma duodenale (1.4%) was the most frequently detected helminth, while Taenia saginata and Strongiloides stercoralis were found in 0.7% of the samples. The results showed that diarrhea was significantly associated with giardiasis and isosporiasis. However, no association was observed between CD4+ cell counts, viral load, and the characteristics of any particular parasite. CONCLUSIONS: Our data may be useful for further comparisons with other Brazilian regions and other developing countries. The data may also provide important clues toward improving the understanding, prevention, and control of enteric parasites around the world.

Osteomyelitis: a current challenge.

Over the last 30 years, the pathogenesis of osteomyelitis has almost been totally elucidated, and many factors responsible for the persistence of this infection have been identified. Numerous antimicrobial agents with distinct spectrums of action, pharmacokinetics, and pharmacodynamics have been used in its treatment. Surgical techniques, including muscle grafts, the Ilizarov technique, and antibiotic bone cements, have been applied. However, bone infections are still a challenge. Despite the importance of isolation and identification of microorganisms to determine the antimicrobial treatment of bone infections, there are few systematic national studies about the etiological profile of these diseases. This article describes the current knowledge of osteomyelitis and summarizes published national data based on the experience of different Orthopedic and Traumatology Services. In general, S. aureus was described as an important etiological agent; however, the difference in design of national studies makes a comparison between the prevalence of bone infection, the associated risk factors, and the different therapeutic approaches difficult. In conclusion, effort is necessary in order to stimulate systematic national studies in different Orthopedics and Traumatology Services to obtain a better consensus on preventive measures and therapies of bone infections.

Catheter-related infections in a northwestern São Paulo reference unit for burned patients care.

Despite improvements in care and rehabilitation of burned patients, infections still remain the main complication and death cause. Catheter-related infections are among the four most common infections and are associated with skin damage and insertion site colonization. There are few studies evaluating this kind of infection worldwide in this special group of patients. Padre Albino Hospital Burn Care Unit (PAHBCU) is the only reference center in the Northwestern São Paulo for treatment of burned patients. This paper presents the results of a retrospective study aiming at describing the epidemiological and clinical features of catheter-related infections at PAHBCU.

Concurrent Dengue and malaria in the Amazon region.

INTRODUCTION: The Amazon region has extensive forested areas and natural ecosystems, providing favorable conditions for the existence of innumerous arboviruses. Over 200 arboviruses have been isolated in Brazil and about 40 are associated with human disease. Four out of 40 are considered to be of public health importance in Brazil: Dengue viruses (1-4), Oropouche, Mayaro and Yellow Fever. Along with these viruses, about 98% of the malaria cases are restricted to the Legal Amazon region. METHODS: This study aimed to investigate the presence of arboviruses in 111 clinical serum samples from patients living in Novo Repartimento (Pará), Plácido de Castro (Acre), Porto Velho (Rondônia) and Oiapoque (Amapá). The viral RNA was extracted and RT-PCR was performed followed by a Multiplex-Nested-PCR, using Flavivirus, Alphavirus and Orthobunyavirus generic and species-specific primers. RESULTS: Dengue virus serotype 2 was detected in two patients living in Novo Repartimento (Pará) that also presented active Plasmodium vivax infection. CONCLUSIONS: Despite scant data, this situation is likely to occur more frequently than detected in the Amazon region. Finally, it is important to remember that both diseases have similar clinical findings, thus the diagnosis could be made concomitantly for dengue and malaria in patients living or returning from areas where both diseases are endemic or during dengue outbreaks.