Validation of cytopathology specimens for digital pathology.

INTRODUCTION: Digital cytopathology is being progressively implemented in centres worldwide, but impediments such as the three-dimensionality of specimens and the size of scanned images have prevented its use from becoming widespread. This study aimed to validate the use of digital whole slide image scanning of cytopathology samples for routine sign-out. METHODS: Specimens were scanned using the Leica Aperio GT 450 System. The following sample types were used: liquid-based cytology, direct conventional smears from fine needle aspirates and cytospins. Cases were validated by the same pathologist who originally rendered the conventional diagnosis, with a washout of at least 3 months. Final digital diagnoses were compared to the original analogical diagnoses, and cases were considered concordant up to a one-degree difference between the original and digital diagnoses. Reasons for the unsuccessful scanning of slides were also noted. The technical procedures followed the College of American Pathologists' guidelines for digital pathology validation. RESULTS: A total of 730 slides from 383 cases (337 female, 51 male; median age 42) were successfully scanned. These cases consisted of the following sample types: 81 (21.1%) conventional smears, 240 (62.7%) liquid-based cytology samples and 62 (16.2%) cytospins. There were only five discordant cases, with a 98.7% agreement between original and digital diagnoses using the difference rate of up to one degree. Seventy-seven slides (10.5%) had to be rescanned due to technical problems. The main reasons for unsuccessful scanning were paucicellular samples (44; 57.1%), the thickness of the smears (18; 23.4%) and issues with the coverslip (15; 19.5%). CONCLUSION: Cytological specimens can be successfully scanned and used for digital pathology, with excellent agreement with the original diagnoses.

Asymptomatic Plasmodium infection in a residual malaria transmission area in the Atlantic Forest region: Implications for elimination.

INTRODUCTION: Elimination of malaria in areas of interrupted transmission warrants careful case assessment to avoid the reintroduction of this disease. Occasional malaria cases are reported among visitors of the Atlantic Forest area of Brazil, while data on residents of this area are scarce. METHODS: A sectional study was carried out to examine 324 individuals living in a municipality where autochthonous cases were detected. RESULTS: Asymptomatic Plasmodium infections were detected in 2.8% of the individuals by polymerase chain reaction (PCR), with one case of P. falciparum (0.3%), two cases of P. vivax (0.6%), and six cases of P. malariae (1.9%). The thick blood smears were negative in all individuals. Serological tests performed in 314 subjects were reactive in 11.1%, with 3.5% for P. falciparum and 7.7% for P. vivax. A subsample of 42 reactive individuals for any Plasmodium species showed P. malariae in 30.9% of specimens. Individuals who entered the Atlantic Forest region were 2.7 times more likely to exhibit reactive serology for P. vivax compared with individuals who did not enter this region (p<0.05). Children <15 years had a higher chance of reactive serology for P. falciparum and P. vivax than individuals ≥15 years of age (p<0.05). Individuals living in the Paraiso district had a higher chance of reactive serology for P. vivax compared to other districts (p<0.05). No associations were found between sex, past exposure to malaria, or serological response to antibodies of any Plasmodium species. CONCLUSIONS: The implications of these results for the elimination of malaria were discussed.

Asymptomatic plasmodium infection in a residual malaria transmission area in the atlantic forest region: implications for elimination

Abstract INTRODUCTION: Elimination of malaria in areas of interrupted transmission warrants careful case assessment to avoid the reintroduction of this disease. Occasional malaria cases are reported among visitors of the Atlantic Forest area of Brazil, while data on residents of this area are scarce. METHODS: A sectional study was carried out to examine 324 individuals living in a municipality where autochthonous cases were detected. RESULTS: Asymptomatic Plasmodium infections were detected in 2.8% of the individuals by polymerase chain reaction (PCR), with one case of P. falciparum (0.3%), two cases of P. vivax (0.6%), and six cases of P. malariae (1.9%). The thick blood smears were negative in all individuals. Serological tests performed in 314 subjects were reactive in 11.1%, with 3.5% for P. falciparum and 7.7% for P. vivax. A subsample of 42 reactive individuals for any Plasmodium species showed P. malariae in 30.9% of specimens. Individuals who entered the Atlantic Forest region were 2.7 times more likely to exhibit reactive serology for P. vivax compared with individuals who did not enter this region (p<0.05). Children <15 years had a higher chance of reactive serology for P. falciparum and P. vivax than individuals ≥15 years of age (p<0.05). Individuals living in the Paraiso district had a higher chance of reactive serology for P. vivax compared to other districts (p<0.05). No associations were found between sex, past exposure to malaria, or serological response to antibodies of any Plasmodium species. CONCLUSIONS: The implications of these results for the elimination of malaria were discussed.

Lipoproteins from vertebrate host blood plasma are involved in Trypanosoma cruzi epimastigote agglutination and participate in interaction with the vector insect, Rhodnius prolixus.

Chagas disease, infecting ca. 8 million people in Central and South America, is mediated by the protozoan parasite, Trypanosoma cruzi. The parasite is transmitted by the bite of blood sucking triatomine insects, such as Rhodnius prolixus, that had previously fed on parasite-infected vertebrate blood and voided their contaminated feces and urine into the wound. The stages of the parasite life cycle in both the insect vector and human host are well-known, but determinants of infection in the insect gut are complex and enigmatic. This paper examines the possible role of the R. prolixus gut agglutinins in the parasite life cycle. The results, derived from gut extracts made from R. prolixus fed on various diets with different vertebrate blood components, and cross adsorption experiments, showed for the first time that R. prolixus has two distinct gut agglutinins originating from their vertebrate blood meal, one for T. cruzi (the parasite agglutinin, PA) and the other for the erythrocytes (the hemagglutinin, HA). Again, uniquely, the results also demonstrate that these two agglutinins are derived, respectively, from the plasma and erythrocyte components of the vertebrate blood. Subsequent experiments, examining in more detail the nature of the plasma components forming the T. cruzi PA, used fractionated extracts of the vertebrate plasma (high density lipoprotein, HDL; low density lipoprotein, LDL, and delipidated plasma) in agglutination assays. The results confirmed the identity of the PA as a high density lipoprotein (HDL) in the plasma of the vertebrate blood meal which agglutinates parasites in the R. prolixus gut. In addition, the use of single or double labeled HDL in fluorescence and confocal microscopy showed the interaction of the labeled HDL with the parasite surface and its internalization at later times. Finally, results of T. cruzi parasitization of R. prolixus, incorporating various vertebrate blood components, resulted in highly significant increases in infectivity in the presence of HDL from the 2nd day of infection, thus confirming the important role of this molecule in T. cruzi infection of R. prolixus.

Malaria in the state of Rio de Janeiro, Brazil, an Atlantic Forest area: an assessment using the health surveillance service.

The lethality of malaria in the extra-Amazonian region is more than 70 times higher than in Amazonia itself. Recently, several studies have shown that autochthonous malaria is not a rare event in the Brazilian southeastern states in the Atlantic Forest biome. Information about autochthonous malaria in the state of Rio de Janeiro (RJ) is scarce. This study aims to assess malaria cases reported to the Health Surveillance System of the State of Rio de Janeiro between 2000-2010. An average of 90 cases per year had parasitological malaria confirmation by thick smear. The number of malaria notifications due to Plasmodium falciparum increased over time. Imported cases reported during the period studied were spread among 51% of the municipalities (counties) of the state. Only 35 cases (4.3%) were autochthonous, which represents an average of 3.8 new cases per year. Eleven municipalities reported autochthonous cases; within these, six could be characterised as areas of residual or new foci of malaria from the Atlantic Forest system. The other 28 municipalities could become receptive for transmission reintroduction. Cases occurred during all periods of the year, but 62.9% of cases were in the first semester of each year. Assessing vulnerability and receptivity conditions and vector ecology is imperative to establish the real risk of malaria reintroduction in RJ.

Malaria in the state of Rio de Janeiro, Brazil, an Atlantic Forest area: an assessment using the health surveillance service

The lethality of malaria in the extra-Amazonian region is more than 70 times higher than in Amazonia itself. Recently, several studies have shown that autochthonous malaria is not a rare event in the Brazilian southeastern states in the Atlantic Forest biome. Information about autochthonous malaria in the state of Rio de Janeiro (RJ) is scarce. This study aims to assess malaria cases reported to the Health Surveillance System of the State of Rio de Janeiro between 2000-2010. An average of 90 cases per year had parasitological malaria confirmation by thick smear. The number of malaria notifications due to Plasmodium falciparum increased over time. Imported cases reported during the period studied were spread among 51% of the municipalities (counties) of the state. Only 35 cases (4.3%) were autochthonous, which represents an average of 3.8 new cases per year. Eleven municipalities reported autochthonous cases; within these, six could be characterised as areas of residual or new foci of malaria from the Atlantic Forest system. The other 28 municipalities could become receptive for transmission reintroduction. Cases occurred during all periods of the year, but 62.9% of cases were in the first semester of each year. Assessing vulnerability and receptivity conditions and vector ecology is imperative to establish the real risk of malaria reintroduction in RJ.

Influence of parasite load on renal function in mice acutely infected with Trypanosoma cruzi.

BACKGROUND: Chagas disease is a neglected tropical disease caused by Trypanosoma cruzi. Despite the vast number of studies evaluating the pathophysiological mechanisms of the disease, the influence of parasite burden on kidney lesions remains unclear. Thus, the main goal of this work was to evaluate the effect of T. cruzi infection on renal function and determine whether there was a correlation between parasite load and renal injury using an acute experimental model of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Low, medium and high parasite loads were generated by infecting C57BL/6 mice with 300 (low), 3,000 (medium) or 30,000 (high) numbers of "Y" strain trypomastigotes. We found that mice infected with T. cruzi trypomastigotes show increased renal injury. The infection resulted in reduced urinary excretion and creatinine clearance. We also observed a marked elevation in the ratio of urine volume to kidney and body weight, blood urea nitrogen, chloride ion, nitric oxide, pro- and anti-inflammatory cytokines and the number of leukocytes in the blood and/or renal tissues of infected mice. Additionally, we observed the presence of the parasite in the cortical/medullary and peri-renal region, an increase of inflammatory infiltrate and of vascular permeability of the kidney. Overall, most renal changes occurred mainly in animals infected with high parasitic loads. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that T. cruzi impairs kidney function, and this impairment is more evident in mice infected with high parasitic loads. Moreover, these data suggest that, in addition to the extensively studied cardiovascular effects, renal injury should be regarded as an important indicator for better understanding the pan-infectivity of the parasite and consequently for understanding the disease in experimental models.

Evaluation of three different DNA extraction methods from blood samples collected in dried filter paper in Plasmodium subpatent infections from the Amazon region in Brazil.

Asymptomatic Plasmodium infection is a new challenge for public health in the American region. The polymerase chain reaction (PCR) is the best method for diagnosing subpatent parasitemias. In endemic areas, blood collection is hampered by geographical distances and deficient transport and storage conditions of the samples. Because DNA extraction from blood collected on filter paper is an efficient method for molecular studies in high parasitemic individuals, we investigated whether the technique could be an alternative for Plasmodium diagnosis among asymptomatic and pauciparasitemic subjects. In this report we compared three different methods (Chelex®-saponin, methanol and TRIS-EDTA) of DNA extraction from blood collected on filter paper from asymptomatic Plasmodium-infected individuals. Polymerase chain reaction assays for detection of Plasmodium species showed the best results when the Chelex®-saponin method was used. Even though the sensitivity of detection was approximately 66% and 31% for P. falciparum and P. vivax, respectively, this method did not show the effectiveness in DNA extraction required for molecular diagnosis of Plasmodium. The development of better methods for extracting DNA from blood collected on filter paper is important for the diagnosis of subpatent malarial infections in remote areas and would contribute to establishing the epidemiology of this form of infection.

Evaluation of three different DNA extraction methods from blood samples collected in dried filter paper in Plasmodium subpatent infections from the Amazon region in Brazil / Avaliação de três métodos de extração de DNA obtidos de amostras de sangue coletadas em papel de filtro em infecções subpatentes de Plasmodium da região Amazônica no Brasil

Asymptomatic Plasmodium infection is a new challenge for public health in the American region. The polymerase chain reaction (PCR) is the best method for diagnosing subpatent parasitemias. In endemic areas, blood collection is hampered by geographical distances and deficient transport and storage conditions of the samples. Because DNA extraction from blood collected on filter paper is an efficient method for molecular studies in high parasitemic individuals, we investigated whether the technique could be an alternative for Plasmodium diagnosis among asymptomatic and pauciparasitemic subjects. In this report we compared three different methods (Chelex®-saponin, methanol and TRIS-EDTA) of DNA extraction from blood collected on filter paper from asymptomatic Plasmodium-infected individuals. Polymerase chain reaction assays for detection of Plasmodium species showed the best results when the Chelex®-saponin method was used. Even though the sensitivity of detection was approximately 66% and 31% for P. falciparum and P. vivax, respectively, this method did not show the effectiveness in DNA extraction required for molecular diagnosis of Plasmodium. The development of better methods for extracting DNA from blood collected on filter paper is important for the diagnosis of subpatent malarial infections in remote areas and would contribute to establishing the epidemiology of this form of infection.

Infecção assintomática por Plasmodium é um novo desafio para a saúde pública no Brasil. A reação em cadeia da polimerase (PCR) é o melhor método para detectar baixas parasitemias presentes em pacientes com infecção assintomática. Nas áreas endêmicas, a coleta de sangue total é dificultada pela distancia geográfica, transporte e adequada armazenagem das amostras. A coleta de sangue em papel de filtro pode ser uma alternativa nessas áreas de difícil acesso. Neste estudo foram comparados três diferentes métodos de extração de ADN a partir de papel de filtro usando como controle extração a partir de sangue total. O protocolo Chelex®-Saponina foi o que obteve o melhor resultado quando comparado com os outros três protocolos. No entanto a sensibilidade foi de 66,7% para o P. falciparum e 31,6% para o P. vivax. Conclui-se que em caso de infecção assintomática o papel de filtro não é ainda uma boa alternativa para coleta de amostras.

Estudo da infecção humana por Plasmodium ssp no município de Guapimirim, estado do Rio de Janeiro / Study of human infection with Plasmodium spp Guapimirim in the county of, state of Rio de Janeiro

No ano 2010, no Brasil mais de 300.000 casos de malária foram detectados, 99,8 por cento deles dos quais, ocorridos na Amazônia legal. Na região extra-amazônica a malária é notificada, embora,a maioria casos são importados das áreas endêmicas do Brasil ou de outros países. Estudos de base populacional realizados em São Paulo e no Espírito Santo têm mostrado a presença de malária autóctone e infecção assintomática em diversas áreas da Mata Atlântica, sendo que os casos apresentam-se com sintomatologia tipicamente leve e baixas densidades parasitárias. De acordo com a Secretaria Estadual de Vigilância Epidemiológica do Rio de Janeiro, no período entre 2001-2008 foram notificados 29 casos de malária autóctones e o município de Nova Friburgo foi responsável por 30 por cento deles. Em 2008, foi diagnosticado um caso autóctone no município de Guapimirim e dois outros casos ocorreram em dezembro de 2009. Com o objetivo de investigar a presença de casos de malária e de infecção assintomática na área peri-urbana e rural do município de Guapimirim e identificar os principais fatores associados à sua ocorrência, foi realizado um estudo observacional misto, que consistiu de uma análise retrospectiva dos dados secundários da Secretaria Estadual de Saúde (2002-2010), uma entrevista com informante chave e um corte seccional no município de Guapimirim no primeiro semestre de 2010. O estudo retrospectivo mostrou uma média anual de 90 casos por ano. Os casos eram importados em sua maioria com predomínio do gênero masculino; a média de idade de 41,96 mais ou menos 14,6 (44,3 mais ou menos 14,56 anos em 2002 e de 37,6 mais ou menos 16 anos em 2010) (p menor que 0,001). As freqüências por espécies de plasmódios foram 49,1 por cento por P. falciparum, 47,3 por cento para P. vivax e 1,0 por cento por P. malariae. P. falciparum foi responsável por 39,4 por cento dos casos em 2002 e por 68,2 por cento em 2010 (menor que 0,001). De 176 indivíduos que tiveram o diagnóstico de malária fechado ou investigação epidemiológica completa, a maioria adquiriu a doença no estado do Amazonas (38,1 por cento), seguido por Rio de Janeiro, (19,9 por cento) e Rondônia (16,5 por cento). O município de Cachoeira de Macacu contribuiu com 22,7 por cento dos casos autóctones do RJ; 63,0 por cento ocorreram no primeiro semestre de cada ano. No estudo seccional foram entrevistadas 324 pessoas maiores de cinco anos de idade, das quais foram coletadas amostras para exames parasitológico e sorológico. Duas gotas espessas foram positivas, uma para Plasmodium spp e a outra para Babesia spp. Nove pessoas foram positivas pela PCR (seis para P. malariae, duas para P. vivax e uma para P. falciparum). Na sorologia, 3,5 por cento das amostras apresentaram anticorpos para IgG anti P. falciparum e 7,7 por cento para o IgG anti P. vivaxObservamos que o ingresso na mata foi um comportamento de risco para infecção plasmodial e as localidades de Garrafão, Orindi e Paraíso (próximas à Cachoeira de Macacu) foram as mais afetadas. Discute-se que a transmissão da malária no estado não está interrompida e que o Rio de Janeiro pode ser considerado como uma área de malária residual com presença de infecção assintomática. Três possíveis cenários diferentes de transmissão são apresentados.