Evaluation of platelet count by automatic blood counter QBC Vet Autoread (R) compared with blood smear estimative and count by hemocytometer

The platelet blood count in laboratorial routine provides to the clinician important information about the hemostasis of the patient. There are many techniques described, however the gold standard techniques realized in hemocytometer spent a lot of time, making this technique impracticable in great routines. This research had the intent to evaluate if the automatic veterinary blood counter QBC Vet Autoread (R), whose results get five minutes to be ready, is capable to offer a trustworthy platelet count number. To this end, were evaluated the correlations among three different forms of platelets count in dogs: count in automatic blood counter QBC Vet Autoread (R), estimative in blood smear and the gold standard method by manual count in hemocytometer. The viability and confidence use of automatic blood counters of the medicine veterinary routine. Seventeen dogs were chosen randomly way, in the medical and surgical routine of HOVET-USP. The analysis revel high correlation between the hemocytometer and the estimative in blood smear (r=0,875) and between the hemocytometer and automatic blood count by QBC Vet Autoread (R) (r=0,939). Conclude that the platelet blood cont by QBC Vet Autoread (R), in addition to be fast, it`s more truthful when compared with estimative in blood smear, although the latter one also had elevated correlation. However, morphological analysis through the smears cannot be dismissed because none of the other two techniques evaluated have the ability to assess platelet morphological changes.

Tentativa de evidenciar o Trypanosoma cruzi no sangue periférico de pacientes com doença de Chagas, em fase crônica, por meio do Quantitative Buffy Coat (QBC)

Valorizando a sensibilidade do sistema Quantitative Buffy Coat (QBC), documentada em modelo experimental murino, estando os animais com infecção aguda pelo Trypanosoma cruzi houve tentativa de evidenciar esse parasita no sangue periférico de 100 pacientes com doença de Chagas, em fase crônica. Com o emprego desse método, nenhuma positividade ocorreu, evidentemente em virtude das pequenas parasitemias, não reveláveis pela técnica, pelo menos conforme o verificado através da casuística considerada.

QBC® for the diagnosis of human and canine american visceral leishmaniasis: preliminary data

"Quantitative Buffy Coat" (QBC®) is a direct and fast fluorescent method used for the identification of blood parasites. Since Leishmania chagasi circulates in blood, we decided to test it in American visceral leishmaniasis (AVL). Bone marrow (BM) and peripheral blood (PB) of 49 persons and PB of 31 dogs were analyzed. QBC® was positive in BM of 11/11 patients with AVL and in 1/6 patients with other diseases. Amastigotes were identified in PB of 18/22 patients with AVL and in none without AVL. The test was positive in 30 out of the 31 seropositive dogs and in 28/28 dogs with Leishmania identified in other tissues. QBC® is a promising method for diagnosis of human AVL, and possibly for the exam of PB of patients with AVL/AIDS, for the control of the cure and for the identification of asymptomatic carriers. Because it is fast and easy to collect and execute, QBC® should be evaluated for programs of reservoir control.

Avaliação da contagem plaquetária pelo contador automático QBC Vet Autoread comparado com estimativa em esfregaço sanguíneo e contagem em hemocitômetro

The platelet blood count in laboratorial routine provides to the clinician important information about the hemostasis of the patient. There are many techniques described, however the gold standard techniques realized in hemocytometer spent a lot of time, making this technique impracticable in great routines. This research had the intent to evaluate if the automatic veterinary blood counter QBC Vet Autoread (R), whose results get five minutes to be ready, is capable to offer a trustworthy platelet count number. To this end, were evaluated the correlations among three different forms of platelets count in dogs: count in automatic blood counter QBC Vet Autoread (R), estimative in blood smear and the gold standard method by manual count in hemocytometer. The viability and confidence use of automatic blood counters of the medicine veterinary routine. Seventeen dogs were chosen randomly way, in the medical and surgical routine of HOVET-USP. The analysis revel high correlation between the hemocytometer and the estimative in blood smear (r=0,875) and between the hemocytometer and automatic blood count by QBC Vet Autoread (R) (r=0,939). Conclude that the platelet blood cont by QBC Vet Autoread (R), in addition to be fast, it's more truthful when compared with estimative in blood smear, although the latter one also had elevated correlation. However, morphological analysis through the smears cannot be dismissed because none of the other two techniques evaluated have the ability to assess platelet morphological changes.

Evaluation of different methods for Plasmodia detection, in well defined population groups in an endemic area of Brazil

In Brazil, more than 500,000 new cases of malaria were notified in 1992. Plasmodium falciparum and P.vivax are the responsible species for 99.3% of the cases. For adequate treatment, precoce diagnosis is necessary. In this work, we present the results of the traditional Plasmodia detection method, thick blood film (TBF), and the results of alternative methods: Immunofluorescence assay (IFA) with polyclonal antibody and Quantitative Buffy Coat method (QBC)® in a well defined population groups. The analysis were done in relation to the presence or absence of malaria clinical symptoms. Also different classes of immunoglobulins anti-P.falciparum were quantified for the global analysis of the results, mainly in the discrepant results. We concluded that alternative methods are more sensitive than TBF and that the association of epidemiological, clinical and laboratory findings is necessary to define the presence of malaria.

A survey of congenital Chagas’ disease, carried out at three Health Institutions in São Paulo City, Brazil

The congenital transmission of Chagas’ disease was evaluated in 57 pregnant women with Chagas’ disease and their 58 offspring. The patients were selected from three Health Institutions in São Paulo City. The maternal clinical forms of Chagas’ disease were: indeterminate (47.4%), cardiac (43.8%) and digestive (8.8%); 55 were born in endemic areas and two in São Paulo City. The transmission of Chagas’ disease at fetal level was confirmed in three (5.17%) of the 58 cases studied and one probably case of congenital Chagas’ disease. Two infected infants were born to chagasic women with HIV infection and were diagnosed by parasitolological assays (microhematocrit, quantitative buffy coat-QBC or artificial xenodiagnosis). In both cases the placenta revealed T. cruzi and HIV p24 antigens detected by immunohistochemistry. In one case, a 14-week old abortus, the diagnosis of congenital T. cruzi infection was confirmed by immunohistochemistry. The other probable infection, a 30-week old stillborn, the parasites were found in the placenta and umbilical cord. The Western blot method using trypomastigote excreted/secreted antigens of T. cruzi (TESA) was positive for IgG antibodies in 54/55 newborns and for IgM in 1/55 newborns. One of the two newborns with circulating parasites had no detectable IgG or IgM antibodies. The assessment of IgG antibodies in the sera of pregnant women and their newborns was performed by ELISA using two different T. cruzi antigens: an alkaline extract of epimastigotes (EAE) and trypomastigote excreted/secreted antigens (TESA). The analysis showed a linear correlation between maternal and newborn IgG antibody titers at birth.

Trypanosoma cruzi parasitemia observed in immunocompromised patients: the importance of the artificial xenodiagnosis

Trypanosoma cruzi parasitemia observed in immunocompromised patients (transplant or positive HIV) occurred more frequently by the artificial xenodiagnosis method (10/38) compared with hemoculture (2/38), given the same quantity of blood. Other ways of diagnosis, like mice inoculation (5/38), QBC and buffy coat (2/38), were evaluated also. This result showed the importance of the artificial xenodiagnosis. The other techniques increased only one more patient positive.

Survival of Trypanosoma cruzi in sugar cane used to prepare juice

Chagas disease can be transmitted to man by many different means, including contact with infected triatomine feces, blood transfusion, laboratory accidents, organ transplants, and congenital or oral routes. The latter mode has received considerable attention recently. In this assay, we evaluate the survival of Trypanosoma cruzi contaminating sugar cane used to prepare juice, as well as the viability and capacity for infection by the parasite after recovery. Thirty triatomines were contaminated with T. cruzi Y strain and 45 days later pieces of sugar cane were contaminated with the intestinal contents of the insects. The pieces were ground at different intervals after contamination (time = 0, 1, 4, 6, 12 and 24 hours) and the juice extracted and analyzed. Different methods were used to show T. cruzi in the juice: direct analysis, hematocrit tube centrifugation and QBC, and experimental inoculation in 47 female BALB/c mice (five control mice and seven mice for each interval examined (five inoculated orally and two intraperitoneally). Positive results were found using the direct analysis and QBC methods for juice prepared up to 12 hours after initial contamination. However, by the centrifugation technique, positivity was found only up to four hours after contamination of the sugar cane. Inoculated animals showed parasitemia during a 14 day observation period, demonstrating the high survival rate of T. cruzi in sugar cane.

Methylene blue vital staining for Trypanosoma cruzi trypomastigotes and epimastigotes

The morphological identification of Trypanosoma cruzi is currently considered to have a high specificity, but its sensitivity, which depends on the volume of the sample examined, is rather low. Trypanosome developmental stages suspended in blood, reduviid feces, and culture media are routinely searched for by means of fresh film examination (about 2 µL). High speed centrifugation of blood samples separates the buffy coat, where most trypomastigotes concentrate. As the parasites are transparent and colorless, their detection is mostly dependent on their motility. The fluorescent vital stain acridine orange has been used to enhance image contrast, as exemplified by the QBC (Quantitative Buffy Coat) technique. Staining blood, buffy coat, reduviid feces, and culture media samples with methylene blue (also a vital dye) is a means of producing sharp, well contrasted images of motile or non-motile T. cruzi developmental stages, only standard laboratory microscopes being required. Slides previously coated with a thin layer of methylene blue are used to stain fresh blood films. Photomicrographs exemplify the results of methylene blue staining applied to living and fixed parasites.

Controle da malária transfusional em região endêmica e não endêmica do Brasil

O presente trabalho teve como objetivo estabelecer critérios adequados à triagem de doadores de sangue de regiões com características epidemiológicas distintas, para malária. Foram estudados 3 locais com critérios de seleção diferentes: São Paulo, SP (sem transmissão vetorial), Belém (baixa transmissão ativa), Matupá, Belém, PA e Peixoto de Azevedo, MT (alta transmissão ativa). A pesquisa de plasmódios foi realizada por gota espessa, QBC Test® e imunofluorescência para pesquisa de antígenos, tendo sido todas as amostras negativas. Houve grande variação na positividade para anticorpos antiplasmodiais por IFI-IgG anti P. vivax e P. falciparum entre doadores aptos nos 3 locais de estudo e entre doadores aptos e inaptos em São Paulo (aptos 1,98%, inaptos 22,3%, p < 0.01) e Belém (aptos 17,2%, inaptos 58,3%, p < 0.01), o que atesta a validade da triagem clínico-epidemiológica realizada. Em Matupá e Peixoto de Azevedo não houve doadores inaptos e a positividade foi de 80,6%. Consideramos que em bancos de sangue a triagem deve seguir critérios clínico-epidemiológicos adequados à situação de cada região. Os métodos laboratoriais de triagem, devem ser para detecção de plasmódios (gota espessa/QBC Test® ou detecção de antígenos parasitários.

Fase aguda da doença de Chagas na Amazônia brasileira: estudo de 233 casos do Pará, Amapá e Maranhão observados entre 1988 e 2005

Foram estudados 233 casos de fase aguda da doença de Chagas, oriundos do Pará, Amapá e Maranhão, observados no período de 1988 a 2005, cento e sessenta deles retrospectivamente de 1988 a 2002 e setenta e três prospectivamente de 2003 a 2005. Entre os casos estudados 78,5% (183/233) faziam parte de surtos provavelmente por transmissão oral, acometendo em média 4 pessoas e 21,5% (50/233) eram casos isolados. Foram considerados casos agudos aqueles que apresentaram exames parasitológicos diretos (a fresco, gota espessa ou Quantitative Buffy Coat - QBC) e/ou IgM anti-Trypanosoma cruzi positivos. Foram feitos ainda xenodiagnósticos em 224 pacientes e hemoculturas em 213. Todos foram avaliados clinica e epidemiologicamente. As manifestações clínicas mais freqüentes foram febre (100%), cefaléia (92,3%), mialgia (84,1%), palidez (67%), dispnéia (58,4%), edema de membros inferiores (57,9%), edema de face (57,5%) dor abdominal (44,2%), miocardite (39,9%) e exantema (27%). O eletrocardiograma mostrou alterações de repolarização ventricular em 38,5% dos casos, baixa voltagem de QRS em 15,4% e desvio de SAQRS em 11,5%, extra-sístoles ventriculares em 5,8%, bradicardia em 5,8% e taquicardia em 5,8%, bloqueio de ramo direito em 4,8% e fibrilação atrial em 4,8%. A alteração mais freqüente vista no ecocardiograma foi o derrame pericárdico em 46,2% dos casos. Treze (5,6%) pacientes evoluíram para o óbito, 10 (76,9%) dos quais por comprometimento cardiovascular, dois por complicações de origem digestiva e um de causa mal definida.

Diagnosis of malaria by acridine orange fluorescent microscopy in an endemic area of Venezuela

Fluorescent (acridine orange) microscopical examination of capillary centrifuged blood (quantitative buffy coat [QBC®] analysis) and Giemsa stained thick blood smears (GTS) were compared for diagnosis of malaria in blood specimens from adults living in malaria transmission areas of the States of Bolivar and Amazonas in southeastern and south Venezuela, respectively. Of a total of 198 GTS examined, 95 subjects (48%) showed parasitaemia. Among the 95 blood films with a positive GTS, 94 were judged positive by the QBC. However, positive QBC tubes were found in 29 out of 103 blood specimens with a negative GTS. Thus, relative to a GTS standard, the sensitivity and specificity of the QBC-test was 99.2% and 72%, respectively. Young trophozoites of Plasmodium vivax and P. falciparum could not be distinguished with certainty. It is confirmed that the QBC offers many advantages compared with the standard diagnosis of malaria parasites, specifically in the speed of staining and ease of interpretation. However, in places where P. falciparum and P. vivax occur, species and stage differentiation should be confirmed with the GTS.

Estudo genotípico de Trypanosoma cruzi: epidemiologia e caracterização molecular de isolados do homem, triatomíneos e mamíferos silvestres do Pará, Amapá e Maranhão

A doença de Chagas aguda (DCA) é endêmica na Amazônia Brasileira sendo a via oral a principal forma de transmissão com surtos familiares ou multifamiliares. Esta via independe da colonização de triatomíneos no domicílio e a ocorrência é regular com média de 100 casos/ano e 5% de óbitos. Apresenta distribuição espaço-temporal bem definida, colocando a enfermidade como emergência de importância em saúde pública nos estados do Pará, Amapá e Amazonas. A presença de mamíferos e triatomíneos silvestres, infectados naturalmente com o c e habitando distintos ecótopos terrestres e arbóreos, mantém um intenso ciclo enzoótico em toda a Amazônia. Perfis moleculares de linhagens de T. cruzi na região estão associados a hospedeiros mamíferos (incluindo o homem), triatomíneos, ecótopos e manifestações clínicas. Foram estudados quatro surtos de DCA ocorridos nos Municípios de Barcarena, Belém e Cachoeira do Arari no Estado do Pará e em Santana, no Estado do Amapá e abordados os aspectos epidemiológicos (parasitológico e sorológico manifestações clínicas, reservatórios e triatomíneos silvestres associados aos surtos). Foi investigado também em São Luís, Estado do Maranhão, o ciclo domiciliar e silvestre do T. cruzi, porém sem a ocorrência de casos de DCA. O estudo incluiu também a genotipagem molecular de T. cruzi pelo gene de mini-exon dos isolados (homem, mamíferos e triatomíneos silvestres) associados aos diferentes ciclos de transmissão. O diagnóstico parasitológico foi confirmado em 63 pacientes com a seguinte sensibilidade nos testes aplicados: 41,3% (26/63) pela gota espessa; 58,7% (37/63) no QBC; 79,4% (50/63) no xenodiagnóstico e 61,9% (39/63) na hemocultura. A sorologia de 2648 pessoas por hemaglutinação indireta (HAI) foi de 3,05% (81/2648) e imunofluorescência indireta IFI apresentaram respectivamente resultados de e 2,49% (66/2648) para IgG e 2,37 (63/2648) para IgM. Os resultados em São Luís foram todos negativos. Foram capturados 24 mamíferos, 13 Didelphis marsupialis, 1 Marmosa cinerea, 5 Philander opossum, 3 Metachirus nudicaudatus, 1 Oryzomys macconnelli, 1 Oecomys bicolor e 433 R. rattus. A taxa de infecção para T. cruzi foi de 7,14% (29/404). Um total de 3279 triatomíneos foi capturado sendo: Triatoma rubrofasciata (n=3008), com taxa de infecção (TI) de 30.46%, (39/128), Rhodnius robustus (n=137), com TI de 76% (79/104), R. pictipes (n=94), TI de 56,9% (49/86%), E. mucronatus (n=6) e P. geniculatus (n=12) com TI de 50% e as demais espécies sem infecção R. neglectus (n=5) e P. lignarius (n=6). As palmeiras foram os principais ecótopos dos triatomíneos silvestres. O urucurizeiro (S. martiana) apresentava infestação de 47,41% (101/213) dos triatomíneos; o “inajazeiro” (Maximiliana regia) 35,21% (75/213); o “babaçueiro” (Orbgnya. speciosa) 5,16% (11/213); o “dendezeiro” (Eleas melanoccoca) 1,87% (4/213) e a “bacabeira” (Oenocarpus bacaba) 10,32% (22/213). Para a genotipagem foram obtidos 46 isolados de tripanossomas de origem humana, 31 isolamentos de mamíferos silvestres e 74 amostras de triatomíneos. Todos os isolados foram caracterizados como da linhagem TcI de T. cruzi. Todos os casos humanos no Pará foram caracterizados como positivos por exame parasitológico. Nem todos os casos de Santana, Amapá, apresentaram casos parasitológicos positivos, pela demora do diagnóstico, mesmo assim estes foram definidos como DCA. Exames como xenodiagnóstico, hemocultura e o QBC® foram mais sensíveis do que a gota espessa. A sorologia por HAI e IFI (IgG e IgM) tiveram excelente sensibilidade para detectar os casos agudos em tempos distintos de infecção. O achado de mamíferos (D. marsupilais) e triatomíneos silvestres (R. pictipes e P. geniculatus) infectados com consideráveis taxas de infecção para T. cruzi no entorno das residências dos pacientes sustentam a importância destes hospedeiros associados à transmissão da DCA. Apesar de na Amazônia circularem vários genótipos de T. cruzi nos diferentes hospedeiros, neste trabalho foi identificada somente a linhagem TCI de T. cruzi, a mais predominante na Região. Em São Luís, Maranhão, embora sem registro de casos de DCA apresenta um ciclo domiciliar associados ao rato doméstico e o triatomíneo da espécie T. rubrofasciata, e um ciclo silvestre mantido por didelfídeos. Nos dois ciclos circulam a linhagem TCI de T. cruzi. Estudos com marcadores de maior resolução com isolados de T. cruzi regionais podem ajudar a esclarecer os ciclos de transmissão, as rotas de contaminação e os hospedeiros envolvidos em casos de DCA na Amazônia.

Funktionelle Rolle von SNARE-Proteinen bei der Myelinisierung

Die Hauptfunktion der Oligodendrozyten im zentralen Nervensystem ist die Myelinisierung. Dabei umwickelt die Zelle mit Ausläufern ihrer Plasmamembran mehrmals die Axone. In den Phasen der aktiven Myelinisierung produziert ein Oligodendrozyt eine Fläche von 5000-50000 µm2 Myelin pro Tag, wobei große Mengen der Myelinkomponenten über vesikulären Transport zur Zelloberfläche transportiert werden müssen. Die Fusion der Vesikel mit ihrer Zielmembran wird duch SNARE-Proteine (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor proteins) kontrolliert, die durch spezifische Interaktionen zwischen R- (Vesikel) und Q- (Zielmembran) SNAREs auch zur Spezifität der Fusion beitragen. Um die SNAREs den oligodendroglialen Transportrouten zuzuordnen, wurde deren Expression, Regulation und subzelluläre Lokalisation in primären Oligodendrozyten, in Oli-neu Zellen und im Myelin untersucht. Die Plasmamembran-Q-SNAREs Syntaxin 3, Syntaxin 4 und SNAP23, sowie das endosomale R-SNAREs VAMP3 zeigten eine zunehmende Expression im Verlauf der Oligodendrozytenreifung, die Expression von SNAP29 hingegen verminderte sich. Zudem akkumulierten die SNARE-Proteine Syntaxin 2, Syntaxin 3, Syntaxin 4 und VAMP7 im adulten Myelin, was für ihre Beteiligung an der Myelinisierung spricht. Co-Immunpräzipitationen ergaben u.a. Interaktionen zwischen den SNARE-Proteinen VAMP3 (R), Syntaxin 4 (Qa) und SNAP23 (Qbc). Anhand der beschriebenen Analyse konnten die SNARE-Proteine den oligodendroglialen Transportwegen zugeordnet werden. Immunzytochemische Analysen zeigten, dass das Hauptmyelinprotein PLP mit dem R-SNARE des Recycling Endosoms VAMP3, und mit dem spät endosomal, lysosomalen SNARE VAMP7 kolokalisiert. Um deren Rolle im PLP-Transport zu untersuchen, wurden verschiedene VAMP3- bzw. VAMP7-Silencing-Experimente durchgeführt. In beiden Fällen führte dies zu einer reduzierten Menge an PLP an der Zelloberfläche. Die Ergebnisse lassen somit auf zwei unabhängige Transportwege für PLP zur Plasmamembran von Oligodendrozyten schließen. Der VAMP7-abhängige PLP-Transport wurde zusätzlich in vivo in AP3-defizienten Mäusen, welche VAMP7 fehlsortieren, überprüft. Die Gehirne der mutanten Mäuse enthielten weniger Myelin, das isolierte Myelin enthielt weniger PLP und CNP. VAMP7 scheint also bei der Myelinisierung die Fusion von PLP- und CNP-enthaltenden Vesikeln mit der Myelinmembran zu vermitteln.