Determination of a cut-off value for the serological diagnosis of scrub typhus by detecting anti-Orientia tsutsugamushi immunoglobulin M

Background & objectives: The diagnosis of scrub typhus (ST) is usually done using enzyme-linked immunosorbent assay (ELISA) due to its ease of performance and reading objectivity. The cut-off value for ELISA needs to be calculated for each geographical location as it depends on zonal endemicity of the disease. This study was, therefore, undertaken to calculate the pan-India cut-off for anti-Orientia tsutsugamushi (OT) immunoglobulin M (IgM) by ELISA. Methods: Samples from cases (cases of ST) and controls (voluntary, consenting, healthy adults) were collected by a network of 29 laboratories across India and tested for anti-OT IgM by immunofluorescence assay (IFA), the considered gold standard test. These samples were retested by ELISA for anti-OT IgM and their optical densities (ODs) were used for cut-off estimation by receiver operating characteristic (ROC) curve. Results: Anti-OT IgM ELISA ODs from 273 controls and 136 cases were used for the cut-off estimation. The ODs of the anti-OT IgM ELISA on healthy individuals and those of confirmed ST cases ranged from 0.1 to 0.75 and 0.5 to 4.718, respectively. ROC curve-based cut-off for ELISA was calculated as 0.554 at a sensitivity of 95.2 per cent and specificity of 95.1 per cent. A value of >1 was noted to have a specificity of 100 per cent in diagnosing ST. Interpretation & conclusions: The cut-off calculated for India was similar to the previous cut-off that was used until now.

Seroprevalence of rickettsial infection in northern Cyprus: A study among hunters

@#This study was conducted to investigate rickettsial seropositivity among hunters, a high-risk population for tick-borne diseases in northern Cyprus. Serum samples were collected from 300 hunters from different locations during the 2017-2018 hunting season (November 2017 - February 2018). The samples were analyzed by indirect immunofluorescence assay (IFA) using slides coated with Rickettsia slovaca, a species belonging to the spotted fever group (SFG). During the sample collection, a questionnaire was also applied to evaluate possible risk factors for rickettsial seropositivity. Of the 300 serum samples, six (2.0%) were found to be IgG-positive with a titer of 1:64. While all seropositive individuals were male, the statistical analysis revealed no significant association of gender with rickettsial seropositivity (p=1.000). Other factors including age (p=0.414), residential places of the participants (p=0.347), hunting years (p=0.694) or hunting abroad (p=1.000) did not significantly affect the IgG positivity. Also, no statistical correlation was found between a history of an arthropod (tick, louse, or flea) bite and rickettsial seropositivity (p=1.000). To our knowledge, this is the first study that demonstrates rickettsial seropositivity among human population in northern Cyprus. Our study suggests that awareness should be raised among the people especially involved in outdoor activities such as hunting, and control programs should be implemented to prevent possible rickettsiosis cases. Further serological studies using other Rickettsia spp. antigens, as well as molecular studies that search for Rickettsia spp. in humans, animals and arthropods are needed to obtain more comprehensive data on rickettsiosis in northern Cyprus.

Seroprevalence and hematological abnormalities associated with Ehrlichia canis in dogs referred to a veterinary teaching hospital in central-western Brazil / Soroprevalência e alterações hematológicas associadas com Ehrlichia canis em cães encaminhados a um hospital veterinário-escola no centro-oeste do Brasil

Ticks are significant parasites of dogs in the tropics, where tick-borne pathogens are highly prevalent, especially in areas where tick control measures are frequently neglected. This study investigated the seroprevalence and hematological abnormalities associated with Ehrlichia canis in dogs referred to a veterinary teaching hospital in Central-western Brazil. Out of 264 dogs tested for anti-Ehrlichia canis antibodies by an indirect immunofluorescence assay (IFA), 59.1% (156/264) were positive. Seropositivity was significantly associated to anemia and thrombocytopenia, alone or in combination, and to leukopenia. Conversely, there were no differences in terms of seroprevalence according to sex, breed and age. This study demonstrated that dogs referred to a veterinary teaching hospital in Central-western Brazil are highly exposed to E. canis and that seropositive dogs are more likely to present hematological abnormalities, particularly anemia, thrombocytopenia and leukopenia. To our knowledge, this is the first study on detection of anti-E. canis antibodies by means of IFA among dogs in the state of Goiás. These findings highlighted the need for increasing awareness among dog owners regarding tick control measures in Central-western Brazil, ultimately to reduce the risk of exposure to E. canis and other tick-borne pathogens.

Carrapatos são importantes parasitos de cães nos trópicos, onde patógenos transmitidos por carrapatos são altamente prevalentes, especialmente em áreas onde as medidas de controle de carrapatos são frequentemente negligenciadas. O estudo investigou a soroprevalência e as anormalidades hematológicas associadas à Ehrlichia canis em cães encaminhados para um hospital veterinário-escola no Centro-oeste do Brasil. Dos 264 cães testados para anticorpos anti-Ehrlichia canis por meio da reação de imunofluorescência indireta (RIFI), 59.1% (156/264) foram positivos. A soropositividade foi associada significativamente à anemia e trombocitopenia, isoladamente ou em combinação, e à leucopenia. Por outro lado, não houve diferenças quanto à soroprevalência segundo sexo, raça e idade. Este estudo demonstrou que cães encaminhados a um hospital veterinário-escola na região Centro-oeste do Brasil são altamente expostos à E. canis, e que cães soropositivos têm maior probabilidade de apresentar alterações hematológicas, principalmente anemia, trombocitopenia e leucopenia. Para o nosso conhecimento, este é o primeiro estudo sobre a detecção de anticorpos anti-E. canis por meio da RIFI em cães do estado de Goiás. Essas descobertas destacam a necessidade de aumentar a conscientização entre os proprietários de cães em relação às medidas de controle do carrapato no Centro-oeste do Brasil, em última análise, para reduzir o risco de exposição ao E. canis e outros patógenos transmitidos por carrapatos.

Molecular diagnosis of a family with May-Hegglin anomaly / 中华医学遗传学杂志

Objective@#To explore the molecular basis for a pedigree affected with May-Hegglin anomaly (MHA).@*Methods@#Peripheral blood samples were collected and subjected to DNA extraction. Exons 1, 10, 16, 24, 25, 26, 30, 31, 33, 38 and 40 and flanking sequences of the MYH9 gene were subjected to PCR amplification and Sanger sequencing. Changes in protein expression were determined by an indirect immunofluorescence assay. Platelet aggregation function of the proband was assessed by thromboelastogram.@*Results@#The proband and his second son both carried a heterozygous 5521G>A (GAG→AAG) missense variant in exon 38 of the MYH9 gene, leading to p. Glu1841Lys substitution at position 1841 of amino acid sequence. Immunofluorescence showed inclusions containing NMMHC-ⅡA. Thromboelastogram suggested enhanced platelet aggregation function of the proband.@*Conclusion@#The c. 5521G>A variant of MYH9 gene has co-segregated with the phenotype of MHA in this pedigree. To assess the aggregation function of platelet by thromboelastogram can predict the risk of bleeding in MHA patients.

Rickettsial pathogens circulating in urban districts of Rio de Janeiro, without report of human Brazilian Spotted Fever / Circulação de patógenos riquetsiais em bairros urbanos do Rio de Janeiro sem relatos humanos da Febre Maculosa Brasileira

Abstract Spotted fever group rickettsioses are emerging diseases. In some of these diseases, domestic dogs act as sentinels. Canine serological studies have demonstrated that rickettsial dispersion is concentrated in rural areas, seroprevalence being higher where human rickettsioses are endemic. In Rio de Janeiro, the Atlantic forest vegetation has been devastated by urbanization. In this context, we aimed to detect Rickettsia spp. in urban areas of the West Zone of Rio de Janeiro. Sera from 130 dogs were tested by Indirect Immunofluorescence Assay, and ticks collected from these dogs were tested by polymerase chain reaction. We found the rate of serological reactions against R. rickettsii and R. parkeri in our study area to exceed those of rural and non-endemic areas, highlighting the importance of dogs as urban sentinels. The possibility of contact with opossums and capybaras increased the chances of exposure to Rickettsia spp., reinforcing the hypothetical link between the landscape and the rickettsial wild cycle. Rhipicephalus sanguineus sensu lato was the tick most frequently observed. PCR-positive samples showed similarity with R. rickettsii and R. felis, an emerging pathogen rarely reported from ticks. We observed that rickettsiae circulate in urban places and ticks from indoor environments, which may be involved in bacterial epidemiology.

Resumo Riquetsioses do Grupo da Febre Maculosa são doenças emergentes. Em algumas destas doenças, os cães domésticos agem como sentinelas. Estudos sorológicos caninos têm demonstrado que a dispersão de patógenos rickettsiais está concentrada em áreas rurais, sendo a soroprevalência maior onde as rickettsioses humanas são endêmicas. Na cidade do Rio de Janeiro, a vegetação de Mata Atlântica vem sendo devastada pela urbanização. Nesse contexto, objetivou-se detectar a presença de Rickettsia spp. em áreas urbanas da Zona Oeste do Rio de Janeiro. Amostras de soro obtidas de 130 cães foram testadas, utilizando-se a Imunofluorescência Indireta. Carrapatos coletados desses cães foram testados, utilizando-se a reação em cadeia da polimerase. Observou-se que as taxas de reações sorológicas contra R. rickettsii e R. parkeri nessa área de estudo excederam a prevalência das áreas rurais e não endêmicas, destacando-se a importância dos cães como sentinelas urbanos das rickettsioses. A possibilidade de contato com capivaras e gambás favoreceu a exposição à Rickettsia spp., reforçando a hipótese de ligação entre a paisagem local e o ciclo silvestre de transmissão riquetsial. O carrapato Rhipicephalus sanguineus sensu lato foi encontrado com maior frequência. Amostras com positividade pela PCR mostraram similaridade com R. rickettsii e R. felis, um patógeno emergente raramente descrito em carrapatos. Observou-se circulação riquetsial em áreas urbanas e em carrapatos obtidos do ambiente doméstico, os quais podem estar envolvidos na epidemiologia dessas bactérias.

Seroprevalence of and Identification of Risk Factors in China / 生物医学与环境科学（英文）

Serum samples were tested for IgG antibodies using indirect immunofluorescence assays. We then analyzed associated risk factors. Serum samples were considered positive when reactive at a dilution of more than 1:320. Differences between groups and risk factors associated with exposure were statistically analyzed using Chi-square tests and the generalized linear model. 122 of 1,260 samples (9.68%) were positive for infection. The infection rate ranged from 0% to 30.43% and differed significantly among age groups ( < 0.01); infection rate in the 50-59 years group was significantly higher than that in other age groups. The seroprevalence of varied significantly among sites within the four provinces, and the infection rate of field workers was significantly higher than that of urban workers.

Bacterial expression, preparation and identification of polyclonal antibody against Escherichia coli FtsZ / 生物工程学报

To prepare polyclonal antibody (PcAb) against Escherichia coli filamentous thermosensitive protein Z (Ec-FtsZ), the artificially synthesized gene fragment coding Ec-FtsZ was subcloned into pET-22b(+) plasmid, and Ec-FtsZ protein was expressed in E. coli BL21(DE3) cell under an optimal bacterial expression condition. Then Ec-FtsZ protein was purified by HisTrap affinity chromatography, and the GTPase (Guanosine triphosphatase) activity of purified Ec-FtsZ protein was further analyzed by malachite green assay. Subsequently, the purified Ec-FtsZ protein was used to immunize rat subcutaneously for preparation of anti-Ec-FtsZ PcAb. The results of enzyme-linked immunosorbent assay (ELISA), Western blotting analysis and immunofluorescence assay showed that the titer of PcAb was 1:256 000, and PcAb exhibited a perfect antigenic specificity against purified and endogenous Ec-FtsZ protein. All these data indicated that the anti-Ec-FtsZ PcAb is successfully prepared, which can be used for further cellular function study and biochemical analysis of Ec-FtsZ protein in vivo.

Brazilian spotted fever serological survey among equids at the Guarapiranga Dam area in the city of São Paulo, Brazil / Investigação sorológica da febre maculosa brasileira entre equídeos na área da represa de Guarapiranga, na cidade de São Paulo, Brasil

In the Guarapiranga dam region located in the metropolitan area of São Paulo, human cases have been reported of Brazilian spotted fever (BSF), a tick-borne disease caused by the bacterium Rickettsia rickettsii. In this area, R. rickettsii is known to be transmitted to humans by Amblyomma aureolatum, a typical dog tick that is not associated with horses. In other BSF-endemic areas, R. rickettsii transmission is associated with Amblyomma sculptum, a tick species that typically infest capybaras and horses. The Guarapiranga Dam bears abundant populations of capybaras and horses. However, since nothing is known about a possible cycle of transmission of R. rickettsii by A. sculptum in this area, this study evaluated such transmission by performing a serosurvey of horses living in the Guarapiranga Dam region. A total of 206 equids living in the margins of the Guarapiranga Dam were serologically tested for antibodies reactive to five Rickettsia species, four of the spotted fever group (R. rickettsii, R. parkeri, R. amblyommatis, R. rhipicephali) and one basal group species, R. bellii. Overall, 171 (83%) equids reacted positively to at least one Rickettsia species. A total of 160 (78%), 123 (60%), 80 (39%), 72 (35%), and 71 (34%), equid sera reacted to R. bellii, R. rickettsii, R. parkeri, R. rhipicephali, and R. amblyommatis, respectively, with endpoint titers ranging from 64 to 1024 for R. bellii, and 64 to 512 for the remaining four Rickettsia species. Endpoint titers to R. bellii (median: 256) was significantly higher (P<0.05) than the endpoint titers to the other four Rickettsia species, for which the median values varied from 64 to 128. A total of 65 (32%) equid sera showed endpoint titers to R. bellii at least 4-fold higher than those to any of the other four antigens, indicating that they have been exposed to R. bellii or a very closely related species. Our results provide serological evidence that the sampled equids were not frequently exposed to R. rickettsii-infected ticks. Since horses are a highly suitable sentinel for R. rickettsii transmission by A. sculptum, we conclude that this tick species has no epidemiological role in the transmission of R. rickettsii in the BSF-endemic area of the Guarapiranga Dam in the metropolitan area of São Paulo.(AU)

Na região da represa de Guarapiranga localizada na área metropolitana de São Paulo, têm sido relatados casos humanos de Febre Maculosa Brasileira (FMB), uma doença transmitida por carrapatos causada pela bactéria Rickettsia rickettsii. Nesta área de estudo, R. rickettsii é conhecida por ser transmitida aos seres humanos pelo Amblyomma aureolatum, um carrapato de cão que não está associado a cavalos. Em outras áreas endêmicas da FMB, a transmissão de R. rickettsii está associada ao Amblyomma sculptum, uma espécie de carrapato que normalmente infesta capivaras e cavalos. A represa de Guarapiranga possui populações abundantes de capivaras e cavalos; no entanto, como nada se sabe sobre um possível ciclo de transmissão de R. rickettsii por A. sculptum nessa área, este estudo avaliou essa transmissão realizando um levantamento sorológico em cavalos que vivem na região da represa de Guarapiranga. Um total de 206 equídeos que vivem nas margens da represa de Guarapiranga foram testados sorologicamente para cinco espécies de Rickettsia, sendo quatro do grupo da FMB (R. rickettsii, R. parkeri, R. amblyommatis, R. rhipicephali) e um do grupo basal (R. bellii). No geral, 171 (83%) equídeos reagiram positivamente a pelo menos uma espécie de Rickettsia. Um total de 160 (78%), 123 (60%), 80 (39%), 72 (35%) e 71 (34%), reagiram a R. bellii, R. rickettsii, R. parkeri, R. rhipicephali e R. amblyommatis, respectivamente, com títulos finais variando de 64 a 1024 para R. bellii e 64 a 512 para as quatro espécies restantes de Rickettsia. Os títulos finais para R. bellii (mediana: 256) foram significativamente maiores (P <0,05) do que os títulos para as outras quatro espécies de Rickettsia, para os quais os valores medianos variaram de 64 a 128. Um total de 65 (32%) equideos, os soros mostraram títulos finais para R. bellii pelo menos quatro vezes maior que os de qualquer um dos outros quatro antígenos, indicando que eles foram expostos a R. bellii ou a uma espécie muito próxima. Os resultados obtidos fornecem evidências sorológicas de que os equídeos amostrados não eram frequentemente expostos a carrapatos infectados por R. rickettsii. Como os cavalos são um sentinela altamente adequado para a transmissão de R. rickettsiipor A. sculptum, a conclusão obtida foi que essa espécie de carrapato não tem papel epidemiológico na transmissão da bactéria na área endêmica de FMB da represa de Guarapiranga na região metropolitana de São Paulo.(AU)

Circulation of spotted fever group rickettsiae among dogs seropositive for Leishmania spp. in an urban area of Brazil

Abstract INTRODUCTION: Dogs play an epidemiological role in several vector-borne diseases that affect human and animal health worldwide. We aimed to identify rickettsial circulation among dogs with canine visceral leishmaniasis (CVL) from a region endemic for both diseases. METHODS: CVL-seropositive dogs were screened for spotted fever group rickettsiae using an indirect immunofluorescence assay. RESULTS: Among the CVL-positive dogs, anti-Rickettsia rickettsii antibodies were identified in one asymptomatic and one oligosymptomatic dog. CONCLUSIONS: This study shows low circulation of antibodies to R. rickettsii in CVL-seropositive dogs. It is recommended that surveillance studies in dogs should continue in order to monitor this scenario.

Prozone effect on indirect immunofluorescence assay for anti-nuclear antibody testing / 国际检验医学杂志

Objective To explore the influence of prozone effect on anti-nuclear antibodies (ANA) testing by indirect immunofluorescence assay (IIFA).Methods The samples with high titer of ANA (≥1∶1 000) were selected from 880fresh serum samples, and were subsequently diluted in 1∶100, 1∶1 000and 1∶10 000ratio.Prozone effect was defined as fluorescence intensity from 1∶1 000dilution was stronger than that from1∶100dilution.The samples with prozone effect were determined manually or by Sprinter XL and EUROPattern.The samples with prozone effect were further characterized by combinations of fluorescence patterns, fluorescence intensities and autoantibody specificities.Results A total of 880samples were tested.Importantly, 34samples displayed prozone effect (3.86％in total and 29.57％in samples with ANA≥1∶1 000).Interestingly, prozone effect was identified by manual detection as well as by Sprinter XL with similar fluorescence patterns and fluorescence intensities.Notably, EUROPattern can only select the central area for identification.Among all samples with prozone effect, 74.42％samples exhibited fluorescence intensities of≥1∶10 000.Speckled pattern was the most prevalent fluorescence patterns in samples with prozone effect (46.51％).In addition, anti-RNP antibodies (62.79％) were the most popular autoantibodies in samples with prozone effect, followed by anti-dsDNA antibodies (51.16％) and anti-SSA antibodies (51.16％).Conclusion Prozone effect was present in ANA testing, especially in samples with high titers, resulting in underestimating the titers.The study highlighted that special attention should be paid to the prozone effect in clinical practice.

Diagnostic performance of serological tests to detect antibodies against acute scrub typhus infection in central India

Background: Differentiating scrub typhus from other acute febrile illness is difficult due to non specificity of clinical symptoms and relative absence of eschar in Indian population. The diagnosis thus relies mainly on laboratory tests. Antibody based serological tests are mainstay of scrub typhus diagnosis. Here, we evaluated the diagnostic performance of IgM ELISA, IgM IFA and ICT to detect antibodies against O. tsutsugamushi in acute serum of febrile patients. Methodology: The serum samples from 600 randomly selected patients suffering from acute undifferentiated fever were tested by all the three tests mentioned above. We used latent class analysis to generate unbiased results as all the tests for scrub typhus diagnosis are imperfect and none of them can be considered as reference standard. Results: We found that IgM ELISA with cutoff titer 0.5 OD has high diagnostic accuracy (sensitivity 99.9% and specificity 99.15) than IgM IFA (sensitivity 96.8% and specificity 99.7%) for scrub typhus diagnosis. ICT used in our study had very high specificity 100% but low sensitivity (38%) which would limit its use for acute serum samples. ICT being a screening or point of care test, has to be more sensitive while some compromise with specificity is affordable. Hence, optimal cutoff for ICT should be evaluated under different settings. Conclusion: IgM ELISA being simple and affordable could be an alternative diagnostic test to IgM IFA which is subjective and costly.

Analysis of the results of 55240 children for detection of seven respiratory viruses in Guangdong Zhongshan district / 国际检验医学杂志

Objective To investigate the epidemic situation of children with respiratory viruses in zhongs-han ,Guangdong to provide evidence for the diagnosis of respiratory virus infections in children .Methods 55 240 cases were collected in a hospital from November 25 ,2011 to September 30 ,2016 ,Influenza virus(IFA , IFB) ,parainfluenza virus (PIV1 ,PIV2 ,PIV3) ,respiratory syncytial virus (RSV) and adenovirus (ADV) were detected by direct immunofluorescent ,and analyzed the results .Results The positive rate of virus infection in 55 240 children was 23 .25%,of which RSV 53 .75%,IFA 13 .83%,ADV10 .81%,PIV3 10 .77%,IFB 6 .49%, PIV1 2 .37%,PIV2 1 .14% and mixed infection 0 .84% .There were statistical significance between male and female (P＜0 .05) .The positive rates of virus infection in children 0- ≤1 years and 1- ≤3 years were higher than those in the other age groups ,the difference was statistically significant (P＜0 .05) .The positive rate of RSV was higher in both age groups (71 .92%,46 .23%) The positive rate of these 7 viruses infection in winter and spring was higher than that in summer and autumn ,the difference was statistically significant (P＜0 .05) , and the positive rate of RSV was the highest .The positive rate of these 7 viruses patients with bronchitis was higher than that of the other patients ,the difference was statistically significant (P＜0 .05) and in 108 patients with mixed infections ,the most cases was patients with RSV (90 cases) .Conclusion The main pathogen is RSV .The infection rate of children under 3 years old is the highest .Winter and spring are the high incidence of respiratory virus infection in children in Guangdong zhongshan district .

Development and verification of indirect immunofluorescence assay for detection of baculovirus titer / 中华微生物学和免疫学杂志

Objective To develop, optimize and preliminarily verify an indirect immunofluores-cence assay ( IFA) for detecting the titer of recombinant baculovirus. Methods Conditions for performing IFA, including cell concentration, co-incubation time, reaction temperature, dilution ratio, reaction time and types of fixative solution, blocking liquid and antibodies, were optimized to establish an IFA method for the detection of baculovirus titer. Specificity, accuracy, reproducibility and intermediate precision of the es-tablished assay were verified. And the results were compared with those of baculovirus rapid titer kit. Re-sults The optimal cell concentration for coating was 0. 6×106 cell/ml, and the optimal reaction time be-tween viruses with cells was 3 d. The optimal conditions for conducting IFA were as follows: formaldehyde buffered acetone (-20℃) was used as fixative, normal goat serum was used as blocking liquid and the first and second generation antibodies at a dilution of 1 : 200 were incubated at 37℃ for 1 h, respectively. Spe-cific fluorescence was observed only in baculovirus but not in others by using the method. No significant difference in virus titers was observed between the established IFA and baculovirus rapid titer kit. The two methods showed a good linear correlation (R2=0. 996). Coefficients of variation for evaluating the reproduc-ibility and intermediate precision were less than 10％. Conclusion IFA for the detection of baculovirus ti-ter was established with good specificity, accuracy, reproducibility and intermediate precision.

Seroprevalence of Rickettsia rickettsii and Rickettsia parkeri in dogs during a Brazilian Spotted Fever outbreak in the State of Rio de Janeiro / Soroprevalência de Rickettsia rickettsii e Rickettsia parkeri em cães durante um surto de Febre Maculosa Brasileira no Estado do Rio de Janeiro

The present paper is the first to perform this evaluation in dogs from the cities of Natividade, Porciuncula and Varre-Sai. The aim of this study is to search for Spotted Fever Group Rickettsia in canine sera using indirect immunofluorescence assay and to identify the probable causative agent of sera reactions in animals. Of the 253 sampled canines, 67.59% (171/253) were seroreactive for Rickettsia rickettsii and 11.07% (28/253) for Rickettsia parkeri, both in dilution 1:64. Titration of tested sera against R. rickettsii antigens reached 1:131.072 and, for R. parkeri, 1:4.096. We conclude that dogs are important sentinels for R. rickettsii infection, and can be infected regardless of sex, age, the habit of visiting woodlands or being in direct contact with equines and capybaras. Serological diagnosis has highlighted many dogs infected by R. rickettsii, and ambient conditions, such as the presence of flowing water bodies, was important for the occurrence of Brazilian Spotted Fever in the northwestern of Rio de Janeiro State.(AU)

O presente trabalho é o primeiro a ser realizado com cães nos municípios de Natividade, Porciúncula e Varre-Sai e tem por objetivo pesquisar Rickettsias do Grupo da Febre Maculosa em soros de cães, por meio da reação de imunofluorescência indireta, e identificar o provável agente causador da reação sorológica nos animais. Dos caninos amostrados, 67,59% (171/253) foram sororreativos para Rickettsia rickettsii e 11,07% (28/253) para Rickettsia parkeri, ambos em diluição de 1:64. A titulação dos soros testados contra antígenos de R. rickettsii chegou a 1:131.072, e para R. parkeri, 1:4.096. Conclui-se que cães são importantes sentinelas para a infecção por R. rickettsii, independente de sexo, idade, hábito de visitar ambientes florestais ou de estarem em contato direto com equinos e capivaras. O diagnóstico sorológico permitiu evidenciar muitos cães infectados por R. rickettsii, e condições ambientais, como a presença de áreas ribeirinhas, foram importantes para a ocorrência de Febre Maculosa Brasileira na região noroeste do Estado do Rio de Janeiro.(AU)

Seropositivity for Rickettsia spp. and Ehrlichia spp. in the human population of Mato Grosso, Central-Western Brazil

Abstract INTRODUCTION The epidemiology of Rickettsia and Ehrlichia species infection is underestimated in Mato Grosso State. METHODS: Serum samples obtained during a Dengue outbreak in 2011-2012 were tested via indirect immunofluorescence and/or ELISA. RESULTS: Samples from 19/506 (3.8%) patients presented antibodies for at least one of three Rickettsia species; 2/506 (0.4%) samples reacted against Ehrlichia canis. Most afflicted patients are residents of cities from the south-central region of the state, where these diseases have been reported in animals. CONCLUSIONS: These results show serological evidence of human exposure to Rickettsia and Ehrlichia species in Mato Grosso State.

Analysis of anti-nuclear antibody and anti-nuclear antibody spectrum in 2 325 patients / 国际检验医学杂志

Objective To investigate the clinical value of anti-nuclear antibody(ANA) and anti-nuclear antibody spectrum(ANAs) detection.Methods A total of 2 325 patients with or suspected with autoimmune diseases(AID) were enrolled and detected for ANA and ANAs by using indirect immunofluorescence assay(IIF) and linear immunoblot assay(LIA) respectively.All detected results were analyzed.Results Among 2 325 patients,896 cases(38.54%) were positive with ANA,with positive rate of 45.46% in female patients,which was higher than the 18.46% of male patients(P<0.05),and the common fluorescence patterns were nuclear particle pattern,nuclear homogeneous pattern and the nucleolus pattern.816 cases(35.10%) were positive with ANAs,and the positive rates of anti-Sjogren's syndrome(SS)-B antibody,anti Ro-52 antibody and anti SS-A antibody were relatively higher.The consistency rate of the two methods was 91.66%.Conclusion ANA and ANAs detection could be with certain correlation,but might be not completely consistent,detection could improve the detection rate and reduce the missed detection rate.

Quantitative detection on different HBsAg levels by chemiluminescence immunoassay and time-resolved immunofluorescence assay / 中国感染控制杂志

Objective To evaluate the accuracy and feasibility of time-resolved immunofluorometric assay (TRI FA) for detection of HBsAg based on Abbott automated chemiluminescence immunoassay(CMIA),so as to carry out this project in primary hospitals,and provide reference for individual antiviral strategy and prediction of therapeutic effect.Methods Serum of 157 patients infected with hepatitis B virus were detected with CMIA and TRIFA,specimens with HBsAg titers exceeding the detection limit were firstly diluted,then performed quantitative analysis.HBsAg levels were divided into 4 groups:≤100 IU/mL,101-1 000 IU/mL,1 001-20 000 IU/mL,and ＞ 20 000 IU/mL,quantitative correlation between two methods was analyzed.Results The linear regression equation of two methods was Y=2.323X-896.3,correlation coefficent r=0.943,P＜0.001.CMIA was as a reference,4 groups were divided for analysis,results showed that when detected specimens was at low concentration of HBsAg,TRIFA value was low compared with CMIA method,while detected specimens was at high concentration of HB sAg,CMIA value was high,two reagents had good consistency in the detection of different concentrations of HBsAg(both P＜0.05),when concentration was at 1 001-20 000 IU/mL,consistency was the best.Conclusion The accuracy of two reagents in the quantitative detection of HBsAg is similar,and the best correlation of detection value is 1 000-20 000 IU/mL.TRIFA assay has wide application for its low-cost and easy to be operated,which is especially suitable for primary hospitals.

Large-Scale in-House Cell-Based Assay for Evaluating the Serostatus in Patients with Neuromyelitis Optica Spectrum Disorder Based on New Diagnostic Criteria

BACKGROUND AND PURPOSE: The detection of aquaporin 4-IgG (AQP4-IgG) is now a critical diagnostic criterion for neuromyelitis optica spectrum disorder (NMOSD). To evaluate the serostatus of NMOSD patients based on the 2015 new diagnostic criteria using a new in-house cell-based assay (CBA). METHODS: We generated a stable cell line using internal ribosome entry site-containing bicistronic vectors, which allow the simultaneous expression of two proteins (AQP4 and green fluorescent protein) separately from the same RNA transcript. We performed in-house CBA using serum from 386 patients: 178 NMOSD patients diagnosed according to the new diagnostic criteria without AQP4-IgG, 63 high risk NMOSD patients presenting 1 of the 6 core clinical characteristics of NMOSD but not fulfilling dissemination in space, and 145 patients with other neurological diseases, including 66 with multiple sclerosis. The serostatus of 111 definite and high risk NMOSD patients were also tested using a commercial CBA kit with identical serum to evaluate the correlation between the 2 methods. All assays were performed by two independent and blinded investigators. RESULTS: Our in-house assay yielded a specificity of 100% and sensitivities of 80% (142 of 178) and 76% (48 of 63) when detecting definite- and high risk NMOSD patients, respectively. The comparison with the commercial CBA kit revealed a correlation for 102 of the 111 patients: no correlation was present in 7 patients who were seronegative using the commercial method but seropositive using the in-house method, and in 2 patients who were seropositive using the commercial method but seronegative using the in-house method. CONCLUSIONS: These results demonstrate that our in-house CBA is a highly specific and sensitive method for detecting AQP4-IgG in NMOSD patients.

Performance of an Automated Fluorescence Antinuclear Antibody Image Analyzer

BACKGROUND: The gold standard for antinuclear antibody (ANA) screening is the indirect immunofluorescence (IIF) assay with human epithelial cells (HEp-2). However, a number of substantial disadvantages of manual IIF assays have highlighted the need for the automation and standardization of fluorescent ANA (FANA) testing. We evaluated the performance of EUROPattern Suite (Euroimmun AG, Germany), an automated FANA image analyzer, with regard to ANA detection and pattern recognition compared with conventional manual interpretation using the fluorescence microscopic IIF assay. METHODS: A total of 104 samples including 70 ANA-positive sera and 34 ANA-negative sera collected from September to October 2015 were included. The sensitivity, specificity, and pattern recognition function were evaluated to determine the performance of EUROPattern Suite compared with the manual IIF assay results. RESULTS: The sensitivity and specificity of EUROPattern Suite for ANA detection were 94.3% and 94.1%, respectively. The concordance rate between the two methods was 94.2%. For pattern recognition, 45.7% of the samples were assigned identical ANA patterns including simple and mixed. When major pattern matching was considered, 83.7% (41/49) and 95.2% (20/21) of the samples with simple and mixed patterns, respectively, showed concordant results between the two methods. CONCLUSIONS: EUROPattern Suite, an automated FANA image analyzer, provides a viable option for distinguishing between positive and negative results, although the ability to assign specific patterns is insufficient to replace manual microscopic interpretation. This automated system may increase efficiency in laboratories, in which a large number of samples need to be processed.

Occurrence of anti-Toxoplasma gondii antibodies and parasite DNA in backyard chicken breeding in Northeast, Brazil / Ocorrência de anticorpos anti-Toxoplasma gondii e DNA do parasita em galinhas de criações domésticas no Nordeste, Brasil

Abstract The aim of the present study was to investigate the occurrence of anti-Toxoplasma gondii antibodies and parasite DNA in backyard chickens bred in the metropolitan area of Recife, Brazil. In total, 212 serum samples were collected from 16 properties, and 12 backyard chickens were collected in the six sanitary districts of Recife. An indirect immunofluorescence assay (IFA) was used to investigate the occurrence of anti-Toxoplasma gondii antibodies. Polymerase chain reaction (PCR) was used to detect T. gondii DNA in brain, heart, liver and lung specimens. Of the samples analyzed by serology, 86/212 (40.56%) were positive; of the samples analyzed by PCR, 2/12 (16.7%) were positive, with both samples positive by both tests (serological and molecular). The presence of antibody anti-T. gondii and parasite DNA in tissues of these animals are worrying aspects for public health because there is a risk of transmission of the parasite to humans through eating undercooked or raw meat. Based on the results, the adoption of preventive measures to prevent the cats access to the chickens creations should be encouraged, since these animals were identified in most of the studied properties.

Resumo O objetivo do presente estudo foi investigar a ocorrência de anticorpos anti-Toxoplasma gondii e de DNA do parasito em galinhas de criações domésticas, na região metropolitana de Recife, Brasil. No total, 212 amostras de soro foram coletadas de aves de 16 estabelecimentos e de 12 galinhas de criações domésticas nos seis distritos sanitários de Recife. Para a pesquisa de anticorpos anti-Toxoplasma gondii foi utilizada a Reação de Imunofluorescência Indireta (RIFI). A Reação em Cadeia da Polimerase (PCR) foi utilizada para detectar o DNA de T. gondii em fragmentos de cérebro, coração, fígado e pulmão. Das amostras analisadas por sorologia, 86/212 (40,56%) foram positivas. Das amostras analisadas por PCR, 2/212 (16,7%) foram positivas, em ambos os testes (sorológicos e moleculares). A presença de anticorpos anti-T. gondii e de DNA parasitário nos tecidos desses animais são aspectos preocupantes para saúde pública, porque há o risco de transmissão do parasita para humanos através da ingestão de carne mal cozida ou crua. Com base nos resultados obtidos, a adoção de medidas preventivas que evitem o acesso de gatos às criações de galinhas deve ser incentivada, uma vez que esses animais foram identificados na maioria das propriedades estudadas.