Identification of respiratory virus in infants with congenital heart disease by comparison of different methods

Respiratory virus infections are the main cause of infant hospitalization and are potentially severe in children with congenital heart disease (CHD). Rapid and sensitive diagnosis is very important to early introduction of antiviral treatment and implementation of precautions to control transmission, reducing the risk of nosocomial infections. In the present study we compare different techniques in the diagnosis of respiratory viruses in CHD infants. Thirty-nine samples of nasopharyngeal aspirate were obtained from CHD infants with symptoms of respiratory infection. The Multiplex PCR (Seeplex® RV 12 ACE Detection) driven to the detection of 12 respiratory viruses was compared with the direct immunofluorescence assay (DFA) and PCR, both targeting seven respiratory viruses. The positivity found by DFA, Multiplex and PCR was 33.3%, 51.3% and 48.7%, respectively. Kappa index comparing DFA and Multiplex, DFA and PCR and PCR and Multiplex PCR was 0.542, 0.483 and 0.539, respectively. The concordance between techniques was considered moderate. Both Multiplex PCR (p = 0.001) and PCR (p = 0.002) detected significantly more respiratory virus than DFA. As the performance of the tests may vary, the combination of two or more techniques may increase diagnostic sensitivity favoring the diagnosis of co-infections, early introduction of antiviral therapy and implementation of appropriate measures.

Use of elisa employing homologous and heterologous antigens for the detection of IgG and subclasses (IgG1 and IgG2) in the diagnosis of Canine visceral leishmaniasis

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.

Evaluation of dengue NS1 antigen detection for diagnosis in public health laboratories, São Paulo State, 2009

The present work evaluated the diagnostic accuracy of detection of Dengue NS1 antigen employing two NS1 assays, an immunochromatographic assay and ELISA, in the diagnostic routine of Public Health laboratories. The results obtained with NS1 assay were compared with virus isolation and, in a subpopulation of cases, they were compared with the IgM-ELISA results obtained with convalescent samples. A total of 2,321 sera samples were analyzed by one of two NS1 techniques from March to October 2009. The samples were divided into five groups: groups I, II and III included samples tested by NS1 and virus isolation, and groups IV and V included patients with a first sample tested by NS1 and a second sample tested by IgM-ELISA. Sensitivity, specificity, positive and negative predictive values, Kappa Index and Kappa Concordance were calculated. The results showed that NS1 testing in groups I, II and III had high sensitivity (98.0%, 99.5% and 99.3%), and predictive values and Kappa index between 0.9 - 1.0. Groups IV and V only had Kappa Concordance calculated, since the samples were analyzed according to the presence of NS1 antigen or IgM antibody. Concordance of 92.1% was observed when comparing the results of NS1-negative samples with IgM-ELISA. Based on the findings, it is possible to suggest that the tests for NS1 detection may be important tools for monitoring the introduction and spread of Dengue serotypes.

Temporal differences in blood meal detection from the midguts of Triatoma infestans

We used genus/species specific PCRs to determine the temporal persistence of host DNA in Triatoma infestans experimentally fed on blood from six common vertebrate species: humans, domestic dogs, guinea pigs, chickens, mice, and pigs. Twenty third or fourth instar nymphs per animal group were allowed to feed to engorgement, followed by fasting-maintenance in the insectary. At 7, 14, 21, or 28 days post-feeding, the midgut contents from five triatomines per group were tested with the respective PCR assay. DNA from all vertebrate species was detected in at least four of five study nymphs at seven and 14 days post-feeding. DNA of humans, domestic dogs, guinea pigs, pigs, and chickens were more successfully detected (80-100%) through day 21, and less successfully (20-100%) at day 28. Findings demonstrate that species-specific PCRs can consistently identify feeding sources of T. infestans within two weeks, a biologically relevant time interval.

Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

Detection of Bartonella henselae DNA in clinical samples including peripheral blood of immune competent and immune compromised patients by three nested amplifications

Bacteria of the genus Bartonella are emerging pathogens detected in lymph node biopsies and aspirates probably caused by increased concentration of bacteria. Twenty-three samples of 18 patients with clinical, laboratory and/or epidemiological data suggesting bartonellosis were subjected to three nested amplifications targeting a fragment of the 60-kDa heat shock protein (HSP), the internal transcribed spacer 16S-23S rRNA (ITS) and the cell division (FtsZ) of Bartonella henselae, in order to improve detection in clinical samples. In the first amplification 01, 04 and 05 samples, were positive by HSP (4.3%), FtsZ (17.4%) and ITS (21.7%), respectively. After the second round six positive samples were identified by nested-HSP (26%), eight by nested-ITS (34.8%) and 18 by nested-FtsZ (78.2%), corresponding to 10 peripheral blood samples, five lymph node biopsies, two skin biopsies and one lymph node aspirate. The nested-FtsZ was more sensitive than nested-HSP and nested-ITS (p < 0.0001), enabling the detection of Bartonella henselae DNA in 15 of 18 patients (83.3%). In this study, three nested-PCR that should be specific for Bartonella henselae amplification were developed, but only the nested-FtsZ did not amplify DNA from Bartonella quintana. We conclude that nested amplifications increased detection of B. henselae DNA, and that the nested-FtsZ was the most sensitive and the only specific to B. henselae in different biological samples. As all samples detected by nested-HSP and nested-ITS, were also by nested-FtsZ, we infer that in our series infections were caused by Bartonella henselae. The high number of positive blood samples draws attention to the use of this biological material in the investigation of bartonellosis, regardless of the immune status of patients. This fact is important in the case of critically ill patients and young children to avoid more invasive procedures such as lymph nodes biopsies and aspirates.

Variability in Galactomannan detection by platelia Aspergillus EIA™ according to the Aspergillus species

Here we investigate the extent to which different Aspergillus species release galactomannan (GM) in vitro. Marked variability was observed in GM reactivity between and within Aspergillus species, with A. terreus strains showing the highest GM indexes. The in vivo significance of these findings remains to be determined.

APPLICATION OF 6-NITROCOUMARIN AS A SUBSTRATE FOR THE FLUORESCENT DETECTION OF NITROREDUCTASE ACTIVITY IN Sporothrix schenckii

Introduction Sporothrix schenckii is a thermal dimorphic pathogenic fungus causing a subcutaneous mycosis, sporotrichosis. Nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several derivatives, already used in several other enzyme assays. The objective of this study was the analysis of 6-nitrocoumarin (6-NC) as a substrate to study the nitroreductase activity in Sporothrix schenckii. Methods Thirty-five samples of S. schenckii were cultivated for seven, 14 and 21 days at 35 °C in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (6-AC) dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a negative control, for posterior examination under an epifluorescence microscope. The organic layer of the seven, 14 and 21-day cultures was analyzed by means of direct illumination with 365 nm UV light and by means of elution on G silica gel plate with hexane:ethyl acetate 1:4 unveiled with UV light. Results All of the strains showed the presence of 6-AC (yellow fluorescence) and 6-hydroxylaminocoumarin (blue fluorescence) in thin layer chromatography, which explains the green fluorescence observed in the fungus structure. Conclusion The nitroreductase activity is widely distributed in the S. schenckii complex and 6-NC is a fluorogenic substrate of easy access and applicability for the nitroreductase activity detection.

MULTIPLEX SYBR® GREEN-REAL TIME PCR (qPCR) ASSAY FOR THE DETECTION AND DIFFERENTIATION OF Bartonella henselae AND Bartonella clarridgeiae IN CATS

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonellaspecies, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

TEMPORAL TRENDS IN THE DETECTION RATES OF HEPATITIS B IN THE SANTA CATARINA STATE, BRAZIL

Hepatitis B is a serious public health problem. The state of Santa Catarina presents areas of high endemicity. The aim of this study was to describe temporal trends in detection rates of hepatitis B in the period from 2002 to 2009 in Santa Catarina and in its regions. A time series study was carried out. Crude rates were calculated and standardized by age using the direct method. Annual variation percentages were estimated by Joinpoint regression. There were two distinct and significant trends in Santa Catarina. From 2002 to 2006 a significant increase of 5.9% per year was observed. From 2006, there was a significant decrease of 6.4% per year. In this same period the southern and far-western regions had significant increases of 15.9% and 4.6% and significant decreases of 7.5% and 4.8%, respectively. Greater Florianópolis and Northeast also showed significant increases until 2006, of 15.4% and 17.4%, respectively. In the following period, non-significant decreases of 5.8% and 9.8% respectively were observed. Foz do Rio Itajaí and Planalto Serrano showed non-significant increases up to half of the studied period of 21.1% and 12.0%, respectively and after, significant decreases of 21.5% and 18.0%, respectively. Vale do Itajaí showed a significant decrease of 9.7%; Planalto Norte showed a non-significant decrease of 0.6% and Midwest a non-significant increase of 2.7% per year, in the period from 2002 to 2009.

MOLECULAR DETECTION OF Leishmania IN PHLEBOTOMINE SAND FLIES IN A CUTANEOUS AND VISCERAL LEISHMANIASIS ENDEMIC AREA IN NORTHEASTERN BRAZIL

Several phlebotomine sand fly species have been regarded as putative or proven vectors of parasites of the genus Leishmania in Brazil, but data for the northeastern region remains incipient. In this study, a total of 600 phlebotomine sand flies were grouped in pools of 10 specimens each and tested by a Leishmania genus-specific PCR and by a PCR targeting Leishmania (Leishmania) infantum. Fourteen out of 60 pools were positive by the genus-specific PCR, being five pools of L. migonei, seven of L. complexa, one of L. sordellii and one of L. naftalekatzi, which correspond to a minimal infection rate of 2.3% (14/600). Our results, associated with their known anthropophily and their abundance, suggest the participation of L. migonei and L. complexa as vectors of Leishmania in northeastern Brazil. Remarkably, this is the first time in this country that the detection of Leishmania DNA in L. sordellii and L. naftalekatzi has been reported, but future studies are necessary to better understand the significance of these findings.

DETECTION OF Leishmania (Viannia) IN Nyssomyia neivai AND Nyssomyia whitmani BY MULTIPLEX POLYMERASE CHAIN REACTION, IN SOUTHERN BRAZIL

Sandflies transmit pathogens of leishmaniasis. The natural infection of sandflies by Leishmania (Viannia) was assessed in municipalities, in the state of Paraná, in Southern Brazil. Sandflies were collected with Falcão and Shannon traps. After dissection in search of flagellates in digestive tubes and identification of the species, female sandflies were submitted to the Multiplex Polymerase Chain Reaction (multiplex PCR) for detection of the fragment of the kDNA of Leishmania (Viannia) and the fragment from the IVS6 cacophony gene region of the phlebotomine insects. The analysis was performed in pools containing seven to 12 guts from females of the same species. A total of 510 female sandflies were analyzed, including nine Migonemyia migonei, 17 Pintomyia fischeri, 216 Nyssomyia neivai, and 268 Nyssomyia whitmani. Although none of the females was found naturally infected by flagellates through dissection, the fragment of DNA from Leishmania (Viannia) was shown by multiplex PCR in one sample of Ny. neivai (0.46%) and three samples of Ny. whitmani (1.12%). It was concluded that Ny. neivai and Ny. whitmani are susceptible to Leishmania infection, and that multiplex PCR can be used in epidemiological studies to detect the natural infection of the sandfly vector, because of its sensitivity, specificity and feasibility.

HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES

This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries.

DETECTION OF VIRULENCE GENES IN ENVIRONMENTAL STRAINS OF Vibrio cholerae FROM ESTUARIES IN NORTHEASTERN BRAZIL

The objectives of this study were to detect the presence of Vibrio cholerae in tropical estuaries (Northeastern Brazil) and to search for virulence factors in the environmental isolates. Water and sediment samples were inoculated onto a vibrio-selective medium (TCBS), and colonies with morphological resemblance to V. cholerae were isolated. The cultures were identified phenotypically using a dichotomous key based on biochemical characteristics. The total DNA extracted was amplified by PCR to detect ompW and by multiplex PCR to detect the virulence genes ctx, tcp, zot and rfbO1. The results of the phenotypic and genotypic identification were compared. Nine strains of V. cholerae were identified phenotypically, five of which were confirmed by detection of the species-specific gene ompW. The dichotomous key was efficient at differentiating environmental strains of V. cholerae. Strains of V. cholerae were found in all four estuaries, but none possessed virulence genes.