Application of molecular techniques in the study of Staphylococcus aureus clonal evolution - A Review

Staphylococcus aureus is an important agent of healthcare-associated and community-acquired infections. A major characteristic of this microorganism is the ability to develop resistance to antimicrobial agents. Several molecular techniques have been applied for the characterization of S. aureus in epidemiological studies. In the present review, we discuss the application of molecular techniques for typing S. aureus strains and describe the nomenclature and evolution of epidemic clones of this important pathogen.

Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99% for both tests) and specificities (100% for both tests) were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Parvovirus B19 infections in state of Rio de Janeiro, Brasil: 526 sera analyzed by IgM-enzyme-linked immunosorbent assay and polymerase chain reaction

In this study were analyzed 526 sera; the patients aged from two days to 65 years old presenting exanthema, which was the most frequent symptom observed, besides fever, adenomegaly, and arthralgia. These sera were negative by enzyme-linked immunosorbent assay (IgM-ELISA) for either rubella (495), toxoplasma (41), cytomegalovirus (12), measles (40), dengue (56), and they were submitted to nested polymerase chain reaction (PCR) for B19 DNA and commercial IgM-ELISA for B19. In 39 abortion cases, IgM or DNA were not detected, therefore they were not took into account for analysis. Specific DNA and IgM were detected respectively in 71 (14.5%) and IgM in 62 (12.7%) sera from 487 sera analyzed. IgM and DNA were simultaneously detected in 43 (8.8%), while agreement among the results by PCR and IgM-ELISA was observed in 440 (90.4%). The sera were collected from January 1999 to December 2000, most of them in 1999 (325), during winter and spring. The major number of clinical cases was observed in the age group from one to ten years old. IgM or DNA were detected in 23 from 51 municipal districts of the state of Rio de Janeiro, where the samples were collected.

Regulating inflammation through the anti-inflammatory enzyme platelet-activating factor-acetylhydrolase

Platelet-activating factor (PAF) is one of the most potent lipid mediators involved in inflammatory events. The acetyl group at the sn-2 position of its glycerol backbone is essential for its biological activity. Deacetylation induces the formation of the inactive metabolite lyso-PAF. This deacetylation reaction is catalyzed by PAF-acetylhydrolase (PAF-AH), a calcium independent phospholipase A2 that also degrades a family of PAF-like oxidized phospholipids with short sn-2 residues. Biochemical and enzymological evaluations revealed that at least three types of PAF-AH exist in mammals, namely the intracellular types I and II and a plasma type. Many observations indicate that plasma PAF AH terminates signals by PAF and oxidized PAF-like lipids and thereby regulates inflammatory responses. In this review, we will focus on the potential of PAF-AH as a modulator of diseases of dysregulated inflammation.

Evaluation of the dot enzyme-linked immunosorbent assay in comparison with standard ELISA for the immunodiagnosis of human toxocariasis

A dot enzyme-linked immunosorbent assay (dot-ELISA) was standardized using excretory-secretory antigens of Toxocara canis for the rapid immunodiagnosis of human toxocariasis. Thirty patients with clinical signs of toxocariasis, 20 cases with other parasitic diseases, and 40 healthy subjects were tested. A total of 0.2 ng of antigen per dot, serum dilution of 1:160 and dilution conjugate of 1:1000 were found optimal. The sensitivity and specificity of the assay were 100 and 95%, respectively. Comparable sensitivity of dot-ELISA and the standard ELISA was obtained, but only 3 cross-reactions occurred in the dot-ELISA, compared with 6 in the standard ELISA. Dot-ELISA is simple to perform, rapid, and low cost. Large-scale screening studies should be done to evaluate its usefulness under field conditions.

Detection and molecular characterization of group A rotavirus from hospitalized children in Rio de Janeiro, Brazil, 2004

Rotavirus is a major cause of infantile acute diarrhea, causing about 440,000 deaths per year, mainly in developing countries. The World Health Organization has been recommending the assessment of rotavirus burden and strain characterization as part of the strategies of immunization programs against this pathogen. In this context, a prospective study was made on a sample of 134 children with acute diarrhea and severe dehydration admitted to venous fluid therapy in two state hospitals in Rio de Janeiro, Brazil, from February to September 2004. Rotavirus where detected by polyacrylamide gel electrophoresis (PAGE) and by an enzyme-linked immunoassay to rotavirus and adenovirus (EIARA) in 48% of the children. Positive samples for group A rotavirus (n = 65) were analyzed by reverse transcription/heminested multiplex polymerase chain reaction to determine the frequency of G and [P] genotypes and, from these, 64 samples could be typed. The most frequent G genotype was G1 (58%) followed by G9 (40%). One mixed infection (G1/G9) was detected. The only [P] genotype identified was [8]. In order to estimate the rotavirus infection frequency in children who acquired diarrhea as hospital infection in those hospitals, we studied 24 patients, detecting the pathogen in 41% of them. This data suggest that genotype G9 is an important genotype in Rio de Janeiro, with implications to the future strategies of vaccination against rotavirus, reinforcing the need of continuous monitoring of circulating strains of the pathogen, in a surveillance context.

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax antibodies and its use in epidemiological surveys

There are data indicating that the distribution of Trypanosoma vivax in the Brazilian territory is expanding with potential to reach other areas, where the vectors are present. The detection of anti-trypanosomal antibodies in serum provides important information of the trypanosomal status in cattle herds. For this reason, an enzyme-linked immunosorbent assay (Tv-ELISA-Ab) with crude antigen from one Brazilian isolate of T. vivax was developed and evaluated. The sensitivity and specificity were respectively 97.6 and 96.9%. In the evaluation of cross-reactions, three calves inoculated with T. evansi trypimastigotes blood forms showed optical densities (OD) under the cut-off during the whole experimental period, except one at 45 days post-inoculation. With relation to Babesia bovis, B. bigemina, and Anaplasma marginale, which are endemic hemoparasites in the studied area, the cross-reactions were shown to be 5.7, 5.3, and 1.1%, respectively. The first serological survey of Pantanal and state of Pará showed that T. vivax is widespread, although regions within both areas had significantly different prevalences. Therefore, this Tv-ELISA-Ab may be a more appropriate test for epidemiological studies in developing countries because the diagnostic laboratories in most countries may be able to perform an ELISA, which is not true for polymerase chain reaction.

The schistosome enzyme that activates oxamniquine has the characteristics of a sulfotransferase

Available evidence suggests that the antischistosomal drug oxamniquine is converted to a reactive ester by a schistosome enzyme that is missing in drug-resistant parasites. This study presents data supporting the idea that the active ester is a sulfate and the activating enzyme is a sulfotransferase. Evidence comes from the fact that the parasite extract loses its activating capability upon dialysis, implying the requirement of some dialyzable cofactor. The addition of the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) restored activity of the dialyzate, a strong indication that a sulfotransferase is probably involved. Classical sulfotransferase substrates like beta-estradiol and quercetin competitively inhibited the activation of oxamniquine. Furthermore, these substrates could be sulfonated in vitro using an extract of sensitive (but not resistant) schistosomes. Gel filtration analysis showed that the activating factor eluted in a fraction corresponding to a molecular mass of about 32 kDa, which is the average size of typical sulfotransferase subunits. Ion exchange and affinity chromatography confirmed the sulfotransferase nature of the enzyme. Putative sulfotransferases present in schistosome databases are being examined for their possible role as oxamniquine activators.

Evaluation of a malaria antibody enzyme immunoassay for use in blood screening

Transfusion-transmitted malaria is rare, but it may produce severe problem in the safety of blood transfusion due to the lack of reliable procedure to evaluate donors potentially exposed to malaria. Here, we evaluated a new enzyme-linked immunosorbent assay malaria antibody test (ELISA malaria antibody test, DiaMed, Switzerland) to detect antibodies to Plasmodium vivax (the indigenous malaria) in the blood samples in the Republic of Korea (ROK). Blood samples of four groups were obtained and analyzed; 100 samples from P.vivax infected patients, 35 from recovery patients, 366 from normal healthy individuals, and 325 from domestic travelers of non-endemic areas residents to risky areas of ROK. P.vivax antibody levels by ELISA were then compared to the results from microscopic examination and polymerase chain reaction (PCR) test. As a result, the ELISA malaria antibody test had a clinical sensitivity of 53.0% and a clinical specificity of 94.0% for P.vivax. Twenty out of 325 domestic travelers (6.2%) were reactive and 28 cases (8.6%) were doubtful. Of the reactive and doubtful cases, only two were confirmed as acute malaria by both microscopy and PCR test. Thus we found that the ELISA malaria antibody test was insufficiently sensitive for blood screening of P.vivax in ROK.

New finding of Giardia intestinalis (Eukaryote, Metamonad) in Old World archaeological site using immunofluorescence and enzyme-linked immunosorbent assays

In this study, nine organic sediment samples from a medieval archaeological site at Pineuilh, France, were examined for Giardia intestinalis using two commercially available immunological kits [enzyme-linked immuno sorbent and immunofluorescence (IFA) assays]. Both techniques detected G. intestinalis in one sample, dated to 1,000 Anno Domini. This is the first time IFA was successfully used to detect protozoa in Old World archaeological samples. Such immunological techniques offer important perspectives concerning ancient protozoa detection and identification.

Cheap, rapid and efficient DNA extraction method to perform multilocus microsatellite genotyping on all Schistosoma mansoni stages

Schistosomes are endoparasites causing a serious human disease called schistosomiasis. The quantification of parasite genetic diversity is an essential component to understand the schistosomiasis epidemiology and disease transmission patterns. In this paper, we propose a novel assay for a rapid, low costly and efficient DNA extraction method of egg, larval and adult stages of Schistosoma mansoni. One euro makes possible to perform 60,000 DNA extraction reactions at top speed (only 15 min of incubation and 5 handling steps).

Evaluation of an enzyme-linked immunoelectrotransfer blot test for the confirmatory serodiagnosis of human toxocariasis

To improve the serodiagnosis of human toxocariasis, a sensitive and specific enzyme-linked immunoelectrotransfer blot (EITB-IgG) test was developed and evaluated using Toxocara canislarvae excretory-secretory antigens for detecting anti-Toxocara IgG antibodies. The EITB-IgG profile of toxocariasis was characterized by comparing 27 sera from patients with toxocariasis, 110 sera from healthy subjects and 186 sera from patients with other helminth diseases (ascariasis, ancylostomiasis, trichuriasis, enterobiasis, strongyloidiasis, hymenolepiasis, diphyllobothriasis, taeniasis, cysticercosis, hydatidosis and fascioliasis). Antigenic bands of 24, 28, 30, 35, 56, 117, 136 and 152 kDa were predominantly recognized in sera from all patients with toxocariasis. However, only bands of 24-35 kDa were highly specific for Toxocara infection (98.3%), whereas other antigenic bands observed displayed cross-reactivity. Additionally, when the results of the EITB-IgG test were compared to those of the ELISA-IgG test, a 100% concordance was observed for positive results in human toxocariasis cases. The concordance for negative results between the two tests for healthy subjects and patients with other helminth diseases were 96.3% and 53.7%, respectively, showing that the EITB-IgG test has a higher specificity than ELISA. In conclusion, the EITB-IgG test is a very useful tool to confirm the serological diagnosis of human toxocariasis.

Pulsed-field gel electrophoresis, virulence determinants and antimicrobial susceptibility profiles of type Ia group B streptococci isolated from humans in Brazil

Group B streptococci (GBS) infections occur worldwide. Although serotyping has been used for epidemiologic purposes, this does not accurately characterize enough members of a genetically heterogeneous bacterial population. The aims of this work were to evaluate the genetic diversity of 45 type Ia GBS strains isolated in Brazil by pulsed-field gel electrophoresis as well as to evaluate antimicrobial susceptibility profiles and identify virulence genes. Twenty-four strains were assigned to cluster A. All strains under study contained the hylB and scpB genes. The bca gene was detected in only 10 strains and none of the streptococci carried the bac gene. Thirty-nine strains were resistant to tetracycline.

Identification of uropathogenic Escherichia coli clonal group A (CgA) in hospitalised patients

This study provides the first description of healthcare-associated infections with Escherichia coli clonal group A (CgA) isolates in Latin America. Isolates were typed by enterobacterial repetitive intergenic consensus-PCR, pulsed-field gel electrophoresis, E. coli phylogenetic grouping, multilocus sequence typing and fimH single nucleotide polymorphism analysis. Out of 42 E. coli hospital isolates studied, three belonged to E. coli phylogenetic group D and ST69 and had fimH sequences identical to that of the CgA reference strain ATCC BAA-457. E. coli CgA is another potential source of resistant infections in hospitals.