Maria Carmen Arroyo Sanchez

Multicenter Evaluation of a Sequence-Based Protocol for Subtyping Shiga Toxins and Standardizing Stx Nomenclature

ABSTRACT When Shiga toxin-producing Escherichia coli (STEC) strains emerged as agents of human disease, two types of toxin were identified: Shiga toxin type 1 (Stx1) (almost identical to Shiga toxin produced by Shigella dysenteriae type 1) and the immunologically distinct type 2 (Stx2). Subsequently, numerous STEC strains have been characterized that express toxins with variations in amino acid sequence, some of which confer unique biological properties. These variants were grouped within the Stx1 or Stx2 type and often assigned names to indicate that they were not identical in sequence or phenotype to the main Stx1 or Stx2 type. A lack of specificity or consistency in toxin nomenclature has led to much confusion in the characterization of STEC strains. Because serious outcomes of infection have been attributed to certain Stx subtypes and less so with others, we sought to better define the toxin subtypes within the main Stx1 and Stx2 types. We compared the levels of relatedness of 285 valid sequence variants of Stx1 and Stx2 and identified common sequences characteristic of each of three Stx/Stx1 and seven Stx2 subtypes. A novel, simple PCR subtyping method was developed, independently tested on a battery of 48 prototypic STEC strains, and improved at six clinical and research centers to test the reproducibility, sensitivity, and specificity of the PCR. Using a consistent schema for nomenclature of the Stx toxins and stx genes by phylogenetic sequence-based relatedness of the holotoxin proteins, we developed a typing approach that should obviate the need to bioassay each newly described toxin and that predicts important biological characteristics.

A Major Secreted Elastase Is Essential for Pathogenicity of Aeromonas hydrophila

ABSTRACT Aeromonas hydrophila is an opportunistic pathogen and the leading cause of fatal hemorrhagic septicemia in rainbow trout. A gene encoding an elastolytic activity, ahyB, was cloned from Aeromonas hydrophila AG2 into pUC18 and expressed inEscherichia coli and in the nonproteolytic speciesAeromonas salmonicida subsp. masoucida. Nucleotide sequence analysis of the ahyB gene revealed an open reading frame of 1,764 nucleotides with coding capacity for a 588-amino-acid protein with a molecular weight of 62,728. The first 13 N-terminal amino acids of the purified protease completely match those deduced from DNA sequence starting at AAG (Lys-184). This finding indicated that AhyB is synthesized as a preproprotein with a 19-amino-acid signal peptide, a 164-amino-acid N-terminal propeptide, and a 405-amino-acid intermediate which is further processed into a mature protease and a C-terminal propeptide. The protease hydrolyzed casein and elastin and showed a high sequence similarity to other metalloproteases, especially with the mature form of thePseudomonas aeruginosa elastase (52% identity),Helicobacter pylori zinc metalloprotease (61% identity), or proteases from several species of Vibrio (52 to 53% identity). The gene ahyB was insertionally inactivated, and the construct was used to create an isogenic ahyB mutant ofA. hydrophila. These first reports of a defined mutation in an extracellular protease of A. hydrophila demonstrate an important role in pathogenesis.

Identification of Staphylococcus spp. by PCR-Restriction Fragment Length Polymorphism of gap Gene

ABSTRACT Oligonucleotide primers specific for the Staphylococcus aureus gap gene were previously designed to identify 12Staphylococcus spp. by PCR. In the present study,AluI digestion of PCR-generated products rendered distinctive restriction fragment length polymorphism patterns that allowed 24 Staphylococcus spp. to be identified with high specificity.

Malaria diagnosis from pooled blood samples: comparative analysis of real-time PCR, nested PCR and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale

Malaria diagnoses has traditionally been made using thick blood smears, but more sensitive and faster techniques are required to process large numbers of samples in clinical and epidemiological studies and in blood donor screening. Here, we evaluated molecular and serological tools to build a screening platform for pooled samples aimed at reducing both the time and the cost of these diagnoses. Positive and negative samples were analysed in individual and pooled experiments using real-time polymerase chain reaction (PCR), nested PCR and an immunochromatographic test. For the individual tests, 46/49 samples were positive by real-time PCR, 46/49 were positive by nested PCR and 32/46 were positive by immunochromatographic test. For the assays performed using pooled samples, 13/15 samples were positive by real-time PCR and nested PCR and 11/15 were positive by immunochromatographic test. These molecular methods demonstrated sensitivity and specificity for both the individual and pooled samples. Due to the advantages of the real-time PCR, such as the fast processing and the closed system, this method should be indicated as the first choice for use in large-scale diagnosis and the nested PCR should be used for species differentiation. However, additional field isolates should be tested to confirm the results achieved using cultured parasites and the serological test should only be adopted as a complementary method for malaria diagnosis.

LABORATORY DIAGNOSIS OF MAIN INFECTIOUS AND AUTOIMUNE DISEASES

The laboratory diagnostic methods have increasingly incorporated fully automated and computerized procedures. In addition to the immunoassays that are available for detection of antigens, antibodies or other markers, the “methods of molecular biology and nanotechnology employed in microarrays or multiplex systems” has been having an increasing use in the clinical practice, as they have defined profiles for different clinical situations. If on one hand this large amount of information helps diagnosis, on the other hand, this diversity of methods and technologies may generate “doubts regarding the interpretation of the clinical value of the results, bewildering the professionals in relation to their initial clinical suspicion”.

Malária humana: padronização e optimização de testes sorológicos para diagnóstico individual e inquéritos soroepidemiológicos

The indirect immunofluorescence antibody test (IFA) is normally employed as reference test in the serology of malaria. In this report we standardized and optimized the test, for our condition, utilizing P. falciparum obtained from human blood on culture and P. vivax obtained from human blood as antigens, for detection of IgG and IgM antibodies. Some technical variables, were tested and best resulto were obtained when sera were diluted in PBS containing 1% Tween-80 and the slides, containing the antigenic preparation were fixed in cold acetone or stabilized on dried air with silica. The ELISA test was standardized for P. falciparum antibodies and the comparison of the IFA and ELISA showed: a) in P. falciparum prime infected patient the sensitivity was 71% for both tests; b) in P. vivax prime infected patients the sensitivity was 40% for both tests; c) in non prime infected patients with P. falciparum malaria the sensitivity was 100% for both tests; d) in non prime infected patients with P. vivax malaria the sensitivity was 85% for ELISA and 92% for IFA; e) in patients with P. vivax and P. falciparum malaria the sensitivity was 100% for both tests. The specificity was 95% for ELISA and 100% for IFA in non malaria individuals. The results showed that the ELISA test could be an alternative for IFA for IgG antibodies in the serology of malaria.O teste de imunofluorescencia indireta (IFI) e considerado teste de referencia na soroiogia da malaria. Neste trabalho procuramos optimizar o teste empregando P. falciparum obtido de sangue humano e de cultura e P. vivax obtido de sangue de paciente como antigenos, para pesquisa de anticorpos IgG e IgM. Das variaveis tecnicas estudadas melhores resultados foram obtidos quando os soros foram diluidos ern PBS contendo 1% de Tween 80 e as lâminas contendo a suspensao antigenica foram estabilizadas em dessecadores ou fixadas com acetona. Foi tambem padronizado o teste imunoenzimatico ELISA com antigenos de P. falciparum obtidos em cultura. O estudo comparativo com o teste de imunofluorescencia indireta para pesquisa de anticorpos IgG mostrou: a) nos pacientes primo infectados por P. falciparum a sensibilidade para ambos os testes foi de 71%; b) nos pacientes primo infectados pelo P. vivax a sensibilidade foi de 40% para ambos os testes; c) nos pacientes nao primo infectados e com malaria atual pelo P. falciparum a sensibilidade para ambos os testes foi de 100%; d) nos pacientes nao primo infectados e com malaria atual pelo P. vivax a sensibilidade foi de 85% para o teste ELISA e de 92% para a IFI; e) nos pacientes com malaria mista a sensibilidade para ambos os testes foi de 100%. A especificidade da IFI foi de 100% e do teste ELISA 95% nos casos de individuos nao malaricos. Os resultados obtidos sugerem ser o teste ELISA, uma boa alternativa para o teste de IFI, para a pesquisa de anticorpos IgG anti P. falciparum, na soroiogia da malaria.

Prevalence and risk factors for quinolone resistance among Escherichia coli strains isolated from males with community febrile urinary tract infection

The purpose of this study was to evaluate the prevalence and clinical risk factors for quinolone resistance (QR) in E. coli strains from males with febrile urinary tract infection (FUTI). An ambispective cross-sectional study was performed in which we evaluated 153 males with a community FUTI caused by E. coli. Among the 153 FUTI episodes, 101 (66%) were due to quinolone susceptible E. coli strains while 52 (34%) were caused by QR E. coli strains. In the univariate analysis QR was associated with older age, higher Charlson scores, dementia, past UTI, urinary tract abnormalities, previous antibiotic use, particularly with fluoroquinolones (FQ), a healthcare-associated (HA)-UTI (HA-UTI) and to four of the components included in the definition of HA-UTI: hospital admission, nursing home residence, indwelling urethral catheter and invasive urinary instrumentation. In the multivariate analysis, HA-UTI (OR 3.82, 95% CI 1.3–11.24; P 0.015) and use of antimicrobials in the previous month (OR 5.82, 95% CI 2.3–14.88; P < 0.001) mainly with FQ (OR 13.97, 95% CI 2.73–71.53; P 0.002) were associated with QR. To have a HA-UTI and a previous use of FQ in the preceding month were strong risk factors for QR E. coli, and thus empirical antimicrobial treatment with quinolones should be avoided in these patients.

Recombinant Leishmania infantum Heat Shock Protein 83 for the Serodiagnosis of Cutaneous, Mucosal, and Visceral Leishmaniases

Routine serological diagnoses for leishmaniases, except in visceral cases, are performed using whole-parasite antigens. We used enzyme-linked immunosorbent assay (ELISA) to evaluate the performance of Leishmania infantum rHsp83 compared with L. major-like total promastigote antigen in the diagnosis of cutaneous (CL), mucosal (ML), and visceral leishmaniases (VL). ELISA-rHsp83 was significantly more sensitive than ELISA–L. major-like when considering either CL/ML (P = 0.041) or all leishmaniasis patients (P = 0.013). When samples from other infectious disease patients were evaluated for cross-reactivity, ELISA-rHsp83 was more specific than ELISA–L. major-like, specifically for Chagas disease samples (P < 0.001). We also evaluated the anti-rHsp83 antibody titers months after treatment and observed no significant difference in ML (P = 0.607) or CL (P = 0.205). We recommend ELISA–L. infantum-rHsp83 as a routine confirmatory serological assay for the diagnosis of Leishmania infection because of the high sensitivity, the specificity, and the insignificant cross-reactivity with other infectious diseases.

Unexpected detection of Plasmodium vivax and Plasmodium falciparum DNA in asymptomatic blood donors: fact or artifact?

A study searching for Plasmodium vivax and Plasmodium falciparum DNA among blood donors from the non-endemic area in Brazil reported a rate of 7.41%. This number is at least three times higher than what has been observed in blood donors from the Amazon, an endemic area concentrating >99% of all malaria cases in Brazil. Moreover, the majority of the donors were supposedly infected by P. falciparum, a rare finding both in men and anophelines from the Atlantic forest. These findings shall be taken with caution since they disagree with several publications in the literature and possibly overestimate the actual risk of malaria transmission by blood transfusion in São Paulo city.

Malaria in pregnant women living in areas of low transmission on the southeast Brazilian Coast: molecular diagnosis and humoural immunity profile

Studies on autochthonous malaria in low-transmission areas in Brazil have acquired epidemiological relevance because they suggest continued transmission in what remains of the Atlantic Forest. In the southeastern portion of the state of São Paulo, outbreaks in the municipality of Juquitiba have been the focus of studies on the prevalence of Plasmodium, including asymptomatic cases. Data on the occurrence of the disease or the presence of antiplasmodial antibodies in pregnant women from this region have not previously been described. Although Plasmodium falciparum in pregnant women has been widely addressed in the literature, the interaction of Plasmodium vivax and Plasmodium malariae with this cohort has been poorly explored to date. We monitored the circulation of Plasmodium in pregnant women in health facilities located in Juquitiba using thick blood film and molecular protocols, as well as immunological assays, to evaluate humoural immune parameters. Through real-time and nested polymerase chain reaction, P. vivax and P. malariae were detected for the first time in pregnant women, with a positivity of 5.6%. Immunoassays revealed the presence of IgG antibodies: 44% for ELISA-Pv, 38.4% for SD-Bioline-Pv and 18.4% for indirect immunofluorescence assay-Pm. The high prevalence of antibodies showed significant exposure of this population to Plasmodium. In regions with similar profiles, testing for a malaria diagnosis might be indicated in prenatal care.