Walter Oelemann

Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus: methicillin-resistant isolates are detected directly in blood cultures by multiplex PCR.

In this study, we standardized and evaluated a multiplex-PCR methodology using specific primers to identify Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus and their methicillin-resistance directly from blood cultures. Staphylococci clinical isolates (149) and control strains (16) previously identified by conventional methods were used to establish the multiplex PCR protocol. Subsequently, this methodology was evaluated using a fast and cheap DNA extraction protocol from 25 staphylococci positive blood cultures. A wash step of the pellet with 0.1% bovine serum albumin (BSA) solution was performed to reduce PCR inhibitors. Amplicons of 154bp (mecA gene), 271bp (S. haemolyticus mvaA gene) and 108 and 124bp (S. aureus and S. epidermidis species-specific fragments, respectively) were observed. Reliable results were obtained for 100% of the evaluated strains, suggesting that this new multiplex-PCR combined with an appropriate DNA-extraction method could be useful in the laboratory for fast and accurate identification of three staphylococci species and simultaneously their methicillin resistance directly in blood cultures.

Humoral response to HspX and GlcB to previous and recent infection by Mycobacterium tuberculosis

BackgroundTuberculosis (TB) remains a major world health problem. Around 2 billions of people are infected by Mycobacterium tuberculosis, the causal agent of this disease. This fact accounts for a third of the total world population and it is expected that 9 million people will become infected each year. Only approximately 10% of the infected people will develop disease. However, health care workers (HCW) are continually exposed to the bacilli at endemic sites presenting increased chance of becoming sick. The objective of this work was to identify LTBI (latent tuberculosis infection) among all asymptomatic HCW of a Brazilian Central Hospital, in a three year follow up, and evaluate the humoral response among HCW with previous and recent LTBI to recombinant HspX and GlcB from M. tuberculosis.MethodsFour hundred and thirty seven HCW were screened and classified into three different groups according to tuberculin skin test (TST) status: uninfected, previous LTBI and recent LTBI. ELISA test were performed to determine the humoral immune response to HspX and GlcB.ResultsThe levels of IgG and IgM against the HspX and GlcB antigens were the same among HCW with recent and previous LTBI, as well as among non infected HCW. However, the IgM levels to HspX was significantly higher among HCW with recent LTBI (OD = 1.52 ± 0.40) than among the uninfected (OD = 1.09 ± 0.50) or subjects with previous LTBI (OD = 0.96 ± 0.51) (p < 0.001).ConclusionIgG and IgM humoral responses to GlcB antigens were similar amongst all studied groups; nevertheless IgM levels against HspX were higher among the recent LTBI/HCW.

Interference of intradermal tuberculin tests on the serodiagnosis of paratuberculosis in cattle

In this experiment 63 animals from a paratuberculosis (PTB) and tuberculosis-free herd were tested by Intradermal Tuberculin Tests (ITT) and blood samples were collected before PPD inoculation and on days 3, 15, 30, 60 and 90 post-inoculation (p.i.). Sera were tested for PTB-specific antibodies by ELISA-PPA and confirmed by a commercial ELISA. Three (4.76%) animals were positive by ELISA-PPA and five (7.93%) in the commercial ELISA, between days 30 and 90 p.i. These results suggest that ITT can interfere in the reliability of ELISAs and that serological testing for PTB should be avoided for 90d after PPD inoculation.

In situ hybridization: A molecular approach for the diagnosis of the microsporidian parasite Enterocytozoon bieneusi

Microsporidia are emerging as opportunistic pathogens in patients with acquired immunodeficiency syndrome (AIDS). Enterocytozoon bieneusi is the most commonly reported microsporidium that is detected in gastrointestinal specimens. This report describes an in situ hybridization technique with a 30-base specific synthetic DNA probe for detection of E bieneusi by light microscopy. Formalin-fixed paraffin-embedded duodenal biopsy specimens from three patients with AIDS, chronic diarrhea, and E bieneusi infection confirmed by electron microscopy were used in this study. Light microscopic examination after colorimetric detection allowed the identification of different stages of the pathogens life cycle in the cytoplasm of enterocytes. No cross-reactivity was noted between the probe and human DNA. Our study underscores the applicability of a synthetic-labeled oligonucleotide for the detection and identification of E bieneusi in clinical samples.

The use of MPB70 and MPB83 to distinguish between bovine tuberculosis and paratuberculosis

In order to demonstrate the potential to distinguish paratuberculosis (PTB) from bovine tuberculosis infection (TB), ELISAs with M. bovis-specific MPB70 or MPB83 as capture antigens were developed and tested on two groups of cattle: Group A comprised 23 animals positive for Mycobacterium avium paratuberculosis (Map) and TB free. Group B comprised 48 animals from a Map free herd during the previous 5 years, but confirmed as tuberculous by positive results on PPD testing and M. bovis culture. Results demonstrated a significant difference (p<0.01) between reactivity of sera from these groups, encouraging the study of purified proteins to differentiate between both diseases.

Detection of Mycobacterium tuberculosis in sputum from Suruí Indian subjects, Brazilian Amazon.

This investigation aimed at the detection of Mycobacterium tuberculosis (MTB) in the sputum of Suruí Indian subjects from Amazonia, Brazil. Polymerase chain reaction analyses were positive for 12 samples, five of which were also culture-positive (N = 147). Four MTB genotypes were identified, one of which showed resistance to rifampicin and isoniazid. The study also highlighted one village complex as of particular importance, considering the relatively high number of tuberculosis cases reported and of MTB isolates obtained.

Detection of human parvovirus B19 infection: a study of 212 suspected cases in the state of Rio de Janeiro, Brazil

BACKGROUND Parvovirus B19 infections are associated with different clinical manifestations that vary from symptom-less to severe. The main clinical manifestations are erythema infectiosum or fifth disease, transient aplastic crisis in individuals with hemoglobinopathies, chronic anemia in the immunocompromised, acute polyarthralgia syndrome in adults, hydrops fetalis, spontaneous abortion and stillbirth. Although the classical features of B19 and rubella infections are distinct, uncommon presentations can lead to misdiagnosis. OBJECTIVE The goal of this study was to assess the occurrence of parvovirus B19 (B19) infection in patients with clinical signs of toxoplasmosis or rubella, both of which were not confirmed by laboratorial techniques. STUDY DESIGN Serum samples from 214 patients were collected between January 1996 and December 1997 in the state of Rio de Janeiro, Brazil, B19 specific IgG and IgM were detected by using commercial enzyme-linked immunosorbent assays (ELISA), and viral nucleic acid was detected employing a nested polymerase chain reaction (PCR) protocol. RESULTS Combining the results obtained by IgM ELISA and PCR, 14.5% of the samples were positive in one or both tests, with a concordance of 92.5% between the two techniques. CONCLUSIONS Specimens collected in 16 out of 22 municipalities were positive in at least one out of the three tests employed, indicating that parvovirus B19 circulates in several regions of the state of Rio de Janeiro.

Improvement of an in-house ELISA for bovine paratuberculosis serology in Brazil

Paratuberculosis (Johnes disease) is a chronic enteritis in cattle caused by infection with Mycobacterium avium subsp. paratuberculosis (Map). In Brazil, few reports describe the isolation of the organism. The gold standard test for Map is its isolation from tissues or feces. Moreover, bacterial growth is slow and test results are available only after three to five months of incubation Furthermore, shedding of bacilli at levels detectable by fecal culture is irregular and does not occur during the early stages of infection, which compromises the sensitivity of this methodology. The most common immunological tests to identify Map infection are complement fixation test (CFT), agar gel immunodiffusion (AGID) and enzyme-linked immunoassay (ELISA). In Brazil, commercial ELISA kits are imported and too expensive to be used as part of diagnostic laboratorial routine. Apart from that, their use has not yet been approved in the country. The aim of the present study was to improve an original assay PPA-ELISA protocol established by our group, and to determine sensitivity and specificity of the modified test. In a first step, we introduced modifications that minimized plate-to-plate and between-well variations, thus making the test more stable and reliable. In the second part of this study, a panel of 106 sera samples was tested by this modified PPA-ELISA protocol in order to estimate its sensitivity and specificity. The new assay presented overall sensitivity of 76.9% and specificity of 70.0%. Our study demonstrated that this assay could be recommended as a valuable tool for diagnosis and control of paratuberculosis in Brazil and other developing countries.

The Mycobacterium avium ssp. paratuberculosis specific mptD gene is required for maintenance of the metabolic homeostasis necessary for full virulence in mouse infections.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johnes disease, a chronic granulomatous enteritis in ruminants. Furthermore, infections of humans with MAP have been reported and a possible association with Crohns disease and diabetes type I is currently discussed. MAP owns large sequence polymorphisms (LSPs) that were exclusively found in this mycobacteria species. The relevance of these LSPs in the pathobiology of MAP is still unclear. The mptD gene (MAP3733c) of MAP belongs to a small group of functionally uncharacterized genes, which are not present in any other sequenced mycobacteria species. mptD is part of a predicted operon (mptABCDEF), encoding a putative ATP binding cassette-transporter, located on the MAP-specific LSP14. In the present study, we generated an mptD knockout strain (MAPΔmptD) by specialized transduction. In order to investigate the potential role of mptD in the host, we performed infection experiments with macrophages. By this, we observed a significantly reduced cell number of MAPΔmptD early after infection, indicating that the mutant was hampered with respect to adaptation to the early macrophage environment. This important role of mptD was supported in mouse infection experiments where MAPΔmptD was significantly attenuated after peritoneal challenge. Metabolic profiling was performed to determine the cause for the reduced virulence and identified profound metabolic disorders especially in the lipid metabolism of MAPΔmptD. Overall our data revealed the mptD gene to be an important factor for the metabolic adaptation of MAP required for persistence in the host.

Sensitivity of AMPLICOR MTB on direct detection of Mycobacterium tuberculosis in smear-negative specimens from outpatients in Rio de Janeiro

We evaluated the performance of the AMPLICOR MTB assay for detection of M. tuberculosis in clinical specimens obtained from 98 smear-negative tuberculous patients (43 with positive and 55 with negative culture results). The sensitivity of the AMPLICOR MTB was 44.9% (44/98) and that of culture was 43.8% (43/98). No significant difference was observed between the results obtained by AMPLICOR MTB and by culture. We conclude that amplification assays could be used for testing smear-negative specimens because a rapid diagnosis will benefit those patients.