Opportunistic Pathogens最新文献

Until recently, the lack of suitable type specific assays has hampered the serological diagnosis of herpes simplex virus (HSV) infections, due to the high crossreactivity between types 1 and 2. The aim of the present paper is the evaluation of new commercial methods for the detection of HSV-1 and HSV-2-specific IgG using glycoprotein G as antigen (BioElisa HSV-1 and BioElisa HSV-2), in their application to viral diagnosis and seroepidemiological studies. Eighty two serum samples from HSV recent infections (30 samples from 13 cases), normal children (28 samples), and patients attended in clinics for sexually transmitted diseases (STD) (24 samples from 20 patients) were studied by such methods and the results compared with those obtained by a conventional indirect ELISA, and by the complement fixation test. The methods gave a type specific identification of the antibody response in nine of the 13 HSV patients. Positive results for anti-HSV-2 IgG were obtained in four cases among the STD patients but in none among the normal children. Nineteen of the former and seven of the latter were positive for anti-HSV-1. Only one sample was reactive in the HSV-1 assay, and negative by the indirect ELISA. Type specific HSV-1 and HSV-2 assays may help the serological diagnosis of HSV infections, since they allow the correct characterization of the antibody response. Bearing in mind the high specificity of the method for HSV-2 IgG, it might be useful in screening of populations for anti-HSV-2 and especially in prevention of the neonatal HSV-2 infection.

A new specific serodiagnosis system for Lyme disease was developed using the highly specific partial peptide of outer surface protein C of Borrelia burgdorferi. Finally three peptides (OspC-I, -II and -III) were selected from the outer surface protein C (OspC) amino acid sequence of Borrelia burgdorferi sensu lato and were synthesized. OspC-I is located in the region that is conserved among species of Lyme disease spirochetes, whereas OspC-II and -III are located in the variable regions of the OspC from B. garinii type strain 20047. An enzyme-linked immunosorbent assay (ELISA) to detect antibodies against these synthetic peptides was carried out using sera from patients with Lyme disease. Furthermore, sera from patients with syphilis, tsutsugamushi disease and rheumatoid arthritis were used as control sera to demonstrate specificity of each peptide in the ELISA. The results showed that the false positive results in control sera of OspC-I, -II and -III ELISA for immunoglobulin M (IgM) antibody were 8, 22 and 16%, and those for IgG antibody were 11, 43 and 35%, respectively. These results suggested that the ELISA using OspC-I was the most specific. Therefore, sera from patients with Lyme disease were tested OspC-I ELISA. Of the 21 patients, 12 were in the acute phase and nine in the convalescent phase, 17 (81%) were positive by IgM or IgG ELISA. The sensitivities of IgM and IgG ELISA were 83 and 33% for acute-phase sera, and 22 and 78% for convalescent-phase sera, respectively, suggesting that the IgM response to OspC-I peptide was often detectable in the early stage of infection. Our data demonstrated that OspC-I was one of the common epitopes among species of Lyme disease spirochetes, and therefore this is a suitable antigen for serodiagnosis of early stage Lyme disease with high specificity.

Pulsed field gel electrophoresis (PFGE) after restriction with the enzyme sfiI, restriction fragment length polymorphism (RFLP) with the enzyme EcoRI, random amplification of polymorphic DNA (RAPD) with the probes 5′−3′ GCTGGTGG and GCG CAC GG and Southern blot hybridization with the species specific DNA probe 27A were used to type 19 fluconazole resistant, MIC ≥ 12.5 μg/ml, isolates of Candida albicans. These were associated with ten HIV positive patients where there had been a clinical failure on fluconazole and who were present concurrently in Monsall Hospital. Seventeen of the isolates were biotype 1. This study demonstrated that each patient was infected by a strain with a unique genotype and there was no evidence of crossinfection. Toothbrushes and, for fluconazole sensitive isolates, bed cups were heavily contaminated emphasising the need for their safe disposal. Low discrimination was produced by PFGE (2 types) based on changes in band pattern after variation in the unstable ribosomal chromosome had been excluded. Better discrimination was obtained with RFLP (10 types) RAPD (10 types) PFGE incorporating changes in band width and position (11 types) of SfiI digestion (11 types) and by Southern blot hybridization (12 types).

A new sandwich enzyme-linked immunosorbent assay (ELISA), capable of detecting 1 ng of galactomannan (GM) per ml, was prospectively evaluated for use in the diagnosis of invasive aspergillosis (IA). In the study, 723 serum samples sequentially collected from 76 immunocompromised children were analysed. The detection of GM was positive in 15 patients, and IA developed in eight of them. For seven patients, the results were considered false positives. No case of IA was observed in patients negative for GM. In this pilot study, the sensitivity, specificity, positive and negative predictive values of the test were 100%, 89%, 53% and 100% respectively. In six patients with IA, antigen detection was positive before the onset of clinical or radiological signs. Seven infected patients who cleared their antigenemia survived to aspergillosis. One patient with continuously high levels of GM died of uncontrolled infection. In spite of the possibility of false positive results, sandwich ELISA is a highly sensitive method, useful for the early diagnosis of IA and for the follow-up of infected patients.

Simple assays which reliably distinguish between past infection with Chlamydia pneumoniae and with Chlamydia trachomatis are needed. We developed an enzyme-linked immunosorbent assay (ELISA) for this purpose by reducing antigen cross reactive lipopolysaccharide epitopes with deoxycholate treatment and releasing antibodies of low affinity with a 6 M urea wash step. Paired serum samples from 212 patients with non-C. pneumoniae-associated community acquired pneumonia and single serum samples from 61 healthy students, 61 women with gynaecological complaints, and 100 blood donors were tested in the assay system. Excellent positive/negative correlation with the microimmunofluorescence (MIF) test was shown. This urea ELISA assay may be useful in assessment of past infection with these organisms, especially in epidemiological studies.

The use of polymerase chain reaction (PCR) for the detection of cytomegalovirus (CMV) in bronchoalveolar lavages (BAL) of immunocompromised patients was evaluated. Firstly, PCR was compared to conventional cell culture (CCC) and shell vial assay (SVA) in 65 BAL received in our laboratory on a routine basis. Sensitivity and specificity of the PCR were 100% and 87.2% respectively. The PCR method was then applied to the monitoring of 55 BAL from nine lung transplant recipients. The data illustrate the excellent sensitivity of PCR. However the predictive value of the test for the occurrence of a symptomatic infection was weak.

Several Microsporum species are common and important human pathogens capable of causing dermatophytosis (also known as tinea or ringworm). The current laboratory procedures involving microscopic examination and in vitro culture are either time-consuming or lacking specificity. In this report, we examined five Microsporum species (i.e., M. canis, M. audouinii, M. cookei, M. gypseum and M. nanum) together with a number of Trichophyton and Epidermophyton dermatophytes in arbitrarily primed PCR. The results obtained indicated that these five Microsporum species formed characteristic DNA band patterns, which were different from each other and also from other dermatophytes such as Trichophyton and Epidermophyton using the random primer 5′-ACGGGCCAGT-3′ in the AP-PCR, allowing their rapid differentiation. In addition, although T. rubrum and T. soudanense had similar bands and were not distinguishable by this primer, other Trichophyton species such as T. mentagrophytes, T. tonsurans, T. verrucosum and T. violaceum displayed distinct band patterns in the AP-PCR. Therefore, this primer is potentially useful for determination of Trichophyton dermatophytes as well.

The ability to determine various mycobacterial species rapidly and precisely is important for the control and prevention of mycobacterial infections in the human population. In the present study, we evaluated the Roche Amplicor Mycobacterium tuberculosis test and the Amplicor M. avium/M. intracellulare test, as well as a multiplex PCR, for diagnosis of mycobacterial infections using 86 clinical (sputum and bronchial washing) and culture specimens from 50 human patients. The Roche Amplicor M. tuberculosis test and the Amplicor M. avium/M. intracellulare test appeared to be highly sensitive and specific for direct detection of M. tuberculosis and M. avium/M. intracellulare, respectively, from clinical specimens. The use of culture derivatives was not necessary. Nonetheless, the Roche Amplicor tests had limited value in identifying other mycobacterial species as these mycobacteria invariably displayed negative reactions in the assays. The multiplex PCR, on the other hand, was capable of differentiating M. tuberculosis, M. avium and M. intracellulare from other mycobacterial species using culture specimens at relatively low cost. However, the multiplex PCR generated inconsistent results when clinical samples were used. Only 42 (48.8%) of the 86 sputum and bronchial washing specimens showed clear, specific electrophoretic bands in the multiplex PCR. Future optimisation of a hybridisation-based detection system is essential for enhanced identification of mycobacteria directly from clinical specimens by the multiplex PCR.