Detection of Legionella pneumophila by real-time PCR for the mip gene

A real-time PCR assay for the mip gene of Legionella pneumophila was tested with 27 isolates of L. pneumophila, 20 isolates of 14 other Legionella species, and 103 non-Legionella bacteria. Eight culture-positive and 40 culture-negative clinical specimens were tested. This assay was 100% sensitive and 100% specific for L. pneumophila.     

Utility of real-time PCR for diagnosis of Legionnaires' disease in routine clinical practice

The main aim of our study was to determine the added value of PCR for the diagnosis of Legionnaires’ disease (LD) in routine clinical practice. The specimens were samples submitted for routine diagnosis of pneumonia from December 2002 to November 2005. Patients were evaluated if, in addition to PCR, the results of at least one of the following diagnostic tests were available: (i) culture for Legionella spp. on buffered charcoal yeast extract agar or (ii) detection of Legionella pneumophila antigen in urine specimens. Of the 151 evaluated patients, 37 (25%) fulfilled the European Working Group on Legionella Infections criteria for a confirmed case of LD (the “gold standard”). An estimated sensitivity, specificity, and overall percent agreement of 86% (32 of 37; 95% confidence interval [CI] = 72 to 95%), 95% (107 of 112; 95% CI = 90 to 98%), and 93% (139 of 149), respectively, were found for 16S rRNA-based PCR, and corresponding values of 92% (34 of 37; 95% CI = 78 to 98%), 98% (110 of 112; 95% CI = 93 to 100%), and 97% (144 of 149), respectively, were found for the mip gene-based PCR. A total of 35 patients were diagnosed by using the urinary antigen test, and 34 were diagnosed by the 16S rRNA-based PCR. With the mip gene PCR one more case of LD (n = 36; not significant) was detected. By combining urinary antigen test and the mip gene PCR, LD was diagnosed in an additional 4 (11%) patients versus the use of the urinary antigen test alone. The addition of a L. pneumophila-specific mip gene PCR to a urinary antigen test is useful in patients with suspected LD who produce sputum and might allow the early detection of a significant number of additional patients.     

Detection of leptospires in urine by PCR for early diagnosis of leptospirosis.

We tested urine samples from patients at different stages of current leptospirosis and thereafter to determine whether use of the PCR for detection of leptospires in urine can be a valuable alternative to culturing. The procedure of DNA extraction and subsequent PCR applied to 15 freshly voided urine samples proved to be twice as sensitive as culturing. Overall, we were able to detect leptospires in approximately 90% (26 of 29) of the urine samples. Urine and serum samples were obtained from seven patients, before the eighth day of illness. Although it is generally assumed that leptospiruria starts approximately in the second week of illness, we were able to detect leptospires in all of these early urine samples. In contrast, only two of seven corresponding serum samples gave positive PCR results, which suggests that PCR analysis of urine can be more successful for early diagnosis of leptospirosis than PCR analysis of serum. Urine samples from six patients who had been treated with antibiotics at the time of illness were positive by PCR, implying that the patients were still shedding leptospires in their urine despite treatment. Some of these samples were even taken years after the infection, indicating that shedding of leptospires in urine may last much longer than is generally assumed. We conclude that detection of leptospires in urine with PCR is a promising approach for early diagnosis of leptospirosis and may also be useful in studying long-term shedding.     

Detection of immunoglobulin heavy chain gene rearrangements in Hodgkin's disease using PCR.

AIM--To detect clonal rearrangements of the immunoglobulin (Ig) heavy chain gene in Hodgkin's disease tissue using the polymerase chain reaction (PCR). METHODS--DNA extracted from 36 samples of Hodgkin's disease was analysed using PCR and primers from conserved sequences in the variable (VH) and joining (JH) regions. RESULTS--Clonal rearrangement was detected only in one case. Evidence of clonal immunoglobulin gene rearrangement had been detected previously in this case using conventional Southern blot analysis. CONCLUSIONS--The sensitivity of the two techniques is equivalent and clonal Ig heavy chain gene rearrangements are rare in Hodgkin's disease.     

Detection of RTX toxin gene in Vibrio cholerae by PCR.

A PCR that amplifies a recently discovered Vibrio cholerae RTX (repeat in toxin) toxin gene was developed. Among 166 clinical and environmental isolates of V. cholerae causing epidemics and sporadic cases of cholera in various parts of the world, all were found to be toxigenic by both PCR and HEp-2 cell cytotoxicity assay. Standard strains of the classical biotype containing a deletion within the gene cluster exhibited negative results by both assays. This is the first rapid genotyping method for differentiation of V. cholerae O1 classical biotype strains from El Tor biotype strains as well as strains of other non-O1 serogroups including serogroup O139. The PCR assay that was developed also specifically detects RTX toxin genes in V. cholerae, as clinical isolates of Vibrio parahaemolyticus, diarrheagenic Escherichia coli, Aeromonas species, and Plesiomonas species were all negative by the RTX toxin-specific PCR as well as the HEp-2 cytotoxicity assay. These findings highlight the characteristics of the RTX toxins in V. cholerae. Their role in the pathogenicity of the bacterium requires further investigation.     

PCR of peripheral blood for diagnosis of meningococcal disease.

Meningococcal disease is normally suspected on clinical grounds and is confirmed by isolation of Neisseria meningitidis bacteria from blood or cerebrospinal fluid or, more recently, by serology or PCR of cerebrospinal fluid. Achieving confirmation of a clinical diagnosis of meningococcal disease has become more difficult in the last few years. The pre-hospitalization administration of parenteral benzylpenicillin normally renders blood cultures sterile, and lumbar puncture is undertaken less frequently, especially in young children. We evaluated PCR for the detection of meningococcal DNA in 80 blood samples taken from patients with known or suspected meningococcal disease or from patients with other diagnoses (negative controls). Both the sensitivity and the specificity of the test were 100% for patients with confirmed cases of meningococcal disease when the blood buffy coat was used (83 to 100% sensitivity and 87 to 100% specificity with 95% confidence limits). Positive PCR results could be obtained from both blood buffy coat and serum samples. Sensitivity was unaffected by prior antibiotic treatment. PCR is a rapid, sensitive test that may be used to confirm a diagnosis of meningococcal disease by using peripheral blood samples. Introduction of this test into clinical laboratories may in some cases obviate the need for lumbar puncture to be performed on patients with suspected meningococcal disease. Our results demonstrate that a substantial number of cases of meningococcal disease are not confirmed by conventional techniques and remain undiagnosed. If the PCR test described here was widely applied, the number of cases of meningococcal disease ascertained might rise by as much as 60% greater than that recognized at present. It is likely that we are in a prevaccination era for meningococcal disease. Better case ascertainment is urgently required to assess the need for vaccines, to determine their costs and benefits, and to monitor their efficacies.     

Using PCR in preimplantation genetic disease diagnosis.

Preimplantation diagnosis of genetic disease can be accomplished by embryo biopsy or polar body analysis using in-vitro gene amplification (PCR). PCR analysis of single cells is subject to a number of errors which decrease the reliability of the diagnosis. Using realistic assumptions about error rates based on experimental data, we analyse some of the practical consequences to be faced by whose wishing to use this diagnostic procedure. We considered both autosomal dominant and recessive diseases. We calculate the probability of making mistakes in the diagnosis, assuming a realistic range in the magnitude of PCR efficiency, cell transfer, and contamination errors. We conclude that, in general, analysing blastomeres is subject to less mis-diagnosis than polar body analysis, except in the case of dominant diseases which are caused by genes which lie extremely close to the centromere. We also show that typing multiple blastomeres from a single embryo or combining polar body typing with blastomere analysis results in significantly lower levels of mis-diagnosis with unacceptable consequences. The preimplantation diagnosis of X-linked diseases based upon Y chromosome sequence analysis is also discussed.     

The role of arbitrarily primed PCR in identifying the source of an outbreak of Legionnaires' disease.

An outbreak of community-acquired Legionnaires' disease (LD) occurred in Providence, R.I., in fall 1993. To find the outbreak source, exposures of 17 case patients were compared to those of 33 matched controls. Case patients were more likely than controls to have visited a section of downtown (area A) during the 2 weeks before illness (11 [65%] versus 9 [27%]; matched odds ratio, 6.5; P = 0.01). Water samples were cultured from 27 aerosol-producing devices within area A. Legionella pneumophila serogroup 1 isolates underwent monoclonal antibody (MAb) subtyping and arbitrarily primed PCR (AP-PCR). All four L. pneumophila serogroup 1 isolates available from case patients who visited area A had identical MAb and AP-PCR patterns. Among 14 environmental isolates, 5 had MAb patterns that matched the case patient isolates, but only 1 had a matching AP-PCR pattern. This investigation implicates a cooling tower in area A as the outbreak source and illustrates the usefulness of AP-PCR for identifying sources of LD outbreaks.     

The morphology of the Legionnaires' disease organism.

The Pennsylvania Department of Health, Bureau of Laboratories, has isolated the Legionnaires disease organism from two patients with Legionnaires' disease proved by serologic techniques. We have studied the morphology which the isolate assumes in yolk sac tissue and on bacteriologic media. The organism was Giménez-positive and gram-variable. Using an indirect immunofluorescent procedure, it was shown to react with convalescent serum samples taken from patients who had Legionnaires' disease. The organism multiplies by binary fission extracellularly and intracellulary; is both coccoid and bacillary in form; and contains characteristic cytoplasm, nucleoids, a cytoplasmic membrane, and a small cell wall of variable size. It may produce spores of unusual appearance. Intracellular replication characteristically occurs within vacuoles. The Legionnaires' disease organism conforms to the morphologic criteria for a prokaryocyte.     

Detection of the 1226 (Jewish) mutation for Gaucher's disease by color PCR. A means for studying the gene frequency of the disorder

The authors applied the polymerase chain reaction- (PCR) based color complementation assay for rapid detection of the 1226 ("Jewish") mutation of the glucocerebrosidase gene. Fifty-seven unrelated patients with Gaucher's disease and 50 unrelated normal Ashkenazi Jewish volunteers were studied. This mutation was identified in more than 75% of the Jewish Gaucher's disease alleles and in 4 (8%) of the 50 normal Jewish volunteers. The reliability of the technique was verified both by DNA sequencing and by leukocyte beta-glucosidase assay. This method is suggested as the simplest and most suitable one for a large-scale screening for the 1226 mutation for Gaucher's disease.     

Application of droplet digital PCR for non-invasive prenatal diagnosis of single gene disease in two familiesCSCD

Objective To assess the value of droplet digital PCR (ddPCR) for non-invasive prenatal diagnosis of single gene disease in two families. Methods Paternal mutation in cell-free DNA derived from the maternal blood and amniotic fluid DNA was detected by ddPCR. Suspected mutation in the amniotic fluid DNA was verified with Sanger sequencing. Reverse The result of ddPCR and Sanger sequencing indicated that the fetuses have carried pathogenic mutations from the paternal side in both families. Conclusion Droplet digital PCR can accurately detect paternal mutation carried by the fetus, and it is sensitive and reliable for analyzing trace samples. This method may be applied for the diagnosis of single gene diseases caused by paternal mutation using peripheral blood sample derived from the mother. © 2018 West China University of Medical Sciences. All rights reserved.     

Urine sample used for congenital toxoplasmosis diagnosis by PCR.

The diagnosis of toxoplasmosis in congenitally infected infants can be difficult; serology is unreliable, and diagnosis must be based on the combination of symptomatology and direct demonstration of the parasite. Four infants suspected of having Toxoplasma gondii infection were studied by serological analysis, tissue culture, and PCR determination. T. gondii was isolated from the urine of one patient. The parasite was detected by PCR in the blood and cerebrospinal fluid of three infants and in the urine in all patients. Because nested PCR proved to be a sensitive, relatively rapid, and specific method and because it can be applied to a variety of different clinical samples, PCR can be a valuable technique for the identification of T. gondii infections in children. The present study indicates that PCR examination of urine, a fluid never before used for diagnosis in this age group, may be valuable in diagnosing cases of congenital toxoplasmosis.     

Legionnaires' disease. Pathological and historical aspects of a 'new' disease

Autopsy tissues and protocols from 26 epidemiologically defined fatal cases of Legionnaires' disease occurring during the 1976 Philadelphia outbreak were reviewed. Consistent pathologic features were limited to the lung, where an acute pneumonia characterized by intra-alveolar exudation of neutrophils, macrophages, and fibrin was observed. An etiologic agent common to most of the victims of Legionnaires' disease was identified within the pneumonic process by application of the Dieterle silver impregnation stain. In some cases, other pulmonary histologic findings were noted, chiefly acute diffuse alveolar damage. However, the importance of acute diffuse alveolar damage is not understood.