Citations

ABSTRACT We developed a rapid thermocycling, real-time detection (also known as real-time PCR) method for the detection of Legionella species directly from clinical specimens. This method uses the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and requires approximately 1 to 2 h to perform. Both a Legionella genus PCR assay and Legionella pneumophila species-specific PCR assay were designed. A total of 43 archived specimens from 35 patients were evaluated, including 19 bronchoalveolar lavage (BAL) specimens and 24 formalin-fixed, paraffin-embedded open lung biopsy specimens. Twenty-five of the specimens were culture-positive for Legionella (9 BAL specimens and 16 tissue specimens). BAL specimens were tested by LightCycler PCR (LC-PCR) methods and by a direct fluorescent antibody (DFA) assay, which detects L. pneumophila serogroups 1 to 6 and several other Legionella species. Tissue sections were tested by the two LC-PCR methods, by DFA, by an in situ hybridization (ISH) assay, specifically designed to detect L. pneumophila , and by Warthin-Starry (WS) staining. The results were compared to the “gold standard” method of bacterial culture. With BAL specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by Legionella genus LC-PCR, 100 and 100%; Legionella genus detection by DFA assay, 33 and 100%; and L. pneumophila detection by L. pneumophila species-specific LC-PCR, 100 and 100%. With open lung biopsy specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by LC-PCR 68.8 and 100%; Legionella genus detection by DFA assay, 44 and 100%; Legionella genus detection by WS staining, 63 and 100%; L. pneumophila species-specific detection by LC-PCR, 17 and 100%; and L. pneumophila species-specific detection by ISH, 100 and 100%. The analytical sensitivity of both LC-PCR assays was <10 CFU/reaction. LC-PCR is a reliable method for the direct detection of Legionella species from BAL specimens. The Legionella genus LC-PCR assay could be performed initially; if positive, L. pneumophila species-specific LC-PCR could then be performed (if species differentiation is desired). The speed with which the LC-PCR procedure can be performed offers significant advantages over both culture-based methods and conventional PCR techniques. In contrast, for the methods evaluated, culture was the best for detecting multiple Legionella species in lung tissue. WS staining, Legionella genus LC-PCR, and L. pneumophila species-specific ISH were useful as rapid tests with lung tissue.

ABSTRACT We evaluated two automated systems, MagNA Pure (Roche Molecular Biochemicals, Indianapolis, Ind.) and BioRobot 9604 (Qiagen, Inc., Chatsworth, Calif.) as effective replacements for the manual IsoQuick method (Orca Research, Inc., Bothell, Wash.) for extraction of herpes simplex virus (HSV) DNA from dermal and genital tract specimens prior to analysis by LightCycler PCR. Of 198 specimens (152 genital, 46 dermal), 92 (46.2%) were positive for HSV DNA by LightCycler PCR after automated extraction of specimens with either the MagNA Pure or BioRobot 9604 instrument. The manual IsoQuick method yielded HSV DNA (total n = 95) from three additional specimens that were negative by the automated method ( P = 0.25, sign test). Although the mean numbers of LightCycler PCR cycles required to reach positivity differed statistically significantly among all three of the methods of extraction, the estimated means differed by no more than 1.5 cycles ( P < 0.05). Seventy (76%) of the 92 specimens that were LightCycler PCR positive by all three extraction methods were also positive by shell vial cell culture assay. HSV DNA was detected by a lower LightCycler PCR cycle number (26.1 cycles) in specimens culture positive for the virus than in culture-negative samples (33.3 cycles) ( P < 0.0001). The manual IsoQuick and automated MagNA Pure and BioRobot 9604 methods provide standardized, reproducible extraction of HSV DNA for LightCycler PCR. The decision to implement a manual versus an automated procedure depends on factors such as costs related to the number of specimens processed rather than on the minimal differences in the technical efficiency of extraction of nucleic acids among these methods.

ABSTRACT Mycobacterium triplex was first named in 1996 as an acid-fast bacillus with features that most resemble Mycobacterium simiae and Mycobacterium avium-intracellulare complex but which possesses a distinct mycolic acid pattern as well as a distinctive 16S rRNA hypervariable region. It has been isolated from lymph node, sputum, and cerebrospinal fluid specimens, but to date only rare clinical cases of this organism have been reported in the literature. The following is a case report of M. triplex that was isolated from the pericardial and peritoneal fluid of a 13-year-old female liver transplant patient.

ABSTRACT Detection of Giardia and Cryptosporidium in clinical stool specimens using the ColorPAC and ProSpecT enzyme immunoassays revealed 98.7% agreement for Giardia detection and 98.1% agreement for Cryptosporidium detection. Sensitivities were uniformly 100%. The specificities of the ColorPAC immunoassay for Giardia and Cryptosporidium detection were 100 and 99.5%, respectively, and those for the ProSpecT assay were 98.4 and 98.6%, respectively. The false-positive reactions with the ProSpecT assay occurred with specimens that were grossly bloody.

ABSTRACT An evaluation was undertaken to determine the utility of the BACTEC Peds Plus/F bottle and the BACTEC 9240 instrument (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) for the detection of clinically significant microorganisms in synovial fluid specimens. The Peds Plus/F bottle was used because in our laboratory the quantity of synovial fluid available for culture is frequently in the range of 0.5 to 3.0 ml. The culture results obtained with the Peds Plus/F bottle were compared to those obtained by a conventional agar plate method for a total of 805 synovial fluid specimens. Microbial growth was produced by 74 cultures (9.2%) from 60 patients, yielding a total of 77 microorganisms. Organisms were classified as pathogens ( n = 62), contaminants ( n = 12), or indeterminate ( n = 3) on the basis of a review of the patients' medical histories. Culture using BACTEC Peds Plus/F bottle detected statistically significantly more pathogens overall (62 versus 51 pathogens [ P = 0.001]) and statistically fewer contaminants overall (1 versus 11 contaminants [ P = 0.006]) than culture by the agar plate method. These results indicate the superior performance of the BACTEC Peds Plus/F bottle over the conventional agar plate method for the detection of clinically significant microorganisms from synovial fluid specimens.

ABSTRACT The BACTEC MYCO/F Lytic blood culture bottle (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) is designed to optimize the recovery of fungi and mycobacteria; however, this bottle also supports the growth of most aerobic bacteria. We compared the MYCO/F Lytic bottle with two other BACTEC bottles and the Isolator system for the recovery of bacteria as well as fungi and mycobacteria from blood. A total of 6,108 blood culture sets were inoculated with blood obtained from adult patients. Twenty-five to 28 ml of blood collected by a phlebotomy team for each blood culture set was randomly distributed into each of four blood culture receptacles: the Isolator tube (Wampole Laboratories, Cranbury, N.J.) and three BACTEC bottles: the MYCO/F Lytic bottle, the BACTEC Plus Aerobic/F bottle, and the BACTEC Anaerobic Lytic/10 bottle. The sediment from the Isolator tube was inoculated onto chocolate agar (CA), brain heart infusion agar (BHI), and Sabouraud dextrose agar (SDA) and into a BACTEC 13A bottle. Incubation durations were as follows: MYCO/F Lytic bottle, 42 days; Plus Aerobic/F bottle, 5 days; Anaerobic Lytic/10 bottle, 5 days; sediment from Isolator tube on CA, 3 days; sediment from Isolator tube on BHI, 30 days; sediment from Isolator tube on SDA, 30 days; and sediment from Isolator tube in a BACTEC 13A bottle, 42 days. Two isolates of Histoplasma capsulatum were recovered from the Isolator tube only. Three isolates of Mycobacterium tuberculosis complex were recovered: two isolates from the MYCO/F Lytic bottle only and one isolate from the Isolator tube (whose sediment was inoculated into the BACTEC 13A bottle) only. Two isolates of Cryptococcus neoformans were recovered: one from the MYCO/F Lytic bottle only and the other from the MYCO/F Lytic bottle and the Isolator tube (whose sediment was inoculated into the BACTEC 13A bottle). For potential pathogens overall, there was a statistical difference in recovery that favored the Isolator system over the MYCO/F Lytic bottle ( P = 0.0015), including statistically significant differences for Staphylococcus aureus ( P = 0.0001) and Streptococcus pneumoniae ( P = 0.0313). However, there was no statistically significant difference between the two blood culture systems when detection of bloodstream infection was considered. The time to detection for all potential pathogens combined was less for the MYCO/F Lytic bottle than for the Isolator system ( P = 0.0004). Overall, the potential pathogen recovery was greater for the BACTEC Plus Aerobic/F bottle than for either the Isolator system ( P = 0.0003) or the MYCO/F Lytic bottle ( P = 0.0001). However, the BACTEC Plus Aerobic/F bottle did not recover M. tuberculosis , H. capsulatum , or C. neoformans isolates. The combination of the Isolator system and MYCO/F Lytic bottle may be useful as a selective blood culture method to optimize the recovery of fungi and mycobacteria from blood. Compared with the manual Isolator system, the MYCO/F Lytic system has the advantage of less preanalytic processing and continuous automated monitoring of bottles for growth by the BACTEC 9240 instrument.

ABSTRACTModifications of mucosal phospholipids have been detected in samples from patients withHelicobacter pylori-positive gastritis. These alterations appear secondary to increased phospholipase A2activity (PLA2). The cytosolic form of this enzyme (cPLA2), normally involved in cellular signaling and growth, has been implicated in cancer pathogenesis. The aim of this study was to investigate cPLA2expression and PLA2activity in the gastric mucosae of patients with and withoutH. pyloriinfection. In gastric biopsies from 10H. pylori-positive patients, cPLA2levels, levels of mRNA as determined by reverse transcriptase PCR, levels of protein as determined by immunohistochemistry, and total PLA2activity were higher than in 10H. pylori-negative gastritis patients. To clarify whetherH. pylorihad a direct effect on the cellular expression of cPLA2, we studied cPLA2expression in vitro with different human epithelial cell lines, one from a patient with larynx carcinoma (i.e., HEp-2 cells) and two from patients with gastric adenocarcinoma (i.e., AGS and MKN 28 cells), incubated with differentH. pyloristrains. The levels of cPLA2, mRNA, and protein expression were unchanged in Hep-2 cells independently of cellular adhesion or invasion of the bacteria. Moreover, no change in cPLA2protein expression was observed in AGS or MKN 28 cells treated with wild-typeH. pylori. In conclusion, our study shows increased cPLA2expression and PLA2activity in the gastric mucosae of patients withH. pyloriinfection and no change in epithelial cell lines exposed toH. pylori.

ABSTRACTAn immunoglobulin G (IgG)-coated surface, such as that found on helminth parasites, is one of the most effective physiologic stimuli for eosinophil activation. The cysteine proteases secreted by tissue-invasive helminth larvae play an important role in evasion of the immune response by degrading the host immunoglobulins. In this study, we investigated whether cysteine proteases in the excretory-secretory product (ESP) produced byParagonimus westermaninewly excysted metacercariae (PwNEM), which cause pulmonary or extrapulmonary paragonimiasis in human beings, could modify effector functions of human eosinophils stimulated with IgG. We coated 96-well plates with human IgG in the absence or presence of the ESP produced by PwNEM. When eosinophils were incubated in the wells coated with IgG in the presence of the ESP, eosinophil degranulation and superoxide production were significantly reduced compared with results for cells incubated in wells coated with IgG alone. This inhibitory effect of the ESP on IgG-induced superoxide production was dose dependent and was significantly abolished by pretreatment of the ESP with heat. These findings suggest that the cysteine proteases secreted by PwNEM attenuate both activation and degranulation of eosinophils stimulated with IgG. Thus, the cysteine proteases produced by tissue-invasive helminth larvae play crucial roles in evasion of IgG-dependent eosinophil helminthotoxicity and in reduction of eosinophil-associated tissue inflammation during the migratory period.

ABSTRACTInfection of severe combined immunodeficient mice withBabesiasp. strain WA1 was studied to assess the contributions of innate and adaptive immunity in resistance to acute babesiosis. Thescidmutation showed little effect in genetically susceptible C3H mice and did not decrease the inherent resistance of C57BL/6 mice to the infection, suggesting that innate immunity plays a central role in determining the course ofBabesiainfection in these strains. In contrast, thescidmutation dramatically impaired resistance in moderately susceptible BALB/c mice, suggesting that acquired immunity may play an important secondary role. In comparison to their female counterparts, male mice of different genetic backgrounds showed increased resistance to the infection, indicating that the gender of the host may influence protection against babesiosis.

ABSTRACT Increased recognition of the prevalence of human babesiosis in the United States, together with rising concern about the potential for transmission of this infection by blood transfusion, has provided motivation to develop definitive serologic and molecular tests for the causative agent, Babesia microti . To develop more sensitive and specific assays for B. microti , we screened a genomic expression library with patient serum pools. This screening resulted in the identification of three classes of novel genes and an additional two novel, unrelated genes, which together encode a total of 17 unique B. microti antigens. The first class (BMN1-2 family) of genes encodes seven closely related antigens with a degenerate six-amino-acid repeat that shows limited homology to Plasmodium sp. merozoite and sporozoite surface antigens. A second class (BMN1-8 family) of genes encodes six related antigens, and the third class (BMN1-17 family) of genes encodes two related antigens. The two remaining genes code for novel and unrelated sequences. Among the three classes of antigens and remaining novel sequences, five were chosen to code for the most immunodominant antigens (BMN1-2, -9, -15, and -17 and MN-10). Western blot analysis with the resulting recombinant proteins indicated that these antigens were targets of humoral immune responses during B. microti infection in humans.