bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of polymerase chain reaction assays for detection of Listeria monocytogenes in clinical cerebrospinal fluid samples
Authors: Jaton K , Sahli R , Bille J
Published: January 2020

In order to improve the diagnosis of Listeria meningitis or meningoencephalitis, especially in patients who have received antibiotics before their cerebrospinal fluid (CSF) has been examined, two assays for the detection of Listeria monocytogenes based on the polymerase chain reaction (PCR) were evaluated. After a standard PCR, the amplified DNA was detected either by a second round of PCR with internal primers followed by gel electrophoresis and ethidium bromide staining (nested PCR) or by dot blot hybridization to an internal digoxigenin-labeled probe (PCR-dot blot). For PCR, two sets of primers within the invasion-associated protein gene (iap gene) were chosen. They allowed for the highly specific detection of all L. monocytogenes reference strains tested (serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, and 7). These primers did not detect amplification products from various other gram-positive or gram-negative bacterial DNAs or human DNA. The sensitivities of both assays were assessed on sterile CSF samples that were artificially seeded with serial dilutions of L. monocytogenes serotype 4b cells. By both methods the limit of detection was less than 10 cells in the initial reaction. Since the nested PCR is more prone to contamination because of manipulation of the amplified products, a standard PCR assay followed by dot blot hybridization was applied to 52 CSF samples in a retrospective study. Of 28 CSF samples which were sterile or positive for bacteria other than Listeria species, 24 were PCR negative. In contrast, from 17 patients with culture-proven Listeria meningitis, 14 of 17 initial CSF samples were PCR positive, as were 3 of 7 culture-negative followup CSF samples taken after patients received antibiotics. These results support the usefulness of this approach in the diagnosis of Listeria meningitis, in particular, when antibiotic administration precedes culture of CSF.
Cytomegalovirus DNA detection in sera from patients with active cytomegalovirus infections
Authors: Brytting M , Xu W , Wahren B , Sundqvist V A
Published: January 2020

Using a specific and sensitive polymerase chain reaction method, we detected reliably the presence of human cytomegalovirus (HCMV) DNA directly in serum samples collected at an early stage of HCMV infection, even before immunoglobulin M (IgM) antibodies were measurable. HCMV DNA was detected in serum from all patients with active HCMV infection; in 91% of these patients, HCMV DNA was found in the acute-phase serum. In 13 of 44 patients, HCMV DNA was found in serum before HCMV-specific IgM. For four kidney transplant recipients, the occurrence of HCMV DNA in serum, virus isolation from urine and leukocytes, and HCMV IgG and IgM serology were determined. We found a correlation between HCMV DNA in serum and positive virus isolation from leukocytes. In three of five congenitally infected infants, HCMV DNA and HCMV IgM were detected in the same sample. Two other infants were HCMV DNA positive, although no HCMV IgM antibodies were measurable. HCMV was found in urine from these infants either by virus isolation or with the polymerase chain reaction. Serum from one of the 22 healthy HCMV-seropositive blood donors was HCMV polymerase chain reaction positive.
Epidemiology of Enterococcus faecalis urinary tract infection in a teaching hospital in London, United Kingdom
Authors: Hall L M , Duke B , Urwin G , Guiney M
Published: January 2020

Enterococcus faecalis is a frequent cause of urinary tract infection in hospitalized patients. Recent reports have suggested that the organism may frequently be acquired by cross-infection from other patients. In this study, we used total DNA restriction patterns to type 135 urine isolates of E. faecalis from four sets of patients. Isolates were placed into types (all bands identical) and into groups (most bands identical). Most isolates were discriminated by the typing method, and the results suggested that direct cross-infection occurred rarely if at all. However, two groups of clonally related isolates occurred frequently in the urine specimens and also in feces from hospital-associated patients and were often associated with antibiotic resistance. Isolates from these two groups were found less frequently in feces from people not associated with the hospital.
Evaluation of commercial and standard methodology for determination of oxacillin susceptibility in Staphylococcus aureus
Authors: Skulnick M , Simor A E , Gregson D , Patel M , Small G W , Kreiswirth B , Hathoway D , Low D E
Published: January 2020

Agar dilution with and without 4% NaCl, broth microdilution with 2% NaCl, the dried MicroScan Rapid Positive MIC 1 panel (Baxter Health Care Corp., West Sacramento, Calif.), the Vitek GPS-SA card (Vitek Systems, Hazelwood, Mo.), and the oxacillin agar screen plate were compared with a DNA probe encoding the mec gene for their abilities to detect oxacillin resistance in 506 clinical isolates of Staphylococcus aureus. The results of testing for the mec gene showed that there were 254 oxacillin-resistant and 252 oxacillin-susceptible isolates of S. aureus. There were 14.2% very major errors with Vitek (a resistant isolate was interpreted as susceptible) and 6.7% very major errors with MicroScan. Fewer major errors were seen: 0.8% with MicroScan (a susceptible isolate was interpreted as resistant) and 0.4% with Vitek. No very major errors but 2.4% major errors occurred by agar dilution with 4% NaCl supplementation, whereas there were 0.8% very major and 0.4% major errors without 4% NaCl supplementation. By broth microdilution there were 2.0% very major and 0.8% major errors. The results of the oxacillin agar screen plate method were 100% concordant with those of the mec gene probe method.
Immunoglobulin A (IgA) and IgG serum antibodies to mycobacterial antigens in Crohn's disease patients and their relatives
Authors: Wayne L G , Hollander D , Anderson B , Sramek H A , Vadheim C M , Rotter J I
Published: January 2020

Sera from patients with Crohn's disease, their relatives, their spouses, and unrelated healthy controls were assayed by enzyme-linked immunosorbent assay for immunoglobulin G (IgG) and IgA antibodies to Mycobacterium tuberculosis, M. avium, and M. gordonae. The patients had significantly higher IgA responses to mycobacterial antigens than did either their relatives or the controls. On the other hand, both the patients and their relatives had significantly higher IgG responses against these antigens than did the controls. The elevated IgA response was more pronounced against isopentanol-extracted whole bacterial cells than it was against soluble protein extracts, and it appeared to be directed against fixed surface antigens that lie under the loosely bound peptidoglycolipid or glycolipid antigens of mycobacteria.
Laboratory diagnosis of Clostridium difficile-associated gastrointestinal disease: comparison of a monoclonal antibody enzyme immunoassay for toxins A and B with a monoclonal antibody enzyme immunoassay for toxin A only and two cytotoxicity assays
Authors: Doern G V , Coughlin R T , Wu L
Published: January 2020

A total of 320 stool specimens obtained from 262 patients suspected of having Clostridium difficile-associated gastrointestinal disease were examined with two cytotoxicity assays (CTAs) and two commercially available enzyme immunoassays (EIAs). The CTAs were an in-house-developed procedure (University of Massachusetts Medical Center [UMMC], Worcester, Mass.) and a commercial test (Bartels CTA; Baxter Healthcare Corp., West Sacramento, Calif.). One EIA was a monoclonal antibody-based assay for C. difficile toxins A and B (Cambridge Biotech Corp. [CBC], Worcester, Mass.). The other EIA employed monoclonal antibodies directed against only toxin A (Meridian Diagnostics, Cincinnati, Ohio). True-positive and true-negative results were defined on the basis of the results of the four assays, clinical assessments of patients, and the results of other laboratory tests. The sensitivities of the four assays were as follows: Bartels CTA, 100%; UMMC CTA, 97.2%; CBC EIA, 84.5%; and Meridian EIA, 69.0%. The Bartels CTA demonstrated a specificity of 99.2%. The other three assays had a specificity of 100%.
Production and characterization of monoclonal antibodies to type 8 lipooligosaccharide of Neisseria meningitidis
Authors: Gu X X , Tsai C M , Karpas A B
Published: January 2020

Eight monoclonal antibodies (MAbs) to lipooligosaccharides (LOSs) of Neisseria meningitidis were produced by immunizing mice with purified LOS from group A meningococcal strain A1. The specificities of the MAbs were examined by enzyme-linked immunosorbent assay (ELISA), immunodot assay, and ELISA inhibition by using the homologous A1 LOS, 12 immunotype LOSs of N. meningitidis (L1 through L12), and LOSs or lipopolysaccharides from other gram-negative bacteria. Two of the MAbs, 4385G7 (immunoglobulin G2b [IgG2b]) and 4387A5 (IgG2a), had the strongest reactivities with the homologous A1 LOS, moderate reactivities with the M978 (L8) LOS, but no reactivity with other LOSs. The other six MAbs (4 IgM and 2 IgG3) reacted with the A1 LOS and with several or many of the 12 LOSs. ELISA inhibition at 50% showed that the inhibitory activities of the LOSs from strains A1 and BB431 (a group B strain) to the specific MAb 4387A5 were about 10 to 20 times greater than that of the M978 (L8) LOS. When compared with MAb 2-1-L8 (L8) by Western blot (immunoblot) analysis and ELISA inhibition, the two specific MAbs recognized a different epitope in the 3.6-kDa LOSs of strains A1 and BB431. We propose that the new epitope is L8a, since the MAbs also reacted with the M978 (L8) LOS. The expression of the L8a epitope in the A1 LOS requires a few monosaccharide residues in its oligosaccharide moiety, and the fatty acid residues in its lipid A moiety also play a role. In a whole-cell ELISA, the two specific MAbs bound specifically to the homologous strain A1 and the L8 prototype strain M978 but not to any other LOS prototype strains. These results suggest that the two specific MAbs can be used for LOS typing of N. meningitidis.
Modified MacConkey medium which allows simple and reliable identification of Providencia stuartii
Authors: Thaller M C , Berlutti F , Pantanella F , Pompei R , Satta G
Published: January 2020

This work describes a modified MacConkey medium (MCP medium) enabling the simple identification of Providencia stuartii, an emerging nosocomial pathogen. A total of 813 strains, belonging to the families Enterobacteriaceae and Pseudomonadaceae, were tested on MCP medium; all P. stuartii strains were phosphatase positive, as were 97.5% of Morganella morganii strains, in contrast with all other tested organisms. A simple discriminating test, such as the ornithine or citrate test, allowed identification of strains of these species. We have also compared the reliabilities of P. stuartii identification by commercial kits (API 20E system) by using a standard MacConkey or MCP medium. Sixteen and three-tenths percent of P. stuartii strains were misidentified by using the former procedure, while with the latter all strains were correctly identified. Finally, the MCP medium was used over a 6-month period in our routine clinical laboratory. Of a total of 1,278 seeded urine samples from elderly patients, we isolated 103 P. stuartii strains which were all correctly identified by coupling MCP medium and the API 20E system. Seventeen and one-half percent of these strains were misidentified when the API 20E system was used in combination with standard MacConkey medium.
Molecular epidemiology of Pseudomonas cepacia determined by polymerase chain reaction ribotyping
Authors: Kostman J R , Edlind T D , LiPuma J J , Stull T L
Published: January 2020

Traditional ribotyping detects genomic restriction fragment length polymorphisms by probing chromosomal DNA with rRNA. Although it is a powerful method for determining the molecular epidemiology of bacterial pathogens, technical difficulties limit its application. As an alternative, polymorphisms were sought in the 16S-23S spacer regions of bacterial rRNA genes by use of the polymerase chain reaction (PCR). Chromosomal DNA from isolates of Pseudomonas cepacia was used as a template in the PCR with oligonucleotide primers complementary to highly conserved sequences flanking the spacer regions of the rRNA genes. Length polymorphisms in the amplified DNA distinguished unrelated isolates of P. cepacia. Isolates of P. cepacia previously implicated in person-to-person transmission were shown to have identical amplification patterns. These data demonstrate the utility of this new PCR ribotyping method for determining the molecular epidemiology of bacterial species.
DNA polymorphisms in methicillin-susceptible and methicillin-resistant strains of Staphylococcus aureus
Authors: Carles-Nurit M J , Christophle B , Broche S , Gouby A , Bouziges N , Ramuz M
Published: January 2020

Restriction fragment length polymorphisms in methicillin-susceptible and methicillin-resistant (MRSA) strains of Staphylococcus aureus isolated in the same hospital over a 4-month period were studied by using SmaI and ApaI digestion of genomic DNA and pulsed-field gel electrophoresis. Each of the 20 methicillin-susceptible strains had a unique SmaI pattern, but the 27 MRSA strains showed only seven SmaI patterns. More than half of the SmaI fragments in all of these seven patterns were identical, as were those in the patterns from two unrelated MRSA strains. Digestion with ApaI, which cuts staphylococcus DNA into at least twice as many fragments, confirmed the results obtained with SmaI. Lastly, the plasmid contents of MRSA strains showing identical SmaI and ApaI electrophoretic patterns were not identical. These results are interpreted as supporting the hypothesis that all MRSA strains arose from a single clone and emphasize the need to use several methods in epidemiological investigations of MRSA outbreaks.
Detection of Babesia microti by polymerase chain reaction
Authors: Persing D H , Mathiesen D , Marshall W F , Telford S R , Spielman A , Thomford J W , Conrad P A
Published: January 2020

Human babesiosis, which is caused by infection with the intraerythrocytic malarialike protozoan Babesia microti, has recently been diagnosed with increasing frequency in residents of New England. Diagnosis is difficult because of the small size of the parasite and the sparse parasitemia that is characteristic of most infections with this pathogen. We generated B. microti-specific DNA sequence information by universal primer amplification of a portion of the eukaryotic 16S-like gene; this was followed by direct DNA sequence analysis. Specific primers were synthesized on the basis of this sequence information for use in the polymerase chain reaction (PCR). The PCR-based system demonstrates a strong bias for detection of B. microti as opposed to Babesia gibsoni and does not amplify vertebrate DNA. The analytical sensitivity of the system is approximately three merozoites. Blood specimens from 12 patients with clinically diagnosed and parasitologically confirmed babesiosis from Nantucket Island, Mass., were PCR positive in a blinded test of this procedure. Thus, DNA amplification may provide an adjunct to conventional methods for the diagnosis of human babesiosis and may provide a new means of monitoring therapy or enhancing epidemiological surveillance for this emerging pathogen.
Use of polymerase chain reaction for detection of toxigenic Vibrio cholerae O1 strains from the Latin American cholera epidemic
Authors: Fields P I , Popovic T , Wachsmuth K , Olsvik O
Published: January 2020

In January 1991, an outbreak of cholera started in Peru and spread throughout most of Latin America within 8 months. As of March 1992, over 450,000 cases and approximately 4,000 deaths have been reported to the Pan American Health Organization. The causative organism is toxigenic Vibrio cholerae O1 of the El Tor biotype and is distinct from the U.S. Gulf Coast strains. A polymerase chain reaction (PCR) that amplifies a 564-bp fragment of the cholera toxin A subunit gene (ctxA) was used to identify toxigenic V. cholerae O1 strains. A total of 150 V. cholerae O1 isolates were tested. They were of unknown toxin status, were associated with recent outbreaks, and were isolated from patients, food, and water. One hundred forty isolates were found to be toxigenic both by PCR and the routine diagnostic enzyme-linked immunosorbent assay. Thirty-eight known toxigenic strains isolated worldwide from 1921 to 1991 were also positive in the PCR. A collection of 18 nontoxigenic V. cholerae O1 strains, 35 Escherichia coli heat-labile-enterotoxin-I-producing strains, 26 Campylobacter strains, and 8 strains of Aeromonas hydrophila, previously reported to produce cholera toxin-like toxin, were all negative in the ctxA PCR. We conclude that this PCR is a diagnostic method that specifically detects toxin genes in V. cholerae O1 strains in a reference laboratory. It is more rapid and less cumbersome than other diagnostic methods for detection of toxicity in these strains.
Comparison of three methods for detection of group A streptococci in throat swabs
Authors: Anhalt J P , Heiter B J , Naumovitz D W , Bourbeau P P
Published: January 2020

Group A streptococci are generally detected in throat swabs by (i) rapid antigen tests, (ii) conventional culture, or (iii) combinations of both. Direct fluorescent-antibody testing of a 2-h enrichment broth (FA/EN) was an accepted method for same-day results before the advent of rapid antigen tests. We compared FA/EN in Todd-Hewitt Broth (THB) with conventional culture and a rapid antigen test, TestPack Strep A (TPS). Nine hundred seventy specimens were evaluated in this study. Cultures were performed for 48 h on sheep blood agar (SBA) incubated aerobically and on a selective agar for group A streptococci (SSA) incubated in 5 to 10% CO2. Following a 2-h incubation, the fluorescent-antibody test was performed. A subculture of the centrifuged sediment from the THB enrichment was also done. In comparison with a positive culture on SBA or SSA or subculture of the THB pellet, the sensitivities and specificities of the different methods were as follows: SBA, 92 and 100%; SSA, 92 and 100%; TPS, 68 and 99%; FA/EN, 88 and 98%. The FA/EN method offers the potential for definitive finalized reports on the same day as specimen collection with greater sensitivity than TPS. This study included sequential plating and rapid antigen testing of a single swab. In a separate set of experiments to validate this study design, it was shown that recovery of streptococci from swabs plated sequentially on five plates did not vary with the order of plating and the actual proportion of organisms recovered from a swab on a single plate was only 1%.
Presence of specific immunoglobulin A-secreting cells in peripheral blood after natural infection with Shigella sonnei
Authors: Orr N , Robin G , Lowell G , Cohen D
Published: January 2020

The appearance of antigen-specific immunoglobulin A (IgA) antibody-secreting cells (ASCs) following natural infection with Shigella sonnei during a common-source outbreak caused by this organism was evaluated in a modified enzyme-linked immunosorbent assay (ELISPOT). A mean IgA ASC value of 2,131.6/10(6) cells against homologous S. sonnei lipopolysaccharide (LPS) was detected in blood samples obtained from patients with bacteriologically proven S. sonnei shigellosis 5 and 10 days after the onset of disease. In the same blood samples, the level of ASC measured against heterologous antigen (Shigella flexneri serotype 2a LPS) was significantly lower than that of the homologous antigen (mean value, 33.12/10(6) cells). Furthermore, the mean number of activated B cells that secreted anti-S. sonnei LPS antibodies was significantly higher among patients with S. sonnei shigellosis than it was among patients with non-Shigella diarrhea (2.5/10(6) cells; standard error, 1.0) and healthy subjects (5.1/10(6) cells; standard error, 2.3) (P less than 0.05). The anti-LPS IgA ASC activity was easily detected within 5 days of the onset of disease, a point at which the levels of anti-S. sonnei LPS IgG and even IgA antibodies were hardly detectable in serum.
DNA fingerprinting of Escherichia coli O157:H7 strains by pulsed-field gel electrophoresis
Authors: Böhm H , Karch H
Published: January 2020

Pulsed-field gel electrophoresis of genomic DNA was carried out on Escherichia coli O157:H7 strains from different geographic locations to determine its value in an epidemiological survey of O157 infections. Pulsed-field gel electrophoresis of XbaI-digested DNA fragments clearly separated E. coli O157:H7 strains from nontoxigenic E. coli O157:H19, O157:H43, and O157:H45 strains and from Shiga-like-toxin-producing E. coli strains of other serogroups. However, among the E. coli O157:H7 strains, the restriction patterns either were identical or differed only by a few fragment bands. In some cases, it was therefore impossible to distinguish among epidemiologically unrelated strains. Hybridization experiments with a DNA probe complementary to Shiga-like toxin II sequences revealed that the Shiga-like toxin II genes were located on DNA fragments of different lengths. Our data show that for a single highly conserved clone, such as E. coli O157:H7, other typing techniques may need to be performed in addition to DNA fingerprinting in epidemiological surveys.
Further consideration of the clonal nature of Salmonella typhi: evaluation of molecular and clinical characteristics of strains from Indonesia and Peru
Authors: Franco A , Gonzalez C , Levine O S , Lagos R , Hall R H , Hoffman S L , Moechtar M A , Gotuzzo E , Levine M M , Hone D M
Published: January 2020

We examined envelope protein profiles, chromosomal restriction endonuclease digest patterns, and immune responses to envelope proteins for collections of Salmonella typhi strains isolated in Peru and Indonesia. Only minor differences in envelope protein patterns were apparent among strains. Strains from 7 of 20 Indonesian patients had a distinct chromosomal digest pattern compared with patterns of Peruvian and other Indonesian strains. Strains with this pattern carried the gene for the j flagellar antigen (H1-j); differences in response to envelope proteins of j and d strains were noted on immunoblot analysis. Our data suggest that there are genotypic and phenotypic differences among S. typhi strains. The clinical importance of these differences remains to be fully evaluated; however, in this study it was not possible to show a clear correlation between strain characteristics and disease severity.
Identification of Ehrlichia chaffeensis morulae in cerebrospinal fluid mononuclear cells
Authors: Dunn B E , Monson T P , Dumler J S , Morris C C , Westbrook A B , Duncan J L , Dawson J E , Sims K G , Anderson B E
Published: January 2020

We report a case of ehrlichiosis in a 72-year-old man who developed extreme lethargy, acute renal failure requiring hemodialysis, and respiratory insufficiency requiring intubation. Lumbar puncture performed on the second day of hospitalization revealed significant cellular pleocytosis. Ehrlichia morulae were tentatively identified in mononuclear cells in routinely processed Wright-stained cytospin preparations of cerebrospinal fluid (CSF). Identification was confirmed by a specific immunocytochemical staining procedure. Subsequent identification specifically as Ehrlichia chaffeensis morulae was established by polymerase chain reaction analysis, which revealed E. chaffeensis-specific DNA in CSF, bone marrow, and blood samples; by indirect fluorescent-antibody analysis, the patient developed an antibody titer of 32,768 against E. chaffeensis antigen. The patient responded to intravenous therapy with doxycycline and dexamethasone. Subsequently, neurologic, hematologic, renal, and pulmonary status had returned to baseline at follow-up 12 weeks after admission. To our knowledge, this is the first identification of E. chaffeensis morulae in CSF cells in an infected patient.
Polymerase chain reaction for detection of Leptospira spp. in clinical samples
Authors: Mérien F , Amouriaux P , Perolat P , Baranton G , Saint Girons I
Published: January 2020

A sensitive assay for Leptospira spp., the causative agent of leptospirosis, was developed on the basis of the polymerase chain reaction (PCR). A 331-bp sequence from the Leptospira interrogans serovar canicola rrs (16S) gene was amplified, and the PCR products were analyzed by DNA-DNA hybridization by using a 289-bp fragment internal to the amplified DNA. Specific PCR products also were obtained with DNA from the closely related nonpathogenic Leptospira biflexa but not with DNA from other spirochetes, such as Borrelia burgdorferi, Borrelia hermsii, Treponema denticola, Treponema pallidum, Spirochaeta aurantia, or more distant organisms such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, and Proteus mirabilis. The assay was able to detect as few as 10 bacteria. Leptospira DNA was detected in urine from experimentally infected mice. In addition, the test was found to be suitable for diagnosing leptospirosis in humans. Cerebrospinal fluid and urine from patients with leptospirosis were positive, whereas samples from control uninfected patients were negative.
Flow cytometric assay for quantifying opsonophagocytosis and killing of Staphylococcus aureus by peripheral blood leukocytes
Authors: Martin E , Bhakdi S
Published: January 2020

We describe a novel flow cytometric method for quantifying opsonophagocytosis and killing of Staphylococcus aureus in cell-rich plasma obtained after dextran sedimentation of erythrocytes. To analyze opsonophagocytosis, phagocytes were labeled with a phycoerythrin-conjugated monoclonal antibody and were incubated with viable staphylococci containing carboxyfluorescein as a vital fluorescent dye. Phagocytosing cells assumed a dual, orange-green fluorescence. The relative numbers of bacteria associating with phagocytes could be determined by quantifying the decrease of free green fluorescent particles. A parallel incubation of fluorescent bacteria with unlabeled cell-rich plasma was performed to assess phagocytic killing. Blood cells were lysed with 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate. This detergent spared viable bacteria, and residual green fluorescent particles were counted. The decrease in the number of these particles relative to the controls yielded the degree of killing. At bacteria-to-phagocyte ratios of 1:1 and 10:1, approximately 36 and 75% of the phagocytes participated in opsonophagocytosis, respectively. Over 90% of the staphylococci were phagocyte associated after 30 to 60 min. Killing rates were on the order of 66% +/- 12% and 80% +/- 7% after 1 and 2 h of incubation, respectively. These numbers, which were confirmed by colony countings, were significantly lower than those reported in the majority of past reports.
Cell-mediated and humoral immune responses induced by scarification vaccination of human volunteers with a new lot of the live vaccine strain of Francisella tularensis
Authors: Waag D M , Galloway A , Sandstrom G , Bolt C R , England M J , Nelson G O , Williams J C
Published: January 2020

Tularemia is a disease caused by the facultative intracellular bacterium Francisella tularensis. We evaluated a new lot of live F. tularensis vaccine for its immunogenicity in human volunteers. Scarification vaccination induced humoral and cell-mediated immune responses. Indications of a positive immune response after vaccination included an increase in specific antibody levels, which were measured by enzyme-linked immunosorbent and immunoblot assays, and the ability of peripheral blood lymphocytes to respond to whole F. tularensis bacteria as recall antigens. Vaccination caused a significant rise (P less than 0.05) in immunoglobulin A (IgA), IgG, and IgM titers. Lymphocyte stimulation indices were significantly increased (P less than 0.01) in vaccinees 14 days after vaccination. These data verify that this new lot of live F. tularensis vaccine is immunogenic.
Evaluation of an immunoenzymatic assay detecting specific anti-Toxocara immunoglobulin E for diagnosis and posttreatment follow-up of human toxocariasis
Authors: Magnaval J F , Fabre R , Maurières P , Charlet J P , de Larrard B
Published: January 2020

In order to complete the immunodiagnosis of human toxocaral disease, an immunoenzymatic assay with excretory-secretory antigens from Toxocara canis larvae was developed for the detection of specific immunoglobulin E (sIgE enzyme-linked immunosorbent assay [ELISA]). The specificity of the assay was evaluated in patients presenting with various allergic or helminthic diseases. The sensitivity was assessed in patients exhibiting clinical and biological symptoms indicative of toxocariasis, serodiagnosis of which was made by the Western blot (WB; immunoblot) procedure that used the same antigen as that used in the sIgE ELISA but that detected specific IgG. The value of the sIgE ELISA for posttreatment follow-up was tested in two groups of patients: one group was treated with diethylcarbamazine; the other group was not treated with DEC. Results showed that the specificity and sensitivity of the sIgE ELISA were moderate. Thus, the sIgE ELISA appeared to be insufficient for properly ensuring the serodiagnosis of toxocariasis when it is used alone. However, sIgE ELISA might be an interesting complementary method for the detection of specific IgG. It was the only assay that was found to be positive in sera from some hypereosinophilic patients. sIgE ELISA values decreased significantly among the patients treated with DEC, indicating that this test would be useful for posttreatment follow-up assessment.
Development of oligonucleotide primers and probes against structural and regulatory genes of human immunodeficiency virus type 1 (HIV-1) and their use for amplification of HIV-1 provirus by using polymerase chain reaction
Authors: Dawood M R , Allan R , Fowke K , Embree J , Hammond G W
Published: January 2020

The polymerase chain reaction is a powerful technique for amplifying a few copies of double-stranded genetic material to millions of copies in a few hours. The sensitivity and specificity of the polymerase chain reaction technique depend to some extent on the nucleotide sequences of the oligonucleotide primer pair used in the amplification. We report new oligonucleotide primers and probes which can be used for the amplification and detection of human immunodeficiency virus type 1 provirus sequences of not only structural but also regulatory genes. These primers are very sensitive and specific and can be used for the detection of African and North American strains of human immunodeficiency virus type 1.
Neisseria spp. and AIDS
Authors: Morla N , Guibourdenche M , Riou J Y
Published: January 2020

Neisseria meningitidis from various serogroups and two commensal neisseriae (N. sicca and N. perflava) were isolated from 15 patients at various stages of human immunodeficiency virus infection in this clinical and bacteriological study. The cases were grouped into the following three classes: (i) infections with an N. meningitidis strain of a serogroup known to be pathogenic (A, B, or C) and apparently independent of the human immunodeficiency virus infection, (ii) infections with a N. meningitidis strain of a serogroup which is normally either commensal or poorly pathogenic (serogroups Y, X, Z, and Z,29E), (iii) pulmonary and disseminated infections occurring in the course of the clinical evolutionary stage of AIDS, in two cases of which commensal neisseriae (N. sicca and N. perflava) were isolated from blood cultures.
Endocarditis caused by Blastoschizomyces capitatus and taxonomic review of the genus
Authors: Polacheck I , Salkin I F , Kitzes-Cohen R , Raz R
Published: January 2020

Blastoschizomyces capitatus Salkin, Gordon, Samsonoff et Rieder was found to be the etiologic agent of endocarditis in a patient with a prosthetic mitral valve. Cultures inoculated with peripheral blood and portions of the valve yielded B. capitatus. Examination of stained tissue sections revealed the presence of fungal filaments morphologically consistent with this organism. The salient characteristics of B. capitatus and the factors contributing to its recognition as a distinct taxon are described.
Cellular and humoral immune responses to rubella virus structural proteins E1, E2, and C
Authors: Chaye H H , Mauracher C A , Tingle A J , Gillam S
Published: January 2020

Better understanding of cell-mediated immune responses to rubella virus would provide the basis for the development of safe and effective vaccines against rubella and would aid in analysis of the pathophysiology of congenital rubella syndrome. We have expressed individual rubella virus structural proteins, E1, E2 and C, via vaccinia virus recombinants. Using the expressed recombinant proteins as antigens, we were able to demonstrate antigen-specific lymphocyte proliferative responses in control individuals and individuals with congenital rubella syndrome. Among the two human groups studied, E1 glycoprotein proved to be a better immunogen than E2 or C. For the control individuals, significant differences in proliferative responses to the structural proteins E1, E2, and C were observed. These differences were not significant in individuals with congenital rubella syndrome. In parallel to the lymphoproliferative responses, immunoglobulin G responses were also found directed mainly to the E1 glycoprotein. These results suggest that E1 may be the most important rubella virus antigen to study in determining the domains required for constructing subunit vaccines against rubella.
Enzymatically active Peptostreptococcus magnus: association with site of infection
Authors: Krepel C J , Gohr C M , Walker A P , Farmer S G , Edmiston C E
Published: January 2020

Fifty-four strains of Peptostreptococcus magnus (11 were recovered from abdominal infections, 18 were from nonpuerperal breast abscesses, and 21 were from diabetic foot infections; the type strain and three other strains were from the American Type Culture Collection, Rockville, Md.) and the type strain of Peptostreptococcus micros were tested for their ability to produce various enzymes, including catalase, hippurate hydrolase, serine dehydratase, threonine dehydratase, collagenase, gelatinase, alkaline phosphatase, and esterase C4. The data were analyzed by cluster analysis. The results showed that all but one strain could be assigned to either of two distinct, valid clusters. The first cluster of 11 strains was composed of strains that were relatively inactive, having produced one or two of the eight strain-dependent enzymes. The second was a large cluster of strains (n = 43) that were considerably more active, all having produced at least three enzymes; the vast majority of strains (89%) produced four or more enzymes. The unclustered strain produced one enzyme that was different from that produced by the strains in the first cluster. The chi 2 test of homogeneity applied to the clustering solution indicated that greater enzyme activity was significantly associated with the site of infection (P less than 0.001). The more enzymatically active P. magnus strains were recovered significantly more often from nonpuerperal breast abscesses and diabetic foot infections than they were from abdominal infections. These results may provide insight into the nature of certain polymicrobial soft tissue infections and suggest that (i) P. magnus may participate more in nonpuerperal breast and diabetic foot infections than in abdominal infections and that (ii) peptostreptococcal production of proteolytic enzymes may have an important adjunctive effect on the pathogenesis of certain soft tissue infections.
Early detection of antibody to human immunodeficiency virus type 1 by using an antigen conjugate immunoassay correlates with the presence of immunoglobulin M antibody
Authors: Gallarda J L , Henrard D R , Liu D , Harrington S , Stramer S L , Valinsky J E , Wu P
Published: January 2020

Sequential plasma samples obtained from 16 individuals who seroconverted were tested for the presence of antibody to human immunodeficiency virus type 1 (HIV-1) by an antigen conjugate enzyme immunoassay (EIA) and a conventional antibody conjugate assay. In 11 of these individuals, the antigen conjugate assay detected antibody to HIV-1 2 to 11 days (mean, 5.5 days) earlier than the antibody conjugate assay. In 11 individuals, HIV-1 p24 antigen was detected a median of 6.5 days (range, 3 to 14 days) prior to positivity by the antigen conjugate EIA. Using class-specific probes, we determined the profiles of immunoglobulin M (IgM), IgG, and IgA antibodies for each individual and correlated these profiles with the EIA signals from both assays. In general, the appearance of IgM exhibited a peak at about 1 week postseroconversion, which was followed by gradually declining levels. Absorbance levels for IgG antibody, however, rose steadily and reached a plateau after 3 to 5 weeks. The levels of IgA were generally low and variable. In contrast to the progressive increase in EIA absorbance observed by the antibody conjugate assay, the antigen conjugate assay displayed a rapid early rise in absorbance which generally coincided with the transient expression of IgM antibody. The subsequent gradual increase coincided with rising levels of IgG. Because the configuration of the antigen conjugate EIA allows for an increased sensitivity for IgM compared with that for other classes of immunoglobulins, these results suggest that earlier detection of antibody to HIV-1 is due to the detection of IgM antibody during the early phase of seroconversion.
Phenotypic variation of Staphylococcus epidermidis isolated from a patient with native valve endocarditis
Authors: Deighton M , Pearson S , Capstick J , Spelman D , Borland R
Published: January 2020

Two colonial variants of Staphylococcus epidermidis were isolated from the valvular tissue of a patient with native valve endocarditis. In addition to differing in colonial morphology, the two variants differed in hemolysis on blood-containing media, in adherence capacity, and in the expression of certain enzymes. Under suitable conditions, both variants were themselves capable of phenotypic variation, although they differed in the rate at which variants were generated. The variants yielded identical profiles on restriction endonuclease analysis of plasmid DNA and pulsed-field gel electrophoresis of whole-cell DNA. This report suggests a possible role for phenotypic variation in coagulase-negative staphylococcal virulence. Congo red agar would be an excellent medium for studying the contribution of variation to the virulence of these organisms.
M protein gene typing of Streptococcus pyogenes by nonradioactively labeled oligonucleotide probes
Authors: Kaufhold A , Podbielski A , Johnson D R , Kaplan E L , Lütticken R
Published: January 2020

A new approach for the typing of Streptococcus pyogenes is described. Oligonucleotide probes of 30 nucleotides in length were derived from currently known sequences of the N-terminal regions of M protein genes (emm genes). The oligonucleotides were labeled with digoxigenin-dUTP and hybridized to dot-blotted genomic DNA from 116 group A streptococcal strains of serotypes M-1, M-2, M-3, M-5, M-6, M-12, M-18, M-19, M-24, and M-49. Hybridization reactions were visualized with a chemiluminescent substrate. In comparison with conventional serological typing of expressed M proteins, the binding of the probes to the corresponding emm genes exhibited 100% sensitivity and specificity. The results emphasize the high degree of type-specific conservation of the N-terminal regions of emm genes from reference strains and epidemiologically unrelated U.S. and European clinical isolates. The existence of two distinct genetic subgroups among eight investigated M-49 strains was unequivocally shown by hybridization assays and further confirmed by nucleotide sequence data obtained from four selected M-49 strains. Because oligonucleotide probes are relatively easy to prepare, easy to handle, and known to give consistent interlaboratory results, the "oligotyping" technique appears to offer potential advantages over conventional serological typing methods.
Legionella lansingensis sp. nov. isolated from a patient with pneumonia and underlying chronic lymphocytic leukemia
Authors: Thacker W L , Dyke J W , Benson R F , Havlichek D H , Robinson-Dunn B , Stiefel H , Schneider W , Moss C W , Mayberry W R , Brenner D J
Published: January 2020

A Legionella-like organism, strain 1677-MI-H, was isolated from the bronchoscopy washings of a patient with pneumonia who had a 2-year history of progressive, chronic lymphocytic leukemia. The growth characteristics, cellular fatty acids, and ubiquinone content of the isolate were consistent with those for Legionella spp. The isolate was serologically distinct in the slide agglutination test with absorbed antisera. DNA hybridization studies showed that strain 1677-MI-H (ATCC 49751) represents a new Legionella species which is named Legionella lansingensis.
High-performance liquid chromatography patterns of Mycobacterium gordonae mycolic acids
Authors: Cage G D
Published: January 2020

An important class of fatty acids contained in the cell envelopes of Mycobacterium organisms is the group of high-molecular-weight, long-chain, alpha-branched, beta-hydroxylated mycolic acids. By using standard saponification techniques and derivatization of the acids to their p-bromophenacyl esters, it is possible to differentiate them by high-performance liquid chromatography. Mycolic acid chromatograms of 63 clinical isolates of Mycobacterium gordonae were compared with conventional biochemical methods for identification. The data show two distinct pattern types for this species, only one of which has been elaborated in the literature by using this protocol. Laboratory workers who intend to use this method as a clinical tool need to be aware that these two pattern types exist.
Identification of Mycobacterium tuberculosis and Mycobacterium avium-M. intracellulare directly from primary BACTEC cultures by using acridinium-ester-labeled DNA probes
Authors: Evans K D , Nakasone A S , Sutherland P A , de la Maza L M , Peterson E M
Published: January 2020

Identification of members of the Mycobacterium tuberculosis complex and the M. avium-M. intracellulare complex (MAC) directly from primary BACTEC cultures was evaluated by using acridinium-ester-labeled DNA probes (AccuProbe; GenProbe, Inc., San Diego, Calif.). In preliminary experiments, blood present in samples was found to interfere with the assay because of nonspecific chemiluminescence, which was measured in relative light units (RLUs). There was a direct relationship between the age of the culture and the number of nonspecific RLUs. A protocol using 1% sodium dodecyl sulfate-5 mM EDTA to treat BACTEC broth cultures which, with specimens containing blood, gave on the average a ninefold reduction in nonspecific chemiluminescence was developed. By using this treatment protocol, 120 specimens were tested directly from BACTEC broth cultures with an AccuProbe for the M. tuberculosis complex and/or the MAC. In order to establish the background of the specimen, the patient sample was assayed without probe. The criteria for the inclusion of BACTEC cultures in the evaluation were a growth index of greater than or equal to 100 and a positive smear for acid-fast bacilli directly from the BACTEC broth. For the 120 cultures tested, if a hybridization result of greater than or equal to 30,000 RLUs was considered positive, the sensitivities for detecting the M. tuberculosis complex and the MAC were 47 and 90%, respectively, with a specificity of 100% for both. However, if a ratio of the RLUs obtained with the MAC or the M. tuberculosis complex probe to those obtained with the specimen background of >/= 20 was considered positive, this gave 77% sensitivity and 100% specificity for BACTEC cultures containing M. tuberculosis complex isolates and 96% sensitivity and 100% specificity for those growing MAC isolates.
Biochemical and antigenic properties of Streptococcus bovis isolated from pigeons
Authors: De Herdt P , Haesebrouck F , Devriese L A , Ducatelle R
Published: January 2020

Biochemical and serological properties of 60 strains of Streptococcus bovis isolated from healthy pigeons and from pigeons that died from S. bovis septicemia were determined. On the basis of the hemolysis of bovine erythrocytes, the production of polysaccharides on saccharose-containing media, and the fermentation of mannitol, inulin, trehalose, and L-arabinose, the isolates were classified in five biotypes and two subbiotypes. Slide agglutination and microagglutination tests using monospecific rabbit antisera allowed the classification of all isolates in five serotypes.
Acanthamoeba keratitis: synergy between amebic and bacterial cocontaminants in contact lens care systems as a prelude to infection
Authors: Bottone E J , Madayag R M , Qureshi M N
Published: January 2020

We encountered a patient with Acanthamoeba keratitis whose contact lens care solution contained numerous trophozoites and cysts admixed with Xanthomonas maltophilia organisms, many of which were adherent to the trophozoite surface and internalized within endocytic vacuoles. Because of this finding, we investigated the role of bacterial cocontaminants in contact lens care systems as substrates for the growth of Acanthamoeba spp. Individual cocultivation of Acanthamoeba castellanii and A. polyphaga with X. maltophilia, Flavobacterium breve, and Pseudomonas paucimobilis showed better enhancement (1.5x) of ameba growth after 96 h than that obtained in the presence of Staphylococcus aureus, S. epidermidis, and Escherichia coli, the standard cocultivation species used for isolation of amebae from clinical specimens. Our data suggest that contamination of contact lens care systems with Acanthamoeba spp. and a bacterial species capable of supporting amebic growth may be the first step in the pathogenesis of ameba-induced keratitis by the provision of large inocula of amebae.
Enzyme immunoassay for Q fever: comparison with complement fixation and immunofluorescence tests and dot immunoblotting
Authors: Cowley R , Fernandez F , Freemantle W , Rutter D
Published: January 2020

Enzyme-linked immunosorbent assays (ELISA) for the detection of specific immunoglobulin G (IgG) and IgM antibodies were developed by using purified Coxiella burnetii cells. Variables, including type of microtiter plate, blocking agent, incubation conditions, antigen stability, and substrate type, were examined to achieve optimal ELISA performance. The reliabilities of the assay systems were compared with those of complement fixation (CF) and enhanced immunofluorescence (EIF) tests with 600 human serum samples from defined clinical cases of Q fever, routine samples, and serum specimens from farmers. ELISA and EIF test results agreed in all cases. Dot immunoblotting was also used to test some of these sera and gave a rapid, qualitative result, which agreed with ELISA and EIF test results in all cases. No instances were found in which both ELISA and EIF test results were negative and the CF test results was positive. However approximately 5% of the sera were positive by ELISA and the EIF test while the CF test result was either negative or unreadable because of serum anticomplementary activity. We conclude that dot immunoblotting is a useful screening test, whereas ELISA and the EIF test are both rapid and sensitive tests when used for the serodiagnosis of Q fever and should be considered to be replacements for the CF test.