bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a method for direct quantification of cytomegalovirus antigenemia by flow cytometry
Authors: Imbert-Marcille B M , Robillard N , Poirier A S , Coste-Burel M , Cantarovich D , Milpied N , Billaudel S
Published: January 2020

Cytomegalovirus (CMV) antigenemia was directly detected in polymorphonuclear leukocytes (PMNLs) from transplant recipients by using flow cytometry (FC). Two fixation and permeabilization methods and seven anti-CMV monoclonal antibodies (MAbs) were evaluated. 1C3, SL20, and NEA-9221 MAbs were more efficacious. The antigenemia detection threshold of FC was 0.05% positive PMNLs, and percentages correlated well with DNA viral load and the appearance of clinical symptoms.
Fatal pulmonary infection caused by the basidiomycete Hormographiella aspergillata
Authors: Verweij P E , van Kasteren M , van de Nes J , de Hoog G S , de Pauw B E , Meis J F
Published: January 2020

A fatal case of a pulmonary infection caused by Hormographiella aspergillata, the anamorph of the mushroom Coprinus cinereus, is reported for a patient receiving treatment for a second relapse of acute lymphoblastic leukemia. The filamentous basidiomycete was identified with restriction fragment length polymorphism patterns of PCR-amplified internally transcribed spacers and small subunit ribosomal DNA with four restriction enzymes. The patient failed to respond to treatment with amphotericin B and itraconazole. The fungus was cultured from the lungs at autopsy: the MIC of amphotericin B for the fungus was low (0.5 mg/liter), and that of itraconazole was high (8 mg/liter).
Sequences of Pneumocystis carinii f. sp. hominis strains associated with recurrent pneumonia vary at multiple loci
Authors: Keely S P , Stringer J R
Published: January 2020

The sequences of the internal transcribed spacer (ITS) of Pneumocystis carinii f. sp. hominis strains from 7 of 15 AIDS patients were found to vary during discrete episodes of P. carinii pneumonia. Changes in the ITS sequence correlated with changes in the mitochondrial large-subunit rRNA sequence. The coincidence of changes in the sequences of the ITS, which is located in the nucleus, with changes in a mitochondrial gene excludes mutation as the cause of the genetic differences between P. carinii f. sp. hominis strains isolated during different episodes of P. carinii pneumonia and supports the hypothesis that recurrent P. carinii pneumonia is caused by reinfection rather than by reactivation of latent organisms. Thus, limiting the exposure of immunocompromised patients to P. carinii f. sp. hominis should help prevent P. carinii pneumonia.
Discrimination of Campylobacter jejuni isolates by fla gene sequencing
Authors: Meinersmann R J , Helsel L O , Fields P I , Hiett K L
Published: January 2020

Comparison of the entire coding sequence of flaA (1,764 nucleotides) from 15 isolates of Campylobacter jejuni showed two regions of high variability, one region approximately from base positions 700 to 1,450 and a short variable region (SVR) from base positions 450 to 600. Parsimony analysis of the SVR sequences yielded a dendrogram similar to that which was derived by analysis of the entire gene. PCR was used to generate templates, and the SVR was sequenced with primers constructed to hybridize to conserved flanking sequences. The SVRs of 22 isolates of C. jejuni from four outbreaks that have been well characterized and a larger panel of isolates from three additional outbreaks were sequenced. Analysis of the nucleotide sequences produced results that grouped the isolates very similarly to other subtyping techniques. Sequence data were also generated for isolates from three additional outbreaks. Categorizing the isolates by fla SVR DNA sequence placed them in epidemiologically relevant groups. Sequence analysis of the C. jejuni flaA SVR may be a useful tool for epidemiologic investigations and could complement or replace serotyping and other subtyping methods.
Production of toxins (enterotoxins, verotoxins, and necrotoxins) and colicins by Escherichia coli strains isolated from septicemic and healthy chickens: relationship with in vivo pathogenicity
Authors: Blanco J E , Blanco M , Mora A , Blanco J
Published: January 2020

Since the mechanism of virulence of Escherichia coli strains pathogenic to birds is not fully understood, the prevalence of toxic factors produced by E. coli strains pathogenic to other animals was investigated. A total of 625 E. coli strains isolated from visceral organs of chickens with colisepticemia and from feces of healthy chickens in Spain were tested for production of enterotoxins (heat labile [LT] and heat stable [STa]), verotoxins (VT1, VT2, and VT2v), cytotoxic necrotizing factors (CNF1 and CNF2), alpha-hemolysin (Hly), enterohemolysin (EntHly), colicin V (Col V) and other types of colicins, and necrotic and lethal activities. Only 45 (7%) of avian E. coli strains were toxigenic: 20 strains produced a cytotoxic response in HeLa but not in Vero cells, indicating the production of a cytotoxin not related to the VTs; 16 were EntHly+; 5 produced a new cytotonic product that causes the appearance of whitish vacuola in Vero and HeLa cells; 3 synthesized soluble factors that cause lethal activity in mice; and 1 elaborated LT. None of 625 avian E. coli strains was positive for production of VTs or CNFs. In contrast, colicinogenicity occurred in 335 (73%) of the 458 septicemic strains and 97 (58%) of 167 fecal isolates (P < 0.01), and this property was correlated with in vivo pathogenicity of strains. Thus, 80% (P < 0.001) and 66% (P < 0.001) of strains producing Col V and other types of colicins were characterized as being of high pathogenicity, whereas only 15% of the noncolicinogenic strains were classified as highly pathogenic. Our results clearly support the special pathogenicity theory, because 60% of the E. coli strains belonging to 18 serogroups (O1, O2, O5, O8, O12, O14, O15, O18, O20, O53, O78, O81, O83, O102, O103, O115, O116, and O132) most frequently identified among clinical septicemic strains were classified as highly pathogenic in in vivo assays, whereas only 24% of the strains with O serogroups less prevalent among diseased chickens were considered highly pathogenic (P < 0.01).
Evaluation of the automated COBAS AMPLICOR hepatitis C virus PCR system
Authors: Poljak M , Seme K , Koren S
Published: January 2020

To evaluate the reliability and feasibility of the automated Roche COBAS AMPLICOR PCR system for routine detection of hepatitis C virus (HCV) RNA, a total of 405 serum samples previously tested by an in-house nested PCR and manual Roche AMPLICOR microwell plate HCV test were examined. Complete concordance was found between the results with the HCV COBAS AMPLICOR system and the previously determined HCV RNA status. The automated HCV COBAS AMPLICOR system provides the clinical microbiology laboratory with a specific and sensitive PCR method for rapid and reliable detection of HCV RNA.
Immunoglobulin A antibodies to Helicobacter pylori
Authors: Jaskowski T D , Martins T B , Hill H R , Litwin C M
Published: January 2020

Serological testing for immunoglobulin G (IgG) antibodies to Helicobacter pylori has proven useful in supporting the diagnosis of infection with this organism, but the clinical value of IgA antibodies in H. pylori-related gastritis remains controversial. The purpose of our study was to determine the frequency of IgA-positive IgG-negative patients with symptoms of gastrointestinal (GI) disorders, thus assessing the clinical utility of IgA testing for H. pylori-related gastritis. It was found previously that the frequency of infected individuals in this category (IgA positive and IgG negative) is about 2%, but a large number of IgG-negative patients with GI disorders suggestive of H. pylori infection have not been investigated until now.
Molecular tracking of Candida albicans in a neonatal intensive care unit: long-term colonizations versus catheter-related infections
Authors: Ruiz-Diez B , Martinez V , Alvarez M , Rodriguez-Tudela J L , Martinez-Suarez J V
Published: January 2020

Nosocomial neonatal candidiasis is a major problem in infants requiring intensive therapy. The subjects of this retrospective study were nine preterm infants admitted to the neonatal intensive care unit of the Hospital Central de Asturias between March 1993 and August 1994. The infants were infected with or colonized by Candida albicans. Five patients developed C. albicans bloodstream infections. A total of 36 isolates (including isolates from catheters and parenteral nutrition) were examined for molecular relatedness by PCR fingerprinting and restriction fragment length polymorphism (RFLP) analysis. The core sequence of phage M13 was used as a single primer in the PCR-based fingerprinting procedure, and RFLP analysis was performed with C. albicans-specific DNA probe 27A. Both techniques were evaluated with a panel of eight C. albicans reference strains, and each technique showed eight different patterns. With the 36 isolates from neonates, each technique enabled us to identify by PCR and RFLP analysis seven and six different patterns, respectively. The combination of these two methods (composite DNA type) identified eight different profiles. A strain with one of these profiles was present in three patients and in their respective catheters. Patients infected with or colonized by this isolate profile were clustered in time. Among the other patients, each patient was infected over time and at multiple anatomic sites with a C. albicans strain with a distinct DNA type. We conclude that C. albicans was most commonly producing long-term colonizations, although horizontal transmission probably due to catheters also occurred.
Comparison of different methods for extraction of DNA of fungal pathogens from cultures and blood
Authors: Löffler J , Hebart H , Schumacher U , Reitze H , Einsele H
Published: January 2020

Five commercially available extraction kits and an in-house DNA extraction method for the release of DNA from Candida albicans and Aspergillus niger cells were assessed for sensitivity, purity, duration, and cost. All commercially available kits helped to shorten the duration of DNA extraction. However, the sensitivity varied from 1 to 1,000 fungal cells/ml and costs varied from $0.10 to 2.30. The QIAmp Tissue kit was the commercially available assay that yielded the same sensitivity and purity of fungal DNA release as the in-house protocol but was the most expensive method. In comparing these two extraction protocols, a 99% concordance of PCR results for 125 blood samples analyzed could be demonstrated.
Identification by spoligotyping of a caprine genotype in Mycobacterium bovis strains causing human tuberculosis
Authors: Gutiérrez M , Samper S , Jiménez M S , van Embden J D , Marín J F , Martín C
Published: January 2020

We have used spoligotyping to characterize 18 Mycobacterium bovis strains isolated from cattle and 23 M. bovis strains isolated from goats. The spoligotypes revealed that caprine strains form a separate and well-differentiated group that we refer to hereafter in this abstract as the caprine genotype. To evaluate the importance of this genotype as a cause of tuberculosis in other animal species, including humans, we applied the spoligotyping method to 112 strains, including to all isolates identified as M. bovis by a Mycobacterial National Reference Laboratory (Majadahonda, Madrid) from 1994 to 1996. Eighty-three of these strains were identified in human isolates. In addition to being identified in three goat isolates and two sheep isolates, the caprine genotype was also found in three isolates causing human tuberculosis. Evidence to support the argument that there is a zoonotic risk of caprine tuberculosis was presented by the identification of the caprine genotype in an isolate from a veterinary worker with a recent history of contact with tuberculous goats.
Detection of antibodies to respiratory syncytial virus attachment and nucleocapsid proteins with recombinant baculovirus-expressed antigens
Authors: Buraphacheep W , Britt W J , Sullender W M
Published: January 2020

The ability to measure antibodies against individual respiratory syncytial virus (RSV) proteins is important in the analysis of immune responses to RSV. We expressed the nucleocapsid (N) protein and the group A and B RSV attachment (G) proteins from recombinant baculoviruses. The three recombinant RSV proteins were used individually in an enzyme-linked immunosorbent assay (ELISA; bac-ELISA for results from assays of all three proteins). The bac-ELISA results were compared to the results obtained by a whole-virus ELISA (RS-ELISA for results from assays of both group A and B viruses). Antibody samples from 113 children were tested. The determination of seronegative or seropositive status by the bac-ELISA was compared to the same determination by the RS-ELISA; the sensitivity of bac-ELISA was 87% (95% confidence interval [CI], 78 to 93%), the specificity was 82% (CI, 59 to 94%), and the positive and negative predictive values were 95% (CI, 86 to 98%) and 60% (CI, 41 to 77%), respectively. The group specificity of the G-protein ELISA was confirmed by testing antibodies from experimentally immunized animals. Thus, the bac-ELISA was shown to be comparable to the whole-virus ELISA in detecting antibody responses to RSV, while it offered the advantage of measuring specific antibody responses to individual RSV proteins.
Performance characteristics of the Gen-Probe Probe Competition Assay used as a supplementary test for the Gen-Probe PACE 2 and 2C assays for detection of Chlamydia trachomatis
Authors: Beebe J L , Sharpton T R , Zanto S N , Steece R S , Rogers C , Mottice S L
Published: January 2020

The performance characteristics of the Gen-Probe Probe Competition Assay (PCA) used in conjunction with the Gen-Probe PACE 2 and 2C direct detection assays for Chlamydia trachomatis were examined. Data collected by five public health laboratories by using the Gen-Probe PACE 2 were pooled and analyzed. Of 25,081 endocervical and male urethral specimens tested by the PACE 2 assay, 773 were tested by PCA. Of 334 specimens initially positive by the PACE 2 assay with an initial PACE 2 result of greater than 2,000 relative light units (RLU), 333 (99.7%) were positive by PCA while 242 of 339 (71.4%) specimens with an initial result between the cutoff and 2,000 RLU were positive by PCA, and 35 of 100 (35%) specimens with initial results between 200 RLU and the cutoff were positive by PCA. An additional 10,938 specimens were tested by the PACE 2C assay. Of these, positive PCA results were obtained for 187 of 188 (99.5%) specimens with initial results of greater than 2,000 RLU, 99 of 163 (60.7%) of specimens in the range of cutoff to 2,000 RLU, and 12 of 100 (12%) in the range of 200 RLU to the cutoff. These results indicate that specimens greater than 2,000 RLU do not require a supplemental test and that additional positive results can be obtained by testing specimens with an initial result below the cutoff.
Sensitive assay for measurement of antibodies to Clostridium botulinum neurotoxins A, B, and E: use of hapten-labeled-antibody elution to isolate specific complexes
Authors: Doellgast G J , Brown J E , Koufman J A , Hatheway C L
Published: January 2020

The measurement of chicken and human antibodies to Clostridium botulinum neurotoxins A, B, and E was accomplished by affinity isolation of complexes containing these antibodies. By this approach, a mixture of toxin with the test antibody, fluoresceinated antibody, and enzyme (Russell's viper venom factor X activator)-labeled antibody is allowed to form a complex in solution phase. This complex is then bound to a matrix containing antifluorescein antibody. All components not bound to the matrix are washed off, and the complex is isolated intact by elution with fluorescein, which competes with the complex for binding to the antifluorescein matrix. The eluted complex is then bound to a matrix which specifically binds the test antibody (anti-chicken immunoglobulin Y [IgY] or anti-human IgG), and the bound complex is measured by using the enzyme label. Using this approach, we were able to measure as little as 1 ng of specific antibody per ml from affinity-isolated, monospecific chicken antibody preparations and to measure antibody specifically from IgY fractions of monospecific chicken antibody preparations. Human antibodies from subjects immunized with pentavalent toxoid preparations were detectable at dilutions as great as 24,300-fold, and undiluted serum from most control subjects showed no measurable antibody. Antibody was also measured in 65 subjects who were receiving preparations of neurotoxin A (BOTOX) for the treatment of spastic disorders. Eighteen of them had toxin-specific antibody reactive with toxin B, and two of them had toxin-specific antibody reactive with toxin A. The two patients having antibody to toxin A were refractory to treatment with this toxin. This approach of isolation of hapten-labeled immune complexes under nondenaturing conditions with hapten is broadly applicable to the specific measurement of antibodies present at very low concentrations in serum.
Differentiation of Mycobacterium tuberculosis isolates by spoligotyping and IS6110 restriction fragment length polymorphism
Authors: Goyal M , Saunders N A , van Embden J D , Young D B , Shaw R J
Published: January 2020

Mycobacterium tuberculosis isolates from 167 patients attending three London hospitals were analyzed by two techniques for strain differentiation. A significant number of isolates that appeared identical with the recently developed spoligotyping system could be distinguished from each other by IS6110 restriction fragment length polymorphism analysis, with the latter technique demonstrating a generally higher level of discrimination. Spoligotyping, on the other hand, was particularly useful for analysis of isolates with low IS6110 copy numbers, and use of the two techniques in tandem provided an optimal approach to the identification of clusters with epidemiological evidence consistent with recent transmission. Spoligotyping can be applied directly to clinical samples by PCR and provides an important tool for the rapid detection of nosocomial transmission of individual strains.
Prenatal diagnosis of rubella virus infection by direct detection and semiquantitation of viral RNA in clinical samples by reverse transcription-PCR
Authors: Revello M G , Baldanti F , Sarasini A , Zavattoni M , Torsellini M , Gerna G
Published: January 2020

A reverse transcription-nested PCR (RT-nPCR) method for prenatal diagnosis of rubella virus (RV) infection was developed. In the first step of RT-nPCR a synthetic RNA molecule (pRRV) differing from the RV target sequence by having a 21-nucleotide insertion was used as the internal control of amplification for the detection of PCR inhibitors. In addition, comparison of pRRV and RV-specific PCR signals allowed for the semiquantitation of RV input target sequences (range, 10 to > and = 1,000 RV genomes). In parallel, a complete RT-nPCR assay was performed with the same samples in the absence of the internal control to confirm the results of the first step and to detect RV RNA-positive samples containing < 10 RV genomes. Subsequently, the RT-nPCR method was used to examine retrospectively clinical samples (direct RT-nPCR) from eight congenitally infected and eight uninfected fetuses for RV RNA. RT-nPCR was also used to detect RV RNA in cell cultures (culture-RT-nPCR) 96 h after inoculation with the same specimens. With amniotic fluid (AF) samples, direct RT-nPCR identified eight of eight cases of RV transmission (sensitivity, 100%), whereas culture-RT-nPCR and virus isolation detected only six of eight cases (sensitivity, 75%). However, when the culture-RT-nPCR results were positive, culture-RT-nPCR confirmed the direct RT-nPCR results 3 days to 3 weeks earlier than virus isolation. The specificity of direct RT-nPCR was 100%, with eight of eight uninfected fetuses being negative. Semiquantitation showed only small amounts (< and = 100 copies) of viral RNA in clinical samples. In conclusion, direct RT-nPCR with AF samples (i) shows 100% sensitivity and specificity for prenatal diagnosis of RV infection and (ii) is a rapid technique, giving results in 24 to 48 h after sampling.
Rapid identification of clinically significant species and taxa of aerobic actinomycetes, including Actinomadura, Gordona, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella isolates, by DNA amplification and restriction endonuclease analysis
Authors: Steingrube V A , Wilson R W , Brown B A , Jost K C , Blacklock Z , Gibson J L , Wallace R J
Published: January 2020

A previously described PCR-restriction fragment length polymorphism (RFLP) identification schema for Nocardia that used an amplified 439-bp segment (amplicon) of the 65-kDa heat shock protein gene was evaluated for potential use with isolates of all clinically significant aerobic actinomycetes. The study included 28 reference (American Type Culture Collection) strains and 198 clinical isolates belonging to 20 taxonomic groups. Of these 198 isolates, 188 could be differentiated by this PCR-RFLP method. Amplicons from all aerobic actinomycete isolates lacked BstEII recognition sites, thereby distinguishing them from those of mycobacteria that contain one or more such sites. Of 29 restriction endonucleases, MspI plus HinfI produced RFLP patterns that differentiated 16 of the 20 taxa. A single RFLP pattern was observed for 15 of 20 taxa that included 65% of phenotypically clustered isolates. Multiple patterns were seen with Gordona bronchialis, Nocardia asteroides complex type VI, Nocardia otitidiscaviarum, Nocardia transvalensis, and Streptomyces spp. Streptomyces RFLP patterns were the most heterogeneous (five patterns among 19 isolates), but exhibited a unique HinfI fragment of > 320 bp. RFLP patterns that matched those from type strains of Streptomyces albus, Streptomyces griseus, or Streptomyces somaliensis were obtained from 14 of 19 Streptomyces isolates. Only 10 of 28 isolates of N. otitidiscaviarum failed to yield satisfactory amplicons, while only 6 of 188 (3.2%) clinical isolates exhibited patterns that failed to match one of the 21 defined RFLP patterns. These studies extended the feasibility of using PCR-RFLP analysis as a rapid method for the identification of all clinically significant species and taxa of aerobic actinomycetes.
Assessment of detection of Candida mannoproteinemia as a method to differentiate central venous catheter-related candidemia from invasive disease
Authors: Girmenia C , Martino P , De Bernardis F , Cassone A
Published: January 2020

The proper management of candidemic patients is controversial because of the difficulties of an early differentiation of central venous catheter (CVC)-related candidemia from deep-seated invasive Candida infection. In particular, more information on possible markers of invasive disease is needed. We performed a retrospective, pilot investigation to assess the diagnostic potential of a dot immunobinding assay for Candida mannoprotein antigen in serial serum samples from 31 candidemic patients in the setting of hematologic malignancy. Mannoproteinemia (antigenemia) was detected in 1 of 14 (7.1%) patients with transient or CVC-related candidemia and in 13 of 17 (76.5%) patients with non-CVC-related persistent candidemia. Of the 11 subjects of this latter group with documented tissue invasion, 10 (91%) were antigenemic. The patients belonging to the different categories did not significantly differ in the duration of candidemia, nor was there any significant difference among the different groups of subjects either in the number of serum samples examined or in their collection time during candidemia. The day of the first antigenemic sample during candidemia greatly varied among subjects with invasive infection, although on average mannoproteinemia was detectable by the first week of candidemia. In summary, our data demonstrate a correlation between mannoproteinemia and tissue invasion by Candida spp. in candidemic patients and suggest that mannoprotein detection by our method has a potential for the diagnosis of invasive candidiasis in these subjects.
Rapid diagnosis of enterovirus infection by a new one-step reverse transcription-PCR assay
Authors: Kessler H H , Santner B , Rabenau H , Berger A , Vince A , Lewinski C , Weber B , Pierer K , Stuenzner D , Marth E , Doerr H W
Published: January 2020

The AMPLICOR Enterovirus Test was evaluated with 103 cerebrospinal fluid (CSF) specimens. Twenty-seven CSF specimens were culture positive. With the AMPLICOR test, enterovirus RNA was detected in 34 specimens. Compared with culture, the AMPLICOR test gave a sensitivity of 96.3% and a specificity of 100%. The sensitivity of culture was 79.4% in comparison with the AMPLICOR test.
Severe keratitis due to Nocardia farcinica
Authors: Eggink C A , Wesseling P , Boiron P , Meis J F
Published: January 2020

Keratitis due to Nocardia farcinica occurred in a 49-year-old female after inappropriate cleaning of her semipermeable rigid contact lenses with basin-stored water during a holiday in France. N. farcinica was differentiated from Nocardia asteroides by its growth at 45 degrees C, acid production from rhamnose, its opacification of Middlebrook 7H10 agar, and its marked degree of resistance to all cephalosporins, aminoglycosides, tetracyclins, macrolides, and trimethoprim-sulfamethoxazole. To the best of our knowledge, this is the first reported case of human N. farcinica keratitis, confirming that this microorganism can be responsible for serious human disease.
Virulent strains of Streptococcus suis serotype 2 and highly virulent strains of Streptococcus suis serotype 1 can be recognized by a unique ribotype profile
Authors: Smith H E , Rijnsburger M , Stockhofe-Zurwieden N , Wisselink H J , Vecht U , Smits M A
Published: January 2020

The ribotype profiles of 42 different Streptococcus suis strains were studied. These strains belonged to five serotypes and differed in their virulence for pigs as well as in the expression of the muramidase-released protein and the extracellular protein factor. For the ribotyping, chromosomal DNAs were digested with EcoRI and were hybridized with a 1,066-bp ribosomal DNA probe. The hybridization patterns showed genetic heterogeneity within and between the serotypes. Pathogenic strains of serotype 2 and highly pathogenic strains of serotype 1 could be recognized by their unique ribotype profiles. Nonpathogenic strains showed a high degree of genetic heterogeneity. Moreover, by comparing the 16S ribosomal DNA sequences of a number of S. suis strains, we were able to design two DNA probes which specifically hybridized with S. suis strains.
Isolation of serotype G8, P6[1] bovine rotavirus from adult cattle with diarrhea
Authors: Sato M , Nakagomi T , Tajima K , Ezura K , Akashi H , Nakagomi O
Published: January 2020

Two electrophoretically identical strains of group A rotavirus were isolated from diarrheic cows aged 3, 4, and 7 years on a dairy farm in Japan. They had a rare serotype combination of G8 and P6[1] and were shown by RNA-RNA hybridization to be most closely related to bovine strain NCDV-Lincoln (G6P6[1]).
Detection of genetically diverse human immunodeficiency virus type 1 group M and O isolates by PCR
Authors: Respess R A , Butcher A , Wang H , Chaowanachan T , Young N , Shaffer N , Mastro T D , Biryahwaho B , Downing R , Tanuri A , Schechter M , Pascu R , Zekeng L , Kaptué L , Gürtler L , Eberle J , Ellenberger D , Fridlund C , Rayfield M , Kwok S
Published: January 2020

A panel of 136 genetically diverse group M and 5 group O adult isolates from outside the United States and Europe were evaluated by PCR with the Roche AMPLICOR HIV-1 test, a modified version of the AMPLICOR HIV-1 test, and a new primer pair/probe system. Detection of some of these isolates was less efficient with the AMPLICOR HIV-1 test; however, the assay was significantly improved by reducing the sample input and lowering the annealing temperature. The new primer pair/probe set detected 140 of 141 isolates, including the 5 group O isolates that were not detected with either of the AMPLICOR HIV-1 test formats.
Diagnosis of toxoplasmosis by joint detection of immunoglobulin A and immunoglobulin M
Authors: Arcavi M , Orfus G , Griemberg G
Published: January 2020

An indirect immunofluorescence test with total anti-human immunoglobulin conjugate (IgG,A,M-IIF) can be used for joint detection of immunoglobulin A (IgA) and IgM antibodies, provided serum IgG is previously absorbed with anti-human IgG. To determine the validity of the IgG,A,M-IIF assay with absorbed sera, the results obtained were compared with those obtained by methods routinely used for the detection of acute-phase markers, IgA and IgM IIF and enzyme immunoassay. Accordingly, 114 serum samples were selected from patients showing titers of > or = 1:1,024 by IgG,A,M-IIF. (i) In 90 of the samples, neither IgA nor IgM was detected by any of the methods employed; (ii) the remaining 24 samples showed IgA and/or IgM. In all cases, the IgG,A,M-IIF assay with absorbed sera was positive. These comparative data support the use of IgG,A,M-IIF, performed with absorbed and unabsorbed sera simultaneously, for determining the presence of specific IgG, IgA, and IgM by employing a single technique (IIF), one conjugate (anti-IgG,A,M), and only one sample (with and without previous absorption), thus providing a useful initial tool for the diagnosis of toxoplasmosis.
Prevalence of granulocytic Ehrlichia infection among white-tailed deer in Wisconsin
Authors: Belongia E A , Reed K D , Mitchell P D , Kolbert C P , Persing D H , Gill J S , Kazmierczak J J
Published: January 2020

Human granulocytic ehrlichiosis (HGE) is caused by an agent that is nearly indistinguishable from the veterinary pathogens Ehrlichia equi and Ehrlichia phagocytophila. The deer tick, Ixodes scapularis, is a vector of the HGE agent, and the white-tailed deer is the primary host for adult Ixodes ticks. We assessed the distribution of granulocytic Ehrlichia infection among deer living within (Wisconsin) and outside (western and southern Iowa) the geographic range of L. scapularis. Whole-blood samples were tested for HGE 16S ribosomal DNA (rDNA) by PCR, and E. equi antibody was detected by indirect immunofluorescence assay (IFA). Antibody titers of > or = 1:64 were defined as positive, and all positive samples were retested with a second lot of substrate antigen. E. equi antibody was present in 14 (8%) of 187 Wisconsin deer and 0 of 60 Iowa specimens (rate ratio undefined; P = 0.025). An additional 30 serum samples from Wisconsin deer were excluded because IFA results were discrepant between substrate lots. The reciprocal antibody titers ranged from 64 to 512 (geometric mean, 141) for positive samples. PCR results were positive for 27 (15%) of 181 Wisconsin deer. The prevalence of infection in northwestern Wisconsin deer was not significantly different from that in central Wisconsin deer, as determined by IFA and PCR. In two samples that were sequenced, the 16S rDNA was nearly identical to that of the granulocytic Ehrlichia species but distinct from that of Anaplasma marginale. The DNA sequences of the samples differed from the published sequences for E. equi, E. phagocytophila, and the HGE agent by 1 or 2 nucleotides (> or = 99.1% homology) at phylogenetically informative sites. Granulocytic Ehrlichia organisms in deer are widely distributed within the geographic range of L. scapularis in Wisconsin. Deer may serve as useful sentinels for areas where HGE transmission to humans may occur.
Multiplex PCR assay for immediate identification of the infecting species in patients with mycobacterial disease
Authors: Kox L F , Jansen H M , Kuijper S , Kolk A H
Published: January 2020

Rapid identification of infecting mycobacterial species enables appropriate medical care decisions to be made. Our aim was to demonstrate the clinical usefulness of the multiplex PCR assay, a test based on PCR, which permits direct identification of 12 mycobacterial species in clinical specimens. A total of 259 specimens from 177 patients who had clinical symptoms of mycobacterial disease but for whom there were difficulties in diagnosis were tested. Specimens were analyzed within 48 h of receipt of the sample. Mycobacteria were identified in 102 specimens; 66 specimens contained nontuberculous mycobacteria, and 36 specimens contained Mycobacterium tuberculosis complex mycobacteria. The PCR assay identified the mycobacterial species in 43 (97.7%) of 44 microscopy- and culture-positive specimens and in 15 (93.8%) of 16 culture-positive, microscopy-negative specimens. It also permitted species identification in infections caused by more than one mycobacterial species. For 56 (96.5%) of the 58 specimens from patients with infections caused by opportunistic mycobacteria, the organisms were identified with the PCR assay. The test was useful also for the identification of fastidious mycobacteria, e.g., M. genavense, and those that cannot be cultured, e.g., M. leprae. After resolution of discrepant results, the sensitivity of the PCR assay was 97.9%, the specificity was 96.9%, the positive predictive value was 95.0%, and the negative predictive value was 98.7%. For culture these values were 60.8, 100, 100, and 81.0%, respectively. Thus, the multiplex PCR assay enables prompt diagnosis when rapid identification of infecting mycobacteria is necessary.
Outbreak of amoxicillin-resistant Haemophilus influenzae type b: variable number of tandem repeats as novel molecular markers
Authors: van Belkum A , Melchers W J , Ijsseldijk C , Nohlmans L , Verbrugh H , Meis J F
Published: January 2020

An outbreak caused by amoxicillin-resistant Haemophilus influenzae type b was noted among patients suffering from chronic obstructive pulmonary disease. Since infections were clustered in time and place, an ongoing outbreak was suspected. The spread of the strain and the course of the outbreak could be followed by random amplification of polymorphic DNA (RAPD) analysis of the different bacterial isolates. In addition, studies were aimed at the determination of length polymorphism in regions of repetitive DNA. By PCR-mediated amplification of variable number of tandem repeat regions (VNTRs), additional insight into the genome composition of the epidemic strain was gained. Our results show that VNTRs comprising repeat units that are 3, 5, or 6 nucleotides in length provided stable genetic markers that can be used for molecular typing of H. influenzae type b. VNTRs built from tetranucleotide units, however, appear to be hypervariable and not suited for epidemiological studies. The observed variability in this latter class of VNTRs might be reminiscent of the bacterium's capacity to deal with unfavorable host factors.
Evaluation of nine immunoassay kits (enzyme immunoassay and direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvum in human fecal specimens
Authors: Garcia L S , Shimizu R Y
Published: January 2020

It is well known that Giardia lamblia and Cryptosporidium parvum can cause severe symptoms in humans, particularly those who are immunologically compromised. Immunoassay procedures offer both increased sensitivity and specificity compared to conventional staining methods. These reagents are also helpful when screening large numbers of patients, particularly in an outbreak situation or when screening patients with minimal symptoms. The data obtained by using 9 diagnostic kits were compared: direct fluorescent-antibody assay (DFA) kits (TechLab Giardia/Crypto IF kit, TechLab Crypto IF kit, and Meridian Merifluor Cryptosporidium/Giardia) and enzyme immunoassay (EIA) kits (Alexon ProSpecT Giardia EZ Microplate Assay, Alexon ProSpecT Cryptosporidium Microplate Assay, Cambridge Giardia lamblia Antigen Microwell ELISA, Meridian Premier Giardia lamblia, Meridian Premier Cryptosporidium, TechLab Giardia CELISA, Trend Giardia lamblia EIA). The test with the Meridian Merifluor Cryptosporidium/Giardia kit was used as the reference method. In various combinations, 60 specimens positive for Giardia, 60 specimens positive for Cryptosporidium, 40 specimens positive for a Giardia-Cryptosporidium mix, and 50 negative fecal specimens were tested. Different species (nine protozoa, three coccidia, one microsporidium, five nematodes, three cestodes, and one trematode) were included in the negative specimens. The sensitivity of EIA for Giardia ranged from 94% (Alexon) to 99% (Trend and Cambridge); the specificity was 100% with all EIA kits tested. The sensitivity of EIA for Cryptosporidium ranged from 98% (Alexon) to 99% (Meridian Premier); specificities were 100%. All DFA results were in agreement, with 100% sensitivity and specificity; however, the TechLab reagents resulted in fluorescence intensity that was generally one level below that seen with the reagents used in the reference method. In addition to sensitivity and specificity, factors such as cost, simplicity, ease of interpretation of results (color, intensity of fluorescence), equipment, available personnel, and number of tests ordered are also important considerations prior to kit selection.
Human disease in Europe caused by a granulocytic Ehrlichia species
Authors: Petrovec M , Lotric Furlan S , Zupanc T A , Strle F , Brouqui P , Roux V , Dumler J S
Published: January 2020

Human granulocytic ehrlichiosis (HGE) was recently described in North America. It is caused by an Ehrlichia species closely related to Ehrlichia phagocytophila and Ehrlichia equi, recognized to infect mostly ruminants and horses, respectively. The vector in North America is the tick Ixodes scapularis, which is also the vector of the Lyme disease agent, Borrelia burgdorferi. Previous serologic studies in patients with a diagnosis of Lyme borreliosis indicate that HGE may exist in Europe. We report the first documented case of HGE in Europe. The diagnosis was established by seroconversion to E. equi and the HGE agent and by PCR with sequence analysis of the gene encoding the HGE agent 16S rRNA. Interestingly, the patient presented with a self-limited but moderately severe illness. Thus, European physicians need to be aware that HGE exists in Europe and that the diagnosis should be considered in febrile patients with tick bites in areas where Lyme disease is endemic.
Dot immunoperoxidase assay for detection of parvovirus B19 antigens in serum samples
Authors: Gentilomi G , Musiani M , Zerbini M , Gallinella G , Venturoli S , Manaresi E
Published: January 2020

We describe a simple and rapid dot immunoperoxidase assay for the direct detection of parvovirus B19 capsid antigens in human sera. The assay was performed with serum specimens dotted onto nylon membranes. VP1 and VP2 B19 antigens, which represent 4 and 96% of the capsid, respectively, were detected with a pool of monoclonal antibodies directed against the two proteins, and the complex was visualized by immunoperoxidase staining. The assay could be performed in about 4 h, and positive results were revealed at the end of the reaction as dark blue spots on the nylon membrane at the site of positive specimens. A total of 541 serum samples from different subjects and with different laboratory evaluations with regard to B19 infection were analyzed. The results obtained by the dot immunoperoxidase assay were compared with the results obtained for the presence of B19 DNA by dot blot hybridization and nested PCR. With optimized working conditions, the dot immunoperoxidase assay was able to detect the presence of B19 with a sensitivity comparable or slightly higher than that achieved by dot blot hybridization but less than that achieved by nested PCR. Since the level of sensitivity of the dot immunoperoxidase assay proved to be appropriate for the detection of acute B19 infection, and since the cost, time to a result, and versatility of the assay are important issues, from our evaluation, the dot immunoperoxidase assay described may be particularly suitable for large-scale screening of samples and a good alternative to DNA detection methods in the routine laboratory evaluation of B19 infection.
Investigation of a pseudo-outbreak of Nocardia asteroides infection by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA PCR
Authors: Louie L , Louie M , Simor A E
Published: January 2020

Molecular strain typing by pulsed-field gel electrophoresis and by randomly amplified polymorphic DNA analysis was used to investigate a cluster of four Nocardia asteroides isolates associated with the BACTEC 460 TB system. An instrument motor drive misalignment resulted in inadequate needle sterilization and cross-contamination of BACTEC vials. This pseudo-outbreak illustrates the importance of proper BACTEC 460 needle sterilization and maintenance and confirms the usefulness of molecular typing methods for epidemiologic investigations.
Rapid identification of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus by restriction enzyme analysis of PCR-amplified 16S rRNA genes
Authors: Riggio M P , Lennon A
Published: January 2020

Restriction enzyme analysis of PCR-amplified 16S rRNA genes was used to distinguish among clinical isolates of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, and Haemophilus paraphrophilus which were originally identified by conventional phenotypic methods. This PCR-based method is a reliable and rapid alternative to conventional methods for identification of these bacterial species.
Microsatellite polymorphism in the promoter sequence of the elongation factor 3 gene of Candida albicans as the basis for a typing system
Authors: Bretagne S , Costa J M , Besmond C , Carsique R , Calderone R
Published: January 2020

The polymorphism of a TTC/TTTC microsatellite in the promoter sequence of the elongation factor 3 gene of Candida albicans was investigated by PCR. One primer was fluorescein labeled, and PCR signals were read with an automatic sequencer. Twenty-nine reference strains and 31 independent clinical isolates were studied. Eleven different alleles were identified, giving 16 different profiles among the 60 strains tested, with a discriminatory power of 0.88. This marker is stable upon subculture, and reproducibility was achieved by automated procedures. When several microsatellite markers are available, many isolates can be rapidly and reproducibly tested for epidemiological questions, such as the prevalence of a given strain in a hospital setting and transmission between patients.
Serological diagnosis of Chagas' disease: a potential confirmatory assay using preserved protein antigens of Trypanosoma cruzi
Authors: Mendes R P , Hoshino-Shimizu S , Moura da Silva A M , Mota I , Heredia R A , Luquetti A O , Leser P G
Published: January 2020

The diagnosis of Chagas' disease relies mostly on data provided by immunologic tests, but inconclusive results often require elucidation, especially in blood banks. When six different types of Trypanosoma cruzi epimastigote antigens were studied by an immunoblotting assay (IBA), a preserved protein antigen (Ag PP) was found to present the most interesting immunochemical features because of its high reactivity with anti-T. cruzi antibodies. Thus, the IBA with Ag PP (PP IBA) was assessed with panels of coded and noncoded serum samples prepared in different laboratories, including the Brazilian Reference Laboratory for Chagas' Disease. It was found that serum samples from patients proved (clinically, eletrocardiographically, serologically, and epidemiologically) to have Chagas' disease consistently recognized 12 bands (140, 100, 85, 78, 59, 57, 46, 35, 27, 23, 20, and 18 kDa) of Ag PP. In contrast, sera from nonchagasic patients, including patients with mucocutaneous leishmaniasis, were negative or reacted weakly, and one serum sample did not have more than five different bands. These bands were 78, 57, 46, 35, 27, 23, 20, or 18 kDa. A criterion was adopted to interpret the results obtained in the PP IBA. The criterion considered positive a serum sample recognizing all 12 bands and considered negative a serum sample that did not recognize any of the bands except the eight nonspecific bands mentioned above. The PP IBA indicated maximum sensitivity and specificity as well as high positive and negative predictive values. The data demonstrate that the PP IBA discriminates chagasic from nonchagasic infections and seems to be applicable as a confirmatory assay for elucidating inconclusive results obtained by standard serology.
A case of tick-borne encephalitis in Japan and isolation of the the virus
Authors: Takashima I , Morita K , Chiba M , Hayasaka D , Sato T , Takezawa C , Igarashi A , Kariwa H , Yoshimatsu K , Arikawa J , Hashimoto N
Published: January 2020

A case of tick-borne encephalitis (TBE) has not been reported for many years in Japan, although a serological survey of sera from domestic animals suggested the presence of TBE foci in Hokkaido, the northern island of Japan. Studies were conducted to prove the presence of an endemic focus of TBE virus in Japan by means of serology and virus isolation. In October 1993 in Hokkaido, a severe case of encephalitis in a dairy farmer's wife was diagnosed as TBE. Serological examination of paired serum specimens showed a rise in the neutralization antibody titer to Russian spring summer encephalitis virus. A seroepizootiological survey of dogs showed that the TBE-related virus was prevalent in the area. Three virus isolates were obtained from the blood of sentinel dogs, and antigenic analysis grouped the isolates into TBE-related viruses. Sequence analysis of the envelope protein gene identified one of the isolates as being of the same subtype as the Russian spring summer encephalitis (Far Eastern TBE) virus. The results provide evidence that TBE is endemic in a certain area of Japan.
Analysis of the thermostable direct hemolysin (tdh) gene and the tdh-related hemolysin (trh) genes in urease-positive strains of Vibrio parahaemolyticus isolated on the West Coast of the United States
Authors: Okuda J , Ishibashi M , Abbott S L , Janda J M , Nishibuchi M
Published: January 2020

Urease-positive (Ure+) and urease-negative (Ure-) strains of Vibrio parahaemolyticus isolated from patients on the West Coast of the United States between 1979 and 1995 were analyzed for the thermostable direct hemolysin (tdh) gene and the tdh-related hemolysin (trh) genes (trh1 and trh2). The DNA colony hybridization method with the polynucleotide probes was used to determine the distribution of the genes. Of 60 Ure+ strains, 59 strains (98%) had the trh (either trh1 or trh2) gene and 54 strains (90%) carried the tdh gene. The absence of the trh gene or a related sequence in an exceptional Ure+ strain was confirmed by Southern blot analyses. The stronger correlation with the trh gene than with the tdh gene was mostly attributable to strains possessing only the trh2 gene. Of 25 Ure- strains, 20 strains (80%) had the tdh gene but none had the trh gene. These results indicate a very strong correlation between the Ure+ phenotype and the trh gene and are consistent with those reported for strains isolated in Asia. The Ure+ strains carrying the trh genes were not restricted to a unique group of the strains. The O4:K12 strains carrying the trh1 gene have predominantly been isolated since 1979. However, strains of various non-O4:K12 serovars carrying either the trh1 or the trh2 gene became predominant after 1992. In addition, analysis by the arbitrarily primed PCR method revealed two subgroups within the selected Ure+ O4:K12 strains. Hybridization tests with oligonucleotide probes demonstrated that the trh1 sequences of the West Coast strains differ to some extent from those of Asian strains. Nevertheless, a PCR method previously established to detect both the trh1 and the trh2 genes in Asian strains could detect 98% of those genes in the West Coast strains.