bluuebunny/crossref_metadata_2025 huggingface dataset:
Development and Evaluation of a Rapid Dipstick Assay for Serodiagnosis of Acute Human Brucellosis
Authors: Smits Henk L. , Basahi M. A. , Díaz Ramon , Marrodan T. , Douglas James T. , Rocha Alice , Veerman Jaques , Zheludkov M. M. , Witte Olav W. M. , de Jong Joop , Gussenhoven George C. , Goris Marga G. A. , van der Hoorn Menno A. W. G.
Published: December 2019

A dipstick assay for the detection of brucella-specific immunoglobulin M antibodies was evaluated with 707 sera from 247 laboratory-confirmed brucellosis patients and 342 control sera from brucellosis-free individuals. These sera were collected from six different countries. The assay was found to be highly sensitive and specific. In addition, the test is easy to use and does not require specialized training or equipment, and the components are stable without a requirement for refrigeration. All of these factors make the test ideal for developing countries and rural settings.
Associations of
Streptococcus suis
Serotype 2 Ribotype Profiles with Clinical Disease and Antimicrobial Resistance
Authors: Rasmussen S. R. , Aarestrup F. M. , Jensen N. E. , Jorsal S. E.
Published: December 2019

A total of 122
Streptococcus suis
serotype 2 strains were characterized thoroughly by comparing clinical and pathological observations, ribotype profiles, and antimicrobial resistance. Twenty-one different ribotype profiles were found and compared by cluster analysis, resulting in the identification of three ribotype clusters. A total of 58% of all strains investigated were of two ribotypes belonging to different ribotype clusters. A remarkable relationship existed between the observed ribotype profiles and the clinical-pathological observations because strains of one of the two dominant ribotypes were almost exclusively isolated from pigs with meningitis, while strains of the other dominant ribotype were never associated with meningitis. This second ribotype was isolated only from pigs with pneumonia, endocarditis, pericarditis, or septicemia. Cluster analysis revealed that strains belonging to the same ribotype cluster as one of the dominant ribotypes came from pigs that showed clinical signs similar to those of pigs infected with strains with the respective dominant ribotype profiles. Furthermore, strains belonging to different ribotype clusters had totally different patterns of resistance to antibiotics because strains isolated from pigs with meningitis were resistant to sulfamethazoxazole and strains isolated from pigs with pneumonia, endocarditis, pericarditis, or septicemia were resistant to tetracycline.
Vancomycin-Resistant
Enterococcus faecium
Colonization in Children
Authors: Singh-Naz Nalini , Sleemi Ambreen , Pikis Andreas , Patel Kantilal M. , Campos Joseph M.
Published: December 2019

Nosocomial vancomycin-resistant
Enterococcus
(VRE) infections have been described in only small numbers of pediatric patients. In none of these studies were multivariate analyses performed to assess which factors were independent risk factors in these patients. In the present cohort study of patients admitted to our hematology/oncology unit, surveillance cultures revealed a colonization rate of 24% and all isolates were identified as
Enterococcus faecium
. Risk factors associated with colonization with VRE identified by multiple logistic regression analysis included young age and chemotherapy with antineoplastic agents, cefotaxime, vancomycin, and ceftazidime. A molecular epidemiological tool, pulsed-field gel electrophoresis, was used to determine the relatedness of the VRE isolates detected. DNA analysis by this method identified two major clusters of VRE isolates. Young children with gastrointestinal colonization with VRE, without evidence of clinical infection, can serve as a reservoir for the spread of VRE.
Simple, Reliable, and Cost-Effective Yeast Identification Scheme for the Clinical Laboratory
Authors: Koehler Ann P. , Chu Kai-Cheong , Houang Elizabeth T. S. , Cheng Augustine F. B.
Published: December 2019

The appearance of colonies on the chromogenic medium CHROMagar Candida combined with observation of morphology on corn meal–Tween 80 agar was used for the identification of 353 clinical yeast isolates. The results were compared with those obtained with API yeast identification kits. The accuracy of identification and the turnaround time were equivalent for each method, and our cultural method was less expensive.
Molecular Characterization of Penicillin-Resistant
Streptococcus pneumoniae
Isolates from Bulgaria
Authors: Setchanova Lena , Tomasz Alexander
Published: December 2019

As part of an ongoing surveillance program of antibiotic-resistant
Streptococcus pneumoniae
in Sofia, Bulgaria, 120 penicillin-resistant strains (PRSP) (most of them recovered from children hospitalized with pneumococcal disease) were analyzed by microbiological and molecular methods. Several unique features of this collection are of particular interest. (i) Most isolates (112 of 120) were also resistant to trimethoprim-sulfamethoxazole (SXT) (97 of 120 isolates, or 80%), and over 70% (86 of 120) of the isolates were resistant to at least three antibiotics in addition to penicillin. (ii) Close to 80% of all isolates were represented by large clusters of bacteria, each with a unique serotype, antibiotype, and chromosomal macrorestriction pattern (determined by pulsed-field gel electrophoresis), as well as unique restriction fragmentation length polymorphisms of the penicillin-binding protein genes
pbp1a
,
pbp2x
, and
pbp2b
. (iii) A large proportion (45 of 120, or 38%) of the strains belonged to two internationally spread epidemic clones of
S. pneumoniae
, the first expressing capsular type 23F and the second expressing serotype 9. (iv) A unique Bulgarian cluster composed of eight serotype 19F isolates was resistant to tetracycline, SXT, cefotaxime, and extremely high levels of penicillin and erythromycin. Nevertheless, this clone did not react with either the
erm
or the
mef
DNA probes, and thus the mechanism of macrolide resistance in this group of PRSP remains to be elucidated.
Detection of the 70-Kilodalton
Histoplasma capsulatum
Antigen in Serum of Histoplasmosis Patients: Correlation between Antigenemia and Therapy during Follow-Up
Authors: Gómez B. L. , Figueroa J. I. , Hamilton A. J. , Diez S. , Rojas M. , Tobón A. , Restrepo A. , Hay R. J.
Published: December 2019

Histoplasmosis is an important systemic fungal infection, particularly among immunocompromised individuals, who may develop a progressive disseminated form which is often fatal if it is untreated. In such patients, the detection of antibody responses for both diagnosis and follow-up may be of limited use, whereas the detection of
Histoplasma capsulatum
var.
capsulatum
antigens may provide a more practical approach. We have recently described an inhibition enzyme-linked immunosorbent assay (ELISA) for the detection in patients’ sera of a 69- to 70-kDa
H. capsulatum
var.
capsulatum
-specific antigen which appears to be useful in diagnosis. To investigate its potential for the follow-up of histoplasmosis patients during treatment, antigen titers in the sera of 16 patients presenting with different clinical forms of histoplasmosis were monitored at regular intervals for up to 80 weeks. Sera from four of five patients with the acute form of the disease showed rapid falls in antigenemia, becoming antigen negative by week 14 (range, weeks 10 to 16). Sera from four patients with disseminated histoplasmosis showed falls in antigen levels; three of them became antigen negative by week 32; the fourth patient became negative by week 48. In contrast, antigen titers in four of six AIDS patients with the disseminated form of the disease remained positive throughout follow-up. Sera from only one patient who presented with the chronic form of the disease were analyzed, and this individual’s serum became antigen negative by week 9. The inhibition ELISA is shown to be of particular use in the monitoring of non-AIDS patients with the acute and disseminated forms of the disease and may complement existing means of follow-up.
Evaluation of Two Commercial Kits and Arbitrarily Primed PCR for Identification and Differentiation of
Actinobacillus actinomycetemcomitans
,
Haemophilus aphrophilus
, and
Haemophilus paraphrophilus
Authors: Doğan Başak , Asikainen Sirkka , Jousimies-Somer Hannele
Published: December 2019

The closely related species
Actinobacillus actinomycetemcomitans
,
Haemophilus aphrophilus
, and
Haemophilus paraphrophilus
are common findings in oral microbiota. The aims of this study were to evaluate the applicability of the Rapid NH and API ZYM kits and arbitrarily primed PCR (AP-PCR) in the identification and differentiation of the three species from each other. The material included 62 clinical isolates and three reference strains of
A. actinomycetemcomitans
representing the 5 serotypes and 18 AP-PCR genotypes.
Haemophilus
species included 12 clinical isolates and 11 reference strains of
H. aphrophilus
,
H. paraphrophilus
, and 5 other species. For the PCR amplification, the oligonucleotide 5′-CAGCACCCAC-3′ was used as a primer. Contrary to the consistent performance of API ZYM, the Rapid NH system was able to identify only 10 of 65 (15%)
A. actinomycetemcomitans
isolates, whereas all
Haemophilus
species were correctly identified. The API ZYM test differentiated
A. actinomycetemcomitans
from
H. aphrophilus
and
H. paraphrophilus
by negative β-galactosidase and α-glucosidase reactions and a positive esterase lipase reaction. However, the API ZYM test was unable to differentiate
H. aphrophilus
from
H. paraphrophilus
, it also could not differentiate
A. actinomycetemcomitans
serotypes from each other. Among the
H. aphrophilus
isolates three AP-PCR genotypes and among
H. paraphrophilus
isolates only one AP-PCR genotype, distinct from those of
A. actinomycetemcomitans
, were found. The Rapid NH test showed poor ability to identify clinical isolates of all
A. actinomycetemcomitans
serotypes. Moreover, AP-PCR genotyping proved to be a rapid method for the species differentiation of
A. actinomycetemcomitans
,
H. aphrophilus
, and
H. paraphrophilus
.
Isolation of
Legionella pneumophila
by Centrifugation of Shell Vial Cell Cultures from Multiple Liver and Lung Abscesses
Authors: La Scola Bernard , Michel Gérard , Raoult Didier
Published: December 2019

A 7-year-old girl was admitted to the hospital with acute lymphoblastic leukemia and was treated with allogenic cord blood transplantation. At day 30 after graft, she developed a fever and multiple nodular lesions disseminated in the liver and lungs. All bacterial cultures attempted on liver and lung biopsy specimens and blood remained sterile on standard axenic media. However, inoculation of liver and lung biopsy specimens on eukaryotic cell monolayers by the centrifugation-shell vial technique (M. Marrero and D. Raoult, Am. J. Trop. Med. Hyg. 40:197–199, 1989) led to the recovery of a strain of
Legionella pneumophila
serogroup 1, identified by 16S rRNA gene amplification and sequencing and serotyping. Our findings demonstrate that the centrifugation-cell culture method, which has previously been useful for the isolation of other strictly or facultatively intracellular bacteria, can also serve as a method for the recovery of
L. pneumophila
from clinical material.
Optimal Detection and Identification of
Mycobacterium haemophilum
in Specimens from Pediatric Patients with Cervical Lymphadenopathy
Authors: Samra Zmira , Kaufmann Lea , Zeharia Avraham , Ashkenazi Shai , Amir Jacob , Bahar Judy , Reischl Udo , Naumann Ludmilla
Published: December 2019

Acid-fast bacilli from pediatric patients with lymphadenopathy were detected in the BACTEC radiometric system and in MB Redox broth, but not on Löwenstein Jensen medium. PCR amplification identified the isolates as
Mycobacterium haemophilum
, which has special nutrition requirements (iron supplements) for growth. Suitable culture medium ensures optimal recovery of this microorganism, avoiding underdiagnosis.
hsp65
Sequencing for Identification of Rapidly Growing Mycobacteria
Authors: Ringuet H. , Akoua-Koffi C. , Honore S. , Varnerot A. , Vincent V. , Berche P. , Gaillard J. L. , Pierre-Audigier C.
Published: December 2019

Partial sequencing of the
hsp65
gene was used for the identification of rapidly growing mycobacteria (RGM). A 441-bp fragment (A. Telenti, F. Marchesi, M. Balz, F. Bally, E. Böttger, and T. Bodmer, J. Clin. Microbiol. 31:175–178, 1993) was amplified and sequenced by an automated fluorescence-based method involving capillary electrophoresis. Type strains of 10 RGM species were first studied. Each species had a unique nucleotide sequence, distinguishing it clearly from the other species. A panel of strains from the four main RGM species responsible for human infections,
Mycobacterium abscessus
,
Mycobacterium chelonae
,
Mycobacterium fortuitum
, and
Mycobacterium peregrinum
, was also studied. There were few sequence differences within each of these species (<2% of bases were different from the type strain sequence), and they had no effect on species assignment.
hsp65
sequencing unambiguously differentiated
M. chelonae
and
M. abscessus
, two species difficult to identify by classical methods and 16S rRNA gene sequencing. The devised procedure is a rapid and reliable tool for the identification of RGM species.
Comparative Study of Susceptibilities of Germinated and Ungerminated Conidia of
Aspergillus fumigatus
to Various Antifungal Agents
Authors: Manavathu Elias K. , Cutright Jessica , Chandrasekar Pranatharthi H.
Published: December 2019

Conidia are used as inocula for the in vitro susceptibility testing of
Aspergillus fumigatus
. Since the MIC is defined on the basis of visible mycelial growth, conidia should germinate and produce sporelings (germinated conidia) for monitoring of the growth inhibition and fungicidal activity of a drug. If a compound is capable of inhibiting germination of conidia while affecting or not affecting the growth of the organism, the MIC obtained will be the concentration of the drug required for the inhibition of conidial germination but not necessarily that required for inhibition of the growth of the organism. We investigated the susceptibility of germinated and ungerminated conidia to amphotericin B, itraconazole, voriconazole, and SCH56592. The MICs of various antifungal agents for germinated conidia were almost identical to those obtained for ungerminated conidia. In addition, both the germinated and ungerminated conidia were killed with almost equal efficiency by all of the compounds tested when exposed to the drugs for 24 h. These results suggest that either germinated or ungerminated conidia could be used as inocula for in vitro susceptibility studies of
A. fumigatus
with identical results.
Identification and Characterization of IS
2404
and IS
2606
: Two Distinct Repeated Sequences for Detection of
Mycobacterium ulcerans
by PCR
Authors: Stinear Timothy , Ross Bruce C. , Davies John K. , Marino Lui , Robins-Browne Roy M. , Oppedisano Frances , Sievers Aina , Johnson Paul D. R.
Published: December 2019

Molecular analysis of
Mycobacterium ulcerans
has revealed two new insertion sequences (ISs), IS
2404
and IS
2606
. IS
2404
was identified by complete sequencing of a previously described repetitive DNA segment from
M. ulcerans
. This element is 1,274 bp long, contains 12-bp inverted repeats and a single open reading frame (ORF) potentially encoding a protein of 327 amino acids (aa), and apparently generates 7-bp direct repeats upon transposition. Amino acid similarity was found between the putative transposase and those encoded by ISs in other bacterial sequences from
Aeromonas salmonicida
(
As
Is
1
),
Escherichia coli
(H repeat element),
Vibrio cholerae
(
Vc
IS
1
), and
Porphyromonas gingivalis
(
PG
IS
2
). The second IS, IS
2606
, was discovered by sequence analysis of a
Hae
III fragment of
M. ulcerans
genomic DNA containing a repetitive sequence. This element is 1,404 bp long, with 12-bp inverted repeats and a single ORF potentially encoding a protein of 445 aa. Database searches revealed a high degree of amino acid identity (70%) with the putative transposase of IS
1554
from
M. tuberculosis
. Significant amino acid identity (40%) was also observed with transposases from several other microorganisms, including
Rhizobium meliloti
(IS
Rm3
),
Burkholderia cepacia
(IS
1356
),
Corynebacterium diphtheriae
, and
Yersinia pestis
. PCR screening of DNA from 45 other species of mycobacteria with primers for IS
2404
confirm that this element is found only in
M. ulcerans
. However, by PCR, IS
2606
was also found in
Mycobacterium lentiflavum
, another slow-growing member of the genus
Mycobacterium
that is apparently genetically distinct from
M. ulcerans
. Testing the sensitivity of PCR based on IS
2404
and IS
2606
primers demonstrated the ability to detect 0.1 and 1
M. ulcerans
genome equivalents, respectively. The ability to detect small numbers of cells by using two gene targets will be particularly useful for analyzing environmental samples, where there may be low concentrations of
M. ulcerans
among large numbers of other environmental mycobacteria.
Antimicrobial Susceptibilities and Plasmid Contents of
Neisseria gonorrhoeae
Isolates from Commercial Sex Workers in Dhaka, Bangladesh: Emergence of High-Level Resistance to Ciprofloxacin
Authors: Bhuiyan Bahar Uddin , Rahman Motiur , Miah Mohammed Ruhul Amin , Nahar Shamsun , Islam Nazrul , Ahmed Monira , Rahman Kazi Masihur , Albert M. John
Published: December 2019

Commercial sex workers (CSWs) serve as the most important reservoir of sexually transmitted diseases (STD), including gonorrhea. Periodic monitoring of the antimicrobial susceptibility profile of
Neisseria gonorrhoeae
in a high-risk population provides essential clues regarding the rapidly changing pattern of antimicrobial susceptibilities. A study concerning the prevalence of gonococcal infection among CSWs was conducted in Bangladesh. The isolates were examined with regards to their antimicrobial susceptibility to, and the MICs of, penicillin, tetracycline, ciprofloxacin, cefuroxime, ceftriaxone, and spectinomycin by disk diffusion and agar dilution methods. The total plasmid profile of the isolates was also analyzed. Of the 224 CSWs, 94 (42%) were culture positive for
N. gonorrhoeae
. There was a good correlation between the results of the disk diffusion and agar dilution methods. Some 66% of the isolates were resistant to penicillin, and 34% were moderately susceptible to penicillin. Among the resistant isolates, 23.4% were penicillinase-producing
N. gonorrhoeae
(PPNG). 60.6% of the isolates were resistant and 38.3% were moderately susceptible to tetracycline, 17.5% were tetracycline-resistant
N. gonorrhoeae
, 11.7% were resistant and 26.6% had reduced susceptibility to ciprofloxacin, 2.1% were resistant and 11.7% had reduced susceptibility to cefuroxime, and 1% were resistant to ceftriaxone. All PPNG isolates contained a 3.2-MDa African type of plasmid, and a 24.2-MDa conjugative plasmid was present in 34.1% of the isolates. Since quinolones such as ciprofloxacin are recommended as the first line of therapy for gonorrhea, the emergence of significant resistance to ciprofloxacin will limit the usefulness of this drug for treatment of gonorrhea in Bangladesh.
Human Herpesvirus 8 Seroprevalence and Evaluation of Nonsexual Transmission Routes by Detection of DNA in Clinical Specimens from Human Immunodeficiency Virus-Seronegative Patients from Central and Southern Italy, with and without Kaposi’s Sarcoma
Authors: Cattani Paola , Capuano Maria , Cerimele Francesca , La Parola Ilaria Lesnoni , Santangelo Rosaria , Masini Cinzia , Cerimele Decio , Fadda Giovanni
Published: December 2019

In order to investigate the seroprevalence of human herpesvirus 8 (HHV-8) infection in central and southern Italy, sera from human immunodeficiency virus (HIV)-seronegative subjects, with and without Kaposi’s sarcoma (KS), were analyzed by immunofluorescence assay, using BC-3, a cell line latently infected with HHV-8. High titers of antibody against HHV-8 lytic and latent antigens were detected in all 50 KS patients studied, while in 50 HIV-seronegative subjects without KS, 32 (64%) were found positive for HHV-8 antibodies. Titers in the sera of these patients were lower than those for KS patients. This data suggests that HHV-8 infection is not restricted to KS patients and that the prevalence of HHV-8 infection in the general population may be correlated with differing rates of prevalence of KS in different parts of the world. In view of these findings, possible nonsexual transmission routes were evaluated. Nested PCR was used to test for the presence of HHV-8 DNA in saliva, urine, and tonsillar swabs from KS and non-KS patients. In KS patients, 14 out of 32 tonsillar swabs (43.7%), 11 out of 24 saliva samples (45.8%), and just 2 out of 24 urine samples (8.3%) tested positive for HHV-8 DNA. In the control group, on the contrary, none of the 20 saliva and 20 urine specimens was positive for HHV-8 DNA; only 1 out of 22 tonsillar swabs gave a positive result. This data supports the hypothesis that HHV-8 infects the general population in a latent form. The reactivation of viral infection may result in salivary shedding of HHV-8, contributing to viral spread by nonsexual transmission routes.
Serological Evidence of Infection with
Ehrlichia
spp. in Red Foxes (
Vulpes vulpes
) in Switzerland
Authors: Pusterla Nicola , Deplazes Peter , Braun Ueli , Lutz Hans
Published: December 2019

Serum samples from 1,550 red foxes in Switzerland were tested for antibodies to the agents of canine granulocytic and monocytic ehrlichiosis by an indirect immunofluorescent technique. Forty-four (2.8%) of the samples were positive for
Ehrlichia phagocytophila
, which is an antigen marker for granulocytic ehrlichiosis. In contrast, none of the samples had antibodies specific to
Ehrlichia canis
, the agent of monocytic ehrlichiosis.
Serotyping ofCryptococcus neoformansIsolates from Clinical and Environmental Sources in Spain
Authors: Baró Teresa , Torres-Rodríguez Josep M. , Morera Yolanda , Alía Concepción , López Olga , Méndez Raul
Published: December 2019

We determined biovars and serotypes of 154 isolates ofCryptococcus neoformansfrom clinical and environmental sources from different areas of Spain. All clinical isolates belonged toC. neoformansvar.neoformans. Serotypes showed an irregular distribution.C. neoformansvar.gattiiserotype B was isolated from necropsy specimens from goats with pulmonary disease.
rpoB
Mutations in Multidrug-Resistant Strains of
Mycobacterium tuberculosis
Isolated in Italy
Authors: Pozzi G. , Meloni M. , Iona E. , Orrù G. , Thoresen O. F. , Ricci M. L. , Oggioni M. R. , Fattorini L. , Orefici G.
Published: December 2019

Mutations of
rpoB
associated with rifampin resistance were studied in 37 multidrug-resistant (MDR) clinical strains of
Mycobacterium tuberculosis
isolated in Italy. At least one mutated codon was found in each MDR strain. It was always a single-base substitution leading to an amino acid change. Nine different
rpoB
alleles, three of which had not been reported before, were found. The relative frequencies of specific mutations in this sample were different from those previously reported from different geographical areas, since 22 strains (59.5%) carried the mutated codon TTG in position 531 (Ser→Leu) and 11 (29.7%) had GAC in position 526 (His→Asp).
Specificity of IS
6110
-Based DNA Fingerprinting and Diagnostic Techniques for
Mycobacterium tuberculosis
Complex
Authors: Githui Willie A. , Wilson Stuart M. , Drobniewski Francis A.
Published: December 2019

Restriction fragment length polymorphism and hybridization of DNA extracted from
Mycobacterium tuberculosis
, nontuberculous mycobacteria, and nonmycobacterial species with a probe derived from IS
6110
confirmed that IS
6110
was specific to
M. tuberculosis
complex. In addition, DNA amplification with IS
6110
-specific primers yielded a 181-bp fragment only in DNA from
M. tuberculosis
complex isolates.
Detection of Cell Wall Mannoprotein Mp1p in Culture Supernatants of
Penicillium marneffei
and in Sera of Penicilliosis Patients
Authors: Cao Liang , Chan King-Man , Chen Daliang , Vanittanakom Nongnuch , Lee Cindy , Chan Che-Man , Sirisanthana Thira , Tsang Dominic N. C. , Yuen Kwok-Yung
Published: December 2019

Mannoproteins are important and abundant structural components of fungal cell walls. The
MP1
gene encodes a cell wall mannoprotein of the pathogenic fungus
Penicillium marneffei
. In the present study, we show that Mp1p is secreted into the cell culture supernatant at a level that can be detected by Western blotting. A sensitive enzyme-linked immunosorbent assay (ELISA) developed with antibodies against Mp1p was capable of detecting this protein from the cell culture supernatant of
P. marneffei
at 10
4
cells/ml. The anti-Mp1p antibody is specific since it fails to react with any protein-form lysates of
Candida albicans
,
Histoplasma capsulatum
, or
Cryptococcus neoformans
by Western blotting. In addition, this Mp1p antigen-based ELISA is also specific for
P. marneffei
since the cell culture supernatants of the other three fungi gave negative results. Finally, a clinical evaluation of sera from penicilliosis patients indicates that 17 of 26 (65%) patients are Mp1p antigen test positive. Furthermore, a Mp1p antibody test was performed with these serum specimens. The combined antibody and antigen tests for
P. marneffei
carry a sensitive of 88% (23 of 26), with a positive predictive value of 100% and a negative predictive value of 96%. The specificities of the tests are high since none of the 85 control sera was positive by either test.
Molecular Epidemiology of Tuberculosis in Malaysia
Authors: Dale Jeremy W. , Nor Rohana Mat , Ramayah Soshila , Tang Thean Hock , Zainuddin Zainul F.
Published: December 2019

Molecular typing with IS
6110
was applied to
Mycobacterium tuberculosis
isolates from all parts of Malaysia. The degree of clustering increased with patient age, suggesting that reactivation may contribute to clustering. Identical banding patterns were also obtained for isolates from widely separate regions. Therefore, the use of clustering as a measure of recent transmission must be treated with caution. Strains related to the Beijing family were common in Peninsular Malaysia but were less common in Sabah and Sarawak, while a distinct group of strains comprised nearly 40% of isolates from East Malaysia but such strains were rare in Peninsular Malaysia. Single-copy strains, common in South and Southeastern Asia, constituted nearly 20% of isolates from the peninsula but were virtually absent in East Malaysia. The marked geographical difference in the prevailing strains indicates not only a restricted dissemination of
M. tuberculosis
but also a considerable degree of stability in the banding patterns.
Recurrent “
Flexispira rappini
” Bacteremia in an Adult Patient Undergoing Hemodialysis: Case Report
Authors: Sorlin P. , Vandamme P. , Nortier J. , Hoste B. , Rossi C. , Pavlof S. , Struelens M. J.
Published: December 2019

A blood culture from a 65-year-old febrile man undergoing hemodialysis revealed, 5 days after inoculation, an unusual gram-negative fusiform rod with darting motility. During another episode of fever 21 days later, this
Campylobacter
-like organism was again recovered from three blood cultures and subcultured under an H
2
-enriched microaerobic atmosphere. The organism was catalase negative and oxidase positive and hydrolyzed urea rapidly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of whole-cell proteins was indistinguishable from that of “
Flexispira rappini
” LMG 8738 described by Archer et al. in 1988 (J. R. Archer, S. Romero, A. E. Ritchier, M. E. Hamacher, B. M. Steiner, J. H. Bryner, and R. F. Schell, J. Clin. Microbiol. 26:101–105, 1988). The analysis of the 16S ribosomal DNA sequence revealed a similarity of 99.3% between the two strains. The patient recovered completely after a 4-week course of meropenem therapy. This is the first reported case of a recurrent “
F. rappini
” bacteremia in an adult patient, which confirms that this organism may be an invasive pathogen in immunocompromised patients, like other newly described
Helicobacter
species.
Quantitative Real-Time PCR for Detection of Members of the
Ehrlichia phagocytophila
Genogroup in Host Animals and
Ixodes ricinus
Ticks
Authors: Pusterla Nicola , Huder Jon B. , Leutenegger Christian M. , Braun Ueli , Madigan John E. , Lutz Hans
Published: December 2019

A TaqMan PCR was established for identification and quantitation of members of the
Ehrlichia phagocytophila
group in experimentally infected cows and in
Ixodes ricinus
ticks. The TaqMan PCR identified a 106-bp section of the 16S rRNA gene by use of a specific fluorogenic probe and two primers. This technique was specific for members of the
E. phagocytophila
group, which include
E. phagocytophila
,
Ehrlichia equi
, and the agent of human granulocytic ehrlichiosis. The TaqMan system identified 10 copies of a cloned section of the 16S rRNA gene of
E. phagocytophila
. The sensitivity and specificity of the TaqMan PCR were similar to those of conventional nested PCR. The numbers of ehrlichiae in leukocytes of the two cows experimentally infected with
E. phagocytophila
were measured daily by TaqMan PCR and had a course similar to that of the percentages of infected leukocytes determined daily by light microscopy. The prevalence of infected free-living ticks, which were collected from areas where bovine ehrlichiosis is endemic and from regions with sporadic occurrences of granulocytic ehrlichiosis in dogs and horses, was identical as determined by nested PCR and TaqMan PCR.
Evidence of the Human Granulocytic Ehrlichiosis Agent in
Ixodes ricinus
Ticks in Switzerland
Authors: Pusterla Nicola , Leutenegger Christian M. , Huder Jon B. , Weber Rainer , Braun Ueli , Lutz Hans
Published: December 2019

A total of 1,667
Ixodes ricinus
ticks were collected from five regions in Switzerland where there have been sporadic occurrences of granulocytic ehrlichiosis in dogs and horses. The ticks were examined for rickettsiae of the
Ehrlichia phagocytophila
group via nested PCR. Twenty-one ticks (1.3%) were positive; 3 (0.5%) were nymphs, 6 (1.3%) were adult males, and 12 (1.9%) were adult females. The number of positive ticks varied with the stage of development and with the geographical origin. Nucleotide sequencing of the isolated PCR products identified these products as part of the 16S rRNA gene of
Ehrlichia
. In addition, these products had 100% homology with the agent of human granulocytic ehrlichiosis. The occurrence of this agent in
I. ricinus
in Switzerland presents a potential danger of transmission of granulocytic ehrlichiosis to dogs, horses, and humans.
Laboratory Diagnosis of Common Viral Infections of the Central Nervous System by Using a Single Multiplex PCR Screening Assay
Authors: Read Steven J. , Kurtz John B.
Published: December 2019

A multiplex PCR assay that detects the four commonest causes of viral meningitis and encephalitis in the United Kingdom (herpes simplex virus [HSV] type 1 [HSV-1], HSV type 2 [HSV-2], varicella-zoster virus [VZV], and enteroviruses) was developed, and its sensitivity was compared with those of similar assays described previously for this application. Compared to the previous assays, this single multiplex PCR assay had higher molecular sensitivities for the detection for each of the viruses and improved utility for routine use in a diagnostic laboratory. The assay was used to test a series of 1,683 consecutive cerebrospinal fluid (CSF) samples between June 1997 and March 1998 inclusively. Viral nucleic acid was detected in 138 (8.2%) of the CSF samples, including enteroviruses in 51 samples, HSV-2 in 33 samples, VZV in 28 samples, and HSV-1 in 25 samples. Compared to the accepted relative incidence of viral etiologies, aseptic meningitis due to HSV-2 infection was high, and in adult female patients with symptoms of aseptic meningitis, HSV-2 was the virus most commonly detected in the CSF.
Discrimination of Subtype B and Non-Subtype B Strains of Human Immunodeficiency Virus Type 1 by Serotyping: Correlation with Genotyping
Authors: Murphy Gary , Belda Francisco J. , Pau Chou-Pong , Clewley Jonathan P. , Parry John V.
Published: December 2019

The ability of a peptide-based serotyping assay to differentiate human immunodeficiency virus (HIV) type 1 (HIV-1) subtype B infections from non-subtype B infections was investigated with 166 anti-HIV-1- and HIV RNA-positive (by PCR) serum or plasma specimens. The specimens were divided genetically into those infected with subtype B and non-subtype B by application of a screening heteroduplex mobility assay (HMA) that used plasmids for subtypes A and B alone. Specimens that were not clearly infected with HIV-1 subtype B by HMA or for which the two methods had discordant results in distinguishing those infected with subtype B from those infected with non-subtype B were then investigated with a full HMA plasmid panel and, for selected specimens,
env
sequencing. For the 141 genotyped and serotypically reactive specimens, the correlation between genotyping and serotyping (all subtypes) was 69%. Of the 67 specimens that reacted monotypically as serotype B, 64 were shown to be infected with genotype B (positive predictive value, 96%). Of the 82 specimens that contained genotype B nucleic acid, 64 reacted monotypically as serotype B (sensitivity, 78%), and 4 specimens reacted with a single non-subtype B peptide; the viruses in 14 specimens could not be assigned a serotype. Initial screening results had indicated that 12 samples had results discordant between restricted HMA and serotyping. The V3 loop amino acids of the infecting HIV strains from the seven specimens with discordant serology results were analyzed. For five specimens discordance occurred when the amino acid sequence of the infecting virus closely resembled those of more than one consensus peptide antigen or when the observed V3 crown motif of the strain was atypical for the genetic subtype present. For the other two specimens no explanation for the discordance was identified. Five specimens gave unclear or discordant results in the initial HMA screen, but the results were resolved when the full plasmid panel was used. Serotyping, although of limited sensitivity, distinguishes between subtype B and non-subtype B infections with a high degree of specificity. However, it poorly differentiates the major non-subtype B subtypes, particularly subtypes A and C. When HIV-1 subtype B predominates, serological typing and/or subtype-restricted HMA screening usefully distinguishes between subtype B and non-subtype B infections.
Relatedness Analyses of
Histoplasma capsulatum
Isolates from Mexican Patients with AIDS-Associated Histoplasmosis by Using Histoplasmin Electrophoretic Profiles and Randomly Amplified Polymorphic DNA Patterns
Authors: Reyes-Montes M. R. , Bobadilla-Del Valle M. , Martínez-Rivera M. A. , Rodríguez-Arellanes G. , Maravilla E. , Sifuentes-Osornio J. , Taylor M. L.
Published: December 2019

The present paper analyzes the histoplasmin electrophoretic profiles and the randomly amplified polymorphic DNA (RAPD) patterns of the fungus
Histoplasma capsulatum
isolated from Mexican patients with AIDS-associated histoplasmosis. Clinical isolates from Guatemala, Colombia, and Panama, as well as
H. capsulatum
isolates from different sources in nature, were also processed. All histoplasmin samples shared four antigenic fractions of 200, 49, 10.5, and 8.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). According to their percentage of relatedness, based on SDS-PAGE histoplasmin electrophoretic image analysis,
H. capsulatum
isolates were divided in two groups: group A contained all AIDS-associated isolates studied and two human reference strains from Mexican histoplasmosis patients without AIDS; group B included bat guano, infected bat, and cock excreta isolates from the State of Guerrero, Mexico, plus three human histoplasmosis strains from Guatemala, Panama, and Colombia. Polymorphic DNA patterns evaluated by RAPD-PCR showed three major bands of 4.4, 3.2, and 2.3 kb in most
H. capsulatum
isolates studied. Four groups were related by DNA polymorphisms: group I was formed by most of the AIDS-associated
H. capsulatum
isolates studied, one human histoplasmosis strain from Colombia, two human reference strains from Mexican patients without AIDS, and one human histoplasmosis strain from Guatemala. Group II consisted of only a single strain from Panama. Group III included three strains: one from a Mexican patient with AIDS and two isolated from nature in Guerrero (cock excreta and bat guano). The last, group IV, consisted of only one strain isolated from an infected bat, captured in Guerrero. A tight relationship between phenotypic and genotypic characterization was observed, and both analyses could be useful tools for typing
H. capsulatum
from different sources and geographic origins.
Distribution of
Porphyromonas gingivalis
Strains with
fimA
Genotypes in Periodontitis Patients
Authors: Amano Atsuo , Nakagawa Ichiro , Kataoka Kosuke , Morisaki Ichijiro , Hamada Shigeyuki
Published: December 2019

Fimbriae (FimA) of
Porphyromonas gingivalis
are filamentous components on the cell surface and are thought to play an important role in the colonization and invasion of periodontal tissues. We previously demonstrated that
fimA
can be classified into four variants (types I to IV) on the basis of the nucleotide sequences of the
fimA
gene. In the present study, we attempted to detect the four different
fimA
genes in saliva and plaque samples isolated from patients with periodontitis using the PCR method. Four sets of
fimA
type-specific primers were designed for the PCR assay. These primers selectively amplified 392-bp (type I), 257-bp (type II), 247-bp (type III), and 251-bp (type IV) DNA fragments of the
fimA
gene. Positive PCR results were observed with reference strains of
P. gingivalis
in a type-specific manner. All other laboratory strains of oral and nonoral bacteria gave negative results. The sensitivity of the PCR assay for
fimA
type-specific detection was between 5 and 50 cells of
P. gingivalis
. Clinical samples were obtained from saliva and subgingival plaque from deep pockets (≥4 mm) of 93 patients with periodontitis. Bacterial genomic DNA was isolated from the samples, and the targeted fragments were amplified by PCR. The presence of
P. gingivalis
was demonstrated in 73 patients (78.5%), and a single
fimA
gene was detected in most patients. The distribution of the four
fimA
types among the
P. gingivalis
-positive patients was as follows: type I, 5.4%; type II, 58.9%; type III, 6.8%; type IV, 12.3%; types I and II, 6.8%; types II and IV, 2.7%; and untypeable, 6.8%.
P. gingivalis
with type II
fimA
was detected more frequently in the deeper pockets, and a significant difference of the occurrence was observed between shallow (4 mm) and deep (≥8 mm) pockets. These results suggest that
P. gingivalis
strains that possess type II
fimA
are significantly more predominant in periodontitis patients, and we speculate that these organisms are involved in the destructive progression of periodontal diseases.
Latent
Pneumocystis carinii
Infection in Commercial Rat Colonies: Comparison of Inductive Immunosuppressants plus Histopathology, PCR, and Serology as Detection Methods
Authors: Weisbroth Steven H. , Geistfeld James , Weisbroth Stephanie P. , Williams Bruce , Feldman Sanford H. , Linke Michael J. , Orr Sally , Cushion Melanie T.
Published: December 2019

Histopathologic evaluation combined with a period of immunosuppression has been the standard procedure for detection of
Pneumocystis carinii
in commercial rat colonies. Variation in induction regimens and in the sensitivity of detection methods may result in underreporting of the presence of
P. carinii
in breeding colonies or delay its detection. In the present study, methylprednisolone and cyclophosphamide were evaluated for the ability to induce
P. carinii
infection in rats from an enzootically infected commercial barrier colony. The presence of
P. carinii
was detected by histopathologic methods and by amplification of a targeted region of the
P. carinii
thymidylate synthase gene by PCR over the 8-week study period. Sera taken from rats prior to either induction regimen were evaluated for the presence of
P. carinii
-specific antibodies by the immunoblotting technique. Few significant differences in ability to induce organism burden or in histopathology were observed between the two immunosuppressive regimens. However, a dramatic loss of weight over the study period was observed in rats treated with methylprednisolone but not in rats treated with cyclophosphamide. Although histopathologic changes attributable to
P. carinii
did not appear before 2 weeks with either immunosuppressant, the presence of the organism in these animals was detected by immunoblotting and PCR. Cyst scores and the intensities of the histopathologic lesions increased during the study period, but the number of rats exhibiting evidence of
P. carinii
infection did not change after week 3. These results suggest that use of the PCR method on postmortem lung tissue of rats without prior induction regimens or identification of anti-
P. carinii
antibodies in antemortem serum samples is a sufficiently sensitive method for detection of the presence of a
P. carinii
carrier state in rodent breeding colonies.
Molecular Cloning and Characterization of the
Ehrlichia chaffeensis
Variable-Length PCR Target: an Antigen-Expressing Gene That Exhibits Interstrain Variation
Authors: Sumner John W. , Childs James E. , Paddock Christopher D.
Published: December 2019

A clone expressing an immunoreactive protein with an apparent molecular mass of 44 kDa was selected from an
Ehrlichia chaffeensis
Arkansas genomic library by probing with anti-
E. chaffeensis
hyperimmune mouse ascitic fluid. Nucleotide sequencing revealed an open reading frame (ORF) capable of encoding a 198-amino-acid polypeptide. The ORF contained four imperfect, direct, tandem 90-bp repeats. The nucleotide and deduced amino acid sequences did not show close homologies to entries in the molecular databases. PCR with primers whose sequences matched the sequences flanking the ORF was performed with DNA samples extracted from cell cultures infected with nine different isolates of
E. chaffeensis
, blood samples from seven patients with monocytic ehrlichiosis, and
Amblyomma americanum
ticks collected in four different states. The resulting amplicons varied in length, containing three to six repeat units. This gene, designated the variable-length PCR target, is useful for PCR detection of
E. chaffeensis
and differentiation of isolates.
Invasive Aspergillosis Caused by
Aspergillus ustus
: Case Report and Review
Authors: Verweij Paul E. , van den Bergh Marjolein F. Q. , Rath Peter M. , de Pauw Ben E. , Voss Andreas , Meis Jacques F. G. M.
Published: December 2019

A case of invasive pulmonary aspergillosis in an allogeneic bone marrow transplant recipient caused by
Aspergillus ustus
is presented.
A. ustus
was also recovered from the hospital environment, which may indicate that the infection was nosocomially acquired. A literature review revealed seven cases of invasive infections caused by
A. ustus
, and three of these were primarily cutaneous infections. In vitro susceptibility testing of 12
A. ustus
isolates showed that amphotericin B and terbinafine had fungicidal activity and that itraconazole and voriconazole had fungistatic activity.
Septicemia Caused by Dysgonic Fermenter 3 in a Severely Immunocompromised Patient and Isolation of the Same Microorganism from a Stool Specimen
Authors: Grob René , Zbinden Reinhard , Ruef Christian , Hackenthal Matthias , Diesterweg Ingrid , Altwegg Martin , von Graevenitz Alexander
Published: December 2019

Dysgonic fermenter 3 (DF-3)-associated bacteremia occurred in a febrile patient with acute myelocytic leukemia during aplasia. Another DF-3 isolate, identical by ribotyping, was grown 10 weeks later from stool collected in the absence of diarrhea. This is the first case in which DF-3 was isolated from blood and stool specimens from the same patient.
Survey of Antibiotic Resistance among Enterococci in North Rhine-Westphalia, Germany
Authors: Reinert R. R. , Conrads G. , Schlaeger J. J. , Werner G. , Witte W. , Lütticken R. , Klare I.
Published: December 2019

A surveillance study on antibiotic resistance of enterococcal isolates (
n
= 730) was carried out in North Rhine-Westphalia, Germany, in 1997. Resistance rates to ampicillin (7.4%), high-level gentamicin (15.0%), high-level streptomycin (27.9%), ciprofloxacin (37.9%), vancomycin (1.5%), and teicoplanin (1.5%) were determined. All vancomycin-resistant enterococci (VRE) carried the
vanA
gene.
Sma
I and
Apa
I macrorestriction patterns indicated an intra- and interhospital spread of VRE.
Controlled Clinical Comparison of bioMérieux VITAL and BACTEC NR-660 Blood Culture Systems for Detection of Bacteremia and Fungemia in Adults
Authors: Wilson Michael L. , Mirrett Stanley , McDonald L. Clifford , Weinstein Melvin P. , Fune Jose , Reller L. Barth
Published: December 2019

A total of 9,446 blood cultures were collected from adult patients at three university-affiliated hospitals. Of these, 8,943 cultures were received with both aerobic bottles filled adequately; 885 yielded 1,016 microorganisms, including 622 isolates (61%) that were the cause of sepsis, 337 isolates (33%) that were contaminants, and 57 isolates (6%) that were indeterminate as the cause of sepsis. With the exception of
Staphylococcus aureus
, which was recovered more often from VITAL aerobic bottles, more pathogenic microorganisms were recovered from BACTEC NR6 (aerobic) bottles than from VITAL aerobic bottles. Growth of pathogenic microorganisms was detected earlier in VITAL aerobic bottles. A total of 8,647 blood cultures were received with both anaerobic bottles filled adequately; 655 yielded 740 microorganisms, including 486 isolates (66%) that were the cause of sepsis, 215 isolates (29%) that were contaminants, and 39 isolates (6%) that were indeterminate as the cause of sepsis. More pathogenic microorganisms were recovered from VITAL anaerobic bottles than from BACTEC NR7 (anaerobic) bottles. Growth of pathogenic microorganisms was detected earlier in VITAL anaerobic bottles. In 8,500 sets all four bottles were received adequately filled. When paired aerobic and anaerobic bottle sets (systems) were compared, more pathogenic microorganisms (again with the exception of
S. aureus
) were recovered from the BACTEC system. For the 304 septic episodes (253 unimicrobial and 51 polymicrobial), significantly more were detected by the BACTEC system. We conclude that VITAL requires modification to improve recovery of pathogenic microorganisms to make it competitive with other commercially available blood culture systems.