bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromosomal Rearrangements in
Salmonella enterica
Serotype Typhi Affecting Molecular Typing in Outbreak Investigations
Authors: Echeita M. A. , Usera M. A.
Published: December 2019

Salmonella enterica
serotype Typhi strains belonging to eight different outbreaks of typhoid fever that occurred in Spain between 1989 and 1994 were analyzed by ribotyping and pulsed-field gel electrophoresis. For three outbreaks, two different patterns were detected for each outbreak. The partial digestion analysis by the intron-encoded endonuclease I-
Ceu
I of the two different strains from each outbreak provided an excellent tool for examining the organization of the genomes of epidemiologically related strains.
S. enterica
serotype Typhi seems to be more susceptible than other serotypes to genetic rearrangements produced by homologous recombinations between
rrn
operons; these rearrangements do not substantially alter the stability or survival of the bacterium. We conclude that genetic rearrangements can occur during the emergence of an outbreak.
Therapeutic Effect of Doxycycline in Experimental Subclinical Canine Monocytic Ehrlichiosis: Evaluation of a 6-Week Course
Authors: Harrus Shimon , Waner Trevor , Aizenberg Itzhak , Bark Hylton
Published: December 2019

The efficacy of doxycycline treatment (10 mg/kg of body weight every 24 h for 42 days) in eliminatingEhrlichia canisfrom four subclinically infected dogs was evaluated. One dog remained PCR positive, suggesting that 6 weeks of doxycycline treatment may not be sufficient to clearE. canisparasites from all subclinically infected dogs. Serology (indirect immunofluorescent antibody assay) was shown to be unreliable in assessing recovery from the carrier state, as anti-E. canisantibodies persisted after elimination of the parasite. Our findings suggest that an increase in the platelet count may be an important indicator for dogs that recover from subclinical ehrlichiosis.
Facklamia ignava
sp. nov., Isolated from Human Clinical Specimens
Authors: Collins Matthew D. , Lawson Paul A. , Monasterio Rafael , Falsen Enevold , Sjöden Berit , Facklam Richard R.
Published: December 2019

Two strains of a hitherto-undescribed gram-positive, catalase-negative coccus isolated from human sources were characterized by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies demonstrated that the unknown strains are genealogically identical and constitute a new line close to, but distinct from,
Facklamia hominis
. The unknown bacterium was readily distinguished from
F. hominis
by biochemical tests and electrophoretic analysis of whole-cell proteins. On the basis of phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as
Facklamia ignava
sp. nov. The type strain of
Facklamia ignava
is CCUG 37419.
Diagnosis of Meningococcal Meningitis by Broad-Range Bacterial PCR with Cerebrospinal Fluid
Authors: Kotilainen Pirkko , Jalava Jari , Meurman Olli , Lehtonen Olli-Pekka , Rintala Esa , Seppälä Olli-Pekka , Eerola Erkki , Nikkari Simo
Published: December 2019

We used broad-range bacterial PCR combined with DNA sequencing to examine prospectively cerebrospinal fluid (CSF) samples from patients with suspected meningitis. Fifty-six CSF samples from 46 patients were studied during the year 1995. Genes coding for bacterial 16S and/or 23S rRNA genes could be amplified from the CSF samples from five patients with a clinical picture consistent with acute bacterial meningitis. For these patients, the sequenced PCR product shared 98.3 to 100% homology with the
Neisseria meningitidis
sequence. For one patient, the diagnosis was initially made by PCR alone. Of the remaining 51 CSF samples, for 50 (98.0%) samples the negative PCR findings were in accordance with the negative findings by bacterial culture and Gram staining, as well as with the eventual clinical diagnosis for the patient. However, the PCR test failed to detect the bacterial rRNA gene in one CSF sample, the culture of which yielded
Listeria monocytogenes
. These results invite new research efforts to be focused on the application of PCR with broad-range bacterial primers to improve the etiologic diagnosis of bacterial meningitis. In a clinical setting, Gram staining and bacterial culture still remain the cornerstones of diagnosis.
Detection and Semiquantitative Analysis of Human Herpesvirus 8 DNA in Specimens from Patients with Kaposi’s Sarcoma
Authors: Mendez J. C. , Procop G. W. , Espy M. J. , Paya C. V. , Smith T. F.
Published: December 2019

Kaposi’s sarcoma (KS) is the most common neoplasm in patients with AIDS. Epidemiologic evidence and the recent identification of herpesvirus-like DNA sequences in patients with KS have suggested a role for viral agents in the etiopathogenesis of this disease. It is unclear if these sequences are present in all types of KS and if the copy number of these sequences has a correlation with disease severity (staging). In order to clarify these issues, we retrospectively analyzed, by PCR and Southern blotting, formalin-fixed, paraffin-embedded biopsy specimens from 12 patients previously diagnosed with KS by histopathologic examination of these specimens between the years of 1977 and 1996. We also analyzed tissue samples from these patients taken from dermal sites without KS lesions and control tissues from healthy subjects. Of the 12 patients, 6 had classic KS, 5 had AIDS-associated KS, and 1 had the endemic type of KS. We tested the specimens for other herpesviruses, including cytomegalovirus, human herpesvirus 6 (HHV-6), HHV-7, HHV-8, and Epstein-Barr virus. Of 20 biopsy specimens from patients previously diagnosed with KS, 19 were positive for HHV-8 sequences (95%), while PCR for the DNAs of other herpesvirus agents was negative. Uninvolved tissue from these patients and control tissue from healthy subjects gave negative results for all viruses. Semiquantitative analysis of Southern blots showed higher levels of HHV-8 DNA in those patients with multicentric and visceral involvement than in those patients with localized involvement. In addition, in patients with localized skin disease, the nodular stage had higher levels of HHV-8 DNA than the patch or plaque stages. Our data confirmed that HHV-8 is involved in the etiopathogenesis of all types of KS and that there is a correlation between HHV-8 DNA load and the severity and staging of this disease.
Canine Intestinal Spirochetes Consist of
Serpulina pilosicoli
and a Newly Identified Group Provisionally Designated “
Serpulina canis
” sp. nov
Authors: Duhamel G. E. , Trott D. J. , Muniappa N. , Mathiesen M. R. , Tarasiuk K. , Lee J. I. , Hampson D. J.
Published: December 2019

The spirochetes inhabiting the large intestines of humans and animals consist of a diverse group of related organisms. Intestinal spirochetosis caused by
Serpulina pilosicoli
is a newly recognized enteric disease of human beings and animals with potential public health significance. The purpose of this study was to determine the species identity of canine intestinal spirochetes by comparing 30 isolates obtained from dogs in Australia (
n
= 25) and the United States (
n
= 5) with reference strains representing
Serpulina
species and
Brachyspira aalborgi
, by phenotypic and genetically based typing methods. All of the canine isolates were indole negative and produced a weak β-hemolysis when cultured anaerobically on agar medium containing blood. Four isolates were identified as
S. pilosicoli
by 16S rRNA-specific PCR assays, rRNA gene restriction fragment length polymorphism or ribotyping, and multilocus enzyme electrophoresis. The remaining 26 isolates formed a cluster related to porcine
Serpulina innocens
as determined by multilocus enzyme electrophoresis but had a unique ribotype pattern. The data suggested the existence of a novel
Serpulina
species, provisionally designated “
Serpulina canis
,” colonizing the intestines of dogs.
Localization of Persistent
Enterocytozoon bieneusi
Infection in Normal Rhesus Macaques (
Macaca mulatta
) to the Hepatobiliary Tree
Authors: Mansfield Keith G. , Carville Angela , Hebert Daniel , Chalifoux Laura , Shvetz Daniel , Lin K. C. , Tzipori S. , Lackner Andrew A.
Published: December 2019

Enterocytozoon bieneusi
is the most common microsporidian parasite recognized in human patients with AIDS. Recently, we identified a virtually identical organism causing a spontaneous infection associated with hepatobiliary and intestinal disease in simian immunodeficiency virus (SIV)-infected macaques. To examine the natural history of the infection, we examined captive rhesus macaques for
E. bieneusi
by PCR, in situ hybridization, and cytochemical techniques. PCR performed on fecal DNA detected enterocytozoon infection in 22 (16.7%) of 131 normal rhesus macaques (
Macaca mulatta
), compared to 18 (33.8%) of 53 rhesus macaques experimentally inoculated with SIV. In normal rhesus macaques, persistence of infection was demonstrated for up to 262 days and was usually not associated with clinical signs. In six of seven normal rhesus animals,
E. bieneusi
was detected by PCR in bile obtained through percutaneous cholecystocentesis but not by in situ hybridization performed on endoscopic biopsies of duodenum and proximal jejunum.
Efficient Isolation of Human Immunodeficiency Virus Type 1 RNA from Cervical Swabs
Authors: Hajjar Adeline M. , Lewis Paul F. , Endeshaw Yohannes , Ndinya-Achola Jackoniah , Kreiss Joan K. , Overbaugh Julie
Published: December 2019

An efficient method for the isolation of human immunodeficiency virus type 1 (HIV-1) nucleic acids from dry cervical swabs was developed. HIV-1
gag
and
env
were detected in 96% (25 of 26) and 81% (21 of 26), respectively, of the samples tested by PCR from HIV-1-seropositive women in a Kenyan cohort study. Eighty-eight percent of the swabs (22 of 25) were positive for
gag
RNA, and 85% (17 of 20) were positive for
env
RNA. Fewer than 1,000 copies of HIV-1
gag
RNA were detected in four swabs in which a competitive quantitative PCR assay was used. The method described here may be useful for both qualitative and quantitative analyses of HIV RNA in mucosal secretions as well as amplification and cloning of full-length viral genes for functional studies.
In Vitro Testing of Susceptibilities of Filamentous Ascomycetes to Voriconazole, Itraconazole, and Amphotericin B, with Consideration of Phylogenetic Implications
Authors: McGinnis Michael R. , Pasarell Lester
Published: December 2019

The in vitro susceptibilities of three hundred eighty-one isolates representing two classes, five orders, nine families, 30 genera, and 51 species of ascomycetous fungi to voriconazole, itraconazole, and amphotericin B were tested by using a modification of the National Committee for Clinical Laboratory Standards M27-A reference method. For those fungi of known phylogenetic relatedness, drug MICs were consistently low for isolates among all clades, except for members of the family Microascaceae. The highest MICs of all drugs tested were consistently for the Microascaceae, supporting the observation of fungal phylogeny and corresponding susceptibility to antifungal drugs. Itraconazole and voriconazole have a broad range of activity against phylogenetically similar agents of hyalohyphomycosis, phaeohyphomycosis, chromoblastomycosis, and mycetoma.
Human Infection with
Hymenolepis diminuta
: Case Report from Spain
Authors: Tena Daniel , Simón Montserrat Pérez , Gimeno Carmen , Pomata María Teresa Pérez , Illescas Soledad , Amondarain Izaskun , González Alejandro , Domínguez Jesús , Bisquert Julia
Published: December 2019

We report a case of
Hymenolepis diminuta
infection in a human. The patient was a 5-year-old girl referred to us through the onset of a cyanotic attack. Treatment with a single dose (10 mg/kg of body weight) of praziquantel was ineffective, but the parasite was eradicated after three treatment cycles with the same drug at dosages of 25 mg/kg/day for 5 days.
Comparison of Broth Microdilution Method Using
Haemophilus
Test Medium and Agar Dilution Method for Susceptibility Testing of
Eikenella corrodens
Authors: Alcalá Luis , García-Garrote Fernando , Cercenado Emilia , Peláez Teresa , Ramos Gema , Bouza Emilio
Published: December 2019

Susceptibility testing of
Eikenella corrodens
is usually performed by a Mueller-Hinton sheep blood agar dilution (AD) method. However, this method is impractical for testing only a few strains. We compared AD with the broth microdilution method using
Haemophilus
test medium (HTM) in order to determine the susceptibility of 36 clinical isolates of
E. corrodens
to eight antimicrobial agents. MICs obtained by the HTM method yielded 95.5 and 84% agreement (within 2 and 1 log
2
dilutions, respectively) with those obtained by AD. The HTM method with incubation in CO
2
for 48 h was highly reproducible and constitutes an easy alternative for antimicrobial susceptibility testing of
E. corrodens
.
Differentiation between
Mycobacterium tuberculosis
and
Mycobacterium avium
by Amplification of the 16S-23S Ribosomal DNA Spacer
Authors: Sansila Arunnee , Hongmanee Poonpilas , Chuchottaworn Charoen , Rienthong Somsak , Rienthong Dhanida , Palittapongarnpim Prasit
Published: December 2019

Differentiation between
Mycobacterium tuberculosis
and
M. avium
is helpful for the treatment of disseminated mycobacterial infection in AIDS patients. This can traditionally be done by time-consuming biochemical tests or with Accuprobe. Previously, PCR restriction enzyme analysis (PCR-REA) of the 16S-23S rRNA gene spacer was shown to be able to identify a limited number of strains of
Mycobacterium
. In this study the method was improved by using more specific primers and was tested with 50 clinical isolates of
M. tuberculosis
and 65 clinical isolates of
M. avium
complex. Probes specific to the spacers of
M. tuberculosis
and
M. avium
were also tested. Both
M. tuberculosis
and
M. avium
could be reliably identified either by PCR-REA or by PCR-hybridization, with the results completely agreeing with those obtained by biochemical tests and with the Accuprobe, respectively. The method may therefore be useful as an alternative in-house method for identification of the bacteria.
Detection of Cytomegalovirus in Plasma and Cerebrospinal Fluid Specimens from Human Immunodeficiency Virus-Infected Patients by the AMPLICOR CMV Test
Authors: Long Christopher M. , Drew Lawrence , Miner Richard , Jekic-McMullen Dragana , Impraim Chaka , Kao Shaw-Yi
Published: December 2019

We have developed the AMPLICOR CMV Test, which is rapid and sensitive for the detection of cytomegalovirus (CMV) in plasma and cerebrospinal fluid (CSF) specimens. The test incorporated an internal control in the reaction mixture to monitor the amplification efficiency and the presence of inhibitors. The AMPLICOR CMV Test was very specific in detecting 12 clinical CMV isolates and four laboratory CMV strains tested. Cross-reactivity with 26 non-CMV pathogens was not observed. The AMPLICOR CMV Test requires only 50 μl of specimen (plasma or CSF) for processing. The performance of the AMPLICOR CMV Test was compared to those of the CMV antigenemia assay and the conventional tube culture method. Among 112 plasma specimens from 43 human immunodeficiency virus-infected patients, CMV was detected in 20 (18%) of the specimens by the AMPLICOR CMV Test, 21 (19%) of the specimens by the CMV antigenemia assay, and 10 (9%) of the specimens by culture. In CSF specimens from AIDS patients, CMV was detected in 10 of 58 (17%) specimens tested by the AMPLICOR CMV Test, 5 of 28 (18%) specimens tested by the antigen assay, and none of the 25 specimens tested by culture. While the performance of the AMPLICOR CMV Test in this study was comparable to that of the CMV antigen assay, processing of specimens by the AMPLICOR CMV Test was much simpler than that by the antigen assay; in addition, the antigen assay requires greater than 10
5
leukocytes from blood or 1 ml of CSF to perform the assay. Our study suggested that the AMPLICOR CMV Test could provide a rapid and sensitive assay for the detection of CMV in plasma and CSF specimens.
Epidemiological Study of Prevalence of Genogroup II Human Calicivirus (Mexico Virus) Infections in Japan and Southeast Asia as Determined by Enzyme-Linked Immunosorbent Assays
Authors: Honma Shinjiro , Nakata Shuji , Numata Kazuko , Kogawa Keiko , Yamashita Teruo , Oseto Mitsuaki , Jiang Xi , Chiba Shunzo
Published: December 2019

Mexico virus (MXV) is a genogroup II human calicivirus (HuCV). We conducted an epidemiological study to determine the prevalence of MXV infection in infants and adults in Japan and Southeast Asia by enzyme-linked immunosorbent assays (ELISAs) developed by using baculovirus-expressed recombinant MXV (rMXV) capsids. Of 155 stool specimens obtained from children younger than 10 years old with acute clinical gastroenteritis (diarrhea and vomiting) associated with small, round-structured viruses in Japan from 1987 to 1989, only 2 were positive for MXV antigen. In 42 outbreaks of acute gastroenteritis in Japan from 1986 to 1994, 1 in an infant home and 1 among adults were positive for MXV antigen. The pattern of acquisition of antibody to rMXV was different from that of acquisition of antibody to group A rotavirus, the prototype HuCV Sapporo virus, and Norwalk virus. The prevalence of antibody to rMXV remained low for the first 3 years of life, showed a steep rise during nursery school age, reaching a prevalence of 50%, and another steep rise during adolescence, reaching 80%; and steadily increased thereafter. A high prevalence of antibody (82 to 88%) was observed in adult populations in Japan and Southeast Asia, suggesting that MXV infection is common in these areas. The discrepancy between the high prevalence of antibody to MXV and a low rate of detection of MXV antigen may be explained by a high specificity of the antigen ELISA for the prototype and closely related MXV strains while serological responses can detect responses to a broader group of viruses.
The Gamma Interferon Gene Knockout Mouse: a Highly Sensitive Model for Evaluation of Therapeutic Agents against
Cryptosporidium parvum
Authors: Griffiths Jeffrey K. , Theodos Cynthia , Paris Melissa , Tzipori Saul
Published: December 2019

Cryptosporidiosis is a serious disease in malnourished children and in people with malignancies or AIDS. Current rodent models for evaluating drug therapy against cryptosporidiosis have many limitations, including the need for a high inoculum, the absence of symptoms resembling those seen in humans, and the need to maintain exogenous immunosuppression. We have developed a gamma interferon knockout (GKO) mouse model with which to evaluate therapies against
C. parvum
and have used paromomycin for evaluation of this model. The GKO model offers considerable improvements over other systems, since it requires no additional immunosuppression and adult mice can be infected with as few as 10 oocysts (compared with 10
7
for SCID mice). Infected mice develop profound gastrointestinal dysfunction due to extensive infection and severe mucosal damage involving the entire small intestine. Clinical symptoms, which include depression, anorexia, weight loss, and wasting, result in death within 2 to 4 weeks. The time of death depends on the oocyst challenge dose. Paromomycin modulated parasitological and clinical parameters in highly predictable and significant ways, including prevention of death. In addition, examination of the extensively infected gut provided an important insight into the dynamics between a specific drug treatment, its impact on the extent and the site of parasite distribution, and clinical outcome. These uniform symptoms of weight loss, wasting, and death are powerful new parameters which bring this model closer to the actual disease seen in humans and other susceptible mammalian species.
Naturally Occurring Orthopoxviruses: Potential for Recombination with Vaccine Vectors
Authors: Sandvik Tore , Tryland Morten , Hansen Hilde , Mehl Reidar , Moens Ugo , Olsvik Ørjan , Traavik Terje
Published: December 2019

Orthopoxviruses are being increasingly used as live recombinant vectors for vaccination against numerous infectious diseases in humans, domestic animals, and wildlife. For risk assessments and surveillance, information about the occurrence, distribution and ecology of orthopoxviruses in western Europe is important but has mainly been based on serological investigations. We have examined kidneys, lungs, spleens, and livers of Norwegian small rodents and common shrews (
Sorex araneus
) for the presence of orthopoxvirus DNA sequences by PCR with primers complementary to the viral thymidine kinase (TK) gene. PCR amplicons were verified as orthopoxvirus specific by hybridization with a vaccinia virus TK-specific probe. A total of 347 animals (1,388 organs) from eight locations in different parts of Norway, collected at different times of the year during 1993 to 1995, were examined. Fifty-two animals (15%) from five locations, up to 1,600 km apart, carried orthopoxvirus DNA in one or more of their organs, most frequently in the lungs. These included 9 of 68 (13%) bank voles (
Clethrionomys glareolus
), 4 of 13 (31%) gray-sided voles (
Clethrionomys rufocanus
), 3 of 11 (27%) northern red-backed voles (
Clethrionomys rutilus
), 16 of 76 (21%) wood mice (
Apodemus sylvaticus
), and 20 of 157 (13%) common shrews. The previous isolation of cowpox virus from two clinical cases of infection (human and feline) at two of the locations investigated suggests that the viruses detected are cowpox and that some of the virus-carrying small mammalian species should be included among the cowpox virus natural reservoir hosts in Scandinavia and western Europe.
Evaluation of the Murex Hybrid Capture Cytomegalovirus DNA Assay versus Plasma PCR and Shell Vial Assay for Diagnosis of Human Cytomegalovirus Viremia in Immunocompromised Patients
Authors: Barrett-Muir Winsome Y. , Aitken Celia , Templeton Kate , Raftery Martin , Kelsey Steve M. , Breuer Judy
Published: December 2019

We evaluated a cytomegalovirus (CMV) 24-hour shell vial assay (SVA), the Murex Hybrid Capture CMV DNA assay (HCA), and a CMV plasma PCR for the detection of CMV viremia in renal and bone marrow transplant recipients and human immunodeficiency virus-infected patients. CMV viremia was detected by at least one method in 125 of 317 evaluable samples (39.4%) from 78 patients and was detected in 19.8% of samples by SVA, 26.8% by HCA, and 32.2% by plasma PCR. There was moderate to substantial agreement between the results of the different tests (kappa coefficient = 0.415 to 0.631). However, HCA and plasma PCR were significantly more sensitive than SVA (
P
= 0.001 and
P
< 0.0001, respectively; McNemar’s test), and plasma PCR was more sensitive than HCA (
P
= 0.031; McNemar’s test). HCA and plasma PCR were more consistently positive than SVA during viremic episodes (
P
= 0.0002 and
P
< 0.0001, respectively; McNemar’s test). The use of HCA or plasma PCR may therefore improve the diagnosis and management of CMV disease in susceptible patient groups.
Prevalence of Astroviruses in a Children’s Hospital
Authors: Shastri Sunita , Doane Anne Martin , Gonzales Jaime , Upadhyayula Usha , Bass Dorsey M.
Published: December 2019

An enzyme immunoassay for astrovirus was used to screen 357 stool samples from 267 symptomatic inpatients at a tertiary-care children’s hospital. Thirty stool samples from 26 patients contained astrovirus antigen, while rotavirus was found in 34 samples and
Clostridium difficile
toxin was found in 40. Half of the astrovirus infections were nosocomial. Additional pathogens were identified in six of the astrovirus antigen-positive stool samples. Most (80%) of the astroviruses recovered were of serotype 1. Astrovirus infections were significantly more common than rotavirus or
C. difficile
infections in very young infants and in those with surgical short-bowel syndrome.
Evaluation of the Etest Method for Determining Fluconazole Susceptibilities of 402 Clinical Yeast Isolates by Using Three Different Agar Media
Authors: Pfaller M. A. , Messer S. A. , Karlsson Å. , Bolmström A.
Published: December 2019

The performance of the Etest for fluconazole susceptibility testing of 402 yeast isolates was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. Etest MICs were determined with RPMI agar containing 2% glucose (RPG), Casitone agar (CAS), and Mueller-Hinton agar (MHA) and were read after incubation for 48 h at 35°C. The yeast isolates included
Candida albicans
(
n
= 161),
Candida glabrata
(
n
= 41),
Candida tropicalis
(
n
= 35),
Candida parapsilosis
(
n
= 29),
Candida krusei
(
n
= 32),
Candida lusitaniae
(
n
= 31),
Candida
species (
n
= 19),
Cryptococcus neoformans
(
n
= 40), and miscellaneous yeast species (
n
= 14). The Etest results correlated well with reference MICs. Overall agreement was 94% with RPG, 97% with CAS, and 53% with MHA. When RPG was used, agreement ranged from 89% for
Candida
spp. to 100% for
C. krusei
. When CAS was utilized, agreement ranged from 93% for
Cryptococcus neoformans
to 100% for
C. tropicalis
,
C. parapsilosis
,
C. lusitaniae
,
Candida
spp., and miscellaneous yeast species. With MHA, agreement ranged from 17% for
C. parapsilosis
to 90% for
C. krusei
. Both RPG and CAS supported growth of all yeast species, whereas growth on MHA was comparatively weaker. Etest results were somewhat easier to read on CAS. The Etest method using either RPG or CAS, but not MHA, appears to be a viable alternative to the NCCLS reference method for determining fluconazole susceptibilities of yeasts.
Phenotypic and Genotypic Characterization of Clinical Strains of CDC Group IVc-2
Authors: Osterhout Gerard J. , Valentine Joan L. , Dick James D.
Published: December 2019

CDC group IVc-2 is a gram-negative, oxidase-positive, nonfermentative bacillus that has been implicated in human infections, including septicemia and peritonitis. Biochemically it most closely resembles
Bordetella bronchiseptica
and
Alcaligenes
sp. Results of cellular fatty acid (CFA) and 16S rRNA gene analysis were combined with biochemical data to assist in identification and classification. The predominant CFAs were hexadecanoic acid (16:0),
cis
-9-hexadecanoic acid (16:1ω7c),
cis
-11-octadecanoic acid (18:1ω7c), and Δ-
cis
-9,10-methylenehexadecanoic acid (17:0cyc). Small amounts (2 to 5%) of 3-hydroxytetradecanoic acid (3-OH-14:0), tetradecanoic acid (14:0), 2-hydroxyhexadecanoic acid (2-OH-16:0), and Δ-
cis
-11,12-methyleneoctadecanoic acid (19:0cyc) were also consistently present. The highest 16S rRNA gene similarity was with
Ralstonia eutropha
and
Ralstonia solanacearum
. The CFA and 16S rRNA gene sequence data support the inclusion of CDC group IVc-2 in the recently created genus
Ralstonia
, which includes
R. eutropha
,
R. pickettii
, and
R. solanacearum
.
Phylogenetic Classification of
Trichophyton mentagrophytes
Complex Strains Based on DNA Sequences of Nuclear Ribosomal Internal Transcribed Spacer 1 Regions
Authors: Makimura Koichi , Mochizuki Takashi , Hasegawa Atsuhiko , Uchida Katsuhisa , Saito Hiuga , Yamaguchi Hideyo
Published: December 2019

Using internal transcribed spacer 1 (ITS1) region ribosomal DNA sequences from 37 stock strains and clinical isolates provisionally termed
Trichophyton mentagrophytes
complex in Japan, we demonstrated the mutual phylogenetic relationships of these strains. Members of this complex were classified into 3 ITS1-homologous groups and 13 ITS1-identical groups by their sequences. ITS1-homologous group I consists of
Arthroderma vanbreuseghemii
,
T. mentagrophytes
human isolates, and several strains of
T. mentagrophytes
animal isolates. Five strains of
Arthroderma simii
form a cluster comprising ITS1-homologous group II. The Americano-European and African races of
Arthroderma benhamiae
,
T. mentagrophytes
var.
erinacei
, and one strain of a
T. mentagrophytes
animal isolate constitute ITS1-homologous group III. According to the phylogenetic tree constructed with
Trichophyton rubrum
as an outgroup, ITS1-homologous groups I and II comprised a monophyletic cluster and ITS1-homologous group III constituted another cluster which was rather distant from the others in the complex. This system was applicable to the phylogenetic analysis of closely related strains. Using this technique, human and animal isolates of
T. mentagrophytes
were also clearly distinguishable from each other.
Branched-DNA Assay for Detection of the
mecA
Gene in Oxacillin-Resistant and Oxacillin-Sensitive Staphylococci
Authors: Kolbert C. P. , Arruda J. , Varga-Delmore P. , Zheng X. , Lewis M. , Kolberg J. , Persing D. H.
Published: December 2019

The identification of methicillin-resistant staphylococcus isolates in the clinical laboratory has typically been performed by using methods that detect phenotypic expression of resistance determinants. However, these methods may be difficult to interpret and some isolates do not express resistance until selective pressure is administered. Assays that detect genetic determinants are not subject to these limitations and have been effective in distinguishing isolates that are capable of expressing the resistance phenotype. In this study, a novel branched-DNA (bDNA) hybridization assay was used to test for the
mecA
gene in 416 clinical staphylococcal isolates. The results were compared with those obtained by a PCR-based assay and oxacillin disk diffusion. For 155
Staphylococcus aureus
and 261 coagulase-negative
Staphylococcus
isolates, the bDNA assay and PCR results were 100% concordant. Among the
S. aureus
isolates, 20 were MecA
+
and 135 were MecA
−
. For the coagulase-negative staphylococci, 150 were MecA
+
and 111 were MecA
−
. The results from the genotypic detection methods were compared with those obtained by oxacillin disk diffusion. No discrepancies were detected among the
S. aureus
isolates; however, 10 coagulase-negative isolates were MecA
+
but oxacillin sensitive and 1 isolate was MecA
−
but oxacillin resistant. Oxacillin resistance was induced in 6 of the 10 MecA
+
isolates previously classified as oxacillin sensitive. These results suggest that the bDNA method described here is a sensitive and efficient method for detection of methicillin resistance in staphylococci and that genetic detection methods may be useful for detection of potential methicillin resistance in the clinical laboratory.
Cloning and Characterization of Multigenes Encoding the Immunodominant 30-Kilodalton Major Outer Membrane Proteins of
Ehrlichia canis
and Application of the Recombinant Protein for Serodiagnosis
Authors: Ohashi Norio , Unver Ahmet , Zhi Ning , Rikihisa Yasuko
Published: December 2019

A 30-kDa major outer membrane protein of
Ehrlichia canis
, the agent of canine ehrlichiosis, is the major antigen recognized by both naturally and experimentally infected dog sera. The protein cross-reacts with a serum against a recombinant 28-kDa protein (rP28), one of the outer membrane proteins of a gene (
omp-1
) family of
Ehrlichia chaffeensis
. Two DNA fragments of
E. canis
were amplified by PCR with two primer pairs based on the sequences of
E. chaffeensis omp-1
genes, cloned, and sequenced. Each fragment contained a partial 30-kDa protein gene of
E. canis
. Genomic Southern blot analysis with the partial gene probes revealed the presence of multiple copies of these genes in the
E. canis
genome. Three copies of the entire gene (
p30
,
p30-1
, and
p30a
) were cloned and sequenced from the
E. canis
genomic DNA. The open reading frames of the two copies (
p30
and
p30-1
) were tandemly arranged with an intergenic space. The three copies were similar but not identical and contained a semivariable region and three hypervariable regions in the protein molecules. The following genes homologous to three
E. canis
30-kDa protein genes and the
E. chaffeensis omp-1
family were identified in the closely related rickettsiae:
wsp
from
Wolbachia
sp.,
p44
from the agent of human granulocytic ehrlichiosis,
msp-2
and
msp-4
from
Anaplasma marginale
, and
map-1
from
Cowdria ruminantium
. Phylogenetic analysis among the three
E. canis
30-kDa proteins and the major surface proteins of the rickettsiae revealed that these proteins are divided into four clusters and the two
E. canis
30-kDa proteins are closely related but that the third 30-kDa protein is not. The
p30
gene was expressed as a fusion protein, and the antibody to the recombinant protein (rP30) was raised in a mouse. The antibody reacted with rP30 and a 30-kDa protein of purified
E. canis
. Twenty-nine indirect fluorescent antibody (IFA)-positive dog plasma specimens strongly recognized the rP30 of
E. canis
. To evaluate whether the rP30 is a suitable antigen for serodiagnosis of canine ehrlichiosis, the immunoreactions between rP30 and the whole purified
E. canis
antigen were compared in the dot immunoblot assay. Dot reactions of both antigens with IFA-positive dog plasma specimens were clearly distinguishable by the naked eye from those with IFA-negative plasma specimens. By densitometry with a total of 42 IFA-positive and -negative plasma specimens, both antigens produced results similar in sensitivity and specificity. These findings suggest that the rP30 antigen provides a simple, consistent, and rapid serodiagnosis for canine ehrlichiosis. Cloning of multigenes encoding the 30-kDa major outer membrane proteins of
E. canis
will greatly facilitate understanding pathogenesis and immunologic study of canine ehrlichosis and provide a useful tool for phylogenetic analysis.
Identification of
Trichosporon asahii
by PCR Based on Sequences of the Internal Transcribed Spacer Regions
Authors: Sugita Takashi , Nishikawa Akemi , Shinoda Takako
Published: December 2019

Trichosporon asahii
is a major causative agent of deep-seated trichosporonosis, which has a high mortality rate. To detect
T. asahii
, we have developed specific oligonucleotide primers based on the internal transcribed spacer regions of this organism’s genome. Amplification products were selectively obtained from only
T. asahii
DNA; the DNAs of other
Trichosporon
species, as well as those of medically relevant yeasts such as
Candida albicans
,
Cryptococcus neoformans
, and
Malassezia furfur
, were not amplified. This detection system will be useful as a microbiological tool for the diagnosis of trichosporonosis.
Phaeohyphomycosis Caused by
Phaeoacremonium inflatipes
Authors: Padhye Arvind A. , Davis Miriam S. , Baer Dale , Reddick Anne , Sinha Kaushal K. , Ott Juliana
Published: December 2019

Phaeoacremonium inflatipes
, one of three species previously classified as strains of
Phialophora parasitica
, was identified as the causal agent of a subcutaneous infection of the left foot of an 83-year-old woman from South Carolina. The patient had a granulomatous growth over the anteromedial aspect of her left foot. It was surgically excised, which led to complete healing without complications. Tissue sections of the excised mass stained with hematoxylin and eosin and Gomori’s methenamine silver strains showed many septate hyphal elements of various lengths, some exhibiting brownish pigment in the cell walls of the hyphae. Portions of the tissue, when cultured, yielded many colonies which were initially glabrous, off white becoming velvety, greyish brown on aging. Microscopically, their hyphae were septate, branched, and phaeoid and bore lateral and terminal, erect, septate conidiophores. The conidiogenous cells (phialides) were terminal or lateral, mostly monophialidic, subcylindrical to spinelike in shape, and constricted at their bases and bore funnel-shaped, inconspicuous collarettes at their tips. The conidia were subhyaline, oblong, and ellipsoid to allantoid.
Houseflies Are an Unlikely Reservoir or Vector for
Helicobacter pylori
Authors: Osato Michael S. , Ayub Kamran , Le Hong-Hahn , Reddy Rita , Graham David Y.
Published: December 2019

The route of transmission of
Helicobacter pylori
from individual to individual remains undefined. It has recently been reported that the domestic housefly,
Musca domestica
, when fed pure cultures of
H. pylori
, was able to harbor the organism in its midgut for up to 30 h (P. Grubel, S. Hoffman, F. K. Chong, N. A. Barstein, C. Mepani, and D. R. Cave, J. Clin. Microbiol. 35:1300–1303, 1997). Our investigation examined whether houseflies could acquire
H. pylori
from fresh human feces. Domestic houseflies (40 flies/group) were exposed for 24 h to feces from an
H. pylori
-positive volunteer, feces from an
H. pylori
-negative volunteer, or feces from an
H. pylori
-negative volunteer to which a known amount of viable
H. pylori
had been added. At various intervals, flies were sacrificed and the midguts were excised, homogenized, and plated in duplicate onto selective horse blood agar plates. All plates were incubated under microaerobic conditions at 37°C for 14 days. Emergent colonies presumptive of
H. pylori
were picked and tested biochemically to confirm the identity as
H. pylori. H. pylori
was not recovered from houseflies fed human feces either naturally infected or artificially infected with
H. pylori
. These results suggest that the domestic housefly is not a vector for transmission or a reservoir for
H. pylori
infection.
Actinobacillus equuliSepticemia: an Unusual Zoonotic Infection
Authors: Ashhurst-Smith Christopher , Norton Robert , Thoreau Wendy , Peel Margaret M.
Published: December 2019

We describe the isolation ofActinobacillus equulifrom the blood of a 53-year-old butcher with septicemia. This species of the genusActinobacillusis primarily associated with animals and animal diseases, especially septicemia in foals. This is the first report of the isolation ofA. equulifrom a human with septicemia.
Typing of
Listeria monocytogenes
Strains by Repetitive Element Sequence-Based PCR
Authors: Jeršek B. , Gilot P. , Gubina M. , Klun N. , Mehle J. , Tcherneva E. , Rijpens N. , Herman L.
Published: December 2019

Listeria monocytogenes
strains possess short repetitive extragenic palindromic (REP) elements and enterobacterial repetitive intergenic consensus (ERIC) sequences. We used repetitive element sequence-based PCR (rep-PCR) to evaluate the potential of REP and ERIC elements for typing
L. monocytogenes
strains isolated from humans, animals, and foods. On the basis of rep-PCR fingerprints,
L. monocytogenes
strains were divided into four major clusters matching origin of isolation. rep-PCR fingerprints of human and animal isolates were different from those of food isolates. Computer evaluation of rep-PCR fingerprints allowed discrimination among the tested serotypes 1/2a, 1/2b, 1/2c, 3b, and 4b within each major cluster. The index of discrimination calculated for 52 epidemiologically unrelated isolates of
L. monocytogenes
was 0.98 for REP- and ERIC-PCR. Our results suggest that rep-PCR can provide an alternative method for
L. monocytogenes
typing.
Efficiencies of Four Versions of the AMPLICOR HIV-1 MONITOR Test for Quantification of Different Subtypes of Human Immunodeficiency Virus Type 1
Authors: Triques K. , Coste J. , Perret J. L. , Segarra C. , Mpoudi E. , Reynes J. , Delaporte E. , Butcher A. , Dreyer K. , Herman S. , Spadoro J. , Peeters M.
Published: December 2019

Three versions of a commercial human immunodeficiency virus (HIV) type 1 (HIV-1) load test (the AMPLICOR HIV-1 MONITOR Test versions 1.0, 1.0+, and 1.5; Roche Diagnostics, Branchburg, N.J.) were evaluated for their ability to detect and quantify HIV-1 RNA of different genetic subtypes. Plasma samples from 96 patients infected with various subtypes of HIV-1 (55 patients infected with subtype A, 9 with subtype B, 21 with subtype C, 2 with subtype D, 7 with subtype E, and 2 with subtype G) and cultured virus from 29 HIV-1 reference strains (3 of subtype A, 6 of subtype B, 5 of subtype C, 3 of subtype D, 8 of subtype E, 3 of subtype F, and 1 of subtype G) were tested. Detection of subtypes A and E was significantly improved with versions 1.0+ and 1.5 compared to that with version 1.0, whereas detection of subtypes B, C, D, and G was equivalent with the three versions. Versions 1.0, 1.0+, and 1.5 detected 65, 98, and 100% of the subtype A-infected samples from patients, respectively, and 71, 100, and 100% of the subtype E-infected samples from patients, respectively. Version 1.5 yielded a significant increase in viral load for samples infected with subtypes A and E (greater than 1 log
10
HIV RNA copies/ml). For samples infected with subtype B, C, and D and tested with version 1.5, only a slight increase in viral load was observed (<0.5 log
10
). We also evaluated a prototype automated version of the test that uses the same PCR primers as version 1.5. The results with the prototype automated test were highly correlated with those of the version 1.5 test for all subtypes, but were lower overall. The AMPLICOR HIV-1 MONITOR Test, version 1.5, yielded accurate measurement of the HIV load for all HIV-1 subtypes tested, which should allow the test to be used to assess disease prognosis and response to antiretroviral treatment in patients infected with a group M HIV-1 subtype.
Quantitative Analysis of Epstein-Barr Virus Load by Using a Real-Time PCR Assay
Authors: Kimura Hiroshi , Morita Makoto , Yabuta Yumi , Kuzushima Kiyotaka , Kato Koji , Kojima Seiji , Matsuyama Takaharu , Morishima Tsuneo
Published: December 2019

To measure the virus load in patients with symptomatic Epstein-Barr virus (EBV) infections, we used a real-time PCR assay to quantify the amount of EBV DNA in blood. The real-time PCR assay could detect from 2 to over 10
7
copies of EBV DNA with a wide linear range. We estimated the virus load in peripheral blood mononuclear cells (PBMNC) from patients with symptomatic EBV infections. The mean EBV-DNA copy number in the PBMNC was 10
3.7
copies/μg of DNA in patients with EBV-related lymphoproliferative disorders, 10
4.1
copies/μg of DNA in patients with chronic active EBV infections, and 10
2.2
copies/μg of DNA in patients with infectious mononucleosis. These numbers were significantly larger than those in either posttransplant patients or immunocompetent control patients without EBV-related diseases. In a patient with infectious mononucleosis, the virus load decreased as the symptoms resolved. The copy number of EBV DNA in PBMNC from symptomatic EBV infections was correlated with the EBV-positive cell number determined by the in situ hybridization assay (
r
= 0.842;
P
< 0.0001). These results indicate that the real-time PCR assay is useful for diagnosing symptomatic EBV infection and for monitoring the virus load.
False-Positive Gen-Probe Direct
Mycobacterium tuberculosis
Amplification Test Results for Patients with Pulmonary
M. kansasii
and
M. avium
Infections
Authors: Jorgensen James H. , Salinas Jesse R. , Paxson Rosemary , Magnon Karen , Patterson Jan E. , Patterson Thomas F.
Published: December 2019

The Gen-Probe Amplified Mycobacterium Tuberculosis Direct (MTD) test has been approved for use in the United States for the rapid diagnosis of pulmonary tuberculosis in patients with acid-fast smear-positive sputum samples since 1996. Four patients infected with human immunodeficiency virus and one chronic pulmonary-disease patient seen in our institutions with abnormal chest radiographs and fluorochrome stain-positive sputa were evaluated for tuberculosis, including performance of the MTD test on expectorated sputum samples. Three of these five patients’ sputa were highly smear-positive (i.e., more than 100 bacilli per high-power field), while two patient’s sputa contained 1 to 10 bacilli per field. MTD results on sputum specimens from these patients ranged from 43,498 to 193,858 relative light units (RLU). Gen-Probe has defined values of at least 30,000 RLU as indicative of a positive test, i.e., the presence of
Mycobacterium tuberculosis
RNA. Four of the patients’ sputum cultures yielded growth of
M. kansasii
within 6 to 12 days, and the fifth produced growth of
M. avium
only. One patient’s culture contained both
M. kansasii
and
M. avium
, but none of the initial or follow-up cultures from these five patients revealed
M. tuberculosis
. However, subsequent cultures from three of the patients again revealed
M. kansasii
. During the period of this study, in which MTD tests were performed on smear-positive sputum specimens from 82 patients, four of seven patients with culture-proven
M. kansasii
pulmonary infections yielded one or more false-positive MTD tests. The MTD sensitivity observed in this study was 93.8%, and the specificity was 85.3%. Five cultures of
M. kansasii
(including three of these patients’ isolates and
M. kansasii
ATCC 12478), and cultures of several other species were examined at densities of 10
5
to 10
7
viable CFU/ml by the MTD test. All five isolates of
M. kansasii
and three of three isolates of
M. simiae
yielded false-positive test results, with readings of 75,191 to 335,591 RLU. These findings indicate that low-level false-positive MTD results can occur due to the presence of
M. kansasii
,
M. avium
, and possibly other
Mycobacterium
species other than
M. tuberculosis
in sputum. Low-level positive MTD results of 30,000 to 500,000 RLU should be interpreted in light of these findings. It remains to be determined if the enhanced MTD test (MTD 2) recently released by Gen-Probe will provide greater specificity than that observed in this report with its first-generation test.
Comparison between PCR and Detection of Antigen in Sera for Diagnosis of Pulmonary Aspergillosis
Authors: Kawamura Sumio , Maesaki Shigefumi , Noda Tetsuhiro , Hirakata Youichi , Tomono Kazunori , Tashiro Takayoshi , Kohno Shigeru
Published: December 2019

We evaluated the usefulness of PCR and antigen detection for the diagnosis of pulmonary aspergillosis. Forty-four serum samples from patients with pulmonary aspergillosis (33 with pulmonary aspergilloma, 4 with allergic bronchopulmonary aspergillosis, 4 with invasive pulmonary aspergillosis, and 3 with aspergillus pyothorax) were used in this study. PCR detection of
Aspergillus
DNA in serum samples was successful in 39 patients. Galactomannan antigen was detected by sandwich enzyme-linked immunosorbent assay in 25 patients and by latex agglutination test in 13 patients. Detection of
Aspergillus
DNA in serum samples by nested PCR had the highest sensitivity of the three methods tested for the diagnosis of pulmonary aspergillosis.
Rapid Identification of
Burkholderia pseudomallei
by Latex Agglutination Based on an Exopolysaccharide-Specific Monoclonal Antibody
Authors: Steinmetz I. , Reganzerowski A. , Brenneke B. , Häussler S. , Simpson A. , White N. J.
Published: December 2019

We recently identified a constitutively expressed exopolysaccharide of
Burkholderia pseudomallei
which is composed of a unique linear tetrasaccharide repeating unit consisting of three galactose residues and one 3-deoxy-
d
-
manno
-2-octulosonic acid residue. In this study we developed a latex agglutination test based on monoclonal antibody 3015, which is specific for this exopolysaccharide, and evaluated this test for rapid identification of
B. pseudomallei
grown on agar plates. All 74 environmental and clinical
B. pseudomallei
strains tested, originating from different areas of Southeast Asia, northern Australia, and Africa, showed a strong and specific agglutination.
B. pseudomallei
-like organisms and a variety of other bacteria did not react. In conclusion this monoclonal antibody-based test is a simple, rapid, and highly specific method for identifying
B. pseudomallei
culture isolates from different geographic areas.
Viral Superinfection in Previously Unrecognized Chronic Carriers of Hepatitis B Virus with Superimposed Acute Fulminant versus Nonfulminant Hepatitis
Authors: Chu Chia-Ming , Yeh Chau-Ting , Liaw Yun-Fan
Published: December 2019

The role of viral superinfection in hepatitis B surface antigen carriers with superimposed fulminant (n= 60) versus nonfulminant (n= 90) acute hepatitis was studied. The frequency of hepatitis A virus (HAV) (0 versus 2.2%), HCV (18.3 versus 21.1%), HDV (15.0 versus 7.8%), and HEV (1.7 versus 4.4%) infection showed no significant difference, while simultaneous HCV and HDV infection was significantly more prevalent in the former (8.3 versus 0%). Only 3.6% of fulminant cases and 3.3% of nonfulminant controls were HGV RNA positive.