bluuebunny/crossref_metadata_2025 huggingface dataset:
Monitoring of Transmission of Tuberculosis between Wild Boars and Cattle: Genotypical Analysis of Strains by Molecular Epidemiology Techniques
Authors: Serraino Andrea , Marchetti Giulia , Sanguinetti Valeria , Rossi Maria Cristina , Zanoni Renato Giulio , Catozzi Lidia , Bandera Alessandra , Dini Walter , Mignone Walter , Franzetti Fabio , Gori Andrea
Published: December 2019

An epidemiological survey for the monitoring of bovine tuberculosis transmission was carried out in western Liguria, a region in northern Italy. Fifteen
Mycobacterium bovis
strains were isolated from 63 wild boar samples (62 from mandibular lymph nodes and 1 from a liver specimen). Sixteen mediastinal lymph nodes of 16 head of cattle were collected, and 15
Mycobacterium bovis
strains were subsequently cultured. All
M. bovis
strains isolated from cattle and wild boars were genotyped by spoligotyping and by restriction fragment length polymorphism (RFLP) analysis with the IS
6110
and IS
1081
probes. All
M. bovis
strains showed the typical spoligotype characterized by the absence of the 39 to 43 spacers in comparison with the number in
M. tuberculosis
. A total of nine different clusters were identified by spoligotyping. The largest cluster included 9 strains isolated from wild boars and 11 strains isolated from cattle, thus confirming the possibility of transmission between the two animal species. Fingerprinting by RFLP analysis with the IS
6110
probe showed an identical single-band pattern for 29 of 30 strains analyzed, and only 1 strain presented a five-band pattern. The use of IS
1081
as a second probe was useful for differentiation of
M. bovis
from
M. bovis
BCG but not for differentiation among
M. bovis
strains, which presented the same undifferentiated genomic profile. In relation to the epidemiological investigation, we hypothesized that the feeding in pastures contaminated by cattle discharges could represent the most probable route of transmission of
M. bovis
between the two animal species. In conclusion, our results confirmed the higher discriminatory power of spoligotyping in relation to that of RFLP analysis for the differentiation of
M. bovis
genomic profiles. Our data showed the presence of a common
M. bovis
genotype in both cattle and wild boars, confirming the possible interspecies transmission of
M. bovis.
Molecular Characterization of the Genera
Proteus
,
Morganella
, and
Providencia
by Ribotyping
Authors: Pignato S. , Giammanco G. M. , Grimont F. , Grimont P. A. D. , Giammanco G.
Published: December 2019

The so-called
Proteus-Providencia
group is constituted at present by three genera and 10 species. Several of the recognized species are common opportunistic pathogens for humans and animals. Different methods based on the study of phenotypic characters have been used in the past with variable levels of efficiency for typing some species for epidemiological purposes. We have determined the rRNA gene restriction patterns (ribotypes) for the type strains of the 10 different species of the genera
Proteus
,
Morganella
, and
Providencia
. Visual inspection of
Eco
RV- and
Hin
cII-digested DNA from the type strains showed remarkably different patterns for both enzymes, but
Eco
RV provided better differentiation. Both endonucleases were retained to study a large number of wild and collection strains belonging to the different species. Clinical isolates of
Proteus mirabilis
,
Proteus penneri
,
Morganella morganii
, and
Providencia heimbachae
showed patterns identical or very similar to those of the respective type strains, so that groups of related patterns (ribogroups) were found to correspond to the diverse species. On the contrary, distinct ribogroups were detected within
Providencia alcalifaciens
(two ribogroups with both enzymes),
Providencia rettgeri
(four ribogroups with
Eco
RV and five with
Hin
cII),
Providencia stuartii
(two ribogroups with
Eco
RV),
Providencia rustigianii
(two ribogroups with
Hin
cII), and
Proteus vulgaris
(two ribogroups with both enzymes). The pattern shown by the ancient
P. vulgaris
type strain NCTC 4175 differed considerably from both
P. vulgaris
ribogroups as well as from the newly proposed type strain ATCC 29905 and from any other strain in this study, thus confirming its atypical nature. Minor differences were frequently observed among patterns of strains belonging to the same ribogroup. These differences were assumed to define ribotypes within each ribogroup. No correlation was observed between ribogroups or ribotypes and biogroups of
P. vulgaris
,
P. alcalifaciens
,
P. stuartii
, and
P. rettgeri
. Since, not only different species showed different rRNA gene restriction patterns, but also different ribogroups and ribotypes have been found in the majority of the species, ribotyping would be a sensitive method for molecular characterization of clinical isolates belonging to the genera
Proteus
,
Morganella
, and
Providencia
.
Immunomagnetic Isolation of
Streptococcus suis
Serotypes 2 and 1/2 from Swine Tonsils
Authors: Gottschalk Marcelo , Lacouture Sonia , Odierno Liliana
Published: December 2019

Isolation of specific serotypes of
Streptococcus suis
from the tonsils, nasal cavities, and genital tract is difficult, since low-pathogenic serotypes and untypeable strains also inhabit these sites. An immunomagnetic separation (IMS) technique for the selective isolation of
S. suis
serotypes 2 and 1/2 was standardized. Superparamagnetic polystyrene beads (immunomagnetic beads or IMB) were coated with either a purified monoclonal antibody (MAb) directed to a capsular sialic acid-containing epitope or purified rabbit immunoglobulin G (polyclonal antibody [PAb]), both specific for
S. suis
serotypes 2 and 1/2. The amount of antibodies required for optimum coating of the beads, the number of IMB required for optimum bacterial recovery, and the nonspecific carryover were considerably higher with the MAb-IMS technique than with the PAb-IMS technique. The sensitivity of the IMS technique was 10
1
CFU/0.1 g of tonsil. The presence of serotype 1/2 bacteria did not considerably affect the recovery rate of a serotype 2 strain and vice versa. To validate the technique, PAb-coated beads were used to study 192 tonsils from animals from
S. suis
serotype 2- or 1/2-infected herds. Results showed that significantly more positive tonsils were detected by the IMS technique than by the standard procedure. This method represents an innovative and highly sensitive approach for the isolation of
S. suis
serotypes 2 and 1/2 from carrier animals.
Quantitation of Hepatitis B Virus Genomic DNA by Real-Time Detection PCR
Authors: Abe Aki , Inoue Kazuaki , Tanaka Takeshi , Kato Junko , Kajiyama Naoki , Kawaguchi Ryuji , Tanaka Satoshi , Yoshiba Makoto , Kohara Michinori
Published: December 2019

Quantitation of hepatitis B virus (HBV) DNA in serum is a useful method for the monitoring of HBV replication. We attempted to develop a quantitative assay system for HBV DNA that is more sensitive, accurate, and reproducible than existing systems. We detected HBV DNA by real-time detection PCR (RTD-PCR) based on Taq Man chemistry. The efficacy of this assay was evaluated by quantitatively measuring sequential levels of synthetic DNA and DNA in clinical serum samples. The detection limit of this system was as few as 10 DNA copies/reaction. A linear standard curve was obtained between 10
1
and 10
8
DNA copies/reaction. The coefficient of variation for both intra- and interexperimental variability indicated remarkable reproducibility. This system detected HBV DNA in 100% of chronic hepatitis B patients tested and never detected HBV DNA in healthy volunteers who were negative for HBV markers. These observations suggest that RTD-PCR is an excellent candidate for a standard HBV quantification method.
International Multicenter Evaluation of the Clinical Utility of a Dipstick Assay for Detection of
Leptospira
-Specific Immunoglobulin M Antibodies in Human Serum Specimens
Authors: Smits Henk L. , Ananyina Yulia V. , Chereshsky Annette , Dancel Louella , Lai-A-Fat Rudy F. M. , Chee Howard D. , Levett Paul N. , Masuzawa Toshiyuki , Yanagihara Yasutake , Muthusethupathi M. A. , Sanders Eduard J. , Sasaki David M. , Domen Harry , Yersin Claude , Aye Tin , Bragg Sandra L. , Gussenhoven George C. , Goris Marga G. A. , Terpstra Wiepko J. , Hartskeerl Rudy A.
Published: December 2019

We performed a multicenter evaluation of a robust and easily performed dipstick assay for the serodiagnosis of human leptospirosis. The assay is aimed at the detection of
Leptospira
-specific immunoglobulin M (IgM) antibodies. The study involved 2,665 serum samples collected from 2,057 patients with suspected leptospirosis in 12 countries on five continents with different levels of endemicity and different surveillance systems. The patients were grouped as laboratory-confirmed leptospirosis case patients and noncase patients based on the results of culturing and the microscopic agglutination test. Paired samples from 27.7% of the subjects were tested. Of the 485 case patients, 87.4% had a positive dipstick result for one or more samples. Of the 1,513 noncase patients, only 7.2% had a positive result. Whereas most (88.4%) of the positive samples from the case patients showed moderate to strong (2+ to 4+) staining in the dipstick assay, most (68.1%) of the positive samples from the noncase patients showed weak (1+) staining. The sensitivity of the dipstick assay increased from 60.1% for acute-phase serum samples to 87.4% for convalescent-phase samples. The specificities for these two groups of samples were 94.1 and 92.7%, respectively. The dipstick assay detected a broad variety of serogroups. The results of the dipstick assay were concordant (observed agreement, 93.2%; kappa value, 0.76) with the results of an enzyme-linked immunosorbent assay for the detection of specific IgM antibodies, a test which is often used in the laboratory diagnosis of current or recent leptospirosis. This study demonstrated that this easily performed dipstick assay is a valuable and useful test for the quick screening for leptospirosis; has a wide applicability in different countries with different degrees of endemicity; can be used at all levels of the health care system, including the field; and will be useful for detecting and monitoring outbreaks of leptospirosis.
Polymorphism of the Human Immunodeficiency Virus Type 1 (HIV-1) Protease Gene and Response of HIV-1-Infected Patients to a Protease Inhibitor
Authors: Bossi Philippe , Mouroux Mireille , Yvon Anne , Bricaire François , Agut Henri , Huraux Jean-Marie , Katlama Christine , Calvez Vincent
Published: December 2019

In order to analyze the impact of protease gene polymorphism on response to regimens containing a protease inhibitor, the entire protease coding domain from 58 human immunodeficiency virus type 1 (HIV-1)-infected patients who were protease inhibitor naive was sequenced before therapy was started. Plasma HIV-1 RNA levels were measured at baseline and at month 3 and month 6 after treatment. All patients were treated with a combination of two reverse transcriptase inhibitors and a protease inhibitor (saquinavir EOF [
n
= 28], ritonavir [
n
= 16], or indinavir [
n
= 14]). Before treatment, 30 different positions whose codons differed from the subtype B consensus sequence were observed. Major mutations associated with protease inhibitor resistance were not observed. No statistical correlation between the number of amino acid differences and the treatment efficacy at month 3 (−2.4 log) or month 6 (−2.7 log) was observed. At baseline, genotypic analysis of the HIV-1 protease gene of patients who have never received a protease inhibitor does not allow prediction of the efficacy of regimens containing a protease inhibitor.
Immunoglobulin M Antibody Test To Detect Genogroup II Norwalk-Like Virus Infection
Authors: Brinker James P. , Blacklow Neil R. , Jiang Xi , Estes Mary K. , Moe Christine L. , Herrmann John E.
Published: December 2019

Sera obtained from adult volunteers inoculated with genogroup II Norwalk-like viruses (NLVs), Hawaii virus, and Snow Mountain virus and from patients involved in outbreaks of gastroenteritis were tested for genogroup II NLV Mexico virus-specific immunoglobulin M (IgM) by use of a monoclonal antibody, recombinant Mexico virus antigen (rMXV)-based IgM capture enzyme-linked immunosorbent assay (ELISA). Sera from genogroup I Norwalk virus (NV)-inoculated volunteers and from patients involved in a genogroup I NLV outbreak were also tested. In sera from those infected with genogroup I NV or NLVs in volunteer and outbreak studies, only 3 of 25 were rMXV IgM positive; in contrast, 24 of 25 were IgM positive for recombinant NV (rNV). In sera from those infected with genogroup II NLVs in volunteer and outbreak studies, 28 of 47 were rMXV IgM positive and none were IgM positive for rNV, showing the specificity of each IgM test for its respective genogroup. In an outbreak of gastroenteritis not characterized as being of viral etiology but suspected to be due to NV, 7 of 13 persons had IgM responses to rMXV, whereas none had IgM responses to rNV, thus establishing the diagnosis as genogroup II NLV infection. The rMXV-based IgM capture ELISA developed is specific for the diagnosis of genogroup II NLV infections.
Immunochromatographic Strip-Based Detection of
Entamoeba histolytica-E. dispar
and
Giardia lamblia
Coproantigen
Authors: Pillai Dylan R. , Kain Kevin C.
Published: December 2019

BIOSITE Triage was 68.3% sensitive and 100% specific for the detection of
Entamoeba histolytica-E. dispar
(
n
= 71) compared to Alexon-Trend’s ProSpecT test (reference standard) using fresh-frozen stool. Neither test is able to distinguish
E. histolytica
from
E. dispar
. Triage was 83.3% sensitive and 100% specific compared to microscopy (formalin-ether concentrates and permanent stains) for the detection of
Giardia lamblia.
Theileria
sp. Infections Associated with Bovine Fatalities in the United States Confirmed by Small-Subunit rRNA Gene Analyses of Blood and Tick Samples
Authors: Chae Joon-seok , Levy Michael , Hunt John , Schlater Jack , Snider Glen , Waghela Suryakant D. , Holman Patricia J. , Wagner G. Gale
Published: December 2019

Theileria
sp.-specific small subunit (SSU) rRNA gene amplification confirmed the presence of the organism in cattle and in
Amblyomma americanum
and
Dermacentor variabilis
ticks collected from a cattle herd in Missouri. Blood from the index animal had type A and type D
Theileria
SSU rRNA genes. The type D gene was also found in blood from two cohort cattle and tick tissues. The type A SSU rRNA gene was previously reported from bovine
Theileria
isolates from Texas and North Carolina; the type D gene was reported from a Texas cow with theileriosis.
Recurrent, Disseminated
Mycobacterium marinum
Infection Caused by the Same Genotypically Defined Strain in an Immunocompromised Patient
Authors: Holmes G. Frank , Harrington Susan M. , Romagnoli Mark J. , Merz William G.
Published: December 2019

An 81-year-old male with myasthenia gravis developed a cutaneous infection with
Mycobacterium marinum
, which apparently resolved following local heat therapy. Five months later, the patient developed new skin lesions and pancytopenia.
M. marinum
was isolated from his bone marrow. Pulsed-field gel electrophoresis was performed to determine if the skin and bone marrow isolates were clonally related. Digestion of the genomic DNA with the restriction enzymes
Spe
I and
Ase
I yielded indistinguishable banding patterns. An epidemiologically unrelated control strain showed significant banding differences. The results suggest that the patient’s recurrent, disseminated infection was due to recrudescence of his initial infection rather than reinfection by another strain.
Elevated Phenotypic Switching and Drug Resistance of
Candida albicans
from Human Immunodeficiency Virus-Positive Individuals prior to First Thrush Episode
Authors: Vargas Kaaren , Messer Shawn A. , Pfaller Michael , Lockhart Shawn R. , Stapleton Jack T. , Hellstein John , Soll David R.
Published: December 2019

Strains of
Candida albicans
obtained from human immunodeficiency virus (HIV)-positive individuals prior to their first episode of oral thrush were already in a high-frequency mode of switching and were far more resistant to a number of antifungal drugs than commensal isolates from healthy individuals. Switching in these isolates also had profound effects both on susceptibility to antifungal drugs and on the levels of secreted proteinase activity. These results suggest that commensal strains colonizing HIV-positive individuals either undergo phenotypic alterations or are replaced prior to the first episode of oral thrush. They also support the suggestion that high-frequency phenotypic switching functions as a higher-order virulence trait, spontaneously generating in colonizing populations variants with alterations in a variety of specific virulence traits.
Increased Sensitivity of the BACTEC 460 Mycobacterial Radiometric Broth Culture System Does Not Decrease the Number of Respiratory Specimens Required for a Definitive Diagnosis of Pulmonary Tuberculosis
Authors: Harvell Jeff D. , Hadley W. Keith , Ng Valerie L.
Published: December 2019

The BACTEC 460 radiometric mycobacterial broth culture system has consistently demonstrated faster and increased recovery of
Mycobacterium tuberculosis
from respiratory specimens of patients with pulmonary tuberculosis than conventional culture methods. We thus questioned whether three sputa were still necessary to definitively diagnose pulmonary tuberculosis if the BACTEC radiometric culture system were in use. We performed a retrospective analysis of 430 sequential respiratory specimens submitted from 143 patients and from which
M. tuberculosis
had been recovered by in vitro culture and simultaneously assessed the diagnostic yield of acid-fast smear in this same cohort.
M. tuberculosis
was recovered from the first specimen for 117 (82%) of the 143 patients, from the second for 14 patients (10%; cumulative rate, 92%), and from the third for 12 patients (8%; cumulative rate, 100%). With the exception of those for bronchial brushings, recovery rates of
M. tuberculosis
were comparable for all respiratory specimen types (expectorated sputum, induced sputum, tracheal aspirates, bronchoalveolar lavage fluids). Only 46 (32%) of these 143 patients had acid-fast bacilli detected in smears; acid-fast bacilli were detected in the first submitted specimen for 44 patients (96%) and in the second for the remaining 2 patients (4%; cumulative rate, 100%). Culture- or smear-positive rates for sequential specimens obtained from AIDS patients were comparable to those for non-AIDS patients. Overall, the diagnostic culture yield of sequentially submitted specimens was not different from previously published studies in which the BACTEC radiometric culture system had not been used. Despite the documented enhanced ability of the BACTEC 460 radiometric mycobacterial culture system to recover
M. tuberculosis
more often and faster than conventional methods, three sequential respiratory specimens (regardless of type) were still necessary to definitively diagnose pulmonary tuberculosis.
Comparison of Danish Isolates of
Salmonella enterica
Serovar Enteritidis PT9a and PT11 from Hedgehogs (
Erinaceus europaeus
) and Humans by Plasmid Profiling and Pulsed-Field Gel Electrophoresis
Authors: Nauerby B. , Pedersen K. , Dietz H. H. , Madsen M.
Published: December 2019

During the years 1994 to 1998, 10 strains of
Salmonella enterica
serovar Enteritidis phage type 11 (PT11) and 6 PT9a strains were isolated from Danish hedgehogs, together with 7 strains that did not yield phage susceptibility patterns conforming with any known phage type (routine dilution no conformity [RDNC]). From 1995 to 1998, five Danish patients were reported infected with serovar Enteritidis PT11 and two with PT9a. All serovar Enteritidis PT11, PT9a, and RDNC isolates from hedgehogs and humans were analyzed by pulsed-field gel electrophoresis (PFGE), plasmid profiling, and restriction fragment length polymorphism (RFLP) of plasmids. By use of S1 nuclease and
Hin
dIII, the PT11 and PT9a isolates had identical plasmid profiles and RFLP patterns, which differed from the RDNC profiles. The PFGE profiles were identical for all serovar Enteritidis PT11 and PT9a strains from hedgehogs, four of five human strains of serovar Enteritidis PT11, and two human strains of serovar Enteritidis PT9a, irrespective of restriction enzyme, whereas the last human strain deviated slightly when
Not
I was used but not when
Xba
I or
Spe
I was used. The results indicate that serovar Enteritidis PT9a and PT11 are closely related and that PT11 and PT9a from Danish hedgehogs and humans belong to the same clonal lineage.
Pneumococcal
pspA
Sequence Types of Prevalent Multiresistant Pneumococcal Strains in the United States and of Internationally Disseminated Clones
Authors: Beall Bernard , Gherardi Giovanni , Facklam Richard R. , Hollingshead Susan K.
Published: December 2019

In a recent genotypic survey of β-lactam-resistant pneumococci recovered in different areas of United States during 1997, eight clonal types that each represented 3 to 40 isolates accounted for 134 of 144 isolates (G. Gherardi, C. Whitney, R. Facklam, and B. Beall, J. Infect. Dis. 181:216–229, 2000). We determined the degree of
pspA
gene diversity among these 134 isolates and for 11 previously characterized internationally disseminated multiresistant strains. Thirty-four different
pspA
restriction profiles were determined for an amplicon encompassing the variable portion of the structural gene that encodes the surface-exposed domain of PspA and a variable-length proline-rich putative cell wall-associated domain. These restriction profiles closely correlated with those of 33 different
pspA
sequence types of an approximately 230-residue region corresponding to residues 182 to 410 of the strain Rx1 PspA. These residues encompass a 100-residue clade-defining region known to contain cross-protective epitopes for which 17 sequence types were found. Distinct, conserved
pspA
sequence types were found for the majority of strains within seven of the eight U.S. clonal types assessed, while one pulsed-field gel electrophoresis type was represented by isolates of three distinct PspA clades. Sequence typing of
pspA
provides an added level of specificity in the subtyping of isolates and is a necessary first step in determining the components needed in a PspA vaccine which could elicit effective cross-protective coverage.
Expression of a Gene Encoding the Major Antigenic Protein 2 Homolog ofEhrlichia chaffeensisand Potential Application for Serodiagnosis
Authors: Alleman A. Rick , Barbet Anthony F. , Bowie Michael V. , Sorenson Heather L. , Wong Susan J. , Bélanger Myriam
Published: December 2019

The major antigenic protein 2 (MAP2) homolog ofEhrlichia chaffeensiswas cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of human monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblotting using antihistidine antibody. However, immune sera failed to react with the recombinant on immunoblots when the antigen was denatured by heat or reduced using β-mercaptoethanol. The recombinant MAP2 (rMAP2) was used in an ELISA format with 60 blinded serum samples. Twenty of the serum samples were previously demonstrated to contain antibodies reactive withE. chaffeensisby indirect immunofluorescence assays (IFAs). The remaining 40 samples were seronegative. All samples negative by IFA were also found to be negative for antibodies against the rMAP2 ofE. chaffeensisby using the ELISA. Only 1 of 20 IFA-positive samples tested negative in the rMAP2 ELISA. There was 100% agreement using IFA-negative samples and 95% agreement using IFA-positive samples, resulting in a 97.5% overall agreement between the two assays. These data suggest that the rMAP2 homolog ofE. chaffeensismay have potential as a test antigen for the serodiagnosis of human monocytic ehrlichiosis. To our knowledge, this recombinant is unique because it is thus far the onlyE. chaffeensisrecombinant antigen that has been shown to work in an ELISA format.
Diagnosis of Cutaneous Leishmaniasis in Colombia: the Sampling Site within Lesions Influences the Sensitivity of Parasitologic Diagnosis
Authors: Ramírez José Robinson , Agudelo Sonia , Muskus Carlos , Alzate Juan Fernando , Berberich Christof , Barker Douglas , Velez Ivan Darío
Published: December 2019

Parasitologic confirmation of cutaneous leishmaniasis is obligatory before chemotherapy can be considered. Direct microscopic examination of scrapings taken from indurated borders of ulcers has been routinely used as primary method of diagnosis. In this report we compared the sensitivity of examination of dermal scrapings taken from the bottoms of ulcers (BDS) with that of dermal scrapings taken from indurated active margins of lesions (MDS) in a total of 115 patients. The sensitivities of the microscopic examination were 90.4 and 78.3% for BDS and MDS samples, respectively. When the PCR method was used with a group of 40 patients, we also observed a higher sensitivity when BDS samples were examined (80.8% in BDS samples versus 57.7% in MDS samples). The improvement of the diagnostic sensitivity in the BDS samples appears to be related to the higher parasite load and more easily detectable morphology of amastigotes in the centers of the ulcers. Other parasitologic diagnostic methods, such as culture and histopathologic examination of biopsies, are less sensitive (67.5 and 64.3%, respectively). Aspirate culture, however, was shown to be the most sensitive method for the diagnosis of patients with chronic ulcers. When microscopic examinations of both MDS and BDS samples are combined, the sensitivity of diagnosis may rise up to 94%. We therefore recommend this method as a primary routine procedure for diagnosis of cutaneous leishmaniasis.
InTray GC Medium Versus Modified Thayer-Martin Agar Plates for Diagnosis of Gonorrhea from Endocervical Specimens
Authors: Beverly Amanda , Bailey-Griffin Jill R. , Schwebke Jane R.
Published: December 2019

A new self-contained medium system for the cultivation of
Neisseria gonorrhoeae
was compared to modified Thayer-Martin medium for the diagnosis of gonorrhea from endocervical specimens. There was no difference in the ability of the two methods to support the growth of
N. gonorrhoeae
.
Evaluation of Etest for Susceptibility Testing of
Mycobacterium tuberculosis
Authors: Joloba Moses L. , Bajaksouzian Saralee , Jacobs Michael R.
Published: December 2019

The Etest method for susceptibility testing of
Mycobacterium tuberculosis
was compared to the agar proportion method using four first-line agents and two fluoroquinolones. Catergorical agreement between the methods was 100% for rifampin, ethambutol, streptomycin, and ofloxacin and 98% for isoniazid. Results were obtained in 6 to 10 days by Etest. The Etest method is suitable for testing the agents evaluated against
M. tuberculosis
.
Vancomycin-Intermediate
Staphylococcus aureus
in Korea
Authors: Kim Mi-Na , Pai Chik Hyun , Woo Jun Hee , Ryu Ji So , Hiramatsu Keiichi
Published: December 2019

Recent reports on some methicillin-resistant
Staphylococcus aureus
(MRSA) with reduced susceptibility to vancomycin have been a major concern in Korea because of the widespread use of vancomycin due to a high prevalence of MRSA in the country. We describe a 45-year-old man with long-standing pelvic abscess due to MRSA. In spite of vancomycin and teicoplanin treatment for a long period of time, the patient died from MRSA sepsis. The blood culture isolate of MRSA exhibited reduced susceptibility to vancomycin (MIC, 8 μg/ml). This is the first report of a vancomycin-intermediate
S. aureus
case from Korea.
Histoplasmosis of the Thyroid
Authors: Goldani Luciano Z. , Klock Clóvis , Diehl Ada , Monteiro Ane C. , Maia Ana Luiza
Published: December 2019

Fungal infection of the thyroid is rare. Most reported cases have involved
Aspergillus
,
Coccidioides
, and
Candida
species in the setting of disseminated disease. Infection of the thyroid with
Histoplasma capsulatum
is rarely reported as part of disseminated disease, even in geographic areas where histoplasmosis is endemic. We report a 52-year-old woman with a previous Hashimoto's disease and non-Hodgkin's lymphoma in which a diffuse enlarged thyroid gland with a large nodule was the only apparent locus of histoplasmosis. Fine-needle aspiration of the thyroid was an important diagnostic tool in establishing the diagnosis of histoplasmosis of the thyroid. The patient was initially treated with itraconazole (400 mg/day) for the fungal infection and six cycles of chemotherapy for the lymphoma. At a 6-month follow-up examination, the patient was doing well on suppressive therapy of itraconazole (200 mg/day), with no symptoms and with regression of the thyroid nodule and cervical adenopathy.
Failure To Detect Circulating
Aspergillus
Markers in a Patient with Chronic Granulomatous Disease and Invasive Aspergillosis
Authors: Verweij Paul E. , Weemaes Corry M. , Curfs Jo H. A. J. , Bretagne Stephane , Meis Jacques F. G. M.
Published: December 2019

We report a patient with chronic granulomatous disease who developed invasive pulmonary aspergillosis and a subphrenic abscess. During treatment, high levels of
Aspergillus
antigen were detected in the abscess, but circulating antigen and
Aspergillus
DNA were undetectable in the serum.
Discrimination between Clinically Relevant and Nonrelevant
Acanthamoeba
Strains Isolated from Contact Lens- Wearing Keratitis Patients in Austria
Authors: Walochnik J. , Haller-Schober E.-M. , Kölli H. , Picher O. , Obwaller A. , Aspöck H.
Published: December 2019

Eighteen cases of
Acanthamoeba
-associated keratitis among contact lens wearers seen at the Department of Ophthalmology, Karl-Franzens-University, Graz, Austria, between 1996 and 1999 are reviewed. The amoebae were proven to be the causative agents in three patients. The aim of our study was to discriminate between clinically relevant and nonrelevant isolates and to assess the relatedness of the isolates to published strains. Altogether, 20 strains of free-living amoebae, including 15
Acanthamoeba
strains, 3
Vahlkampfia
strains, and 2
Hartmannella
strains, were isolated from clinical specimens. The virulent
Acanthamoeba
strains were identified as
A. polyphaga
and two strains of
A. hatchetti
. To our knowledge this is the first determination of keratitis-causing
Acanthamoeba
strains in Austria. Clinically relevant isolates differed markedly from nonrelevant isolates with respect to their physiological properties. 18S ribosomal DNA sequence types were determined for the three physiologically most-divergent strains including one of the keratitis-causing strains. This highly virulent strain exhibited sequence type T6, a sequence type not previously associated with keratitis. Sequence data indicate that
Acanthamoeba
strains causing keratitis as well as nonpathogenic strains of
Acanthamoeba
in Austria are most closely related to published strains from other parts of the world. Moreover, the results of our study support the assumption that pathogenicity in
Acanthamoeba
is a distinct capability of certain strains and not dependent on appropriate conditions for the establishment of an infection.
Identification of Ciprofloxacin-Resistant
Campylobacter jejuni
by Use of a Fluorogenic PCR Assay
Authors: Wilson David L. , Abner Sheila R. , Newman Thomas C. , Mansfield Linda S. , Linz John E.
Published: December 2019

Fluoroquinolones are one class of antimicrobial agents commonly used to treat severe
Campylobacter jejuni
infection.
C. jejuni
strains resistant to high levels of the fluoroquinolone ciprofloxacin (MIC ≥16 μg/ml) have been predominantly characterized with a C→T transition in codon 86 of
gyrA
. The
gyrA
gene encodes one subunit of DNA gyrase, which is a primary target for fluoroquinolone antibiotics. This study establishes a rapid PCR-based TaqMan method for identifying ciprofloxacin-resistant
C. jejuni
strains that carry the C→T transition in codon 86 of
gyrA
. The assay uses real-time detection, eliminating the need for gel electrophoresis. Optimization of the assay parameters using purified
Campylobacter
DNA resulted in the ability to detect femtogram levels of DNA. The method should be useful for monitoring the development of ciprofloxacin resistance in
C. jejuni
. Compiled nucleotide sequence data on the quinolone resistance-determining region of
gyrA
in
Campylobacter
indicate that sequence comparison of this region is a useful method for tentative identification of
Campylobacter
isolates at the species level.
Analytical Performance and Clinical Utility of a Nucleic Acid Sequence-Based Amplification Assay for Detection of Cytomegalovirus Infection
Authors: Witt Donald J. , Kemper M. , Stead Andrew , Sillekens P. , Ginocchio Christine C. , Espy Mark J. , Paya Carlos V. , Smith Thomas F. , Roeles Frits , Caliendo Angela M.
Published: December 2019

A nucleic acid sequence-based amplification (NASBA) assay for qualitative detection of human cytomegalovirus (CMV) pp67 mRNA was evaluated in a multicenter study. Negative results were obtained for all specimens from 50 CMV-seronegative and 50 CMV-seropositive low-risk whole-blood donors. No interference with CMV mRNA amplification was observed in the testing of 288 specimens containing various potential interfering substances, nonspecifically reacting substances (including mRNA from other herpesviruses), and three anticoagulants. A total of 95% (50 of 51) of CMV-positive (cell culture- and antigenemia immunofluorescence [AG-IFA]-positive) clinical specimens were positive by the NASBA assay. Results from different operators over multiple testing days were consistent for each of four panel members containing different concentrations of CMV mRNA, indicating the reproducibility of the assay. The estimated 95% reliable upper detection limit of the assay was 600 mRNA copies; the lower limit of detection was less than 25 mRNA copies. The clinical utility of the assay was evaluated with longitudinally collected specimens from solid-organ transplant patients (
n
= 21). A total of 98% (81 of 83) of the specimens from CMV-negative patients were negative by the NASBA assay, while 90% (10 of 11) of patient specimens that were positive by cell culture or AG-IFA were positive by the NASBA assay. Positive NASBA assay results were obtained earlier than AG-IFA or cell culture results for 55% of the patients and at the same time for the remainder of the patients (45%). The overall agreement between the NASBA assay and current reference tests was 86% when active CMV infection was present. These studies indicate that the CMV pp67 mRNA NASBA assay has reproducible and sensitive performance characteristics that should enable more rapid diagnosis of CMV infection.
Emergence and Rapid Spread of Carbapenem Resistance during a Large and Sustained Hospital Outbreak of MultiresistantAcinetobacter baumannii
Authors: Corbella Xavier , Montero Abelardo , Pujol Miquel , Domínguez M. Angeles , Ayats Josefina , Argerich M. José , Garrigosa Frederic , Ariza Javier , Gudiol Francesc
Published: December 2019

Beginning in 1992, a sustained outbreak of multiresistantAcinetobacter baumanniiinfections was noted in our 1,000-bed hospital in Barcelona, Spain, resulting in considerable overuse of imipenem, to which the organisms were uniformly susceptible. In January 1997, carbapenem-resistant (CR)A. baumanniistrains emerged and rapidly disseminated in the intensive care units (ICUs), prompting us to conduct a prospective investigation. It was an 18-month longitudinal intervention study aimed at the identification of the clinical and microbiological epidemiology of the outbreak and its response to a multicomponent infection control strategy. From January 1997 to June 1998, clinical samples from 153 (8%) of 1,836 consecutive ICU patients were found to contain CRA. baumannii. Isolates were verified to beA. baumanniiby restriction analysis of the 16S-23S ribosomal genes and the intergenic spacer region. Molecular typing by repetitive extragenic palindromic sequence-based PCR and pulsed-field gel electrophoresis showed that the emergence of carbapenem resistance was not by the selection of resistant mutants but was by the introduction of two new epidemic clones that were different from those responsible for the endemic. Multivariate regression analysis selected those patients with previous carriage of CRA. baumannii(relative risk [RR], 35.3; 95% confidence interval [CI], 7.2 to 173.1), those patients who had previously received therapy with carbapenems (RR, 4.6; 95% CI, 1.3 to 15.6), or those who were admitted into a ward with a high density of patients infected with CRA. baumannii(RR, 1.7; 95% CI, 1.2 to 2.5) to be at a significantly greater risk for the development of clinical colonization or infection with CRA. baumanniistrains. In accordance, a combined infection control strategy was designed and implemented, including the sequential closure of all ICUs for decontamination, strict compliance with cross-transmission prevention protocols, and a program that restricted the use of carbapenem. Subsequently, a sharp reduction in the incidence rates of infection or colonization withA. baumannii, whether resistant or susceptible to carbapenems, was shown, although an alarming dominance of the carbapenem-resistant clones was shown at the end of the study.
Rapid Detection of Contagious Caprine Pleuropneumonia Using a
Mycoplasma capricolum
subsp.
capripneumoniae
Capsular Polysaccharide-Specific Antigen Detection Latex Agglutination Test
Authors: March J. B. , Gammack C. , Nicholas R.
Published: December 2019

Latex microspheres (diameter, 8 μm) were coated with anti-
Mycoplasma capricolum
subsp.
capripneumoniae
polyclonal immunoglobulin G (IgG) antiserum (anti-F38 biotype). The coated microspheres, when used in a latex agglutination test (LAT), detected
M. capricolum
subsp.
capripneumoniae
antigen in the serum of goats with contagious caprine pleuropneumoniae (CCPP). Beads also agglutinated strongly in the presence of purified
M. capricolum
subsp.
capripneumoniae
capsular polysaccharide (CPS). Preabsorption of CPS-specific antibodies prior to coating of the beads removed agglutinating activity in the presence of
M. capricolum
subsp.
capripneumoniae
, strongly suggesting that CPS is the likely soluble antigen recognized by the test. In addition, the specificity of the LAT exactly mirrored that of an
M. capricolum
subsp.
capripneumoniae
CPS-specific monoclonal antibody (WM25): of the 8 other mycoplasma species tested, agglutination was observed only with bovine serogroup 7. The LAT detected all 11 strains of
M. capricolum
subsp.
capripneumoniae
examined in this study, with a sensitivity level of 2 ng of CPS, or the equivalent of 1.7 × 10
4
CFU, in a reaction volume of 0.03 ml of serum. With field sera from goats with CCPP, the results of the LAT exhibited a 67% correlation with the results of the currently used complement fixation test (CFT), with the main discrepancy in diagnosis resulting from the increased sensitivity of the LAT compared to that of CFT. This antigen-detection LAT should prove particularly useful in identifying animals in the earliest stages of CCPP and combines sensitivity and low cost with ease of application in the field, without the need for any specialist training or equipment.
Production of Bovine Herpesvirus Type 1-Seronegative Latent Carriers by Administration of a Live-Attenuated Vaccine in Passively Immunized Calves
Authors: Lemaire Mylène , Meyer Gilles , Baranowski Eric , Schynts Frédéric , Wellemans Guy , Kerkhofs Pierre , Thiry Etienne
Published: December 2019

The consequences of the vaccination of neonatal calves with the widely used live-attenuated temperature-sensitive (
ts
) bovine herpesvirus type 1 (BHV-1) were investigated. The
ts
strain established acute and latent infections in all vaccinated calves either with or without passive immunity. Four of seven calves vaccinated under passive immunity became clearly BHV-1 seronegative by different serological tests, as did uninfected control calves after the disappearance of maternal antibodies, and they remained so for long periods. A cell-mediated immune response was detected by a BHV-1 gamma interferon assay, but this test failed to detect the seronegative latent carriers (SNLCs). While they are not detected, SNLCs represent a threat for BHV-1-free herds or countries. This study demonstrates that SNLCs can be easily obtained by inoculation with a live-attenuated BHV-1 under passive immunity and that latent carrier animals without any antibody do exist. Consequently, this situation could represent a good model to experimentally produce SNLCs.
Evaluation of a New Culture Medium for
Borrelia burgdorferi
Authors: Marques Adriana R. , Stock Frida , Gill Vee
Published: December 2019

We evaluated the new MPM medium for the growth of
Borrelia burgdorferi
. All 18 blood samples from 17 patients with Lyme disease were negative. Growth studies showed that by day 4, most organisms in MPM were not viable. Our results reinforce the use of BSK medium as the primary choice for growing
B. burgdorferi.
Improved Pulsed-Field Gel Electrophoresis for Typing Vancomycin-Resistant Enterococci
Authors: Turabelidze Durmishkhan , Kotetishvili Mamuka , Kreger Arnold , Morris J. Glenn , Sulakvelidze Alexander
Published: December 2019

A rapid protocol for subtyping vancomycin-resistant enterococci by pulsed-field gel electrophoresis is reported. The procedure is simple and potentially cost-effective and allows reproducible subtyping of the strains in approximately 1 day.
Quantitative Gram Stain Interpretation Criteria Used by Microbiology Laboratories in Alberta, Canada
Authors: Church Deirdre , Melnyk Elizabeth , Unger Barbara
Published: December 2019

Microbiology laboratories in Alberta, Canada, were surveyed to determine the quantitative interpretive criteria used to routinely read and report Gram stains. There was a wide variability in the quantitative reporting criteria cited for both cells and bacteria, with only 11 of 32 (34.4%) laboratories surveyed using the criteria recommended by the external proficiency-testing program. Lack of standardized criteria not only poses a problem in the grading of proficiency testing results but may also impact the quality of patient care.
Is
Chlamydia pneumoniae
Present in Brain Lesions of Patients with Multiple Sclerosis?
Authors: Hammerschlag Margaret R. , Ke Zheng , Lu Fengmin , Roblin Patricia , Boman Jens , Kalman Bernadette
Published: December 2019

We investigated the presence of
Chlamydia pneumoniae
in 81 normal and pathological specimens obtained from postmortem brain tissues of patients with multiple sclerosis and with other neurological or nonneurological diseases. The assays used included PCR amplification of all DNA samples in the initial study. Culture and a second PCR amplification of the organism in a subset of 19 brain specimens were also performed in two separate laboratories. All results were negative. Thus, this study on a large number of brain tissues suggests that
C. pneumoniae
is not involved in inflammatory demyelination.
Acinetobacter baumannii-Infected Vascular Catheters Collected from Horses in an Equine Clinic
Authors: Vaneechoutte Mario , Devriese Luc A. , Dijkshoorn Lenie , Lamote Benedicte , Deprez Piet , Verschraegen Gerda , Haesebrouck Freddy
Published: December 2019

Acinetobacter baumanniiwas isolated from tips clipped from seven intravenous jugular catheters collected from horses in the Ghent University equine clinic. They originated from seven different horses. Three of the seven showed evidence of local infection.
Citrobacter rodentium
, the Causative Agent of Transmissible Murine Colonic Hyperplasia, Exhibits Clonality: Synonymy of
C. rodentium
and Mouse-Pathogenic
Escherichia coli
Authors: Luperchio Steven A. , Newman Joseph V. , Dangler Charles A. , Schrenzel Mark D. , Brenner Don J. , Steigerwalt Arnold G. , Schauer David B.
Published: December 2019

Citrobacter rodentium
(formerly
Citrobacter freundii
biotype 4280 and
Citrobacter
genomospecies 9) was described on the basis of biochemical characterization and DNA-DNA hybridization data and is the only
Citrobacter
species known to possess virulence factors homologous to those of the human pathogens enteropathogenic
Escherichia coli
and enterohemorrhagic
E. coli
. These virulence factors are encoded on the locus of enterocyte effacement (LEE), a pathogenicity island required for the characteristic attaching and effacing (AE) pathology seen in infection with these three pathogens.
C. rodentium
, which apparently infects only mice, provides a useful animal model for studying the molecular basis of AE pathology. No work has been done to assess differences in pathogenicity between
C. rodentium
isolates from diverse sources. Here, we report the examination of 15
C. rodentium
isolates using a battery of genetic and biochemical approaches. No differences were observed between the isolates by repetitive-element sequence-based PCR analysis, biochemical analysis, and possession of LEE-specific virulence factors. These data suggest that members of the species are clonal. We further characterized an atypical
E. coli
strain from Japan called mouse-pathogenic
E. coli
(MPEC) that, in our hands, caused the same disease as
C. rodentium
. Applying the same battery of tests, we found that MPEC possesses LEE-encoded virulence factors and is indistinguishable from the previously characterized
C. rodentium
isolate DBS100. These results demonstrate that MPEC is a misclassified
C. rodentium
isolate and that members of this species are clonal and represent the only known attaching and effacing bacterial pathogen of mice.
Evaluation of Nucleic Acid Amplification Tests as Reference Tests for
Chlamydia trachomatis
Infections in Asymptomatic Men
Authors: Johnson Robert E. , Green Timothy A. , Schachter Julius , Jones Robert B. , Hook Edward W. , Black Carolyn M. , Martin David H. , St. Louis Michael E. , Stamm Walter E.
Published: December 2019

Urine ligase chain reaction (LCR) and PCR tests and urethral swab culture were compared for their abilities to detect
Chlamydia trachomatis
infection in 3,639 asymptomatic men by using one-, two-, and three-test reference standards. Frozen urine at four of five participating centers was also tested by a transcription-mediated amplification assay which was used as a reference test. LCR increased the yield of positive results by 27% and PCR increased the yield of positive results by 26% over the yield of positive results by culture (
n
= 295). LCR and PCR sensitivities were similar, ranging from 80.4 to 93.5%, depending on the reference standard. Culture sensitivity was substantially less. A multiple-test standard yielded LCR, PCR, and culture specificities of 99.6%, with or without discrepant analysis. Test performance varied among centers partly due to different interpretations of the testing protocols. The study confirms that urine LCR and PCR for the detection of
C. trachomatis
have substantially improved sensitivities over that of urethral swab culture for testing of asymptomatic men, enabling screening of this important target group. These tests, perhaps in combination, are also candidate reference tests for the conduct of test evaluation studies.