bluuebunny/crossref_metadata_2025 huggingface dataset:
Amplification of Reiterated Sequences of Herpes Simplex Virus Type 1 (HSV-1) Genome To Discriminate between Clinical HSV-1 Isolates
Authors: Maertzdorf J. , Remeijer L. , Van Der Lelij A. , Buitenwerf J. , Niesters H. G. M. , Osterhaus A. D. M. E. , Verjans G. M. G. M.
Published: December 2019

Herpes simplex virus type 1 (HSV-1)-related disease ranges from a localized, self-limiting illness to fatal disease in immunocompromised individuals. The corneal disease herpetic keratitis may develop after reactivation of a latent virus or reinfection with an exogenous herpesvirus. Molecular analysis of the virus involved may allow distinction between these two options. The HSV-1 genome contains several hypervariable regions that vary in numbers of reiterating regions (reiterations I to VIII [ReI to ReVIII]) between individual strains. Twenty-four HSV-1 clones, derived by subcloning of HSV-1 (strain F) twice in limiting dilutions, were tested in a PCR-based assay to analyze the stabilities of ReI, ReIII, ReIV, and ReVII. ReI and ReIII proved to vary in size upon subcloning, whereas ReIV and ReVII were stable. Subsequently, 37 unrelated isolates and 10 sequential isolates from five patients, all with HSV-1-induced keratitis, were genotyped for ReIV and ReVII. Of the 37 unrelated samples, 34 (92%) could be discriminated, while the genotypes of the viruses in sequential samples were identical for each individual. Conclusively, the data show that the approach presented allows the rapid and accurate discrimination of HSV-1 strains in studies that address the transmission and pathogenesis of HSV-1 infections.
Detection of Decreased Fluoroquinolone Susceptibility in Salmonellas and Validation of Nalidixic Acid Screening Test
Authors: Hakanen Antti , Kotilainen Pirkko , Jalava Jari , Siitonen Anja , Huovinen Pentti
Published: December 2019

We evaluated 1,010
Salmonella
isolates classified as fluoroquinolone susceptible according to the National Committee for Clinical Laboratory Standards guidelines for susceptibility to nalidixic acid and three fluoroquinolones. These isolates were divided into two distinct subpopulations, with the great majority (
n
= 960) being fully ciprofloxacin susceptible and a minority (
n
= 50) exhibiting reduced ciprofloxacin susceptibility (MICs ranging between 0.125 and 0.5 μg/ml). The less ciprofloxacin-susceptible isolates were uniformly resistant to nalidixic acid, while only 12 (1.3%) of the fully susceptible isolates were nalidixic acid resistant. A similar association was observed between resistance to nalidixic acid and decreased susceptibility to ofloxacin or norfloxacin. A mutation of the
gyrA
gene could be demonstrated in all isolates for which the ciprofloxacin MICs were ≥0.125 μg/ml and in 94% of the nalidixic acid-resistant isolates but in none of the nalidixic acid-susceptible isolates analyzed. Identification of nalidixic acid resistance by the disk diffusion method provided a sensitivity of 100% and a specificity of 87.3% as tools to screen for isolates for which the MICs of ciprofloxacin were ≥0.125 μg/ml. We regard it as important that microbiology laboratories endeavor to recognize these less susceptible
Salmonella
strains, in order to reveal their clinical importance and to survey their epidemic spread.
Genotypic and Phenotypic Relationships between Clinical and Environmental Isolates of
Stenotrophomonas maltophilia
Authors: Berg Gabriele , Roskot Nicolle , Smalla Kornelia
Published: December 2019

While the gram-negative bacterium
Stenotrophomonas maltophilia
is used in biotechnology (e.g., for biological control of plant pathogens and for bioremediation), the number of
S. maltophilia
diseases in humans has dramatically increased in recent years. A total of 40
S. maltophilia
isolates from clinical and environmental sources (plant associated and water) was investigated to determine the intraspecies diversity of the group and to determine whether or not the strains could be grouped based on the source of isolation. The isolates were investigated by phenotypic profiling (enzymatic and metabolic activity and antibiotic resistance patterns) and by molecular methods such as temperature-gradient gel electrophoresis of the 16S rRNA gene fragment, PCR fingerprinting with BOX primers, and pulsed-field gel electrophoresis (PFGE) after digestion with
Dra
I. Results of the various methods revealed high intraspecies diversity. PFGE was the most discriminatory method for typing
S. maltophilia
when compared to the other molecular methods. The environmental strains of
S. maltophilia
were highly resistant to antibiotics, and the resistance profile pattern of the strains was not dependent on their source of isolation. Computer-assisted cluster analysis of the phenotypic and genotypic features did not reveal any clustering patterns for either clinical or environmental isolates.
Comparison of the OptiMAL Test with PCR for Diagnosis of Malaria in Immigrants
Authors: Iqbal Jamshaid , Sher Ali , Hira Parsotam R. , Al-Owaish Rashed
Published: December 2019

The OptiMAL test (Flow Inc., Portland, Oreg.), which detects a malaria parasite lactate dehydrogenase (pLDH) antigen, has not been evaluated for its sensitivity in the diagnosis of malaria infection in various epidemiological settings. Using microscopy and a PCR as reference standards, we performed a comparison of these assays with the OptiMAL test for the detection of
Plasmodium falciparum
and
Plasmodium vivax
infection in 550 immigrants who had come from areas where malaria is endemic to reside in Kuwait, where malaria is not endemic. As determined by microscopy, 125 (23%) patients had malaria, and of these, 84 (67%) were infected with
P. vivax
and 36 were infected with
P. falciparum
; in 5 cases the parasite species could not be determined due to a paucity of the parasites. The PCR detected malaria infection in 145 (26%) patients; 102 (70%) of the patients had
P. vivax
infection and 43 had
P. falciparum
infection. Of the five cases undetermined by microscopy, the PCR detected
P. falciparum
infection in two cases,
P. vivax
infection in two cases, and mixed (
P. falciparum
plus
P. vivax
) infection in one case. Correspondingly, the OptiMAL test detected malaria infection in 95 patients (17%); of these, 70 (74%) had
P. vivax
infection and 25 were infected with
P. falciparum
. In this study, 61 (49%) of the 125 malaria cases, as confirmed by microscopy, had a degree of parasitemia of <100 parasites per μl, and 23 (18%) of the cases had a degree of <50 parasites per μl. Our results show that the sensitivity of the OptiMAL test is high (97%) at a high level of parasitemia (>100 parasites/μl) but drops to 59% when the level is <100 parasites/μl and to 39% when it is <50 parasites/μl. In addition, the OptiMAL test failed to identify four patients whose blood smears contained
P. falciparum
gametocytes only. We conclude that the sensitivity and specificity of the OptiMAL test are comparable to those of microscopy in detecting malaria infection at a parasitemia level of >100 parasites/μl; however, the test failed to identify more than half of the patients with a parasitemia level of <50 parasites/μl. Thus, the OptiMAL test should be used with great caution, and it should not replace conventional microscopy in the diagnosis of malaria infection.
Ability of the Digene Hybrid Capture II Test To Identify
Chlamydia trachomatis
and
Neisseria gonorrhoeae
in Cervical Specimens
Authors: Schachter Julius , Hook Edward W. , McCormack William M. , Quinn Thomas C. , Chernesky Max , Chong Sylvia , Girdner Jennifer I. , Dixon Paula B. , DeMeo Lynette , Williams Eva , Cullen Allison , Lorincz Attila
Published: December 2019

The Digene Hybrid Capture II (HCII CT/GC) test is a combination test designed to detect
Chlamydia trachomatis
and
Neisseria gonorrhoeae
in a single specimen. It is a nucleic acid hybridization test which uses signal amplification to increase sensitivity. We compared its performance to that of culture on cervical specimens from 1,370 women. Direct fluorescent-antibody assay was used to resolve discrepant results for
C. trachomatis
. Samples were collected with a proprietary cervical brush or with endocervical swabs. The HCII CT/GC test proved to be sensitive and specific in detecting these organisms. Compared to
N. gonorrhoeae
culture, it had a sensitivity of 93% (87/94) and a specificity of 98.5% (1,244/1,263). Compared to
C. trachomatis
culture, the sensitivity was 97.7% (129/132) and specificity was 98.2% (1,216/1,238). Testing of some specimens with discrepant results by PCR suggested that the test would actually prove to be even more specific if it were compared to a nucleic acid amplification test (NAAT). The sensitivity of
C. trachomatis
culture was somewhat less, at 88.6% (117/132). The endocervical brush appeared to be better than Dacron swabs for collecting specimens. The HCII CT/GC test offers an attractive format that allows simultaneous detection of
C. trachomatis
and
N. gonorrhoeae
with a single specimen. An initial positive result is followed by repeat tests with probes to identify chlamydiae or gonococci. This test is more sensitive than
C. trachomatis
culture and is at least as sensitive as culture for gonococci. It deserves further evaluation and comparison with NAATs and may well offer an attractive alternative for diagnosis and screening of these infections.
Detection of Human Herpesvirus 6 by Reverse Transcription-PCR
Authors: Norton Renee A. , Caserta Mary T. , Hall Caroline Breese , Schnabel Kenneth , Hocknell Peter , Dewhurst Stephen
Published: December 2019

The role of human herpesvirus 6 (HHV-6) in disease beyond primary infection remains unclear. We have developed and validated a new reverse transcription-PCR (RT-PCR) assay for HHV-6 that can determine the presence of HHV-6 in clinical specimens and differentiate between latent and replicating virus. Peripheral blood mononuclear cells from 109 children were evaluated for HHV-6 by RT-PCR, DNA PCR, and viral culture. Of these samples, 106 were suitable for analysis. A total of 20 samples were positive for HHV-6 by culture and DNA PCR, of which 19 were positive by RT-PCR (sensitivity, 95%). All 28 samples from children that were negative by viral culture, but positive by DNA PCR, were negative for viral transcripts by our RT-PCR assay. One positive RT-PCR result was observed in 56 samples that were negative by tissue culture and DNA PCR. This indicates a low rate of false-positive results (1.2%) and a specificity of 98.8%. This RT-PCR assay can reliably differentiate between latent and actively replicating HHV-6 and should allow insight into the pathogenesis of this ubiquitous virus.
Phenotypic and Genotypic Heterogeneity among Cultivable Pathogen-Related Oral Spirochetes and
Treponema vincentii
Authors: Riviere G. R. , Smith K. S. , Willis S. G. , Riviere K. H.
Published: December 2019

Recent findings challenge the assumption that pathogen-related oral spirochetes (PROS) are related to
Treponema pallidum. Treponema vincentii
, grown in OMIZ-Pat media, cross-reacted with monoclonal antibody H9-2 against
T. pallidum
, and cultivable PROS had 16S rRNA gene sequences similar to those of
T. vincentii
(C.-B. Choi, C. Wyss, and U. B. Göbel. J. Clin. Microbiol. 34:1922–1925, 1996). Aims of the present study were to determine whether antigen phenotypes of oral treponemas were influenced by growth conditions and to evaluate the genetic relatedness of cultivable PROS to
T. pallidum
and
T. vincentii
. Results show that three
T. pallidum
monoclonal antibodies (H9-1, H9-2, and F5) cross-reacted with whole cells from four
Treponema
species grown in modified OMIZ-Pat medium, but not with treponemas grown in NOS medium. Only H9-2 reacted in immunoblots with reduced proteins from cultivable PROS and
T. vincentii
. Three of five PROS isolates were amplified by
T. vincentii
-specific PCR, and one was amplified by
Treponema medium
-specific PCR. None were amplified by
T. pallidum
-specific PCR. Three of five PROS isolates had 16S ribosomal DNA restriction fragment length polymorphism patterns identical to that of
T. vincentii
, and the patterns of two isolates resembled that of
T. medium
. Arbitrarily primed-PCR profiles from whole genomic DNA were distinct among five PROS isolates and two
T. vincentii
strains. Thus, PROS isolates represent a heterogeneous group of treponemas that share some 16S rRNA gene sequences with
T. vincentii
and
T. medium
, but not with
T. pallidum
. It is proposed that the PROS nomenclature be dropped.
Field Evaluation of the Determine Rapid Human Immunodeficiency Virus Diagnostic Test in Honduras and the Dominican Republic
Authors: Palmer Carol J. , Dubon Jose M. , Koenig Ellen , Perez Eddy , Ager Arba , Jayaweera Dushyantha , Cuadrado Raul R. , Rivera Ada , Rubido Alex , Palmer Dennis A.
Published: December 2019

Rapid detection of human immunodeficiency virus (HIV) infection can result in improved patient care and/or faster implementation of public health preventive measures. A new rapid test, Determine (Abbott, Abbott Park, Ill.), detects HIV type 1 (HIV-1) and HIV-2 antibodies within 15 min by using 50 μl of serum or plasma. No specialized equipment or ancillary supplies are required, and results are read visually. A positive result is noted by the appearance of a red line. An operational control (red line) indicates proper test performance. We evaluated the Determine rapid HIV detection test with a group of well-characterized serum samples (CD4 counts and viral loads were known) and serum samples from HIV-positive individuals at field sites in Honduras and the Dominican Republic. In the field evaluations, the results obtained by the Determine assay were compared to those obtained by local in-country HIV screening procedures. We evaluated serum from 100 HIV-positive patients and 66 HIV-negative patients. All samples gave the expected results. In a companion study, 42 HIV-positive samples from a Miami, Fla., serum bank were tested by the Determine assay. The samples had been characterized in terms of CD4 counts and viral loads. Fifteen patients had CD4 counts <200 cells/mm
3
, while 27 patients had CD4 counts >200 cells/mm
3
. Viral loads ranged from 630 to 873,746 log
10
copies/ml. All samples from the Miami serum bank were positive by the Determine test. Combined results from the multicenter studies indicated that the correct results were obtained by the Determine assay for 100% (142 of 142) of the HIV-positive serum samples and 100% (66 of 66) of the HIV-negative serum samples. The Determine test was simple to perform and the results were easy to interpret. The Determine test provides a valuable new method for the rapid identification of HIV-positive individuals, especially in developing countries with limited laboratory infrastructures.
Similar Signature of the Prion Protein in Natural Sheep Scrapie and Bovine Spongiform Encephalopathy-Linked Diseases
Authors: Baron Thierry G. M. , Madec Jean-Yves , Calavas Didier
Published: December 2019

It has been suggested that specific molecular features could characterize the protease-resistant prion protein (PrP
res
) detected in animal species as well as in humans infected by the infectious agent strain that causes bovine spongiform encephalopathy (BSE). Studies of glycoform patterns in such diseases in French cattle and cheetahs, as well as in mice infected by isolates from both species, revealed this characteristic molecular signature. Similar studies of 42 French isolates of natural scrapie, from 21 different flocks in different regions of France, however, showed levels of the three glycoforms comparable to those found in BSE-linked diseases. Moreover, the apparent molecular size of the unglycosylated form was also indistinguishable among all different sheep isolates, as well as isolates from BSE in cattle. Overall results suggest that scrapie cases with features similar to those of BSE could be found more frequently in sheep than previously described.
Comparison of MB/BacT and BACTEC 460 TB Systems for Recovery of Mycobacteria in a Routine Diagnostic Laboratory
Authors: Roggenkamp Andreas , Hornef Mathias W. , Masch Adelheid , Aigner Bettina , Autenrieth Ingo B. , Heesemann Jürgen
Published: December 2019

MB/BacT, BACTEC 460 TB, and Löwenstein-Jensen (LJ) medium were evaluated in parallel for recovery of mycobacteria from 3,700 continuous clinical specimens in a routine laboratory. Mycobacteria were identified from 123 (3.3%) specimens. The recovery rates for all mycobacteria by the different systems were 91.0, 73.0, and 53.6% for BACTEC 460 TB, MB/BacT, and LJ medium, respectively. The recovery rates for
Mycobacterium tuberculosis
complex were 97.1, 80.2, and 67.6%, respectively. The lack of sensitivity of the MB/BacT system was more pronounced with smear-negative specimens and resulted in a failure to detect three patients with infectious tuberculosis.
Molecular Markers Reveal Exclusively Clonal Reproduction inTrichophyton rubrum
Authors: Gräser Y. , Kühnisch J. , Presber W.
Published: December 2019

Genotypic variability among 96Trichophyton rubrumstrains which displayed different colony morphologies and were collected from four continents was investigated. Twelve markers representing 57 loci were analyzed by PCR fingerprinting, amplified fragment length polymorphism, and random amplified monomorphic DNA markers. Interestingly, none of the methods used revealed any DNA polymorphism, indicating a strictly clonal mode of reproduction and a strong adaptation to human skin.
Isolation of
Rickettsia prowazekii
from Blood by Shell Vial Cell Culture
Authors: Birg Marie-Laure , La Scola Bernard , Roux Véronique , Brouqui Philippe , Raoult Didier
Published: December 2019

A blood sample from a patient who returned from Algeria with a fever inoculated on human embryonic lung fibroblasts by the shell vial cell culture technique led to the recovery of
Rickettsia prowazekii
. The last clinical strain was isolated 30 years ago. Shell vial cell culture is a versatile method that could replace the classic animal and/or embryonated egg inoculation.
Fatal Case of
Trichoderma harzianum
Infection in a Renal Transplant Recipient
Authors: Guarro Josep , Antolín-Ayala María Isabel , Gené Josepa , Gutiérrez-Calzada Jesús , Nieves-Díez Carlos , Ortoneda Montserrat
Published: December 2019

We describe the second known case of human infection by
Trichoderma harzianum
. A disseminated fungal infection was detected in the postmortem examination of a renal transplant recipient and confirmed in culture. The only other reported infection by this fungus caused peritonitis in a diabetic patient. The in vitro antifungal susceptibilities of the clinical strain and three other strains of
Trichoderma
species to six antifungal drugs are provided. This case illustrates the widening spectrum of opportunistic
Trichoderma
spp. in immunocompromised patients and emphasizes the problems in diagnosing invasive fungal diseases.
Septicemia in Neutropenic Patients Infected with
Clostridium tertium
Resistant to Cefepime and Other Expanded-Spectrum Cephalosporins
Authors: Steyaert Sophia , Peleman Renaat , Vaneechoutte Mario , De Baere Thierry , Claeys Geert , Verschraegen Gerda
Published: December 2019

Clostridium tertium
was isolated from two immunocompromised patients with septicemia, fever, and gastrointestinal symptoms. The strains were resistant to ceftazidime, cefepime, and clindamycin; intermediately resistant to penicillin; and susceptible to metronidazole, quinolones, and vancomycin.
Identity of a Novel Swine Hepatitis E Virus in Taiwan Forming a Monophyletic Group with Taiwan Isolates of Human Hepatitis E Virus
Authors: Hsieh Sen-Yung , Meng Xiang-Jin , Wu Ying-Hua , Liu Shih-Tung , Tam Albert W. , Lin Dneg-Yn , Liaw Yun-Fan
Published: December 2019

Recently, we found that more than 10% of the cases of acute non-A, non-B, non-C hepatitis in Taiwan were caused by a novel strain of hepatitis E virus (HEV). Since none of these patients had a history of travel to areas where HEV is endemic, the source of transmission remains unclear. The recent discovery of a swine HEV in herd pigs in the United States has led us to speculate that HEV may also circulate in herd pigs in Taiwan and may serve as a reservoir for HEV in Taiwan. Of 275 herd pigs obtained from 10 pig farms in Taiwan, 102 (37%) were seropositive for serum anti-HEV immunoglobulin G (IgG). A 185-bp genomic sequence within the ORF-2 of the HEV genome was amplified and cloned from serum samples of an anti-HEV positive pig and subsequently from serum samples of a patient with acute hepatitis E. Sequence comparison revealed that the swine and human isolates of HEV share 97.3% identity. Phylogenetic analyses further showed that the Taiwan swine and human isolates of HEV form a distinct branch divergent from all other known strains of HEV, including the U.S. swine strain. To examine the potential risk of cross-species transmission of swine HEV to humans, the seroprevalences of anti-HEV IgG in 30 swine handlers, 20 pork dealers, and 50 control subjects were assessed and were found to be 26.7, 15, and 8%, respectively (for swine handlers versus controls,
P
= 0.048). Our findings may help provide an understanding of the modes of HEV transmission and may also raise potential public health concerns for HEV zoonosis.
Specific Detection of
Aspergillus
Species in Blood and Bronchoalveolar Lavage Samples of Immunocompromised Patients by Two-Step PCR
Authors: Skladny Heyko , Buchheidt Dieter , Baust Corinna , Krieg-Schneider Frank , Seifarth Wolfgang , Leib-Mösch Christine , Hehlmann Rüdiger
Published: December 2019

The increasing incidence of aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the currently limited diagnostic tools. We developed a two-step PCR assay that specifically amplifies a region of the 18S rRNA gene that is highly conserved in
Aspergillus
species. A number of primers with the least homology to equivalent human or
Candida
gene sequences were screened for the pairs that gave the highest sensitivity and specificity. No cross-reaction with the wide range of fungal and bacterial pathogens so far tested was observed. This assay allows direct and rapid detection of down to 10 fg of
Aspergillus
DNA corresponding to 1 to 5 CFU per ml of blood. A total of 315 blood and bronchoalveolar lavage samples from 140 subjects, including 93 patients at risk for invasive fungal disease, were screened. The result was a 100% correlation between positive histology, culture, or high-resolution computed tomography findings and PCR results. The test specificity was 89%. Our data point to the considerable potential clinical value of this simple, specific, rapid, and inexpensive PCR assay for improving the means of early diagnosis of systemic aspergillosis in high-risk patients.
Western and Dot Blotting Analyses ofEhrlichia chaffeensisIndirect Fluorescent-Antibody Assay-Positive and -Negative Human Sera by Using Native and RecombinantE. chaffeensisandE. canisAntigens
Authors: Unver Ahmet , Rikihisa Yasuko , Ohashi Norio , Cullman Louis C. , Buller Richard , Storch Gregory A.
Published: December 2019

Human monocytic ehrlichiosis is an emerging infectious disease caused byEhrlichia chaffeensis, a gram-negative obligatory intracellular bacterium closely related toE. canis. The immunoreactive recombinant fusion proteins rP28 and rP30 have become available after cloning and expressing of the 28- and 30-kDa major outer membrane protein genes ofE. chaffeensisandE. canis, respectively. Western immunoblotting was performed to analyze the antibody responses of the 37E. chaffeensisindirect fluorescent-antibody assay (IFA)-positive and 20 IFA-negative serum specimens with purified whole organisms, rP28, and rP30. All IFA-negative sera were negative with purified whole organisms, rP28, or rP30 by Western immunoblot analysis (100% relative diagnostic specificity). Of 37 IFA-positive sera, 34 sera reacted with any native proteins ofE. chaffeensisranging from 44 to 110 kDa, and 30 sera reacted with 44- to 110-kDa nativeE. canisantigens. The 28-kDaE. chaffeensisand 30-kDaE. canisnative proteins were recognized by 25 IFA-positive sera. Fifteen IFA-positive sera reacted with rP28 by Western blot analysis, whereas 34 IFA-positive sera reacted with rP30 (92% relative diagnostic specificity), indicating that rP30 is more sensitive than rP28 for detecting the antibodies in IFA-positive sera. These 34 IFA-positive sera were positive by the dot blot assay with rP30, distinguishing them from IFA-negative sera. Except for three rP30-negative but IFA-positive specimens that instead showed anE. ewingiiinfection-like profile by Western immunoblotting, the results of Western and dot blot assays with rP30 matched 100% with the IFA test results. Densitometric analysis of dot blot reactions showed a positive correlation between the dot density and the IFA titer. These results suggest that rP30 antigen would provide a simple, consistent, and rapid serodiagnosis for human monocytic ehrlichiosis.
Multiplex PCR for Detection and Typing of Porcine Circoviruses
Authors: Ouardani M. , Wilson L. , Jetté R. , Montpetit C. , Dea S.
Published: December 2019

Sets of oligonucleotide primers were designed according to the sequences of the open reading frames (ORFs) ORF1 and ORF2 of the prototype nonpathogenic PK-15 strain of porcine circovirus (PCV) type 1 (PCV-1). By the PCR performed with the various primer sets, genomic DNA or RNA from other bacterial or viral pathogens of the respiratory tracts of pigs could not be amplified. A positive amplification reaction could be visualized with DNA extracted from a viral suspension containing as few as 10 viral particles per ml. No DNA fragment could be amplified from lysates of continuous porcine cell lines (PT, ST, and PFT cells) known to be negative for PCV. When tested with clinical samples from pigs, the results of the single PCR method showed nearly 93% (13 of 14 samples) correlation with histopathological and immunohistochemical findings. Interestingly, subclinical PCV infections could be detected by single PCR with clinical samples that have been submitted from animals with irrelevant cases of respiratory and/or enteric problems. On the basis of the nucleotide sequences of PCV strains (PCV-2) recently associated with outbreaks of postweaning multisystemic wasting syndrome (PWMS) in Quebec, Canada, pig farms, other primers were designed from the PCV-1 genome, and these primers failed to amplify genomic fragments specific to the ORF1 or ORF2 genes of clinical isolates associated with PWMS but amplified DNA from the PCV-1 strain. Two rapid multiplex PCR (mPCR) methods have been developed to distinguish between both genotypes of PCV. By those two mPCR methods, (i) species-specific primer pairs were used to amplify a DNA fragment of 488 bp specific for the ORF2 genes of both genotypes, whereas a 375-bp fragment was amplified from the ORF1 gene of the PCV-1 strain only, or (ii) species-specific primer pairs were used to amplify a DNA fragment of 646 bp specific for the ORF1 genes of both genotypes, whereas a 425-bp fragment was amplified from the ORF2 gene of the PCV-1 strain only. By both mPCR methods, a PCV-2 infection was demonstrated in tissues of 94.2% (33 of 35) of the sick pigs tested, in agreement with previous findings showing the close association of this new genotype of PCV with outbreaks of PMWS in Europe and North America. On the other hand, a PCV-1 infection was confirmed in only 5.7% (2 of 35) of the pigs, and confirmation of a mixed infection with PCV-2 was obtained by a single PCR with PCV-2-specific primers.
Comparison of Four Clinical Specimen Types for Detection of Influenza A and B Viruses by Optical Immunoassay (FLU OIA Test) and Cell Culture Methods
Authors: Covalciuc Kristi A. , Webb Kenneth H. , Carlson Curtis A.
Published: December 2019

Although laboratory diagnosis of respiratory viruses has been widely studied, there is a relative insufficiency of literature examining the impact of specimen type on the laboratory diagnosis of influenza A and B. In a clinical study comparing the FLU OIA test with 14-day cell culture, clinical specimens from nasopharyngeal swabs, throat swabs, nasal aspirates, and sputum were obtained from patients experiencing influenza-like symptoms. A total of 404 clinical specimens were collected from 184 patients. Patients were defined as influenza positive if the viral culture of a specimen from any sample site was positive. Patients were defined as influenza negative if the viral cultures of specimens from all sample sites were negative. By this gold standard, culture and FLU OIA test results for each sample type were compared. For each of the four specimen types, the viral culture and FLU OIA test demonstrated equal abilities to detect the presence of influenza A or B virus or viral antigen. Sputum and nasal aspirate samples were the most predictive of influenza virus infection. Throat swabs were the least predictive of influenza virus infection, with both tests failing to detect influenza virus in nearly 50% of the throat samples studied.
Transmission Dynamics of Tuberculosis in a High-Incidence Country: Prospective Analysis by PCR DNA Fingerprinting
Authors: Haas W. H. , Engelmann G. , Amthor B. , Shyamba S. , Mugala F. , Felten M. , Rabbow M. , Leichsenring M. , Oosthuizen O. J. , Bremer H. J.
Published: December 2019

We have prospectively analyzed the DNA fingerprints of
Mycobacterium tuberculosis
strains from a random sample of patients with newly diagnosed tuberculosis in Windhoek, Namibia. Strains from 263 smear-positive patients in whom tuberculosis was diagnosed during 1 year were evaluated, and the results were correlated with selected epidemiological and clinical data. A total of 163 different IS
6110
fingerprint patterns were observed among the 263 isolates. Isolates from a high percentage of patients (47%) were found in 29 separate clusters, with a cluster defined as isolates with 100% matching patterns. The largest cluster included isolates from 39 patients. One predominant strain of
M. tuberculosis
caused 15% of cases of smear-positive pulmonary tuberculosis in Windhoek. That strain was also prevalent in the north of the country, suggesting that in contrast to other African countries with isolates with high levels of diversity in their DNA fingerprint patterns, only a restricted number of different strains significantly contribute to the tuberculosis problem in Namibia.
Confirmation of Low-Titer, Herpes Simplex Virus-Positive Specimen Results by the Enzyme-Linked Virus-Inducible System (ELVIS) Using PCR and Repeat Testing
Authors: Patel Navin , Kauffmann Lynn , Baniewicz Geri , Forman Michael , Evans Martin , Scholl David
Published: December 2019

The ELVIS HSV
Id
test kit (an enzyme-linked virus-inducible system) (Diagnostic Hybrids, Inc.) uses genetically engineered BHK cells to produce a detectable enzyme, beta-galactosidase, upon infection with either herpes simplex virus (HSV) type 1 (HSV-1) or HSV-2. Twenty six ELVIS-positive clinical specimens were selected for study by PCR and with monoclonal antibodies because they were originally low-titer HSV-positive specimens by ELVIS but HSV antibody nonreactive upon follow-up staining of the ELVIS monolayer. Twenty-one of 26 specimens were frozen, thawed, and retested with ELVIS without removing the cellular debris from the specimen; 18 were ELVIS positive and 3 were ELVIS negative on retesting. A typing result was provided upon retesting for 14 of 18 ELVIS-positive specimens (11 were HSV-1 and 3 were HSV-2) with HSV-specific monoclonal antibodies; no antibody signal was observed for 4 of 18 ELVIS-positive specimens. Sixteen of 26 specimens were subjected to blinded PCR analysis with two different primer sets, including all those that were repeat tested with ELVIS without success and those that had insufficient quantity for repeat testing. All 16 specimens analyzed were PCR positive with primer set 1; 15 of 16 were also positive with primer set 2, with the HSV type identified for all specimens (7 were HSV-1 and 8 were HSV-2). These results indicate that the original ELVIS result with these low-titer specimens was correct and further confirm the sensitivity and specificity of ELVIS HSV
Id
as a rapid, cell culture-based kit for the detection of HSV.
Sensitive and Specific Serodiagnosis of Lyme Disease by Enzyme-Linked Immunosorbent Assay with a Peptide Based on an Immunodominant Conserved Region ofBorrelia burgdorferiVlsE
Authors: Liang Fang Ting , Steere Allen C. , Marques Adriana R. , Johnson Barbara J. B. , Miller James N. , Philipp Mario T.
Published: December 2019

VlsE, the variable surface antigen ofBorrelia burgdorferi, contains an immunodominant conserved region named IR6. In the present study, the diagnostic performance of a peptide enzyme-linked immunosorbent assay (ELISA) based on a 26-mer synthetic peptide (C6) with the IR6sequence was explored. Sensitivity was assessed with serum samples (n= 210) collected from patients with clinically defined Lyme disease at the acute (early localized or early disseminated disease), convalescent, or late disease phase. The sensitivities for acute-, convalescent-, and late-phase specimens were 74% (29 of 39), 85 to 90% (34 of 40 to 35 of 39), and 100% (59 of 59), respectively. Serum specimens from early neuroborreliosis patients were 95% positive (19 of 20), and those from an additional group of patients with posttreatment Lyme disease syndrome yielded a sensitivity of 62% (8 of 13). To assess the specificity of the peptide ELISA, 77 serum samples from patients with other spirochetal or chronic infections, autoimmune diseases, or neurologic diseases and 99 serum specimens from hospitalized patients in an area where Lyme disease is not endemic were examined. Only two potential false positives from the hospitalized patients were found, and the overall specificity was 99% (174 of 176). Precision, which was assessed with a panel of positive and negative serum specimens arranged in blinded duplicates, was 100%. Four serum samples with very high anti-OspA antibody titers obtained from four monkeys given the OspA vaccine did not react with the C6peptide. This simple, sensitive, specific, and precise ELISA may contribute to alleviate some of the remaining problems in Lyme disease serodiagnosis. Because of its synthetic peptide base, it will be inexpensive to manufacture. It also will be applicable to serum specimens from OspA-vaccinated subjects.
Predominant
Staphylococcus aureus
Isolated from Antibiotic-Associated Diarrhea Is Clinically Relevant and Produces Enterotoxin A and the Bicomponent Toxin LukE-LukD
Authors: Gravet Alain , Rondeau Murielle , Harf-Monteil Colette , Grunenberger Fabienne , Monteil Henri , Scheftel Jean-Michel , Prévost Gilles
Published: December 2019

Staphylococcus aureus
was isolated as the predominant or only isolate from cultures of stools of 60 patients over 2 years in a university hospital, leading to the collection of 114 isolates. Diarrhea was observed in 90% of the patients. Ninety-eight percent of the patients had received antibiotics in the month before the diarrhea. Ninety-two percent of the
S. aureus
isolates were methicillin resistant.
S. aureus
was encountered with antibiotic-associated diarrhea among 47 quite elderly patients affected or not affected by a gastrointestinal disease. Among the antimicrobial treatments, cessation of the previous therapy when possible or rapid application of oral vancomycin therapy was the most appropriate. Analysis of total DNA by pulsed-field gel electrophoresis revealed 27 different
Sma
I pulsotypes distributed in 15 clusters. The pulsotypes never differed for related isolates from a single patient, even if they originated from patients with bacteremia.
S. aureus
was not isolated as the predominant isolate in cultures of stools of 57 patients who received an antimicrobial treatment for more than 5 days without diarrhea. Occurence of production of both enterotoxin A and the bicomponent leucotoxin LukE-LukD by the
S. aureus
isolates was significantly different from that by random isolates. The results strongly suggest that when predominant in stool samples,
S. aureus
should be considered a possible etiologic agent for some cases of antibiotic-associated diarrhea.
Performance Criteria of DNA Fingerprinting Methods for Typing ofHelicobacter pyloriIsolates: Experimental Results and Meta-Analysis
Authors: Burucoa Christophe , Lhomme Vincent , Fauchere Jean Louis
Published: December 2019

Typing systems are used to discriminate between isolates ofHelicobacter pylorifor epidemiological and clinical purposes. Discriminatory power and typeability are important performance criteria of typing systems. Discriminatory power refers to the ability to differentiate among unrelated isolates; it is quantitatively expressed by the discriminatory index (DI). Typeability refers to the ability of the method to provide an unambiguous result for each isolate analyzed; it is quantitatively expressed by the percentage of typeable isolates. We evaluated the discriminatory power and the typeability of the most currently used DNA fingerprinting methods for the typing ofH. pyloriisolates: ribotyping, PCR-based restriction fragment length polymorphism (PCR-RFLP) analysis, and random amplified polymorphism DNA (RAPD) analysis. Forty epidemiologically unrelated clinical isolates were selected to constitute a test population adapted to the evaluation of these performance criteria. A meta-analysis of typeability and discriminatory power was conducted retrospectively with raw data from published studies in which ribotyping, PCR-RFLP, RAPD, repetitive extragenic palindromic DNA sequence-based PCR (REP-PCR), or pulsed-field gel electrophoresis (PFGE) was used. Experimental results and the meta-analysis demonstrated the optimal typeability (100%) and the excellent discriminatory powers of PCR-based typing methods: RAPD analysis, DIs, 0.99 to 1; REP-PCR, DI, 0.99; and PCR-RFLP analysis, DIs, 0.70 to 0.97). Chromosome restriction-based typing methods (ribotyping and PFGE) are limited by a low typeability (12.5 to 75%) that strongly decreases their discriminatory powers: ribotyping, DI, 0.92; PFGE, DIs, 0.24 to 0.88. We do not recommend the use of ribotyping and PFGE for the typing ofH. pyloriisolates. We recommend the use of PCR-based methods.
Characteristics of
Streptococcus pyogenes
Serotype M1 and M3 Isolates from Patients in Japan from 1981 to 1997
Authors: Murase Toshiyuki , Suzuki Rieko , Osawa Ro , Yamai Shiro
Published: December 2019

Streptococcus pyogenes
isolates obtained in 1981 to 1997 from patients and healthy subjects were characterized by pulsed-field gel electrophoresis (PFGE) patterns, biotyping, and the presence of
spe
genes encoding streptococcal pyrogenic exotoxins. Changes in the profiles were shown in the serotype M1/T1 isolates from pharyngitis over this period, but not in serotype M3/T3 isolates. The characteristics of isolates from patients with toxic shock-like syndrome (TSLS) were comparable to those of the other isolates, including those from healthy subjects. This finding suggests that further phenotypic and molecular characterization, such as investigating the genomic difference represented by the pathogenicity island, of isolates with apparently the same profiles would be necessary to determine the etiology of diseases caused by
S. pyogenes
, including TSLS.
Unusual, High Genetic Diversity of Aleutian Mink Disease Virus
Authors: Olofsson Anders , Mittelholzer Christian , Treiberg Berndtsson Louise , Lind Lars , Mejerland Torbjörn , Belák Sándor
Published: December 2019

The genetic diversity of Aleutian mink disease virus (AMDV) was examined. Sequences obtained from 35 clinical samples were compared with five published sequences. An unusual, high genetic variability was revealed. Three phylogenetic subgroups of AMDV were identified, and the presence of more than one genotype at some farms was detected.
Rapid Identification of
Campylobacter
,
Arcobacter
, and
Helicobacter
Isolates by PCR-Restriction Fragment Length Polymorphism Analysis of the 16S rRNA Gene
Authors: Marshall Stephen M. , Melito Pasquale L. , Woodward David L. , Johnson Wendy M. , Rodgers Frank G. , Mulvey Michael R.
Published: December 2019

A rapid two-step identification scheme based on PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S rRNA gene was developed in order to differentiate isolates belonging to the
Campylobacter
,
Arcobacter
, and
Helicobacter
genera. For 158 isolates (26 reference cultures and 132 clinical isolates), specific RFLP patterns were obtained and species were successfully identified by this assay.
Molecular Epidemiology of a Cluster of Cases Due to
Klebsiella pneumoniae
Producing SHV-5 Extended-Spectrum β-Lactamase in the Premature Intensive Care Unit of a Hungarian Hospital
Authors: Szabó Dóra , Filetóth Zsolt , Szentandrássy Julianna , Némedi Mária , Tóth Erzsébet , Jeney Csaba , Kispál Gyula , Rozgonyi Ferenc
Published: December 2019

Fifteen nosocomial cases of extended-spectrum β-lactamase-producing
Klebsiella pneumoniae
occurred among 132 neonates in a premature intensive care unit in Hungary in June through November 1998. Fourteen strains were indistinguishable by molecular biological typing and harbored the same single conjugative extended-spectrum β-lactamase-encoding plasmid that was spontaneously found in a
Serratia marcescens
strain in the same patient.
Urinary Tract Infections Associated with Nontyphoidal
Salmonella
Serogroups
Authors: Abbott Sharon L. , Portoni Barbara A. , Janda J. Michael
Published: December 2019

In an analysis of over 23,000 nontyphoidal strains of
Salmonella
submitted to the Microbial Diseases Laboratory between 1992 and 1996, two groups (C
1
and E) were significantly recovered more often from the urinary tract than stool compared to more common groups such as B and D. An analysis of >60 urine isolates from 1996 suggests that most of these represent true urinary tract infections, as opposed to colonization or fecal contamination, by virtue of being isolated in pure culture and in high concentrations (>100,000 CFU/ml).
Characterization of Blood Culture Isolates of
Streptococcus dysgalactiae
subsp.
equisimilis
Possessing Lancefield's Group A Antigen
Authors: Brandt Claudia M. , Haase Gerhard , Schnitzler Norbert , Zbinden Reinhard , Lütticken Rudolf
Published: December 2019

For three human blood culture isolates of beta-hemolytic streptococci with Lancefield's serogroup A antigen, phylogenetic analysis of the 16S rRNA genes confirmed biochemical identification as
Streptococcus dysgalactiae
subsp.
equisimilis
. Genes encoding M or M-like proteins, which are considered to be major virulence determinants in streptococci, were detected in all of these strains. Our data clearly demonstrate that for beta-hemolytic streptococci, the species assignment should not be based on the results of serogrouping alone.
Quantitative Analysis of mRNA as a Marker for Viability of
Mycobacterium tuberculosis
Authors: Hellyer T. J. , DesJardin L. E. , Hehman G. L. , Cave M. D. , Eisenach K. D.
Published: December 2019

Numerous assays which use conserved DNA or rRNA sequences as targets for amplification have been described for the diagnosis of tuberculosis. However, these techniques have not been applied successfully to the monitoring of therapeutic efficacy owing to the persistence of amplifiable nucleic acid beyond the point at which smears and cultures become negative. Semiquantitative analysis of rRNA has been used to reduce the time required for antimicrobial susceptibility testing of
Mycobacterium tuberculosis
, although growth for up to 5 days in the presence of some drugs is still required to discriminate resistant strains. The purpose of the present study was to determine whether quantitative analysis of
M. tuberculosis
mRNA could be used to assess bacterial viability and to illustrate the application of this technique to rapid determination of drug susceptibility. Levels of mRNA encoding the 85B protein (α-antigen), IS
6110
DNA, and 16S rRNA were compared in parallel cultures of
M. tuberculosis
that were treated with either no drug, 0.2 μg of isoniazid per ml, or 1 μg of rifampin per ml. Exposure of sensitive strains to isoniazid or rifampin for 24 h reduced the levels of 85B mRNA to <4 and <0.01%, respectively, of those present in control cultures without drug. In contrast, the levels of IS
6110
DNA and 16S rRNA did not diminish over the same period. Strains which were resistant to either isoniazid or rifampin demonstrated no reduction in 85B mRNA in the presence of the drug to which they were nonresponsive. Quantitative analysis of 85B mRNA offers a potentially useful tool for the rapid determination of
M. tuberculosis
drug susceptibility and for the monitoring of therapeutic efficacy.
Usefulness of Spoligotyping in Molecular Epidemiology of
Mycobacterium bovis
-Related Infections in South America
Authors: Zumárraga Martín J. , Martin Carlos , Samper Sofia , Alito Alicia , Latini Omar , Bigi Fabiana , Roxo Eliana , Cicuta María Elena , Errico Francisco , Ramos Miguel Castro , Cataldi Angel , van Soolingen Dick , Romano María Isabel
Published: December 2019

Two hundred twenty-four
Mycobacterium bovis
isolates, mainly from South American countries, were typed by spoligotyping, and 41 different spoligotypes were identified. A total of 202
M. bovis
isolates (90%) were grouped into 19 different clusters. The largest cluster contained 96 isolates (42.8%) on the basis of the most frequently observed spoligotype, spoligotype 34. Nineteen
M. bovis
isolates from humans in Argentina had spoligotypes and polymorphic GC-rich repetitive sequence (PGRS) types that represented the most common types found among isolates from cattle. All five isolates from Uruguay and three of the six isolates from Paraguay had spoligotypes that were also detected for isolates from Argentina. The spoligotypes of isolates from Brazil, Costa Rica, and Mexico and of some of the isolates from Paraguay could not be found in Argentina. A total of 154
M. bovis
isolates were selected in order to compare the discriminative power of spoligotyping and restriction fragment length polymorphism (RFLP) analysis with direct repeat (DR) and PGRS probes. By spoligotyping, 31 different types were found, while
Alu
I-digested DR probe-associated RFLP analysis identified 42 types, and RFLP analysis with the PGRS probe also detected 42 types; these were partly independent of the DR types. By combining the results obtained by spoligotyping and by RFLP analysis with the DR and PGRS probes, 88 different types were obtained. Although the differentiation of
M. bovis
by spoligotyping was less discriminatory than differentiation by RFLP analysis with the DR and PGRS probes, spoligotyping is easier to perform and its results are easier to interpret. Therefore, for the purpose of typing of
M. bovis
isolates, spoligotyping could be performed first and the isolates could be grouped into clusters and then analyzed by RFLP analysis with the DR and PGRS probes.