bluuebunny/crossref_metadata_2025 huggingface dataset:
Simple and Inexpensive but Highly Discriminating Method for Computer-Assisted DNA Fingerprinting of
Pseudomonas aeruginosa
Authors: Al-Samarrai Taha H. , Zhang Ningxin , Lamont Iain L. , Martin Lois , Kolbe John , Wilsher Margaret , Morris Arthur J. , Schmid Jan
Published: December 2019

We describe here a method for computer-assisted fingerprinting of
Pseudomonas aeruginosa
. In this method, DNA is digested with
Sal
I, and bands with molecular sizes of ≥9.7 kb are visually scored after electrophoresis on agarose gels. Pattern scores are entered into a Microsoft Excel database. In scoring, the number of bands within each of a set of molecular size ranges is scored, rather than the absolute molecular size of each band, substantially enhancing the speed and reproducibility of the method, while eliminating the need for using expensive gel scanning equipment and software. Pattern scores are used to generate matrices of genetic distance values, which can be visualized in neighbor-joining trees. The method reliably distinguishes two epidemiologically unrelated isolates in 99.3% of all comparisons. The genetic relationships between isolates observed with the method were consistent with those obtained by analysis of two
P. aeruginosa
genes, indicating that it provides valid estimates of genetic divergence between isolates. Using the method, respiratory tract isolates from cystic fibrosis patients in Green Lane Hospital in Auckland, New Zealand, were shown to be genetically less diverse than epidemiologically unrelated isolates from other patients. This finding was not due to the existence of clusters of related strains specialized toward colonization of the respiratory tract and thus was indicative of transmission between patients. Analysis of multiple isolates from individual cystic fibrosis patients suggested that up to five separate clusters of genetically related strains may simultaneously be present in a patient. The method described should significantly enhance our ability to investigate the epidemiology of
P. aeruginosa.
Evaluation of Etest for Susceptibility Testing of Multidrug-Resistant Isolates of
Mycobacterium tuberculosis
Authors: Hazbón Manzour Hernando , del Socorro Orozco Maria , Labrada Luz Angela , Tovar Rafael , Weigle Kristen A. , Wanger Audrey
Published: December 2019

To prescribe effective treatment schemes for patients with tuberculosis, more-efficient susceptibility testing techniques for
Mycobacterium tuberculosis
are needed, especially in regions with multidrug resistance. Etest (AB BIODISK, Solna, Sweden) is a simple technique that provides quantitative drug susceptibility results for
M. tuberculosis
in 5 to 10 days from a culture grown at low cost. The performance of Etest was compared to that of the reference proportion method, using 95
M. tuberculosis
clinical isolates of which 42.1% (40 of 95) were resistant to at least one antibiotic by the reference method. Overall agreement between Etest and the reference method was 98.9% (94 of 95) for detection of multidrug resistance; for resistance to individual drugs, agreement was 97.9% (93 of 95) for rifampin, 96.0% (92 of 95) for ethambutol, 94.7% (90 of 95) for isoniazid, and 85.3% (81 of 95) for streptomycin. This study supports the utility of Etest for timely detection of drug resistance in
M. tuberculosis
and for use in tuberculosis control programs.
Evaluation of the Oxyrase OxyPlate Anaerobe Incubation System
Authors: Wiggs Lois S. , Cavallaro Joseph J. , Miller J. Michael
Published: December 2019

The Oxyrase OxyPlate anaerobe incubation system was evaluated for its ability to support the growth of clinically significant anaerobic bacteria previously identified by the Anaerobe Reference Laboratory at the Centers for Disease Control and Prevention. The results were compared with those obtained with conventional anaerobe blood agar plates incubated in an anaerobe chamber. We tested 251 anaerobic bacterial strains. Plates were read at 24, 48, and 72 h; growth was scored by a numerical coding system that combines the degree of growth and the colony size. Organisms (number of strains tested) used in this study were
Actinomyces
(32),
Anaerobiospirillum
(8),
Bacteroides
(39),
Campylobacter
(8),
Clostridium
(96),
Fusobacterium
(12),
Leptotrichia
(8),
Mobiluncus
(8),
Peptostreptococcus
(16), and
Propionibacterium
(24). At 24 h, 101 (40.2%) of the 251 strains tested showed better growth with the anaerobe chamber than with the OxyPlate system, 10 (4.1%) showed better growth with the OxyPlate system, and the remaining 140 (55.8%) showed equal growth with both systems. At 48 h, 173 (68.9%) showed equal growth with both systems, while 78 (31.1%) showed better growth with the anaerobe chamber. At 72 h, 176 (70.1%) showed equal growth with both systems, while 75 (29.9%) showed better growth with the anaerobe chamber. The OxyPlate system performed well for the most commonly isolated anaerobes but was inadequate for some strains. These results indicate that the Oxyrase OxyPlate system was effective in creating an anaerobic atmosphere and supporting the growth of anaerobic bacteria within 72 h. OxyPlates would be a useful addition to the clinical microbiology laboratory lacking resources for traditional anaerobic culturing techniques.
Detection of Carbapenemase-Producing
Acinetobacter baumannii
in a Hospital
Authors: Takahashi Ayako , Yomoda Sachie , Kobayashi Isao , Okubo Toyoji , Tsunoda Mitsuko , Iyobe Shizuko
Published: December 2019

Acinetobacter baumannii
strains resistant to both imipenem (IPM) and ceftazidime (CAZ) were isolated from 1994 through 1996 at Gunma University Hospital. Nine isolates from different inpatients were examined for carbapenem-hydrolyzing activity and for the carbapemase gene
bla
IMP
by the PCR method. All nine isolates were carbapenemase-producing strains that hydrolyzed IPM and that harbored
bla
IMP
. The
bla
IMP
gene was transmissible by conjugation to an IPM-susceptible recipient strain of
A. baumannii
and conferred resistance to IPM, CAZ, cefotaxime (CTX), ampicillin (AMP), and piperacillin (PIP). Either intermediate or high-level resistance to amikacin (AMK) was transferred from two and five strains, respectively, concomitantly with
bla
IMP
, and gentamicin (GEN) resistance was also transferred in one instance of high-level AMK resistance. Comparative examination of clinical isolates for resistance patterns to nine drugs, IPM, CAZ, CTX, aztreonam, AMP, PIP, AMK, GEN, and norfloxacin, in addition to pulsed-field gel electrophoresis patterns with
Not
I-digested genomic DNA, confirmed nosocomial transmission of infections involving carbapenemase-producing
A. baumannii
strains.
Comparison of Sorbitol MacConkey Agar and a Two-Step Method Which Utilizes Enzyme-Linked Immunosorbent Assay Toxin Testing and a Chromogenic Agar To Detect and Isolate Enterohemorrhagic
Escherichia coli
Authors: Novicki Thomas J. , Daly Judy A. , Mottice Susan L. , Carroll Karen C.
Published: December 2019

Enterohemorrhagic
Escherichia coli
(EHEC) and specifically serotype O157:H7 are a significant cause of hemorrhagic gastrointestinal disease and the hemolytic uremic syndrome. Methods currently used in clinical microbiology labs, such as sorbitol-MacConkey (SMAC) agar, reliably detect only O157:H7. We have evaluated a two-step method that has the potential to identify and isolate all EHEC serotypes, including serotype O157:H7. This method utilizes a chromogenic selective-differential medium for the isolation of
E. coli
together with an enzyme-linked immunosorbent assay (ELISA) that detects the Shiga-like toxins Stx1 and Stx2. Both are commercially available and usable in a wide range of clinical microbiology laboratories. Compared to a Vero cell cytotoxic assay, SMAC had sensitivities of 23.5% for the identification of all EHEC serotypes and of 50.0% for the identification of O157:H7 alone. The two-step method had sensitivities of 76.5 and 100%, respectively. The ELISA alone had a sensitivity of 82.4% in the detection of Stx1 and Stx2. The specificity was 100% in all cases. Overall, 14 EHEC isolates were obtained: 8 (58%) O157:H7, 2 (14%) O26, 2 (14%) O111:NM, 1 (7%) O103:H2, and 1 (7%) O121:H19. All but one were isolated during the months of May to September. The two-step method was found to be considerably more expensive than SMAC for both positive and negative samples.
Characterization of
Mycobacterium tuberculosis
Isolates from Patients in Houston, Texas, by Spoligotyping
Authors: Soini Hanna , Pan Xi , Amin Amol , Graviss Edward A. , Siddiqui Anees , Musser James M.
Published: December 2019

Mycobacterium tuberculosis
isolates (
n
= 1,429) from 1,283 patients collected as part of an ongoing population-based tuberculosis epidemiology study in Houston, Texas, were analyzed by spoligotyping and IS
6110
profiling. The isolates were also assigned to one of three major genetic groups on the basis of nucleotide polymorphisms located at codons 463 and 95 in the genes (
katG
and
gyrA
) encoding catalase-peroxidase and the A subunit of DNA gyrase, respectively. A total of 225 spoligotypes were identified in the 1,429 isolates. There were 54 spoligotypes identified among 713 isolates (
n
= 623 patients) assigned to 73 IS
6110
clusters. In addition, among 716 isolates (
n
= 660 patients) with unique IS
6110
profiles, 200 spoligotypes were identified. No changes were observed either in the IS
6110
profile or in the spoligotype for the 281 isolates collected sequentially from 133 patients. Five instances in which isolates with slightly different spoligotypes had the same IS
6110
profile were identified, suggesting that in rare cases isolates with different spoligotypes can be clonally related. Spoligotypes correlated extremely well with major genetic group designations. Only three very similar spoligotypes were shared by isolates from genetic groups 2 and 3, and none was shared by group 1 and group 2 organisms or by group 1 and group 3 organisms. All organisms belonging to genetic groups 2 and 3 failed to hybridize with spacer probes 33 to 36. Taken together, the results support the existence of three distinct genetic groups of
M. tuberculosis
organisms and provide new information about the relationship between IS
6110
profiles, spoligotypes, and major genetic groups of
M. tuberculosis
.
Sequence Variation in the
ftsZ
Gene of
Bartonella henselae
Isolates and Clinical Samples
Authors: Ehrenborg C. , Wesslén L. , Jakobson Å. , Friman G. , Holmberg M.
Published: December 2019

In a search for methods for subtyping of
Bartonella henselae
in clinical samples, we amplified and sequenced a 701-bp region in the 3′ end of the
ftsZ
gene in 15
B. henselae
isolates derived from cats and humans in the United States and Europe. The
ftsZ
sequence variants that were discovered were designated variants Bh
ftsZ
1, 2, and 3 and were compared with 16S rRNA genotypes I and II of the same isolates. There was no
ftsZ
gene variation in the strains of 16S rRNA type I, all of which were Bh
ftsZ
1. The type II strains constituted two groups, with nucleotide sequence variation in the
ftsZ
gene resulting in amino acid substitutions at three positions, one of which was shared by the two groups. One 16S rRNA type II isolate had an
ftsZ
gene sequence identical to those of the type I strains. Variants Bh
ftsZ
1 and 2 were detected in tissue specimens from seven Swedish patients with diagnoses such as chronic multifocal osteomyelitis, cardiomyopathy, and lymphadenopathy. Patients with similar clinical entities displayed either Bh
ftsZ
variant. The etiological role of
B. henselae
in these patients was supported by positive
Bartonella
antibody titers and/or amplification and sequencing of a part of the
B. henselae gltA
gene.
B. henselae ftsZ
gene sequence variation may be useful in providing knowledge about the epidemiology of various
B. henselae
strains in clinical samples, especially when isolation attempts have failed. This report also describes manifestations of atypical
Bartonella
infections in Sweden.
Use of Epidemiologically Well-Defined Subjects and Existing Immunofluorescence Assays To Calibrate a New Enzyme Immunoassay for Human Herpesvirus 8 Antibodies
Authors: Martin Jeffrey N. , Amad Zahwa , Cossen Cynthia , Lam Phung K. , Kedes Dean H. , Page-Shafer Kimberly A. , Osmond Dennis H. , Forghani Bagher
Published: December 2019

Agreement between assays for the detection of human herpesvirus 8 (HHV-8) antibodies has been limited. In part, this disagreement has been because assay calibration (i.e., differentiating positive from negative results) has not been done in a standardized fashion with reference to a wide spectrum of HHV-8-infected (true-positive) and HHV-8-uninfected (true-negative) persons. To describe the performance of an assay for HHV-8 antibodies more accurately, we used epidemiologically well-characterized subjects in conjunction with testing on two existing immunofluorescence assays for HHV-8 antibodies to define two groups: a group of 135 HHV-8-infected individuals (true positives), including Kaposi's sarcoma patients and those asymptomatically infected, and a group of 234 individuals with a high likelihood of being HHV-8 uninfected (true negatives). A new enzyme immunoassay (EIA), using lysed HHV-8 virion as the antigen target, was then developed. With the above true positives and true negatives as references, the sensitivity and specificity of the EIA associated with different cutoff values were determined. At the cutoff that maximized both sensitivity and specificity, sensitivity was 94% and specificity was 93%. When the EIA was used to test a separate validation group, a distribution of seropositivity that matched that predicted for the agent of Kaposi's sarcoma was observed: 55% of homosexual men were seropositive, versus 6% seropositivity in a group of children, women, and heterosexual men. It is proposed that the EIA has utility for large-scale use in a number of settings and that the calibration method described can be used for other assays, both to more accurately describe the performance of these assays and to permit more-valid interassay comparison.
Comparison of Three Assays for Cytomegalovirus Detection in AIDS Patients at Risk for Retinitis
Authors: Wattanamano Pornthep , Clayton John L. , Kopicko Jeffrey J. , Kissinger Patricia , Elliot Steven , Jarrott Christine , Rangan Setlur , Beilke Mark A.
Published: December 2019

The purpose of this study was to determine the sensitivity and specificity of three different methods of cytomegalovirus (CMV) detection for AIDS patients at risk for CMV retinitis. Patients with CD4
+
counts of <100/μl and negative baseline screening eye examinations were tested for CMV infection by (i) pp65 antigenemia expression in leukocytes, (ii) the Digene Hybrid Capture CMV DNA System, and (iii) the Roche Amplicor Qualitative PCR Test. The incidence of CMV retinitis in our study of 296 patients at the Medical Center of Louisiana—New Orleans HIV Outpatient Clinic was 7.2 per 100 person-years (a total of 20 episodes in 18 patients from April 1997 to February 1999). Receiver operating characteristic curves were calculated for each assay to determine optimal cutoff points which maximized the sensitivity and specificity of each assay. The sensitivities of the assays compared to the eye examinations were 80% for the pp65 antigenemia assay (cutoff, >0 cell per 1.5 × 10
5
leukocytes), 85% for the Digene assay (cutoff, 1,400 genome copies/ml of whole blood), and 60% for the Amplicor assay. The specificities of the assays were 84, 84, and 87%, respectively. The Digene assay with a cutoff of ≥1,400 genome copies/ml gave optimal sensitivity and specificity and was found to have predictive values equal to those of the more technically cumbersome antigenemia assay.
Differential Diagnosis of
Taenia saginata
and
Taenia solium
Infection by PCR
Authors: González Luis Miguel , Montero Estrella , Harrison Leslie J. S. , Parkhouse R. Michael E. , Garate Teresa
Published: December 2019

We have designed species-specific oligonucleotides which permit the differential detection of two species of cestodes,
Taenia saginata
and
Taenia solium
. The oligonucleotides contain sequences established for two previously reported, noncoding DNA fragments cloned from a genomic library of
T. saginata
. The first, which is
T. saginata
specific (fragment HDP1), is a repetitive sequence with a 53-bp monomeric unit repeated 24 times in direct tandem along the 1,272-bp fragment. From this sequence the two oligonucleotides that were selected (oligonucleotides PTs4F1 and PTs4R1) specifically amplified genomic DNA (gDNA) from
T. saginata
but not
T. solium
or other related cestodes and had a sensitivity down to 10 pg of
T. saginata
gDNA. The second DNA fragment (fragment HDP2; 3,954 bp) hybridized to both
T. saginata
and
T. solium
DNAs and was not a repetitive sequence. Three oligonucleotides (oligonucleotides PTs7S35F1, PTs7S35F2, and PTs7S35R1) designed from the sequence of HDP2 allowed the differential amplification of gDNAs from
T. saginata
,
T. solium
, and
Echinococcus granulosus
in a multiplex PCR, which exhibits a sensitivity of 10 pg.
Rapid Identification of Bacteria in Blood Cultures by Using Fluorescently Labeled Oligonucleotide Probes
Authors: Jansen G. J. , Mooibroek M. , Idema J. , Harmsen H. J. M. , Welling G. W. , Degener J. E.
Published: December 2019

The applicability of whole-cell hybridization for the identification of pathogenic bacteria in blood from septic patients was examined. Oligonucleotide probes, fluorescently labeled with fluorescein isothiocyanate, directed against the variable regions of the 16S rRNAs of the following bacterial species and/or genera were used:
Streptococcus
spp.,
Enterococcus faecalis
,
Staphylococcus aureus
, coagulase-negative staphylococci (CoNS),
Escherichia coli
,
Pseudomonas aeruginosa
, and the
Enterobacteriaceae
family. A probe specific for the rRNAs of almost all bacteria and its complementary, reversed counterpart was used as positive and negative control, respectively. The probes were used in conjunction with a fast and simple-to-use protocol for whole-cell hybridization. This protocol yields an identification after 25 to 45 min, depending on whether the bacterium is gram positive or gram negative. A total of 182 blood samples which tested positive in a blood culture machine were investigated. All probes except for the ones for
S. aureus
and the CoNS showed sensitivities and specificities of 1.000. It was concluded that whole-cell hybridization is well suited for the fast screening of septic blood containing streptococci and/or enterococci or gram-negative rods.
Fluorescent In Situ Hybridization Allows Rapid Identification of Microorganisms in Blood Cultures
Authors: Kempf Volkhard A. J. , Trebesius Karlheinz , Autenrieth Ingo B.
Published: December 2019

Using fluorescent in situ hybridization (FISH) with rRNA-targeted fluorescently labelled oligonucleotide probes, pathogens were rapidly detected and identified in positive blood culture bottles without cultivation and biotyping. In this study, 115 blood cultures with a positive growth index as determined by a continuous-reading automated blood culture system were examined by both conventional laboratory methods and FISH. For this purpose, oligonucleotide probes that allowed identification of approximately 95% of those pathogens typically associated with bacteremia were produced. The sensitivity and specificity of these probes were 100%. From all 115 blood cultures, microorganisms were grown after 1 day and identification to the family, genus, or species level was achieved after 1 to 3 days while 111 samples (96.5%) were similarly identified by FISH within 2.5 h. Staphylococci were identified in 62 of 62 samples, streptococci and enterococci were identified in 19 of 20 samples, gram-negative rods were identified in 28 of 30 samples, and fungi were identified in two of two samples. Thus, FISH is an appropriate method for identification of pathogens grown in blood cultures from septicemic patients.
Heterogeneous Vancomycin Resistance in Methicillin-Resistant
Staphylococcus aureus
Strains Isolated in a Large Italian Hospital
Authors: Marchese A. , Balistreri G. , Tonoli E. , Debbia E. A. , Schito G. C.
Published: December 2019

Of 179 methicillin-resistant
Staphylococcus aureus
strains isolated from 1997 to 1998, two strains (1.1%) gave subclones for which the vancomycin MICs were 8 mg/liter. Pulsed-field gel electrophoresis showed identical restriction patterns for both isolates, suggesting transfer of a single clone between two different patients.
Genetic Characterization of
Campylobacter jejuni
O:41 Isolates in Relation with Guillain-BarréSyndrome
Authors: Wassenaar Trudy M. , Fry Benjamin N. , Lastovica Albert J. , Wagenaar Jaap A. , Coloe Peter J. , Duim Birgitta
Published: December 2019

Campylobacter jejuni
O:41 strains are found in association with Guillain-Barré syndrome in South Africa. Strains of this serotype collected over 17 years were characterized by amplified fragment length polymorphism and flagellin typing to determine their clonal nature. Despite minor variation in GM1 expression, all of the strains were genetically indistinguishable, indicating that they are representative of a genetically stable clone.
Epidemiology of Glycopeptide-Resistant Enterococci Colonizing High-Risk Patients in Hospitals in Johannesburg, Republic of South Africa
Authors: von Gottberg Anne , van Nierop Wim , Dusé Adriano , Kassel Marlene , McCarthy Kerrigan , Brink Adrian , Meyers Marilyn , Smego Raymond , Koornhof Hendrik
Published: December 2019

Recent cases of infections caused by glycopeptide-resistant enterococci (GRE) have highlighted the emergence of these organisms in the Republic of South Africa. During May 1998 we conducted a prevalence study in four hospitals in Johannesburg and obtained 184 rectal swabs from patients identified as being at high risk for GRE colonization. Twenty enterococcal isolates showing various glycopeptide resistance genotypes were recovered: 3
Enterococcus faecium vanA
isolates, 10
E. faecium vanB
isolates, 6
E. gallinarum vanC1
isolates, and 1
E. avium vanA
isolate. Macrorestriction analysis was used to demonstrate the clonal spread of GRE strains within hospitals. Evidence also demonstrated the likely persistence of the original
E. faecium vanA
isolate associated with the first confirmed death contributed to by GRE infection in South Africa in March 1997.
Diagnostically and Experimentally Useful Panel of Strains from the
Burkholderia cepacia
Complex
Authors: Mahenthiralingam Eshwar , Coenye Tom , Chung Jacqueline W. , Speert David P. , Govan John R. W. , Taylor Peter , Vandamme Peter
Published: December 2019

Two new species,
Burkholderia multivorans
and
Burkholderia vietnamiensis
, and three genomovars (genomovars I, III, and IV) currently constitute the
Burkholderia cepacia
complex. A panel of 30 well-characterized strains representative of each genomovar and new species was assembled to assist with identification, epidemiological analysis, and virulence studies on this important group of opportunistic pathogens.
Comparative Evaluation of Two Branched-DNA Human Immunodeficiency Virus Type 1 RNA Quantification Assays with Lower Detection Limits of 50 and 500 Copies per Milliliter
Authors: Manegold Christoph , Krempe Catharina , Jablonowski Helmut , Kajala Lasse , Dietrich Manfred , Adams Ortwin
Published: December 2019

We have comparatively evaluated Quantiplex version 3.0 and version 2.0 on 133 plasma samples and a repetitive dilution series. Version 3.0 yielded higher human immunodeficiency virus RNA values, and the ratio of version 3.0 results to version 2.0 results decreased from 3.47 below 1,000 copies/ml to 1.97 above 50,000 copies/ml [linear regression, log (version 3.0) = 0.915 + 0.871 × log (version 2.0);
r
2
= 0.952].
Infection of a Hip Prosthesis by
Actinomyces naeslundii
Authors: Wüst Jürg , Steiger Ulrich , Vuong Hanh , Zbinden Reinhard
Published: December 2019

We present the case of a 77-year-old woman who developed an
Actinomyces naeslundii
infection of a hip prosthesis. The isolate grew well aerobically with 5% CO
2
. Possible diagnostic problems may arise in the microbiological laboratory because aerobic growth is not sufficiently accounted for in some of the traditional identification schemes and commercial test kits. Therefore, besides presenting an unusual pathogen in this setting, this report focuses on possible diagnostic problems in the microbiological laboratory.
Multiplex PCR for Detection of Genes for
Staphylococcus aureus
Enterotoxins, Exfoliative Toxins, Toxic Shock Syndrome Toxin 1, and Methicillin Resistance
Authors: Mehrotra Manisha , Wang Gehua , Johnson Wendy M.
Published: December 2019

A multiplex PCR assay for detection of genes for staphylococcal enterotoxins A to E (
entA
,
entB
,
entC
,
entD
, and
entE
), toxic shock syndrome toxin 1 (
tst
), exfoliative toxins A and B (
etaA
and
etaB
), and intrinsic methicillin resistance (
mecA
) was developed. Detection of
femA
was used as an internal positive control. The multiplex PCR assay combined the primers for
sea
to
see
and
femA
in one set and those for
eta
,
etb
,
tst
,
mecA
, and
femA
in the other set. Validation of the assay was performed using 176 human isolates of
Staphylococcus aureus
. This assay offers a very specific, quick, reliable, and inexpensive alternative to conventional PCR assays used in clinical laboratories to identify various staphylococcal toxin genes.
Evaluation of the Oricult-N Dipslide for Laboratory Diagnosis of Vaginal Candidiasis
Authors: Carlson Petteri , Richardson Malcolm , Paavonen Jorma
Published: December 2019

The Oricult-N semiquantitative dipslide (Orion Diagnostica, Espoo, Finland) was evaluated for the laboratory diagnosis of vaginal candidiasis. It was compared with broth culture (Vagicult; Orion Diagnostica). Oricult-N was positive for 14.5% of 124 symptomatic patients and 12% of 50 asymptomatic controls. The results for broth cultures were 17 and 22%, respectively. Thus, the test group and the control group did not differ significantly by either method. High vaginal yeast counts (≥10
5
CFU/ml) were detected by Oricult-N in 7% of patients and in 0% of controls, but both groups harbored low numbers of yeasts. An accurate quantitative cutoff point separating a level of yeast associated with infection from vaginal yeast carriage could not be defined in the study. Nevertheless, the easy semiquantitation allowed by the Oricult-N method could be helpful because, especially in low-count carriers of
Candida
, other potential causes of vaginal symptoms should be considered. The Oricult-N method was technically simple and could be applied in primary health care. Further studies are required, however, before Oricult-N can be recommended as a routine diagnostic tool.
Detection of “CandidatusHelicobacter suis” in Gastric Samples of Pigs by PCR: Comparison with Other Invasive Diagnostic Techniques
Authors: De Groote D. , Ducatelle R. , van Doorn L. J. , Tilmant K. , Verschuuren A. , Haesebrouck F.
Published: December 2019

Recently, a new 16S ribosomal DNA-based PCR assay was developed for the specific detection of “CandidatusHelicobacter suis” (former “Gastrospirillum suis”) in porcine gastric samples. In the present study, this PCR assay was compared to three other invasive diagnostic methods (rapid urease test, immunohistochemistry, histologic analysis by Giemsa staining). Antral stomach samples from 200 slaughterhouse pigs from Belgium and The Netherlands were examined. Bacterial presence was determined in 77% (154 of 200) of the samples by PCR in combination with Southern blot hybridization, 56% (111 of 200) of the samples by immunohistochemistry, 61% (122 of 200) of the samples by urease testing (20 h postinoculation [p.i.]), 36% (71 of 200) of the samples by urease testing (3 h p.i.), and 33% (65 of 200) of the samples by Giemsa staining. The intrinsic specificity of the PCR assay was assessed by Southern blot analysis with an “CandidatusH. suis”-specific probe and sequencing of PCR products. Interassay sensitivity and specificity values were assessed for each test by pairwise comparisons between tests. Agreement between tests was evaluated by calculating Cohen's kappa coefficient. From that analysis, the PCR assay was considered the most reliable benchmark. Microscopic detection of immunohistochemically labeled or Giemsa-stained “CandidatusH. suis” cells in stomach sections proved to be highly specific (100%) but relatively insensitive (72 and 42%, respectively) compared to the PCR assay. A longer incubation time of the urease test improved its sensitivity considerably (74 versus 55%) but was accompanied by a loss of specificity (72 versus 93%). In conclusion, we found the “CandidatusH. suis”-specific PCR assay to be a sensitive and reliable diagnostic method for the detection of “CandidatusH. suis” in the stomachs of pigs and could prove to be a valuable tool for further epidemiological studies both for “CandidatusH. suis”- and for “Helicobacter heilmannii” type 1-related research.
Comparison of Classic and Molecular Approaches for the Identification of Untypeable Enteroviruses
Authors: Oberste M. Steven , Maher Kaija , Flemister Mary R. , Marchetti George , Kilpatrick David R. , Pallansch Mark A.
Published: December 2019

Members of the familyPicornaviridaeare the most common viruses infecting humans, and species in several genera also infect a wide variety of other mammals. Picornaviruses have traditionally been classified by antigenic type, based on a serum neutralization assay. However, this method is time-consuming and labor-intensive, is sensitive to virus aggregation and antigenic variation, and requires a large number of antisera to identify all serotypes, even when antiserum pools are used. We developed generic reverse transcription (RT)-PCR primers that will amplify all human enterovirus serotypes, as well as many rhinoviruses and other picornaviruses, and used RT-PCR amplification of the VP1 gene and amplicon sequencing to identify enteroviruses that were refractory to typing by neutralization with pooled antisera. Enterovirus serotypes determined by sequencing were confirmed by neutralization with monospecific antisera. Of 55 isolates tested, 49 were of known enterovirus serotypes, two were rhinoviruses, and four were clearly picornaviruses but did not match any known picornavirus sequence. All four untyped picornaviruses were closely related to one another in sequence, suggesting that they are of the same serotype. RT-PCR, coupled with amplicon sequencing, is a simple and rapid method for the typing and classification of picornaviruses and may lead to the identification of many new picornavirus serotypes.
Plasmodium falciparum
Histidine-Rich Protein 2-Based Immunocapture Diagnostic Assay for Malaria: Cross-Reactivity with Rheumatoid Factors
Authors: Iqbal J. , Sher A. , Rab A.
Published: December 2019

Recently introduced rapid nonmicroscopic immunocapture assays for the diagnosis of malaria infection are being evaluated for their sensitivity and specificity in various epidemiological settings. A
Plasmodium falciparum
histidine-rich protein 2 (PfHRP-2)-based assay (ICT) and a
Plasmodium
-specific lactate dehydrogenase test (OptiMAL) were evaluated for their specificities in different groups of patients who tested negative for malaria infection by microscopy. The patients were selected from different disease groups: rheumatoid arthritis, hepatitis C, toxoplasmosis, schistosomiasis, and hydatid disease. One hundred thirty-three of the 225 patients were positive for rheumatoid factor. Thirty-five of the 133 (26%) rheumatoid factor-positive patients gave a false-positive reaction with the ICT assay, but only 4 of these gave false-positive reactions with the OptiMAL test. Thirty-three of the 35 false-positive specimens became negative when repeat tested with the ICT assay after absorbing out the rheumatoid factor activity. Our study shows that the PfHRP-2-based ICT assay gave a false-positive reaction in 26% of the patients who had rheumatoid factors, but were negative for malaria by microscopy.
Comparison of Two Commercial Methods for Measurement of Cytomegalovirus Load in Blood Samples after Renal Transplantation
Authors: Tong C. Y. William , Cuevas Luis E. , Williams Helen , Bakran Ali
Published: December 2019

A cohort of 77 renal transplant recipients was prospectively studied for comparison of two commercially available cytomegalovirus (CMV) load assays, i.e., the COBAS AMPLICOR CMV Monitor test (Amplicor), using plasma samples, and the Murex Hybrid Capture System (HCS), using whole blood. The manufacturer of the HCS assay changed the version of the test from 1.0 (HCS-1) to 2.0 (HCS-2) after the first 37 patients had been tested. Despite the differences in principle and type of specimen used, the Amplicor and HCS assays gave comparable results. The regression line correlating the HCS-1 assay to the Amplicor assay was similar to that correlating the HCS-2 assay to the Amplicor assay. The HCS results could be converted to Amplicor-equivalent units by using linear-regression equations [log
10
HCS-1 result = 0.49 (log
10
Amplicor result) + 2.58, and log
10
HCS-2 result = 0.61 (log
10
Amplicor result) + 2.18]. The HCS-2 assay appeared to have the lowest detection limit, followed by the Amplicor assay and then the HCS-1 assay. When a sliding scale of cutoff values in Amplicor-equivalent units (>1,000, >2,500, >6,000, >16,000, >40,000, and >100,000 copies/ml) was applied to diagnose CMV disease, similar patterns of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were observed with the Amplicor and HCS assays. A cutoff value of >40,000 copies/ml has a low sensitivity (Amplicor, 29.4%; HCS, 41.2%) but is specific (Amplicor, 96.7%; HCS, 93.3%) and can be used for the differential diagnosis of CMV disease (PPV, 71.4% [Amplicor] or 63.6% [HCS]; NPV, 82.9% [Amplicor] or 84.8% [HCS]). A lower cutoff value of >1,000 copies/ml improves the sensitivity (Amplicor, 76.5%; HCS, 82.4%) and has a high NPV (Amplicor, 91.8%; HCS, 94.2%) but, due to the low PPV (Amplicor, 46.2%; HCS, 56%), is useful only for exclusion of CMV disease.
Etiology of Sexually Transmitted Infections among Street-Based Female Sex Workers in Dhaka, Bangladesh
Authors: Rahman Motiur , Alam Ashraful , Nessa Khairun , Hossain Anowar , Nahar Shamsun , Datta Dilip , Alam Khan Shahnewaz , Amin Mian Ruhul , Albert M. John
Published: December 2019

An etiological study of sexually transmitted infections (STIs) was conducted among female sex workers (FSWs) in Dhaka, Bangladesh. Endocervical swab and blood samples from 269 street-based FSWs were examined for
Neisseria gonorrhoeae
,
Chlamydia trachomatis
, and
Trichomonas vaginalis
as well as for antibodies to
Treponema pallidum
and herpes simplex virus 2 (HSV-2). Sociodemographic data and data regarding behavior were also collected. A total of 226 of the 269 FSWs (84%) were positive for the STI pathogens studied. Among the 269 FSWs, 35.5% were positive for
N. gonorrhoeae
, 25% were positive for
C. trachomatis
, 45.5% were positive for
T. vaginalis
, 32.6% were seropositive for
T. pallidum
, 62.5% were seropositive for HSV-2, and 51% had infections with two or more pathogens.
Molecular Characterization of
Brucella
Strains Isolated from Marine Mammals
Authors: Bricker Betsy J. , Ewalt Darla R. , MacMillan Alastair P. , Foster Geoff , Brew Simon
Published: December 2019

Recently, gram-negative bacteria isolated from a variety of marine mammals have been identified as
Brucella
species by conventional phenotypic analysis. This study found the 16S rRNA gene from one representative isolate was identical to the homologous sequences of
Brucella abortus
,
B. melitensis
,
B. canis
, and
B. suis
. IS
711
-based DNA fingerprinting of 23 isolates from marine mammals showed all the isolates differed from the classical
Brucella
species. In general, fingerprint patterns grouped by host species. The data suggest that the marine mammal isolates are distinct types of
Brucella
and not one of the classical species or biovars invading new host species. In keeping with historical precedent, the designation of several new
Brucella
species may be appropriate.
Detection of
Giardia lamblia
and
Cryptosporidium parvum
Antigens in Human Fecal Specimens Using the ColorPAC Combination Rapid Solid-Phase Qualitative Immunochromatographic Assay
Authors: Garcia Lynne S. , Shimizu Robyn Y.
Published: December 2019

The ColorPAC
Giardia/Cryptosporidium
(Becton Dickinson) is a solid-phase qualitative immunochromatographic assay that detects and distinguishes between
Giardia lamblia
and
Cryptosporidium parvum
in human stool. Agreement between the Alexon-Trend ProSpecT
Giardia
Rapid EIA and the ColorPAC assay was 166 of 172 (96.5%). Agreement between the Alexon-Trend ProSpecT
Cryptosporidium
Rapid EIA and the ColorPAC assay was 169 of 171 (98.8%). No cross-reactions were seen with other parasites or human cells.
Experimental Inoculation with Human Granulocytic Ehrlichia Agent Derived from High- and Low-Passage Cell Culture in Horses
Authors: Pusterla Nicola , Madigan John E. , Asanovich Kristin M. , Chae Joon-Seok , Derock Elfriede , Leutenegger Christian M. , Pusterla Jeannine Berger , Lutz Hans , Dumler J. Stephen
Published: December 2019

We report the successful infection throughout intravenous inoculation with low and high passage of in vitro-grown human granulocytic ehrlichiosis (HGE) agent in horses. Differences in disease severity but not in incubation time, hematological changes, PCR detection, ehrlichial load, seroconversion time, and titer range were noted between horses infected with a low and a high passage of in vitro-grown HGE agent.
Potential Errors in Recognition of
Erysipelothrix rhusiopathiae
Authors: Dunbar Sherry A. , Clarridge Jill E.
Published: December 2019

Here we describe four isolations of
Erysipelothrix rhusiopathiae
associated with polyarthralgia and renal failure, septic arthritis, classic erysipeloid, and peritonitis. Although the biochemical identification was straightforward in each case, recognition presented a challenge to the clinical microbiologist, since in three cases
E. rhusiopathiae
was not initially considered due to unusual clinical presentations, in two cases the significance might not have been appreciated because growth was in broth only, and in one case the infection was thought to be polymicrobic. Because the Gram stain can be confusing, abbreviated identification schemes that do not include testing for H
2
S production could allow
E. rhusiopathiae
isolates to be misidentified as
Lactobacillus
spp. or
Enterococcus
spp. in atypical infections.
Evaluation of the Wider System, a New Computer-Assisted Image-Processing Device for Bacterial Identification and Susceptibility Testing
Authors: Cantón Rafael , Pérez-Vázquez María , Oliver Antonio , Sánchez Del Saz Begoña , Gutiérrez M. Olga , Martínez-Ferrer Manuel , Baquero Fernando
Published: December 2019

The Wider system is a newly developed computer-assisted image-processing device for both bacterial identification and antimicrobial susceptibility testing. It has been adapted to be able to read and interpret commercial MicroScan panels. Two hundred forty-four fresh consecutive clinical isolates (138 isolates of the family
Enterobacteriaceae
, 25 nonfermentative gram-negative rods [NFGNRs], and 81 gram-positive cocci) were tested. In addition, 100 enterobacterial strains with known β-lactam resistance mechanisms (22 strains with chromosomal AmpC β-lactamase, 8 strains with chromosomal class A β-lactamase, 21 broad-spectrum and IRT β-lactamase-producing strains, 41 extended-spectrum β-lactamase-producing strains, and 8 permeability mutants) were tested. API galleries and National Committee for Clinical Laboratory Standards (NCCLS) microdilution methods were used as reference methods. The Wider system correctly identified 97.5% of the clinical isolates at the species level. Overall essential agreement (±1 log
2
dilution for 3,719 organism-antimicrobial drug combinations) was 95.6% (isolates of the family
Enterobacteriaceae
, 96.6%; NFGNRs, 88.0%; gram-positive cocci, 95.6%). The lowest essential agreement was observed with
Enterobacteriaceae
versus imipenem (84.0%), NFGNR versus piperacillin (88.0%) and cefepime (88.0%), and gram-positive isolates versus penicillin (80.4%). The category error rate (NCCLS criteria) was 4.2% (2.0% very major errors, 0.6% major errors, and 1.5% minor errors). Essential agreement and interpretive error rates for eight β-lactam antibiotics against isolates of the family
Enterobacteriaceae
with known β-lactam resistance mechanisms were 94.8 and 5.4%, respectively. Interestingly, the very major error rate was only 0.8%. Minor errors (3.6%) were mainly observed with amoxicillin-clavulanate and cefepime against extended-spectrum β-lactamase-producing isolates. The Wider system is a new reliable tool which applies the image-processing technology to the reading of commercial trays for both bacterial identification and susceptibility testing.
Intraspecies Diversity of
Cryptococcus laurentii
as Revealed by Sequences of Internal Transcribed Spacer Regions and 28S rRNA Gene and Taxonomic Position of
C. laurentii
Clinical Isolates
Authors: Sugita Takashi , Takashima Masako , Ikeda Reiko , Nakase Takashi , Shinoda Takako
Published: December 2019

The intraspecies diversity of an opportunistic yeast pathogen,
Cryptococcus laurentii
, was revealed by analysis of the sequences of the internal transcribed spacer regions and the 28S rRNA gene. Ten strains of
C. laurentii
were grouped into two major phylogenetic groups and were further divided into at least seven species. Four of the strains isolated from patients did not represent a single species but showed heterogeneity. These results suggest that
C. laurentii
is a genetically heterogeneous species, and this must be taken into consideration when identifying
C. laurentii
clinical isolates.
A Simple PCR Method for Rapid Genotype Analysis of
Mycobacterium ulcerans
Authors: Stinear Timothy , Davies John K. , Jenkin Grant A. , Portaels Françoise , Ross Bruce C. , OppEdIsano Frances , Purcell Maria , Hayman John A. , Johnson Paul D. R.
Published: December 2019

Two high-copy-number insertion sequences, IS
2404
and IS
2606
, were recently identified in
Mycobacterium ulcerans
and were shown by Southern hybridization to possess restriction fragment length polymorphism between strains from different geographic origins. We have designed a simple genotyping method that captures these differences by PCR amplification of the region between adjacent copies of IS
2404
and IS
2606
. We have called this system 2426 PCR. The method is rapid, reproducible, sensitive, and specific for
M. ulcerans
, and it has confirmed previous studies suggesting a clonal population structure of
M. ulcerans
within a geographic region.
M. ulcerans
isolates from Australia, Papua New Guinea, Malaysia, Surinam, Mexico, Japan, China, and several countries in Africa were easily differentiated based on an array of 4 to 14 PCR products ranging in size from 200 to 900 bp. Numerical analysis of the banding patterns suggested a close evolutionary link between
M. ulcerans
isolates from Africa and southeast Asia. The application of 2426 PCR to total DNA, extracted directly from
M. ulcerans
-infected tissue specimens without culture, demonstrated the sensitivity and specificity of this method and confirmed for the first time that both animal and human isolates from areas of endemicity in southeast Australia have the same genotype.
Diversity in a Variable-Number Tandem Repeat from
Yersinia pestis
Authors: Adair D. M. , Worsham P. L. , Hill K. K. , Klevytska A. M. , Jackson P. J. , Friedlander A. M. , Keim P.
Published: December 2019

We have identified a tetranucleotide repeat sequence, (CAAA)
N
, in the genome of
Yersinia pestis
, the causative agent of plague. This variable-number tandem repeat (VNTR) region has nine alleles and great diversity (calculated as 1 minus the sum of the squared allele frequencies) (diversity value, 0.82) within a set of 35 diverse
Y. pestis
strains. In contrast, the nucleotide sequence of the
lcrV
(low-calcium-response) gene differed only slightly among these strains, having a haplotype diversity value of 0.17. Replicated cultures, phenotypic variants of particular strains, and extensively cultured replicates within strains did not differ in VNTR allele type. Thus, while a high mutation rate must contribute to the great diversity of this locus, alleles appear stable under routine laboratory culture conditions. The classic three plague biovars did not have single identifying alleles, although there were allelic biases within biovar categories. The antiqua biovar was the most diverse, with four alleles observed in 5 strains, while the orientalis and mediaevalis biovars exhibited five alleles in 21 strains and three alleles in 8 strains, respectively. The CAAA VNTR is located immediately adjacent to the transcriptional promoters for flanking open reading frames and may affect their activity. This VNTR marker may provide a high-resolution tool for epidemiological analyses of plague.
Development of a Serological Test for
Haemophilus ducreyi
for Seroprevalence Studies
Authors: Elkins Christopher , Yi Kyungcheol , Olsen Bonnie , Thomas Christopher , Thomas Kevin , Morse Stephen
Published: December 2019

We developed a new enzyme immunoassay (rpEIA) for use in determining the seroprevalence of chancroid. Three highly conserved outer membrane proteins from
Haemophilus ducreyi
strain 35000 were cloned, overexpressed, and purified from
Escherichia coli
for use as antigens in the rpEIA. Serum specimens from patients with and without chancroid were assayed to determine optimum sensitivity and specificity and to establish cutoff values. On the basis of these data, rpEIA was found to be both sensitive and specific when used to test a variety of serum specimens from patients with genital ulcers and urethritis and from healthy blood donors.
Borrelia burgdorferi
B31 Erp Proteins That Are Dominant Immunoblot Antigens of Animals Infected with Isolate B31 Are Recognized by Only a Subset of Human Lyme Disease Patient Sera
Authors: Miller Jennifer C. , El-Hage Nazira , Babb Kelly , Stevenson Brian
Published: December 2019

Sera from animals infected with
Borrelia burgdorferi
isolates yield intense immunoblot signals from the B31 ErpA/I/N and ErpB/J/O proteins, which have apparent molecular masses of 19 and 60 kDa, respectively. Since
B. burgdorferi
proteins with those molecular masses are of immunodiagnostic importance, Lyme disease patient sera were used in studies of B31 lysates and recombinant B31 ErpA/I/N and ErpB/J/O proteins. Immunoblot analyses indicated that only a minority of the patients produced antibodies that recognized the tested B31 Erp proteins. Southern blot analyses of Lyme disease spirochetes cultured from 16 of the patients indicated that all these bacteria contain genes related to the B31
erpA/I/N
and
erpB/J/O
genes, although signal strengths indicated only weak similarities in many cases, suggestive of genetic variability of
erp
genes among these bacteria. These data indicate that Erp proteins are generally not the 19- and 60-kDa antigens observed on serodiagnostic immunoblots.
Desulfovibrio desulfuricans
Bacteremia in a Dog
Authors: Shukla Sanjay K. , Reed Kurt D.
Published: December 2019

Desulfovibrio desulfuricans
was isolated from the blood of a dog presenting with fever, anorexia, and rear limb stiffness. The isolate was identified by 16S rRNA gene amplification and sequencing.