bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative Study of Different Standardization Concepts in Quantitative Competitive Reverse Transcription-PCR Assays
Authors: Haberhausen Gerd , Pinsl Judith , Kuhn Carl-Christoph , Markert-Hahn Christine
Published: December 2019

Four different standardization approaches based on a competitive reverse transcription (RT)-PCR assay were compared with a noncompetitive assay based on an external standard curve. Criteria for assessment were accuracy in quantitation, correctness of recovery, sensitivity, dynamic range, reproducibility, throughput, and convenience of sample handling. As a model system, we used the 5′-noncoding region of hepatitis C virus (HCV) for amplification in all quantitative RT-PCRs. A computer program that allowed parallel data processing was developed. Surprisingly, all methods were found suitable for accurate quantitation and comparable with respect to the criterion correctness of recovery. All results differed only by a factor of about 2. The reason for this finding might be that all of our mimics, as well as the wild-type genome of HCV, exhibited exactly the same amplification and hybridization efficacy. Moreover, minimal competition occurred in our experiments over a 5-log dynamic range. A further topic of our investigation was the comparison of two different competitive RNA fragments, mimics, with regard to their suitability as internal standards. One was a heterologous mimic, in which only the primer binding sites were identical to the wild type. The second one was a homologous mimic identical to the wild type except for a small region used for differential hybridization, which was replaced by a permutated sequence of the same length. Both the homologous and heterologous internal mimics were found appropriate for an accurate competitive RT-PCR assay, provided that amplification efficacy, as well as capture efficacy, is proven identical for both analyte and mimic.
Detection and Prevalence of
Helicobacter
Infection in Pet Cats
Authors: Neiger R. , Dieterich C. , Burnens A. , Waldvogel A. , Corthésy-Theulaz I. , Halter F. , Lauterburg B. , Schmassmann A.
Published: December 2019

The presence of spiral bacteria in the feline stomach has been recognized for over a century, but the identities and degrees of prevalence of such organisms in privately owned cats are still poorly documented. The aims of this study were (i) to adapt different diagnostic tools and evaluate their practicality for diagnosing feline gastric
Helicobacter
colonization, (ii) to determine the prevalence of gastric
Helicobacter
-like organisms in pet cats, (iii) to identify the feline species, and (iv) to correlate the presence of a
Helicobacter
infection with gastritis. Biopsy samples were taken gastroscopically from the antra and the corpora of clinically healthy pet cats.
Helicobacter
-like organisms were detected by Gram staining, Warthin-Starry staining, and rapid urease testing in biopsy specimens and by [
13
C]urea breath testing in 79, 77, 78, and 85% of cases, respectively. PCR analysis revealed that 78% of the cats (38 of 49) were infected by
Helicobacter heilmannii
; however, none of them was harboring
Helicobacter pylori
or
Helicobacter felis
. Culture was positive for one cat; the organism was identified as
Helicobacter pametensis
by dot blot DNA hybridization. By a combination of the detection methods, 91% of the pet cats were found to be
Helicobacter
positive. For 46 cats (79%) diagnostic tests were concordant. All cats showed mild to moderate gastritis in either the antrum or the corpus, regardless of the presence or density of gastric bacteria. In summary, pet cats are frequently colonized by
H. heilmannii
without a significant correlation between infection and degree of gastritis.
Serologic Evidence of Infection with
Ehrlichia
spp. in Wild Rodents (Muridae: Sigmodontinae) in the United States
Authors: Nicholson William L. , Muir Susie , Sumner John W. , Childs James E.
Published: December 2019

Rodent (Muridae: Sigmodontinae) blood and sera collected from 14 states were tested for seroreactivity to a cultured isolate of the human granulocytic ehrlichiosis (HGE) agent by using an indirect immunofluorescence assay. Of the 1,240 samples tested, 136 (11%) were found to be reactive at titers of ≥32. Rodents with HGE agent-specific antibodies were found in New York (23% of 491 samples; geometric mean endpoint titer [GMT] = 441), Connecticut (11% of 100 samples; GMT = 481), California (9% of 32 samples; GMT = 323), Colorado (2% of 212 samples; GMT = 256), Florida (7% of 27 samples; GMT = 362), Maryland (7% of 15 samples; titer = 64), New Jersey (4% of 76 samples; titer = 256), and Wisconsin (13% of 8 samples; titer = 128). Samples from Georgia (
n
= 16), Illinois (
n
= 27), Nevada (
n
= 27), North Carolina (
n
= 52), Ohio (
n
= 57), and Utah (
n
= 100) were not reactive. The earliest seroreactive sample was from a
Peromyscus leucopus
mouse collected in June 1986 in Connecticut, and the majority of the seroreactive samples (68%) were from this species. Samples from other
Peromyscus
species (
P. boylii
,
P. maniculatus
, and
P. gossypinus
) were also found to be reactive, with a GMT for the genus of 410. Several species of
Neotoma
woodrats (
N. fuscipes
,
N. lepida
,
N. albigula
, and
N. mexicana
) from California and Colorado had antibodies that reacted with the HGE agent (genus GMT = 194), suggesting that enzootic cycles of
Ehrlichia
spp. exist outside of the areas of confirmed human disease. Attempts to amplify and detect ehrlichial DNA from the limited tissues available (
n
= 40 animals) were unsuccessful. Further studies are needed to determine the identity of the organisms inducing antibody production in these rodent species and to elucidate the epidemiology and public health importance of these agents.
Lyme Disease in Taiwan: First Human Patient with Characteristic Erythema Chronicum Migrans Skin Lesion
Authors: Shih Chien-Ming , Wang Jin-Choung , Chao Li-Lian , Wu Trong-Neng
Published: December 2019

We report herein the first laboratory-diagnosed case of Lyme disease in a human in Taiwan. A 45-year-old Taiwanese man living in Taipei, in northern Taiwan, had an expanding skin lesion (measuring 23 by 15 cm) on his abdomen for 2 to 3 weeks and recurrent attacks of pain and swelling of the knee joint. Serologic tests indicated a significantly elevated titer of antibody to
Borrelia burgdorferi
. After appropriate antibiotic treatment for 3 weeks, the skin lesion was cured and the joint swelling was improved. Although several strains of
Borrelia
spirochetes had been isolated from rodents (
Rattus losea
) in Taiwan, the tick vector responsible for the transmission remains to be identified.
Epidemiologic Evaluation of Antimicrobial Resistance in Community-Acquired Enterococci
Authors: Silverman J. , Thal L. A. , Perri M. B. , Bostic G. , Zervos M. J.
Published: December 2019

Fecal samples from 200 consecutive patients admitted to a community hospital yielded 107 enterococci. High-level gentamicin resistance occurred in 10 (14%) of the
Enterococcus faecalis
isolates. Ampicillin resistance occurred in two (3%) of the
E. faecalis
isolates and six (23%) of the
Enterococcus faecium
isolates. There were no vancomycin-resistant enterococci. Risk factors for enterococci with high-level aminoglycoside (gentamicin) or ampicillin resistance included prior hospitalization and previous antibiotic use.
Intra-Assay Performance Characteristics of Five Assays for Quantification of Human Immunodeficiency Virus Type 1 RNA in Plasma
Authors: Lin Hsiang Ju , Pedneault Louise , Hollinger F. Blaine
Published: December 2019

Three kits (Roche AMPLICOR human immunodeficiency virus type 1 [HIV-1] Monitor, Chiron enhanced-sensitivity bDNA, and Organon Teknika NASBA HIV-1 QT) and two in-house assays (from National Genetics Institute and Baylor College of Medicine) were compared with a blinded panel. The results were evaluated as to intra-assay sensitivity, precision, and ability to detect differences in a dilution series.
Detection and Direct Typing of Herpes Simplex Virus in Perianal Ulcers of Patients with AIDS by PCR
Authors: do Nascimento Maria C. , Sumita Laura M. , de Souza Vanda A. U. F. , Pannuti Cláudio S.
Published: December 2019

The presence of herpes simplex virus type 1 (HSV-1) and HSV-2 in perianal ulcerations of 41 AIDS patients was assessed by virus culture and a type-specific PCR-based assay. HSV was isolated from the lesion site in 24 of 41 (58.5%) patients, and HSV DNA was detected by PCR in all 24 (100%) of these specimens. Additionally, PCR was used to detect HSV DNA in 12 of 17 (70.5%) HSV culture-negative samples. Thus, HSV genomic sequences could be demonstrated in 36 of 41 (87.8%) perianal ulcers in this series. Full agreement in HSV typing by either immunodot assay or PCR was seen in 24 samples that were positive by both virus culture and PCR. HSV-2 was demonstrated in 35 of 36 (97.2%) HSV-positive samples.
Evaluation of PCR for Diagnosis of Melioidosis
Authors: Haase Antje , Brennan Maree , Barrett Siobhan , Wood Yvonne , Huffam Sarah , O’Brien Daniel , Currie Bart
Published: December 2019

Previously published PCR-based diagnostic tests for melioidosis were evaluated for clinical usefulness. A
Burkholderia pseudomallei
16S rRNA-derived primer set had a sensitivity approaching 100% for clinical samples from 22 culture-confirmed cases of melioidosis and enabled diagnosis of 3 culture-negative cases. However, samples from 10 of 30 inpatients from Royal Darwin Hospital with other diagnoses were positive by PCR, giving a specificity of 67% and a positive predictive value of only 70%. Although there are a number of intriguing possible explanations for our results, concerns of inappropriate therapy resulting from a positive result by PCR have led us to forgo the advantage of rapid PCR diagnosis for melioidosis until a better system is validated.
Detection of
Mycobacterium tuberculosis
Complex in Cattle by PCR Using Milk, Lymph Node Aspirates, and Nasal Swabs
Authors: Vitale Fabrizio , Capra Giuseppina , Maxia Letizia , Reale Stefano , Vesco Gesualdo , Caracappa Santo
Published: December 2019

The PCR technique was applied to the diagnosis of tuberculosis in live cattle, and both skin-test-negative and skin-test-positive animals were studied. DNA was taken from various sources including specimens of lymph node aspirates, milk, and nasal swabs. After slaughter and visual inspection, tissues such as lymph nodes, lungs, and udders from tuberculin reactors were tested by the same technique. Specific oligonucleotide primers internal to the IS
6110
insertion element were used to amplify a 580-bp fragment. A 182-bp fragment was obtained by designating a nested PCR from the first amplification product. This fragment was cloned and sequenced, and after being labeled it was employed in dot blot hybridization. A total of 100 cattle were tested, and PCR analysis was performed using nasal swab, milk, and lymph node aspirate. Sixty skin-test-positive cows were also tested to detect mycobacterial DNA in tissue samples from lymph nodes, lungs, and udders, and the infection was confirmed in all of the animals. Using PCR analysis of tissue samples from slaughtered animals as a “gold standard” we calculated 100% values for sensitivity, specificity, and positive and negative predictive values for milk and lymph node aspirate samples. The respective values for nasal swab samples were 58, 100, 100, and 28%. The respective values for all of the samples were 74, 100, 100, and 35%, while for visual inspection the values were 81, 100, 100, and 58%, respectively. PCR analysis of specimens of lymph node aspirates, milk, and nasal swabs from skin-test-negative animals showed that 52% of these skin test results were false negatives. These animals, not being removed from the farms, represent a potential source of further infection.
Sensitivity and Specificity of an Improved Rapid Latex Agglutination Test for Identification of Methicillin-Sensitive and -Resistant
Staphylococcus aureus
Isolates
Authors: Smole Sandra C. , Aronson Elyssa , Durbin Annette , Brecher Stephen M. , Arbeit Robert D.
Published: December 2019

The performance of a second-generation rapid agglutination kit, Slidex Staph Plus (SSP; bioMérieux), was compared to those of the Slidex Staph (SS; bioMérieux), Staphaurex (SRX; Murex Diagnostics), and BBL Staphyloslide (BBL; Becton Dickinson) kits by using 508 clinical isolates composed of 150 methicillin-sensitive
Staphylococcus aureus
(MSSA) organisms, 154 methicillin-resistant
S. aureus
(MRSA) organisms, and 204 non-
S. aureus Staphylococcus
spp. Of the 508 isolates tested, 75% were fresh clinical isolates, with the remainder taken from five different freezer collections. All four agglutination tests had comparable sensitivities for MSSA and MRSA. However, the SS kit was significantly less specific (93.1%) than the three other tests (
P
> 0.05, McNemar test). These results demonstrate that the new rapid latex agglutination kit, SSP, was more specific for the identification of
S. aureus
than the previous version and performed comparably to the SRX and BBL kits.
Group A Streptococcal Vir Types Are M-Protein Gene (emm) Sequence Type Specific
Authors: Gardiner Don L. , Goodfellow Alison M. , Martin Diana R. , Sriprakash Kadaba S.
Published: December 2019

The M-protein genes (emmgenes) of 103 separate impetiginousStreptococcus pyogenesisolates were sequenced and the sequence types were compared to the types obtained by Vir typing. Vir typing is based on restriction fragment length polymorphism (RFLP) analysis of a 4- to 7-kb pathogenicity island encodingemmand other virulence genes. By using bothHaeIII andHinfI to generate RFLP profiles, complete concordance between Vir type andemmsequence type was found. Comparison of theemmsequences with those in GenBank revealed new sequence types sharing less than 90% identity with known types. Diversity in theemmsequence was generated by corrected frameshift mutations, point mutations, and small in-frame mutations.
Epidemiology of a Dominant Clonal Strain of Vancomycin-Resistant
Enterococcus faecium
at Separate Hospitals in Boston, Massachusetts
Authors: Fridkin Scott K. , Yokoe Deborah S. , Whitney Cynthia G. , Onderdonk Andrew , Hooper David C.
Published: December 2019

In 1996, the dominant (43%) strain of vancomycin-resistant enterococci (VRE; type A) at Massachusetts General Hospital was identified at Brigham and Women’s Hospital (BWH). To characterize the epidemiology of infection with type A isolates of VRE at BWH, we collected demographic and clinical data for all patients from whom VRE were isolated from a clinical specimen through September 1996. The first clinical isolates from all BWH patients from whom VRE were isolated were typed by pulsed-field gel electrophoresis of
Sma
I digests of chromosomal DNA. Among patients hospitalized after the first patient at BWH infected with a type A isolate of VRE was identified, exposures were compared between patients who acquired type A isolates of VRE and those who acquired other types of VRE. Isolates from 99 patients identified to have acquired VRE were most commonly from blood (
n
= 27), urine (
n
= 19), or wounds (
n
= 19). Three months after the index patient arrived at BWH and at a time when ≥12 types of strains of VRE were present, type A isolates of VRE became dominant; 39 of 75 (52%) of the study cohort had acquired type A isolates of VRE. We found no association between the acquisition of type A isolates of VRE and transfer from another institution or temporal overlap by service, ward, or floor with patients known to have acquired type A isolates of VRE. By multivariate analysis, only residence in the medical intensive care unit (adjusted odds ratio [OR], 3.2; 95% confidence interval [CI], 1.4 to 107) and the receipt of two or more antibiotics per patient-day (adjusted OR, 12.2; 95% CI, 1.2 to 9.0) were associated with the acquisition of strain A. This strain of VRE, dominant at two Boston hospitals, was associated with intensity of antibiotic exposures (i.e., two or more antibiotics per patient-day). We hypothesize that this strain may have unidentified properties providing a mechanism favoring its spread and dominance over other extant isolates, and further studies are needed to define these properties.
Panfungal PCR Assay for Detection of Fungal Infection in Human Blood Specimens
Authors: Van Burik Jo-Anne , Myerson David , Schreckhise Randall W. , Bowden Raleigh A.
Published: December 2019

A novel panfungal PCR assay which detects the small-subunit rRNA gene sequence of the two major fungal organism groups was used to test whole-blood specimens obtained from a series of blood or bone marrow transplant recipients. The 580-bp PCR product was identified after amplification by panfungal primers and hybridization to a 245-bp digoxigenin-labeled probe. The lower limit of detection of the assay was approximately four organisms per milliliter of blood. Multiple whole-blood specimens from five patients without fungal infection or colonization had negative PCR results. Specimens from 11 infected patients had positive PCR results. Blood from three patients with pulmonary aspergillosis had positive PCR results: one patient’s blood specimen obtained in the week prior to the diagnosis of infection by a positive bronchoalveolar lavage fluid culture result was positive by PCR, and blood specimens obtained from two patients 1 to 2 days after lung biopsy and which were sterile by culture were positive by PCR. The blood of four patients with candidemia, three patients with mixed fungal infections, and one patient with fusariosis also had positive PCR signals. The panfungal PCR assay can detect multiple fungal genera and may be used as an adjunct to conventional methods for the detection of fungal infection or for describing the natural history of fungal infection. Further studies are needed to define the sensitivity and specificity of this assay for the diagnosis of fungal infection prior to the existence of other clinical or laboratory indications of invasive fungal infection.
Immunoglobulin A-Specific Capture Enzyme-Linked Immunosorbent Assay for Diagnosis of Dengue Fever
Authors: Talarmin Antoine , Labeau Bhety , Lelarge Josiane , Sarthou Jean-Louis
Published: December 2019

Dengue fever (DF) is usually diagnosed by testing for dengue virus immunoglobulin M (IgM) by a capture enzyme-linked immunosorbent assay (ELISA) (MAC-ELISA). However, IgM can last for months, and its presence might reflect a previous infection. We have tested the use of anti-dengue virus IgA capture ELISA (AAC-ELISA) for the diagnosis of DF by comparing the results of MAC-ELISAs and AAC-ELISAs for 178 serum samples taken from patients with confirmed cases of DF. IgM appears more rapidly (mean delay of positivity, 3.8 days after the onset of DF) than IgA (4.6 days) but lasts longer; the peak IgA titer is obtained on day 8. The specificity and the positive predictive value of AAC-ELISA are 100%; its sensitivity and negative predictive value (NPV) are also 100% between days 6 and 25 after the onset of DF, but they decrease drastically when data for tests conducted with specimens from the first days of infection are included, because the IgA titers, like the IgM titers, have not yet risen. AAC-ELISA is a simple method that can be performed together with MAC-ELISA and that can help in interprating DF serology.
Limitations of the Current Microbial Identification System for Identification of Clinical Yeast Isolates
Authors: Kellogg James A. , Bankert David A. , Chaturvedi Vishnu
Published: December 2019

The ability of the rapid, computerized Microbial Identification System (MIS; Microbial ID, Inc.) to identify a variety of clinical isolates of yeast species was compared to the abilities of a combination of tests including the Yeast Biochemical Card (bioMerieux Vitek), determination of microscopic morphology on cornmeal agar with Tween 80, and when necessary, conventional biochemical tests and/or the API 20C Aux system (bioMerieux Vitek) to identify the same yeast isolates. The MIS chromatographically analyzes cellular fatty acids and compares the results with the fatty acid profiles in its database. Yeast isolates were subcultured onto Sabouraud dextrose agar and were incubated at 28°C for 24 h. The resulting colonies were saponified, methylated, extracted, and chromatographically analyzed (by version 3.8 of the MIS YSTCLN database) according to the manufacturer’s instructions. Of 477 isolates of 23 species tested, 448 (94%) were given species names by the MIS and 29 (6%) were unidentified (specified as “no match” by the MIS). Of the 448 isolates given names by the MIS, only 335 (75%) of the identifications were correct to the species level. While the MIS correctly identified only 102 (82%) of 124 isolates of
Candida glabrata
, the predictive value of an MIS identification of unknown isolates as
C. glabrata
was 100% (102 of 102) because no isolates of other species were misidentified as
C. glabrata
. In contrast, while the MIS correctly identified 100% (15 of 15) of the isolates of
Saccharomyces cerevisiae
, the predictive value of an MIS identification of unknown isolates as
S. cerevisiae
was only 47% (15 of 32), because 17 isolates of
C. glabrata
were misidentified as
S. cerevisiae
. The low predictive values for accuracy associated with MIS identifications for most of the remaining yeast species indicate that the procedure and/or database for the system need to be improved.
Ultrastructure, Immunofluorescence, Western Blot, and PCR Analysis of Eight Isolates ofEncephalitozoon(Septata)intestinalisEstablished in Culture from Sputum and Urine Samples and Duodenal Aspirates of Five Patients with AIDS
Authors: del Aguila C. , Croppo G. P. , Moura H. , Da Silva A. J. , Leitch G. J. , Moss D. M. , Wallace S. , Slemenda S. B. , Pieniazek N. J. , Visvesvara G. S.
Published: December 2019

Microsporidia are ancient, intracellular, eukaryotic protozoan parasites that form spores and that lack mitochondria. Currently, as many as eight species included under six genera are known to infect humans, mostly patients with AIDS. Among these,Enterocytozoon bieneusi, the agent of gastrointestinal (GI) disease, is the most frequently identified microsporidian in clinical laboratories in the United States.Encephalitozoon(Septata)intestinalis, the agent that causes a disseminated infection including infection of the GI tract, is the second most frequently identified microsporidian parasite. In spite of this, not many isolates ofE. intestinalishave been established in culture. We describe here the continuous cultivation of eight isolates ofE. intestinalisobtained from different samples including the urine, sputum, and duodenal aspirate or biopsy specimens from five AIDS patients originating from California, Colorado, and Georgia. The specific identification was made on the bases of ultrastructural, antigenic, and PCR analyses.
Three Cases of
Anaerobiospirillum succiniciproducens
Bacteremia Confirmed by 16S rRNA Gene Sequencing
Authors: Tee Wee , Korman Tony M. , Waters Mary Jo , Macphee Andrew , Jenney Adam , Joyce Linda , Dyall-Smith Michael L.
Published: December 2019

We describe three cases of
Anaerobiospirillum succiniciproducens
bacteremia from Australia. We believe one of these cases represents the first report of
A. succiniciproducens
bacteremia in a human immunodeficiency virus (HIV)-infected individual. The other two patients had an underlying disorder (one patient had bleeding esophageal varices complicating alcohol liver disease and one patient had non-Hodgkin’s lymphoma). A motile, gram-negative, spiral anaerobe was isolated by culturing blood from all patients. Electron microscopy showed a curved bacterium with bipolar tufts of flagella resembling
Anaerobiospirillum
spp. Sequencing of the 16S rRNA genes of the isolates revealed no close relatives (organisms likely to be in the same genus) in the sequence databases, nor were any sequence data available for
A. succiniciproducens
. This report presents for the first time the 16S rRNA gene sequence of the type strain of
A. succiniciproducens
, strain ATCC 29305. Two of the three clinical isolates have sequences identical to that of the type strain, while the sequence of the other strain differs from that of the type strain at 4 nucleotides.
Detection of
Ureaplasma urealyticum
in Endotracheal Tube Aspirates from Neonates by PCR
Authors: Nelson S. , Matlow A. , Johnson G. , Th’ng C. , Dunn M. , Quinn P.
Published: December 2019

A PCR-based test was optimized for the detection of
Ureaplasma urealyticum
from neonatal respiratory specimens, with primers directed against the multiple-banded antigen gene (L. J. Teng, X. Zheng, J. I. Glass, H. Watson, J. Tsai, and G. H. Cassell, J. Clin. Microbiol. 32:1464–1469, 1994). Endotracheal tube aspirates (225) from 103 low-birth-weight neonates (<1,250 g) were taken, when possible, at days 0, 4, and 14 after birth and examined by culture and by PCR. Of 77 specimens positive by either method, 73 were detected by PCR and 60 were detected by culture. Overall, 36% of the neonates were positive for
U. urealyticum
by either method. Of 16 patients with PCR-positive–culture-negative results, 13 had positive cultures at another sampling point, and one additional patient had a twin with positive cultures. Of 11 patients with day 0 specimens positive by PCR alone, 9 subsequently became culture positive, demonstrating the utility of this test in early detection. Multiple serovars were present in over 50% of positive specimens, with serovars 3 and 14 in combination being most prevalent. The amplicon size generated from the specimen by PCR correctly predicted the biovars isolated in over 85% of positive specimens. Thus, this PCR test was valuable in allowing early detection of
U. urealyticum
in neonatal respiratory specimens, as well as in providing biovar information.
Effects of Ammonia Inhalation and Acetic Acid Pretreatment on Colonization Kinetics of Toxigenic
Pasteurella multocida
within Upper Respiratory Tracts of Swine
Authors: Hamilton T. D. C. , Roe J. M. , Hayes C. M. , Webster A. J. F.
Published: December 2019

Pigs reared in intensive production systems are continuously exposed to ammonia released by the microbial degradation of their excrement. Exposure to this gas has been shown to increase the severity of the disease progressive atrophic rhinitis by facilitating colonization of the pig’s upper respiratory tract by
Pasteurella multocida
. The etiological mechanism responsible for this synergy was investigated by studying the colonization kinetics of
P. multocida
enhanced by ammonia and comparing them with those evoked by an established disease model. Three-week-old Large White piglets were weaned and allocated to five experimental groups (groups A to E). Pigs in groups A and B were exposed continuously to ammonia at 20 ppm for the first 2 weeks of the study. Pigs in group C were pretreated with 0.5 ml of 1% acetic acid per nostril on days −2 and −1 of the study. On day 0 all the pigs in groups A, C, and D were inoculated with 1.4 × 10
8
toxigenic
P. multocida
organisms given by the intranasal route. The kinetics of
P. multocida
colonization were established by testing samples obtained at weekly intervals throughout the study. The study was terminated on day 37, and the extent of turbinate atrophy was determined by using a morphometric index. The results of the study showed that exposure to aerial ammonia for a limited period had a marked effect on the colonization of toxigenic
P. multocida
in the nasal cavities of pigs, which resulted in the almost total exclusion of commensal flora. In contrast, ammonia had only a limited effect on
P. multocida
colonization at the tonsil. The exacerbation of
P. multocida
colonization by ammonia was restricted to the period of ammonia exposure, and the number of
P. multocida
organisms colonizing the upper respiratory tract declined rapidly upon the cessation of exposure to ammonia. During the exposure period, the ammonia levels in mucus recovered from the nasal cavity and tonsil were found to be 7- and 3.5-fold higher, respectively, than the levels in samples taken from unexposed controls. Acetic acid pretreatment also induced marked colonization of the nasal cavity which, in contrast to that induced by ammonia, persisted throughout the time course of the study. Furthermore, acetic acid pretreatment induced marked but transient colonization of the tonsil. These findings suggest that the synergistic effect of ammonia acts through an etiological mechanism different from that evoked by acetic acid pretreatment. A strong correlation was found between the numbers of
P. multocida
organisms isolated from the nasal cavity and the severity of clinical lesions, as determined by using a morphometric index. The data presented in the paper highlight the potential importance of ammonia as an exacerbating factor in respiratory disease of intensively reared livestock.
Identification and Analysis of a New
vacA
Genotype Variant of
Helicobacter pylori
in Different Patient Groups in Germany
Authors: Strobel Sonja , Bereswill Stefan , Balig Peter , Allgaier Peter , Sonntag Hans-Günther , Kist Manfred
Published: December 2019

The vacuolating cytotoxin of
Helicobacter pylori
(VacA) is known to cause cell damage to mammalian cells and is suspected to give rise to gastric epithelial lesions that might lead to peptic ulcer disease. As shown recently, the gene encoding VacA exhibits genetic variation, with three different families of signal sequences (s1a, s1b, and s2) and two families of midregion sequences (m1 and m2). In order to investigate the relationship between the presence of specific
vacA
genotypes and peptic ulceration, the
vacA
genotypes of 158 clinical isolates of
H. pylori
were determined. The study group consisted of 106 patients with duodenal ulceration; 52 patients with nonulcer dyspepsia (NUD) were used as controls.
H. pylori
of genotype s1 was isolated from 96% of the patients with ulcerations, whereas genotype s2 was only present in 4%, indicating a strong correlation between the
vacA
genotype and peptic ulceration (
P
< 0.001). In contrast, 31% of the patients from the NUD control group were infected with strains of
vacA
genotype s2. Particular midregion genotypes (m1 and m2) were not associated with clinical manifestations. The midregions from 18% of the isolates could not be classified by the proposed scheme. DNA sequencing revealed high homology between the untypeable midregions and that of genotype m1, with multiple base pair exchanges, some affecting the primer annealing site. Compared to those of m1 and m2 alleles, the divergent midregions from untypeable strains showed clustering, indicating the presence of a further subfamily of sequences in the midregion of
vacA
in German isolates, for which we propose the term “m1a.” A new specific primer that we designed for typing m1a isolates might be useful in other studies.
Comparison of Nine Commercially Available Enzyme-Linked Immunosorbent Assays for Detection of
Giardia lamblia
in Fecal Specimens
Authors: Aldeen William E. , Carroll K. , Robison A. , Morrison M. , Hale D.
Published: December 2019

Overall performance, including ease of use, total hands-on time, incubation and processing times, sensitivity, and specificity, of each of nine enzyme-linked immunosorbent assays (ELISAs) were compared by using 222 individual fecal samples submitted for the detection of
Giardia lamblia
. The assays evaluated were manufactured by Alexon, Inc., Cambridge Biotech Corp., Meridian, Inc., and Trend Scientific, Inc. All assays used polyclonal antibodies except the “new and improved” Microplate (direct and diluted methods) by Alexon, which is a monoclonal antibody assay. Seventy specimens were positive for
G. lamblia
by ELISA, ova and parasite test, and/or direct fluorescent-antibody assay. One hundred fifty two were negative by all three methods. Sensitivities and specificities ranged from 88.6 to 100% and 99.3 to 100%, respectively. The total hands-on time needed to run one specimen ranged from 1 min to 2 min 17 s per specimen. All except one commercially available ELISA were found to be rapid, sensitive, and specific for the detection of
G. lamblia
in fecal specimens.
Presence of Multiple “
Helicobacter heilmannii
” Strains in an Individual Suffering from Ulcers and in His Two Cats
Authors: Dieterich Christine , Wiesel Paul , Neiger Reto , Blum André , Corthésy-Theulaz Irène
Published: December 2019

Circumstantial evidence suggests that “
Helicobacter heilmannii
” infection is an example of zoonosis. The presence of “
H. heilmannii
” strains in a human subject with acute gastric erosions, in his two cats, and in two unrelated cats was analyzed, and the genetic relatedness of the human and feline strains was assessed. A 580-bp, PCR-amplified sequence of “
H. heilmannii
” urease B gene (
ureB
) obtained from biopsies from the human subject and his two cats was restricted with
Alu
I and cloned for sequencing. Analysis of the restriction fragment length polymorphism of the
ureB
-amplified product suggested the presence of different individual “
H. heilmannii
” strains in the cats and of three distinct strains in the human subject. One of the “
H. heilmannii
”
ureB
sequences amplified from the human subject’s biopsies was identical to that derived from one of his cats. The degree of similarity between the other “
H. heilmannii
” human and feline nucleotide sequences was higher than 97%. Most of the base substitutions were conservative. We conclude that human and animal “
H. heilmannii
” strains are closely related and that humans can be infected by more than one “
H. heilmannii
” strain, as has been observed for
Helicobacter pylori.
Molecular Epidemiology of Oral Treponemes Associated with Periodontal Disease
Authors: Moter Annette , Hoenig Carina , Choi Bong-Kyu , Riep Birgit , Göbel Ulf B.
Published: December 2019

Periodontitis, a disease responsible for tooth loss worldwide, is characterized by chronic inflammation of the periodontium, eventually leading to destruction of periodontal ligaments and supporting alveolar bone. Spirochetes, identified by dark-field microscopy as being the most predominant bacteria in advanced lesions, are thought to play a causative role. Various spirochetal morphotypes were observed, but most of these morphotypes are as yet uncultivable. To assess the role of these organisms we designed oligonucleotide probes for the identification of both cultivable and so far uncultivable spirochetes in periodontitis patients. Subgingival plaque specimens taken from diseased sites (
n
= 200) and healthy control sites (
n
= 44) from 53 patients with rapidly progressive periodontitis (RPP) were submitted to direct in situ hybridization or dot blot hybridization after prior amplification with eubacterial primers. Spirochetes were found in all patients, but their distributions varied considerably. Parallel use of oligonucleotide probes specific for cultivable or so far uncultivable treponemes suggested the presence of novel yet unknown organisms at a high frequency. These uncultivable treponemes were visualized by fluorescence in situ hybridization, and their morphologies, sizes, and numbers could be estimated. All RPP patients included in this study harbored oral treponemes that represent either novel species, e.g.,
Treponema maltophilum
, or uncultivable phylotypes. Therefore, it is necessary to include these organisms in etiologic considerations and to strengthen efforts to cultivate these as yet uncultivable treponemes.
Novel Enzyme-Linked Immunoassay To Determine Nanogram Levels of Pneumocandins in Human Plasma
Authors: Karkhanis Y. D. , Schmatz D. M.
Published: December 2019

A binding enzyme-linked immunosorbent assay (ELISA) has been developed for measuring nanogram concentrations of semisynthetic pneumocandin antifungal agents in human plasma. Semisynthetic pneumocandin L-733,560 was conjugated to succinylated hemocyanin by water-soluble carbodiimide and was used as an immunogen to produce polyclonal antibodies in rabbits. Pneumocandins were used to directly coat the wells of a microtiter plate, and quantitation was achieved by using rabbit polyclonal antibodies to pneumocandin L-733,560 and goat anti-rabbit immunoglobulin G conjugated to either alkaline phosphatase or horseradish peroxidase. Maximum binding of L-733,560 and most related analogs to the wells of the microtiter plate was found to occur in the first 5 min of incubation at 4°C. Once bound to the plate, these pneumocandins could not be removed from the plate, either by treatment with 4.0 to 6.0 M urea or by treatment with 4.0 to 6.0 M guanidine hydrochloride for 24 h at 4°C. The binding ELISA is linear with drug concentration and can detect levels of L-733,560 as low as 5 ng/ml in human plasma. The assay is also useful for quantitating plasma levels of related semisynthetic pneumocandins including clinical candidate MK-0991.
Routine Use of PCR–Reverse Cross-Blot Hybridization Assay for Rapid Identification of
Mycobacterium
Species Growing in Liquid Media
Authors: Sanguinetti M. , Posteraro B. , Ardito F. , Zanetti S. , Cingolani A. , Sechi L. , De Luca A. , Ortona L. , Fadda G.
Published: December 2019

A PCR–reverse cross-blot hybridization assay procedure that is able to rapidly identify 13 species of clinically relevant mycobacteria was evaluated for routine use in the identification of acid-fast isolates growing in BACTEC 460 TB (12B and 13A) and BACTEC 9000 MB (Myco/F) liquid media. Eight of the probes used were already described by Kox et al. (L. F. F. Kox et al., J. Clin. Microbiol. 33:3225–3233, 1995). In addition, we used six other probes specific for
M. chelonae
,
M. malmoense
or
M. szulgai
,
M. genavense
,
M. gordonae
,
M. terrae
, and
M. marinum/M. ulcerans
that we designed ourselves. This procedure allowed us to identify 459 mycobacterial species directly from broth cultures of 5,466 clinical samples collected over 1 year and processed with the radiometric or nonradiometric BACTEC system. Our results were in agreement with those obtained by conventional identification methods and also with those obtained by mycolic acid analysis by high-performance liquid chromatography. This assay seems to be a reliable procedure for the routine identification of mycobacteria, providing an accurate identification of mycobacterial isolates more rapidly than conventional tests, with remarkable implications for an efficacious specific antimycobacterial therapy.
Suitability of Repetitive-DNA-Sequence-Based PCR Fingerprinting for Characterizing Epidemic Isolates of
Salmonella enterica
Serovar Saintpaul
Authors: Beyer W. , Mukendi F. M. , Kimmig P. , Böhm R.
Published: December 2019

Three molecular typing methods, repetitive-sequence-based PCR (rep-PCR) fingerprinting, plasmid profiling, and arbitrarily primed PCR fingerprinting, were used to characterize isolates of
Salmonella enterica
serovar Saintpaul. Most of the isolates were obtained from epidemic human cases of food-borne salmonellosis, together with some from the food material suspected to be the source of infection, and a few were obtained from other cases apparently not related to the epidemic. All three methods adequately discriminated the epidemic strain from other strains of the serovar. In addition several isolates from human cases which are not identical to the epidemic strain were found. These isolates therefore must have been responsible for some sporadic infections, which were only temporally related to the epidemic. These strains showed a high degree of similarity to a strain isolated from a turkey. rep-PCR fingerprinting with REP-Dt primers and primer ERIC1R, applicable even to crude cell lysates, offers an attractive choice as a primary method for the discrimination of various
Salmonella
serotypes as well as isolates within serotype Saintpaul.
Flow Cytometric Testing of Susceptibilities of
Mycobacterium tuberculosis
Isolates to Ethambutol, Isoniazid, and Rifampin in 24 Hours
Authors: Kirk Scott M. , Schell Ronald F. , Moore Andrea V. , Callister Steven M. , Mazurek Gerald H.
Published: December 2019

Susceptibility testing of
Mycobacterium tuberculosis
is seriously limited by the time required to obtain results. We show that susceptibility testing of clinical isolates of
M. tuberculosis
can be accomplished rapidly with acceptable accuracy by using flow cytometry. The susceptibilities of 35 clinical isolates of
M. tuberculosis
to various concentrations of isoniazid, rifampin, and ethambutol were tested by the agar proportion method and by flow cytometry. Agreement between the results from the two methods was 95, 92, and 83% for isoniazid, ethambutol, and rifampin, respectively. Only 11 discrepancies were detected among 155 total tests. The results of flow cytometric susceptibility tests were available within 24 h of inoculation of drug-containing medium, while the proportion method required 3 weeks to complete. The flow cytometric method is also simple to perform.
Mapping and Serodiagnostic Application of a Dominant Epitope within the Human Herpesvirus 8 ORF 65-Encoded Protein
Authors: Pau Chou-Pong , Lam Lee L. , Spira Thomas J. , Black Jodi B. , Stewart John A. , Pellett Philip E. , Respess Richard A.
Published: December 2019

A dominant epitope within the human herpesvirus 8 (HHV8) ORF 65-encoded protein was mapped to an 8-amino-acid (aa) sequence (RKPPSGKK [aa 162 to 169]) by an amino acid replacement method. Using a 14-aa peptide (P4) encompassing this epitope as the antigen, we developed an enzyme immunoassay for HHV8 antibodies. The presence of P4 antibodies in a panel of 61 human serum specimens was highly correlated with biopsy-confirmed Kaposi’s sarcoma. The homologous Epstein-Barr virus peptide derived from BFBR3-encoded protein did not interfere with the assay, suggesting that P4 is specific for HHV8.
“
Flexispira rappini
” Bacteremia in a Child with Pneumonia
Authors: Tee Wee , Leder Karin , Karroum Elena , Dyall-Smith Michael
Published: December 2019

We describe a case of “
Flexispira rappini
” bacteremia from a 9-year-old girl who presented with a 5-day history of fever, productive cough, and malaise. A chest X-ray result was compatible with right middle lobe pneumonia. Blood culture grew a gram-negative spiral fusiform bacterium 2 days after the inoculation. Biochemical tests showed the organism to be catalase negative, oxidase positive, sodium hippurate hydrolysis negative, and urea hydrolysis negative. 16S rRNA gene sequencing identified this organism as “
F. rappini
,” showing a six-base substitution from the type strain. This is the first report of “
F. rappini
” bacteremia in a human, suggesting that this organism has the potential of causing invasive infection, but its role in pneumonia is uncertain and could be unrelated to the bacteremia.
Hepatitis C Virus Infections in Dialysis Centers in The Netherlands: a National Survey by Serological and Molecular Methods
Authors: Schneeberger Peter M. , Keur Ingrid , van der Vliet Walter , van Hoek Kitty , Boswijk Henk , van Loon Anton M. , van Dijk Willemien C. , Kauffmann Robert H. , Quint Wim , van Doorn Leen-Jan
Published: December 2019

A national survey of hepatitis C virus (HCV) infections among dialysis patients in The Netherlands was performed. The study involved 2,653 patients (2,108 hemodialysis patients and 545 chronic ambulatory peritoneal dialysis [CAPD] patients) from 39 of the 49 dialysis centers in the country. Patient sera were analyzed by both serological and molecular methods. Screening by a third-generation enzyme immunoassay (EIA) yielded 79 reactive sera. The presence of anti-HCV antibodies was confirmed in 70 patients by a line immunoassay. All seropositive samples were tested by reverse transcriptase PCR, and 57 samples were found to contain HCV RNA. Of the nine EIA-positive and line immunoassay-negative or indeterminate samples, four were HCV RNA positive. All seronegative samples were screened for the presence of HCV RNA in pools of five sera. Of 2,576 antibody-negative samples, 6 contained HCV RNA. All antibody-positive and RNA-positive samples were also tested by a second serological assay. The prevalence of HCV infections among Dutch dialysis patients as determined by serology or the presence of HCV RNA was 3% (80 of 2,653), i.e., 3.5% (73 of 2,108) in patients treated on hemodialysis and 1.3% (7 of 545) in patients on CAPD. Of these 80 HCV-infected dialysis patients, 67 (84%) were HCV RNA positive. Serological screening alone would have diagnosed only 70 infected patients. Therefore, antibody screening combined with detection of HCV RNA should be considered as the “gold standard” for diagnosing HCV infection in dialysis patients. The prevalence of HCV-infected patients in Dutch dialysis centers ranged from 0 to 8%, suggesting the existence of local risk factors for acquiring HCV infection. Genotyping analysis by reverse hybridization line probe assay revealed the presence of genotypes 1a (23%), 1b (46%), 2 (3%), 2a (13%), 2b (1%), 3a (7%), and 4a (4%). In four (6%) samples multiple genotypes were detected. The genotype distribution of HCV isolates among Dutch dialysis patients was similar to the distribution among nondialysis patients from the Benelux, except for subtype 1a, which was significantly more prevalent among dialysis patients. In only one center, a high prevalence of an uncommon genotype was suggestive of infection from a common source.
Isolation of
Lautropia mirabilis
from Oral Cavities of Human Immunodeficiency Virus-Infected Children
Authors: Rossmann Susan Norton , Wilson Patricia H. , Hicks John , Carter Bruce , Cron Stanley G. , Simon Cara , Flaitz Catherine M. , Demmler Gail J. , Shearer William T. , Kline Mark W.
Published: December 2019

Lautropia mirabilis
, a pleomorphic, motile, gram-negative coccus, has been isolated from the oral cavities of 32 of 60 (53.3%) children infected with human immunodeficiency virus (HIV) and 3 of 25 (12.0%) HIV-uninfected controls; the association of
L. mirabilis
isolation with HIV infection is significant (
P
< 0.001). All children in the study, both HIV-infected children and controls, were born to HIV-infected mothers. The presence of this bacterium was not associated with clinical disease in these children. The HIV-infected children with
L. mirabilis
did not differ from the HIV-infected children without
L. mirabilis
in immunological status, clinical status, or systemic medications. The role of HIV infection itself or concomitant factors in the establishment of
L. mirabilis
in the oral cavity remains to be elucidated.
Peritonitis Associated with Vancomycin-Resistant
Lactobacillus rhamnosus
in a Continuous Ambulatory Peritoneal Dialysis Patient: Organism Identification, Antibiotic Therapy, and Case Report
Authors: Klein Günter , Zill Edith , Schindler Ralf , Louwers Jacobus
Published: December 2019

A case of
Lactobacillus rhamnosus
-associated peritonitis in a patient undergoing continuous ambulatory peritoneal dialysis is reported. The patient was treated with vancomycin after isolation of glycopeptide-susceptible coagulase-negative staphylococci. After a skin rash developed, vancomycin was discontinued and replaced with teicoplanin. Seven weeks after the glycopeptide therapy was discontinued, a
Lactobacillus
strain was isolated in pure cultures. The isolate was identified first incorrectly as
L. acidophilus
but later correctly as
L. rhamnosus
. Antibiotic susceptibility testing showed that the isolate was resistant to glycopeptides but susceptible to several other antibiotics. The antibiotic treatment was then switched to imipenem and was successful.
High Human Herpesvirus 8 Seroprevalence in the Homosexual Population in Switzerland
Authors: Regamey Nicolas , Cathomas Gieri , Schwager Martine , Wernli Marion , Harr Thomas , Erb Peter
Published: December 2019

The seroprevalence of human herpesvirus 8 (HHV-8) in the Swiss population was investigated. By enzyme-linked immunosorbent assay, sera reactive to the recombinant HHV-8 antigen orf 65.2 were found in 24% of human immunodeficiency virus (HIV)-positive patients without and in 92% of HIV-positive patients with Kaposi’s sarcoma. Surprisingly, 20% of homosexual HIV-negative men, versus only 7% of heterosexual HIV-negative individuals and 5% of blood donors, had antibodies to HHV-8.