bluuebunny/crossref_metadata_2025 huggingface dataset:
Multisite Reproducibility of Results Obtained by the Broth Microdilution Method for Susceptibility Testing of
Mycobacterium abscessus
,
Mycobacterium chelonae
, and
Mycobacterium fortuitum
Authors: Woods Gail L. , Bergmann John S. , Witebsky Frank G. , Fahle Gary A. , Wanger Audrey , Boulet Betty , Plaunt Marianne , Brown Barbara A. , Wallace Richard J.
Published: December 2019

A multicenter study was conducted to assess the interlaboratory reproducibility of broth microdilution testing of the more common rapidly growing pathogenic mycobacteria. Ten isolates (four
Mycobacterium fortuitum
group, three
Mycobacterium abscessus
, and three
Mycobacterium chelonae
isolates) were tested against amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, sulfamethoxazole, and tobramycin (
M. chelonae
only) in four laboratories. At each site, isolates were tested three times on each of three separate days (nine testing events per isolate) with a common lot of microdilution trays. Agreement among MICs (i.e., mode ± 1 twofold dilution) varied considerably for the different drug-isolate combinations and overall was best for cefoxitin (91.7 and 97.2% for one isolate each and 100% for all others), followed by doxycycline, amikacin, and ciprofloxacin. Agreement based on the interpretive category, using currently suggested breakpoints, also varied and overall was best for doxycycline (97.2% for one isolate and 100% for the rest), followed by ciprofloxacin and clarithromycin. Reproducibility among MICs and agreement by interpretive category was most variable for imipenem. Based on results reported from the individual sites, it appears that inexperience contributed significantly to the wide range of MICs of several drugs, especially clarithromycin, ciprofloxacin, and sulfamethoxazole. New interpretive guidelines are presented for the testing of
M. fortuitum
against clarithromycin;
M. abscessus
and
M. chelonae
against the aminoglycosides; and all three species against cefoxitin, doxycycline, and imipenem.
Effect of Multiple Freeze-Thaw Cycles on Hepatitis B Virus DNA and Hepatitis C Virus RNA Quantification as Measured with Branched-DNA Technology
Authors: Krajden Mel , Minor James M. , Rifkin Oretta , Comanor Lorraine
Published: December 2019

Quantification of hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA often is performed in specimens that have been frozen and thawed more than once. To ensure optimal therapeutic and prognostic value, it is important to establish whether viral load measurements are affected by repeated freeze-thaw (FT) cycles. We therefore evaluated the effect of multiple FT cycles on HBV DNA and HCV RNA quantification by testing serum specimens subjected to one (baseline), two, four, and eight FT cycles with the appropriate Chiron Quantiplex assay. Linear regression analysis showed minor increases of 1.7% per FT cycle for both HBV DNA and HCV RNA. The rise in HCV RNA levels was more pronounced among low-concentration samples, since further analysis revealed an increase of 3.2% per FT cycle among samples with 0.2 to 3.86 Meq of HCV RNA per ml. Given that the coefficient of variation for the Quantiplex assays is generally 10 to 15%, the minor increases in HBV DNA and HCV RNA levels with progressive FT cycles for the specimens tested were recognized only because analysis of variance revealed a statistically significant trend (
P
< 0.05). Due to the minor statistical trend, the clinical impact for individual patient specimens is likely to be limited, but it may deserve further study. In conclusion, the concentration of HBV DNA and HCV RNA in serum specimens subjected to up to eight short-term FT cycles was stable.
VP4 and VP7 Genotyping of Rotavirus Samples Recovered from Infected Children in Ireland over a 3-Year Period
Authors: O’Mahony Jim , Foley Barbara , Morgan Sheila , Morgan John G. , Hill Colin
Published: December 2019

Between September 1995 and August 1998, the incidence and diversity of the main human rotavirus genotypes (G1, G2, G3, and G4 and P[8], P[4], P[6], and P[9]) among Irish children were determined by using established and adapted reverse transcriptase PCR-based genotyping methods. From a total of 193 rotavirus-positive specimens collected from nine hospitals we successfully identified the P type in 182 (94%) of the samples and the G type in 165 (85.5%) of the samples. Only four samples could not be assigned a G or P type. Two P types existed in Ireland, P[8] (78%) and P[4] (16%), and their relative incidence varied over the 3 years of this study. No P[6] or P[9] types were detected. G1 was the most predominant G type (55%), and the incidences of G2, G3, and G4 isolates were 15.5, 1, and 11%, respectively. Three percent of the samples tested had a mixed G type. A P and G type was assigned to 158 (81.8%) of samples. Of the typeable samples, G1 P[8] was the most prevalent (65%), whereas G2 P[4] (17%), G3 P[8] (1%), G4 P[8] (12%), and mixed types (all G1/ G4 P[8]) (4%) were detected less frequently. In the third year a significant genotypic shift from G1 P[8] to G2 P[4] and G4 P[8] was observed. During the study, we noticed that the inclusion of random primers during cDNA synthesis greatly increased the specificity of the PCR typing assays. No correlation was seen between the contributing hospitals and a specific genotype. In conclusion, the coverage of infection given by the recently licensed tetravalent vaccine would be significantly high in Ireland, although future monitoring of genotypic changes among Irish isolates should be encouraged.
Multidrug-Resistant
Streptococcus pneumoniae
in Poland: Identification of Emerging Clones
Authors: Overweg Karin , Hermans Peter W. M. , Trzcinski Krzysztof , Sluijter Marcel , de Groot Ronald , Hryniewicz Waleria
Published: December 2019

Penicillin resistance among
Streptococcus pneumoniae
isolates has rapidly emerged in Poland during the last decade and has reached prevalence levels of up to 14.4% in 1997. In order to investigate the nature of this increase, a molecular epidemiological analysis of non-penicillin-susceptible multidrug-resistant pneumococci isolated in 1995 and 1996 was conducted. Thirty-seven patients who suffered mainly from upper respiratory tract infections and pneumococcal pneumonia were enrolled in this study. The medical centers to which the patients were admitted were located in 16 Polish towns across the country. Eight distinct BOX PCR types were observed, representing 14 subtypes. Restriction fragment end labeling (RFEL) analysis divided the pneumococcal strains into 16 distinct types. By combining the BOX PCR and RFEL data, four genetically distinct clusters of strains were identified. Two clusters represented the genetic clones 23F and 9V, which have recently emerged all over the world. The two other genetic clusters, which represented serotypes 23F and 6B, clearly predominated in the analyzed collection of Polish non-penicillin-susceptible pneumococcal strains. Since the latter clusters did not match any of the 133 RFEL types of non-penicillin-susceptible pneumococci collected in 15 other countries, their Polish clonal origin is most likely.
Molecular Epidemiologic Evaluation of Transmissibility and Virulence of
Mycobacterium tuberculosis
Authors: Rhee Jeanne T. , Piatek Amy S. , Small Peter M. , Harris Lisa M. , Chaparro Sandra V. , Kramer Fred Russell , Alland David
Published: December 2019

Discovery of genotypic markers associated with increased transmissibility in
Mycobacterium tuberculosis
would represent an important step in advancing mycobacterial virulence studies.
M. tuberculosis
strains may be classified into one of three genotypes on the basis of the presence of specific nucleotide substitutions in codon 463 of the
katG
gene (
katG
-463) and codon 95 of the
gyrA
gene (
gyrA
-95). It has previously been reported that two of these three genotypes are associated with increased IS
6110
-based clustering, a potential proxy of virulence. We designed a case-control analysis of U.S.-born patients with tuberculosis in San Francisco, Calif., between 1991 and 1997 to investigate associations between
katG
-463 and
gyrA
-95 genotypes and epidemiologically determined measures of strain-specific infectivity and pathogenicity and IS
6110
-based clustering status. We used a new class of molecular probes called molecular beacons to genotype the isolates rapidly. Infectivity was defined as the propensity of isolates to cause tuberculin skin test conversions among named contacts, and pathogenicity was defined as their propensity to cause active disease among named contacts. The molecular beacon assay was a simple and reproducible method for the detection of known single nucleotide polymorphisms in large numbers of clinical
M. tuberculosis
isolates. The results showed that no genotype of the
katG
-463- and
gyrA
-95-based classification system was associated with increased infectivity and pathogenicity or with increased IS
6110
-based clustering in San Francisco during the study period. We speculate that molecular epidemiologic studies investigating clinically relevant outcomes may contribute to the knowledge of the significance of laboratory-derived virulence factors in the propagation of tuberculosis in human communities.
Simultaneous Detection of Bovine
Theileria
and
Babesia
Species by Reverse Line Blot Hybridization
Authors: Gubbels J. M. , de Vos A. P. , van der Weide M. , Viseras J. , Schouls L. M. , de Vries E. , Jongejan F.
Published: December 2019

A reverse line blot (RLB) assay was developed for the identification of cattle carrying different species of
Theileria
and
Babesia
simultaneously. We included
Theileria annulata
,
T. parva
,
T. mutans
,
T. taurotragi
, and
T. velifera
in the assay, as well as parasites belonging to the
T. sergenti-T. buffeli-T. orientalis
group. The
Babesia
species included were
Babesia bovis
,
B. bigemina
, and
B. divergens
. The assay employs one set of primers for specific amplification of the rRNA gene V4 hypervariable regions of all
Theileria
and
Babesia
species. PCR products obtained from blood samples were hybridized to a membrane onto which nine species-specific oligonucleotides were covalently linked. Cross-reactions were not observed between any of the tested species. No DNA sequences from
Bos taurus
or other hemoparasites (
Trypanosoma
species,
Cowdria ruminantium
,
Anaplasma marginale
, and
Ehrlichia
species) were amplified. The sensitivity of the assay was determined at 0.000001% parasitemia, enabling detection of the carrier state of most parasites. Mixed DNAs from five different parasites were correctly identified. Moreover, blood samples from cattle experimentally infected with two different parasites reacted only with the corresponding species-specific oligonucleotides. Finally, RLB was used to screen blood samples collected from carrier cattle in two regions of Spain.
T. annulata
,
T. orientalis
, and
B. bigemina
were identified in these samples. In conclusion, the RLB is a versatile technique for simultaneous detection of all bovine tick-borne protozoan parasites. We recommend its use for integrated epidemiological monitoring of tick-borne disease, since RLB can also be used for screening ticks and can easily be expanded to include additional hemoparasite species.
Development of a Simple and Highly Sensitive Enzyme Immunoassay for Hepatitis C Virus Core Antigen
Authors: Aoyagi Katsumi , Ohue Chiharu , Iida Kumiko , Kimura Tatsuji , Tanaka Eiji , Kiyosawa Kendo , Yagi Shintaro
Published: December 2019

A highly sensitive enzyme immunoassay (EIA) for the hepatitis C virus (HCV) core antigen (HCVcAg) was developed, and its performance was compared with that of the AMPLICOR HCV test (Roche Molecular Systems). The developed one-step pretreatment method, 30-min incubation of the specimen with a solution containing three different types of detergents (Triton X-100, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate [CHAPS], and sodium dodecyl sulfate), does not require any special device. Because the interfering anti-core antibody in the sample was sufficiently inactivated by the pretreatment, HCVcAg in the sample could be detected. The immunoreactivity on gel filtration was shifted from void fractions to those corresponding to the molecular mass range from 20 to 25 kDa, which is equal to the estimated molecular mass of HCVcAg, after the pretreatment. By the recovery test with HCVcAg-positive serum, the recovery rate was 93.5 to 106.5%. There was no interference with the EIA by anticoagulants or blood components in the serum. When the cutoff value was tentatively set at 0.5 mU/ml based on the distribution of healthy subjects’ sera, the sera of all healthy subjects (
n
= 125) and patients with hepatitis B (
n
= 50) were negative. HCVcAg was detected in sera from 57 of 73 individuals (78.1%) with anti-HCV antibody. Similarly, HCV RNA was detected in sera from 59 individuals (80.8%) with the AMPLICOR HCV as the qualitative test (AMPLICOR HCV test) and in sera from 54 individuals (74.0%) by the AMPLICOR HCV Monitor as the quantitative test (AMPLICOR Monitor test). Concentrations of HCVcAg and HCV RNA (measured by the AMPLICOR Monitor test) correlated significantly (
r
= 0.8,
P
< 0.001). On seroconversion panels, HCVcAg was detected during the early stage of infection, when anti-HCV antibodies had not been produced. This assay for HCVcAg is simpler than assays for HCV RNA based on gene technology and shows specificity and sensitivity equivalent to those of the AMPLICOR HCV test.
Molecular Typing of
Vibrio parahaemolyticus
Isolates, Obtained from Patients Involved in Food Poisoning Outbreaks in Taiwan, by Random Amplified Polymorphic DNA Analysis
Authors: Wong Hin-Chung , Liu Chi-Chang , Pan Tze-Ming , Wang Tien-Kuei , Lee Chih-Lung , Shih Daniel Yang-Chih
Published: December 2019

Vibrio parahaemolyticus
is one of the most important food-borne pathogens in Taiwan, Japan, and other countries with long coastlines. This paper reports on the development of a new random amplified polymorphic DNA (RAPD) method for the molecular typing of this pathogen. The 10-mer primer 284 (5′-CAG GCG CAC A-3′) was selected to generate polymorphic amplification profiles of the genomic DNA at an annealing temperature of 38°C. A total of 308 clinical isolates of
V. parahaemolyticus
collected during food poisoning outbreaks in Taiwan, mostly occurring between 1993 and 1995, plus 11 environmental and clinical reference strains were analyzed by this RAPD method. A total of 41 polymorphic RAPD patterns were recognized, and these patterns were arbitrarily grouped into 16 types (A to P). Types A, B, C, D, and E were the major types, and subtypes C3, C5, E1, B1, D2, and A2 were the major patterns. The major types were phylogenetically more closely related to each other than to any of the minor types.
Use of PCR in Diagnosis of Human American Tegumentary Leishmaniasis in Rio de Janeiro, Brazil
Authors: Pirmez Claude , da Silva Trajano Valéria , Neto Manoel Paes-Oliveira , da-Cruz Alda Maria , Gonçalves-da-Costa Sylvio Celso , Catanho Marcos , Degrave Wim , Fernandes Octavio
Published: December 2019

In Brazil, the most common etiological agent of American tegumentary leishmaniasis isLeishmania(Viannia)braziliensis. In general, diagnostic techniques envisage the visualization of the parasite, but that technique has a low sensitivity. The main purpose of the present work was to evaluate the PCR as a routine tool for the diagnosis of leishmaniasis. Biopsy specimens from cutaneous or mucosal lesions were taken from 230 individuals from areas whereLeishmaniais endemic: 216 patients who had a clinical picture suggestive ofl leishmaniasis and 14 individuals with cutaneous lesions due to other causes. Each specimen was processed for histopathologic examination, culture, touch preparation, and DNA isolation. Oligonucleotides that amplify the conserved region of the minicircle molecules ofLeishmaniawere used in a hot-start PCR. While at least one conventional technique was positive forLeishmaniafor 62% (134 of 216) of the patients, PCR coupled to hybridization was positive for 94% (203 of 216) of the patients. The 14 patients whose clinical picture was not suggestive of leishmaniasis had negative results by all techniques. The impact of the PCR was striking in mucosal disease. While the disease in only 17% (4 of 24) of the patients could be diagnosed by conventional techniques, PCR was positive for 71% (17 of 24) of the patients. Hybridization showed that all cases of disease were caused by parasites belonging to theVianniasubgenus. Altogether, the results indicate that PCR is a valuable tool for the diagnosis of leishmaniasis on a routine basis and is likely to provide valuable epidemiological information about the disease in countries where it is endemic.
Prevalence of Corynebacterial 16S rRNA Sequences in Patients with Bacterial and “Nonbacterial” Prostatitis
Authors: Tanner Michael A. , Shoskes Daniel , Shahed Asha , Pace Norman R.
Published: December 2019

The etiology of chronic prostatitis syndromes in men is controversial, particularly when positive cultures for established uropathogens are lacking. Although identification of bacteria in prostatic fluid has relied on cultivation and microscopy, most microorganisms in the environment, including some human pathogens, are resistant to cultivation. We report here on an rRNA-based molecular phylogenetic approach to the identification of bacteria in prostate fluid from prostatitis patients. Positive bacterial signals were seen for 65% of patients with chronic prostatitis overall. Seven of 11 patients with bacterial signals but none of 6 patients without bacterial signals were cured with antibiotic-based therapy. Results indicate the occurrence in the prostate fluid of a wide spectrum of bacterial species representing several genera. Most rRNA genes were closely related to those of species belonging to the genera
Corynebacterium
,
Staphylococcus
,
Peptostreptococcus
,
Streptococcus
, and
Escherichia
. Unexpectedly, a wide diversity of
Corynebacterium
species was found in high proportion compared to the proportions of other bacterial species found. A subset of these 16S rRNA sequences represent those of undescribed species on the basis of their positions in phylogenetic trees. These uncharacterized organisms were not detected in control samples, suggesting that the organisms have a role in the disease or are the consequence of the disease. These studies show that microorganisms associated with prostatitis generally occur as complex microbial communities that differ between patients. The results also indicate that microbial communities distinct from those associated with prostatitis may occur at low levels in normal prostatic fluid.
Highly Reproducible Bactericidal Activity Test Results by Using a Modified National Committee for Clinical Laboratory Standards Broth Macrodilution Technique
Authors: Hacek Donna M. , Dressel Dana C. , Peterson Lance R.
Published: December 2019

Bactericidal testing historically has exhibited variable reproducibility, even when prior standardized methods were employed. Several modifications to the National Committee for Clinical Laboratory Standards (NCCLS) broth macrodilution method are proposed to improve reproducibility. Recommended changes from the approved NCCLS guidelines (M21-A and M26-A) include omitting serum supplementation of Mueller-Hinton broth, incubating tubes at 35°C for 24 h with no agitation until they are sampled, running all tests in duplicate with six dilutions instead of nine, reincubating the test for an additional 24 h to resolve discrepant bactericidal activity test results, using a single 0.1-ml sample from each clear tube for subculture, and adopting an alternate method for calculating endpoint determination. In order to test these recommendations in a clinical laboratory setting, we used the modified methodology on 224 separate tests for bactericidal activity. There were 102 serum bactericidal titer (SBT) and 122 minimum bactericidal concentration (MBC) assays performed. By defining reproducibility as agreement between duplicate tests ± 1 dilution, we found 207 of 224 tests (92%) were reproducible at the 24-h subculture point (94% for the SBT assay and 91% for the MBC assay). When the 17 assays with discrepant results were incubated an additional 24 h for a second subculture, only 1 of 224 tests (0.4%) remained discrepant. The method used is practical for a clinical laboratory that chooses to perform bactericidal activity testing and assures a high level of reproducibility between duplicate assays. The total cost of a test was approximately $25.00.
Quantitative Detection of
Borrelia burgdorferi
by Real-Time PCR
Authors: Pahl Andreas , Kühlbrandt Uta , Brune Kay , Röllinghoff Martin , Gessner André
Published: December 2019

Currently, no easy and reliable methods allowing for the quantification of
Borrelia burgdorferi
in tissues of infected humans or animals are available. Due to the lack of suitable assays to detect
B. burgdorferi
CFU and the qualitative nature of the currently performed PCR assays, we decided to exploit the recently developed real-time PCR. This technology measures the release of fluorescent oligonucleotides during the PCR. Flagellin of
B. burgdorferi
was chosen as the target sequence. A linear quantitative detection range of 5 logs with a calculated detection limit of one to three spirochetes per assay reaction mixture was observed. The fact that no signals were obtained with closely related organisms such as
Borrelia hermsii
argues for a high specificity of this newly developed method. A similar method was developed to quantify mouse actin genomic sequences to allow for the standardization of spirochete load. The specificity and sensitivity of the
B. burgdorferi
and the actin real-time PCR were not altered when samples were spiked with mouse cells or spirochetes, respectively. To evaluate the applicability of the real-time PCR, we used the mouse model of Lyme disease. The fate of
B. burgdorferi
was monitored in different tissues from inbred mice and from mice treated with antibiotics. Susceptible C3H/HeJ mice had markedly higher burdens of bacterial DNA than resistant BALB/c mice, and penicillin G treatment significantly reduced the numbers of spirochetes. Since these results show a close correlation between clinical symptoms and bacterial burden of tissues, we are currently analyzing human biopsy specimens to evaluate the real-time PCR in a diagnostic setting.
Rapid and Reliable Identification of
Streptococcus pneumoniae
Isolates by Pneumolysin-Mediated Agglutination
Authors: Cima-Cabal M. D. , Vázquez F. , de los Toyos J. R. , Méndez F. J.
Published: December 2019

A pneumolysin-based agglutination test which allows an easy, rapid, cost-effective, and accurate (100% specific and 95% sensitive) discrimination between pneumococci and other related human and animal pathogenic bacterial strains has been assayed.
Comparison of Three Commercial Systems for Identification of Yeasts Commonly Isolated in the Clinical Microbiology Laboratory
Authors: Wadlin Jill K. , Hanko Gayle , Stewart Rebecca , Pape John , Nachamkin Irving
Published: December 2019

We evaluated three commercial systems (RapID Yeast Plus System; Innovative Diagnostic Systems, Norcross, Ga.; API 20C Aux; bioMerieux-Vitek, Hazelwood, Mo.; and Vitek Yeast Biochemical Card, bioMerieux-Vitek) against an auxinographic and microscopic morphologic reference method for the ability to identify yeasts commonly isolated in our clinical microbiology laboratory. Two-hundred one yeast isolates were compared in the study. The RapID Yeast Plus System was significantly better than either API 20C Aux (193 versus 167 correct identifications;
P
< 0.0001) or the Vitek Yeast Biochemical Card (193 versus 173 correct identifications;
P
= 0.003) for obtaining correct identifications to the species level without additional testing. There was no significant difference between results obtained with API 20C Aux and the Vitek Yeast Biochemical Card system (
P
= 0.39). The API 20C Aux system did not correctly identify any of the
Candida krusei
isolates (
n
= 23) without supplemental testing and accounted for the major differences between the API 20C Aux and RapID Yeast Plus systems. Overall, the RapID Yeast Plus System was easy to use and is a good system for the routine identification of clinically relevant yeasts.
PCR Detection of
Yersinia pestis
in Fleas: Comparison with Mouse Inoculation
Authors: Engelthaler David M. , Gage Kenneth L. , Montenieri John A. , Chu May , Carter Leon G.
Published: December 2019

The “gold standard” for identifying
Yersinia pestis
-infected fleas has been inoculation of mice with pooled flea material. Inoculated mice are monitored for 21 days, and those that die are further analyzed for
Y. pestis
infection by fluorescent-antibody assay and/or culture. PCR may provide a more rapid and sensitive alternative for identifying
Y. pestis
in fleas. To compare these assays, samples were prepared from 381 field-collected fleas. Each flea was analyzed individually by both PCR and mouse inoculation. Sixty of the 381 flea samples were positive for
Y. pestis
by PCR; 48 of these PCR-positive samples caused death in mice (80.0% agreement). None of the 321 PCR-negative samples caused death. Among the 12 mice that survived inoculation with PCR-positive samples, 10 were later demonstrated by serology or culture to have been infected with
Y. pestis
. This suggests that death of inoculated mice is less reliable than PCR as an indicator of the presence of
Y. pestis
in flea samples. Mouse inoculation assays produce results that are comparable to PCR only when surviving as well as dead mice are analyzed for infection. The rapidity and sensitivity (10 to 100 CFU of
Y. pestis
) of PCR suggest that it could serve as a useful alternative to mouse inoculation for routine plague surveillance and outbreak investigations.
Two Cases of
Chromobacterium violaceum
Infection after Injury in a Subtropical Region
Authors: Lee J. , Kim J. S. , Nahm C. H. , Choi J. W. , Kim J. , Pai S. H. , Moon K. H. , Lee K. , Chong Y.
Published: December 2019

Chromobacterium violaceum
is a gram-negative rod and is isolated from soil and water in tropical and subtropical regions. The species have pigmented and nonpigmented colony types. Infections caused by nonpigmented strains are rare. We report on two cases of infection caused by both pigmented and nonpigmented strains of
C. violaceum
. Two 24-year-old Korea Airline stewardesses were admitted to Inha University Hospital, Inchon, South Korea, on 9 August 1997, 3 days after an airplane accident in Guam. Both had multiple lacerations on exposed parts of their bodies. There was swelling, tenderness, and pus discharge. The wounds contained many small fragments of stones and weeds. A pigmented strain was isolated from the left hand and a nonpigmented strain was isolated from the left knee of one patient. For the other patient only a nonpigmented strain was isolated from a foot wound. The nonpigmented colonies from the left-knee and the left-foot wounds did not produce any pigment even after an extended period of incubation. The biochemical characteristics were the same for each strain except for oxidase and indole reactions. The pigmented strain was oxidase negative and indole positive, whereas the nonpigmented strains were oxidase positive and indole negative. The patients were successfully treated by debridement and with appropriate antibiotics.
Borrelia burgdorferiin Tick Cell Culture Modulates Expression of Outer Surface Proteins A and C in Response to Temperature
Authors: Obonyo Marygorret , Munderloh Ulrike G. , Fingerle Volker , Wilske Bettina , Kurtti Timothy J.
Published: December 2019

The Lyme disease spirocheteBorrelia burgdorferisensu stricto downregulates outer surface protein A (OspA) and upregulates outer surface protein C (OspC) during tick feeding. The switching of these proteins correlates with increased spirochetal infectivity for the mammal. We examined the effect of temperature on differential expression of OspA and OspC byB. burgdorfericocultivated with a cell line isolated from the vector tickIxodes scapularis. The effect of incubation at 31, 34, or 37°C on expression of OspA and OspC byB. burgdorferiJMNT and N40 was analyzed by indirect fluorescent-antibody microscopy, polyacrylamide gel electrophoresis, and immunoblotting. The amount of OspA relative to the amount of flagellin was highest in spirochetes cocultivated with tick cells at 31°C and declined with increasing temperature in both strains. OspC production was enhanced in spirochetes cocultivated with tick cells at 37°C. Spirochetes grown axenically in BSK-H medium also produced more OspC at 37°C, but OspA content was not appreciably affected by temperature. Our findings indicate that temperature, along with cultivation in a tick cell culture system, plays a role in the differential expression of OspA and enhances differential expression of OspC by spirochetes.
Detection and Identification ofEhrlichia,Borrelia burgdorferiSensu Lato, andBartonellaSpecies in DutchIxodes ricinusTicks
Authors: Schouls Leo M. , Van De Pol Ingrid , Rijpkema Sjoerd G. T. , Schot Corrie S.
Published: December 2019

A sensitive and specific PCR hybridization assay was developed for the simultaneous detection and identification ofEhrlichiaandBorrelia burgdorferisensu lato. In separate assays the 16S rRNA gene ofEhrlichiaspecies and the 23S-5S rRNA spacer region ofB. burgdorferisensu lato were amplified and labeled by PCR. These PCR products were used in a reverse line blot hybridization assay in which oligonucleotide probes are covalently linked to a membrane in parallel lines. Hybridization of the samples with the oligonucleotide probes on this membrane enabled the simultaneous detection and identification ofEhrlichia,B. burgdorferi, andBartonellaspecies in 40 different samples. The application of the assay to DNA extracts from 121Ixodes ricinusticks collected from roe deer demonstrated that 45% of these ticks carriedEhrlichiaDNA. More than half of these positive ticks carried species with 16S rRNA gene sequences closely related to those ofE. phagocytophilaand the human granulocytic ehrlichiosis agent. The majority of the other positive ticks were infected with a newly identifiedEhrlichia-like species. In addition, 13% of the ticks were infected with one or moreB. burgdorferigenospecies. In more than 70% of the ticks 16S rRNA gene sequences forBartonellaspecies or other species closely related toBartonellawere found. In five of the ticks bothEhrlichiaandB. burgdorferispecies were detected.
Relationship between
Helicobacter pylori iceA
,
cagA
, and
vacA
Status and Clinical Outcome: Studies in Four Different Countries
Authors: Yamaoka Yoshio , Kodama Tadashi , Gutierrez Oscar , Kim Jong G. , Kashima Kei , Graham David Y.
Published: December 2019

There is continuing interest in identifying
Helicobacter pylori
virulence factors that might predict the risk for symptomatic clinical outcomes. It has been proposed that
iceA
and
cagA
genes are such markers and can identify patients with peptic ulcers. We compared
H. pylori
isolates from four countries, looking at the
cagA
and
vacA
genotypes,
iceA
alleles, and presentation of the infection. We used PCR to examine
iceA
,
vacA
, and
cagA
status of 424
H. pylori
isolates obtained from patients with different clinical presentations (peptic ulcer, gastric cancer, and atrophic gastritis). The
H. pylori
isolates examined included 107 strains from Bogota, Colombia, 70 from Houston, Tex., 135 from Seoul, Korea, and 112 from Kyoto, Japan. The predominant genotype differed among countries: the
cagA
-positive
iceA1 vacA
s1c-m1 genotype was predominant in Japan and Korea, the
cagA
-positive
iceA2 vacA
s1b-m1 genotype was predominant in the United States, and the
cagA
-positive
iceA2 vacA
s1a-m1 genotype was predominant in Colombia. There was no association between the
iceA
,
vacA
, or
cagA
status and clinical outcome in patients in the countries studied.
iceA
status shows considerable geographic differences, and neither
iceA
nor combinations of
iceA
,
vacA
, and
cagA
were helpful in predicting the clinical presentation of an
H. pylori
infection.
Evaluation of Mycology Laboratory Proficiency Testing
Authors: Reilly Andrew A. , Salkin Ira F. , McGinnis Michael R. , Gromadzki Sally , Pasarell Lester , Kemna Maggi , Higgins Nancy , Salfinger Max
Published: December 2019

Changes over the last decade in overt proficiency testing (OPT) regulations have been ostensibly directed at improving laboratory performance on patient samples. However, the overt (unblinded) format of the tests and regulatory penalties associated with incorrect values allow and encourage laboratorians to take extra precautions with OPT analytes. As a result OPT may measure optimal laboratory performance instead of the intended target of typical performance attained during routine patient testing. This study addresses this issue by evaluating medical mycology OPT and comparing its fungal specimen identification error rates to those obtained in a covert (blinded) proficiency testing (CPT) program. Identifications from 188 laboratories participating in the New York State mycology OPT from 1982 to 1994 were compared with the identifications of the same fungi recovered from patient specimens in 1989 and 1994 as part of the routine procedures of 88 of these laboratories. The consistency in the identification of OPT specimens was sufficient to make accurate predictions of OPT error rates. However, while the error rates in OPT and CPT were similar for
Candida albicans
, significantly higher error rates were found in CPT for
Candida tropicalis
,
Candida glabrata
, and other common pathogenic fungi. These differences may, in part, be due to OPT’s use of ideal organism representatives cultured under optimum growth conditions. This difference, as well as the organism-dependent error rate differences, reflects the limitations of OPT as a means of assessing the quality of routine laboratory performance in medical mycology.
Evaluation of Statens Serum Institut Enteric Medium for Detection of Enteric Pathogens
Authors: Blom Marianne , Meyer Aase , Gerner-Smidt Peter , Gaarslev Knud , Espersen Frank
Published: December 2019

The efficacy of the Statens Serum Institut (SSI) enteric medium for isolation and direct identification of enteric pathogens was evaluated. Six different biochemical reactions can be read by using the SSI enteric medium, allowing direct identification of a range of enteric pathogens. All 248 gram-negative bacterial species that were tested grew on the SSI enteric medium. Only 10 of 248 bacteria (4%) showed discrepant results in the biochemical reactions, and none of these were enteric pathogens. Forty-three of 47 enteric pathogens (92%) produced identical rates of semiquantitative growth on the SSI enteric medium and 5% blood agar, whereas three
Vibrio
spp. and one
Aeromonas
spp. showed reduced growth. Gram-positive bacteria did not grow on the SSI enteric medium. Most enteric pathogens had a detection limit of 50 bacteria per ml of feces, but higher numbers of
Vibrio
spp. and some
Shigella
spp. were required for detection. The growth rates of 125 enteric pathogens and 12
Yersinia
spp. on the SSI enteric medium, xylose lysine deoxycholate (XLD), Hektoen enteric (HE),
Salmonella-Shigella
(SS), and cefsulodin-irgasan-novobiocin (CIN) agar were compared. Detection rates after application of 200 CFU were 99% for SSI enteric medium, 92% for XLD, 88% for HE, and 82% for SS agar. The 12
Yersinia
spp. grew excellently on both the SSI enteric medium and CIN agar. We conclude that the performance of the SSI enteric medium compares favorably to those of other media tested. Its ability to detect
Yersinia
spp. may limit the number of media needed in the typical laboratory. The direct identification of enteric pathogens on the medium may also provide a more rapid diagnosis.
Salmonella entericaSerotype Dublin Infection: an Emerging Infectious Disease for the Northeastern United States
Authors: McDonough Patrick L. , Fogelman David , Shin Sang J. , Brunner Michael A. , Lein Donald H.
Published: December 2019

Salmonella entericasubspeciesentericaserotype Dublin (S. entericaDublin) emerged for the first time in New York, Pennsylvania, and Ohio in 1988. Since that time this host-adapted serotype has spread throughout the veal- and dairy beef-raising operations in the region; very few dairy farms have experienced clinicalS. entericaDublin infections. This study details the epidemiology of the outbreaks in cattle. During the period 1988 through 1995, nine New York and four Pennsylvania counties have been affected; 13 different locations were involved in New York, and 10 were involved in Pennsylvania. The morbidity and mortality and seasonal distribution of outbreaks, which totaled 35, is described. The antimicrobial susceptibility pattern of isolates revealed that many of the strains were resistant to a number of commonly used drugs. Clinical case details and pathology information are provided, with a caution to clinicians and microbiologists presented with suspect animals, i.e., most cases occurred in older calves, which is atypical for salmonellosis for this region (calves were 8 or more weeks old) and presented as pneumonia and septicemia rather than the primarily diarrheal syndrome that is more typically recognized for the region. The epidemiology of cases is analyzed through cluster analysis of bacterial isolates and their fatty acid methyl ester profiles; at least six clones appeared in the region during the study period. Results of the epidemiology analysis are used to support a hypothesis regarding the source ofS. entericaDublin for the region and its manner of dissemination.
Helicobacter pylori
Infection in Indigenous Families of Central America: Serostatus and Oral and Fingernail Carriage
Authors: Dowsett S. A. , Archila L. , Segreto V. A. , Gonzalez C. R. , Silva A. , Vastola K. A. , Bartizek R. D. , Kowolik M. J.
Published: December 2019

Helicobacter pylori
infection remains one of the most common in humans, but the route of transmission of the bacterium is still uncertain. This study was designed to elucidate possible sources of infection in an isolated, rural population in Guatemala. A total of 242 subjects in family units participated in the study. A medical history, including a history of dyspepsia, was taken by a physician and immunoglobulin G antibodies to
H. pylori
were detected with the QuickVue (Quidel, San Diego, Calif.) onsite serology test. Overall, 58% of subjects were seropositive, with a positive relationship between mother and child (
P
= 0.02) and a positive correlation between the serostatuses of siblings (intraclass correlation coefficient = 0.63). There was no association between serostatus and gastric symptoms. Oral
H. pylori
was detected from periodontal pockets of various depths and the dorsum of the tongue by nested PCR. Eighty-seven percent of subjects had at least one oral site positive for
H. pylori
, with the majority of subjects having multiple positive sites. There was no association between periodontal pocket depth and the detection of
H. pylori
. Nested PCR was also used to detect
H. pylori
from beneath the nail of the index finger of each subject’s dominant hand. Overall, 58% of subjects had a positive fingernail result, with a significant positive relationship between fingernail and tongue positivity (
P
= 0.002). In conclusion, the results of this study suggest that oral carriage of
H. pylori
may play a role in the transmission of infection and that the hand may be instrumental in transmission.
Development and Clinical Evaluation of a Highly Sensitive PCR-Reverse Hybridization Line Probe Assay for Detection and Identification of Anogenital Human Papillomavirus
Authors: Kleter Bernhard , van Doorn Leen-Jan , Schrauwen Lianne , Molijn Anco , Sastrowijoto Suprapto , ter Schegget Jan , Lindeman Jan , ter Harmsel Bram , Burger Matthé , Quint Wim
Published: December 2019

Human papillomavirus (HPV) can be detected by amplification of viral DNA. A novel PCR primer set generating a short PCR fragment (SPF PCR) was used for amplification of a fragment of only 65 bp from the L1 region and permitted ultrasensitive detection of a broad spectrum of HPV genotypes. The intra- and intertypic sequence variations of the 22-bp interprimer region of this amplimer were studied. Among 238 HPV sequences from GenBank and clinical specimens, HPV genotypes were correctly identified based on the 22-bp sequence in 232 cases (97.2%). Genotype-specific probes for HPV genotypes 6, 11, 16, 18, 31, 33 to 35, 39, 40, 42 to 45, 51 to 54, 56, 58, 59, 66, 68, 70, and 74 were selected, and a reverse hybridization line probe assay (LiPA) (the INNO-LiPA HPV prototype research assay) was developed. This LiPA permits the use of amplimers generated by the SPF as well as the MY 09/11 primers. The assay was evaluated with a total of 1,354 clinical specimens, comprising cervical scrapes (classifications ranging from normal cytology to severe dyskaryosis) and formalin-fixed, paraffin-embedded cervical carcinoma samples. LiPA results were highly concordant with sequence analysis of the SPF amplimer, genotype-specific PCR, and sequence analysis of amplimers generated by MY 09/11 primers. The sensitivity of the SPF primers was higher than that of the GP5
+
/6
+
primers over a broad range of HPV types, especially when multiple HPV genotypes were present. In conclusion, the SPF LiPA method allows extremely sensitive detection of HPV DNA as well as reliable identification of HPV genotypes in both cervical smears and paraffin-embedded materials.
Detection of Dengue Virus RNA in Patients after Primary or Secondary Dengue Infection by Using the TaqMan Automated Amplification System
Authors: Laue Thomas , Emmerich Petra , Schmitz Herbert
Published: December 2019

In consecutive serum samples from 25 tourists with acute dengue fever, virus-specific RNA was detected by using fully automated TaqMan reverse transcriptase PCR. For this amplification technique new primers and special fluorochrome-labeled probes had to be synthesized. During amplification the increasing amount of viral DNA could simultaneously be measured in the tightly sealed tubes. Dengue virus RNA was found in almost all patients (17 of 18), if the samples had been taken soon after the onset of symptoms and before anti-dengue virus antibody had been produced. RNA was detectable in only one of five persons who had anti-dengue virus immunoglobulin M (IgM) antibodies but not yet IgG antibodies. In 30 late samples with both IgG and IgM antibodies viral RNA was no longer demonstrable. In two early samples from two frequent travelers obtained 1 and 2 days after the onset of symptoms significant IgG antibody titers were present but there were no anti-dengue virus IgM antibodies. In these samples a viral load of >5 × 10
6
dengue virus RNA copies (dengue types 1 and 2) was detectable. These findings of a high viral load in the presence of anti-dengue virus IgG antibody are suggestive of a secondary dengue virus infection. In the 20 tourists (17 plus 1 plus 2) in whom viral RNA was found, the dengue virus serotype could be related to the area where the infection had taken place. Most of our patients came from southeast Asia and most frequently had dengue virus type 1 infections (8 of 20).
Development of Calibrated Viral Load Standards for Group M Subtypes of Human Immunodeficiency Virus Type 1 and Performance of an Improved AMPLICOR HIV-1 MONITOR Test with Isolates of Diverse Subtypes
Authors: Michael Nelson L. , Herman Steven A. , Kwok Shirley , Dreyer Kimberly , Wang June , Christopherson Cindy , Spadoro Joanne P. , Young Karen K. Y. , Polonis Victoria , McCutchan Francine E. , Carr Jean , Mascola John R. , Jagodzinski Linda L. , Robb Merlin L.
Published: December 2019

Accurate determination of plasma human immunodeficiency virus type 1 (HIV-1) RNA levels is critical for the effective management of HIV-1 disease. The AMPLICOR HIV-1 MONITOR Test, a reverse transcription-PCR-based test for quantification of HIV-1 RNA in plasma, was developed when little sequence information on HIV-1 isolates from outside North America was available. It has since become apparent that many non-subtype B isolates, particularly subtypes A and E, are detected inefficiently by the test. We describe here the AMPLICOR HIV-1 MONITOR Test, version 1.5, an upgraded test developed to minimize subtype-related variation. We also developed a panel of HIV-1 standards containing 30 HIV-1 isolates of subtypes A through G. The virus particle concentration of each cultured viral stock was standardized by electron microscopic virus particle counting. We used this panel to determine the performance of the original AMPLICOR HIV-1 MONITOR Test and version 1.5 of the test with HIV-1 subtypes A through G. The original test underestimated the concentration of HIV-1 subtype A, E, F, and G RNA by 10-fold or more, whereas version of the 1.5 test yielded equivalent quantification of HIV-1 RNA regardless of the subtype. In light of the increasing intermixing of HIV-1 subtypes worldwide, standardization of PCR-based tests against well-characterized viral isolates representing the full range of HIV-1 diversity will be essential for the continued utility of these important clinical management tools.
Identification of Two Novel
Mycobacterium avium
Allelic Variants in Pig and Human Isolates from Brazil by PCR-Restriction Enzyme Analysis
Authors: Leão Sylvia Cardoso , Briones Marcelo R. S. , Sircili Marcelo Palma , Balian Simone Carvalho , Mores Nelson , Ferreira-Neto José Soares
Published: December 2019

Mycobacterium avium
complex (MAC) is composed of environmental mycobacteria found widely in soil, water, and aerosols that can cause disease in animals and humans, especially disseminated infections in AIDS patients. MAC consists of two closely related species,
M. avium
and
M. intracellulare
, and may also include other, less-defined groups. The precise differentiation of MAC species is a fundamental step in epidemiological studies and for the evaluation of possible reservoirs for MAC infection in humans and animals. In this study, which included 111 pig and 26 clinical MAC isolates, two novel allelic
M. avium
PCR-restriction enzyme analysis (PRA) variants were identified, differing from the
M. avium
PRA prototype in the
Hae
III digestion pattern. Mutations in
Hae
III sites were confirmed by DNA sequencing. Identification of these isolates as
M. avium
was confirmed by PCR with DT1-DT6 and IS
1245
primers, nucleic acid hybridization with the AccuProbe system, 16S ribosomal DNA sequencing, and biochemical tests. The characterization of
M. avium
PRA variants can be useful in the elucidation of factors involved in mycobacterial virulence and routes of infection and also has diagnostic significance, since they can be misidentified as
M. simiae
II and
M. kansasii
I if the PRA method is used in the clinical laboratory for identification of mycobacteria.
Identification of Human Rotavirus Strains with the P[14] Genotype by PCR
Authors: Arista Serenella , Vizzi Esmeralda , Alaimo Cristina , Palermo Daniela , Cascio Antonio
Published: December 2019

A seminested PCR typing assay has been extended to identify rotavirus strains with the P[14] genotype. The specificity of the method was confirmed by Southern hybridization and by restriction analysis with the enzyme
Alu
I. One out of four human rotavirus (HRV) strains with unusual subgroup-electropherotype linkage but none out of 50 HRV strains with usual linkage was typed as P[14].