bluuebunny/crossref_metadata_2025 huggingface dataset:
Usefulness of Spoligotyping To Discriminate IS
6110
Low-Copy-Number
Mycobacterium tuberculosis
Complex Strains Cultured in Denmark
Authors: Bauer Jeanett , Andersen Åse B. , Kremer Kristin , Miörner Håkan
Published: December 2019

Mycobacterium tuberculosis
complex strains cultured in Denmark have been analyzed by IS
6110
restriction fragment length polymorphism (RFLP) on a routine basis from 1992 and onwards. Due to the influx of immigrants with tuberculosis, the number of strains harboring only one to five copies of IS
6110
has increased steadily. Since the discriminatory power of IS
6110
fingerprinting for such strains is poor, we have performed additional genotyping of all low-copy-number strains by the recently described PCR-based method known as spoligotyping. A total of 311 clinical strains were typed: 14
Mycobacterium bovis
BCG, 48
M. bovis
, and 249
M. tuberculosis
strains. Spoligotyping correctly differentiated
M. bovis
and
M. bovis
BCG from
M. tuberculosis
strains, but it did not differentiate
M. bovis
from
M. bovis
BCG. All
M. bovis
BCG strains exhibited identical spoligotype patterns. The discriminatory power of spoligotyping of low-copy-number
M. tuberculosis
strains was higher than that of IS
6110
fingerprinting. Based on RFLP typing solely, 83% of the low-copy-number
M. tuberculosis
strains were found to form part of a cluster, and 75% were found to form a cluster on the basis of spoligotyping. When the two techniques were combined, the amount of clustering decreased to 55%. The combination of these two techniques might be valuable in studying the epidemiology of
M. tuberculosis
strains harboring few copies of the IS
6110
element.
Improved Detection of Hepatitis B Virus Surface Antigen by a New Rapid Automated Assay
Authors: Weber Bernard , Bayer Anja , Kirch Peter , Schlüter Volker , Schlieper Dietmar , Melchior Walter
Published: December 2019

The performance of hepatitis B virus (HBV) surface antigen (HBsAg) screening assays is continuously improved in order to reduce the residual risk of transfusion-associated hepatitis B. In a multicenter study, a new automated rapid screening assay, Elecsys HBsAg (Roche Diagnostics), was compared to well-established tests (Auszyme Monoclonal [overnight incubation] version B and IMx HBsAg [Abbott]). Included in the evaluation were 23 seroconversion panels; sera from the acute and chronic phases of infection; dilution series of various HBsAg standards, HBV subtypes, and S gene mutants; and isolated anti-HBV core antigen-positive samples. To challenge the specificity of the new assay, sera from HBsAg-negative blood donors, pregnant women, and dialysis and hospitalized patients and potentially cross-reactive samples were investigated. Elecsys HBsAg showed a higher sensitivity for HBsAg subtypes ad, ay, adw2, adw4, ayw1, ayw2, ayw4, and adr detection in dilution series of different standards or sera than Auszyme Monoclonal version B and/or IMx HBsAg. Acute hepatitis B was detected in 11 to 16 of 23 seroconversion panels between 2 and 16 days earlier with Elecsys HBsAg than with the alternative assays. Elecsys HBsAg and Auszyme Monoclonal version B detected HBsAg surface mutants with equal sensitivity. The sensitivity and specificity of Elecsys HBsAg were 100%. Auszyme Monoclonal version B had a 99.9% specificity, and its sensitivity was 96.6%. IMx HBsAg showed a poorer sensitivity and specificity than the other assays. In conclusion, Elecsys HBsAg permits earlier detection of acute hepatitis B and different HBV subtypes than the alternative assays. By using highly sensitive HBsAg screening assays, low-level HBsAg carriers among isolated anti-HBV core antigen-positive individuals can be detected.
Mutations in the
rpoB
Gene of Rifampin-Resistant
Mycobacterium tuberculosis
Strains Isolated Mostly in Asian Countries and Their Rapid Detection by Line Probe Assay
Authors: Hirano Kazue , Abe Chiyoji , Takahashi Mitsuyoshi
Published: December 2019

Mutations in the
rpoB
gene of 90 rifampin-resistant
Mycobacterium tuberculosis
isolates mostly from Asian countries were analyzed. Ten distinct single-nucleotide substitutions were found among the isolates by automated sequencing. A 3-nucleotide insertion was found in two isolates, and no mutation was found in five isolates (5.6%). A reverse hybridization-based line probe assay (INNO-LiPA Rif TB) for rapid detection of the mutations was evaluated with these isolates. Concordance rates with sequencing results for five wild-type probes (S probes) and four probes for specific mutations (R probes) were 96.7 and 100%, respectively. The overall concordance rate with the in vitro susceptibility testing results was 92.2% (83 of 90 isolates). These results indicate that a commercial line probe assay kit may be useful for rapid diagnosis of rifampin-resistant tuberculosis.
Detection of
Staphylococcus aureus
and
Staphylococcus epidermidis
in Clinical Samples by 16S rRNA-Directed In Situ Hybridization
Authors: Krimmer Vanessa , Merkert Hilde , von Eiff Christof , Frosch Matthias , Eulert Jochen , Löhr Jochen F. , Hacker Jörg , Ziebuhr Wilma
Published: December 2019

Staphylococcus epidermidis
and
Staphylococcus aureus
are the most common causes of medical device-associated infections, including septicemic loosenings of orthopedic implants. Frequently, the microbiological diagnosis of these infections remains ambiguous, since at least some staphylococci have the capacity to reduce their growth rate considerably. These strains exhibit a small-colony phenotype, and often they are not detectable by conventional microbiological techniques. Moreover, clinical isolates of
S. aureus
and
S. epidermidis
adhere to polymer and metal surfaces by the generation of thick, multilayered biofilms consisting of bacteria and extracellular polysaccharides. This study reports improved detection and identification of
S. aureus
and
S. epidermidis
by an in situ hybridization method with fluorescence-labeled oligonucleotide probes specific for staphylococcal 16S rRNA. The technique has proven to be suitable for the in situ detection of staphylococci, which is illustrated by the identification of
S. epidermidis
in a connective tissue sample obtained from a patient with septicemic loosening of a hip arthroplasty. We also show that this technique allows the detection of intracellularly persisting bacteria, including small-colony variants of
S. aureus
, and the differentiation of
S. epidermidis
from other clinically relevant staphylococci even when they are embedded in biofilms. These results suggest that the 16S rRNA in situ hybridization technique could represent a powerful diagnostic tool for the detection and differentiation of many other fastidious microorganisms.
Use of Granada Medium To Detect Group B Streptococcal Colonization in Pregnant Women
Authors: Rosa-Fraile Manuel , Rodriguez-Granger Javier , Cueto-Lopez Marina , Sampedro Antonio , Gaye Enrique Biel , Haro José Manuel , Andreu Antonia
Published: December 2019

Direct inoculation onto Granada medium (GM) in plates and tubes was compared to inoculation into a selective Todd-Hewitt broth (with 8 μg of gentamicin per ml and 15 μg of nalidixic acid per ml) for detection of group B streptococci (GBS) in pregnant women with 800 vaginal and 450 vaginoanorectal samples. Comparatively, GM was found to be as sensitive as the selective broth for the detection of GBS in vaginal specimens and more sensitive than selective broth for the detection of GBS in vaginoanorectal samples (96 versus 82%). The use of GM improved the time to reporting of a GBS-positive result by at least 24 h and reduced the direct cost of screening. We have also found that the inconvenience of anaerobic incubation of GM plates can be avoided when a cover slide is placed upon the inoculum, because aerobic incubation in GM plates with cover slides causes GBS to develop the same pigmentation that it develops with incubation under anaerobic conditions. These data support the routine use of GM plates or tubes as a more accurate, easier, and cheaper method of identification of GBS-colonized women compared to the enrichment broth technique.
Versatile Fluorescent Staining of Fungi in Clinical Specimens by Using the Optical Brightener Blankophor
Authors: Rüchel R. , Schaffrinski Meike
Published: December 2019

Fluorescent staining of fungi in clinical specimens with the optical brightener Blankophor can be performed concomitantly with maceration of surrounding tissue and may be accelerated by heating. The procedure is suitable for disclosing fungi in gram-stained microscopical mounts and can be used for screening of tissue sections prior to immunofluorescence.
Indigenous Disseminated
Penicillium marneffei
Infection in the State of Manipur, India: Report of Four Autochthonous Cases
Authors: Singh P. Narendra , Ranjana K. , Singh Y. Indiver , Singh K. Priyokumar , Sharma S. Surchandra , Kulachandra M. , Nabakumar Y. , Chakrabarti A. , Padhye A. A. , Kaufman L. , Ajello L.
Published: December 2019

We describe four cases of disseminated infection caused by endemic
Penicillium marneffei
in human immunodeficiency virus (HIV)-infected patients from the Manipur state of India. The most common clinical features observed were fever, anorexia, weight loss, hepatosplenomegaly, and, more importantly, skin lesions resembling molluscum contagiosum. The diagnosis in each of the four cases was achieved by direct examination of smears, observance of intracellular yeast-like cells multiplying by fission in biopsied tissue from skin lesions, and isolation of the dimorphic
P. marneffei
in pure culture in each case. In one case, fluorescent antibody studies allowed specific diagnosis. This report documents a new area in which
P. marneffei
is endemic, located in eastern India, and describes the first occurrence in India of
P. marneffei
in HIV-infected patients as well as the extension of the areas of
P. marneffei
endemicity westward to the northeastern state of Manipur.
TT Virus Infection Is Widespread in the General Populations from Different Geographic Regions
Authors: Abe Kenji , Inami Tomoko , Asano Kazue , Miyoshi Chiaki , Masaki Naohiko , Hayashi Shigeki , Ishikawa Ko-ichi , Takebe Yutaka , Win Khin Maung , El-Zayadi Abdel Rahman , Han Kwang-Hyub , Zhang David Y.
Published: December 2019

By PCR screening, we found an extremely high prevalence of TT virus (TTV) in the general populations from different geographic regions. This suggests that TTV may be a common DNA virus with no clear disease association in humans. TTV genotyping by phylogenetic analysis was also performed.
An Uncommon
Helicobacter
Isolate from Blood: Evidence of a Group of
Helicobacter
spp. Pathogenic in AIDS Patients
Authors: Weir Susan C. , Gibert Cynthia L. , Gordin Fred M. , Fischer Steven H. , Gill Vee J.
Published: December 2019

An unusual
Helicobacter
sp. was isolated from the blood of a human immunodeficiency virus (HIV)-infected patient. This organism had spiral morphology, with single amphitrichous flagella, and was negative for hippurate hydrolysis, production of urease, and reduction of nitrate. 16S rRNA gene sequence analysis verified that the isolate was a species of
Helicobacter
, most closely related to an undescribed
Helicobacter
-like isolate from Vancouver, British Columbia, Canada, and to
Helicobacter westmeadii
, a recently described species from Australia. Both organisms had also been isolated from the blood of HIV-infected patients. These blood isolates, along with
Helicobacter cinaedi
, form a cluster of closely related
Helicobacter
spp. that may represent an emerging group of pathogens in immunocompromised patients.
Detection of Porcine Rotavirus Type G9 and of a Mixture of Types G1 and G5 Associated with Wa-Like VP4 Specificity: Evidence for Natural Human-Porcine Genetic Reassortment
Authors: Santos Norma , Lima Rita C. C. , Nozawa Carlos M. , Linhares Rosa E. , Gouvea Vera
Published: December 2019

Rotavirus type G5 is a primarily porcine pathogen that has caused frequent and widespread diarrhea in children in Brazil and in piglets elsewhere. Initial results on the rotavirus types circulating in diarrheic piglets in Brazil disclosed a high diversity of strains with distinct G types including G1, G4, G5, and G9 and the novelty of P[8], the predominant human P specificity type. Those results add strong evidence for the emergence of new strains through natural reassortment between rotaviruses of human and porcine origins.
Outbreak of Mupirocin-Resistant Staphylococci in a Hospital in Warsaw, Poland, Due to Plasmid Transmission and Clonal Spread of Several Strains
Authors: Łe˛ski Tomasz A. , Gniadkowski Marek , Skoczyńska Anna , Stefaniuk Elz˙bieta , Trzciński Krzysztof , Hryniewicz Waleria
Published: December 2019

An outbreak of mupirocin-resistant (MuR) staphylococci was investigated in two wards of a large hospital in Warsaw, Poland. Fifty-three MuR isolates of
Staphylococcus aureus
,
S. epidermidis
,
S. haemolyticus
,
S. xylosus
, and
S. capitis
were identified over a 17-month survey which was carried out after introduction of the drug for the treatment of skin infections. The isolates were collected from patients with infections, environmental samples, and carriers; they constituted 19.5% of all staphylococcal isolates identified in the two wards during that time. Almost all the MuR isolates were also resistant to methicillin (methicillin-resistant
S. aureus
and methicillin-resistant coagulase-negative staphylococci). Seven of the outbreak isolates expressed a low-level-resistance phenotype (MuL), whereas the remaining majority of isolates were found to be highly resistant to mupirocin (MuH). The
mupA
gene, responsible for the MuH phenotype, has been assigned to three different polymorphic loci among the strains in the collection analyzed. The predominant polymorph, polymorph I (characterized by a
mupA
-containing
Eco
RI DNA fragment of about 16 kb), was located on a specific plasmid which was widely distributed among the entire staphylococcal population. All MuR
S. aureus
isolates were found to represent a single epidemic strain, which was clonally disseminated in both wards. The
S. epidermidis
population was much more diverse; however, at least four clusters of closely related isolates were identified, which suggested that some strains of this species were also clonally spread in the hospital environment. Six isolates of
S. epidermidis
were demonstrated to express the MuL and MuH resistance mechanisms simultaneously, and this is the first identification of such dual MuR phenotype-bearing strains. The outbreak was attributed to a high level and inappropriate use of mupirocin, and as a result the dermatological formulation of the drug has been removed from the hospital formulary.
Prospective Cohort Study of EnterotoxigenicEscherichia coliInfections in Argentinean Children
Authors: Viboud Gloria I. , Jouve Mabel J. , Binsztein Norma , Vergara Marta , Rivas Marta , Quiroga Marina , Svennerholm Ann-Mari
Published: December 2019

In a follow-up study, enterotoxigenicEscherichia coli(ETEC) infections in 145 children from two communities located in northeastern Argentina were monitored for 2 years. The occurrence of diarrhea was monitored by weekly household visits. Of 730 fecal specimens collected, 137 (19%) corresponded to diarrheal episodes. ETEC was isolated from a significantly higher proportion of symptomatic (18.3%) than asymptomatic (13.3%) children (P= 0.04541). Individuals of up to 24 months of age were found to have a higher risk of developing ETEC diarrhea than older children (odds ratio [OR], 3.872;P= 0.00021). When the toxin profiles were considered, only heat stable enterotoxin (ST)-producing ETEC was directly associated with diarrhea (P= 0.00035). Fifty-five percent of the ETEC isolated from symptomatic children and 19% of the ETEC isolated from asymptomatic children expressed one of the colonization factors (CFs) investigated, i.e., CF antigen I (CFA/I), CFA/II, CFA/III, and CFA/IV; coli surface antigens CS7 and CS17; and putative CFs PCFO159, PCFO166, and PCFO20, indicating a clear association between diarrhea and ETEC strains that carry these factors (P= 0.0000034). The most frequently identified CFs were CFA/IV (16%), CFA/I (10%), and CS17 (9%). CFs were mostly associated with ETEC strains that produce ST and both heat-labile enterotoxin and ST. Logistic regression analysis, applied to remove confounding effects, revealed that the expression of CFs was associated with illness independently of the toxin type (OR, 4.81;P= 0.0003). When each CF was considered separately, CS17 was the only factor independently associated with illness (OR, 16.6;P= 0.0151). Most CFs (the exception was CFA/IV) fell within a limited array of serotypes, while the CF-negative isolates belonged to many different O:H types. These results demonstrate that some CFs are risk factors for the development of ETEC diarrhea.
Comparison of PCR, Culture, and Direct Fluorescent-Antibody Testing for Detection of
Bordetella pertussis
Authors: Loeffelholz Mike J. , Thompson Curt J. , Long Karla S. , Gilchrist Mary J. R.
Published: December 2019

We prospectively compared the performance of culture, direct fluorescent-antibody testing (DFA), and an in-house-developed PCR test targeting the repeated insertion sequence IS
481
for the detection of
Bordetella pertussis
in nasopharyngeal swab specimens. We tested 319 consecutive paired specimens on which all three tests were performed. A total of 59 specimens were positive by one or more tests. Of these, 5 were positive by all three tests, 2 were positive by culture and PCR, 16 were positive by PCR and DFA, 28 were positive by PCR only, and 8 were positive by DFA only. Any specimen positive by culture was considered to be a true positive, as were specimens positive by both PCR and DFA. Specimens positive only by PCR or DFA were considered discrepant, and their status was resolved by review of patient histories. Patients with symptoms meeting the Centers for Disease Control and Prevention clinical case definition for pertussis and who had a specimen positive by PCR or DFA were considered to have true
B. pertussis
infections. Of the 28 patients positive by PCR only, 20 met the clinical case definition for pertussis, while 3 of the 8 patients positive by DFA only met the clinical case definition. After resolution of the status of discrepant specimens, the sensitivity, specificity, positive predictive value, and negative predictive value were 15.2, 100, 100, and 87.5%, respectively, for culture; 93.5, 97.1, 84.3, and 98.9%, respectively, for PCR; and 52.2, 98.2, 82.8, and 92.4%, respectively, for DFA. The actual positive predictive value of PCR was probably greater, as several PCR-positive patients who did not meet the clinical case definition had symptoms consistent with typical or atypical pertussis. PCR is a sensitive and specific method for the detection of
B. pertussis.
Value of Prenatal Diagnosis and Early Postnatal Diagnosis of Congenital Toxoplasmosis: Retrospective Study of 110 Cases
Authors: Robert-Gangneux Florence , Gavinet Marie-Françoise , Ancelle Thierry , Raymond Josette , Tourte-Schaefer Claudine , Dupouy-Camet Jean
Published: December 2019

We reviewed the files of 110 women with
Toxoplasma
seroconversion during pregnancy. Prenatal diagnosis was attempted for 94 women by amniotic fluid sampling.
Toxoplasma gondii
was detected by PCR, with or without tissue culture and mouse inoculation. The early neonatal diagnostic procedure included placental testing by PCR and/or mouse inoculation, cord blood serological testing, and comparison of maternal and newborn antibodies by Western blotting (WB). Serological follow-up of the infants was conducted during the first year of life or until the diagnosis of congenital toxoplasmosis (CT) could be ruled out. Congenital infection was diagnosed in 27 individuals (20 live births) in the prenatal and/or neonatal period. The sensitivity and specificity of prenatal diagnosis were 81 and 100%, respectively. Placental examination was positive for 66.7% of individuals with CT and was always negative for neonates without CT. Cord blood serology detected immunoglobulin M (IgM) and/or IgA in 80% of infected newborns, with respective specificities of 91.2 and 87.7%. By WB we detected bands on IgG and IgM blots recognized by the newborn serum but not by the maternal serum (neosynthesized IgG and/or IgM) for 88.2% of infected infants within the first 2 months of life with a specificity of 100%. Early postnatal diagnosis was negative for 2 of the 20 neonates with CT. Both of these newborns had a negative prenatal diagnosis and were asymptomatic, suggesting a very low parasite load. In conclusion, despite the use of advanced methods, some cases of congenital toxoplasmosis cannot be detected early, which underlines the importance of careful follow-up of newborns who are at risk.
Evaluation of Reference Dilution Test Methods for Antimicrobial Susceptibility Testing of
Pseudomonas aeruginosa
Strains Isolated from Patients with Cystic Fibrosis
Authors: Saiman Lisa , Burns Jane L. , Whittier Susan , Krzewinski Jay , Marshall Steve A. , Jones Ronald N.
Published: December 2019

The development of multidrug-resistant
Pseudomonas aeruginosa
in patients with cystic fibrosis (CF) is most likely a consequence of increasing life expectancy and more prolonged exposure to antibiotics. The optimal method for antibiotic susceptibility testing of CF strains, particularly mucoid
P. aeruginosa
strains, is unknown. Antimicrobial susceptibilities of 48 CF strains (25 mucoid) and 50 non-CF strains to 12 anti-
Pseudomonas
agents were tested by both agar dilution and commercially custom-prepared broth microdilution plates (PML Microbiologicals, Portland, Oreg.) in three laboratories simultaneously to determine if broth microdilution could substitute for agar dilution as the reference method in subsequent studies. Comparison of MICs generated by agar dilution and broth microdilution demonstrated correlation coefficients (
r
) exceeding 0.85 for all agents tested; correlation was excellent for aminoglycosides (
r
≥ 0.92) and very good for β-lactam agents including agents paired with a β-lactamase inhibitor (
r
≥ 0.87) and for ciprofloxacin (
r
= 0.86). Correlation was not improved by 48-h readings, but correlation between 24- and 48-h readings ranged between 0.91 and 0.98 for both methods. Interlaboratory variations were minimal, as the percentage of acceptable variations was 94% for both methods, and serious discords were infrequent (<2% of comparisons). However, CF strains were more likely to have serious discords than were non-CF strains (
P
< 0.0001), although mucoid strains were not more likely to have serious discords than were nonmucoid strains. In this study, MICs determined by custom-prepared broth microdilution compared favorably with MICs determined by agar dilution. Thus, this broth microdilution assay can serve as a reference method and facilitate future studies to determine the optimal method for antibiotic susceptibility testing of CF strains.
Detection of thePlasmodium falciparumAntigen Histidine-Rich Protein 2 in Blood of Pregnant Women: Implications for Diagnosing Placental Malaria
Authors: Leke Rose F. G. , Djokam Rosine R. , Mbu Robinson , Leke Robert J. , Fogako Josephine , Megnekou Rosette , Metenou Simon , Sama Grace , Zhou Yuan , Cadigan Timothy , Parra Marcela , Taylor Diane Wallace
Published: December 2019

Pregnant women have an increased susceptibility to infection byPlasmodium falciparum. Parasites may be present in the placenta yet not detectable in peripheral blood smears by routine light microscopy. In order to determine how frequently misdiagnosis occurs, peripheral blood and placental samples were collected from 1,077 Cameroonian women at the time of giving birth and examined for the presence of malarial parasites by using light microscopy. Results showed that 20.1% of the women who had placental malaria were peripheral blood smear negative. Thus, malarial infection was not detected by microscopic examination of peripheral blood smears from approximately one out of five malaria-infected women. SinceP. falciparumparasites secrete histidine-rich protein 2 (HRP-2), we sought to determine if detecting HRP-2 in either peripheral plasma or whole blood might be used to diagnose the presence of parasites “hidden” in the placenta. Samples of peripheral plasma from 127 women with different levels of placental malarial infection were assayed by HRP-2-specific enzyme-linked immunosorbent assay. HRP-2 was detected in 88% of the women with placental malaria who tested negative by blood smear. Additionally, whole blood was obtained from 181 women and tested for HRP-2 with a rapid, chromatographic strip test (ICT). The ICT test accurately detected malarial infection in 89.1% ofP. falciparum-infected women. Furthermore, 94% of women with malaria were accurately diagnosed by using a combination of microscopy and the ICT test. Thus, detection of HRP-2 in conjunction with microscopy should improve diagnosis of malaria in pregnant women.
Relevance of CagA Positivity to Clinical Course of
Helicobacter pylori
Infection in Children
Authors: Yahav J. , Fradkin A. , Weisselberg B. , Diver-Haver A. , Shmuely H. , Jonas A.
Published: December 2019

A potential virulence determinant of
Helicobacter pylori
is the
cagA
gene product. To determine the relevance of the expression of CagA to the clinical picture and outcome of
H. pylori
infection in children, we examined 104 consecutive children diagnosed with
H. pylori
infection. Serum samples were collected to test for the presence of immunoglobulin G (IgG) anti-CagA antibodies. Forty-five patients (43%) had antibodies to the CagA protein (group I), and 59 did not (group II). Seropositive patients had a longer prediagnostic history of abdominal pain (
P
= 0.02), more severe abdominal pain (defined as ulcer pain) (
P
= 0.05), a higher prevalence of duodenal ulcer (38 versus 7%;
P
< 0.01), more active chronic gastritis (82 versus 32%;
P
< 0.001), and a higher titer of serum IgG anti-
H. pylori
antibodies (
P
< 0.001). Ninety percent of the patients were monitored for 27 ± 18 months. On multivariate analysis, CagA-negative patients had a 3.8-fold-higher chance of achieving a disease-free state than CagA-positive patients (95% confidence interval, 1.5- to 9.5-fold). We conclude that infection with CagA-producing strains of
H. pylori
is a risk factor for severe clinical disease and ongoing infection.
Genotyping of Hepatitis C Virus Isolates using CLIP Sequencing
Authors: Ross R. S. , Viazov S. O. , Holtzer C. D. , Beyou A. , Monnet A. , Mazure C. , Roggendorf M.
Published: December 2019

Determination of hepatitis C virus (HCV) genotypes and subtypes has become increasingly important for the clinical management and prognosis of HCV infections. The aim of the present study was to assess the specificity and reliability of a newly developed, commercially available HCV genotyping kit (TRUGENE
HCV 5′NC
genotyping kit). This technique utilizes PCR fragments previously generated by the diagnostic Roche AMPLICOR HCV test, which are subsequently subjected to simultaneous PCR amplification and direct sequencing (CLIP sequencing) of the 5′ noncoding region (5′NCR). HCV isolates from 100 randomly chosen patients were genotyped by both the TRUGENE
HCV 5′NC
genotyping kit and DNA enzyme immunoassay (DEIA). Typing results obtained by both methods were in complete concordance in 91% of the cases. HCV RNA from the samples with discordant genotype assignment in both assays was additionally amplified with primers from the HCV core and NS5B regions. Phylogenetic analysis of the obtained sequences supported the results obtained from DEIA in six cases and CLIP sequencing in two cases. In the former six cases, the TRUGENE
HCV 5′NC
genotyping kit could not correctly differentiate between subtypes of genotypes 1 and 2 due to the high conservation of the 5′NCR. However, since there was not any misclassification between HCV genotypes 1 and non-1 types, the results obtained with this system are, in general, reliable and can be used in clinical practice. The TRUGENE
HCV 5′NC
genotyping kit in our hands proved to be a fast and convenient technique that might be an attractive option for HCV genotyping in laboratories already using the Roche AMPLICOR HCV test for diagnostic reverse transcription-PCR.
Comparative Immunoglobulin G Antibody Profiles between Mother and Child (CGMC Test) for Early Diagnosis of Congenital Toxoplasmosis
Authors: Gross Uwe , Lüder Carsten G. K. , Hendgen Vera , Heeg Cornelia , Sauer Irmtraud , Weidner Andrea , Krczal Doris , Enders Gisela
Published: December 2019

Early diagnosis of congenital toxoplasmosis is rendered difficult when specific immunoglobulin M (IgM) and/or IgA antibodies are absent in the blood of the newborn infant. Since maternal IgG antibodies can cross the placenta, determination of IgG antibodies in newborn infants has hitherto not been used routinely for the diagnosis of congenital infection. The aim of this study was to assess the diagnostic usefulness of an immunoblot assay which compares the early IgG profiles between the mother and her child (comparative IgG profile between mother and child; CGMC test) directed against a total cell lysate of
Toxoplasma gondii
tachyzoites. Serum samples from 97 newborn infants at risk of toxoplasma infection were obtained from umbilical cord blood at birth or postnatally until 3 months of life and were directly compared with serum samples from the respective mothers. Congenital toxoplasmosis was diagnosed only when IgG-reactive protein bands that were present in any newborn serum samples were absent in the corresponding maternal serum sample. Congenital infection was defined by conventional serological assays when IgM and/or IgA antibodies were present in newborn infant blood or when IgG titers rose within the first 12 months or were persistently stable for more than 8 months. Using these criteria, congenital infection was definitely confirmed in 11 cases. Three additional cases were diagnosed based on indicative data. The CGMC test, which was performed without knowledge of the results of conventional serologal assays, had sensitivity and specificity of 82.4 and 93.0%, respectively, and positive and negative predictive values of 73.7 and 95.7%, respectively. When true positives and true negatives were considered, the comparative IgG profile had a ratio of 90.9% true results. The CGMC test thus is useful as an additional assay for the rapid diagnosis of congenital toxoplasmosis when paired serum samples from mother and child are available.
Helicobacter pylori
: Clonal Population Structure and Restricted Transmission within Families Revealed by Molecular Typing
Authors: Han Shan-Rui , Zschausch Hans-Christoph E. , Meyer Heinz-Georg W. , Schneider Thomas , Loos Michael , Bhakdi Sucharit , Maeurer Markus J.
Published: December 2019

Helicobacter pylori
infects up to 50% of the human population worldwide. The infection occurs predominantly in childhood and persists for decades or a lifetime.
H. pylori
is believed to be transmitted from person to person. However, tremendous genetic diversity has been reported for these bacteria. In order to gain insight into the epidemiological basis of this phenomenon, we performed molecular typing of
H. pylori
isolates from different families. Fifty-nine
H. pylori
isolates from 27 members of nine families were characterized by using restriction fragment length polymorphism analysis of five PCR-amplified genes, by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA, and by
vacA
and
cagA
genotyping. The 16S rRNA gene exhibited little allelic variation, as expected for a unique bacterial species. In contrast, the
vacA
,
flaA
,
ureAB
, and
lspA-glmM
genes were highly polymorphic, with a mean genetic diversity of 0.83, which exceeds the levels recorded for all other bacterial species. In conjunction with PFGE, 59
H. pylori
isolates could be differentiated into 21 clonal types. Each individual harbored only one clone, occasionally with a clonal variant. Identical strains were always found either between siblings or between a mother and her children. Statistical analysis revealed clonality of population structure in all isolates. The results of this study suggest the possible coexistence of a large array of clonal lineages that are evolving in each individual in isolation from one another. Transmission appears to occur primarily from mother to child and perhaps between siblings.
Genotypic Determination of
Mycobacterium tuberculosis
Antibiotic Resistance Using a Novel Mutation Detection Method, the Branch Migration Inhibition
M. tuberculosis
Antibiotic Resistance Test
Authors: Liu Y. P. , Behr M. A. , Small P. M. , Kurn N.
Published: December 2019

A novel method for the detection of any alteration within a defined sequence has recently been demonstrated (A. Lishanski, N. Kurn, and E. F. Ullman, Nucleic Acids Res. 28:E42, 2000; A. Lishanski, Clin. Chem. 46:9, 2000). Essential to this method are the generation of partial duplexes that are capable of forming four-stranded structures and the ability to detect inhibition of branch migration in these structures (I. G. Panyutin and P. Hsieh, J. Mol. Biol. 230:413–424, 1993). Inhibition of branch migration indicates the presence of sequence alteration. This mutation detection method, termed branch migration inhibition (BMI), is suitable for the detection of drug resistance in
M. tuberculosis
, which is frequently associated with multiple mutations within known genes. We describe the genotypic determination of the rifampin (RMP) and pyrazinamide (PZA) susceptibilities of
M. tuberculosis
isolates, using BMI coupled with the luminescence oxygen channeling immunoassay (LOCI) (E. F. Ullman et al., Proc. Natl. Acad. Sci. USA 91:5426–5430, 1994). RMP and PZA resistances are associated with multiple mutations within the
rpoB
and
pncA
genes, respectively.
M. tuberculosis
genomic DNA samples prepared from 46 clinical isolates were used for genotypic determination of RMP resistance in a “blind study.” Similarly, PZA resistance was determined using genomic DNA samples prepared from 37 clinical isolates. Full agreement of the genotypic and phenotypic determinations of drug susceptibility was demonstrated. RMP susceptibility determination directly from cells of 10 clinical isolates grown in culture was also demonstrated. The genotypic result of only 1 out of 10 isolates did not agree with the phenotypic susceptibility testing result. Sequence analysis of the
rpoB
gene of this clinical isolate revealed a single base substitution, most likely a silent point mutation. The new BMI-LOCI mutation detection method is a rapid and accurate procedure for the genotypic determination of the RMP and PZA susceptibilities of
M. tuberculosis
clinical isolates. BMI can also be detected by using commercially available automated enzyme-linked immunosorbent assay plate formats (Lishanski et al., Nucleic Acids Res. 28:E42, 2000).
Helicobacter aurati
sp. nov., a Urease-Positive
Helicobacter
Species Cultured from Gastrointestinal Tissues of Syrian Hamsters
Authors: Patterson M. M. , Schrenzel M. D. , Feng Y. , Xu S. , Dewhirst F. E. , Paster B. J. , Thibodeau S. A. , Versalovic J. , Fox J. G.
Published: December 2019

A novel helicobacter with the proposed name
Helicobacter aurati
(type strain MIT 97-5075c) has been isolated from the inflamed stomachs and ceca of adult Syrian hamsters. The new species is fusiform with multiple bipolar sheathed flagella and periplasmic fibers; it contains urease and gamma-glutamyl transpeptidase. By 16S rRNA sequencing and repetitive element PCR-based DNA fingerprinting, it was found that
H. aurati
represents a distinct taxon and clusters with
Helicobacter muridarum
,
Helicobacter hepaticus
, and
Helicobacter
sp. MIT 94-022.
H. aurati
was recovered from hamsters housed in various research and vendor facilities. Further studies are necessary to define its association with disease and other microbiota in hamsters, as well as its impact on research projects involving hamsters.
H. aurati
(GenBank accession number
AF297868
) can be used in animal experiments to define the factors that are important for gastric helicobacter pathogenesis.
Class 1 Integron-Borne, Multiple-Antibiotic Resistance Encoded by a 150-Kilobase Conjugative Plasmid in Epidemic
Vibrio cholerae
O1 Strains Isolated in Guinea-Bissau
Authors: Dalsgaard Anders , Forslund Anita , Petersen Andreas , Brown Derek J. , Dias Francisco , Monteiro Serifo , Mølbak Kåre , Aaby Peter , Rodrigues Amabelia , Sandström Anita
Published: December 2019

In the 1996–1997 cholera epidemic in Guinea-Bissau, surveillance for antimicrobial resistance showed the emergence of a multidrug-resistant strain of
Vibrio cholerae
O1 during the course of the epidemic. The strain was resistant to ampicillin, erythromycin, tetracycline, furazolidone, aminoglycosides, trimethoprim, and sulfamethoxazole. Concomitant with the emergence of this strain, we observed a resurgence in the number of registered cholera cases as well as an increase in the case fatality rate from 1.0% before the emergence of the multiple-drug-resistant strain to 5.3% after the emergence of the strain. Our study shows that the strain contained a 150-kb conjugative multiple-antibiotic resistance plasmid with class 1 integron-borne gene cassettes encoding resistance to trimethoprim (
dhfrXII
) and aminoglycosides [
ant
(
3"
)-
1a
]). The finding of transferable resistance to almost all of the antibiotics commonly used to treat cholera is of great public health concern. Studies should be carried out to determine to what extent the strain or its resistance genes have been spread to other areas where cholera is endemic.
Prevalence of Enterotoxin Genes in
Aeromonas
spp. Isolated From Children with Diarrhea, Healthy Controls, and the Environment
Authors: Albert M. John , Ansaruzzaman M. , Talukder Kaisar A. , Chopra Ashok K. , Kuhn Inger , Rahman Motiur , Faruque A. S. G. , Islam M. Sirajul , Sack R. Bradley , Mollby Roland
Published: December 2019

Aeromonads are causative agents of a number of human infections. Even though aeromonads have been isolated from patients suffering from diarrhea, their etiological role in gastroenteritis is unclear. In spite of a number of virulence factors produced by
Aeromonas
species, their association with diarrhea has not been clearly linked. Recently, we have characterized a heat-labile cytotonic enterotoxin (Alt), a heat-stable cytotonic enterotoxin (Ast), and a cytotoxic enterotoxin (Act) from a diarrheal isolate of
Aeromonas hydrophila
. Alt and Ast are novel enterotoxins which are not related to cholera toxin; Act is aerolysin related and has hemolytic, cytotoxic, and enterotoxic activities. We studied the distribution of the
alt
,
ast
, and
act
enterotoxin genes in 115 of 125 aeromonads isolated from 1,735 children with diarrhea, in all 27 aeromonads isolated from 830 control children (
P
= 7 × 10
−4
for comparison of rates of isolation of aeromonads from cases versus those from controls), and in 120 randomly selected aeromonads from different components of surface water in Bangladesh.
Aeromonas
isolates which were positive only for the presence of the
alt
gene had similar distributions in the three sources; the number of isolates positive only for the presence of the
ast
gene was significantly higher for the environmental samples than for samples from diarrheal children; and isolates positive only for the presence of the
act
gene were not found in any of the three sources. Importantly, the number of isolates positive for both the
alt
and
ast
genes was significantly higher for diarrheal children than for control children and the environment. Thus, this is the first study to indicate that the products of both the
alt
and
ast
genes may synergistically act to induce severe diarrhea. In 26 patients,
Aeromonas
spp. were isolated as the sole enteropathogen. Analysis of clinical data from 11 of these patients suggested that isolates positive for both the
alt
and
ast
genes were associated with watery diarrhea but that isolates positive only for the
alt
gene were associated with loose stools. Most of the isolates from the three sources could be classified into seven phenospecies and eight hybridization groups. For the first time,
Aeromonas eucrenophila
was isolated from two children, one with diarrhea and another without diarrhea.
Distribution of a Specific 500-Base-Pair Fragment in
Mycobacterium bovis
Isolates from Sardinian Cattle
Authors: Sechi Leonardo A. , Duprè Ilaria , Leori Guido , Fadda Giovanni , Zanetti Stefania
Published: December 2019

Amplification of a specific, 500-bp fragment from
Mycobacterium bovis
isolates and use of the fragment to differentiate between
Mycobacterium tuberculosis
and
M. bovis
was previously reported (J. G. Rodriguez, G. A. Meja, P. Del Portillo, M. E. Patarroyo, and L. A. Murillo, Microbiology 141:2131–2138, 1995). In the present study, 30
M. bovis
isolates from Sardinian cattle were examined for the presence of this 500-bp fragment; 4 of the 30 isolates lacked the fragment. This result indicates that identification of
M. bovis
strains by amplification of the 500-bp sequence may lead to false-negative results.
G Protein Variation in Respiratory Syncytial Virus Group A Does Not Correlate with Clinical Severity
Authors: Brandenburg A. H. , van Beek R. , Moll H. A. , Osterhaus A. D. M. E. , Claas E. C. J.
Published: December 2019

Respiratory syncytial virus group A strain variations of 28 isolates from The Netherlands collected during three consecutive seasons were studied by analyzing G protein sequences. Several lineages circulated repeatedly and simultaneously during the respective seasons. No relationships were found between lineages on the one hand and clinical severity or age on the other.
Absence of Serotype-Specific Surface Antigen and Altered Teichoic Acid Glycosylation among Epidemic-Associated Strains of
Listeria monocytogenes
Authors: Clark Elizabeth E. , Wesley Irene , Fiedler Franz , Promadej Nattawan , Kathariou Sophie
Published: December 2019

Outbreaks of food-borne listeriosis have often involved strains of serotype 4b. Examination of multiple isolates from three different outbreaks revealed that ca. 11 to 29% of each epidemic population consisted of strains which were negative with the serotype-specific monoclonal antibody c74.22, lacked galactose from the teichoic acid of the cell wall, and were resistant to the serotype 4b-specific phage 2671.
Identification of
Enterococcus
Species and Phenotypically Similar
Lactococcus
and
Vagococcus
Species by Reverse Checkerboard Hybridization to Chaperonin 60 Gene Sequences
Authors: Goh Swee Han , Facklam Richard R. , Chang Michelle , Hill Janet E. , Tyrrell Gregory J. , Burns Emma C. M. , Chan David , He Cheng , Rahim Tazim , Shaw Carol , Hemmingsen Sean M.
Published: December 2019

Data from four recent studies (S. H. Goh et al., J. Clin. Microbiol. 36:2164–2166, 1998; S. H. Goh et al., J. Clin. Microbiol. 34:818–823, 1996; S. H. Goh et al., J. Clin. Microbiol. 35:3116–3121, 1997; A. Y. C. Kwok et al., Int. J. Syst. Bacteriol. 49:1181–1192, 1999) suggest that an approximately 600-bp region of the chaperonin 60 (Cpn60) gene, amplified by PCR with a single pair of degenerate primers, has utility as a potentially universal target for bacterial identification (ID). This Cpn60 gene ID method correctly identified isolates representative of numerous staphylococcal species and
Streptococcus iniae
, a human and animal pathogen. We report herein that this method enabled us to distinguish clearly between 17
Enterococcus
species (
Enterococcus asini
,
Enterococcus rattus
,
Enterococcus dispar
,
Enterococcus gallinarum
,
Enterococcus hirae
,
Enterococcus durans
,
Enterococcus cecorum
,
Enterococcus faecalis
,
Enterococcus mundtii
,
Enterococcus casseliflavus
,
Enterococcus faecium
,
Enterococcus malodoratus
,
Enterococcus raffinosus
,
Enterococcus avium
,
Enterococcus pseudoavium
,
Enterococcus
new sp. strain Facklam, and
Enterococcus saccharolyticus
), and
Vagococcus fluvialis
,
Lactococcus lactis
, and
Lactococcus garvieae
. From 123 blind-tested samples, only two discrepancies were observed between the Facklam and Collins phenotyping method (R. R. Facklam and M. D. Collins, J. Clin. Microbiol. 27:731–734, 1989) and the Cpn60 ID method. In each case, the discrepancies were resolved in favor of the Cpn60 ID method. The species distributions of the 123 blind-tested isolates were
Enterococcus
new sp. strain Facklam (ATCC 700913), 3;
E. asini
, 1;
E. rattus
, 4;
E. dispar
, 2;
E. gallinarum
, 20;
E. hirae
, 9;
E. durans
, 9;
E. faecalis
, 12;
E. mundtii
, 3;
E. casseliflavus
, 8;
E. faecium
, 25;
E. malodoratus
, 3;
E. raffinosus
, 8;
E. avium
, 4;
E. pseudoavium
, 1; an unknown
Enterococcus
clinical isolate, sp. strain R871;
Vagococcus fluvialis
, 4;
Lactococcus garvieae
, 3;
Lactococcus lactis
, 3;
Leuconostoc
sp., 1; and
Pediococcus
sp., 1. The Cpn60 gene ID method, coupled with reverse checkerboard hybridization, is an effective method for the identification of
Enterococcus
and related organisms.
Detection of Cytomegalovirus DNA in Human Specimens by LightCycler PCR
Authors: Schaade Lars , Kockelkorn Peter , Ritter Klaus , Kleines Michael
Published: December 2019

Detection of human cytomegalovirus (CMV) DNA in clinical specimens is considered a cornerstone in the diagnosis of CMV disease. The aim of this study was to evaluate a newly designed LightCycler-based quantitative CMV PCR. Specimens of human origin (
n
= 200) were tested using the LightCycler PCR, the quantitative COBAS AMPLICOR CMV MONITOR (CACM) assay, and a qualitative in-house PCR assay for the presence of CMV DNA. Samples that were reactive in at least two of the three assays were considered CMV DNA positive (
n
= 95 [47.5%]), while samples that were nonreactive in two of the three assays were considered CMV DNA negative (
n
= 105 [52.5%]). Using the LightCycler assay, CMV DNA was detected in 91 of the 95 CMV DNA-positive human specimens (sensitivity, 95.8%; 95% confidence interval [CI], 89.6 to 98.8) and in 1 of the CMV DNA-negative specimens (specificity, 99%; 95% CI, 94.8 to 99.8). Results of CMV load determination as assessed by both quantitative test systems were correlated (
r
= 0.73;
P
< 0.0001; 95% CI, 0.61 to 0.81). Results for undiluted samples containing a high CMV load were more accurate with the LightCycler test than were results obtained with the CACM test, which underestimated the viral load of samples containing high DNA copy numbers. The high level of sensitivity, specificity, accuracy, and rapidity provided by the LightCycler technology are favorable for the use of this system in the detection of CMV DNA in clinical specimens.