bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of Enzyme Immunoassay and Reverse Transcriptase PCR for Identification of Serotype G9 Rotaviruses
Authors: Coulson Barbara S. , Gentsch Jon R. , Das Bimal K. , Bhan M. K. , Glass Roger I.
Published: December 2019

While only four globally important rotavirus G serotypes (1 to 4) have been documented, many studies suggest that serotype G9 viruses may be widely distributed and more important than previously recognized. We have evaluated 10 serotype G9 rotavirus-neutralizing monoclonal antibodies (MAbs) directed to VP7, which bound by direct enzyme immunoassay (EIA) to P1A[8], G9 rotaviruses F45, WI61, and AU32, for their ability to recognize the New Delhi G9 rotavirus 116E. Only one MAb (MAb F45:1) bound to P[11], G9 virus 116E to a high titer by EIA. This MAb was incorporated into an indirect EIA for G serotyping, which was validated with prototype cultivable human rotaviruses of G types 1 to 4 and 9. The EIA was compared with genotyping by reverse transcriptase PCR (RT-PCR) under code for the determination of the G types of rotaviruses obtained from neonates in New Delhi, India. The sensitivities of RT-PCR and EIA (after two additional freeze-thaw cycles) for the typing of G9 rotaviruses were 91 and 86%, respectively, for 24 culture-adapted rotavirus strains. The untypeable culture-adapted rotavirus samples also were unreactive with VP7 group antigen-reactive MAb 60. After two additional freeze-thaw cycles, only 26 of 42 (62%) of stools containing rotavirus typed as G9 by RT-PCR were positive for G9 rotavirus by EIA. Stools containing rotavirus untypeable by EIA contained significantly less MAb 60-reactive VP7 antigen (
P
= 0.0001) than the stools containing typeable rotavirus. Thus, RT-PCR genotyping was the more sensitive method for determination of G9 type, but a serotype was readily determined in rotavirus samples containing MAb 60-reactive VP7 antigen by an EIA that incorporates MAb F45:1.
The Three Major Spanish Clones of Penicillin-Resistant
Streptococcus pneumoniae
Are the Most Common Clones Recovered in Recent Cases of Meningitis in Spain
Authors: Enright Mark C. , Fenoll Asunción , Griffiths David , Spratt Brian G.
Published: December 2019

One hundred six isolates of
Streptococcus pneumoniae
recovered in Spain from patients with meningitis in 1997 and 1998 were characterized by multilocus sequence typing. A heterogeneous collection of genotypes was associated with meningitis in Spain: 65 different sequence types were resolved and, even at a genetic distance of 0.43, there were 37 distinct lineages. Thirty-eight percent of the isolates, including all isolates of serotypes 6B, 9V, 14, and 23F, were resistant to penicillin, and 24% of the isolates were members of the three major Spanish penicillin-resistant or multidrug-resistant clones of serotypes 6B, 9V, and 23F or serotype variants of these clones. These three clones (MICs, 1 to 2 μg of penicillin/ml) were the most common clones associated with pneumococcal meningitis in Spain during 1997 and 1998. Only two of the other clones associated with meningitis were penicillin resistant (MICs, 0.12 to 0.5 μg/ml). One of the two most prevalent penicillin-susceptible clones causing meningitis (serotype 3) has not been detected outside of Spain, whereas the other (serotype 18C) has been recovered from patients with meningitis in the United Kingdom, The Netherlands, and Denmark. The prevalence of meningitis caused by isolates of the three major Spanish penicillin-resistant or multiply antibiotic-resistant clones, which are now globally distributed, is disturbing and clearly establishes their ability to cause life-threatening disease.
Diphtheria in the Republic of Georgia: Use of Molecular Typing Techniques for Characterization of
Corynebacterium diphtheriae
Strains
Authors: Sulakvelidze Alexander , Kekelidze Merab , Gomelauri Tsaro , Deng Yingkang , Khetsuriani Nino , Kobaidze Ketino , De Zoysa Aruni , Efstratiou Androulla , Morris J. Glenn , Imnadze Paata
Published: December 2019

Sixty-six
Corynebacterium diphtheriae
strains (62 of the gravis biotype and 4 of the mitis biotype) isolated during the Georgian diphtheria epidemic of 1993 to 1998 and 13 non-Georgian
C. diphtheriae
strains (10 Russian and 3 reference isolates) were characterized by (i) biotyping, (ii) toxigenicity testing with the Elek assay and PCR, (iii) the randomly amplified polymorphic DNA (RAPD) technique, and (iv) pulsed-field gel electrophoresis (PFGE). Fifteen selected strains were ribotyped. Six RAPD types and 15 PFGE patterns were identified among all strains examined, and 12 ribotypes were found among the 15 strains that were ribotyped. The Georgian epidemic apparently was caused by one major clonal group of
C. diphtheriae
(PFGE type A, ribotype R1), which was identical to the predominant epidemic strain(s) isolated during the concurrent diphtheria epidemic in Russia. A dendrogram based on the PFGE patterns revealed profound differences between the minor (nonpredominant) epidemic strains found in Georgia and Russia. The methodologies for RAPD typing, ribotyping, and PFGE typing of
C. diphtheriae
strains were improved to enable rapid and convenient molecular typing of the strains. The RAPD technique was adequate for biotype differentiation; however, PFGE and ribotyping were better (and equal to each other) at discriminating between epidemiologically related and unrelated isolates.
Quantification of Human Cytomegalovirus DNA in Amniotic Fluid of Mothers of Congenitally Infected Fetuses
Authors: Revello Maria Grazia , Zavattoni Maurizio , Furione Milena , Baldanti Fausto , Gerna Giuseppe
Published: December 2019

A quantitative PCR assay was used to quantitate human cytomegalovirus DNA in amniotic fluid of mothers of 21 fetuses with congenital infection. Seven fetuses presented ultrasound abnormalities or were born with symptoms, whereas 14 fetuses were subclinically infected. Although the median DNA level was higher in symptomatic fetuses, the difference was not statistically significant (
P
= 0.09).
Specific PCR Identification and Differentiation of the Thermophilic Campylobacters,
Campylobacter jejuni
,
C. coli
,
C. lari
, and
C. upsaliensis
Authors: Fermér Christian , Engvall Eva Olsson
Published: December 2019

A sensitive PCR assay that detects the thermophilic campylobacters
C. jejuni
,
C. coli
,
C. lari
, and
C. upsaliensis
is reported. Furthermore, by digestion of the PCR products with two restriction enzymes, species differentiation was demonstrated. Thus, the present method has the potential to be used for both detection and identification of thermophilic
Campylobacter
species.
Capillary Electrophoresis–Single-Strand Conformation Polymorphism Analysis for Rapid Identification of
Pseudomonas aeruginosa
and Other Gram-Negative Nonfermenting Bacilli Recovered from Patients with Cystic Fibrosis
Authors: Ghozzi Rafiaa , Morand Philippe , Ferroni Agnes , Beretti Jean-Luc , Bingen Edouard , Segonds Christine , Husson Marie-Odile , Izard Daniel , Berche Patrick , Gaillard Jean-Louis
Published: December 2019

We used capillary electrophoresis–single-strand conformation polymorphism (CE-SSCP) analysis of PCR-amplified 16S rRNA gene fragments for rapid identification of
Pseudomonas aeruginosa
and other gram-negative nonfermenting bacilli isolated from patients with cystic fibrosis (CF). Target sequences were amplified by using forward and reverse primers labeled with various fluorescent dyes. The labeled PCR products were denatured by heating and separated by capillary gel electrophoresis with an automated DNA sequencer. Data were analyzed with GeneScan 672 software. This program made it possible to control lane-to-lane variability by standardizing the peak positions relative to internal DNA size markers. Thirty-four reference strains belonging to the genera
Pseudomonas
,
Brevundimonas
,
Burkholderia
,
Comamonas
,
Ralstonia
,
Stenotrophomonas
, and
Alcaligenes
were tested with primer sets spanning 16S rRNA gene regions with various degrees of polymorphism. The best results were obtained with the primer set P11P-P13P, which spans a moderately polymorphic region (
Escherichia coli
16S rRNA positions 1173 to 1389 [M. N. Widjojoatmodjo, A. C. Fluit, and J. Verhoef, J. Clin. Microbiol. 32:3002–3007, 1994]). This primer set differentiated the main CF pathogens from closely related species but did not distinguish
P. aeruginosa
from
Pseudomonas alcaligenes-Pseudomonas pseudoalcaligenes
and
Alcaligenes xylosoxidans
from
Alcaligenes denitrificans
. Two hundred seven CF clinical isolates (153 of
P. aeruginosa
, 26 of
Stenotrophomonas maltophilia
, 15 of
Burkholderia
spp., and 13 of
A. xylosoxidans
) were tested with P11P-P13P. The CE-SSCP patterns obtained were identical to those for the corresponding reference strains. Fluorescence-based CE-SSCP analysis is simple to use, gives highly reproducible results, and makes it possible to analyze a large number of strains. This approach is suited for the rapid identification of the main gram-negative nonfermenting bacilli encountered in CF.
Molecular Evidence of Coinfection of Ticks with
Borrelia burgdorferi
Sensu Lato and the Human Granulocytic Ehrlichiosis Agent in Switzerland
Authors: Leutenegger Christian M. , Pusterla Nicola , Mislin Caroline N. , Weber Rainer , Lutz Hans
Published: December 2019

Adult
Ixodes ricinus
ticks were collected in Switzerland and tested for the presence of coinfection with
Borrelia burgdorferi
sensu lato and the human granulocytic ehrlichiosis (HGE) agent by real-time PCR. Of 100 ticks, 49% were positive for
B. burgdorferi
and 2% were positive for the HGE agent. The two HGE agent-positive ticks were also found to be positive for
B. burgdorferi
.
Use of Multiplex PCR To Detect Classical and Newly Described Pyrogenic Toxin Genes in Staphylococcal Isolates
Authors: Monday Steven R. , Bohach Gregory A.
Published: December 2019

Staphylococcus aureusmay contain one or more genes that encode a variety of immunomodulatory pyrogenic toxins (PTs), including the staphylococcal enterotoxins and toxic shock syndrome toxin (TSST). The PTs interact with several cellular targets to produce disease, such as food poisoning and toxic shock syndrome. At present, nine serologically distinct enterotoxins and one immunoreactive form of TSST have been identified and characterized. As isolates ofS. aureusare further assessed, it is anticipated that this number will increase. To facilitate screening, a multiplex PCR was designed to simultaneously determine which of these 10 currently known PT genes an individualS. aureusisolate possesses. We show here, usingS. aureusisolates with characterized PT phenotypes, that this novel PCR technique reliably detects each of the known PTs in a single reaction.
Restriction Fragment Length Polymorphisms of Virulence Plasmids in
Rhodococcus equi
Authors: Takai Shinji , Shoda Masato , Sasaki Yukako , Tsubaki Shiro , Fortier Guillaume , Pronost Stephane , Rahal Karim , Becu Teotimo , Begg Angela , Browning Glenn , Nicholson Vivian M. , Prescott John F.
Published: December 2019

Virulent
Rhodococcus equi
, which is a well-known cause of pyogranulomatous pneumonia in foals, possesses a large plasmid encoding virulence-associated 15- to 17-kDa antigens. Foal and soil isolates from five countries—Argentina, Australia, Canada, France, and Japan—were investigated for the presence of 15- to 17-kDa antigens by colony blotting, using the monoclonal antibody 10G5, and the gene coding for 15- to 17-kDa antigens by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases
Bam
HI,
Eco
RI,
Eco
T22I, and
Hin
dIII, and the digestion patterns that resulted divided the plasmids of virulent isolates into five closely related types. Three of the five types had already been reported in Canadian and Japanese isolates, and the two new types had been found in French and Japanese isolates. Therefore, we tentatively designated these five types 85-kb type I (pREAT701), 85-kb type II (a new type), 87-kb type I (
Eco
RI and
Bam
HI type 2 [V. M. Nicholson and J. F. Prescott, J. Clin. Microbiol. 35:738–740, 1997]), 87-kb type II (a new type), and 90-kb (pREL1) plasmids. The 85-kb type I plasmid was found in isolates from Argentina, Australia, Canada, and France. Plasmid 87-kb type I was isolated in specimens from Argentina, Canada, and France. The 85-kb type II plasmid appeared in isolates from France. On the other hand, plasmids 87-kb type II and 90-kb were found only in isolates from Japan. These results revealed geographic differences in the distribution of the virulence plasmids found in the five countries and suggested that the restriction fragment length polymorphism of virulence plasmids might be useful to elucidate the molecular epidemiology of virulent
R. equi
in the world.
Intravenous Immunoglobulin Therapy: Confounding Effects on Serological Screening for Toxoplasmosis during Pregnancy
Authors: Pelloux Hervé , Fricker-Hidalgo Hélène , Brochier Gilbert , Goullier-Fleuret Andrée , Ambroise-Thomas Pierre
Published: December 2019

The serological diagnosis of toxoplasmic infection during pregnancy is intended to prevent congenital infection of the fetus. However, in the context of recurrent pregnancy loss intravenous immunoglobulin therapy can create a biological trap for the interpretation of serological results, with potentially serious consequences for the outcome of the pregnancy.
Emergence and Spread in French Hospitals of Methicillin-Resistant
Staphylococcus aureus
with Increasing Susceptibility to Gentamicin and Other Antibiotics
Authors: Lelièvre Hervé , Lina Gerard , Jones Mark E. , Olive Claude , Forey Françoise , Roussel-Delvallez Micheline , Nicolas-Chanoine Marie-Hélène , Bébéar Cécile M. , Jarlier Vincent , Andremont Antoine , Vandenesch François , Etienne Jerome
Published: December 2019

Oxacillin (methicillin) resistance in methicillin-resistant
Staphylococcus aureus
(MRSA) is associated with an increased incidence of resistance to other antibiotics, which has increased since it was first reported in 1969. In 1992 a new phenotype of MRSA arose in France; this was characterized by a heterogeneous expression of resistance to oxacillin and susceptibility to various antibiotics, including gentamicin but also tetracycline, minocycline, lincomycin, pristinamycin, co-trimoxazole, rifampin, and fusidic acid. In French hospitals a longitudinal nationwide surveillance of antibiotic resistance in
S. aureus
has allowed for the detection of changes in antibiotic susceptibility profiles. Seven French clinical laboratories (six from the mainland and one from the West Indies) reported the results of susceptibility testing of 57,347
S. aureus
strains isolated in their institutes between 1992 and 1998. Over a 7-year period the incidence of isolation of gentamicin-susceptible MRSA (GS-MRSA) strains has steadily increased to represent, in 1998, 46.8 to 94.4% of the MRSA strains, irrespective of the overall incidence of MRSA. Two predominant types recognized by pulsed-field gel electrophoresis (PFGE) accounted for the majority of the GS-MRSA in different mainland hospitals, both differing from the predominant type observed in the French West Indies. Some GS-MRSA and gentamicin-resistant MRSA (GR-MRSA) strains had closely related PFGE profiles, and hybridization studies confirmed the lack in GS-MRSA of the
aac6′-aph2
" gene, which confers resistance to all aminoglycosides, with conservation of the
ant4
′ gene, which confers resistance to kanamycin, tobramycin, and amikacin. Thus, it is likely that certain GS-MRSA strains could have emerged from GR-MRSA strains by excision or deletion of the
aac6′-aph2
" gene.
Development of a PCR Assay for Rapid Detection of Enterococci
Authors: Ke Danbing , Picard François J. , Martineau Francis , Ménard Christian , Roy Paul H. , Ouellette Marc , Bergeron Michel G.
Published: December 2019

Enterococci are becoming major nosocomial pathogens, and increasing resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus level by targeting the
tuf
gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the
tuf
gene from several bacterial species (available in public databases) and designed degenerate PCR primers derived from conserved regions. These primers were used to amplify a target region of 803 bp from four enterococcal species (
Enterococcus avium
,
E. faecalis
,
E. faecium
, and
E. gallinarum
). Subsequently, the complete nucleotide sequences of these amplicons were determined. The analysis of a multiple alignment of these sequences revealed regions conserved among enterococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 species of enterococci tested (
E. solitarius
DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial species, except for 2
Abiotrophia
species and several
Listeria
species. Furthermore, this assay efficiently amplified all 159 clinical isolates of enterococci tested (61
E. faecium
, 77
E. faecalis
, 9
E. gallinarum
, and 12
E. casseliflavus
isolates). Interestingly, the preliminary sequence comparison of the amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific internal probes. In conclusion, this rapid PCR-based assay is capable of detecting all clinically important enterococci and has potential for use in clinical microbiology laboratories.
Near Absence of Vancomycin-Resistant Enterococci but High Carriage Rates of Quinolone-Resistant Ampicillin-Resistant Enterococci among Hospitalized Patients and Nonhospitalized Individuals in Sweden
Authors: Torell Erik , Cars Otto , Olsson-Liljequist Barbro , Hoffman Britt-Marie , Lindbäck Johan , Burman Lars G. ,   The Enterococcal Study Group
Published: December 2019

Rates of colonization with enterococci with acquired resistance to vancomycin (vancomycin-resistant enterococci [VRE]) and ampicillin (ampicillin-resistant enterococci [ARE]) were determined by using fecal samples from 670 nonhospitalized individuals and 841 patients in 27 major hospitals. Of the hospitalized patients, 181 (21.5%) were carriers of ARE and 9 (1.1%) were carriers of VRE. In univariate analyses, length of hospital stay (odds ratio [OR], 4.6; 95% confidence interval [CI], 2.5 to 8.9) and antimicrobial therapy (OR, 4.7; 95% CI, 3.3 to 6.7) were associated with ARE colonization, as were prior treatment with penicillins (OR, 3.1; 95% CI, 1.8 to 5.5), cephalosporins (OR, 2.9; 95% CI, 1.7 to 5.0), or quinolones (OR, 2.7; 95% CI, 1.5 to 4.7). In logistic regression analysis, antimicrobial therapy for at least 5 days was independently associated with ARE carriage (adjusted OR, 3.8; 95% CI, 2.6 to 5.4). Over 90% of the ARE isolates were fluoroquinolone resistant, whereas 14% of the ampicillin-susceptible
Enterococcus faecium
isolates were fluoroquinolone resistant. ARE carriage rates correlated with the use of fluoroquinolones (
P
= 0.04) but not with the use of ampicillin (
P
= 0.68) or cephalosporins (
P
= 0.40). All nine VRE isolates were
E. faecium vanB
and were found in one hospital. Seven of these isolates were related according to their types as determined by pulsed-field gel electrophoresis. Among the nonhospitalized individuals, the ARE carriage rate was lower (6%;
P
< 0.05), and only one person, who had recently returned from Africa, harbored VRE (
E. faecium vanA
). The absence of VRE colonization in nonhospitalized individuals reflects an epidemiological situation in Sweden radically different from that in countries in continental Europe where glycopeptides have been widely used for nonmedical purposes.
Evaluation of a Bacteriophage-Based Assay (Phage Amplified Biologically Assay) as a Rapid Screen for Resistance to Isoniazid, Ethambutol, Streptomycin, Pyrazinamide, and Ciprofloxacin among Clinical Isolates of
Mycobacterium tuberculosis
Authors: Eltringham I. J. , Wilson S. M. , Drobniewski F. A.
Published: December 2019

Rapid molecular assays for the detection of mutations associated with rifampin resistance in
Mycobacterium tuberculosis
are commercially available. However, they are complex and expensive and have predictive values of 90 to 95%. Molecular assays for other drugs are less predictive of resistance. Ideally, assays based on phenotypic markers should be used for susceptibility testing, but these can take weeks to complete. We previously described a rapid phenotypic assay, the phage amplified biologically (PhaB) assay, for the rapid determination of rifampin and isoniazid susceptibility in clinical isolates of
M. tuberculosis
. In this study, we extended the assay to the study of ethambutol, pyrazinamide, streptomycin, and ciprofloxacin. After the optimization of antibiotic concentrations and incubation conditions, the assay was applied to each drug for a total of 157 isolates. The correlations between the results of the PhaB assay and the resistance ratio method were 94% for isoniazid, 96% for streptomycin, 100% for ciprofloxacin, 88% for ethambutol, and 87% for pyrazinamide. For ciprofloxacin, ethambutol, and pyrazinamide, significantly better correlations were found when a 90% reduction in plaque count was used as the cutoff. Turnaround times for the PhaB assay were 2 to 3 days, compared with 10 days for the resistance ratio method. We believe that this low-cost assay may have widespread applicability for the rapid screening of drug resistance in
M. tuberculosis
isolates, especially in developing countries.
Identification of
Fusarium
Species Involved in Human Infections by 28S rRNA Gene Sequencing
Authors: Hennequin C. , Abachin E. , Symoens F. , Lavarde V. , Reboux G. , Nolard N. , Berche P.
Published: December 2019

Fusarium
spp. have emerged as major opportunistic fungal agents. Since new antifungal agents exhibit variable activity against
Fusarium
isolates depending on the species, rapid identification at the species level is required. Conventional culture methods are difficult, fastidious, and sometimes inconclusive. In this work, we sequenced a 440-bp fragment encoding the 28S rRNA from 33
Fusarium
isolates belonging to six
Fusarium
species associated with human infections. The data were then analyzed by the neighbor-joining method. By using distance matrix analysis and constructing the phylogram, we could easily distinguish the different species for all but one isolate. The method also allowed differentiation between the closely related genera
Acremonium
and
Cylindrocarpon
. In contrast to the case with conventional methods, the results could be obtained within 48 h from a 3-day culture and are independent of mycologist experience, making this method rapid and reliable for identification of
Fusarium
species isolated from patients.
Glycopeptide-Intermediate
Staphylococcus aureus
: Evaluation of a Novel Screening Method and Results of a Survey of Selected U.S. Hospitals
Authors: Hubert Susannah K. , Mohammed Jasmine M. , Fridkin Scott K. , Gaynes Robert P. , McGowan John E. , Tenover Fred C.
Published: December 2019

Isolates of
Staphylococcus aureus
with decreased susceptibilities to glycopeptide antimicrobial agents, such as vancomycin and teicoplanin, have emerged in the United States and elsewhere. Commercially prepared brain heart infusion agar (BHIA) supplemented with 6 μg of vancomycin per ml was shown in a previous study to detect glycopeptide-intermediate
S. aureus
(GISA) with high sensitivity and specificity; however, this medium, when prepared in-house, occasionally showed growth of vancomycin-susceptible control organisms. This limitation could significantly impact laboratories that prepare media in-house, particularly if they wished to conduct large surveillance studies for GISA. Therefore, a pilot study to detect GISA was performed with vancomycin-containing Mueller-Hinton agar (MHA) prepared in-house in place of commercially prepared BHIA. MHA was selected for this study because this medium is widely available and well standardized. The results of the pilot study showed that supplementation of MHA with 5 μg of vancomycin per ml was both a sensitive and a specific method for screening for GISA isolates. This method was used to screen for GISA among 630 clinical isolates of methicillin-resistant
S. aureus
collected during 1997 from 33 U.S. hospitals. Although 14
S. aureus
isolates grew on the screening agar, all were vancomycin susceptible (MICs were ≤1 μg/ml) by broth microdilution testing. Population analyses of five isolates revealed two with a subpopulation for which vancomycin MICs were 8 μg/ml. In summary, the MHA screen plate containing 5 μg of vancomycin per ml prepared in-house provides a sensitive and cost-effective method for large-scale screening for GISA for which vancomycin MICs are 8 μg/ml. However, confirmation of isolates as vancomycin resistant is critical. This study suggests that GISA was not a widespread problem in the United States in 1997.
Rapid Identification of
Burkholderia pseudomallei
in Blood Cultures by a Monoclonal Antibody Assay
Authors: Pongsunk Supinya , Thirawattanasuk Nittaya , Piyasangthong Nuanchan , Ekpo Pattama
Published: December 2019

Burkholderia pseudomallei
is the causative agent of melioidosis. In northeast Thailand, this gram-negative bacterium is a major cause of mortality from septicemia. The definitive diagnosis of this disease is made by bacterial culture. In this study, we produced a monoclonal antibody (MAb) specific to the 30-kDa protein of
B. pseudomallei
by in vivo and in vitro immunization of BALB/c mice with a crude culture filtrate antigen. The MAb could directly agglutinate with all 243 clinical isolates of
B. pseudomallei
but not with other gram-negative bacteria, except for one strain of
Burkholderia mallei
. However, the MAb cross-reacted with the gram-positive
Bacillus
sp. and
Streptococcus pyogenes. B. pseudomallei
in brain heart infusion broth (BHIB) subcultured from a BacT/Alert automated blood culture system could be identified by simple agglutination with this MAb assay. The sensitivity and specificity of direct agglutination compared to the “gold standard,” the culture method, were 94.12 and 98.25%, respectively. However, the MAb adsorbed to polystyrene beads or latex particles directly identified the bacterium in blood culture specimens and in BHIB subcultured from a BacT/Alert automated blood culture system. The sensitivity of the latex agglutination test was 100% for both blood culture and BHIB specimens. The specificity was 85.96 and 96.49% for the blood culture and BHIB specimens, respectively. The specificity could be increased if the nonspecific materials in the blood culture broths were eradicated by centrifugation at low speeds. Thus, a combination of blood culture and the agglutination method could be used for the rapid diagnosis of melioidosis in the routine bacteriological laboratory. This method could speed up detection of the bacterium in blood culture by at least 2 days, compared to the conventional bacterial culture method. In addition, the MAb is stable at room temperature for 2 weeks and at 4, −20, and −70°C for at least 1 year. The latex reagent was stable for at least 6 months at 4°C.
Genotypic Survey of Recent β-Lactam-Resistant Pneumococcal Nasopharyngeal Isolates from Asymptomatic Children in Chile
Authors: Gherardi Giovanni , Inostrozo Jaime S. , O'ryan Miguel , Prado Valeria , Prieto Susana , Arellano Carolina , Facklam Richard R. , Beall Bernard
Published: December 2019

To assess pneumococcal strain variability among young asymptomatic carriers in Chile, we used serotyping, antibiotic susceptibility testing, and genotyping to analyze 68 multidrug-resistant pneumococcal isolates recovered from 54 asymptomatic children 6 to 48 months of age. The isolates represented capsular serotypes 19F (43 isolates), 14 (14 isolates), 23F (7 isolates), 6B (3 isolates), and 6A (1 isolate). Genotypic analysis, which included pulsed-field gel electrophoresis (PFGE) of chromosomal digests, penicillin binding protein (PBP) gene fingerprinting, and
dhf
gene fingerprinting, revealed that the isolates represented six different genetic lineages. Clear circumstantial evidence of capsular switching was seen within each of four of the genetically related sets. The majority of the isolates, consisting of the 43 19F isolates and 2 type 6B isolates, appeared to represent a genetically highly related set distinct from previously characterized pneumococcal strains. Each of three other genetically defined lineages was closely related to one of the previously characterized clones Spain
6B
-2, France
9V
-3, or Spain
23F
-1. A fifth lineage was comprised of four type 23F isolates that, by the techniques used for this study, were genetically indistinguishable from three recent type 19F sterile-site isolates from the United States. Finally, a sixth lineage was represented by a single type 23F isolate which had a unique PFGE type and unique PBP and
dhf
gene fingerprints.
Case of Peritonitis Caused by
Ewingella americana
in a Patient Undergoing Continuous Ambulatory Peritoneal Dialysis
Authors: Kati Chryssa , Bibashi Evangelia , Kokolina Elisabeth , Sofianou Danai
Published: December 2019

Reports of serious infections caused by
Ewingella americana
have been rare. A case of
E. americana
peritonitis in a patient receiving continuous ambulatory peritoneal dialysis is described. This is the first report of
E. americana
causing such an infection.
Identification and PCR-Restriction Fragment Length Polymorphism Analysis of a Variant of the Ibaraki Virus from Naturally Infected Cattle and Aborted Fetuses in Japan
Authors: Ohashi Seiichi , Yoshida Kazuo , Watanabe Youichirou , Tsuda Tomoyuki
Published: December 2019

One hundred fourteen field isolates of the Ibaraki virus (IBAV), a member of the epizootic hemorrhagic disease virus serotype 2 (EHDV-2), were isolated from blood samples of affected and apparently healthy cattle and
Culicoides
biting midges and from blood samples of dams and internal organs of aborted fetuses during an outbreak of Ibaraki disease in the southern part of Japan in 1997. In this outbreak, 242 cattle showed typical symptoms of the disease, and several hundred dams had miscarriages or stillbirths. The viruses that induced typical Ibaraki disease and reproductive problems among cattle were identical and were antigenically closely related to but distinct from previous isolates of IBAV and EHDV-2. The virus was considered to be a putative agent of this outbreak. Reverse transcription-PCR based on segment 3 of the RNA genome of EHDV-2 and restriction fragment length polymorphism analysis of the PCR products were conducted to compare the genomes of the viruses. The results suggested that the virus isolated in 1997 was a variant of IBAV and might be exotic.
Evolution and Clonal Traits of
Mycobacterium tuberculosis
Complex in Guinea-Bissau
Authors: Källenius Gunilla , Koivula Tuija , Ghebremichael Solomon , Hoffner Sven E. , Norberg Renée , Svensson Erika , Dias Francisco , Marklund Britt-Inger , Svenson Stefan B.
Published: December 2019

Two hundred twenty-nine consecutive isolates of
Mycobacterium tuberculosis
complex from patients with pulmonary tuberculosis in Guinea-Bissau, which is located in West Africa, were analyzed for clonal origin by biochemical typing and DNA fingerprinting. By using four biochemical tests (resistance to thiophene-2-carboxylic acid hydrazide, niacin production, nitrate reductase test, and pyrazinamidase test), the isolates could be assigned to five different biovars. The characteristics of four strains conformed fully with the biochemical criteria for
M. bovis
, while those of 85 isolates agreed with the biochemical criteria for
M. tuberculosis
. The remaining 140 isolates could be allocated into one of three biovars (biovars 2 to 4) representing a spectrum between the classical bovine (biovar 1) and human (biovar 5) tubercle bacilli. By using two genotyping methods, restriction fragment length polymorphism analysis with IS
6110
(IS
6110
RFLP analysis) and spoligotyping, the isolates could be separated into three groups (groups A to C) of the
M. tuberculosis
complex. Group A (
n
= 95), which contained the majority of classical human
M. tuberculosis
isolates, had large numbers of copies of IS
6110
elements (mean number of copies, 9) and a distinctive spoligotyping pattern that lacked spacers 33 to 36. Isolates of the major group, group B (
n
= 119), had fewer IS
6110
copies (mean copy number, 5) and a spoligotyping pattern that lacked spacers 7 to 9 and 39 and mainly comprised isolates of biovars 1 to 4. Group C isolates (
n
= 15) had one to three IS
6110
copies, had a spoligotyping pattern that lacked spacers 29 to 34, and represented biovar 3 to 5 isolates. Four isolates whose biochemical characteristics conformed with those of
M. bovis
clustered with the group B isolates and had spoligotype patterns that differed from those previously reported for
M. bovis
, in that they possessed spacers 40 to 43. Interestingly, isolates of group B and, to a certain extent, also isolates of group C showed a high degree of variability in biochemical traits, despite genotypic identity in terms of IS
6110
RFLP and spoligotype patterns. We hypothesize that isolates of groups B and C have their evolutionary origin in West Africa, while group A isolates are of European descent.
Disseminated Infection with
Mycobacterium genavense
: a Challenge to Physicians and Mycobacteriologists
Authors: Thomsen Vibeke Østergaard , Dragsted Ulrik Bak , Bauer Jeanett , Fuursted Kurt , Lundgren Jens
Published: December 2019

In the present study we compared the clinical presentations of patients with a clinical diagnosis of AIDS and disseminated
Mycobacterium genavense
infection (
n
= 12) with those of patients with AIDS and disseminated
M. avium
complex (MAC) infection (
n
= 24). Abdominal pain was seen more frequently in the group of patients infected with
M. genavense
than in patients infected with MAC (
P
= 0.003). Analysis of microbiological data revealed that stool specimens from patients infected with
M. genavense
were more often smear positive than stool specimens from patients infected with MAC (
P
= 0.00002). However,
M. genavense
could be cultured on solid media from only 15.4% of the stool specimens, whereas MAC could be cultured from 71.4% of the specimens. Bone marrow and liver biopsy specimens yielded growth of
M. genavense
within a reasonably short time, allowing species identification by DNA technology. Microbiological data clearly demonstrated the importance of acidic liquid medium for primary culture, the avoidance of pretreatment and the use of additives in culture, and the necessity for prolonged incubation if
M. genavense
is suspected. Susceptibility testing showed that
M. genavense
is sensitive to rifamycins, fluoroquinolones, and macrolides, whereas it is resistant to isoniazid. Susceptibility to ethambutol and clofazimine could not be evaluated. The mean survival times of patients in the two groups were similar.
Identification of
Porphyromonas gingivalis
Strains by Heteroduplex Analysis and Detection of Multiple Strains
Authors: Leys Eugene J. , Smith James H. , Lyons Sharon R. , Griffen Ann L.
Published: December 2019

Heteroduplex analysis has been used extensively to identify allelic variation among mammalian genes. It provides a rapid and reliable method for determining and cataloging minor differences between two closely related DNA sequences. We have adapted this technique to distinguish among strains or clonal types of
Porphyromonas gingivalis
. The ribosomal intergenic spacer region (ISR) was amplified directly from a subgingival plaque sample by PCR with species-specific primers, avoiding the need for culturing the bacteria. The PCR products were then directly compared by heteroduplex analysis with known strains of
P. gingivalis
for identification. We identified 22 distinct but closely related heteroduplex types of
P. gingivalis
in 1,183 clinical samples. Multiple strains were found in 34% of the samples in which
P. gingivalis
was detected. Heteroduplex types were identified from these multistrain samples without separating them by culturing or molecular cloning. PCR with species-specific primers and heteroduplex analysis makes it possible to reliably and sensitively detect and identify strains of
P. gingivalis
in large numbers of samples.
Acute-Phase-Specific Heptapeptide Epitope for Diagnosis of Parvovirus B19 Infection
Authors: Kaikkonen Leena , Lankinen Hilkka , Harjunpää Irene , Hokynar Kati , Söderlund-Venermo Maria , Oker-Blom Christian , Hedman Lea , Hedman Klaus
Published: December 2019

The major capsid protein VP2 of human parvovirus B19, when studied in a denatured form exhibiting linear epitopes, is recognized exclusively by immunoglobulin G (IgG) antibodies of patients with acute or recent B19 infection. By contrast, conformational epitopes of VP2 are recognized both by IgG of the acute phase and by IgG of past immunity. In order to localize the VP2 linear epitope(s) specific for acute-phase IgG, the entire B19 capsid protein sequence was mapped by peptide scanning using well-characterized acute-phase and control sera. A unique heptapeptide epitope showing strong and selective reactivity with the acute-phase IgG was detected and characterized. By using this linear epitope (VP2 amino acids 344 to 350) and virus-like particles exhibiting conformational VP2 epitopes, an innovative approach, second-generation epitope-typing enzyme immunoassay, was set up for improved diagnosis of primary infections by human parvovirus B19.
Phylogeny and PCR Identification of Clinically Important Zygomycetes Based on Nuclear Ribosomal-DNA Sequence Data
Authors: Voigt Kerstin , Cigelnik Elizabeth , O'donnell Kerry
Published: December 2019

A molecular database for all clinically important Zygomycetes was constructed from nucleotide sequences from the nuclear small-subunit (18S) ribosomal DNA and domains D1 and D2 of the nuclear large-subunit (28S) ribosomal DNA. Parsimony analysis of the aligned 18S and 28S DNA sequences was used to investigate phylogenetic relationships among 42 isolates representing species of Zygomycetes reported to cause infections in humans and other animals, together with commonly cultured contaminants, with emphasis on members of the Mucorales. The molecular phylogeny provided strong support for the monophyly of the Mucorales, exclusive of
Echinosporangium transversale
and
Mortierella
spp., which are currently misclassified within the Mucorales.
Micromucor ramannianus
, traditionally classified within
Mortierella
, and
Syncephalastrum racemosum
represent the basal divergences within the Mucorales. Based on the 18S gene tree topology,
Absidia corymbifera
and
Rhizomucor variabilis
appear to be misplaced taxonomically.
A. corymbifera
is strongly supported as a sister group of the
Rhizomucor miehei-Rhizomucor pusillus
clade, while
R. variabilis
is nested within
Mucor
. The aligned 28S sequences were used to design 13 taxon-specific PCR primer pairs for those taxa most commonly implicated in infections. All of the primers specifically amplified DNA of the size predicted based on the DNA sequence data from the target taxa; however, they did not cross-react with phylogenetically related species. These primers have the potential to be used in a PCR assay for the rapid and accurate identification of the etiological agents of mucormycoses and entomophthoromycoses.
Human Antibody Responses to VlsE Antigenic Variation Protein ofBorrelia burgdorferi
Authors: Lawrenz M. B. , Hardham J. M. , Owens R. T. , Nowakowski J. , Steere A. C. , Wormser G. P. , Norris S. J.
Published: December 2019

VlsE is a 35-kDa surface-exposed lipoprotein ofBorrelia burgdorferithat was shown previously to undergo antigenic variation through segmental recombination of silentvlscassettes withvlsEduring experimental mouse infections. Previous data had indicated that sera from North American Lyme disease patients and experimentally infected animals contained antibodies reactive with VlsE. In this study, sera from patients with Lyme disease, syphilis, and autoimmune conditions as well as from healthy controls were examined for reactivity with VlsE by Western blotting and enzyme-linked immunosorbent assay (ELISA). Strong Western blot reactivity to a recombinant VlsE cassette region protein was obtained consistently with Lyme disease sera. Although sera from Lyme disease patients also reacted with a band corresponding to VlsE inB. burgdorferiB31-5A3, interpretation was complicated by low levels of VlsE expression in in vitro-culturedB. burgdorferiand by the presence of comigrating bands. An ELISA using recombinant VlsE was compared with an ELISA using sonically disruptedB. burgdorferias the antigen. For a total of 93 Lyme disease patient sera examined, the VlsE ELISA yielded sensitivities of 63% for culture-confirmed erythema migrans cases and 92% for later stages, as compared to 61 and 98%, respectively, for the “whole-cell” ELISA. The specificities of the two assays with healthy blood donor sera were comparable, but the VlsE ELISA was 90% specific with sera from syphilis patients, compared to 20% specificity for the whole-cell ELISA with this group. Neither assay showed reactivity with a panel of sera from 20 non-Lyme disease arthritis patients or 20 systemic lupus erythematosus patients. Our results indicate that VlsE may be useful in the immunodiagnosis of Lyme disease and may offer greater specificity than ELISAs using wholeB. burgdorferias the antigen.
Genetic Relatedness among Nontypeable Pneumococci Implicated in Sporadic Cases of Conjunctivitis
Authors: Barker Jason H. , Musher Daniel M. , Silberman Ronald , Phan Hoang M. , Watson David A.
Published: December 2019

Nontypeable
Streptococcus pneumoniae
is a common cause of epidemic conjunctivitis. A previous molecular fingerprinting study identified a clone of nontypeable pneumococcus that was responsible for a recent outbreak of conjunctivitis. In the present study, we examined the extent to which pneumococci that cause sporadic cases of conjunctivitis are related to this epidemic strain. Using arbitrarily primed BOX-PCR, we have determined that, of 10 nontypeable pneumococci causing sporadic conjunctivitis, 5 were clonal and closely related to a previous outbreak strain, whereas 5 others were genetically diverse.
Scored Antibody Reactivity Determined by Immunoblotting Shows an Association between Clinical Manifestations and Presence ofBorrelia burgdorferisensu stricto,B. garinii,B. afzelii, andB. Valaisianain Humans
Authors: Ryffel Karine , Péter Olivier , Rutti Bernard , Suard André , Dayer Eric
Published: December 2019

An immunoglobulin G immunoblot was developed with antigenic extracts ofBorrelia burgdorferisensu stricto,B. garinii,B. afzelii, andB. valaisianagenospecies and was reacted with sera from patients with neuroborreliosis, acrodermatitis, and Lyme arthritis. A detailed analysis of the reactivities of the protein bands was performed, and a two-step scoring procedure was selected to determine the preferential reactivity of sera to one particular genospecies. The discriminative potential of 5 proteins (12-kDa, 16-kDa, 18-kDa, OspA, and 66-kDa proteins) was used as a rapid first-step scoring method, followed by scoring of 14 additional protein bands if necessary. The advantage of this procedure is the low percentage of serum samples with inconclusive results for one of the four species (10% for patients with neuroborreliosis, 6% for patients with acrodermatitis chronica atrophicans, and 6% for patients with Lyme arthritis). Among 31 serum samples from patients with neuroborreliosis, 16 were more reactive toB. garinii, 7 were more reactive toB. afzelii, 3 were more reactive toB. valaisiana, and 2 were more reactive toB. burgdorferisensu stricto. Of 31 serum samples from patients with acrodermatitis, 26 showed a higher level of reactivity toB. afzelii. Of 34 serum samples from patients with Lyme arthritis, 21 were more reactive toB. burgdorferisensu stricto, 10 were more reactive toB. afzelii, and 1 was more reactive toB. valaisiana. Our results suggest an organotropism ofBorreliaspecies and provide some evidence of a pathogenic potential ofB. valaisianain humans.
Evaluation of Streck Tissue Fixative, a Nonformalin Fixative for Preservation of Stool Samples and Subsequent Parasitologic Examination
Authors: Nace Eva K. , Steurer Frank J. , Eberhard Mark L.
Published: December 2019

We undertook a study to evaluate Streck tissue fixative (STF) as a substitute for formalin and polyvinyl alcohol (PVA) in fecal preservation. A comparison of formalin, PVA, (mercuric chloride based), and STF was done by aliquoting fecal samples into each fixative. Stool specimens were collected in Haiti, and parasites included
Cyclospora cayetanensis
,
Giardia intestinalis
,
Entamoeba coli
,
Iodamoeba butschlii
,
Endolimax nana
,
Ascaris lumbricoides
,
Trichuris trichiura
,
Strongyloides stercoralis
, and
Necator americanus
. Preserved stools were examined at various predetermined times (1 week, 1 month, and 3 months) to establish the quality of the initial preservation as well as the suitability of the fixative for long-term storage. At each time point, stool samples in fixatives were examined microscopically as follows: (i) in wet mounts (with bright-field and epifluorescence microscopy), (ii) in modified acid-fast-, trichrome-, and safranin-stained smears, and (iii) with two commercial test kits. At the time points examined, morphologic features remained comparable for samples fixed with 10% formalin and STF. For comparisons of STF- and 10% formalin-fixed samples, specific findings showed that
Cyclospora
oocysts retained full fluorescence, modified acid-fast- and safranin-stained smears of
Cryptosporidium
and
Cyclospora
oocysts were equal in staining quality, and results were comparable in the immunofluorescence assay and enzyme immunoassay commercial kits. Stool fixed in STF and stained with trichrome showed less-than-acceptable staining quality compared with stool fixed in PVA. STF provides an excellent substitute for formalin as a fixative in routine examination of stool samples for parasites. However, modifications to the trichrome staining procedures will be necessary to improve the staining quality for protozoal cysts fixed in STF to a level comparable to that with PVA.
Simultaneous Infection with Two Drug-Susceptible
Mycobacterium tuberculosis
Strains in an Immunocompetent Host
Authors: Pavlic Marion , Allerberger Franz , Dierich Manfred P. , Prodinger Wolfgang M.
Published: December 2019

An important assumption for DNA fingerprinting of
Mycobacterium tuberculosis
is that patients are infected with only one strain at a time. Nonetheless, we demonstrate a case of simultaneous infection with two drug-susceptible strains of
M. tuberculosis
in an immunocompetent patient by IS
6110
restriction fragment length polymorphism and spoligotyping. Epidemiological data prove the patient's involvement in two independent clusters. Thus, double infections should be suspected with fingerprints showing divergent band intensities.
Rahnella aquatilis
Sepsis in an Immunocompetent Adult
Authors: Chang Chulhun Ludgerus , Jeong Joseph , Shin Jeong Hwan , Lee Eun Yup , Son Han Chul
Published: December 2019

Rahnella aquatilis
, a rare enteric gram-negative rod which is infrequently isolated in immunocompromised patients, was isolated as a causative organism of sepsis in a 26-year-old immunocompetent male patient. The contaminated intravenous fluid was confirmed to be the source of the organism.