bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Anticoagulant, Processing Delay, and Assay Method (Branched DNA versus Reverse Transcriptase PCR) on Measurement of Human Immunodeficiency Virus Type 1 RNA Levels in Plasma
Authors: Kirstein Lynn M. , Mellors John W. , Rinaldo Charles R. , Margolick Joseph B. , Giorgi Janis V. , Phair John P. , Dietz Edith , Gupta Phalguni , Sherlock Christopher H. , Hogg Robert , Montaner J. S. G. , Muñoz Alvaro
Published: December 2019

We conducted two studies to determine the potential influence of delays in blood processing, type of anticoagulant, and assay method on human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma. The first was an experimental study in which heparin- and EDTA-anticoagulated blood samples were collected from 101 HIV-positive individuals and processed to plasma after delays of 2, 6, and 18 h. HIV-1 RNA levels in each sample were then measured by both branched-DNA (bDNA) and reverse transcriptase PCR (RT-PCR) assays. Compared to samples processed within 2 h, the loss (decay) of HIV-1 RNA in heparinized blood was significant (
P
< 0.05) but small after 6 h (bDNA assay, −0.12 log
10
copies/ml; RT-PCR, −0.05 log
10
copies/ml) and after 18 h (bDNA assay, −0.27 log
10
copies/ml; RT-PCR, −0.15 log
10
copies/ml). Decay in EDTA-anticoagulated blood was not significant after 6 h (bDNA assay, −0.002 log
10
copies/ml; RT-PCR, −0.02 log
10
copies/ml), but it was after 18 h (bDNA assay, −0.09 log
10
copies/ml; RT-PCR, −0.09 log
10
copies/ml). Only 4% of samples processed after 6 h lost more than 50% (≥0.3 log
10
copies/ml) of the HIV-1 RNA, regardless of the anticoagulant or the assay that was used. The second study compared HIV-1 RNA levels in samples from the Multicenter AIDS Cohort Study (MACS; samples were collected in heparin-containing tubes in 1985, had a 6-h average processing delay, and were assayed by bDNA assay) and the British Columbia Drug Treatment Program (BCDTP) (collected in EDTA- or acid citrate dextrose-containing tubes in 1996 and 1997, had a 2-h maximum processing delay, and were assayed by RT-PCR). HIV-1 RNA levels in samples from the two cohorts were not significantly different after adjusting for CD4
+
-cell count and converting bDNA assay values to those corresponding to the RT-PCR results. In summary, the decay of HIV-1 RNA measured in heparinized blood after 6 h was small (−0.05 to −0.12 log
10
copies/ml), and the minor impact of this decay on HIV-1 RNA concentrations in archived plasma samples of the MACS was confirmed by the similarity of CD4
+
-cell counts and assay-adjusted HIV-1 RNA concentrations in the MACS and BCDTP.
Recurrent Bacteremia Caused by a “
Flexispira
”-Like Organism in a Patient with X-Linked (Bruton’s) Agammaglobulinemia
Authors: Weir Susan , Cuccherini Brenda , Whitney Anne M. , Ray Marsha L. , MacGregor John P. , Steigerwalt Arnold , Daneshvar Maryam I. , Weyant Robbin , Wray Betty , Steele John , Strober Warren , Gill Vee J.
Published: December 2019

Helicobacter
spp., except for
Helicobacter cinaedi
, have only rarely been reported in cases of septicemia. A patient with X-linked (Bruton’s) agammaglobulinemia was found to have persistent sepsis with a
Helicobacter
-like organism despite multiple courses of antibiotics. His periods of sepsis were associated with leg swelling thought to be consistent with cellulitis. The organism was fastidious and required a microaerophilic environment containing H
2
for growth. Optimal growth was observed at 35 to 37°C on sheep blood, CDC anaerobe, and Bordet-Gengou agars. Serial subcultures every 4 to 5 days were required to maintain viability. The organism was strongly urease positive and showed highest relatedness to
Helicobacter
-like organisms with the vernacular name “
Flexispira rappini
” by 16S rRNA gene sequence analysis. Genomic DNA hybridization studies, however, found 24 to 37% relatedness to “
F. rappini
” and even less to other
Helicobacter
spp. Although the organism phenotypically resembles “
Flexispira
” and
Helicobacter
, it is thought to represent a new taxon. The patient’s infection was eventually cleared with a prolonged (5-month) course of intravenous imipenem and gentamicin.
Novel Human Erythrovirus Associated with Transient Aplastic Anemia
Authors: Nguyen Quang Tri , Sifer Christophe , Schneider Véronique , Allaume Xavier , Servant Annabelle , Bernaudin Françoise , Auguste Véronique , Garbarg-Chenon Antoine
Published: December 2019

Erythrovirus (formerly parvovirus) B19 causes a wide range of diseases in humans, including anemia due to aplastic crisis. Diagnosis of B19 infection relies on serology and the detection of viral DNA by PCR. These techniques are usually thought to detect all erythrovirus field isolates, since the B19 genome is known to undergo few genetic variations. We have detected an erythrovirus (V9) markedly different from B19 in the serum and bone marrow of a child with transient aplastic anemia. The B19 PCR assay yielded a product that hybridized only very weakly to the B19-specific probe and whose sequence diverged more from those of 24 B19 viruses (11 to 14%) than the divergence found within the B19 group (≤6.65%). Restriction enzyme analysis of the V9 genome revealed that this genetic divergence extended beyond the amplified region. Interestingly, serological tests failed to demonstrate a response characteristic of acute B19 infection. V9 could be a new erythrovirus, and new diagnostic tests are needed for its detection.
Isolation of a New Subspecies,
Bartonella vinsonii
subsp.
arupensis
, from a Cattle Rancher: Identity with Isolates Found in Conjunction with
Borrelia burgdorferi
and
Babesia microti
among Naturally Infected Mice
Authors: Welch David F. , Carroll Karen C. , Hofmeister Erik K. , Persing David H. , Robison Denise A. , Steigerwalt Arnold G. , Brenner Don J.
Published: December 2019

Bacteremia with fever due to a novel subspecies of
Bartonella vinsonii
was found in a cattle rancher. The subspecies shared major characteristics of the genus
Bartonella
in terms of most biochemical features and cellular fatty acid profile, but it was distinguishable from other subspecies of
B. vinsonii
by good growth on heart infusion agar supplemented with X factor and by its pattern of enzymatic hydrolysis of peptide substrates. DNA relatedness studies verified that the isolate belonged to the genus
Bartonella
and that it was genotypically related to
B. vinsonii
. The highest level of relatedness was observed with recently characterized strains from naturally infected mice that were coinfected with
Borrelia burgdorferi
and
Babesia microti
. We propose the name
Bartonella vinsonii
subsp.
arupensis
subsp. nov. as the new subspecies to accommodate these human and murine isolates.
Disseminated Zygomycosis Due to
Rhizopus schipperae
after Heatstroke
Authors: Anstead Gregory M. , Sutton Deanna A. , Thompson Elizabeth H. , Weitzman Irene , Otto Randal A. , Ahuja Sunil K.
Published: December 2019

A 21-year-old woman suffered heatstroke and developed diarrhea while trekking across south Texas. The heatstroke was complicated by seizures, rhabdomyolysis, pneumonia, renal failure, and disseminated intravascular coagulation. The patient’s stool and blood cultures grew
Campylobacter jejuni
. The patient subsequently developed paranasal and gastrointestinal zygomycosis and required surgical debridement and a prolonged course of amphotericin B. The zygomycete cultured was
Rhizopus schipperae
. This is only the second isolate of
R. schipperae
that has been described.
R. schipperae
is characterized by the production of clusters of up to 10 sporangiophores arising from simple but well-developed rhizoids. These asexual reproductive propagules are produced on Czapek Dox agar but are absent on routine mycology media, where only chlamydospores are observed. Despite multiorgan failure, bacteremia, and disseminated zygomycosis, the patient survived and had a good neurological outcome. Heatstroke has not been previously described as a risk factor for the development of disseminated zygomycosis.
Sensitive Assays for Isolation and Detection of Simian Foamy Retroviruses
Authors: Khan Arifa S. , Sears Johnna F. , Muller Jacqueline , Galvin Teresa A. , Shahabuddin Muhammad
Published: December 2019

Simian foamy viruses (SFVs) are highly prevalent in a variety of nonhuman primate species ranging from prosimians to apes. SFVs possess a broad host range, and human infections can occur by cross-species transfer (W. Heneine et al., Nat. Med. 4:403–407, 1998). Retrovirus screening of potential sources of infection, such as laboratory research animals and simian-derived biological products, could minimize human exposure to SFVs by reducing the risk of potential retrovirus infection in humans. We describe a variety of sensitive assays for SFV isolation and detection which were developed with a prototype strain of SFV serotype 2. The
Mus dunni
cell line (M. R. Lander and S. K. Chattopadhyay, J. Virol. 52:695–698, 1984) was found to be highly sensitive for SFV production on the basis of various general and specific retrovirus detection assays such as reverse transcriptase assay, transmission electron microscopy, immunofluorescence assay, and Western blotting. A highly sensitive PCR assay was developed on the basis of the sequences in primary SFV isolates obtained from pig-tailed macaques (
Macaca nemestrina
) and rhesus macaques (
Macaca mulatta
). Analysis of naturally occurring SFV infection in macaques indicated that analysis by a combination of assays, including both highly sensitive, specific assays and less sensitive, broadly reactive assays, is important for evaluation of retrovirus infection.
Rapid Slide Latex Agglutination Test for Detection of Methicillin Resistance in
Staphylococcus aureus
Authors: van Griethuysen Arjanne , Pouw Miranda , van Leeuwen Nan , Heck Max , Willemse Piet , Buiting Anton , Kluytmans Jan
Published: December 2019

The MRSA screen test (Denka Seiken Co., Ltd.), a commercially available, rapid (20-min) slide latex agglutination test for the determination of methicillin resistance by detection of PBP 2a in
Staphylococcus aureus
, was compared with the oxacillin agar screen test and PCR detection of the
mecA
gene. A total of 563
S. aureus
isolates were tested. Two hundred ninety-six of the isolates were methicillin-susceptible isolates from cultures of blood from consecutive patients. Also, 267 methicillin-resistant isolates that comprised 248 different phage types were tested. Methicillin resistance was defined as the presence of the
mecA
gene. Of the 267
mecA
gene-positive isolates, 263 were positive by the MRSA screen test (sensitivity, 98.5%), and all the
mecA
-gene negative strains were negative by the MRSA screen test (specificity, 100%). The oxacillin agar screen test detected methicillin resistance in 250 of the
mecA
gene-positive isolates (sensitivity, 93.6%). The sensitivity of the MRSA screen test was statistically significantly higher than the sensitivity of the oxacillin agar screen test (
P
< 0.05). The MRSA screen test is a highly sensitive and specific test for the detection of methicillin resistance. Also, it offers results within half an hour and is easy to perform, which makes this test a valuable tool in the ongoing battle against methicillin-resistant
S. aureus
.
Evidence of Clonal Dissemination of Multidrug-Resistant
Streptococcus pneumoniae
in Hong Kong
Authors: Ip Margaret , Lyon Donald J. , Yung Raymond W. H. , Chan Colin , Cheng Augustine F. B.
Published: December 2019

The relationship between the phenotypic and genotypic characteristics of 105 penicillin-intermediate or -resistant
Streptococcus pneumoniae
isolates saved during 1994 to 1997 at the Prince of Wales Hospital and Pamela Youde Nethersole Eastern Hospital, Hong Kong, was studied. The
pbp
genes for penicillin-binding proteins 1a, 2b, and 2x for each isolate were amplified by PCR, and the products were digested with restriction enzymes
Hin
fI and
Alu
I. A combination of the pulsed-field gel electrophoresis (PFGE) profiles,
pbp
fingerprints, and phenotypic characteristics of capsular types and antibiograms enabled these isolates to be divided into four major groups. Seventy-four percent (78 of 105) of the strains, belonging to serotypes 23F, 19F, and 14, showed indistinguishable
pbp
fingerprint patterns (group A1, 1-1-1, 1-1-1), with PFGE patterns belonging to group A and its subtypes, suggesting that these strains were closely related. Eighty-three percent (65 of 78) of these isolates were also resistant to tetracycline, erythromycin, chloramphenicol, and trimethoprim. The type 23F isolates were indistinguishable from representative strains of the Spanish 23F clone by these molecular methods, indicating that these strains may be variants of the Spanish 23F clone. Serotype 6B accounted for 19% (20 of 105) of the isolates with reduced penicillin susceptibility and was made up of variants belonging to four different
pbp
fingerprint groups with the PFGE pattern group B, the predominant group being indistinguishable from that of the Spanish 6B clone. Other PFGE and fingerprint groups were mainly obtained from penicillin-susceptible strains of various serotypes. The results suggest that the rapid emergence of drug-resistant
S. pneumoniae
in Hong Kong has been due to the rapid dissemination of several successful clones.
Value of a Single-Tube Widal Test in Diagnosis of Typhoid Fever in Vietnam
Authors: Parry Christopher M. , Hoa Nguyen Thi Tuyet , Diep To Song , Wain John , Chinh Nguyen Tran , Vinh Ha , Hien Tran Tinh , White Nicholas J. , Farrar Jeremy J.
Published: December 2019

The diagnostic value of an acute-phase single-tube Widal test for suspected typhoid fever was evaluated with 2,000 Vietnamese patients admitted to an infectious disease referral hospital between 1993 and 1998. Test patients had suspected typhoid fever and a blood culture positive for
Salmonella typhi
(
n
= 1,400) or
Salmonella paratyphi
A (
n
= 45). Control patients had a febrile illness for which another cause was confirmed (malaria [
n
= 103], dengue [
n
= 76], or bacteremia due to another microorganism [
n
= 156] or tetanus (
n
= 265). An O-agglutinin titer of ≥100 was found in 18% of the febrile controls and 7% of the tetanus patients. Corresponding values for H agglutinins were 8 and 1%, respectively. The O-agglutinin titer was ≥100 in 83% of the blood culture-positive typhoid fever cases, and the H-agglutinin titer was ≥100 in 67%. The disease prevalence in investigated patients in this hospital was 30.8% (95% confidence interval, 26.8 to 35.1%); at this prevalence, an elevated level of H agglutinins gave better positive predictive values for typhoid fever than did O agglutinins. With a cutoff titer of ≥200 for O agglutinin or ≥100 for H agglutinin, the Widal test would diagnose correctly 74% of the blood culture-positive cases of typhoid fever. However, 14% of the positive results would be false-positive, and 10% of the negative results would be false-negative. The Widal test can be helpful in the laboratory diagnosis of typhoid fever in Vietnam if interpreted with care.
Molecular Characterization and Antibiotic Susceptibilities of Ocular Isolates of
Staphylococcus epidermidis
Authors: Sechi Leonardo A. , Pinna Antonio , Pusceddu Cinzia , Fadda Giovanni , Carta Francesco , Zanetti Stefania
Published: December 2019

Nineteen isolates of
Staphylococcus epidermidis
from patients with ocular infections were analyzed. Patients were selected in retrospect, by choosing cases in which
S. epidermidis
was the sole isolate. Twelve different patterns were obtained after hybridization with a probe with high-level homology to insertion sequences found in
S. epidermidis
. Susceptibilities to penicillin, methicillin, gentamicin, tetracycline, erythromycin, ciprofloxacin, vancomycin, and teicoplanin were determined. Six strains were resistant to three or more antibiotics.
Replacement for 30-Milliliter Flat-Bottomed, Glass-Stoppered, Round Bottles Used in VDRL Antigen Preparation
Authors: Pope Victoria , Castro Arnold
Published: December 2019

When the flat-bottomed, glass-stoppered, round bottle traditionally used to make VDRL antigen was discontinued, an appropriate substitute was needed. Although many laboratories have switched to one of the other nontreponemal tests for syphilis serology screening, the VDRL test remains the only approved procedure for testing spinal fluids of patients with possible neurosyphilis. We tested 25-ml glass-stoppered, convex-bottomed Erlenmeyer flasks to determine if these could be used as appropriate substitutes. We tested 52 reactive sera and 54 nonreactive sera by using one reference antigen prepared in the traditional flat-bottomed bottles and five antigens prepared in the Erlenmeyer flasks. Results with all serum samples were comparable. We also tested two lots of a commercial antigen plus an additional lot of reference antigen. Again there was no difference in the reactivity of the antigens. Therefore, we conclude that 25-ml glass-stoppered Erlenmeyer flasks can be used as an appropriate substitute for glass-stoppered, flat-bottomed, round glass bottles in the making of VDRL antigen.
Molecular Epidemiology of Ceftazidime-Resistant Gram-Negative Bacilli on Inanimate Surfaces and Their Role in Cross-Transmission during Nonoutbreak Periods
Authors: D’Agata Erika M. C. , Venkataraman Lata , DeGirolami Paola , Samore Matthew
Published: December 2019

We described the molecular epidemiology of expanded-spectrum cephalosporin-resistant gram-negative bacilli (RGN) recovered from inanimate surfaces. RGN were isolated from 9% of environmental cultures. Numerous species, each with multiple unique strains, were recovered. Epidemiological links between environmental, personnel, and patient strains suggested the exogenous acquisition of RGN from the hospital environment.
Detection of
Chlamydia trachomatis
Endocervical Infections by Ligase Chain Reaction versus ACCESS
Chlamydia
Antigen Assay
Authors: Waites K. B. , Smith K. R. , Crum M. A. , Hockett R. D. , Wells A. H. , Hook E. W.
Published: December 2019

Ligase chain reaction (LCR) was compared with ACCESS immunoassay for detection of chlamydial infections in females. Despite efforts to improve ACCESS performance by evaluation of specimens that were in the test performance “grey zone,” LCR remained more sensitive and was less expensive to perform. ACCESS had a sensitivity of 83.9%, a specificity of 99.7%, a positive predictive value of 96.3%, and a negative predictive value of 98.5%.
Characterization ofStaphylococcus aureusCoagulase Type VII Isolates from Staphylococcal Food Poisoning Outbreaks (1980–1995) in Tokyo, Japan, by Pulsed-Field Gel Electrophoresis
Authors: Shimizu Akira , Fujita Manabu , Igarashi Hideo , Takagi Michihiro , Nagase Naoko , Sasaki Asako , Kawano Junichi
Published: December 2019

Staphylococcus aureuscoagulase type VII strains have been the strains most frequently isolated from staphylococcal food poisoning outbreaks in Tokyo, Japan. We applied pulsed-field gel electrophoresis (PFGE) of chromosomal DNA digested withSmaI to characterize 129 coagulase type VII strains. These were isolated from 129 cases occurring in outbreaks in 35 districts during a 16-year period (1980–1995). The 129 outbreak strains were classified into three types, designated A (n= 115), B (n= 10), and C (n= 4). Types A and C were further divided into 33 (A1 to A33) and 4 (C1 to C4) subtypes, respectively. Strains of the same subtypes were isolated from food poisoning cases in the same districts at time intervals of 1 or 2 to 5 years. PFGE typing appears to be a useful method for subdividing strains ofS. aureuscoagulase type VII. A combination of coagulase typing and PFGE typing would provide more detailed information than the former method alone in epidemiologic investigations of staphylococcal food poisoning.
Prevalence of O1/K1- and O2/K3-Reactive
Actinobacillus suis
in Healthy and Diseased Swine
Authors: Slavić ĐurĐa , Toffner Tania L. , Monteiro Mario A. , Perry Malcolm B. , MacInnes Janet I.
Published: December 2019

A cell surface antigen-typing system was devised for the swine pathogen
Actinobacillus suis
and used to examine the prevalence of different lipopolysaccharide (O) types in healthy and diseased pigs. The strains examined in this study were isolated from a variety of locations in Canada and from Kansas. Lipopolysaccharide preparations of 151 isolates of
A. suis
were characterized by immunoblotting using polyclonal antisera generated to strains SO4 (O1/K1), H89-1173 (O2/K3), and VSB 3714, a rough strain. Approximately 54% (62 of 114) of
A. suis
isolates from diseased pigs, all (11 of 11) isolates from healthy pigs, and all (4 of 4) reference strains reacted with O1/K1 antiserum. More than 80% (18 of 22) of
A. suis
strains used for bacterin production and approximately 41% (47 of 114) of isolates from diseased pigs bound O2/K3 antiserum. One isolate appeared to be rough, and five were untypeable. O1/K1- and O2/K3-reactive strains were equally prevalent in Kansas, whereas O2/K3-reactive strains were more common in Québec and western Canada and O1/K1 strains were most common in Ontario. The fact that virtually all of the strains submitted for bacterin production were O2/K3-reactive strains is consistent with the notion that these strains may be more virulent than O1/K1 strains; alternatively, this may reflect geographic or other biases. In addition, we observed cross-reactivity between
A. suis
cell surface antigens and swine antisera to several other important pathogens. This finding may explain why previous attempts to develop a simple serodiagnostic test for
A. suis
have been unsuccessful.
Evaluation of Phenotypic and Genotypic Methods for SubtypingCampylobacter jejuniIsolates from Humans, Poultry, and Cattle
Authors: Nielsen Eva Møller , Engberg Jørgen , Fussing Vivian , Petersen Lise , Brogren Carl-Henrik , On Stephen L. W.
Published: December 2019

Six methods for subtyping ofCampylobacter jejuniwere compared and evaluated with a collection of 90 isolates from poultry, cattle, and sporadic human clinical cases as well as from a waterborne outbreak. The applied methods were Penner heat-stable serotyping; automated ribotyping (RiboPrinting); random amplified polymorphic DNA typing (RAPD); pulsed-field gel electrophoresis (PFGE); restriction fragment length polymorphisms of the flagellin gene,flaA(fla-RFLP); and denaturing gradient gel electrophoresis offlaA(fla-DGGE). The methods were evaluated and compared on the basis of their abilities to identify isolates from one outbreak and discriminate between unrelated isolates and the agreement between methods in identifying clonal lines. All methods identified the outbreak strain. For a collection of 80 supposedly unrelated isolates, RAPD and PFGE were the most discriminatory methods, followed byfla-RFLP and RiboPrinting.fla-DGGE and serotyping were the least discriminative. All isolates included in this study were found to be typeable by each of the methods. Thirteen groups of potentially related isolates could be identified using a criterion that at least four of the methods agreed on clustering of isolates. None of the subtypes could be related to only one source; rather, these groups represented isolates from different sources. Furthermore, in two cases isolates from cattle and human patients were found to be identical according to all six methods.
Detection and Quantitation of Human Immunodeficiency Virus Type 1 in the Female Genital Tract
Authors: Baron Penny , Bremer James , Wasserman Steven S. , Nowicki Marek , Driscoll Barbara , Polsky Bruce , Kovacs Andrea , Reichelderfer Patricia S.
Published: December 2019

Human immunodeficiency virus type 1 (HIV-1) was detected in the genital tracts of 59% of 225 women by RNA PCR and in 7% of the women by culture. In a comparison of two sampling methods, endocervical swabs were more sensitive than cervicovaginal lavage for HIV-1 RNA detection by PCR but not by culture and their sensitivity was independent of the concentration of HIV-1 RNA.
Performance of Five Agar Media for Recovery of Fungi from Isolator Blood Cultures
Authors: Procop G. W. , Cockerill F. R. , Vetter E. A. , Harmsen W. S. , Hughes J. G. , Roberts G. D.
Published: December 2019

We studied the recovery of 1,270 fungal isolates from 176,144 Isolator blood cultures (0.72% positive) on bacterial and fungal media, under routine and differing incubation conditions. Except with
Histoplasma capsulatum
, chocolate agar incubated for only 3 days proved to be an excellent medium for the recovery of fungi from the Isolator system.
Evaluation of Different Preservation and Storage Methods forMalasseziaspp
Authors: Crespo M. J. , Abarca M. L. , Cabañes F. J.
Published: December 2019

Freezing at −80°C, lyophilization, preservation in distilled water, and storage in different culture media were performed in order to find a suitable method that allowed a prolonged storage ofMalasseziaspp. Freezing at −80°C was the only method successful at maintaining all species.
VP4 and VP7 Genotyping by Reverse Transcription-PCR of Human Rotavirus in Mexican Children with Acute Diarrhea
Authors: Rodríguez Castillo Araceli , Villa Andrés Velasco , Ramírez González JoséErnesto , Mayén Pimentel Elvira , Melo Munguía Martín , Díaz de Jesús Benita , Olivera Díaz Hiram , García Lozano Herlinda
Published: December 2019

Dual typing (VP4 and VP7) of rotavirus obtained from 257 Mexican children during three epidemiological seasons was performed by reverse transcription-PCR. The P1G1 genotype was the most prevalent (40%), followed by P1G3 (19%) and P2G2 (16%). Thirty-one specimens (12%) presented mixed infections, while some genotypes were not found. This is the first dual typing of isolates from diarrhea cases in Mexico.
Granulomatous Amebic Encephalitis in a Patient with AIDS: Isolation of
Acanthamoeba
sp. Group II from Brain Tissue and Successful Treatment with Sulfadiazine and Fluconazole
Authors: Seijo Martinez M. , Gonzalez-Mediero G. , Santiago P. , Rodriguez de Lope A. , Diz J. , Conde C. , Visvesvara G. S.
Published: December 2019

A patient with AIDS, treated with highly active antiretroviral therapy and trimethoprim-sulfamethoxazole, presented with confusion, a hemifield defect, and a mass lesion in the right occipital lobe. A brain biopsy confirmed granulomatous amebic encephalitis (GAE) due to
Acanthamoeba castellanii
. The patient was treated with fluconazole and sulfadiazine, and the lesion was surgically excised. This is the first case of AIDS-associated GAE responding favorably to therapy. The existence of a solitary brain lesion, absence of other sites of infection, and intense cellular response in spite of a very low CD4 count conditioned the favorable outcome. We review and discuss the diagnostic microbiologic options for the laboratory diagnosis of infections due to free-living amebae.
Recognition of Two Novel Phenons of the Genus
Acinetobacter
among Non-Glucose-Acidifying Isolates from Human Specimens
Authors: Nemec Alexandr , Dijkshoorn Lenie , Ježek Petr
Published: December 2019

Genomic species diversity among 147
Acinetobacter
clinical isolates not belonging to the
A. calcoaceticus- A. baumannii
(ACB) complex was investigated by phenotypic and genotypic identification methods. The isolates were obtained between 1991 and 1999 from numerous diagnostic laboratories in the Czech Republic and were studied by numerical probabilistic identification using two biochemical frequency matrices and amplified rDNA restriction analysis (ARDRA). Their final identification was derived from the combined phenotypic and ARDRA results. In total, 102 isolates were unambiguously (
n
= 89) or presumptively (
n
= 13) identified as
A. lwoffii
(
n
= 63), genomic species 13BJ/14TU (
n
= 9),
A. johnsonii
(
n
= 7),
A. haemolyticus
(
n
= 6),
A. junii
(
n
= 5), and other genomic species (
n
< 5 isolates each). Forty-five isolates could not be identified as belonging to any described species. Among the unidentified isolates two large groups of non-glucose-acidifying, nonhemolytic, and non-gelatinase-producing isolates were distinguished. These groups, designated phenon 1 (
n
= 17) and phenon 2 (
n
= 15), had distinctive phenotypic features and novel ARDRA profiles, which suggests that they represent hitherto undescribed
Acinetobacter
species. Phenon 2 included mainly clinically insignificant isolates from outpatients, while phenon 1 comprised clinically relevant isolates mostly from the blood of hospitalized patients, and its precise taxonomic definition may therefore be of medical importance. Overall, the development of practical methods for identification required for the elucidation of the biological significance of the (genomic) species within the genus
Acinetobacter
remains a challenging task.
Outbreak of
Bacillus cereus
Infections in a Neonatal Intensive Care Unit Traced to Balloons Used in Manual Ventilation
Authors: Van Der Zwet Wil C. , Parlevliet Gerard A. , Savelkoul Paul H. , Stoof Jeroen , Kaiser Annie M. , Van Furth A. Marceline , Vandenbroucke-Grauls Christina M.
Published: December 2019

In 1998, an outbreak of systemic infections caused by
Bacillus cereus
occurred in the Neonatal Intensive Care Unit of the University Hospital Vrije Universiteit, Amsterdam, The Netherlands. Three neonates developed sepsis with positive blood cultures. One neonate died, and the other two neonates recovered. An environmental survey, a prospective surveillance study of neonates, and a case control study were performed, in combination with molecular typing, in order to identify potential sources and transmission routes of infection. Genotypic fingerprinting by amplified-fragment length polymorphism (AFLP) showed that the three infections were caused by a single clonal type of
B. cereus
. The same strain was found in trachea aspirate specimens of 35 other neonates. The case control study showed mechanical ventilation with a Sensormedics ventilation machine to be a risk factor for colonization and/or infection (odds ratio, 9.8; 95% confidence interval, 1.1 to 88.2). Prospective surveillance showed that colonization with
B. cereus
occurred exclusively in the respiratory tract of mechanically ventilated neonates. The epidemic strain of
B. cereus
was found on the hands of nursing staff and in balloons used for manual ventilation. Sterilization of these balloons ended the outbreak. We conclude that
B. cereus
can cause outbreaks of severe opportunistic infection in neonates. Typing by AFLP proved very useful in the identification of the outbreak and in the analysis of strains recovered from the environment to trace the cause of the epidemic.
Rapid Method for Species-Specific Identification of
Vibrio cholerae
Using Primers Targeted to the Gene of Outer Membrane Protein OmpW
Authors: Nandi Bisweswar , Nandy Ranjan K. , Mukhopadhyay Sarmishtha , Nair G. Balakrish , Shimada Toshio , Ghose Asoke C.
Published: December 2019

The distribution of genes for an outer membrane protein (OmpW) and a regulatory protein (ToxR) in
Vibrio cholerae
and other organisms was studied using respective primers and probes. PCR amplification results showed that all (100%) of the 254
V. cholerae
strains tested were positive for
ompW
and 229 (∼98%) of 233 were positive for
toxR
. None of the 40 strains belonging to other
Vibrio
species produced amplicons with either
ompW
- or
toxR
-specific primers, while 80 bacterial strains from other genera tested were also found to be negative by the assay. These studies were extended with representative number of strains using
ompW
- and
toxR
-specific probes in DNA dot blot assay. While the
V. cholerae
strains reacted with
ompW
probe, only one (
V. mimicus
) out of 60 other bacterial strains tested showed weak recognition. In contrast, several strains belonging to other
Vibrio
species (e.g.,
V. mimicus
,
V. splendidus
,
V. alginolyticus
,
V. fluvialis
,
V. proteolyticus
,
V. aestuarianus
,
V. salmonicida
,
V. furnissii
, and
V. parahaemolyticus
) showed weak to strong reactivity to the
toxR
probe. Restriction fragment length polymorphism analysis and nucleotide sequence data revealed that the
ompW
sequence is highly conserved among
V. cholerae
strains belonging to different biotypes and/or serogroups. All of these results suggest that the
ompW
gene can be targeted for the species-specific identification of
V. cholerae
strains. The scope of this study was further extended through the development of a one-step multiplex PCR assay for the simultaneous amplification of
ompW
and
ctxA
genes which should be of considerable value in the screening of both toxigenic and nontoxigenic
V. cholerae
strains of clinical as well as environmental origin.
Comparison of the BACTEC MGIT 960 and ESP Culture System II for Growth and Detection of Mycobacteria
Authors: Williams-Bouyer Natalie , Yorke Rebecca , Lee Hung I. , Woods Gail L.
Published: December 2019

The performances of two continuously monitoring mycobacterial culture systems—ESP Culture System II (ESP II; Trek Diagnostics, Inc., Westlake, Ohio) and BACTEC MGIT 960 (BD Biosciences, Sparks, Md.)—were compared. In addition to both liquid media, all specimens were plated onto Middlebrook 7H11/7H11 selective agar. A total of 3,151 specimens of all types (56.3% were respiratory specimens) were cultured; 231 (7.3%) yielded mycobacteria. The most common species recovered were
Mycobacterium avium
complex (69 isolates) and
Mycobacterium tuberculosis
complex (MTBC; 65 isolates). The recovery rates for ESP II, BACTEC MGIT 960, and Middlebrook agar, respectively, were 71.2, 63.9, and 61.8% for all mycobacteria; 70.2, 72.6, and 66.3% for all mycobacteria except
Mycobacterium gordonae
; and 73.8, 84.6, and 87.7% for MTBC. For liquid plus solid medium combinations, recovery rates for all mycobacteria and for MTBC, respectively, were 84.1 and 92.3% for ESP II plus Middlebrook agar and 81.5 and 98.5% for BACTEC MGIT 960 plus Middlebrook agar. The differences in recovery of all mycobacteria by ESP II and by BACTEC MGIT 960 were not significant; for the individual species, the only significant difference was recovery of more isolates of
M. gordonae
by ESP II. For those isolates recovered in both automated systems, mean times to detection of all mycobacteria and MTBC, respectively, were 15.8 and 17.4 days for ESP II and 12.5 and 11.9 days for BACTEC MGIT 960 (
P
< 0.05). False-positive signals occurred with 23 (0.7%) BACTEC MGIT 960 cultures and 84 (2.7%) ESP II cultures (
P
< 0.01). Overall contamination rates were 17.1% for BACTEC MGIT 960, 18.9% for ESP II, and 11.0% for Middlebrook agar. In summary, the ESP II and BACTEC MGIT 960 systems performed comparably with regard to growth and detection of mycobacteria, and the contamination rates were similar. However, with ESP II, times to detection of all mycobacteria and of MTBC were significantly longer, the recovery rate of
M. gordonae
was significantly higher, and the number of false-positive signals was greater than with BACTEC MGIT 960.
Comparison of Intertypic Antigenicity of Aino Virus Isolates by Dot Immunobinding Assay Using Neutralizing Monoclonal Antibodies
Authors: Yoshida Kazuo , Ohashi Seiichi , Kubo Tomomi , Tsuda Tomoyuki
Published: December 2019

Neutralizing monoclonal antibodies (MAbs) against the Aino virus were prepared, and the neutralizing epitopes of the virus were defined by competitive binding assay. Seven continuous and overlapping neutralizing epitopes existed on the G1 glycoprotein of the Aino virus. Two antigenic domains were identified and were designated I and II, with domain II consisting of six epitopes. Dot immunobinding assays (DIAs) were performed with MAbs that recognized these seven neutralizing epitopes. DIAs were performed with 1 Australian strain and 21 isolates found in Japan between the years 1964 and 1995. The MAb response patterns of all isolates were divided into four groups. The Japanese isolates did not show large differences in antigenicity, but the antigenicity of the Australian strain collected in 1968 was significantly different from that of the Japanese strains; the Australian strain lacked reactivity to three epitopes and showed only low reactivity to one epitope.
Extensive Sequence Divergence among Bovine Respiratory Syncytial Viruses Isolated during Recurrent Outbreaks in Closed Herds
Authors: Larsen L. E. , Tjørnehøj K. , Viuff B.
Published: December 2019

The nucleotides coding for the extracellular part of the G glycoprotein and the full SH protein of bovine respiratory syncytial virus (BRSV) were sequenced from viruses isolated from numerous outbreaks of BRSV infection. The isolates included viruses isolated from the same herd (closed dairy farms and veal calf production units) in different years and from all confirmed outbreaks in Denmark within a short period. The results showed that identical viruses were isolated within a herd during outbreaks and that viruses from recurrent infections varied by up to 11% in sequence even in closed herds. It is possible that a quasispecies variant swarm of BRSV persisted in some of the calves in each herd and that a new and different highly fit virus type (master and consensus sequence) became dominant and spread from a single animal in connection with each new outbreak. Based on the high level of diversity, however, the most likely explanation was that BRSV was (re)introduced into the herd prior to each new outbreak. These findings are highly relevant for the understanding of the transmission patterns of BRSV among calves and human respiratory syncytial virus among humans.
Rapid-Cycle PCR for Detection and Typing of
Mycoplasma pneumoniae
in Clinical Specimens
Authors: Kong Fanrong , Gordon Susanna , Gilbert Gwendolyn L.
Published: December 2019

We designed several new primers and modified previously described species- and type-specific primers targeting the
Mycoplasma pneumoniae
P1 adhesin gene. Optimized thermal profiles allowed one-step or nested PCR to be completed in less than 1 h. In 10 patients with pneumonia,
M. pneumoniae
type 1 was identified in 3 and type 2 in 7.
Persistent
Legionella
Infection in a Patient after Bone Marrow Transplantation
Authors: Schindel C. , Siepmann U. , Han S.-R. , Ullmann A. J. , Mayer E. , Fischer T. , Maeurer M.
Published: December 2019

We report on a patient who developed
Legionella
pneumonia after bone marrow transplantation. Despite appropriate antibiotic treatment, disease progressed. The patient developed a lung abscess from which
Legionella
and
Prevotella
were isolated. Cure was achieved by surgical resection. The resected material was sterile, but 16S ribosomal DNA analysis revealed
Legionella
DNA.
Diarrhea in Children under 5 Years of Age from Ifakara, Tanzania: a Case-Control Study
Authors: Gascón J. , Vargas M. , Schellenberg D. , Urassa H. , Casals C. , Kahigwa E. , Aponte J. J. , Mshinda H. , Vila J.
Published: December 2019

A matched case-control study was conducted in the Maternal and Child Health Clinic (MCH) in Ifakara, Tanzania, during the rainy season in order to elucidate the risk factors for and etiology of diarrheal diseases in children under 5 years of age. Cases (103) and controls (206) were matched for sex and age group. Precoded questionnaires with demographic details, clinical history, and physical signs were completed. Stools samples were collected for bacterial, parasitological, and viral studies. A high number of siblings (odds ratio [OR], 0.86;
P
= 0.027), the number of siblings surviving (OR, 0.82;
P
= 0.007), the birth order (OR, 0.85;
P
= 0.018) and the distance from the house to the water source (OR, 0.33;
P
= 0.011) were associated with the risk of diarrhea. There were high rates of enteropathogen isolates in stool samples from children without diarrhea (52.23%).
Shigella
species were the only enteropathogen statistically related with diarrhea (OR, 2.90;
P
< 0.029). Enterotoxigenic, enteropathogenic, and enteroaggregative strains of
Escherichia coli
were not related with diarrhea, and neither were
Giardia lamblia
or
Salmonella
species.
Carried Meningococci in the Czech Republic: a Diverse Recombining Population
Authors: Jolley K. A. , Kalmusova J. , Feil E. J. , Gupta S. , Musilek M. , Kriz P. , Maiden M. C. J.
Published: December 2019

Population and evolutionary analyses of pathogenic bacteria are frequently hindered by sampling strategies that concentrate on isolates from patients with invasive disease. This is especially so for the gram-negative diplococcus
Neisseria meningitidis
, a cause of septicemia and meningitis worldwide. Meningococcal isolate collections almost exclusively comprise organisms originating from patients with invasive meningococcal disease, although this bacterium is a commensal inhabitant of the human nasopharynx and very rarely causes pathological effects. In the present study, molecular biology-based techniques were used to establish the genetic relationships of 156 meningococci isolated from healthy young adults in the Czech Republic during 1993. None of the individuals sampled had known links to patients with invasive disease. Multilocus sequence typing (MLST) showed that the bacterial population was highly diverse, comprising 71 different sequence types (STs) which were assigned to 34 distinct complexes or lineages. Three previously identified hyperinvasive lineages were present: 26 isolates (17%) belonged to the ST-41 complex (lineage 3); 4 (2.6%) belonged to the ST-11 (electrophoretic type [ET-37]) complex, and 1 (0.6%) belonged to the ST-32 (ET-5) complex. The data were consistent with the view that most nucleotide sequence diversity resulted from the reassortment of alleles by horizontal genetic exchange.
Transfusion-Acquired, Autochthonous Human Babesiosis in Japan: Isolation of
Babesia microti
-Like Parasites with hu-RBC-SCID Mice
Authors: Saito-Ito Atsuko , Tsuji Masayoshi , Wei Qiang , He Shenyi , Matsui Toshimitsu , Kohsaki Masatoshi , Arai Satoru , Kamiyama Tsuneo , Hioki Kyoji , Ishihara Chiaki
Published: December 2019

We have isolated piroplasms from a patient who developed the first case of human babesiosis in Japan by using NOD/shi-
scid
mice whose circulating erythrocytes (RBCs) had been replaced with human RBCs (hu-RBC-SCID mice). Following inoculation of the patient's blood specimen into hu-RBC-SCID mice, parasites proliferated within the human RBCs in the mice, resulting in a high level of parasitemia. Parasite DNA was prepared from blood samples of the patient and the mice, and the nuclear small-subunit rRNA gene (rDNA) was amplified and sequenced. Both DNA samples gave rise to identical sequences which showed the highest degree of homology (99.2%) with the
Babesia microti
rDNA. Because the patient had received a blood transfusion before the onset of babesiosis, we investigated the eight donors who were involved. Their archived blood samples were analyzed for specific antibody and parasite DNA; only a single donor was found to be positive by both tests, and the parasite rDNA sequence from the donor coincided with that derived from the patient. The donor's serum exhibited a high antibody titer against the isolate from the patient, whereas it exhibited only a weak cross-reaction against
B. microti
strains isolated in the United States. We conclude that the first Japanese babesiosis case occurred due to a blood transfusion and that the etiological agent is an indigenous Japanese parasite which may be a geographical variant of
B. microti
. Our results also demonstrated the usefulness of hu-RBC-SCID mice for isolation of parasites from humans and for maintenance of the parasite infectivity for human RBCs.
Genomic and Phylogenetic Analysis of Hepatitis C Virus Isolates from Argentine Patients: a Six-Year Retrospective Study
Authors: Quarleri J. F. , Robertson B. H. , Mathet V. L. , Feld M. , Espı́nola L. , Requeijo M. P. , Mandó O. , Carballal G. , Oubiña J. R.
Published: December 2019

Typing of hepatitis C virus (HCV) isolates from Argentine patients was performed by using different methodologies in a population of 243 patients. HCV subtype was assigned based upon restriction fragment length polymorphism (RFLP). HCV RNA genomes obtained from serum samples were classified as belonging to clade 1 (53.5%), 2 (23.0%), or 3 (8.6%); 14.8% of samples showed HCV mixed infections, more frequently implying different subtypes within the same clade. In addition to RFLP typing, phylogenetic relatedness among sequences from both 5′ untranslated region (
n
= 50) and nonstructural 5B coding region (
n
= 15) was established.
Vibrio parahaemolyticus
Serovar O3:K6 as Cause of Unusually High Incidence of Food-Borne Disease Outbreaks in Taiwan from 1996 to 1999
Authors: Chiou Chien-Shun , Hsu Shih-Yuan , Chiu Shiou-Ing , Wang Tien-Kuei , Chao Cheng-Shun
Published: December 2019

The occurrence of food-borne disease outbreaks in Taiwan increased dramatically in 1996, and the incidence has since remained elevated. This increase in outbreaks is correlated with a high rate of isolation of
Vibrio parahaemolyticus
, which caused between 61 and 71% of the total outbreaks for the period 1996 to 1999. By serotyping, 40 serovars were identified from 3743
V. parahaemolyticus
isolates, of which O3:K6 was the most frequently detected. The O3:K6 serovar could have emerged in Taiwan as early as October 1995 and at that time accounted for only 0.6% of the
V. parahaemolyticus
infections. This level increased suddenly to 50.1% in 1996 and reached a peak (83.8%) in 1997. Comparison of the outbreak profiles for the etiology groups indicates that the high incidence of food-borne disease outbreaks during 1996 to 1999 can be attributed to the extraordinarily high O3:K6 infections. In 1999, the O3:K6 serovar was still prevalent, and accounted for 61.3% of all
V. parahaemolyticus
infections. Due to its extraordinarily high infection frequency and its capability to spread globally, this organism needs to be intensively monitored internationally.
Comparison of Human Papillomavirus Detection and Typing by Cycle Sequencing, Line Blotting, and Hybrid Capture
Authors: Vernon Suzanne D. , Unger Elizabeth R. , Williams Deon
Published: December 2019

We compared the results of human papillomavirus (HPV) detection and typing from 781 cervical samples assayed by three methods: L1 consensus PCR followed by cycle sequencing, L1 consensus PCR with biotinylated primers followed by hybridization to a line blot, and Hybrid Capture assay. Both PCR assays used L1 consensus PCR with primers MY09 and MY11. We evaluated the amplification efficiencies of both PCR assays and also compared the specific HPV types detected by each method. The samples positive by the Hybrid Capture assay were compared to the specific types detected by the PCR-based assays. The concordance between the two PCR assays in producing an HPV amplicon visible by gel electrophoresis or in detecting any HPV type was moderate: kappa values were 0.61 (95% confidence interval [CI] = 0.56 to 0.67) and 0.51 (95% CI = 0.46 to 0.58), respectively. The McNemar test for correlated proportions indicated that biotinylated PCR was less likely to produce a band (
P
= 0.001) and to detect an HPV type (
P
= 0.001) than the other PCR assay. In comparing the Hybrid Capture assay results with the HPV types detected by the PCR-based assays, we found that positivity by the Hybrid Capture assay for a number of samples may be due to cross-hybridization with HPV types not included in the Hybrid Capture assay probe cocktails.