bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycoplasma hyopneumoniaePotentiation of Porcine Reproductive and Respiratory Syndrome Virus-Induced Pneumonia
Authors: Thacker Eileen L. , Halbur Patrick G. , Ross Richard F. , Thanawongnuwech Roongroje , Thacker Brad J.
Published: December 2019

An experimental model that demonstrates a mycoplasma species acting to potentiate a viral pneumonia was developed.Mycoplasma hyopneumoniae, which produces a chronic, lymphohistiocytic bronchopneumonia in pigs, was found to potentiate the severity and the duration of a virus-induced pneumonia in pigs. Pigs were inoculated withM. hyopneumoniae21 days prior to, simultaneously with, or 10 days after inoculation with porcine reproductive and respiratory syndrome virus (PRRSV), which induces an acute interstitial pneumonia in pigs. PRRSV-induced clinical respiratory disease and macroscopic and microscopic pneumonic lesions were more severe and persistent inM. hyopneumoniae-infected pigs. At 28 or 38 days after PRRSV inoculation,M. hyopneumoniae-infected pigs still exhibited lesions typical of PRRSV-induced pneumonia, whereas the lungs of pigs which had received only PRRSV were essentially normal. On the basis of macroscopic lung lesions, it appears that PRRSV infection did not influence the severity ofM. hyopneumoniaeinfection, although microscopic lesions typical ofM. hyopneumoniaewere more severe in PRRSV-infected pigs. These results indicate thatM. hyopneumoniaeinfection potentiates PRRSV-induced disease and lesions. Most importantly,M. hyopneumoniae-infected pigs with minimal to nondetectable mycoplasmal pneumonia lesions manifested significantly increased PRRSV-induced pneumonia lesions compared to pigs infected with PRRSV only. This discovery is important with respect to the control of respiratory disease in pigs and has implications in elucidating the potential contribution of mycoplasmas in the pathogenesis of viral infections of other species, including humans.
Pneumolysin PCR-Based Diagnosis of Invasive Pneumococcal Infection in Children
Authors: Toikka Pia , Nikkari Simo , Ruuskanen Olli , Leinonen Maija , Mertsola Jussi
Published: December 2019

Blood-based pneumolysin PCR was compared to blood culture and detection of pneumolysin immune complexes, as well as to detection of antibodies to pneumolysin and to C polysaccharide, in the diagnosis of pneumococcal infection in 75 febrile children. Invasive pneumococcal infection was suspected on clinical grounds in 67 of the febrile children, and viral infection was suspected on clinical grounds in 8 of the febrile children. In addition, 15 healthy persons were examined to test the specificity of the PCR assay. Plasma, serum, and leukocyte fractions were analyzed by PCR. The combination of all test results led to the diagnosis of pneumococcal infection in 25 patients. Pneumolysin PCR was positive in 44% of these children, an increase occurred in the pneumolysin antibodies in 39% and in the C polysaccharide antibodies in 30% of the patients; pneumolysin immune complexes were found in convalescent serum in 30%, pneumolysin immune complexes occurred in acute-phase serum samples in 16%, and a positive blood culture was found in 20% of the patients. None of the healthy controls had positive results by PCR. The results suggest that the diagnosis of
Streptococcus pneumoniae
infection from blood samples necessitates the use of several different assays. Pneumolysin PCR was the most sensitive assay, but its clinical value is reduced by the fact that three blood fractions are needed.
Validation of Binary Typing for
Staphylococcus aureus
Strains
Authors: van Leeuwen Willem , Verbrugh Henri , van der Velden Jos , van Leeuwen Nan , Heck Max , van Belkum Alex
Published: December 2019

Most of the DNA-based methods for genetic typing of
Staphylococcus aureus
strains generate complex banding patterns. Therefore, we have developed a binary typing procedure involving strain-differentiating DNA probes which were generated on the basis of randomly amplified polymorphic DNA (RAPD) analysis. We present and validate the usefulness of 15 DNA probes, according to generally accepted performance criteria for molecular typing systems. RAPD analysis with multiple primers was performed on 376
S. aureus
strains of which 97% were methicillin resistant (MRSA). Among the 1,128 RAPD patterns generated, 66 were selected which identified 124 unique DNA fragments. From these amplicons, only 12% turned out to be useful for isolate-specific binary typing. The nature of the RAPD-generated DNA fragments was investigated by partial DNA sequence analysis. Several homologies with known
S. aureus
sequences and with genes from other species were discovered; however, 87% of the probe sequences are of previously unknown origin. The locations of most of the DNA probes on the chromosome of
S. aureus
NCTC 8325 were determined by hybridization. Seven fragments were randomly dispersed along the genome, five were clustered within the 2500- to 2600-kb position of the genome, and the remaining four did not recognize complementary sequences in
S. aureus
NCTC 8325. A total of 103
S. aureus
strains (69% MRSA) were used for the validation of the binary typing technique. The 15 DNA probes provided stable epidemiological markers, both in vitro (type consistency after serial passages on culture media) and in vivo (comparison of sequential isolates recovered from cases of persistent colonization). The discriminatory power of binary typing (
D
= 0.998) exceeded that of pulsed-field gel electrophoresis (
D
= 0.966) and RAPD analysis (
D
= 0.949). Reproducibility, measured by analyzing multiple strains belonging to a multitude of different epidemiological clusters, was comparable to that of other genotyping techniques used. Contribution of the DNA probes to the discriminatory power of the system was analyzed by comparison of dendrograms. This study demonstrates that binary typing is a robust tool for the genetic typing of
S. aureus
isolates.
PCR Detection of Adenovirus in a Bone Marrow Transplant Recipient: Hemorrhagic Cystitis as a Presenting Manifestation of Disseminated Disease
Authors: Echavarria Marcela S. , Ray Stuart C. , Ambinder Richard , Dumler J. Stephen , Charache Patricia
Published: December 2019

Adenoviruses (AdV), causing fatal disseminated infections in bone marrow transplant (BMT) recipients, are associated not only with hemorrhagic cystitis (HC) but also with hepatitis, conjunctivitis, and viral interstitial pneumonia. The importance of this virus as a cause of disseminated disease, however, has remained underappreciated. AdV infection has been diagnosed primarily through the use of cell culture. The fact that cell culture is insensitive for detecting this virus has hindered recognition of the role that AdV may play in morbidity and mortality in BMT recipients. To emphasize these points, we describe a patient who presented with HC due to AdV serotype 11, genotype c, and died with disseminated infection. In addition to cell culture, this study used a newly developed PCR-based method, capable of detecting all AdV serotypes tested, including different genotypes of serotype 11. The PCR result was positive in all culture-positive samples, including samples of urine, conjunctiva, and bronchoalveolar lavage (BAL). Importantly, the PCR method provided evidence of urinary shedding of AdV in a pretransplant, culture-negative specimen and showed dissemination in a subset of culture-negative specimens, including BAL, blood, and bone marrow samples. The lack of widespread awareness of the fact that localized infections may presage dissemination, and the previous associated lack of rapid, sensitive diagnostic assays, has impaired recognition of AdV infections in patients undergoing BMT. Early detection may contribute to therapy modification and avoidance of unwarranted diagnostic procedures. It may also assist in epidemiologic control of this highly infectious pathogen and lead to a renewed interest in preventive and therapeutic approaches.
In Vitro Culture and Drug Sensitivity Assay of
Plasmodium falciparum
with Nonserum Substitute and Acute-Phase Sera
Authors: Ringwald Pascal , Meche Fleurette Solange , Bickii Jean , Basco Leonardo K.
Published: December 2019

The short-term in vitro growth of
Plasmodium falciparum
parasites in the asexual erythrocytic stage and the in vitro activities of eight standard antimalarial drugs were assessed and compared by using RPMI 1640 medium supplemented with 10% nonimmune human serum, 10% autologous or homologous acute-phase serum, or 0.5% Albumax I (lipid-enriched bovine serum albumin). In general, parasite growth was maximal with autologous (or homologous) serum, followed by Albumax I and nonimmune serum. The 50% inhibitory concentrations (IC
50
s) varied widely, depending on the serum or serum substitute. The comparison of IC
50
s between assays with autologous and nonimmune sera showed that monodesethylamodiaquine, halofantrine, pyrimethamine, and cycloguanil had similar IC
50
s. Although the IC
50
s of chloroquine, monodesethylamodiaquine, and dihydroartemisinin were similar with Albumax I and autologous sera, the IC
50
s of all test compounds obtained with Albumax I differed considerably from the corresponding values obtained with nonimmune serum. Our results suggest that Albumax I and autologous and homologous sera from symptomatic, malaria-infected patients may be useful alternative sources of serum for in vitro culture of
P. falciparum
isolates in the field. However, autologous sera and Albumax I do not seem to be suitable for the standardization of isotopic in vitro assays for all antimalarial drugs.
Comparative Evaluation of First- and Second-Generation Digene Hybrid Capture Assays for Detection of Human Papillomaviruses Associated with High or Intermediate Risk for Cervical Cancer
Authors: Poljak Mario , Brenčič Anja , Seme Katja , Vince Adriana , Marin Irena J.
Published: December 2019

In the present study we comparatively evaluated the first- and second-generation Digene Hybrid Capture assays for detection of human papillomaviruses (HPV) associated with high or intermediate risk for cervical cancer in cervical specimens. Concordant results were obtained with 468 of 483 (96.8%) specimens. All 15 specimens which gave repeatedly discordant results were positive by the second-generation test only, and 14 of them tested PCR positive. The enhanced sensitivity of the second-generation assay is mainly a result of the reformulation of hybridization reagents and, to a lesser extent, a result of the addition of new HPV probes.
Antimicrobial Resistance and Serotype Distribution of
Streptococcus pneumoniae
Strains Causing Childhood Infections in Bangladesh, 1993 to 1997
Authors: Saha Samir K. , Rikitomi N. , Ruhulamin M. , Masaki H. , Hanif M. , Islam Maksuda , Watanabe K. , Ahmed K. , Matsumoto K. , Sack R. B. , Nagatake T.
Published: December 2019

Three hundred sixty-two
Streptococcus pneumoniae
strains were isolated from children under 5 years of age at Dhaka Shishu (Children) Hospital from 1993 to 1997. The strains were isolated from blood (
n
= 105), CSF (
n
= 164), ear swab (
n
= 61), eye swab (
n
= 20), and pus (
n
= 12). Of the 362 isolates, 42 (11.6%) showed intermediate resistance (MIC, <0.1 μg/ml) and only 4 (1.1%) showed complete resistance (MIC, >2.0 μg/ml) to penicillin. Penicillin resistance exhibited a strong relationship with serotype 14; 47.8% of the penicillin-resistant strains belonged to this type. A remarkably high (64.1%) resistance to co-trimoxazole was observed, along with a significant increase during the time period studied; there was no relationship to capsular type. By way of contrast, penicillin resistance did not show any significant change during the study period. Resistance to chloramphenicol (2.2%) and erythromycin (1.1%) was rare. The high resistance to co-trimoxazole and its increasing trend demand elucidation of the clinical impact of pneumonia treatment by this antimicrobial and reconsideration of the World Health Organization recommendation for co-trimoxazole administration to children with community-acquired pneumonia at the health care worker level in Bangladesh.
Prevalence of Enterotoxigenic
Bacteroides fragilis
in Children with Diarrhea in Japan
Authors: Kato Naoki , Liu Chengxu , Kato Haru , Watanabe Kunitomo , Nakamura Haruhi , Iwai Naoichi , Ueno Kazue
Published: December 2019

In age-matched controlled studies performed in Japan, enterotoxigenic
Bacteroides fragilis
was isolated from 14.9% of 114 children aged 1 to 14 years with antibiotic-unassociated diarrhea (AUD) and 6.5% of 108 children aged 1 to 6 years with antibiotic-associated diarrhea (AAD). The difference in comparison with control children, was significant for AUD children but not AAD children.
Justification for Use of a Single Trichrome Stain as the Sole Means for Routine Detection of Intestinal Parasites in Concentrated Stool Specimens
Authors: Kellogg James A. , Elder Carol J.
Published: December 2019

Of 12,321 stool samples analyzed over a 6-year interval, 870 (7.1%) were positive for a total of 1,019 parasites, of which 1,011 (99.2%) were found in trichrome-stained smears of unconcentrated specimens while only 479 (47.0%) were detected in iodine-stained smears of concentrated samples. Stool specimens were next analyzed by trichrome staining of both unconcentrated and concentrated specimens preserved in either mercury-polyvinyl alcohol (PVA) or cupric PVA. Of 2,198 specimens, 171 (7.8%) were positive for a total of 208 parasites, 192 (92.3%) and 204 (98.1%) of which were found in the unconcentrated and concentrated specimens, respectively (
P
< 0.05). In our patient population, examination of a single trichrome-stained smear of a concentrated stool specimen is a cost-effective alternative to routinely analyzing both concentrated and unconcentrated specimens for parasites.
Evaluation of the Fungitest Kit by Using Strains from Human Immunodeficiency Virus-Infected Patients: Study of Azole Drug Susceptibility
Authors: Witthuhn F. , Toubas D. , Béguinot I. , Aubert D. , Rouger C. , Remy G. , Pinon J. M.
Published: December 2019

One hundred eighteen
Candida
clinical isolates from human immunodeficiency virus-infected patients were tested for their susceptibilities to fluconazole and itraconazole by Fungitest and the National Committee for Clinical Laboratory Standards MIC method. Fungitest results depended on both yeast species and antifungal agents. This test is able to detect sensitive strains (97% agreement with results of the MIC method in tests with fluconazole and 84% agreement in tests with itraconazole) but has a poor capacity to detect resistant strains (26% agreement in tests with fluconazole and 5% agreement in tests with itraconazole).
Coronary Angioplasty Induces Rise in
Chlamydia pneumoniae
-Specific Antibodies
Authors: Tiran Andreas , Tio Rene A. , Ossewaarde Jacobus M. , Tiran Beate , den Heijer Peter , The T. Hauw , Wilders-Truschnig Martie M.
Published: December 2019

Chlamydia pneumoniae
is frequently found in atherosclerotic lesions, and high titers of specific antibodies are associated with increased risk for acute myocardial infarction. However, a causative relation has not been established yet. We performed a prospective study of 93 patients undergoing percutaneous transluminal coronary angioplasty (PTCA) to investigate whether angioplasty influences
Chlamydia
-specific antibody titers and whether there is an association with restenosis. Blood samples were obtained before and 1 and 6 months after angioplasty. Antibodies against chlamydial lipopolysaccharide and against purified
C. pneumoniae
elementary bodies were measured by enzyme-linked immunosorbent assay (ELISA). After angioplasty, the prevalence of antibodies to lipopolysaccharide rose from 20 to 26% for immunoglobulin A (IgA), from 53 to 64% for IgG, and from 2 to 7% for IgM (
P
= 0.021, 0.004, and 0.046, respectively). There was a rapid increase of mean antibody titers of all antibody classes within 1 month of PTCA. During the following 5 months, antibody titers decreased slightly but were still higher than baseline values. Results of the
C. pneumoniae
-specific ELISA were essentially the same. The rise of anti-
Chlamydia
antibodies was not caused by unspecific reactivation of the immune system, as levels of antibodies against cytomegalovirus did not change. Neither seropositivity nor antibody titers were related to restenosis. However, increases in mean IgA and IgM titers were restricted to patients who had suffered from myocardial infarction earlier in their lives. In conclusion, we show that PTCA induces a stimulation of the humoral immune response against
C. pneumoniae
. These data support the idea that plaque disruption during angioplasty might make hidden chlamydial antigens accessible to the immune system.
Development and Characterization of Complex DNA Fingerprinting Probes for the Infectious Yeast
Candida dubliniensis
Authors: Joly Sophie , Pujol Claude , Rysz Michal , Vargas Kaaren , Soll David R.
Published: December 2019

Using a strategy to clone large genomic sequences containing repetitive elements from the infectious yeast
Candida dubliniensis
, the three unrelated sequences Cd1, Cd24, and Cd25, with respective molecular sizes of 15,500, 10,000, and 16,000 bp, were cloned and analyzed for their efficacy as DNA fingerprinting probes. Each generated a complex Southern blot hybridization pattern with endonuclease-digested genomic DNA. Cd1 generated an extremely variable pattern that contained all of the bands of the pattern generated by the repeat element RPS of
Candida albicans
. We demonstrated that Cd1 does not contain RPS but does contain a repeat element associated with RPS throughout the
C. dubliniensis
genome. The Cd1 pattern was the least stable over time both in vitro and in vivo and for that reason proved most effective in assessing microevolution. Cd24, which did not exhibit microevolution in vitro, was highly variable in vivo, suggesting in vivo-dependent microevolution. Cd25 was deemed the best probe for broad epidemiological studies, since it was the most stable over time, was the only truly
C. dubliniensis
-specific probe of the three, generated the most complex pattern, was distributed throughout all
C. dubliniensis
chromosomes, and separated a worldwide collection of 57
C. dubliniensis
isolates into two distinct groups. The presence of a species-specific repetitive element in Cd25 adds weight to the already substantial evidence that
C. dubliniensis
represents a bona fide species.
Comparison ofEhrlichia murisStrains Isolated from Wild Mice and Ticks and Serologic Survey of Humans and Animals withE. murisas Antigen
Authors: Kawahara Makoto , Ito Tadahiko , Suto Chiharu , Shibata Shinichiro , Rikihisa Yasuko , Hata Kazuhisa , Hirai Katsuya
Published: December 2019

In metropolitan Tokyo, theEhrlichia murisseropositivity rate of 24 wild mice was 63% in Hinohara Village, but in the surrounding areas, it was 0 to 5%. This finding suggests that the reservoir ofE. murisis focal. Among the 15 seropositive mice, ehrlichiae were isolated from 9Apodemus speciosusmice and 1A. argenteusmouse, respectively. Five ehrlichial isolates were obtained from 10 ticks (Haemaphysalis flava) collected in Asuke Town, Aichi Prefecture, where theE. muristype strain had been isolated. These new isolates were compared with theE. muristype strain. The mouse virulence and ultrastructure of the new isolates were similar to those of the type strain, and all of them were cross-reactive with each other, as well as with the type strain, by indirect immunofluorescent-antibody test. The levels of similarity of the base sequences of the 16S rRNA gene of one of theA. speciosusisolates and one of the tick isolates to that of theE. muristype strain were 99.79 and 99.93%, respectively. We suggest that all of these isolates areE. muris; thatE. murisis not limited toEothenomys kageusbut infects other species of mice; and thatE. murisis present at locations other than Aichi Prefecture. It appears thatH. flavais a potential vector ofE. muris. Twenty (1%) of 1803 humans from metropolitan Tokyo were found to be seropositive forE. murisantibodies. A serological survey revealed that exposure toE. murisor organisms antigenically cross-reactive toE. murisoccurred among dogs, wild mice, monkeys, bears, deer, and wild boars in Gifu Prefecture, nearby prefectures, and Nagoya City, central Japan. However, human beings andRattus norvegicusrats in this area were seronegative. These results indicate broader geographic distribution of and human and animal species exposure toE. murisor relatedEhrlichiaspp. in Japan.
Evidence for Nasal Carriage of Methicillin-Resistant Staphylococci Colonizing Intravascular Devices
Authors: Frebourg Noëlle Barbier , Cauliez Bruno , Lemeland Jean-François
Published: December 2019

Nasal surveillance cultures were performed for 54 patients exhibiting ≥10
3
CFU of methicillin-resistant coagulase-negative staphylococci per ml in central venous catheter (CVC) rinse cultures over a 6-month period. Forty-two of the nasal cultures yielded growth of methicillin-resistant coagulase-negative staphylococci, and 33 of the 42 cultures contained organisms that belonged to the same species as the CVC isolates. Of the 33 same-species isolates, 20 appeared to be identical strains by pulsed-field gel electrophoresis analysis. These data suggest that measures should be taken to reduce cross-contamination between the respiratory tract and intravascular devices. However, the potential interest in detecting methicillin-resistant coagulase-negative staphylococcus carriage in high-risk patients is hampered by the lack of sensitivity of nasal surveillance cultures.
Central Line Sepsis in a Child Due to a Previously Unidentified Mycobacterium
Authors: Hogg Geoffrey G. , Schinsky Mark F. , McNeil Michael M. , Lasker Brent A. , Silcox Vella A. , Brown June M.
Published: December 2019

A rapidly growing mycobacterium similar to strains in the present
Mycobacterium fortuitum
complex (
M. fortuitum
,
M. peregrinum
, and
M. fortuitum
third biovariant complex [sorbitol positive and sorbitol negative]) was isolated from a surgically placed central venous catheter tip and three cultures of blood from a 2-year-old child diagnosed with metastatic hepatoblastoma. The organism’s unique phenotypic profile and ribotype patterns differed from those of the type and reference strains of the
M. fortuitum
complex and indicate that this organism may represent a new pathogenic taxon.
Isolation of a Human Rotavirus Strain with a Super-Short RNA Pattern and a New P2 Subtype
Authors: Nakagomi Toyoko , Horie Yasuo , Koshimura Yumi , Greenberg Harry B. , Nakagomi Osamu
Published: December 2019

Super-short rotavirus strains that have a rearranged gene segment 11 are rarely found in humans, and only five isolates, all from Southeast Asia, have been described in the literature. We report the first isolation in Japan from an infant with severe diarrhea of a rotavirus possessing a super-short RNA pattern. This strain, designated AU19, had a G1 VP7 and is also the first isolate in Japan that possesses a P2[6] VP4. Furthermore, the P2[6] VP4 carried by AU19 was divergent in the hypervariable region of the amino acid sequence from the P2A[6] VP4s carried by asymptomatic neonatal strains or from the P2B[6] VP4 carried by porcine rotavirus strain Gottfried. Thus, AU19 is likely to represent a new VP4 subtype, which we propose to call P2C. Given the recent emergence of the P2[6] VP4s in India, Brazil, and the United States and the role of VP4 in protective immunity, further scrutiny is justified to see whether the emergence of the previously underrepresented P2[6] VP4 serotype is related to this new P2 subtype.
Production of Specific Monoclonal Antibodies to
Aspergillus
Species and Their Use in Immunohistochemical Identification of Aspergillosis
Authors: Fenelon L. E. , Hamilton A. J. , Figueroa J. I. , Bartholomew M. A. , Allen M. H. , McCarthy P. , Hay R. J.
Published: December 2019

Two anti-
Aspergillus
murine monoclonal antibodies (MAbs), designated 164G and 611F, have been produced; both specifically recognize cytoplasmic antigens of
A. fumigatus
,
A. flavus
, and
A. niger
by enzyme-linked immunosorbent assay. The MAbs can identify
Aspergillus
spp. both in frozen sections by immunofluorescence and in paraffin-embedded clinical specimens by immunofluorescence and immunoperoxidase staining.
Case of Sepsis Caused by
Bifidobacterium longum
Authors: Ha Gyoung Yim , Yang Chang Heon , Kim Heesoo , Chong Yunsop
Published: December 2019

We report a case of sepsis caused by
Bifidobacterium longum
in a 19-year-old male who had developed high fever, jaundice, and hepatomegaly after acupuncture therapy with small gold needles. Anaerobic, non-spore-forming, gram-positive bacilli were isolated from his blood and finally identified as
B. longum
. He recovered completely after treatment with ticarcillin and metronidazole. To our knowledge, this is the first report of incidental sepsis caused by
B. longum
.
Extensive Cross-Contamination of Specimens with
Mycobacterium tuberculosis
in a Reference Laboratory
Authors: de C. Ramos Marcelo , Soini Hanna , Roscanni Glaucia C. , Jaques Monica , Villares Maria C. , Musser James M.
Published: December 2019

A striking increase in the numbers of cultures positive for
Mycobacterium tuberculosis
was noticed in a mycobacterial reference laboratory in Campinas, Sao Paulo State, Brazil, in May 1995. A contaminated bronchoscope was the suspected cause of the increase. All 91
M. tuberculosis
isolates grown from samples from patients between 8 May and 18 July 1995 were characterized by spoligotyping and IS
6110
fingerprinting. Sixty-one of the 91 isolates had identical spoligotype patterns, and the pattern was arbitrarily designated S36. The 61 specimens containing these isolates had been processed and cultured in a 21-day period ending on 1 June 1995, but only 1 sample was smear positive for acid-fast bacilli. The patient from whom this sample was obtained was considered to be the index case patient and had a 4+ smear-positive lymph node aspirate that had been sent to the laboratory on 10 May. Virtually all organisms with spoligotype S36 had the same IS
6110
fingerprint pattern. Extensive review of the patients’ charts and investigation of laboratory procedures revealed that cross-contamination of specimens had occurred. Because the same strain was grown from all types of specimens, the bronchoscope was ruled out as the outbreak source. The most likely source of contamination was a multiple-use reagent used for specimen processing. The organism was cultured from two of the solutions 3 weeks after mock contamination. This investigation strongly supports the idea that
M. tuberculosis
grown from smear-negative specimens should be analyzed by rapid and reliable strain differentiation techniques, such as spoligotyping, to help rule out laboratory contamination.
Phylogenetic Classification and Species Identification of Dermatophyte Strains Based on DNA Sequences of Nuclear Ribosomal Internal Transcribed Spacer 1 Regions
Authors: Makimura Koichi , Tamura Yoshiko , Mochizuki Takashi , Hasegawa Atsuhiko , Tajiri Yoshito , Hanazawa Ryo , Uchida Katsuhisa , Saito Hiuga , Yamaguchi Hideyo
Published: December 2019

The mutual phylogenetic relationships of dermatophytes of the genera
Trichophyton
,
Microsporum
, and
Epidermophyton
were demonstrated by using internal transcribed spacer 1 (ITS1) region ribosomal DNA sequences.
Trichophyton
spp. and
Microsporum
spp. form a cluster in the phylogenetic tree with
Epidermophyton floccosum
as an outgroup, and within this cluster, all
Trichophyton
spp. except
Trichophyton terrestre
form a nested cluster (100% bootstrap support). Members of dermatophytes in the cluster of
Trichophyton
spp. were classified into three groups with ITS1 homologies, with each of them being a monophyletic cluster (100% bootstrap support). The
Arthroderma vanbreuseghemii-Arthroderma simii
group consists of
A. vanbreuseghemii
,
A. simii
,
Trichophyton mentagrophytes
isolates from humans,
T. mentagrophytes
var.
quinckeanum
,
Trichophyton tonsurans
, and
Trichophyton schoenleinii. Arthroderma benhamiae
,
T. mentagrophytes
var.
erinacei
, and
Trichophyton verrucosum
are members of the
Arthroderma benhamiae
group.
Trichophyton rubrum
and
Trichophyton violaceum
form the
T. rubrum
group. This suggests that these “species” of dermatophytes have been overclassified. The ITS1 sequences of 11 clinical isolates were also determined to identify the species, and all strains were successfully identified by comparison of their base sequences with those in the ITS1 DNA sequence database.
Evaluation of Accuracy and Repeatability of Identification of Food-Borne Pathogens by Automated Bacterial Identification Systems
Authors: Odumeru Joseph A. , Steele Marina , Fruhner Lynne , Larkin Carolyn , Jiang Jiangdong , Mann Elroy , McNab W. Bruce
Published: December 2019

The performances of five automated microbial identification systems, relative to that of a reference identification system, for their ability to accurately and repeatedly identify six common food-borne pathogens were assessed. The systems assessed were the MicroLog system (Biolog Inc., Hayward, Calif.), the Microbial Identification System (MIS; MIDI Inc., Newark, Del.), the VITEK system (bioMérieux Vitek, Hazelwood, Mo.), the MicroScan WalkAway 40 system (Dade-MicroScan International, West Sacramento, Calif.), and the Replianalyzer system (Oxoid Inc., Nepean, Ontario, Canada). The sensitivities and specificities of these systems for the identification of food-borne isolates of
Bacillus cereus
,
Campylobacter jejuni
,
Listeria monocytogenes
,
Staphylococcus aureus
,
Salmonella
spp., and verotoxigenic
Escherichia coli
were determined with 40 reference positive isolates and 40 reference negative isolates for each pathogen. The sensitivities of these systems for the identification of these pathogens ranged from 42.5 to 100%, and the specificities of these systems for the identification of these pathogens ranged from 32.5 to 100%. Some of the systems had difficulty correctly identifying the reference isolates when the results were compared to those from the reference identification tests. The sensitivity of MIS for the identification of
S. aureus
,
B. cereus
,
E. coli
, and
C. jejuni
, for example, ranged from 47.5 to 72.5%. The sensitivity of the Microlog system for the identification of
E. coli
was 72.5%, and the sensitivity of the VITEK system for the identification of
B. cereus
was 42.5%. The specificities of four of the five systems for the identification of all of the species tested with the available databases were greater than or equal to 97.5%; the exception was MIS for the identification of
C. jejuni
, which displayed a specificity of 32.5% when it was tested with reference negative isolates including
Campylobacter coli
and other
Campylobacter
species. All systems had >80% sensitivities for the identification of
Salmonella
species and
Listeria
species at the genus level. The repeatability of these systems for the identification of test isolates ranged from 30 to 100%. Not all systems included all six pathogens in their databases; thus, some species could not be tested with all systems. The choice of automated microbial identification system for the identification of a food-borne pathogen would depend on the availability of identification libraries within the systems and the performance of the systems for the identification of the pathogen.
Identification of
Mycobacterium kansasii
by Using a DNA Probe (AccuProbe) and Molecular Techniques
Authors: Richter Elvira , Niemann Stefan , Rüsch-Gerdes Sabine , Hoffner Sven
Published: December 2019

The newly formulated
Mycobacterium kansasii
AccuProbe was evaluated, and the results obtained with the new version were compared to the results obtained with the old version of this test by using 116
M. kansasii
strains, 1
Mycobacterium gastri
strain, and 19 strains of several mycobacterial species. The sensitivity of this new formulation was 97.4% and the specificity was 100%. Still, three
M. kansasii
strains were missed by this probe. To evaluate the variability within the species, genetic analyses of the
hsp65
gene, the spacer sequence between the 16S and 23S rRNA genes, and the 16S rRNA gene of several
M. kansasii
AccuProbe-positive strains as well as all AccuProbe-negative strains were performed. Genetic analyses of the one
M. gastri
strain from the comparative assay and of two further
M. gastri
strains were included because of the identity of the 16S rRNA gene in
M. gastri
to that in
M. kansasii
. The data confirmed the genetic heterogeneity of
M. kansasii
. Furthermore, a subspecies with an unpublished
hsp65
restriction pattern and spacer sequence was described. The genetic data indicate that all
M. kansasii
strains missed by the AccuProbe test belong to one subspecies, the newly described subspecies VI, as determined by the
hsp65
restriction pattern and the spacer sequence. Since the
M. kansasii
strains that are missed are rare and all
M. gastri
strains are correctly negative, the new formulated AccuProbe provides a useful tool for the identification of
M. kansasii
.
Evaluation of Three Glycoprotein G2-Based Enzyme Immunoassays for Detection of Antibodies to Herpes Simplex Virus Type 2 in Human Sera
Authors: Eis-Hübinger Anna Maria , Däumer Martin , Matz Bertfried , Schneweis Karl Eduard
Published: December 2019

Three new glycoprotein G-based enzyme immunoassays (ETI-HSVK-G 2, Sorin Diagnostics Biomedica [assay A]; HSV Type 2 Specific IgG ELISA, Gull Laboratories, Inc. [assay B]; Cobas Core HSV-2 IgG EIA, Roche [assay C]) for the detection of herpes simplex virus (HSV) type 2 (HSV-2)-specific antibodies were evaluated. By testing sera from 25 individuals with culture-proven HSV-2 infection, the assays showed a sensitivity of 96%. The specificities, evaluated with sera from 70 HSV antibody-negative children, 75 HSV antibody-positive children, and 69 HSV antibody-negative adults, were 100% for assay A, 96.2% for assay B, and 97.8% for assay C, respectively. Discrepant results by any of the three assays, i.e., reactivity of a specimen in only one or two assays, occurred with similar frequencies for HSV-seronegative individuals as well as HSV-seropositive children and adults. For sera with discrepant results, the positive reactivity was mostly low. Thus, for determination of the prevalence of HSV-2 antibodies, only concordantly positive results were considered. On the basis of the results obtained with sera from 41 adults with culture-proven HSV-1 infection and from 173 HSV-antibody-positive pregnant women, the HSV-2 seroprevalence was 9.8%. The results show that the new glycoprotein G2-based enzyme immunoassays are useful tools for the detection of type-specific HSV-2 antibodies. However, if only one assay is performed, careful interpretation of the results is indicated, especially if the exhibited reactivity is low, and for determination of the definitive HSV-2 serostatus, confirmatory assays may still be necessary.
Highly Sensitive Methods for Quantitation of Human Immunodeficiency Virus Type 1 RNA from Plasma, Cells, and Tissues
Authors: Fischer Marek , Huber Werner , Kallivroussis Alex , Ott Peter , Opravil Milos , Lüthy Ruedi , Weber Rainer , Cone Richard W.
Published: December 2019

Precise and sensitive quantitation of viral RNA in specimens from human immunodeficiency virus (HIV) type 1 (HIV-1)-infected individuals has become an indispensable tool for the monitoring of the efficacy of highly active antiretroviral combination therapy. The present report describes reproducible and efficient protocols with enhanced sensitivity for quantitation of HIV-1 RNA from plasma, peripheral blood mononuclear cells, and tissues with Qiagen silica columns for RNA purification combined with the Roche Amplicor HIV-1 Monitor test for quantitative reverse transcription-PCR (RT-PCR). Extraction of RNA from 0.5 ml of plasma resulted in the detection of fewer than 20 HIV RNA copies/ml of plasma, equivalent to the centrifugation-based boosted RT-PCR assay. Silica extraction of cellular RNA resulted in the detection of fewer than 3 HIV-1 RNA copies/μg of total RNA. These techniques facilitate direct comparisons of viral loads between liquid and cellular specimens. Application of these sensitive methods may improve the assessment of the response to new antiretroviral regimens.
Serological Discrimination of Dogs Infected with GastricHelicobacterspp. and Uninfected Dogs
Authors: Strauss-Ayali Dalit , Simpson Kenneth W. , Schein Amy H. , McDonough Patrick L. , Jacobson Richard H. , Valentine Beth A. , Peacock Jeff
Published: December 2019

Characterization of the humoral immune responses of people toHelicobacter pyloriinfection has facilitated the investigation of the host response to bacterial virulence factors and the development of sensitive and specific diagnostic tests. Dogs are commonly infected with gastricHelicobacterspp., but the presence of multipleHelicobacterspp. and possible coinfection in individual dogs have complicated serological evaluation. Evaluation of the antigenic homology ofHelicobacterspp. revealed that the major protein bands ofHelicobacter felisandHelicobacter bizzozeronii, twoHelicobacterspp. that infect dogs, were very similar to UreA (29 to 31 kDa), UreB (63 to 66 kDa), and HSP (58 to 60 kDa) ofH. pylori, and sera from infected and uninfected dogs bound in a similar way to each antigen. Immunoblotting and an enzyme-linked immunosorbent assay (ELISA) withH. felisATCC 49179 antigen were performed with 101 serum samples (from 78 infected dogs and 23 uninfected dogs). Samples from uninfected dogs (median = 8) had fewer bands on immunoblotting than samples from infected dogs (median = 16) (P< 0.05). Combinations of the presence of any two of the low-molecular-mass bands (19, 25, 30, 32, and 37 kDa) or the high-molecular-mass bands (86 and 94 kDa) were found almost solely in samples from infected dogs (P< 0.0001). Kinetic ELISA results were significantly higher for samples from infected dogs (median = 0.0802 optical density unit [OD]/min) than for samples from uninfected dogs (median = 0.01428 OD/min). The combination of ELISA and immunoblotting results gave a specificity of 95.6% and a sensitivity of 79.8%. No correlation between ELISA results, colonization density, degree of inflammation, and presence of lymphoid follicles was observed. The results indicate substantial antigenic homology betweenH. felis,H. pylori, andH. bizzozeronii. The combination of ELISA and immunoblotting was a highly specific and moderately sensitive indicator of infection. The degree of seropositivity assessed by ELISA was not related to bacterial colonization density, the degree of gastric inflammation, or the presence of lymphoid follicles.
The
Cryptosporidium
“Mouse” Genotype Is Conserved across Geographic Areas
Authors: Morgan Una M. , Sturdee Anthony P. , Singleton Grant , Gomez M. Soledad , Gracenea Mercedes , Torres Jordi , Hamilton Steven G. , Woodside D. P. , Thompson R. C. Andrew
Published: December 2019

A 298-bp region of the
Cryptosporidium parvum
18S rRNA gene and a 390-bp region of the acetyl coenzyme A synthetase gene were sequenced for a range of
Cryptosporidium
isolates from wild house mice (
Mus domesticus
), a bat (
Myotus adversus
), and cattle from different geographical areas. Previous research has identified a distinct genotype, referred to as the “mouse”-derived
Cryptosporidium
genotype, common to isolates from Australian mice. Comparison of a wider range of Australian mouse isolates with United Kingdom and Spanish isolates from mice and cattle and also an Australian bat-derived
Cryptosporidium
isolate revealed that the “mouse” genotype is conserved across geographic areas. Mice are also susceptible to infection with the “cattle”
Cryptosporidium
genotype, which has important implications for their role as reservoirs of infection for humans and domestic animals.
Comparison of Direct Plating and Broth Enrichment Culture for the Detection of Intestinal Colonization by Glycopeptide-Resistant Enterococci among Hospitalized Patients
Authors: Ieven M. , Vercauteren E. , Descheemaeker P. , van Laer F. , Goossens H.
Published: December 2019

The results of prevalence studies on glycopeptide-resistant enterococci (GRE) in the intestine may be influenced by the detection methods applied. In most studies different media, different concentrations of antibiotics, and different methods are used, and these differences result in differences in recovery rates. In this cross-sectional study on the carrier state of GRE among patients at the University Hospital Antwerp, Antwerp, Belgium, performed on 21 May 1996, direct plating and broth enrichment were compared by using the same media. Stool samples (
n
= 213) or rectal swabs (
n
= 122) were plated directly on Enterococcosel agar (bioMérieux) and after enrichment in Enterococcosel broth. The prevalence of GRE was 12.8%. Direct plating recovered 53.4% of the GRE isolates, and broth enrichment recovered an additional 46.5% of them; in the latter test the isolates were thus present at less than 10
3
CFU per g of feces. The prevalence of GRE among dialysis patients was higher than among the other patients, but the difference was not significant (
P
= 0.06), possibly as a result of the small numbers of dialysis patients examined. The GRE species isolated included 19
E. gallinarum
(44.2%), 13
E. faecium
(30.2%), 6
E. faecalis
(13.9%), and 5
E. casseliflavus
(11.6%) isolates. All
E. faecalis
and
E. faecium
strains isolated carried the
vanA
gene, and
E. gallinarum
and
E. casseliflavus
carried the
vanC1
and
vanC2
gene, respectively. The majority of isolates were polyclonal. Our data indicate that the rate of detection of GRE from both stool samples and rectal swabs is significantly increased with enrichment cultures.
Mycobacterium malmoense
-Specific Nested PCR Based on a Conserved Sequence Detected in Random Amplified Polymorphic DNA Fingerprints
Authors: Kauppinen Juha , Mäntyjärvi Rauno , Katila Marja-Leena
Published: December 2019

Mycobacterium malmoense
is an opportunistic human pathogen of increasing clinical importance. Since it is difficult to detect and identify the organism by conventional techniques, it was decided to seek a nucleic acid amplification method specific for
M. malmoense
. The method was based on detection of a conserved band in random amplified polymorphic DNA (RAPD) fingerprints of 45
M. malmoense
strains. This band was a 1,046-bp product which was proven to be
M. malmoense
specific in dot blot hybridization analysis with a panel of mycobacterial strains belonging to 39 other species. The fragment was sequenced, and oligonucleotide primers were synthesized to evaluate the specificity of the PCR. Two primer pairs were found to be specific and sensitive in the nested PCR that was developed. All 49
M. malmoense
strains analyzed produced a PCR product of the expected size. In contrast, no strains belonging to the other mycobacterial species tested produced amplicons with these primers under specified reaction conditions. The results of the electrophoresis were confirmed by the hybridization with the
M. malmoense
-specific oligonucleotide probe. This method could be applied to the analysis of clinical or environmental samples, permitting the rapid detection of
M. malmoense
.
Pulsed-Field Gel Electrophoresis Used To Investigate Genetic Diversity ofHaemophilus influenzaeType b Isolates in Australia Shows Differences between Aboriginal and Non-Aboriginal Isolates
Authors: Moor Patricia Ezekiel , Collignon Peter C. , Gilbert Gwendolyn L.
Published: December 2019

We used pulsed-field gel electrophoresis to study the epidemiology and population structure ofHaemophilus influenzaetype b. DNAs from 187 isolates recovered between 1985 and 1993 from Aboriginal children (n= 76), non-Aboriginal children (n= 106), and non-Aboriginal adults (n= 5) in urban and rural regions across Australia were digested with theSmaI restriction endonuclease. Patterns of 13 to 17 well-resolved fragments (size range, ∼8 to 500 kb) defining 67 restriction fragment length polymorphism (RFLP) types were found. Two types predominated. One type (n= 37) accounted for 35 (46%) of the isolates from Aboriginals and 2 (2%) of the isolates from non-Aboriginals, and the other type (n= 41) accounted for 2 (3%) of the isolates from Aboriginals and 39 (35%) of the isolates from non-Aboriginals. Clustering revealed seven groups at a genetic distance of ∼50% similarity in a tree-like dendrogram. They included two highly divergent groups representing 50 (66%) isolates from Aboriginals and 6 (5%) isolates from non-Aboriginals and another genetically distinct group representing 7 (9%) isolates from Aboriginals and 81 (73%) isolates from non-Aboriginals. The results showed a heterogeneous clonal population structure, with the isolates of two types accounting for 42% of the sample. There was no association between RFLP type and the diagnosis of meningitis or epiglottitis, age, sex, date of collection, or geographic location, but there was a strong association between the origin of isolates from Aboriginal children and RFLP type F2a and the origin of isolates from non-Aboriginal children and RFLP type A8b. The methodology discriminated well among the isolates (D= 0.91) and will be useful for the monitoring of postvaccine isolates ofH. influenzaetype b.
Second Generation of the Automated Cobas Amplicor HCV Assay Improves Sensitivity of Hepatitis C Virus RNA Detection and Yields Results That Are More Clinically Relevant
Authors: Doglio Alain , Laffont Catherine , Caroli-Bosc François X. , Rochet Pascale , Lefebvre J.-Claude
Published: December 2019

The first and second generations of the Cobas Amplicor HCV assay were compared among patients at risk of hepatitis C virus (HCV) infection. The second-generation test was found to be of greater sensitivity and of good specificity among clinical specimens containing HCV RNA of different genotypes. Finally, this new test is shown to predict the outcome of interferon therapy better.
Molecular Cloning and Sequencing of thearoAGene fromActinobacillus pleuropneumoniaeand Its Use in a PCR Assay for Rapid Identification
Authors: Moral Carmen Hernanz , Soriano Alberto Cascón , Salazar María Sánchez , Marcos Javier Yugueros , Ramos Susana Suárez , Carrasco German Naharro
Published: December 2019

The gene (aroA) ofActinobacillus pleuropneumoniae, serotype 2, encoding 5-enolpyruvylshikimate-3-phosphate synthase was cloned by complementation of thearoAmutation inEscherichia coliK-12 strain AB2829, and the nucleotide sequence was determined. A pair of primers from the 5′ and 3′ termini were selected to be the basis for development of a specific PCR assay. A DNA fragment of 1,025 bp was amplified from lysedA. pleuropneumoniaeserotypes 1 to 12 of biovar 1 or from isolated DNA. No PCR products were detected when chromosomal DNAs from other genera were used as target DNAs; however, a 1,025-bp DNA fragment was amplified whenActinobacillus equulichromosomal DNA was used as a target, which could be easily differentiated by its NAD independence. The PCR assay developed was very sensitive, with lower detection limits of 12 CFU withA. pleuropneumoniaecells and 0.8 pg with extracted DNA. Specificity and sensitivity make this PCR assay a useful method for the rapid identification and diagnosis ofA. pleuropneumoniaeinfections.
False-Positive Results Obtained with the Alexon ProSpecT
Cryptosporidium
Enzyme Immunoassay
Authors: Doing Kirk M. , Hamm Jill L. , Jellison Jo Ann , Marquis Jessica A. , Kingsbury Cindy
Published: December 2019

Cryptosporidium
is known to cause diarrhea in immunocompromised patients and is also associated with outbreaks of disease due to food-borne and waterborne parasites. Traditional procedures, involving iodine staining of wet mounts of stool sediments and trichrome staining, lack the sensitivity to detect
Cryptosporidium
. Special staining procedures, such as the modified acid-fast and safranin stains, are generally employed. Less labor-intensive antigen detection assays have simplified detection; however, careful attention to local epidemiology is important because false-positive tests occur. Here, we report two incidents involving 62 false-positive results obtained with the Alexon ProSpecT
Cryptosporidium
enzyme immunoassay, which were deemed false-positive based on negative results obtained from extensive microscopic examinations.