bluuebunny/crossref_metadata_2025 huggingface dataset:
Microscopic Examination and Broth Culture of Cerebrospinal Fluid in Diagnosis of Meningitis
Authors: Dunbar Sherry A. , Eason Rachel A. , Musher Daniel M. , Clarridge Jill E.
Published: December 2019

We reviewed the results of microscopic Gram stain examination and routine culture for 2,635 cerebrospinal fluid (CSF) samples processed in an adult hospital microbiology laboratory during 55 months. There were 56 instances of bacterial or fungal meningitis (16 associated with central nervous system [CNS] shunt infection), four infections adjacent to the subarachnoid space, four cases of sepsis without meningitis, and an additional 220 CSF specimens with positive cultures in which the organism isolated was judged to be a contaminant. Because 121 of these contaminants were isolated in broth only, elimination of the broth culture would decrease unnecessary work. However, 25% of the meningitis associated with CNS shunts would have been missed by this practice. The most common cause of meningitis was
Cryptococcus neoformans
, followed by
Streptococcus pneumoniae
and
Neisseria meningitidis
. In 48 of 56 (88%) of cases, examination of the Gram-stained specimen revealed the causative organism. If patients who had received effective antimicrobial therapy prior to lumbar puncture are excluded, the CSF Gram stain is 92% sensitive. Microscopic examination incorrectly suggested the presence of organisms in only 3 of 2,635 (0.1%) CSF examinations. Thus, microscopic examination of Gram-stained, concentrated CSF is highly sensitive and specific in early diagnosis of bacterial or fungal meningitis.
Use of PCR for Diagnosis of Post-Kala-Azar Dermal Leishmaniasis
Authors: Osman Omran F. , Oskam Linda , Kroon Nel C. M. , Schoone Gerard J. , Khalil El-Tahir A. G. , El-Hassan Ahmed M. , Zijlstra Ed E. , Kager Piet A.
Published: December 2019

Microscopy and PCR were compared for use in the diagnosis of post-kala-azar dermal leishmaniasis (PKDL) in 63 patients. Aspirates of lymph nodes (samples from 52 patients), skin (23 samples), and bone marrow (18 samples) were used. For 11 patients lymph node aspiration could be repeated 6 months after they recovered from PKDL. During active PKDL, PCR was positive for 42 of 52 (80.8%) lymph node aspirates and 19 of 23 (82.7%) skin aspirates, whereas microscopy was positive for only 9 of 52 (17.3%) lymph node aspirates and 7 of 23 (30.4%) skin aspirates. PCR was always positive when parasites were seen by microscopy. When the results obtained with lymph node and skin aspirates from the same patient (
n
= 16) were compared, there was complete agreement. Bone marrow samples were negative by microscopy and PCR for 16 patients and positive by both methods for 1 patient; for one sample only the PCR was positive. PCR confirmed the co-occurrence of visceral leishmaniasis and PKDL in one patient and confirmed the suspicion of this co-occurrence in the other patient. After recovery, no parasites were found by microscopy, but 2 of 11 (18.2%) samples were still positive by PCR. Thirty negative controls were all found to be PCR negative, and 15 positive controls were all PCR positive. Cross-reactions with
Mycobacterium leprae
could be ruled out. In conclusion, PCR with inguinal lymph node or skin aspirates is suitable for confirming the clinical diagnosis of PKDL. In some patients, lymph node aspirates are probably preferred because aspiration of material from the skin may leave scars.
Cloning and Expression of the 44-Kilodalton Major Outer Membrane Protein Gene of the Human Granulocytic Ehrlichiosis Agent and Application of the Recombinant Protein to Serodiagnosis
Authors: Zhi N. , Ohashi N. , Rikihisa Y. , Horowitz H. W. , Wormser G. P. , Hechemy K.
Published: December 2019

A 44-kDa major outer membrane protein of the human granulocytic ehrlichiosis (HGE) agent is an immunodominant antigen in human infection. A gene encoding this protein was cloned and sequenced. Southern blot results revealed the existence of multigenes homologous to the P44 gene in the genome of the HGE agent. The recombinant 44-kDa protein (rP44) was expressed by using expression vector pET30a. The reactivity of the affinity-purified rP44 was evaluated by Western immunoblot analysis and dot blot immunoassay. Western immunoblot analysis showed that mouse anti-rP44 serum reacted with 44- to 42-kDa proteins in six different HGE agent strains tested except strain 2, in which three proteins of 42, 40, and 38 kDa were recognized. Eleven HGE patient serum samples, a horse anti-HGE serum, and a horse anti-
Ehrlichia equi
serum recognized the rP44 protein. This suggests that rP44 is an HGE-
E. equi
group-specific antigen. Neither human anti-
Ehrlichia chaffeensis
serum nor rabbit anti-
Borrelia burgdorferi
serum reacted with rP44. Sera from two patients coinfected with the HGE agent and
B. burgdorferi
reacted positively with rP44 and the HGE agent. Sera from 20 HGE patients with indirect fluorescent-antibody (IFA) titers ranging from 1:20 to 1:2,560 gave distinct positive reactions in a dot immunoblot assay. There was a positive correlation between the color densities of the dot reactions and the IFA titers when greater than 50 ng of recombinant antigen per dot was used. The use of the affinity-purified rP44 protein as antigen would provide a more specific, consistent, and simpler serodiagnosis for HGE than the use of whole infected cells or purified HGE agents.
CDC Group O-3: Phenotypic Characteristics, Fatty Acid Composition, Isoprenoid Quinone Content, and In Vitro Antimicrobic Susceptibilities of an Unusual Gram-Negative Bacterium Isolated from Clinical Specimens
Authors: Daneshvar M. I. , Hill B. , Hollis D. G. , Moss C. W. , Jordan J. G. , Macgregor J. P. , Tenover F. , Weyant R. S.
Published: December 2019

Between 1983 and 1994, 13 phenotypically similar unidentified clinical isolates were received by the Special Bacteriology Reference Laboratory, Centers for Disease Control and Prevention (CDC). Sources included blood (four strains), lung (three strains), knee fluid and duodenal tissue (one strain each), bone, and lymph node tissue (two strains each). All were aerobic glucose-oxidizing, slender, long, curved gram-negative rods that utilized xylose, sucrose, and maltose; did not grow on MacConkey agar in 1 to 2 days; were oxidase positive; hydrolyzed esculin; and grew on
Campylobacter
selective medium. All were negative for urease, indole, nitrate reduction, and gelatin hydrolysis. All were motile by means of a single polar flagellum with a noticeably short wavelength; however, motility was sometimes difficult to demonstrate. The cellular fatty acid compositions of these strains, as analyzed by gas-liquid chromatography, were unique, characterized by relatively large amounts of 16:1ω7c, 16:0, and 18:1ω7c with smaller amounts of 12:0, 3-OH-12:1, 14:0, 15:0, 18:0, Br-19:1, and 19:0cyc
11–12
. High-performance liquid chromatography and mass spectrometry of the quinone extracts of three representative strains showed ubiquinone-10 as the major component. Based on the breakpoints for the family
Enterobacteriaceae
, all the strains were susceptible in vitro to aminoglycosides, sulfamethoxazole-trimethoprim, and chloramphenicol but were resistant to most beta-lactams except imipenem. The MICs of amoxicillin-clavulanate and ciprofloxacin for these strains clustered around the breakpoints, which makes it difficult to predict the strains’ response in vivo to these agents. This group has been designated CDC oxidizer group 3 (O-3).
Quantitation of Bacteria in Blood of Typhoid Fever Patients and Relationship between Counts and Clinical Features, Transmissibility, and Antibiotic Resistance
Authors: Wain John , Diep To Song , Ho Vo Anh , Walsh Amanda M. , Hoa Nguyen Thi Tuyet , Parry Christopher M. , White Nicholas J.
Published: December 2019

Salmonella typhi
was isolated from 369 and
Salmonella paratyphi
A was isolated from 6 of 515 Vietnamese patients with suspected enteric fever. Compared with conventional broth culture of blood, direct plating of the buffy coat had a diagnostic sensitivity of 99.5% (95% confidence interval [CI], 97.1 to 100%). Blood bacterial counts were estimated by the pour plate method. The median
S. typhi
count in blood was 1 CFU/ml (range, <0.3 to 387 CFU/ml), of which a mean of 63% (95% CI, 58 to 67%) were intracellular. The mean number of bacteria per infected leukocyte was 1.3 (interquartile range [IQR], 0.7 to 2.4) CFU/cell (
n
= 81). Children (<15 years old;
n
= 115) had higher median blood bacterial counts than adults (
n
= 262): 1.5 (range, <0.3 to 387) versus 0.6 (range, <0.3 to 17.7) CFU/ml (
P
= 0.008), and patients who excreted
S. typhi
in feces had higher bacteremias than those who did not: a median of 3 (range, <0.3 to 32) versus 1 (range, <0.3 to 68) CFU/ml (
P
= 0.02). Blood bacterial counts declined with increasing duration of illness (
P
= 0.002) and were higher in infections caused by multidrug-resistant
S. typhi
(1.3 [range, <0.3 to 387] CFU/ml;
n
= 313) than in infections caused by antibiotic-sensitive
S. typhi
(0.5 [range, <0.3 to 32] CFU/ml;
n
= 62) (
P
= 0.006). In a multivariate analysis this proved to be an independent association, suggesting a relationship between antibiotic resistance and virulence in
S. typhi.
Detection of Circulating
Paracoccidioides brasiliensis
Antigen in Urine of Paracoccidioidomycosis Patients before and during Treatment
Authors: Salina Margarete Aparecida , Shikanai-Yasuda Maria Aparecida , Mendes Rinaldo Poncio , Barraviera Benedito , Mendes Giannini Maria José Soares
Published: December 2019

For the diagnosis and follow-up of paracoccidioidomycosis patients undergoing therapy, we evaluated two methods (immunoblotting and competition enzyme immunoassay) for the detection of circulating antigen in urine samples. A complex pattern of reactivity was observed in the immunoblot test. Bands of 70 and 43 kDa were detected more often in urine samples from patients before treatment. The immunoblot method detected gp43 and gp70 separately or concurrently in 11 (91.7%) of 12 patients, whereas the competition enzyme immunoassay detected antigenuria in 9 (75%) of 12 patients. Both tests appeared to be highly specific (100%), considering that neither fraction detectable by immunoblotting was present in urine samples from the control group. gp43 remained present in the urine samples collected during the treatment period, with a significant decrease in reactivity in samples collected during clinical recovery and increased reactivity in samples collected during relapses. Reactivity of some bands was also detected in urine specimens from patients with “apparent cure.” The detection of
Paracoccidioides brasiliensis
antigens in urine appears to be a promising method for diagnosing infection, for evaluating the efficacy of treatment, and for detecting relapse.
Invasive Infection with
Fusarium chlamydosporum
in a Patient with Aplastic Anemia
Authors: Segal Brahm H. , Walsh Thomas J. , Liu Johnson M. , Wilson Jon D. , Kwon-Chung Kyung J.
Published: December 2019

We report the first case of invasive disease caused by
Fusarium chlamydosporum
. The patient had aplastic anemia with prolonged neutropenia and was treated with immunosuppressive therapy. While she was receiving empirical amphotericin B, a dark crusted lesion developed on her nasal turbinate. Histologic analysis revealed invasive hyaline hyphae and some darkly pigmented structures that resembled conidia of dematiaceous molds. Only after the mold was grown in culture were characteristic colonial morphology, phialides, conidia, and chlamydospores evident, thus permitting the identification of
F. chlamydosporum
. This case illustrates the ever-increasing spectrum of pathogenic
Fusarium
spp. in immunocompromised patients and emphasizes the potential pitfalls in histologic diagnosis, which may have important treatment implications.
Antimicrobial Susceptibility Testing of
Bilophila wadsworthia
Isolates Submitted for Routine Laboratory Examination
Authors: Mochida Chikako , Hirakata Yoichi , Matsuda Junichi , Iori Fumiaki , Ozaki Yumi , Nakano Michiko , Hamaguchi Koichi , Izumikawa Koichi , Yamaguchi Toshiyuki , Tomono Kazunori , Maesaki Shigefumi , Yamada Yasuaki , Kohno Shigeru , Kamihira Shimeru
Published: December 2019

MICs of antibiotics against
Bilophila wadsworthia
isolates were measured by agar and broth microdilution with pyruvic acid and by Etest. The inoculum size influenced greatly agar dilution. Despite discrepancies in MICs depending on the measurement method used, clindamycin consistently showed potent activity. Broth microdilution and Etest appear to be candidates for laboratory susceptibility testing.
Multiplex PCR for Enterotoxigenic, Attaching and Effacing, and Shiga Toxin-ProducingEscherichia coliStrains from Calves
Authors: Franck Sophia M. , Bosworth Brad T. , Moon Harley W.
Published: December 2019

A multiplex PCR was developed to identify enterotoxigenic, attaching and effacing, and Shiga toxin-producingEscherichia colistrains by amplifying genes encoding K99 and F41 fimbriae, heat-stable enterotoxin a, intimin, and Shiga toxins 1 and 2. This multiplex PCR was specific and sensitive. It will be useful for identification ofE. colistrains which cause diarrhea in calves.
Diagnosis of
Mycobacterium microti
Infections among Humans by Using Novel Genetic Markers
Authors: van Soolingen Dick , van der Zanden Adri G. M. , de Haas Petra E. W. , Noordhoek Gerda T. , Kiers Albert , Foudraine Norbert A. , Portaels Francoise , Kolk Arend H. J. , Kremer Kristin , van Embden Jan D. A.
Published: December 2019

As a result of DNA typing of
Mycobacterium microti
isolates from animals in the United Kingdom and The Netherlands, we diagnosed four human
M. microti
infections. These are the first
M. microti
infections among humans to be reported. Three of the patients were immunocompromised and suffered from generalized forms of tuberculosis. The fourth patient was a 34-year-old immunocompetent male with a persistent cough and undefined X-ray abnormalities. Two of the
M. microti
infections were recognized by their IS
6110
restriction fragment length polymorphism (RFLP) patterns, which showed a high degree of similarity with those of
M. microti
strains isolated from a pig and a ferret in The Netherlands. The two other human
M. microti
infections were recognized by using the recently developed DNA fingerprinting method, “spoligotyping,” directly on clinical material. All
M. microti
isolates from the United Kingdom and The Netherlands were found to contain an exceptionally short genomic direct repeat region, resulting in identical two-spacer sequence reactions in spoligotyping. In contrast, the highly similar IS
6110
RFLP patterns of the vole strains from the United Kingdom differed considerably from the RFLPs of all
M. microti
strains isolated in The Netherlands, suggesting that geographic isolation led to divergent strains in the United Kingdom and on the continent.
Determination of Types of
Enterocytozoon bieneusi
Strains Isolated from Patients with Intestinal Microsporidiosis
Authors: Liguory Olivier , David Felicia , Sarfati Claudine , Derouin Francis , Molina Jean-Michel
Published: December 2019

To determine the types of
Enterocytozoon bieneusi
strains associated with intestinal microsporidiosis, we developed a rapid and efficient approach for typing parasites obtained from stool specimens by PCR-restriction fragment length polymorphism (PCR-RFLP). Typing was based on DNA polymorphism of the ribosomal DNA internal transcribed spacer (ITS) region of
E. bieneusi
. RFLPs generated with two restriction enzymes (
Nla
III and
Fnu
4HI) in PCR-amplified ITS products were used to classify strains into different lineages. This approach was successfully used to differentiate 78 strains that had been obtained from the stools of 65 patients with intestinal microsporidiosis. Among the 78 strains, we found four genetically unrelated lineages, showing the genetic diversity of
E. bieneusi
. Type I strains of
E. bieneusi
were found in a majority of the samples, accounting for 51 (78%) of the 65 microsporidiosis cases. In contrast, type II, III, and IV strains were found in only 8 (12%), 3 (5%), and 3 (5%) cases, respectively. All strains of
E. bieneusi
we have tested so far fall into one of four different lineages, and this study shows that human intestinal microsporidiosis is most often associated with type I strains. PCR-RFLP analysis of the ITS region of
E. bieneusi
should be useful for epidemiological studies.
Identification and Molecular Characterization of Beta-Hemolytic Streptococci Isolated from Harbor Porpoises (
Phocoena phocoena
) of the North and Baltic Seas
Authors: Swenshon M. , Lämmler C. , Siebert U.
Published: December 2019

The present study was designed to identify and comparatively investigate 35 beta-hemolytic streptococci isolated from stranded harbor porpoises or from animals caught in fishing nets of the North and Baltic seas. According to biochemical and serological data and to lectin agglutination tests with the lectin of
Arachis hypogaea
, all 35 isolates could be classified in Lancefield’s serological group L and could be identified as
Streptococcus dysgalactiae
subsp.
dysgalactiae
. All 35 group L streptococci were uniformly sensitive to most of the antibiotics tested. To further analyze the epidemiological relationship, the isolates were subjected to macrorestriction analysis of their chromosomal DNA by pulsed-field gel electrophoresis. Digestion of the chromosomal DNA with the restriction enzymes
Sma
I and
Apa
I revealed that most of the group L streptococci seemed to be apparently identical or related. These results indicate that one clone or at least related group L streptococcal clones play an important role for infections of harbor porpoises of the North and Baltic seas. This might possibly be caused by a direct transfer of the bacteria from animal to animal.
Antigenic and Molecular Characterization of Wild Type 1 Poliovirus Causing Outbreaks of Poliomyelitis in Albania and Neighboring Countries in 1996
Authors: Fiore L. , Genovese D. , Diamanti E. , Catone S. , Ridolfi B. , Ibrahimi B. , konomi R. , van der Avoort H. G. A. M. , Hovi T. , Crainic R. , Simeoni P. , Amato C.
Published: December 2019

Mass vaccination has led poliomyelitis to become a rare disease in a large part of the world, including Western Europe. However, in the past 20 years wild polioviruses imported from countries where polio is endemic have been responsible for outbreaks in otherwise polio-free European countries. We report on the characterization of poliovirus isolates from a large outbreak of poliomyelitis that occurred in Albania in 1996 and that also spread to the neighboring countries of Yugoslavia and Greece. The epidemics involved 145 subjects, mostly young adults, and caused persisting paralysis in 87 individuals and 16 deaths. The agent responsible for the outbreak was isolated from 74 patients and was identified as wild type 1 poliovirus by both immunological and molecular methods. Sequence analysis of the genome demonstrated the involvement of a single virus strain throughout the epidemics, and genotyping analysis showed 95% homology of the strain with a wild type 1 poliovirus strain isolated in Pakistan in 1995. Neutralization assays with both human sera and monoclonal antibodies were performed to analyze the antigenic structure of the epidemic strain, suggesting its peculiar antigenic characteristics. The presented data underline the current risks of outbreaks due to imported wild poliovirus and emphasize the need to improve vaccination efforts and also the need to implement surveillance in countries free of indigenous wild poliovirus.
Blood Cultures Positive for Coagulase-Negative Staphylococci: Antisepsis, Pseudobacteremia, and Therapy of Patients
Authors: Souvenir David , Anderson Donald E. , Palpant Samuel , Mroch Henry , Askin Susan , Anderson Jeffrey , Claridge Jerry , Eiland John , Malone Connie , Garrison Mark W. , Watson Patrice , Campbell Douglas M.
Published: December 2019

A blood culture cohort study investigating issues related to isolation of coagulase-negative staphylococci (CoNS) and other skin microflora is reported. Data were collected over 12 weeks to determine the incidence of significant CoNS bacteremia versus that of pseudobacteremia (contaminants) and to evaluate drug therapy in patients with cultures positive for CoNS. In addition, the effectiveness of 0.2% chlorine peroxide as a bactericidal disinfectant was compared to that of 10% providone iodine. A total of 3,276 cultures of blood from 1,433 patients were evaluated in the study. Eighty-nine cultures were positive for skin flora, with 81 of 89 (91%) involving CoNS. The incidence of significant CoNS bacteremia was 20 of 81 (24.7%), that of indeterminate bacteremia was 10 of 81 (12.3%), and that of contamination was 59 of 81 (72.8%). The incidence of significant bacteremia involving CoNS was double the 10 to 12% rate based on previous estimations at our institutions. In tests with the two bactericidal disinfectants, 22 of 1,639 cultures (1.3%) in the chlorine peroxide group versus 37 of 1,637 (2.3%) in the providone iodine group were considered contaminated (
P
= 0.065). Rates of contamination for venipuncture versus catheter collection were not significantly different (
P
= 0.46). The overall contamination rate was 59 of 3,276 (1.8%), which is consistent with the lower end of published quality assurance benchmark standards. The low rate was believed to be due to the professional phlebotomy staff in our institutions. There was excellent agreement between retrospective analysis by reviewers, when formal criteria were used, and the attending physicians’ intuitive clinical impressions in the classification of significant bloodstream infections (100% agreement) or contamination (95% agreement). However, physicians still used antimicrobial agents to treat nearly one-half of the patients with contaminated blood cultures, with vancomycin being misused in 34% of patients. In addition, 10% of patients with significant bacteremia were treated with inappropriate agents. There were no significant adverse events or prolonged hospital stays due to the unnecessary use of vancomycin; however, the additional costs of treating patients whose cultures contained CoNS contaminants was estimated to be $1,000 per patient. Measures to limit the unnecessary use of vancomycin (and other agents) are important.
Extremely High Prevalence of Nasopharyngeal Carriage of Penicillin-Resistant
Streptococcus pneumoniae
among Children in Kaohsiung, Taiwan
Authors: Chiou Chen-Chia Christine , Liu Yung-Ching , Huang Tsi-Shu , Hwang Wen-Kuei , Wang Jen-Hsien , Lin Hsi-Hsun , Yen Muh-Yong , Hsieh Kai-Sheng
Published: December 2019

Resistance (intermediate and high) to penicillin among
Streptococcus pneumoniae
strains is an emerging problem worldwide. From 1995 to 1997, isolates of
S. pneumoniae
not susceptible to penicillin were seen with increasing frequency from blood, cerebrospinal fluid, pleural fluid, and middle ear fluid from pediatric patients at the Veterans General Hospital-Kaohsiung. To determine the prevalence of carriage of these penicillin-nonsusceptible
S. pneumoniae
isolates, we obtained nasopharyngeal swab specimens from 2,905 children (ages, 2 months to 7 years) attending day-care centers or kindergartens or seen in our outpatient clinic.
S. pneumoniae
was isolated from 611 children, and 584 strains were available for analysis. The oxacillin disc test was used as a screening test to evaluate penicillin susceptibility. The MICs of 11 antibiotics (penicillin, cefaclor, cefuroxime, ceftriaxone, cefotaxime, imipenem, chloramphenicol, clarithromycin, rifampin, vancomycin, and teicoplanin) were determined by the E-test. Only 169 (29%) of the strains were susceptible to penicillin; 175 (30%) strains were intermediately resistant and 240 (41%) were highly resistant. The isolates also demonstrated high rates of resistance to other β-lactams (46% were resistant to cefaclor, 45% were resistant to cefuroxime, 45% were resistant to ceftriaxone, 31% were resistant to cefotaxime, and 46% were resistant to imipenem). The rate of resistance to macrolide antimicrobial agents was strikingly high; 95% of the isolates were not susceptible to clarithromycin. However, 97% were susceptible to rifampin and 100% were susceptible to the two glycopeptides (vancomycin and teicoplanin). While reports of penicillin-resistant
S. pneumoniae
increased worldwide through the 1980s, the high prevalence (71%) of resistance reported here is astonishing. Surveillance of nasopharyngeal swab specimen cultures may provide useful information on the prevalence of nonsusceptible strains causing invasive disease. Such information could be used to guide therapy of pneumococcal infections.
Survival ofAcinetobacter baumanniion Dry Surfaces: Comparison of Outbreak and Sporadic Isolates
Authors: Jawad A. , Seifert H. , Snelling A. M. , Heritage J. , Hawkey P. M.
Published: December 2019

Acinetobacterspp. are important nosocomial pathogens reported with increasing frequency in outbreaks of cross-infection during the past 2 decades. The majority of such outbreaks are caused byAcinetobacter baumannii. To investigate whether desiccation tolerance may be involved in the ability of certain strains ofA. baumanniito cause hospital outbreaks, a blind study was carried out with 39 epidemiologically well-characterized clinical isolates ofA. baumanniifor which survival times were determined under simulated hospital conditions. The survival times on glass coverslips of 22 strains isolated from eight well-defined hospital outbreaks in a German metropolitan area were compared with the survival times of 17 sporadic strains not involved in outbreaks but rather isolated from inpatients in the same geographic area. All sporadic isolates have been shown by pulsed-field gel electrophoresis to represent different strain types. There was no statistically significant difference between the survival times of sporadic strains ofA. baumanniiand outbreak strains (27.2 versus 26.5 days, respectively;P≤ 0.44) by the Wilcoxon-Mann-Whitney test. All investigatedA. baumanniistrains, irrespective of their areas of endemicity or epidemic occurrence, have the ability to survive for a long time on dry surfaces. Antimicrobial susceptibility testing showed thatA. baumanniioutbreak strains were significantly more resistant to various broad-spectrum antimicrobial agents than sporadic strains. Both desiccation tolerance and multidrug resistance may contribute to their maintenance in the hospital setting and may explain in part their propensity to cause prolonged outbreaks of nosocomial infection.
Epidemiological and Environmental Investigations of
Legionella pneumophila
Infection in Cattle and Case Report of Fatal Pneumonia in a Calf
Authors: Fabbi Massimo , Pastoris Maddalena Castellani , Scanziani Eugenio , Magnino Simone , Di Matteo Luigi
Published: December 2019

A fatal pneumonia due to
Legionella pneumophila
was diagnosed in a young calf reared in a dairy herd located in northern Italy. Clinical symptoms consisted of watery diarrhea, hyperthermia, anorexia, and severe dyspnea. The pathological and histological findings were very similar to those observed in human legionellosis.
Legionella pneumophila
serogroup 1 (SG1) and SG10 were isolated from the calf’s lung, and
L. pneumophila
SG1 was isolated from the calf’s liver.
L. pneumophila
SG1 was also demonstrated in the lung tissue by immunofluorescence and immunohistochemical examinations. Nine of 10
L. pneumophila
SG1 isolates belonged to the Olda subtype, and 1 belonged to the Camperdown subtype. A very low prevalence of antibodies to
Legionella
was detected in cows and calves reared in the same herd. Cultures of aqueous sediment of an old electric water heater which supplied hot water for the feeding of the calves yielded
L. pneumophila
SG1. Four of the colonies tested belonged to the Olda subtype. Ten clinical and four environmental isolates were examined for the presence of plasmids. Nine of them were also examined by pulsed-field gel electrophoresis assay, and the same patterns were found for
L. pneumophila
SG1 Olda strains isolated from the calf and from the electric heater. This is the first report of a documented case of a naturally occurring
Legionella
pneumonia in an animal. Cattle probably act as accidental hosts for legionellae, much the same as humans.
Molecular Epidemiology of
Stenotrophomonas maltophilia
Isolated from Clinical Specimens from Patients with Cystic Fibrosis and Associated Environmental Samples
Authors: Denton Miles , Todd Neil J. , Kerr Kevin G. , Hawkey Peter M. , Littlewood James M.
Published: December 2019

Stenotrophomonas maltophilia
was isolated from the respiratory tracts of 41 (25%) of 163 children attending our pediatric cystic fibrosis unit between September 1993 and December 1995. The extents of
S. maltophilia
contamination of environmental sites frequented by these patients were investigated with a selective medium incorporating vancomycin, imipenem, and amphotericin B. Eighty-two isolates of
S. maltophilia
were cultured from 67 different environmental sites sampled between January and July 1996. The organism was widespread in the home environment, with 20 (36%) and 25 (42%) of sampled sites positive in the homes of colonized and noncolonized patients, respectively. In the nosocomial setting, it was isolated from 18 (32%) sites in the hospital ward and from 4 (17%) sites in the outpatient clinic area. The most common sites of contamination were sink drains, faucets, and other items frequently in contact with water. All environmental and clinical isolates were genotyped with enterobacterial repetitive intergenic consensus sequences as primers. A total of 33 of the 41 patients were colonized with unique strains, and four pairs of patients shared strains. Further characterization by pulsed-field gel electrophoresis after digestion with
Xba
I found that there was no evidence of patient-to-patient transmission; however, there was some evidence that a small number of patients may have acquired the organism from the hospital environment. Resampling of environmental sites in the hospital ward in January 1997 revealed evidence of genetic drift, complicating the accurate determination of environmental sources for clinical strains. The source of the majority of
S. maltophilia
strains colonizing the respiratory tracts of these patients with cystic fibrosis remained uncertain but may have represented multiple, independent acquisitions from a variety of environmental sites both within and outside the hospital.
Evaluation of Intestinal Protozoan Morphology in Human Fecal Specimens Preserved in EcoFix: Comparison of Wheatley’s Trichrome Stain and EcoStain
Authors: Garcia Lynne S. , Shimizu Robyn Y.
Published: December 2019

As a result of disposal problems related to the use of mercury compounds, many laboratories have switched from mercuric chloride-based Schaudinn’s and polyvinyl alcohol (PVA) stool preservatives to other, non-mercury-based preservatives. A comparison of organism recoveries and morphologies of the intestinal protozoa was undertaken with PVA containing the EcoFix zinc-based Schaudinn’s preservative (Meridian Diagnostics, Inc.); both Wheatley’s modification of Gomori’s trichrome stain (WT) and EcoStain (ES) were used to stain 51 human fecal specimens. Morphology, clarity of nuclear and cytoplasmic detail, overall color differences, and the ease or difficulty in detecting intestinal protozoa in fecal debris were assessed for the two permanent stained smears. Overall, organism morphology of the intestinal protozoa stained with WT and that of protozoa stained with ES were not equal in nuclear and cytoplasmic detail or range of color. However, the same organisms were identified in stained fecal smears with either WT or ES, with the exception of situations in which organism numbers were characterized as rare. Included were 67 protozoan challenges (number of organisms):
Entamoeba histolytica-Entamoeba dispar
(5),
Entamoeba coli
(9),
Entamoeba hartmanni
(6),
Endolimax nana
(12),
Iodamoeba bütschlii
(8),
Blastocystis hominis
(19),
Giardia lamblia
(6),
Dientamoeba fragilis
(2), yeast (2), and leukocytes (2). Five specimens were negative for parasites but contained fecal debris that was compared for morphologic detail and color range. The ES produces a more gray-green monotone with very little pink or red tone; contrast among the various colors is less than that seen with WT. Stain intensity for all organisms was acceptable, and there were no problems with stain deposition. The quality of the protozoan morphology with ES was often comparable to that with WT (36 of 67 [53.7%]) and, in some cases, better (24 of 67 [35.8%]). Organisms on the WT-stained smear exhibited better morphology in a few instances (4 of 67 [6%]), and in three instances, there were discrepant organism numbers.
Detection ofMycoplasma hyopneumoniaein Bronchoalveolar Lavage Fluids of Pigs by PCR
Authors: Baumeister A. Katrin , Runge Martin , Ganter Martin , Feenstra Anne A. , Delbeck Friedrich , Kirchhoff Helga
Published: December 2019

In the present investigation we developed a method for the detection ofMycoplasma hyopneumoniaein bronchoalveolar lavage fluid (BALF) of pigs by PCR with a primer pair flanking a DNA fragment of 853 bp specific forM. hyopneumoniae. Several methods were tested to eliminate the amplification inhibitors present in BALFs. The best results were obtained by the extraction of the DNA from the BALFs. By the PCR performed with the extracted DNA, 102CFU ofM. hyopneumoniaecould be detected in 1 ml of BALF from specific-pathogen-free swine experimentally inoculated withM. hyopneumoniae. DNA from 11 other mycoplasma species and 17 cell-walled bacterial species colonizing the respiratory tracts of pigs was not amplified. In a field study BALFs from 40 pigs from farms with a history of chronic pneumonia were tested forM. hyopneumoniaeby cultivation and by PCR (i) with BALFs incubated in Friis medium and (ii) with DNA extracted from the BALFs. In addition, PCR was performed with postmortem lung washings from 19 of the 40 pigs, and immunofluorescence tests were carried out with sections of lungs from 18 of the 40 pigs.M. hyopneumoniaecould not be detected in 18 of the 40 pigs by any of the five methods tested. The remaining 22 pigs showed a positive reaction by the PCR with DNA extracted from the BALFs and variable positive reactions by the other tests. A complete correspondence could be observed between the immunofluorescence test result and the result of PCR with DNA. The investigation shows that the PCR with DNA extracted from BALFs is a suitable technique for the sensitive and specific in vivo detection ofM. hyopneumoniae.
Processing Respiratory Specimens with C
18
-Carboxypropylbetaine: Development of a Sediment Resuspension Buffer That Contains Lytic Enzymes To Reduce the Contamination Rate and Lecithin To Alleviate Toxicity
Authors: Thornton Charles G. , MacLellan Kerry M. , Brink Thomas L. , Wolfe David M. , Llorin Oscar J. , Passen Selvin
Published: December 2019

The C
18
-carboxypropylbetaine (CB-18) procedure for processing respiratory specimens for the detection of mycobacteria was shown to provide significant increases in sensitivity by smear and culture. However, the procedure also produced increased contamination, a loss in liquid culture sensitivity, and a reduction in smear specificity. Because of these observations, the toxicity of CB-18 and the nature of the contamination were characterized. Preincubation in 1 mM CB-18 impacted viability in a time-dependent fashion, but the magnitude of the loss was species and isolate dependent.
Mycobacterium tuberculosis
isolates were the most susceptible, losing 20 to 30% of the CFU within 30 min and 30 to 60% after 3 h, whereas
Mycobacterium avium
and
Mycobacterium fortuitum
isolates were unaffected by CB-18. In liquid culture, when the concentration of CB-18 exceeded 5 μg/ml, there was an impact on growth characteristics for the most susceptible
M. tuberculosis
isolate. In contrast,
M. fortuitum
isolates were able to grow in 100 μg of CB-18 per ml. In liquid culture, the deleterious effects of CB-18 were enhanced in the presence of antibiotics, whereas growth on solid media was not similarly affected. Supplementation of the resuspension buffer with 0.15% lecithin alleviated toxicity. Initial attempts to modify the CB-18 procedure to control contamination incorporated acids or alkalis; however, losses in culture sensitivity occurred. Studies to identify these contaminants led to the development of a sediment resuspension buffer that contained lytic enzymes to combat contamination and lecithin to alleviate toxicity. This formulation included lysozyme, zymolyase, and
Cytophaga
and
Trichoderma
extracts and was seen to reduce contamination to acceptable levels (<5%).
Recombinant Hepatitis A Virus Antigen: Improved Production and Utility in Diagnostic Immunoassays
Authors: LaBrecque F. D. , LaBrecque D. R. , Klinzman D. , Perlman S. , Cederna J. B. , Winokur P. L. , Han J.-Q. , Stapleton J. T.
Published: December 2019

Hepatitis A virus (HAV) immunoassays use cell culture-derived HAV antigen to detect HAV-specific antibodies. The current method of production of HAV antigen in tissue culture is time-consuming and expensive. We previously expressed the HAV open reading frame in recombinant vaccinia viruses (rV-ORF). The recombinant HAV polyprotein was accurately processed and was assembled into subviral particles. These particles were bound by HAV-neutralizing antibodies and were able to elicit antibodies which were detected by commercial immunoassays. The present investigation compared the production of HAV antigen by standard tissue culture methods to the production of HAV antigen with the recombinant vaccinia virus system. In addition, HAV and rV-ORF antigens were assessed for their utility in diagnostic immunoassays. Serum or plasma samples from HAV antibody-positive and antibody-negative individuals were evaluated by immunoassay that used either HAV or rV-ORF antigen. All samples (86 of 86) in which HAV antibody was detected by a commercial enzyme-linked immunosorbent assay (ELISA) also tested positive by the recombinant antigen-based immunoassay (VacRIA). Similarly, all samples (50 of 50) that were HAV antibody negative also tested negative by the VacRIA. The lower limit of detection of HAV antibody was similar among immunoassays with either HAV or rV-ORF antigen. Thus, in the population studied, the sensitivity and specificity of the VacRIA were equivalent to those of the commercial ELISA. Since production of recombinant antigen is faster and less expensive than production of traditional HAV antigen, the development of diagnostic HAV antibody tests with recombinant HAV antigen appears warranted.
Characterization of Serologically Nontypeable
Actinobacillus actinomycetemcomitans
Isolates
Authors: Paju Susanna , Saarela Maria , Alaluusua Satu , Fives-Taylor Paula , Asikainen Sirkka
Published: December 2019

Our previous studies have shown that
Actinobacillus actinomycetemcomitans
isolates of a given arbitrarily primed PCR (AP-PCR) genotype belong to the same serotype (of serotypes a through e). In the present study we investigated whether the AP-PCR genotypes of nonserotypeable
A. actinomycetemcomitans
isolates match those of the serotypeable isolates. The isolates were additionally characterized by restriction analysis of the
apaH
PCR amplification products. The material included 75 nonserotypeable and 18 serotypeable
A. actinomycetemcomitans
isolates from 34 epidemiologically unrelated subjects. The serotypeable isolates were obtained from subjects who also harbored nonserotypeable isolates. Eight AP-PCR genotypes were distinguished among the isolates; six genotypes matched those detected in our previous studies, whereas two genotypes were new. Intraindividually, the
A. actinomycetemcomitans
isolates produced identical AP-PCR banding patterns, regardless of whether they were serotypeable or nonserotypeable, in 22 of 23 subjects participating with multiple isolates. AP-PCR genotype 3, corresponding to serotype c, was by far the most common among the nonserotypeable isolates (62% of subjects). Results obtained with the
apaH
restriction analysis confirmed the results obtained with AP-PCR for 31 of the 34 subjects. The results suggest that nonserotypeable
A. actinomycetemcomitans
isolates originate from serotypeable isolates, especially from serotype c isolates, and the likelihood of the existence of additional serotypes is small.
Rapid Diagnosis of Japanese Encephalitis by Using an Immunoglobulin M Dot Enzyme Immunoassay
Authors: Solomon Tom , Thao Le Thi Thu , Dung Nguyen Minh , Kneen Rachel , Hung Nguyen The , Nisalak Ananda , Vaughn David W. , Farrar Jeremy , Hien Tran Tinh , White Nicholas J. , Cardosa Mary Jane
Published: December 2019

Japanese encephalitis (JE) occurs in rural settings in southern and eastern Asia, where diagnostic facilities are limited. For the diagnosis of JE virus (JEV) infection, we developed a nitrocellulose membrane-based immunoglobulin M (IgM) capture dot enzyme immunoassay (MAC DOT) that is rapid, simple to use, requires no specialized equipment, and can distinguish JEV from dengue infection. In a prospective field study in southern Vietnam, 155 cerebrospinal fluid (CSF) and 341 serum samples were collected from 111 children and 83 adults with suspected encephalitis. The JEV MAC DOT, performed on site, was scored visually from negative to strongly positive by two observers, and the results were compared subsequently with those of the standard IgM capture enzyme-linked immunosorbent assay. For the 179 patients with adequate specimens, the MAC DOT correctly identified 59 of 60 JEV-positive patients and 118 of 119 JEV-negative patients (sensitivity [95% confidence intervals], 98.3% [92.1 to 99.9%]; specificity, 99.2% [95.9 to 100.0%]; positive predictive value, 0.98; negative predictive value, 0.99). The MAC DOT also correctly identified three patients with dengue encephalopathy. Admission specimens were positive for 73% of JE patients. Interobserver agreement for MAC DOT diagnosis was excellent (kappa = 0.94). The JEV MAC DOT is a simple and reliable rapid diagnostic test for JE in rural hospitals.
Q Fever in the Greek Island of Crete: Detection, Isolation, and Molecular Identification of Eight Strains of
Coxiella burnetii
from Clinical Samples
Authors: Spyridaki Ioanna , Gikas Achilleas , Kofteridis Diamantis , Psaroulaki Anna , Tselentis Yannis
Published: December 2019

Over a period of 6 years (1989 to 1995), serum samples from 3,300 patients suspected to be infected by
Coxiella burnetii
were assayed for the presence of antibodies against antigen phase II of the microorganism by the indirect immunofluorescence antibody technique (IFAT). One hundred fifty-two cases were recorded, and blood samples from 17 patients were cultured for the isolation of the pathogen. By a centrifugation shell vial technique, eight strains were isolated from patients suffering from acute Q fever. The microorganism was detected in the cultures by IFAT, by Gimenez staining, and by the cytopathogenic effect on Vero and human embryonic lung (HEL) cells. PCR followed by restriction fragment length polymorphism analysis was used to confirm the diagnosis and identify the
Coxiella burnetii
strains within the cell cultures as well as to compare them with reference strains. In order to avoid time-consuming cultures, to achieve direct detection of
Coxiella burnetii
in clinical samples (blood, buffy coat, etc.), and to increase the specificity and sensitivity of the detection, nested PCR was performed. The first step of DNA extraction was performed with the QIAamp blood kit 250. For the second step of the PCR assays, the conditions of temperature and times of recycling were properly modified, and the microorganism was detected within 4 h. Our study demonstrates that Q fever is an endemic disease in Crete and that the diagnosis of
Coxiella burnetii
infection can be rapidly achieved by the detection of the microorganism in buffy coat samples by nested PCR. Although the presenting symptoms of the disease in this study differed from those in other studies, the Cretan strains do not differ genotypically from the reference strains (Nine Mile and Q212).
Diagnostic Use of PCR for Detection of
Pneumocystis carinii
in Oral Wash Samples
Authors: Helweg-Larsen Jannik , Skov Jensen Jørgen , Benfield Thomas , Gerner Svendsen Ulrik , Lundgren Jens D. , Lundgren Bettina
Published: December 2019

There is a need to develop noninvasive methods for the diagnosis of
Pneumocystis carinii
pneumonia in patients unable to undergo bronchoscopy or induction sputum. Oral wash specimens are easily obtained, and
P. ca- rinii
nucleic acid can be amplified and demonstrated by PCR. In routine clinical use, easy sample processing and single-round PCR are needed to ensure rapid analysis and to reduce the risk of contamination. We developed a single-round Touchdown PCR (TD-PCR) protocol with the ability to detect PCR inhibition in the specimen. The TD-PCR was evaluated in a routine diagnostic laboratory and was compared to a previously described PCR protocol (mitochondrial RNA) run in a research laboratory. Both PCR methods amplified a sequence of the mitochondrial rRNA gene of
P. carinii
. Paired bronchoalveolar lavage (BAL) and oral wash specimens from 76 consecutive human immunodeficiency virus type 1-infected persons undergoing a diagnostic bronchoscopy were included. The TD-PCR procedure was quicker than the mitochondrial PCR procedure (<24 versus 48 h) and, compared to microscopy, had sensitivity, specificity, and positive and negative predictive values of 89, 94, 93, and 91%, respectively, for oral wash specimens and 100, 91, 90, and 100%, respectively, for BAL specimens. Our results suggest that oral wash specimens are a potential noninvasive method to obtain a diagnostic specimen during
P. carinii
pneumonia infection and that it can be applied in a routine diagnostic laboratory.
Assessment of Spontaneous Fluctuations of Viral Load in Untreated Patients with Chronic Hepatitis C by Two Standardized Quantitation Methods: Branched DNA and Amplicor Monitor
Authors: Halfon Philippe , Bourlière Marc , Halimi Gilles , Khiri Hacène , Bertezene Patrice , Portal Isabelle , Botta-Fridlund Danielle , Gauthier AndréPierre , Jullien Monique , Feryn Jean Marc , Gerolami Victoria , Cartouzou Guy
Published: December 2019

Quantitation of hepatitis C virus (HCV) RNA in serum has been used to predict and monitor the efficacy of interferon therapy in chronic HCV infection. We prospectively studied the fluctuation of viremia by a longitudinal follow-up of HCV RNA levels for 2 months in six untreated patients. Spontaneous fluctuations of HCV RNA ranged from 2.8- to 5.7-fold with branched DNA assay and from 2.9- to 5.6-fold with Monitor. These large spontaneous fluctuations (up to 0.75 log), observed daily, weekly, and monthly, raise doubt about the clinical value of a single assessment of pretherapeutic viremia.
Molecular Diagnosis of Human Rhinovirus Infections: Comparison with Virus Isolation
Authors: Hyypiä Timo , Puhakka Tuomo , Ruuskanen Olli , Mäkelä Mika , Arola Anita , Arstila Pertti
Published: December 2019

To compare the sensitivity and specificity of RT-PCR with that of virus isolation in the detection of human rhinoviruses, we tested nasopharyngeal aspirates from 200 patients on the 1st and 7th days after the onset of the common cold. An assay utilizing a short amplicon in the conserved 5′ noncoding region was found highly sensitive. Of 192 positive samples altogether, 65 were found positive by RT-PCR only, 6 were positive by isolation exclusively, and 121 gave positive results in both tests.
Effects of Various Handling and Storage Conditions on Stability of
Treponema pallidum
DNA in Cerebrospinal Fluid
Authors: Villanueva A. V. , Podzorski R. P. , Reyes M. P.
Published: December 2019

Treponema pallidum
DNA from even small numbers of organisms was detectable in cerebrospinal fluid (CSF) stored at room temperature or at 4°C for several hours and in CSF subjected to three freeze-thaw cycles. These results suggest that negative PCR results for
T. pallidum
from patients diagnosed with
T. pallidum
invasion of the central nervous system are probably not due to the loss of target DNA prior to testing.