bluuebunny/crossref_metadata_2025 huggingface dataset:
Diagnosis of cutaneous leishmaniasis and species discrimination of parasites by PCR and hybridization
Authors: Rodríguez N , Guzman B , Rodas A , Takiff H , Bloom B R , Convit J
Published: January 2020

The aim of this study was to assess the efficacy of PCR methodology in establishing the diagnosis of cutaneous leishmaniasis in patients from areas of endemicity in Venezuela. Biopsies from 233 patients with cutaneous ulcers suggestive of leishmaniasis were analyzed by PCR, employing oligonucleotides directed against conserved regions of kinetoplast DNA (kDNA), and the PCR products were then hybridized to nonradioactively labeled, species-specific, cloned kDNA fragments. The ability of PCR to detect Leishmania cells was compared with those of the conventional methodologies: skin testing with killed promastigotes (Montenegro test), examination of Giemsa-stained biopsy smears, and in vitro culture of biopsy tissue. The PCR-hybridization technique detected the presence of Leishmania cells in 98% of patients clinically diagnosed as having leishmaniasis and also positive by the Montenegro skin test. In comparison, leishmania positivity was found in only 42% of cultures and 64% of biopsy smears. By hybridizing the PCR product to new kDNA probes specific for either Leishmania mexicana or Leishmania braziliensis, we found that both species are major causes of cutaneous leishmaniasis in Venezuela, and the species identification was confirmed by restriction enzyme analysis of kDNA from biopsy cultures. This work demonstrates that PCR coupled with hybridization is useful not only for the diagnosis of cutaneous leishmaniasis but also for the taxonomic discrimination essential for both epidemiology and therapy. This technique can be used to diagnose leishmaniasis in a country in which the disease is endemic and can perhaps be adapted for use in a rural clinic.
Bordetella holmesii sp. nov., a new gram-negative species associated with septicemia
Authors: Weyant R S , Hollis D G , Weaver R E , Amin M F , Steigerwalt A G , O'Connor S P , Whitney A M , Daneshvar M I , Moss C W , Brenner D J
Published: January 2020

CDC nonoxidizer group 2 (NO-2) currently consists of 15 gram-negative, rod-shaped, oxidase-negative, asaccharolytic, brown soluble pigment-producing strains isolated from blood cultures, usually from young adults. On the basis of their cellular fatty acid profiles, NO-2 strains formed a single group that was identical with the profile of Bordetella avium. 16S rRNA sequencing of one NO-2 strain and the type strains of B. pertussis, B. parapertussis, B. bronchiseptica, and B. avium showed a high degree of homology (> or = 98% over 1,525 bases). The NO-2 guanine-plus-cytosine content (61.5 to 62.3 mol%) and major ubiquinone analysis (ubiquinone-8) results were both consistent with those for the genus Bordetella. DNA relatedness studies (hydroxyapatite method) confirmed a close relatedness between NO-2 and Bordetella species and demonstrated that NO-2 strains were a single new species. The name B. holmesii sp. nov. is proposed for CDC group NO-2.
Detection by PCR of wild-type canine parvovirus which contaminates dog vaccines
Authors: Senda M , Parrish C R , Harasawa R , Gamoh K , Muramatsu M , Hirayama N , Itoh O
Published: January 2020

A method for detecting wild-type canine parvovirus (CPV) strains which contaminate vaccines for dogs has been developed by PCR. PCR primers which distinguish vaccine strains from the most common, recent strains of wild-type CPV in many countries, including Japan and the United States, were developed. This PCR is based on the differences in nucleotide sequences which determine the two antigenic types of this virus. CPV vaccine strains derived from antigenically old-type virus prevalent in former times were not detected by PCR with differential primers. Detection sensitivity of PCR was 100- to 10,000-fold higher than that of the culture method in Crandell feline kidney cells.
Patients with enteric adenovirus gastroenteritis admitted to an Australian pediatric teaching hospital from 1981 to 1992
Authors: Grimwood K , Carzino R , Barnes G L , Bishop R F
Published: January 2020

During the period 1981 to 1992, 4,473 fecal specimens collected from children hospitalized with acute gastroenteritis at the Royal Children's Hospital, Melbourne, Australia, were examined by electron microscopy. A monoclonal antibody enzyme immunoassay for enteric adenovirus (EAd) types 40 (Ad40) and 41 (Ad41) was used when adenoviruses were visualized. Fecal samples were positive for adenovirus by both electron microscopy and enzyme immunoassay in 138 patients (3.1%). Ad40 was identified in 19 children (14%), and Ad41 was identified in 119 children (86%). These EAd were identified during each of the 12 years surveyed. EAd were present year-round, but the annual number of hospitalizations was not constant. Yearly prevalence varied from 0.7% (1981) to 6.5% (1985). This was associated with monthly fluctuations in Ad41 activity, with overall peak monthly prevalence in May (late autumn). By contrast, Ad40 numbers remained low and constant year-round. The frequency of Ad41 relative to Ad40 increased from 25% in 1981 to exceed 75% after 1983. Children admitted with EAd infection were more likely to have diarrhea for more than 5 days (P < 0.001) but less likely to be febrile or dehydrated (P < 0.05) than children with rotavirus infection. EAd are responsible for enteric symptoms of only a fraction of hospitalized children with infectious diarrhea but result in a more-protracted illness than rotavirus. Their relationship to persistent diarrhea requires further investigation.
Value of plasmid profiling, ribotyping, and detection of IS200 for tracing avian isolates of Salmonella typhimurium and S. enteritidis
Authors: Millemann Y , Lesage M C , Chaslus-Dancla E , Lafont J P
Published: January 2020

Seventy selected strains of Salmonella typhimurium and S. enteritidis isolated from related poultry flocks in three independent geographical areas were characterized by phenotypic and genotypic methods to compare the usefulness of the methods in epidemiological studies. The 56 S. typhimurium isolates were poorly discriminated by their biotypes, resistance patterns, and plasmid profiles. Nine different ribotypes were obtained after DNA digestion by BglII, PvuII, and SmaI. Seven IS200 types, characterized by six to nine copies of IS200 on the chromosome, were detected after digestion of genomic DNA by PstI. These studies resulted in the definition of 15 clonal lineages distributed in three clusters. The 14 S. enteritidis strains were not discriminated either by ribotyping or by detection of IS200 (IS200 typing), but were separated on the basis of antibiotic resistance and plasmid profiling. The stability of the insertion sequence type was confirmed by inoculation of an S. typhimurium strain to axenic chickens reared for 15 weeks in sterile isolators.
Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR
Authors: Dutka-Malen S , Evers S , Courvalin P
Published: January 2020

A PCR assay that allows simultaneous detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci (Enterococcus faecium, E. faecalis, E. gallinarum, and E. casseliflavus) was developed. This assay was based on specific amplification of internal fragments of genes encoding D-alanine:D-alanine ligases and related glycopeptide resistance proteins. The specificity of the assay was tested on 5 well-characterized glycopeptide-resistant strains and on 15 susceptible enterococcal type strains. Clinical isolates of enterococci that could not be identified to the species level by conventional methods were identified by the PCR test. This assay offers a specific and rapid alternative to antibiotic susceptibility tests, in particular for detection of low-level vancomycin resistance.
Evaluation of a new immunologic test kit for rapid detection of group A streptococci, the Abbott Testpack Strep A plus
Authors: Laubscher B , van Melle G , Dreyfuss N , de Crousaz H
Published: January 2020

We compared the Testpack Strep A plus (TPSAP) enzyme-linked immunosorbent assay test for group A beta-hemolytic streptococcal (GAS) antigen rapid detection with blood agar culture in 454 pediatric patients with clinical pharyngitis. Of the 454 patients, 118 (25.9%) had positive oropharyngeal cultures for GAS. TPSAP sensitivity was 89.9% (106 of 118) and specificity was 95.8% (322 of 336). We conclude that the TPSAP is specific enough to indicate treatment for a patient with a positive test but that a negative test should be confirmed by culture.
Simple and rapid detection of Mycobacterium tuberculosis complex organisms in bovine tissue samples by PCR
Authors: Liébana E , Aranaz A , Mateos A , Vilafranca M , Gomez-Mampaso E , Tercero J C , Alemany J , Suarez G , Domingo M , Dominguez L
Published: January 2020

Mycobacterium bovis is a slowly growing microorganism, and confirmation of the diagnosis by conventional culture is a lengthy process. A simple, rapid method for the extraction of DNA from bovine tissue samples was developed and used in a PCR designed for the diagnosis of tuberculosis. Tissues from 81 cattle from tuberculosis-infected herds (group 1) and 19 cattle from tuberculosis-free herds (group 2) were tested in this PCR, and the results were compared with those of conventional culture. The PCR assay detected 71.4% of the culture-positive animals from group 1. Tissue from all animals in group 2 were negative in the PCR assay and by culture. The described method could be used as a rapid screening technique which would be complementary to culture of tissue specimens for the routine diagnosis of bovine tuberculosis. The PCR technique is much faster than culture and reduces the time for diagnosis from several months to 2 days. It also provides for the detection of M. bovis when rapidly growing Mycobacterium spp. are present in the sample and may be able to detect the presence of M. bovis in samples even when organisms have become nonviable.
Detection of Mycobacterium leprae infection by PCR
Authors: Wichitwechkarn J , Karnjan S , Shuntawuttisettee S , Sornprasit C , Kampirapap K , Peerapakorn S
Published: January 2020

PCR amplification of the 531-bp fragment of the Mycobacterium leprae pra gene in fresh biopsy and slit skin smear samples was evaluated for its usefulness in the detection of leprosy bacilli in patients in Thailand. In multibacillary patients, 87.1% (27 of 31) of biopsy specimens and 41.9% (13 of 31) of slit skin smear specimens were positive by PCR, whereas in paucibacillary patients, 36.4% (8 of 22) of biopsy specimens and 18.2% (4 of 22) of slit skin smear specimens yielded detectable PCR amplification. Compared with other diagnostic procedures, PCR showed a clear advantage over both microscopic examination of slit skin smears and serologic detection of anti-phenolic glycolipid 1 antibody, especially in paucibacillary patients when bacterial indexes were 0 and seropositivity was only 6.25%. PCR was also evaluated for its potential to help monitor bacterial clearance in some of these patients during chemotherapeutic treatment. The PCR results on slit skin smear samples at 1, 3, and 6 months of chemotherapy showed that the number of PCR-positive cases of both multibacillary and paucibacillary types decreased sequentially. The results of this study are encouraging. However, investigation of a larger number of clinical specimens with an improvement in PCR methods, especially on slit skin smears, needs to be done before PCR can be established as a diagnostic procedure for leprosy patients and subclinical cases or as a tool for drug assessment.
Rapid identification of Staphylococcus aureus from blood culture bottles by a classic 2-hour tube coagulase test
Authors: McDonald C L , Chapin K
Published: January 2020

The rapid, reliable identification of Staphylococcus aureus from positive blood cultures provides important information. While multiple methodologies for detection of S. aureus from blood culture broths exist, none is satisfactory. Immunologic tests have shown varied sensitivities, the thermonuclease test, while sensitive, is not practical for routine use, and probe tests are expensive. Few studies have addressed using the tube coagulase test (TCT). This study compared two immunologic methods, the Staph Latex kit (Remel Laboratories) and the Staphaurex kit (Wellcome Diagnostics), with a rabbit plasma TCT (Difco Laboratories) to identify S. aureus within 2 h directly from blood culture broths and pelleted supernatants from BACTEC (Johnston Laboratories) bottles. One hundred twelve unique clinical blood culture isolates consistent with a Gram stain for staphylococci and 68 negative blood culture bottles seeded with a variety of gram-positive organisms were evaluated. Sensitivity and specificity among clinical specimens for the 2-h TCT were 79.5 and 100%, respectively. Sensitivities for the immunologic methods were 12.8 and 10.2% for the Staphaurex and Remel Staph Latex, respectively, and specificities for both were 100%. These results contradict previously reported results for both immunologic and TCT methods and dictate that a specific as well as a sensitive method be employed. The 2-h TCT was found to be a cost-effective, reliable, and rapid method for identifying S. aureus from positive blood cultures.
Detection and differentiation of antigenically distinct small round-structured viruses (Norwalk-like viruses) by reverse transcription-PCR and southern hybridization
Authors: Ando T , Monroe S S , Gentsch J R , Jin Q , Lewis D C , Glass R I
Published: January 2020

Application of reverse transcription (RT)-PCR to detect small round-structured viruses (SRSVs) from fecal specimens of patients with gastroenteritis has been insensitive because of the tremendous sequence heterogeneity between strains. We have designed two RT-PCR primer sets (G-1 and G-2) based on the nucleotide sequence diversity in the RNA polymerase gene of SRSVs belonging to two distinct genogroups represented by Norwalk virus (primers G-1) and Snow Mountain agent (primers G-2). All 22 SRSV strains examined that had been classified previously by solid-phase immune electron microscopy into four antigenic types (UK1, UK2, UK3, and UK4) could be detected by RT-PCR with these two primer sets. The G-1 primer set detected 6 UK2 strains, and the G-2 primers detected 16 strains, including 7 UK1, 5 UK3, and 4 UK4 strains. On the basis of nucleotide sequences of 81-bp fragments of the RT-PCR products from 13 strains determined in this study, together with those previously reported for 17 SRSV strains, we designed four sets of internal oligonucleotide probes (P1-A, P1-B, P2-A, and P2-B) for Southern hybridization, using chemiluminescent detection. The P1-A probe hybridized with PCR products from the UK2 strains; the P1-B probe, with products from two of the seven UK1 strains; the P2-A probe, with four of the remaining five UK1 strains; and the P2-B probe, with products from both UK3 and UK4 strains, as well as with one strain originally typed as UK1 which showed cross-reactivity with UK4 upon retesting by solid-phase immune electron microscopy. RT-PCR with both the G-1 and the G-2 primer sets can increase the detection rate of the many antigenically distinct SRSVs and, when combined with Southern hybridization, may predict the antigenic type of the SRSV associated with infection.
Detection of p24 antigen with and without immune complex dissociation for longitudinal monitoring of human immunodeficiency virus type 1 infection
Authors: Henrard D R , Wu S , Phillips J , Wiesner D , Phair J
Published: January 2020

Sequential specimens obtained from 87 multicenter AIDS cohort study participants were tested by three p24 antigen tests. They included a polyclonal enzyme immunoassay (EIA), a monoclonal EIA, and a monoclonal EIA after immune complex dissociation (ICD) of specimens. Subjects were grouped into two categories defined by real-time testing with the polyclonal EIA: 39 had become positive for p24 antigen (antigen converters) during follow-up, and 48 had progressed to AIDS without detectable antigenemia. Twenty-four (61%) antigen converters were positive by ICD-monoclonal EIA about 1 year earlier than by monoclonal EIA. In contrast, only 12 (25%) patients who progressed to AIDS without detectable antigenemia became positive by ICD-p24 EIA before developing AIDS. Thus, the main benefit of ICD treatment may be to detect p24 antigenemia approximately 1 year before the regular assay rather than to identify additional antigenemic people. Quantitative plasma RNA levels were also determined in longitudinal samples from 20 antigen converters and 7 men who developed AIDS without antigenemia. Although mean human immunodeficiency virus type 1 RNA levels were higher in antigen-positive than in antigen-negative samples (P = 0.002), more than half (11 of 20) of the antigen converters had no measurable change in human immunodeficiency virus type 1 RNA associated with change to antigen positivity.
Use of yeast killer system to identify species of the Nocardia asteroides complex
Authors: Provost F , Polonelli L , Conti S , Fisicaro P , Gerloni M , Boiron P
Published: January 2020

We evaluated the ability of the yeast killer system to differentiate members belonging to the Nocardia asteroides complex (Nocardia asteroides, Nocardia farcinica, and Nocardia nova). Nocardia strains were selected randomly from clinical isolates. Type strains of each Nocardia species and recognized killer yeasts were taken from different collections. A clear area of inhibition surrounding the yeast cells demonstrated a positive killer effect on Nocardia spp. Two yeast strains, Pichia mrakii (K9) and Pichia lynferdii (K76), showed different killer activities against each Nocardia species. The group N. asteroides was identified as K9+ K76-, the group N. farcinica was identified as K9- K76-, and the group N. nova was identified as K9+ K76+. The three killer identifications correlated with specific taxonomic groups determined by using classical methods. The yeast killer system may be a useful means for identifying organisms within the N. asteroides complex.
Involvement of multiple Cryptococcus neoformans strains in a single episode of cryptococcosis and reinfection with novel strains in recurrent infection demonstrated by random amplification of polymorphic DNA and DNA fingerprinting
Authors: Haynes K A , Sullivan D J , Coleman D C , Clarke J C , Emilianus R , Atkinson C , Cann K J
Published: January 2020

We compared the abilities of random amplification of polymorphic DNA and DNA fingerprinting, with oligonucleotide probes, to type five pairs of Cryptococcus neoformans clinical isolates recovered from five separate human immunodeficiency virus-positive patients in London, England. The two techniques had comparable discriminatory abilities when applied to these isolates. A total of eight different isolate types were demonstrated in these patients. No isolate type was observed in more than one patient. Two of the isolate pairs recovered from single episodes of cryptococcosis within 1 day of each other were genotypically indistinguishable by both methods. The other three pairs of isolates were all distinguishable. One of these isolate pairs was obtained from a single episode of cryptococcosis, while the other two were obtained from recurrent infections. These results indicate that multiple strains of C. neoformans may be responsible for a single episode of cryptococcosis and that recurrent infection may occur as a result of reinfection with a novel strain.
Rapid diagnosis of Entamoeba infection by using Entamoeba and Entamoeba histolytica stool antigen detection kits
Authors: Haque R , Neville L M , Hahn P , Petri W A
Published: January 2020

Humans are infected by two morphologically identical species of Entamoeba: Entamoeba histolytica causes amebic colitis and liver abscess, and Entamoeba dispar is noninvasive. Several weeks of culture and isoenzyme (zymodeme) analysis are required to differentiate E. histolytica from E. dispar. Here we report a field trial of commercial antigen detection kits designed to rapidly detect and differentiate E. histolytica from E. dispar in stool specimens. Stool specimens from 202 patients with diarrhea were examined for E. histolytica and E. dispar by microscopy, culture, and antigen detection. Compared with culture, microscopic identification of the E. histolytica-E. dispar complex was 60% sensitive and 79% specific, while the screening antigen detection test for the E. histolytica-E. dispar complex was 80% sensitive and 99% specific. Differentiation of E. dispar from E. histolytica by the E. histolytica-specific test was 95% sensitive and 93% specific compared with zymodeme analysis. We conclude that the antigen detection test for the E. histolytica-E. dispar complex is more sensitive and specific than microscopy and that the E. histolytica-specific antigen detection test is as reliable and much more rapid than zymodeme analysis for the differentiation of E. histolytica from E. dispar.
Sensitivity of bacteriologic culture for detection of Escherichia coli O157:H7 in bovine feces
Authors: Sanderson M W , Gay J M , Hancock D D , Gay C C , Fox L K , Besser T E
Published: January 2020

The sensitivities of several plating and broth enrichment methods for the detection of Escherichia coli O157:H7 in (i) bovine fecal samples directly inoculated with E. coli O157:H7, (ii) fecal samples from cattle in herds previously positive for E. coli O157:H7, and (iii) fecal samples from calves shedding E. coli O157:H7 after experimental oral inoculation were compared. Three enrichment protocols and three plating protocols were evaluated with directly inoculated fecal samples. All broth enrichment methods were superior to direct plating when they were combined with subsequent plating on sorbitol-MacConkey with cefixime and tellurite (SMACct). SMACct was the most sensitive plating medium, and the three alternative broth enrichment methods gave similar improvements in sensitivity. Of 351 fecal samples from known positive herds, 24 samples (6.8%) were positive by one or more methods. By the most sensitive plating method, cultures of 10-g samples were slightly more sensitive (19 of 351 [5.4%]) than cotton-tipped swab fecal samples (14 of 351 [4.0%]); however, this difference was not significant. For samples from calves orally inoculated with E. coli O157:H7, separation by immunomagnetic beads was slightly more sensitive (79%) than broth enrichment followed by plating at two dilutions (10(-3) and 10(-4)) (71%); however, this difference was not significant. The combination of overnight enrichment of swab fecal samples (0.1 g) and plating on SMACct at two dilutions (10(-3) and 10(-4)) appears to be a sensitive method for detection in large-scale studies involving hundreds of samples per week.
Comparison of MIDI Sherlock system and pulsed-field gel electrophoresis in characterizing strains of methicillin-resistant Staphylococcus aureus from a recent hospital outbreak
Authors: Leonard R B , Mayer J , Sasser M , Woods M L , Mooney B R , Brinton B G , Newcomb-Gayman P L , Carroll K C
Published: January 2020

An outbreak of methicillin-resistant Staphylococcus aureus infections at the University of Utah Health Sciences Center occurred over a 7-month period. While the isolates phenotypically appeared to be similar in gross morphology and have similar Vitek antibiotic susceptibility patterns, two additional methods of strain characterization were evaluated to enhance the epidemiological investigation: pulsed-field gel electrophoresis and gas chromatography with the MIDI Sherlock system. Sherlock uses gas chromatography to qualitatively and quantitatively analyze the cellular fatty acid composition of organisms and creates two-dimensional plots based on principal-component analysis to define groups of closely related organisms. All isolates were also evaluated by digesting their chromosomal DNAs with the low-frequency-cutting enzyme SmaI and separating the restriction fragments by contour-clamped homogeneous electric field gel electrophoresis. Sample preparation for this pulsed-field gel electrophoresis included a novel cell lysis procedure involving achromopeptidase, greatly reducing the turnaround time. Isolates tested were recovered from the following: 45 suspected outbreak patients, 6 hospitalized patients believed to be unrelated to the outbreak, 6 patients from outside the hospital, and one health care practitioner implicated in the outbreak. Of 45 phenotypically similar suspect strains, 43 clustered tightly on the Sherlock two-dimensional plot. All outbreak patient isolates were also identical by pulsed-field gel electrophoresis with the exception of the same two outliers identified by Sherlock. In this epidemiologic investigation, we found an excellent correlation between the Sherlock and pulsed-field gel electrophoresis results for strain characterization of methicillin-resistant S. aureus.
Sera from OspA-vaccinated dogs, but not those from tick-infected dogs, inhibit in vitro growth of Borrelia burgdorferi
Authors: Straubinger R K , Chang Y F , Jacobson R H , Appel M J
Published: January 2020

Dogs were challenged with Borrelia burgdorferi by exposure to ticks, with or without prior protection from infection by recombinant OspA (rOspA) vaccination. Sera from these dogs were tested for their capability to inhibit the growth of B. burgdorferi in vitro. Bacterial growth was detected by a color change in the culture medium, and the optical density was measured with a spectrophotometer in microtiter plates. By growth inhibition, which was complement dependent, the color change was lacking after 5 days of incubation. Over a 1-year study, nonvaccinated dogs infected by exposure to ticks showed high antibody titers to B. burgdorferi by kinetic enzyme-linked immunosorbent assay (KELA). The same sera did not inhibit spirochetal growth or did so only at a low dilution. These results corresponded to the lack of OspA and OspB antibodies seen in Western blots (immunoblots), and these dogs were not protected from infection or disease. In contrast, dogs immunized with rOspA prior to challenge with infected ticks produced high antibody titers, as determined by KELA, but their sera also had high growth-inhibiting antibody titers. Western blot analysis showed a strong band in the 32-kDa region when the sera of these dogs were tested. When adjuvant was administered with rOspA, antibody titers by both KELA and growth inhibition were higher and persisted longer in the immunized dogs. All dogs immunized with rOspA were protected from infection and disease.
Specific detection of methicillin-resistant Staphylococcus species by multiplex PCR
Authors: Vannuffel P , Gigi J , Ezzedine H , Vandercam B , Delmee M , Wauters G , Gala J L
Published: January 2020

In Staphylococcus aureus, mecA and femA are the genetic determinants of methicillin resistance. By using a multiplex PCR strategy, 310- and 686-bp regions of the mecA and femA genes, respectively, were coamplified to identify susceptible (lacking mecA) and resistant (mecA+) staphylococci and to differentiate S. aureus (femA+) from coagulase-negative staphylococci (lacking femA). A third staphylococcal genomic sequence, corresponding to IS431 and spanning 444 bp, was used as a PCR control. One hundred sixty-five staphylococcal strains were tested. All 72 methicillin-resistant strains were found to be mecA+, and 92 of the 93 susceptible isolates lacked mecA. Only one coagulase-negative Staphylococcus isolate carrying the mecA gene was highly susceptible to oxacillin. The femA determinant was a unique feature of S. aureus; it was found in 100% of the S. aureus strains tested but was undetectable in all of the coagulase-negative staphylococci tested. The possibility of directly detecting the mecA and femA genes in blood samples was also investigated. After two amplification steps, a sensitivity of 50 microorganisms per ml of freshly collected spiked blood was achieved. In conclusion, coamplification of mecA and femA determinants proved to be very reliable both for rapid detection of methicillin resistance and differential diagnosis between S. aureus and other staphylococci. This technique, which can be successfully performed with blood samples, could be a useful tool in the diagnosis and treatment monitoring of staphylococcal infections.
Combined PCR-oligonucleotide ligation assay for rapid detection of Salmonella serovars
Authors: Stone G G , Oberst R D , Hays M P , McVey S , Chengappa M M
Published: January 2020

We have developed a rapid and sensitive assay for the detection of Salmonella serovars in veterinary clinical specimens. This method utilizes a short cultivation period followed by PCR. For detection of the amplified product, an enzyme-linked immunosorbent assay (ELISA)-based oligonucleotide ligation assay (OLA) was used. In this study, the PCR-OLA technique was compared with conventional culture and membrane hybridization for the detection of Salmonella bacteria. In evaluating the PCR-OLA with Salmonella serovars and non-Salmonella strains of bacteria, A490 readings for 51 Salmonella strains, representing 28 serovars, were significantly higher (P < 0.05) than those for 25 non-Salmonella bacteria. With serial 10-fold dilutions of Salmonella CFU or with known concentrations of purified chromosomal DNA from Salmonella typhimurium ATCC 29946, the PCR-OLA was able to detect > or = 20 CFU per assay or > or = 80 fg of chromosomal DNA (corresponding to 160 molecules of DNA). Of 102 suspect clinical specimens screened, 15 were positive for Salmonella bacteria by both culture and the PCR-OLA procedure (100% sensitivity), and 3 samples were positive only by PCR-OLA (96.6% specificity), indicating a positive predictive value of 83.3% and a negative predictive value of 100%. In all experiments, the PCR-OLA was as sensitive as membrane hybridization. These results indicate that a limited enrichment cultivation and PCR-OLA could be used as a presumptive screening test for the detection of Salmonella serovars from any sample that currently requires extensive cultivation and that this assay would be adaptable to automation.
Identification of widespread Helicobacter hepaticus infection in feces in commercial mouse colonies by culture and PCR assay
Authors: Shames B , Fox J G , Dewhirst F , Yan L , Shen Z , Taylor N S
Published: January 2020

The identification of a new murine pathogen, Helicobacter hepaticus, and its association with chronic active hepatitis and liver tumors prompted an evaluation of the prevalence of H. hepaticus in commercially available mice. Of the 28 different strains or stocks, totaling 160 mice from four major commercial vendors, cultured for H. hepaticus, 100% of mice from two outbred strains from one vendor were infected with H. hepaticus, whereas 9 of 13 inbred mouse strains from another vendor were infected. This high prevalence of H. hepaticus established a need for a rapid and reproducible, noninvasive assay for the screening of colony-maintained mice being used for biomedical research. The culturing of fecal material by using 0.45-microns-pore- size filtration for H. hepaticus consistently yielded reproducible results but required extended periods of time. (1 to 3 weeks) to obtain a definitive answer. Although it is rapid, the use of a direct PCR-based detection assay with fecal specimens is restricted by inhibitory agents. to circumvent these inhibitory agents and to augment our H. hepaticus culture technique, we have developed a novel PCR system in which the bacteria are isolated from fecal material in the presence of polyvinylpyropyrollidone and lysed by treatment with Chelex 100. The PCR is performed with Tth polymerase supplemented with a polymerase enhancer. By this PCR method, 24 H. hepaticus culture-positive and 30 H. hepaticus culture-negative fecal samples were correctly identified. Moreover, two samples which were PCR positive and culture negative initially were positive by both methods upon retesting of fresh material. Southern blot hybridizations and sequencing of PCR products showed them to be H. hepaticus specific. A comparison of results obtained under identical conditions indicated a 100-fold increase in sensitivity with Tth polymerase over Taq polymerase. This PCR method can be used as a noninvasive means of rapidly screening large numbers of colony mice for H. hepaticus.
Molecular characterization and multilaboratory evaluation of Enterococcus faecalis ATCC 51299 for quality control of screening tests for vancomycin and high-level aminoglycoside resistance in enterococci
Authors: Swenson J M , Clark N C , Sahm D F , Ferraro M J , Doern G , Hindler J , Jorgensen J H , Pfaller M A , Reller L B , Weinstein M P
Published: January 2020

Studies were conducted to validate the use of Enterococcus faecalis ATCC 51299 (which is vancomycin resistant and resistant to high levels of gentamicin and streptomycin) and E. faecalis ATCC 29212 (which is susceptible to vancomycin and against which gentamicin or streptomycin and cell wall-active agents have synergistic kill activity) as controls in an agar screening test for vancomycin resistance and high-level streptomycin and gentamicin resistance and a broth microdilution screening test for high-level streptomycin and gentamicin resistance. Both organisms performed as expected in these tests and will serve as appropriate controls. However, E. faecalis ATCC 29212 was occasionally noted to produce light growth on the vancomycin screening plate with certain lots of agar. Quality control ranges for disk diffusion tests with disks with large amounts of streptomycin (300 micrograms) and gentamicin (120 micrograms) were established for E. faecalis ATCC 29212; zone limits are 16 to 22 mm for gentamicin and 14 to 19 mm for streptomycin. No zones for inhibition were seen when E. faecalis ATCC 51299 was tested with these high-content disks.
PCR as a confirmatory test for human immunodeficiency virus type 1 infection in individuals with indeterminate western blot (immunoblot) profiles
Authors: Sethoe S Y , Ling A E , Sng E H , Monteiro E H , Chan R K
Published: January 2020

A combination of two PCR systems which amplify human immunodeficiency virus type 1 (HIV-1) gag and pol DNA was used to elucidate the HIV-1 infection status of a group of individuals screening positive for anti-HIV but having indeterminate Western blot (immunoblot) profiles. The results obtained suggest PCR could be used to resolve such cases.
Single-step PCR for detection of Brucella spp. from blood and milk of infected animals
Authors: Leal-Klevezas D S , Martínez-Vázquez I O , López-Merino A , Martínez-Soriano J P
Published: January 2020

A versatile method for the extraction of Brucella DNA and PCR are presented as reliable tools for the detection of Brucella spp. from body fluids of infected animals. Two oligonucleotides homologous to regions of the gene encoding for an outer membrane protein (omp-2) were designed to detect the pathogen from milk and/or blood of infected goats, bovines, and human patients. The sensitivity of our test and its ability to detect the pathogen in samples from the field reveal a promising advance in the diagnosis of brucellosis in animals and humans.
Meningococcal carriage, alcohol consumption, and campus bar patronage in a serogroup C meningococcal disease outbreak
Authors: Imrey P B , Jackson L A , Ludwinski P H , England A C , Fella G A , Fox B C , Isdale L B , Reeves M W , Wenger J D
Published: January 2020

Community outbreaks of serogroup C invasive meningococcal disease are increasing in North America (L. H. Harrison, JAMA 273:419-421, 1995; L. A. Jackson, A. Schuchat, M. W. Reeves, and J. D. Wenger, JAMA 273:382-389, 1995; C. M. Whalen, J. C. Hockin, A. Ryan, and F. Ashton, JAMA 273:390-394). In a recent 15-month university outbreak, disease was linked to patronage of a specific campus-area bar, suggesting that aspects of a campus bar environment might promote meningococcal transmission (P. B. Imrey, L. A. Jackson, P. H. Ludwinski, et al., Am. J. Epidemiol., in press). To investigate this hypothesis, oropharyngeal carriage results from samples taken from 867 university health service clients and 85 campus-area bar employees during the last 3 months of the outbreak were analyzed to determine factors correlated with carriage of any strain of Neisseria meningitidis. Results were validated with data from samples from 344 health center clients and 211 campus bar employees taken 8 months after the last outbreak case. Recent alcohol consumption (adjusted prevalence odds ratio = 3.8 for > 15 versus 0 drinks in last week [P = 0.0012]) and campus bar patronage (adjusted odds ratio = 1.9 for any versus no patronage in last 2 weeks [P = 0.0122]) showed separate effects in both univariate and multiple logistic regression analyses of data from the 1992 health center clients. Prevalence of meningococcal carriage among 1992 campus bar workers was 3.8 times that among health center clients; this prevalence ratio was roughly 2.5 after adjustment for alcohol consumption and bar patronage. Recent antibiotic usage was protective (prevalence odds ratio = 0.3) among health center clients and bar workers. These findings were generally supported by the validation samples. If alcohol consumption and other aspects of the campus bar environment facilitate transmission of and/or colonization by N. meningitidis, then the introduction of a highly pathogenic substrain into the campus bar environment may provide an unusual opportunity for invasive meningococcal disease within a campus community.
Comparison of antigen detection and PCR assay using bronchoalveolar lavage fluid for diagnosing invasive pulmonary aspergillosis in patients receiving treatment for hematological malignancies
Authors: Verweij P E , Latgé J P , Rijs A J , Melchers W J , De Pauw B E , Hoogkamp-Korstanje J A , Meis J F
Published: January 2020

The performance of a sandwich enzyme-linked immunosorbent assay (ELISA) which detects Aspergillus galactomannan (GM) was evaluated in bronchoalveolar lavage (BAL) fluid samples from 19 patients who were treated for hematological malignancies and who were suspected of having invasive pulmonary aspergillosis (IPA). All patients had fever and pulmonary infiltrates on the chest roentgenogram on the day that the BAL fluid was obtained. The ELISA results were compared with the results of culture and Aspergillus genus-specific PCR analysis of BAL fluid samples. ELISA was also performed with serum samples. Aspergillus species were detected by PCR or ELISA with BAL fluid samples from five of seven patients who had radiological evidence of IPA. Serum ELISA results were positive for all patients with ELISA-positive BAL fluid, and for four patients the serum ELISA was positive before the BAL fluid was obtained. PCR and ELISA were positive for 2 and 1 of 10 BAL fluid samples, respectively, obtained from patients without radiological evidence of IPA, and 5 and 2 of 35 BAL fluid samples, respectively, obtained from nonneutropenic patients. This preliminary investigation suggests that GM may be detected by ELISA in BAL fluid samples from patients at risk of IPA, but that monitoring of serum GM levels may allow for the earlier diagnosis of IPA. However, further evaluation in prospective studies is required.
Evaluation of E-Test for determination of antimicrobial MICs for Pseudomonas aeruginosa isolates from cystic fibrosis patients
Authors: Marley E F , Mohla C , Campos J M
Published: January 2020

We determined the E-Test and National Committee for Clinical Laboratory Standards standardized agar dilution MICs of ceftazidime, ciprofloxacin, piperacillin, and tobramycin for Pseudomonas aeruginosa during tests of 100 rough and mucoid P. aeruginosa isolates from cystic fibrosis patients. The levels of agreement (+/- 1 log2 dilution) between quantitative E-Test and agar dilution MIC results were 80, 97, 73, and 89% for ceftazidime, ciprofloxacin, piperacillin, and tobramycin, respectively. Comparison of the results after converting the MIC data to qualitative categories (susceptible, intermediate, and resistant) yielded levels of agreement of 84, 96, 88, and 93% for the same agents, respectively. Of the 39 qualitative discrepancies, 36 were minor and 3 were very major. We conclude that use of the E-Test is easier and more practical than use of the agar dilution method for most laboratories and that the E-Test furnishes results which are at least as accurate as those obtained by the agar dilution method. However, the higher cost of the E-Test method would likely discourage most laboratories from selecting it over disk diffusion for routine antimicrobial susceptibility testing of P. aeruginosa isolates from cystic fibrosis patients.
Quantitation of human immunodeficiency virus type 1 DNA by two PCR procedures coupled with enzyme-linked oligosorbent assay
Authors: Mallet F , Hebrard C , Livrozet J M , Lees O , Tron F , Touraine J L , Mandrand B
Published: January 2020

Two quantitative PCR methods with our nonisotopic enzyme-linked oligosorbent assay (ELOSA) in microtiter plate format were developed for quantitation of human immunodeficiency virus type 1 (HIV-1). Quantitative competitive PCR (QC-PCR) was based on the coamplification of the wild-type nef region with a mimic competitive nef gene template carrying mutations in the capture region. Correlation of wild-type HIV-1 nef DNA to mimic template copy number permitted quantitation of HIV-1 copy numbers in the range of 20 to 2,000 copies per micrograms of DNA. Internally controlled PCR (IC-PCR) was based on coamplification of the nef region and the ras gene as an internal endogenous standard. Correlation to known amounts of HIV-1 DNA permitted quantitation by IC-PCR of HIV-1 copy numbers in the range of 10 to 2,000 copies per microgram of DNA. QC- and IC-PCR-ELOSA were performed on a panel of 53 seropositive patients and 12 seronegative controls. The methods showed similar coefficients of variation below 24%. Quantitations by QC- and IC-PCR-ELOSA were identical for 77% of patient samples. The copy level ranged between 443 +/- 156 and 21,453 +/- 13,511 copies per 10(5) CD4 cells for asymptomatic and AIDS patients, respectively. The simplicity and reliability of QC- and IC-PCR-ELOSA methods make them appropriate for routine laboratory use in the quantitation of viral and bacterial DNAs.
Nosocomial outbreak caused by Scedosporium prolificans (inflatum): four fatal cases in leukemic patients
Authors: Alvarez M , Lopez Ponga B , Rayon C , Garcia Gala J , Roson Porto M C , Gonzalez M , Martinez-Suarez J V , Rodriguez-Tudela J L
Published: January 2020

Four cases of fatal disseminated Scedosporium prolificans (inflatum) infection occurring in neutropenic patients are reported. Because of hospital renovation, the patients were cared for in a temporary hematologic facility. S. prolificans (inflatum) was isolated from blood cultures of these four patients, two of whom underwent full necropsy, and revealed abundant vegetative hyphae and ovoid conida with truncate bases in many organs. In vitro susceptibility testing of fungal strains showed all isolates to be resistant to amphotericin B, flucytosine, miconazole, ketoconazole, fluconazole, and itraconazole, with MICs greater than 16 micrograms/ml. The reported infections, two in each of two rooms, occurred over a period of 1 month, with very similar clinical outcomes. Circumstancial evidence suggested a nosocomial outbreak, but the environmental samples collected from the rooms, corridors, and adjacent areas did not yield S. prolificans (inflatum). Nevertheless, circumstantial evidence suggested a nosocomial outbreak of S. prolificans (inflatum) infection.
Sexual and in-contact transmission of asinine strain of equine arteritis virus among donkeys
Authors: Paweska J T , Volkmann D H , Barnard B J , Chirnside E D
Published: January 2020

Two in a group of five naturally seropositive donkey stallions were found to shed equine arteritis virus (EAV) in their semen as demonstrated by virus isolation. Direct intramuscular inoculation of sonicated semen from one virus-shedding stallion (S3) caused clinical disease in two donkeys from which virus was recovered and in which seroconversion was detected. Sexual transmission was confirmed in two mares mated to S3 when after a febrile response during which EAV was isolated from huffy coats and nasal and ocular exudates, both mares were found to have seroconverted. In-contact transmission in a susceptible stallion was demonstrated after its exposure to a sexually infected mare. The 3' end of the asinine virus was amplified directly from donkey semen with EAV-specific primers, and its nucleotide sequence was found to be homologous to that of the prototype Bucyrus virus isolated from horses. These results indicate that EAV and its disease transmission are analogous in donkeys and horses.
Importance in diagnosis of gastritis of detection by PCR of the cagA gene in Helicobacter pylori strains isolated from children
Authors: Husson M O , Gottrand F , Vachee A , Dhaenens L , de la Salle E M , Turck D , Houcke M , Leclerc H
Published: January 2020

The cagA gene has been detected by PCR and DNA hybridization in 45 Helicobacter pylori strains isolated from children. For each child, clinical symptoms, endoscopic aspect of the gastric mucosa, and histological gastritis were evaluated. Gene-positive strains were associated with hemorrhagic gastritis in 66.6% of the children, while gene-negative strains were associated with hemorrhagic gastritis in 11.2% of the children (P = 0.0001). In addition, 88.8% of gene-positive strains were associated with severe histological gastritis (scores of 3 and 4), and gene-negative strains were collected from the gastric mucosa with the same type of infiltration of neutrophils and lymphocytes in the lamina propia in 55.5% of the children. These differences were statistically significant (P = 0.017). Gene-positive strains were also isolated more frequently from children with vomiting (P = 0.04), while the absence of clinical signs was not significantly different in cagA gene-positive or -negative patients. All of these observations confirmed the role of this cagA gene as a marker of gastric inflammation in children. The detection of this gene might be helpful to determine the degree of inflammation of the gastric mucosa in the absence of abdominal symptoms. We might better understand the natural history of H. pylori infection if we studied the evolution of gastritis in children with regard to the cagA status of isolated strains.
Purification of a baculovirus-expressed hepatitis E virus structural protein and utility in an enzyme-linked immunosorbent assay
Authors: He J , Ching W M , Yarbough P , Wang H , Carl M
Published: January 2020

We report on the purification of the full-length structural protein encoded by open reading frame 2 (ORF-2) of hepatitis E virus. The ORF-2 protein, expressed in Sf9 cells by using a recombinant baculovirus vector system, was successfully purified to homogeneity. Gel electrophoresis of the purified ORF-2 protein showed a single polypeptide of 75 kDa by Coomassie blue staining and by Western blot (immunoblot) analysis. We demonstrated that the partially purified ORF-2 protein could be used successfully in a sensitive and specific enzyme-linked immunosorbent assay for the detection of antibodies to hepatitis E virus.
A novel insertion element from Mycobacterium avium, IS1245, is a specific target for analysis of strain relatedness
Authors: Guerrero C , Bernasconi C , Burki D , Bodmer T , Telenti A
Published: January 2020

The insertion sequence IS1245 is a novel mycobacterial repetitive element identified in Mycobacterium avium. It encodes a transposase which exhibits a 64% amino acid similarity with IS1081, an insertion element present in the M. tuberculosis complex. The host range of IS1245 appears limited to M. avium as this element was not identified in M. intracellulare or in any other of 18 mycobacteria species tested. When IS1245 was used for restriction fragment length polymorphism (RFLP) analysis, human isolates characteristically presented a high number of copies (median, 16; range, 3 to 27) and a diversity of RFLP patterns comparable to that found by pulsed-field gel electrophoresis. Isolates from nonhuman sources differed both in number of copies and in RFLP pattern diversity: while swine isolates shared the characteristics of human strains, those from several avian sources exhibited a very low copy number of IS1245 and appeared clonal on the basis of RFLP.