bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of agar dilution, broth microdilution, disk diffusion, E-test, and BACTEC radiometric methods for antimicrobial susceptibility testing of clinical isolates of the Nocardia asteroides complex
Authors: Ambaye A , Kohner P C , Wollan P C , Roberts K L , Roberts G D , Cockerill F R
Published: January 2020

An evaluation was undertaken to determine the optimal method for the in vitro susceptibility testing of 26 Nocardia asteroides complex isolates to the following antimicrobial agents: amikacin, ampicillin, amoxicillin-clavulanate, ceftriaxone, ciprofloxacin, erythromycin, imipenem, minocycline, and trimethoprim-sulfamethoxazole. Five testing methods were studied including the agar dilution, broth microdilution, and disk diffusion methods, the epsilometer test (E-test), and the BACTEC radiometric method. Results for each antimicrobial agent and each testing method were interpreted as indicating susceptibility, intermediate susceptibility, or resistance according to current guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) for bacteria that grow aerobically and were then compared to a "gold standard" susceptibility test result. The gold standard result for each Nocardia isolate was established by a consensus of the results of the majority of testing methods used in the study. When the results were combined for all antimicrobial agents tested against all Nocardia isolates by all methods, the BACTEC radiometric method produced the highest level of agreement (97.9%) with the consensus results and had the fewest very major (n = 1), major (n = 2), and minor (n = 2) errors. In contrast, the results of the agar dilution method were in least agreement (93.2%) with the consensus results, and this method also produced the most very major (n = 8), major (n = 4), and, along with the disk diffusion method, minor (n = 6) errors. For all test methods, interpretive errors were most frequent when testing ampicillin or amoxicillin-clavulanate. Moreover, for all Nocardia nova isolates tested, ampicillin susceptibility results by any of the testing methods were not in agreement with the results of testing for beta-lactamase by the nitrocefin (Cefinase) disk method. We conclude that among the methods evaluated, the BACTEC radiometric method appeared to be the best for determining the in vitro susceptibilities of members of the N. asteroides complex to a panel of nine antimicrobial agents. However, none of the test methods, including the BACTEC method, accurately predicted the ampicillin resistance of the N. nova isolates tested, all of which produced beta-lactamase. Presuming that this beta-lactamase hydrolyzes ampicillin, this disparity may relate to the NCCLS breakpoints that were used, which may require modification for this antimicrobial agent when tested against N. nova isolates.
Detection and characterization of the coli surface antigen 6 of enterotoxigenic Escherichia coli strains by using monoclonal antibodies
Authors: Helander A , Grewal H M , Gaastra W , Svennerholm A M
Published: January 2020

We describe, for the first time, the production of monoclonal antibodies (MAbs) against coli surface antigen 6 (CS6) of enterotoxigenic Escherichia coli (ETEC) and their use for characterization and diagnosis of CS6. Two MAbs, MAbs CS6-20:11:9 and CS6-2A:14, were produced by immunizing mice with purified CS6 or CS6-containing bacterial extracts. The MAb specificity was demonstrated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunoelectron microscopy, which showed that the MAbs bound to CS6-expressing bacteria as well as to purified CS6 and CS6 structural subunits but not to CS6-negative bacteria or other purified ETEC colonization factors. By using bacterial recombinants, i.e., strains with a complete CS6 operon or parts thereof, it was found that both MAbs were specific for CssB, one of the two structural subunits of CS6. Although the MAbs bound specifically to the entire surface of CS6-expressing bacteria, no structure of CS6 could be identified. The usefulness of the MAbs for the detection of CS6 was evaluated in an inhibition ELISA and in a dot blot test. Ninety-two ETEC strains with known colonization factors were analyzed, and all CS6-positive strains were identified by either assay with MAb CS6-2A:14, whereas MAb CS6-20:11:9 failed to identify two CS6-positive strains; in no instance was any CS6-negative strain identified by either of the MAbs. Parallel analyses of 48 strains with a gene probe specific for the other structural subunit of CS6, i.e., CssA, and the MAb-based assays gave identical results, suggesting the simultaneous presence of both subunits.
Detection of human immunodeficiency virus type 1 (HIV-1) DNA and RNA sequences in HIV-1 antibody-positive blood donors in Uganda by the Roche AMPLICOR assay
Authors: Jackson J B , Piwowar E M , Parsons J , Kataaha P , Bihibwa G , Onecan J , Kabengera S , Kennedy S D , Butcher A
Published: January 2020

The ability of commercially available PCR-based assays to accurately detect or quantitate human immunodeficiency virus type 1 (HIV-1) DNA or RNA in individuals predominantly infected with HIV-1 subtypes A and D is not known. Therefore, peripheral leukocytes from 43 individuals in Kampala, Uganda, positive for HIV by the Western blot (immunoblot) assay were tested by using the Roche AMPLICOR HIV-1 assay for the detection of DNA gag sequences. Plasma from these same individuals was tested by using the Roche HIV-1 AMPLICOR MONITOR HIV-1 assay for the quantitation of HIV-1 RNA gag sequences. In addition, peripheral leukocytes were tested for HIV-1 DNA by using a lower annealing temperature or a different primer pair for the HIV-1 pol region. The proportions of individuals with detectable HIV-1 DNA and RNA gag sequences by the Roche assays were 74 and 90%, respectively. The proportions positive for HIV-1 DNA sequences by using a 50 degrees C annealing temperature or the pol primer pair were 71 and 98%, respectively. In summary, the standard Roche assay did not detect HIV-1 DNA sequences in a significant number of HIV-1-infected individuals in Uganda. However, use of a pol primer pair increased the sensitivity of the assay to 98%. The sensitivity of the Roche AMPLICOR MONITOR assay for the detection and quantitation of HIV-1 RNA sequences was significantly higher than that of the DNA-based assay, but the efficiency of the assay, and hence, the accuracy of the values obtained with RNA, is not known. Modifications to existing assays are needed to enhance the sensitivities and accuracies of these commercially available assays for use in developing countries where non-B HIV-1 subtypes predominate.
Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology
Authors: Kamerbeek J , Schouls L , Kolk A , van Agterveld M , van Soolingen D , Kuijper S , Bunschoten A , Molhuizen H , Shaw R , Goyal M , van Embden J
Published: January 2020

Widespread use of DNA restriction fragment length polymorphism (RFLP) to differentiate strains of Mycobacterium tuberculosis to monitor the transmission of tuberculosis has been hampered by the need to culture this slow-growing organism and by the level of technical sophistication needed for RFLP typing. We have developed a simple method which allows simultaneous detection and typing of M. tuberculosis in clinical specimens and reduces the time between suspicion of the disease and typing from 1 or several months to 1 or 3 days. The method is based on polymorphism of the chromosomal DR locus, which contains a variable number of short direct repeats interspersed with nonrepetitive spacers. The method is referred to as spacer oligotyping or "spoligotyping" because it is based on strain-dependent hybridization patterns of in vitro-amplified DNA with multiple spacer oligonucleotides. Most of the clinical isolates tested showed unique hybridization patterns, whereas outbreak strains shared the same spoligotype. The types obtained from direct examination of clinical samples were identical to those obtained by using DNA from cultured M. tuberculosis. This novel preliminary study shows that the novel method may be a useful tool for rapid disclosure of linked outbreak cases in a community, in hospitals, or in other institutions and for monitoring of transmission of multidrug-resistant M. tuberculosis. Unexpectedly, spoligotyping was found to differentiate M. bovis from M. tuberculosis, a distinction which is often difficult to make by traditional methods.
Isolation of Mycoplasma hominis from a brain abscess
Authors: Zheng X , Olson D A , Tully J G , Watson H L , Cassell G H , Gustafson D R , Svien K A , Smith T F
Published: January 2020

Mycoplasma hominis is a commensal in the genital tract of women and has been associated with urogenital and extragenital infections. However, central nervous system infections with this organism in adults are very rare. Here we describe the recovery of M. hominis from a brain abscess associated with a postpartum infection. Seroconversion to the isolated strain was detected by both a metabolic inhibition test and an immunoblotting assay. This case demonstrates the pathogenic potential of M. hominis and the need for rapid recognition of the organism so that appropriate chemotherapeutic intervention can occur.
Molecular epidemiology of multiply antibiotic-resistant Shigella flexneri in Fortaleza, Brazil
Authors: Lima A A , Sidrim J J , Lima N L , Titlow W , Evans M E , Greenberg R N
Published: January 2020

In northeastern Brazil, strains of Shigella flexneri resistant to multiple antibiotics are often found in patients in both urban areas and community hospitals. This study used pulsed-field gel electrophoresis (PFGE) and plasmid analysis to further analyze the molecular epidemiology of Shigella flexneri strains isolated from hospitals and an urban community in Fortaleza, Brazil. Twenty-six strains of S. flexneri from three distinct areas in the city of Fortaleza, Brazil, were examined: 14 strains from people with diarrhea who lived in an urban community of 2,000 persons, 5 strains from patients in the university hospital, and 7 strains from children in a pediatric hospital. PFGE identified six unique groups of S. flexneri circulating among patients during the 45-month study. Seven strains were further studied for antibiotic resistance plasmid profiles. Three unique antibiotic resistance plasmid profiles were found. Strains collected from the hospitalized patients demonstrated the variety of PFGE and antibiotic resistance patterns in the area. Strains collected from the patients living in the urban community setting demonstrated the persistence of certain PFGE patterns as well as the acquisition of multiple antibiotic resistance plasmids. Effective interventional strategies for such geographic locations as Fortaleza, Brazil, will be more complex than those for single-strain outbreak situations.
Measurement of levels of human immunodeficiency virus type 1 reverse transcriptase (RT) and RT activity-blocking antibody in human serum by a new standardized colorimetric assay
Authors: Awad R J , Corrigan G E , Ekstrand D H , Thorstensson R , Källander C F , Gronowitz J S
Published: January 2020

Standardization and calibration of a new colorimetric assay for detection of reverse transcriptase (RT) was carried out for optimal detection of RT activity-blocking antibody (RTb-Ab) in serum. A total of 99 of 100 Swedish and 54 of 54 African human immunodeficiency virus type 1 (HIV-1) antibody-positive individuals had RTb-Ab. The one RTb-Ab-negative HIV-1 serum sample from a Swedish individual was obtained early during seroconversion. Five of 615 HIV-1-negative sera from tumor patients, pregnant women, patients undergoing routine viral diagnostics, and blood donors gave false-positive results. In addition, 3 of 126 HIV-1-negative African serum samples and 2 of 91 serum samples selected because of false reactivity in other commercially available HIV antibody assays were positive for RTb-Ab. RT activity and RTb-Ab were measured in sera from newly HIV-1-infected individuals during seroconversion. Peak RT activity was usually detected between days 8 and 13 after the onset of symptoms of primary infection. In addition, HIV-1 RTb-Ab was detected in the same recently infected individuals in most cases within 1 month and in some cases as early as 10 to 12 days after the onset of symptoms. A cross-reactivity study involving HIV-1 and HIV-2 RTb-Abs and their homologous RT showed HIV-1 RTb-Ab to be highly type specific. None of 10 serum samples from HIV-1-infected individuals showed cross-reacting RTb-Ab toward HIV-2 RT, whereas 4 of 10 serum samples from HIV-2-infected patients showed cross-reactivity toward HIV-1 RT; however, the cross-reactivity toward HIV-1 RT was 3,000 times lower than that toward its homologous RT. Future uses for the assay with reference to the recent World Health Organization proposal for other methods instead of Western blotting (immunoblotting) for confirming HIV-1 infection and for methods for the diagnosis of infection as follow-up in vaccine trials are also discussed.
Direct detection and identification of Mycobacterium ulcerans in clinical specimens by PCR and oligonucleotide-specific capture plate hybridization
Authors: Portaels F , Agular J , Fissette K , Fonteyne P A , De Beenhouwer H , de Rijk P , Guédénon A , Lemans R , Steunou C , Zinsou C , Dumonceau J M , Meyers W M
Published: January 2020

We compared various diagnostic tests for their abilities to detect Mycobacterium ulcerans infection in specimens from patients with clinically active disease. Specimens from 10 patients from the area of Zangnanado (Department of Zou, Benin) with advanced, ulcerated active M. ulcerans infections were studied by direct smear, histopathology, culture, PCR, and oligonucleotide-specific capture plate hybridization (OSCPH). A total of 27 specimens, including 12 swabs of exudate collected before debridement and 15 fragments of tissue obtained during debridement, were submitted to bacteriologic and histopathologic analysis. The histopathologic evaluation of tissues from all six patients so tested revealed changes typical of those caused by M. ulcerans infection. Five specimens were contaminated, and M. ulcerans was cultivated on Löwenstein-Jensen medium from 12 of the remaining 22 (54.5%) specimens. Detection of mycobacteria was performed by PCR, and M. ulcerans was detected by OSCPH with a new probe (5'-CACGGGATTCATGTCCTGT-3') reacting with M. ulcerans and Mycobacterium marinum. In 10 of 22 (45.5%) specimens, M. ulcerans was identified by PCR-OSCPH. There was no statistically significant difference between the detection of M. ulcerans by culture and by PCR-OSCPH (P > 0.05). This is the first demonstration of an amplification system (PCR-OSCPH) with a sensitivity similar to that of culture for the direct and rapid recognition of M. ulcerans in clinical specimens. This system is capable of identifying M. ulcerans, even in paucibacillary lesions. Our findings suggest that PCR-OSCPH should be used in the quest for the elusive environmental reservoir(s) of M. ulcerans.
Serological diagnosis of hantavirus infections by an enzyme-linked immunosorbent assay based on detection of immunoglobulin G and M responses to recombinant nucleocapsid proteins of five viral serotypes
Authors: Elgh F , Lundkvist A , Alexeyev O A , Stenlund H , Avsic-Zupanc T , Hjelle B , Lee H W , Smith K J , Vainionpää R , Wiger D , Wadell G , Juto P
Published: January 2020

Worldwide, hantaviruses cause more than 100,000 human infections annually. Rapid and accurate methods are important both in monitoring acute infections and for epidemiological studies. We and others have shown that the amino termini of hantavirus nucleocapsid proteins (Ns) are sensitive tools for the detection of specific antibodies in hantavirus disease. Accordingly, we expressed truncated Ns (amino acids 1 to 117) in Escherichia coli from the five hantaviruses known to be pathogenic to man; Hantaan (HTN), Seoul (SEO), Dobrava (DOB), Sin Nombre (SN), and Puumala (PUU) viruses. In order to obtain pure antigens for use in an enzyme-linked immunosorbent assay (ELISA), the recombinant proteins were purified by polyhistidine-metal chelate affinity chromatography. Polyclonal animal antisera and a panel of serum specimens from hantavirus-infected individuals from Scandinavia, Slovenia, Russia, Korea, China, and the United States were used to evaluate the usefulness of the method. With both human and animal sera, it was possible to designate the antibody response into two groups: those with HTN, SEO, and DOB virus reactivity on the one hand and those with SN and PUU virus reactivity on the other. In sera from Scandinavia, European Russia, and the United States, the antibody response was directed mainly to the PUU and SN virus group. The sera from Asia reacted almost exclusively with the HTN, SEO, and DOB types of viruses. This was true for both the immunoglobulin M (IgM) and IgG antibody responses, indicating that this type of discrimination can be done during the acute phase of hantavirus infections. Both the HTN, SEO, and DOB virus and the PUU and SN virus types of antibody response patterns were found in patients from the Balkan region (Solvenia).
Use of the Treponema pallidum-specific captia syphilis IgG assay in conjunction with the rapid plasma reagin to test for syphilis
Authors: Reisner B S , Mann L M , Tholcken C A , Waite R T , Woods G L
Published: January 2020

The Captia Syphilis IgG enzyme immunoassay (EIA) was evaluated for use in conjunction with the rapid plasma reagin test (RPR) as a method to test for syphilis. A total of 1,288 serum specimens were tested by the routine laboratory protocol of the RPR followed by microhemagluttination assay for Treponema pallidum (MHA-TP) testing of RPR-reactive sera as well as the EIA-RPR protocol in which the automated EIA followed by a manual RPR test for EIA-positive specimens is used. When using the routine protocol, 131 specimens were initially reactive by the RPR, and 113 of these were reactive by MHA-TP. When using the EIA-RPR protocol, 170 specimens were initially positive by EIA, and of these, 112 were RPR reactive, indicating active disease. When compared to the routine protocol, the EIA-RPR protocol had sensitivity, specificity, and positive and negative predictive values of 96.5, 99.7, 97.3, and 99.7%, respectively. After resolution of discrepancies by additional testing, the adjusted sensitivity, specificity, and positive and negative predictive values were 100, 99.8, 98.3, and 100%, respectively. This evaluation demonstrates that when used in conjunction with the RPR, the Captia Syphilis EIA is a reliable method by which to test for syphilis.
Helicobacter westmeadii sp. nov., a new species isolated from blood cultures of two AIDS patients
Authors: Trivett-Moore N L , Rawlinson W D , Yuen M , Gilbert G L
Published: January 2020

A slowly growing anaerobic Helicobacter species was isolated from the blood cultures of two human immunodeficiency virus-positive patients admitted to Westmead Hospital, Westmead, Australia, with fevers. The morphology of the isolates was consistent with Helicobacter cinaedi or Helicobacter fennelliae. The results of culture growth conditions, biochemical tests, gas chromatography data, ribotyping, and 16S rDNA sequencing showed that these isolates represent a new Helicobacter species, for which the name Helicobacter westmeadii has been proposed.
Single-tube, nested, reverse transcriptase PCR for detection of viable Mycobacterium tuberculosis
Authors: Jou N T , Yoshimori R B , Mason G R , Louie J S , Liebling M R
Published: January 2020

Several problems remain before molecular biology-based techniques, such as PCR, are widely accepted for the detection of infectious agents. Among the most formidable of these problems are the inability of the tests to distinguish between viable and nonviable organisms. We approached this problem by using the fact that bacterial mRNA has an extremely short half-life, averaging only a few minutes. We reasoned that by targeting bacterial mRNA by a reverse transcriptase PCR (RT-PCR), a positive signal would indicate the presence of a recently viable organism. To test our hypothesis, we chose to target the mRNA coding for the ubiquitous 85B antigen of mycobacteria. After partially sequencing the gene coding for 85B, we developed primers that were specific for Mycobacterium tuberculosis. In a single-tube, nested, RT-PCR (STN RT-PCR), these primers detected fewer than 40 CFU in spiked sputum samples and as few as 12 CFU in clinical sputum specimens. The sensitivity of STN RT-PCR with smear-negative samples was as good as that of culture. The specificity was 100%. More importantly, when M. tuberculosis was cultured with and without 1 microgram of isoniazid per ml, this assay could distinguish between those cultures which contained the antibiotic and those which did not. Subcultures on Lowenstein-Jensen agar confirmed the viability assessments of the STN RT-PCR. Control experiments demonstrated that isoniazid did not inhibit the RT-PCR. In addition, when an IS6110-targeted, DNA PCR was used to examine the same samples, all samples though 13 days (the last sample) continued to be positive, irrespective of whether isoniazid was present, thereby demonstrating the superiority of an mRNA target in the detection of mycobacterial viability.
Association of Ureaplasma urealyticum biovars with clinical outcome for neonates, obstetric patients, and gynecological patients with pelvic inflammatory disease
Authors: Abele-Horn M , Wolff C , Dressel P , Pfaff F , Zimmermann A
Published: January 2020

In this prospective study, the prevalence of the two Ureaplasma urealyticum biovars, parvo and T960, was determined in pregnant women and in gynecological patients colonized by ureaplasmas. Furthermore, we investigated the association of these biovars with gynecological complications and adverse pregnancy outcome. Isolates of U. urealyticum from 254 women were biotyped by a PCR method recently developed. The parvo biovar was found in 81% (206 of 254) of the patients, and the T960 biovar was found in 30% (76 of 254) of the patients; 6% (14 of 254) of the women were coinfected. Identical biovars were detected in mothers and their infants. Serial isolations or cultures from different sampling sites of the same individual revealed the same biovar. T960 was dominant in patients with pelvic inflammatory disease (57%) and patients who had had a miscarriage (42%), showed a higher rate of tetracycline resistance than did parvo isolates (55 versus 18%), and seemed to have more adverse effects on pregnancy outcome with regard to birth weight (2,500 versus 1,720 g), gestational age (35 versus 30 weeks), and preterm delivery (35 versus 77%).
Use of monoclonal antibodies to facilitate identification, cloning, and purification of Chlamydia trachomatis hsp10
Authors: LaVerda D , Byrne G I
Published: January 2020

As a requisite for a physiological and immunological investigation, reagents were developed that facilitated the identification and purification of Chlamydia trachomatis hsp10 (chsp10). Monoclonal antibodies that specifically recognize chsp10 were generated with multiple-antigen peptides (MAPs) to promote recognition of Chlamydia-specific epitopes. MAP2, containing amino acids 54 to 69 of the hsp10 sequence, elicited strong antibody responses after immunization of BALB/c mice. Monoclonal antibodies from several cloned hybridomas reacted on immunoblots with an approximately 15-kDa chlamydial protein and recombinant chsp10. Because of its strict specificity for chsp10, monoclonal antibody M1.2 was selected for routine use. M1.2 reacted by immunoblot with the hsp10s of several C. trachomatis strains but not with Chlamydia psittaci hsp10 or Escherichia coli homolog GroES, suggesting that M1.2 recognizes a species-specific epitope. Recombinant chsp10 was purified by immunoaffinity chromatography with M1.2. For large-scale purification, chsp10 was appended with a C-terminal six-histidine tag for purification by nickel chelate affinity chromatography. The hypA gene encoding the chsp10 of C. trachomatis serovar E/Bour was cloned into the pQE-60 vector (QIAGEN, Inc.) following PCR amplification from genomic DNA. E. coli DH5 transformants were screened for chsp10 expression by colony immunoblotting with M1.2, were tested for nickel matrix binding, and were sequenced. The sequence of serovar E/Bour chsp10 was found to be closely homologous to those of hsp10s of other chlamydiae. Purified chsp10 and specific anti-chsp10 monoclonal antibodies will be useful for investigating the biological and immunological roles of hsp10 in chlamydial infections.
Persistent JC virus (JCV) infection is demonstrated by continuous shedding of the same JCV strains
Authors: Kitamura T , Sugimoto C , Kato A , Ebihara H , Suzuki M , Taguchi F , Kawabe K , Yogo Y
Published: January 2020

The polyomavirus JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy, is ubiquitous in the human population, infecting children asymptomatically. JCV is often detected in normal renal tissue and in the urine of healthy individuals. We demonstrate that renal JCV represents that which persists after primary infection.
A rapid and simple method for extracting human immunodeficiency virus type 1 RNA from plasma: enhanced sensitivity
Authors: Mulder J , Resnick R , Saget B , Scheibel S , Herman S , Payne H , Harrigan R , Kwok S
Published: January 2020

We present here a rapid and simple technique for processing human immunodeficiency virus (HIV)-infected plasma by high-speed centrifugation. HIV type 1 virions are pelleted from up to 1 ml of plasma, gently lysed with a nonionic detergent, and directly amplified. The procedure has few manipulations and requires approximately 1.5 h for the processing of 24 samples. Viral recovery ranges from 80 to 90%, with an analytical sensitivity approaching 20 copies/ml.
Evaluation of a line probe assay kit for characterization of rpoB mutations in rifampin-resistant Mycobacterium tuberculosis isolates from New York City
Authors: Cooksey R C , Morlock G P , Glickman S , Crawford J T
Published: January 2020

A commercial line probe assay kit (Inno-LiPA Rif.TB) for rapid identification of mutations in the rpoB gene associated with rifampin resistance in Mycobacterium tuberculosis was evaluated with a collection of 51 rifampin-resistant strains. Nine distinct rpoB mutations were identified. Concordances with automated sequence results for five wild-type kit probes and four probes for specific mutations were 94.1 and 100%, respectively. Overall concordance of the line probe assay kit with phenotypic rifampin susceptibility testing results was 90.2%.
Four-day incubation period for blood culture bottles processed with the Difco ESP blood culture system
Authors: Doern G V , Brueggemann A B , Dunne W M , Jenkins S G , Halstead D C , McLaughlin J C
Published: January 2020

Blood culture records from 1994 to 1995 from five U.S. medical centers all using the Difco ESP continuous monitoring blood culture system were reviewed retrospectively. Among a total of 7,362 isolates of bacteria and yeasts, only 0.1% of possibly significant isolates would have been missed had blood cultures been routinely incubated for 4 days instead of the 5 days recommended by the manufacturer. Conversely, numerous contaminants, detected only on day 5, would have been eliminated by a 4-day incubation period.
Vector potential of houseflies (Musca domestica) for Helicobacter pylori
Authors: Grübel P , Hoffman J S , Chong F K , Burstein N A , Mepani C , Cave D R
Published: January 2020

The mode of transmission of Helicobacter pylori is unknown. Since viable bacteria have been shown to be excreted in feces from infected individuals and houseflies habitually develop and feed on excrement, we hypothesized that flies ingest and harbor H. pylori and, in turn, contaminate the human environment. This study examined the possible vector potential of houseflies (Musca domestica) for H. pylori. Caged houseflies were exposed to freshly grown H. pylori on agar plates. After a 6-h feeding period, the plates were removed and were replaced with sterile petri dishes containing a droplet of sterile brucella broth. At regular intervals, small numbers of houseflies were removed for microbiological and histological analysis, and the petri dishes were replaced with fresh sterile plates with fresh drops of brucella broth. The flies' bodies, the flies' dissected alimentary tracts, and excreta on the petri dishes were cultured for H. pylori, whose identity was confirmed by the urease, catalase, and oxidase reactions and Gram staining. In contrast to control flies, viable H. pylori could be isolated from external surfaces for up to 12 h and from gut and excreta for as long as 30 h after the initial feeding period. After 30 h other gram-negative bacteria overgrew the cultures of samples from all locations tested, rendering the selective culture of H. pylori colonies impossible. Histological analysis revealed Helicobacter-like organisms in the gut lumen and attached to intestinal epithelial cells. We conclude that houseflies can harbor viable H. pylori on their bodies and in their intestinal tracts. They are also able to disseminate viable H. pylori in excreta, and they may therefore present a significant reservoir and be a vector in the transmission of H. pylori.
Determination of genotypes of Toxoplasma gondii strains isolated from patients with toxoplasmosis
Authors: Howe D K , Honoré S , Derouin F , Sibley L D
Published: January 2020

To determine the genotypes of Toxoplasma gondii strains associated with human toxoplasmosis, we developed a sensitive approach for typing parasites grown from clinical samples by short-term in vitro culture. A newly described nested PCR assay was capable of amplifying genomic DNA from as few as five parasites in the presence of host tissues. Typing was based on DNA polymorphisms at the SAG2 locus, encoding tachyzoite surface antigen p22. Restriction fragment length polymorphisms in PCR-amplified SAG2 products were used to classify strains into one of the three major lineages of T. gondii. This approach was successfully used to determine the genotypes of 68 of 72 samples that had been previously isolated from patients with congenital, cerebral, and disseminated toxoplasmosis. Type II strains of T. gondii were found in a majority of the samples, accounting for 55 (81%) of the 68 toxoplasmosis cases. In contrast, type I and III strains were found in only 7 (10%) and 6 (9%) of the 68 cases, respectively. The results of this study support the previous finding that type II strains are most often associated with human toxoplasmosis. Nested PCR analysis at the SAG2 locus provides rapid assignment of T. gondii to a specific genotype that should be useful in analyzing a variety of clinical samples.
A simple method for determining metronidazole resistance of Helicobacter pylori
Authors: Henriksen T H , Brorson O , Schöyen R , Thoresen T , Lia A
Published: January 2020

The reliability of methods for determination of Helicobacter pylori resistance to metronidazole has been found to depend upon the incubation time. Because the disk diffusion method is more vulnerable than other methods to prolonged incubation, this method has not been recommended for H. pylori. However, because media designed for rapid growth of H. pylori have been introduced, the time has come to look at the clinical usefulness of this inexpensive and simple method again. The correlation of readings obtained with the E test (AB Biodisk, Solna, Sweden) and Rosco's (Taastrup, Denmark) disk diffusion method for in vitro metronidazole resistance determination for H. pylori with a short incubation time (24 to 31 h) was studied. Plates which could not be read after 24 to 31 h were reincubated for another night. Fifty-seven consecutive clinical strains were tested. Because the rate of regrowth of H. pylori depends upon the age of the colonies inoculated, the reproducibility of resistance test results for young colonies versus old colonies was also studied. Resistance plates could be read after 24 to 31 h of incubation for 28 of 29 strains when the inoculum consisted of young colonies (3 to 4 days old). For these 29 strains, a high correlation (r = -0.937) was found between results obtained with the E test and those obtained with the disk diffusion test. A poorer correlation was found for old colonies (> or = 5 days old) (r = -0.742), which required a prolonged incubation for 8 of 23 strains. In conclusion, short incubation was successfully applied with young colonies. Results obtained with the simple and inexpensive disk diffusion method correlated well with those obtained with the E test.
Optimal combination of media for primary isolation of Helicobacter pylori from gastric biopsy specimens
Authors: Piccolomini R , Di Bonaventura G , Festi D , Catamo G , Laterza F , Neri M
Published: January 2020

The aim of the present study was to compare eight media, four nonselective and four selective media, to determine the best combination of media for the primary isolation of Helicobacter pylori. Over a period of 5 months, mucosal antral biopsy specimens were obtained from 222 consecutive dyspeptic patients undergoing endoscopy. Biopsy samples were plated in parallel on all eight media. Egg yolk emulsion agar (EYE), Skirrow's medium, Dent's medium, and modified Thayer-Martin medium were used as selective media; modified chocolate agar (MCHOC), Triptycase soy agar (TSA), brucella agar, and brain heart infusion agar were used as nonselective media. Overall, by using these eight media, H. pylori was recovered from biopsy specimens from 114 of 222 patients, yielding an isolation rate of 51%. Comparison of all possible combinations of the eight media showed that the highest rate of isolation of H. pylori was 100% (114 of 114) with EYE-MCHOC, followed by 96.5% (110 of 114) when EYE-TSA was used. Conversely, it was found that none of the media used alone yielded a 100% rate of recovery (the maximum recovery rate was 95%, which was achieved with EYE). These results indicate that the association of EYE and MCHOC yielded the maximum recovery of H. pylori from gastric biopsy specimens. Therefore, the use of selective and nonselective media in parallel offers optimal recovery rates with only a slight increase in costs.
Validity of self-obtained vaginal specimens for diagnosis of trichomoniasis
Authors: Schwebke J R , Morgan S C , Pinson G B
Published: January 2020

A comparison of self- and clinician-collected vaginal specimens for the diagnosis of trichomoniasis was conducted. The sensitivities of culture methods using self- and clinician-collected specimens were 84.6 and 88.5%, respectively. There was no significant difference between the sensitivities of culture methods using self- and clinician-collected vaginal specimens for the diagnosis of trichomoniasis.
Analysis and typing of the vacA gene from cagA-positive strains of Helicobacter pylori isolated in Japan
Authors: Ito Y , Azuma T , Ito S , Miyaji H , Hirai M , Yamazaki Y , Sato F , Kato T , Kohli Y , Kuriyama M
Published: January 2020

Approximately 50% of Helicobacter pylori strains produce a cytotoxin that is encoded by vacA and that induces vacuolation of eukaryotic cells. Mosaicism in vacA alleles was reported, and there are three different families of vacA signal sequences (s1a, s1b, and s2) and two different families of middle-region alleles (m1 and m2). In addition, the vacA genotype of a strain is associated with its cytotoxin phenotype and its capacity to induce peptic ulceration. To clarify the strain diversity of H. pylori in Japan, 87 Japanese clinical isolates of H. pylori (40 from patients with chronic atrophic gastritis, 25 from patients with duodenal ulcer, 16 from patients with gastric ulcer, 3 from patients with both duodenal and gastric ulcers, and 3 from patients with intestinal type gastric cancer) were characterized by vacA typing by PCR and DNA sequencing. Eighty-four of the 87 isolates were s1a/m1, one was s1b/m1, and two could not be typed. Moreover, all isolates in this study were cagA positive. There were no distinct differences between the cytotoxin-producing strains and cytotoxin-nonproducing strains within the 0.73-kb middle region. Japanese strains were highly homologous, with more than 96% identity in this region, in which maximum divergence has been reported. In addition, there were no associations between the specific vacA types and the level of in vitro cytotoxin activity or the clinical consequences. These results indicate that the cagA-positive, s1a/m1-type strains are common in Japan, regardless of the vacA phenotype or clinical outcome.
Variations in fluconazole susceptibility and DNA subtyping of multiple Candida albicans colonies from patients with AIDS and oral candidiasis suffering one or more episodes of infection
Authors: Redding S W , Pfaller M A , Messer S A , Smith J A , Prows J , Bradley L L , Fothergill A W , Rinaldi M G
Published: January 2020

Five Candida albicans colonies from each infection in AIDS patients receiving fluconazole therapy for oropharyngeal candidiasis over a 2-year period were evaluated by antifungal susceptibility testing and DNA subtyping, and the results were correlated with clinical response to determine the occurrence of clinically significant selection of more-resistant C. albicans over multiple infections. A total of 534 C. albicans isolates were obtained from 38 patients who exhibited 84 episodes of infection. Antifungal susceptibility testing revealed that the MICs for 93% of the isolates were < or = 8.0 microg/ml and the MICs for 7% of the isolates were > or = 64 microg/ml. DNA subtyping revealed 70 different subtypes, with 78% of patients with one infection exhibiting one DNA subtype and 80% of patients with more than one infection exhibiting multiple DNA subtypes. Also, patients who had multiple infections had lower CD4 counts than those with single infections. Differences between the single-infection group and the multiple-infection group regarding the number of DNA subtypes and CD4 counts were both statistically significant. Of the 74 evaluable infections all were successfully treated with regular-dose (100-mg/day) fluconazole, except for three patients who ultimately responded to higher-dose fluconazole. Only one patient may have shown clinically significant selection of a more-resistant C. albicans strain over multiple courses of treatment. Interestingly, MICs reached only 8.0 microg/ml, even though doses of 400 mg of fluconazole were necessary for clinical cure.
Detection of endocervical anti-Chlamydia trachomatis immunoglobulin A in pregnant women by a rapid, 6-minute enzyme-linked immunosorbent assay: comparison with PCR and chlamydial antigen detection methods
Authors: Witkin S S , Bongiovanni A M , Inglis S R
Published: January 2020

There is a need for a rapid, uncomplicated, and inexpensive test for Chlamydia trachomatis infection in women. We evaluated the ability of a 6-min enzyme-linked immunosorbent assay (ELISA) that requires no laboratory equipment (IgA Rapid SeroTest; Savyon Diagnostics) to detect C. trachomatis immunoglobulin A (IgA) in the endocervices of 167 inner-city pregnant women and compared the results with DNA amplification (Amplicor PCR; Roche Diagnostics) and antigen detection (Chlamydiazyme; Abbott Laboratories) performed on the same women. Anti-C. trachomatis IgA was detected in the cervices of 32 women (19.2%). Samples from 23 women (13.8%) were PCR positive, while chlamydial antigen was present in 20 women (12.0%). There was only 1 sample (4.3%) that was positive by PCR but negative by ELISA; 10 samples were ELISA positive and PCR negative. In contrast, seven samples (30.4%) were PCR positive but Chlamydiazyme negative and four were Chlamydiazyme positive and PCR negative. Compared to PCR, the IgA ELISA had a sensitivity of 95.7%, a specificity of 93.1%, a positive predictive value of 68.8%, and a negative predictive value of 99.3%. The antigen assay had a sensitivity of only 69.6%, a specificity of 97.2%, a positive predictive value of 80.0%, and a negative predictive value of 95.2%. In high-risk groups where laboratory testing is not available, or where the patient might not return to obtain her testing result and be treated, the Rapid IgA SeroTest is a viable alternative for detection of cervical C. trachomatis in pregnant women.
Evidence for numerous omp1 alleles of porcine Chlamydia trachomatis and novel chlamydial species obtained by PCR
Authors: Kaltenböck B , Schmeer N , Schneider R
Published: January 2020

A nested PCR for genus-specific amplification of the Chlamydia omp1 locus was established. This PCR detected single template molecules in 200-microl specimen aliquots. Amplified chlamydial omp1 alleles were typed by heminested species PCRs and allele PCRs. We applied this method to 407 specimens from several host animals with various clinical conditions, and we detected prevalences of chlamydiae from 6 to 50%. Amplicons from peacock enteritis and equine infertility specimens were not typeable according to present omp1 allelic criteria for the chlamydial species. DNA sequencing revealed novel omp1 alleles which were 29.9 and 47.6% divergent in the deduced peptide sequences from the most closely related chlamydiae. Phylogenetic reconstruction indicated segregation of these alleles from the current four chlamydial species (90 and 97% bootstrap support), thus strongly suggesting the existence of additional chlamydial species. Allele typing of amplicons from swine with intestinal, urogenital, and respiratory infections demonstrated several unique omp1 allelic variants of Chlamydia trachomatis. These novel alleles had deduced peptide sequences which were 11.6 to 19% divergent from porcine C. trachomatis S45. Mutations were clustered in the C-terminal region of variable segment IV of the omp1 locus encoding subspecies and serovar determinants of the chlamydial major outer membrane protein, thus implying that there are numerous serovars of porcine C. trachomatis. These results demonstrate the need for routine application of sensitive genus-specific detection of chlamydiae in animal specimens and suggest a more prominent role than anticipated for chlamydiae in animal diseases.
Comparative evaluation of the E test, agar dilution, and broth microdilution for testing susceptibilities of Helicobacter pylori strains to 20 antimicrobial agents
Authors: Piccolomini R , Di Bonaventura G , Catamo G , Carbone F , Neri M
Published: January 2020

The Epsilometer test (E test; AB Biodisk, Solna, Sweden), a new quantitative technique for the determination of antimicrobial susceptibility, was compared to reference methods (agar dilution and broth microdilution) for the antimicrobial susceptibility testing of Helicobacter pylori. Seventy-one H. pylori strains isolated from patients with duodenal ulcers were tested against 20 antimicrobial agents. The E test and the agar dilution method were carried out on Mueller-Hinton agar; the broth microdilution method was performed with Mueller-Hinton broth. The E-test results showed excellent correlation with the agar dilution results, with 91.3 and 98.8% agreement within 1 and 2 log2 dilution steps, respectively, in a total of 1,350 tests. The correlation between the E-test results and the broth microdilution results was slightly higher, with 91.6 and 99.1% agreement within 1 and 2 log2 dilution steps, respectively, in a total of 1,317 tests. There were six major errors and two very major errors by the metronidazole E test compared to the results obtained by reference methods. Excellent agreement between E-test, agar dilution, and broth microdilution results was found for resistance to erythromycin (8%), clarithromycin (6%), and tetracycline (6%). Our results confirm that the E test is comparable to standardized methods for susceptibility testing. Therefore, the E test is a reliable and alternative method for testing H. pylori susceptibility to a wide range of antimicrobial agents in clinical practice.
Study of transmission routes of Helicobacter pylori in relation to seroprevalence of hepatitis A virus
Authors: Furuta T , Kamata T , Takashima M , Futami H , Arai H , Hanai H , Kaneko E
Published: January 2020

The seroprevalence of Helicobacter pylori in a group of 1,043 healthy Japanese people was compared with that of hepatitis A virus (HAV), which was used as a marker of fecal-oral exposure. No statistically significant relationship was observed between seropositivity for HAV and that for H. pylori. Therefore, the fecal-oral spread of H. pylori is of limited relevance in Japan.
Molecular detection and identification of Paracoccidioides brasiliensis
Authors: Sandhu G S , Aleff R A , Kline B C , da Silva Lacaz C
Published: January 2020

Nearly 800 nucleotides from the 5' terminus of the 28S ribosomal gene of Paracoccidioides brasiliensis were sequenced, and a 14-base DNA probe specific for this species was identified. Hybridization results showed that the probe identified P. brasiliensis ribosomal DNA in a panel of ribosomal DNAs representing a total of 48 species of fungi.
Pitfalls and fallacies of cat scratch disease serology: evaluation of Bartonella henselae-based indirect fluorescence assay and enzyme-linked immunoassay
Authors: Bergmans A M , Peeters M F , Schellekens J F , Vos M C , Sabbe L J , Ossewaarde J M , Verbakel H , Hooft H J , Schouls L M
Published: January 2020

The diagnostic value of the detection of immunoglobulin G (IgG) and IgM by Bartonella henselae-based indirect fluorescence assay (IFA) and enzyme-linked immunoassay (EIA) for the diagnosis of cat scratch disease (CSD) was evaluated. The IFA was performed either with B. henselae that was cocultivated for a few hours with Vero cells or with noncocultivated B. henselae as the antigen. Additionally, the performance of a Bartonella PCR hybridization assay based on the 16S rRNA gene was determined and compared with those of the serologic assays. The study group consisted of 45 patients suspected of suffering from CSD by fulfilling one or more of the classical criteria. The specificities of the immunoassays were set at > or = 95% by analysis of sera from 60 healthy blood donors. It is shown that the sensitivities of the IgG assays are very low (40.9% for the IFA with noncocultivated B. henselae as antigen) and that those of the IgM assays are higher (71.4% for the EIA) for patients who fulfilled two or more criteria for CSD. The IgM EIA showed the highest sensitivity: 71.4% in patients with two or more criteria for CSD and 80.6% for patients with a positive Bartonella PCR result. The results indicate that the specificities of both IFA and EIA IgG serologies and the sensitivity of the IFA IgM serology need to be improved. The PCR hybridization assay showed a sensitivity of 86.4% for patients who fulfilled two or more criteria for CSD and 100% for seven patients who fulfilled three or more criteria. The kinetics of IgG and IgM antibody production were studied in 18 patients with CSD on the basis of a positive B. henselae IFA IgM serology. The results indicate that there is no standard course of anti-B. henselae IgG and IgM production in patients with CSD, because some patients produced high levels of both IgG and IgM, others produced only high levels of IgM, and a few patients produced only low levels of antibodies.
Pseudo-outbreak of septicemia due to rapidly growing mycobacteria associated with extrinsic contamination of culture supplement
Authors: Ashford D A , Kellerman S , Yakrus M , Brim S , Good R C , Finelli L , Jarvis W R , McNeil M M
Published: January 2020

Between April and December 1994, 23 blood cultures from human immunodeficiency virus-infected patients grew rapidly growing mycobacteria suspected to be Mycobacterium chelonae at a hospital in New Jersey. The isolates were later identified as M. abscessus. Several bacterial species, including M. abscessus, were cultured from an opened multidose supplement vial (BBL Septi-Chek AFB Supplement) that had been used for mycobacterial blood cultures. The M. abscessus isolates from case patients and the supplement vial had identical multilocus enzyme electrophoresis and antimicrobial susceptibility patterns. Finding a contaminated vial of supplement, together with the lack of a distinct syndrome in case patients, was consistent with a pseudo-outbreak.