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PubMed2000

Impacts of Molecular Diagnostic Technologies on Plant Disease Management.

Detection and diagnosis of plant viruses has included serological laboratory tests since the 1960s. Relatively little work was done on serological detection of plant pathogenic bacteria and fungi prior to the development of ELISA and monoclonal antibody technologies. Most applications for laboratory-based tests were directed at virus detection with relatively little emphasis on fungal and bacterial pathogens, though there was some good work done with other groups of plant pathogens. With the advent of molecular biology and the ability to compare regions of genomic DNA representing conserved sequences, the development of laboratory tests increased at an amazing rate for all groups of plant pathogens. Comparison of ITS regions of bacteria, fungi, and nematodes has proven useful for taxonomic purposes. Sequencing of conserved genes has been used to develop PCR-based detection with varying levels of specificity for viruses, fungi, and bacteria. Combinations of ELISA and PCR technologies are used to improve sensitivity of detection and to avoid problems with inhibitors or PCR often found in plants. The application of these technologies in plant pathology has greatly improved our ability to detect plant pathogens and is increasing our understanding of, their ecology and epidemiology.

ELISA↗

PubMed2005

Passive and active components of neonatal innate immune defenses.

Innate immune defenses are crucial for survival in the first days and weeks of life. At birth, newborns are confronted with a vast array of potentially pathogenic microorganisms that were not encountered in utero. At this age, cellular components of the adaptive immune system are in a naive state and are slow to respond. Antibodies received from the dam are essential for defense, but represent a finite and dwindling resource. Innate components of the immune system detect pathogen-associated molecular patterns (PAMPs) on microorganisms (and their products) by means of pattern-recognition receptors (PRRs). Soluble mediators of the innate system such as complement proteins, pentraxins, collectins, ficolins, defensins, lactoferrin, lysozyme etc. can bind to structures on pathogens, leading to agglutination, interference with receptor binding, opsonization, neutralization, direct membrane damage and recruitment of additional soluble and cellular elements through inflammation. Cell-associated receptors such as the Toll-like receptors (TLRs) can activate cells and coordinate responses (both innate and adaptive). In this paper, accumulated knowledge of the receptors, soluble and cellular elements that contribute to innate defenses of young animals is reviewed. Research interest in this area has been intermittent, and the literature varies in quantity and quality. It is hoped that documentation of the limitations of our knowledge base will lead to more extensive and enlightening studies.

Animals↗

PubMed2026

Emerging food- and waterborne pathogen Arcobacter in wastewater: diversity and antibiotic resistance.

Arcobacter spp. are emerging food- and waterborne pathogens frequently detected in wastewater. Despite their high abundance in wastewater, Arcobacter diversity, antibiotic resistance, and genomic traits remain poorly characterized. To address these knowledge gaps, we conducted a comprehensive study of Arcobacter spp. in influent, effluent, and activated sludge from a Finnish wastewater treatment plant using full-length 16S rRNA gene sequencing, isolate-based genomics, and phenotypic antibiotic susceptibility testing. Arcobacter spp. were highly abundant in raw sewage but substantially removed during treatment. Four Arcobacter species were identified, dominated by Arcobacter cryaerophilus and Arcobacter suis. A proportion of amplicon sequence variants unclassified to species-level revealed potentially unexplored Arcobacter diversity. For the first time, we observed intragenomic variability in 16S rRNA gene copies of A. cryaerophilus, highlighting the importance of integrating culture-based and culture-independent approaches. Phenotypic testing revealed high proportions of non-wild-type isolates for clinically relevant antibiotics, including ampicillin, cefotaxime, tetracycline, and erythromycin. Genomic analyses showed that antibiotic resistance profiles were primarily mediated by chromosomally encoded determinants, including β-lactamases, efflux systems, and point mutations. Additionally, a broad arsenal of chromosomal and plasmid-borne resistance genes to heavy metals, biocides, and organic solvents was detected, reflecting adaptations to the wastewater environment. These findings provide novel insights into Arcobacter species-level diversity, resistance mechanisms, and ecological adaptations in anthropogenically influenced environments. The study highlights the significance of Arcobacter for public health and establishes a foundation for further research.IMPORTANCEArcobacter spp. are emerging human and animal pathogens that exhibit increasing resistance to clinically relevant antibiotics. Most community-acquired infections are linked to exposure through contaminated food and water, yet studies investigating their occurrence and diversity in wastewater remain scarce. Here, we focus on wastewater as an abundant source of Arcobacter spp. and a potential dissemination route contributing to downstream contamination of surface waters, irrigated soils, and possibly the food chain. By characterizing the species-level diversity, genomic traits, and antibiotic resistance profiles of Arcobacter spp. in wastewater, this study provides critical insights into the ecology and epidemiology of this ubiquitous genus.

Arcobacter↗

PubMed1995

Differentiating between Besnoitia besnoiti from cattle and Sarcocystis hoarensis from rodents.

To provide a biological basis for studies designed to establish the mode of transmission of the veterinary pathogen Besnoitia besnoiti, we compared salient features of this pathogen in cattle with those of Sarcocystis hoarensis in rodents. The cysts and cystozoites of these organisms can readily be distinguished morphologically. In contrast to S. hoarensis, which is well adapted to rodents, B. besnoiti fails to mature in jirds or mice and generally is lethal in jirds. Serological reagents discriminately detect these pathogens. B. besnoiti, therefore, can unambiguously be differentiated from S. hoarensis either by morphological or serological methods or on the basis of experimental comparisons of virulence in laboratory rodents.

Animals↗

PubMed2001

Detection of antibodies to a pathogenic mycoplasma in American alligators (Alligator mississippiensis), broad-nosed Caimans (Caiman latirostris), and Siamese crocodiles (Crocodylus siamensis).

An epidemic of pneumonia with fibrinous polyserositis and multifocal arthritis emerged in captive American alligators (Alligator mississippiensis) in Florida, United States, in 1995. Mycoplasma alligatoris sp. nov. was cultured from multiple organs, peripheral blood, synovial fluid, and cerebrospinal fluid of affected alligators. In a subsequent experimental inoculation study, the Henle-Koch-Evans postulates were fulfilled for M. alligatoris as the etiological agent of fatal mycoplasmosis of alligators. That finding was remarkable because mycoplasmal disease is rarely fatal in animals. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies produced by alligators in response to M. alligatoris exposure was developed by using plasma obtained from naturally infected alligators during the original epidemic. The assay was validated by using plasma obtained during an experimental dose-response study and applied to analyze plasma obtained from captive and wild crocodilian species. The ELISA reliably detected alligator seroconversion (P < 0.05) beginning 6 weeks after inoculation. The ELISA also detected seroconversion (P < 0.05) in the relatively closely related broad-nosed caiman Caiman latirostris and the relatively distantly related Siamese crocodile Crocodylus siamensis following experimental inoculation with M. alligatoris. The ELISA may be used to monitor exposure to the lethal pathogen M. alligatoris among captive, repatriated, and wild crocodilian species.

Alligators and Crocodiles↗

PubMed2005

How Toll-like receptors and Nod-like receptors contribute to innate immunity in mammals.

Innate immune detection of pathogens relies on specific classes of microbial sensors called pattern-recognition molecules (PRM). In mammals, such PRM include Toll-like receptors (TLRs) and the intracellular proteins NOD1 and NOD2, which belong to the family of Nod-like receptors (NLRs). Over the last decade as these molecules were discovered, a function in innate immunity has been assigned for the majority of them and, for most, the microbial motifs that these molecules detect were identified. One of the next challenges in innate immunity is to establish a better understanding of the complex interplay between signaling pathways induced simultaneously by distinct PRMs and how this affects tailoring first-line responses and the induction of adaptive immunity to a given pathogen.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Evaluation of universal preenrichment broth for growth of heat-injured pathogens.

Universal preenrichment broth (UPB) was developed to enable enrichment of injured foodborne pathogens of different genera simultaneously in lieu of having to undergo separate simultaneous enrichment cultures for subsequent detection or isolation of each pathogen. Enrichment conditions in UPB for growth of injured pathogens to populations that will enable pathogen detection by rapid immuno-based or polymerase chain reaction (PCR)-based assays have not been defined. Hence, studies were done to determine recovery and growth rates of heat-injured Escherichia coli O157:H7, Salmonella enterica ser. Typhimurium, Salmonella enterica ser. Enteritidis. and Listeria monocytogenes in UPB. Bacterial cells were heat injured in tryptic phosphate broth at 57.2 degrees C and inoculated at populations of ca. 0.17 to 63 injured cells per ml with raw ground beef, fresh chicken, lettuce, and environmental sponge samples. Enrichment cultures were sampled at 1, 2, 3, 4, 5, 6, and 24 h at 37 degrees C postinoculation, and pathogens were enumerated on appropriate selective media. Results revealed that recovery and growth of pathogens during the first 6 h of enrichment were not sufficient to ensure adequate numbers of bacteria (> 10(3) CFU/ ml) for detection by most immunoassays or PCR assays. Cells often required 3 to 4 h for recovery before growth was initiated. Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes cell populations in enrichment cultures with ground beef or lettuce at 6 h were 0.5 to 2.9 log10 CFU/ml. At 24 h of incubation, cell counts of enrichment samples for the three pathogens from all food and environmental sponge samples ranged from 4.0 to 8.3 log10 CFU/ml. Enrichment in UPB at 37 degrees C of foods or environmental sponge samples containing heat-injured cells of Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes reliably provides at 24 h of incubation-but not at 6 h-sufficient cell populations for detection by rapid immunoassay or PCR assay procedures that can detect at least 4 log10 CFU/ml. These results raise questions regarding the sensitivity of rapid detection methods that employ an abbreviated enrichment protocol of 6 h or less.

Animals↗

PubMed2002

Experimental Escherichia coli epididymitis in rats: a model to assess the outcome of antibiotic treatment.

OBJECTIVE: To assess the effect of initial antimicrobial therapy with a new highly potent quinolone (sparfloxacin) on the outcome of infection, especially acute and chronic inflammation, in a rat model of unilateral Escherichia coli epididymitis. MATERIALS AND METHODS: The study included 60 Sprague-Dawley rats, each of which received 0.1 mL of an E. coli (0:6 strain) suspension (106 colony forming units/mL) injected into the right ductus deferens. At 24 h after infection an oral antimicrobial treatment with sparfloxacin was initiated in half of the animals. The rats were killed 14 days, 3 and 6 months after infection, and both epididymes and the prostate gland cultured to re-isolate E. coli. To evaluate the grade of inflammation in both epididymes, histological variables, including acute and chronic inflammation and scar formation, were evaluated and a total inflammatory score, representing the sum of all variables, computed. RESULTS: Whereas antimicrobial therapy eradicated the pathogen, in untreated animals the pathogen was detectable for up to 6 months after infection in the infected epididymis and/or the prostate gland, while the contralateral epididymis was sterile. The inflammatory reaction in the infected epididymis was significantly less in treated animals (P < 0.001). Subclinical nonbacterial inflammation was present in the contralateral epididymis. CONCLUSIONS: Although adequate antimicrobial treatment eradicated the pathogen and reduced the grade of epididymal damage, inflammation was not avoided. Subclinical inflammation of the contralateral epididymis may contribute to impaired fertility. These results indicate that an inflammatory reaction initiated by bacteria might persist as a nonbacterial process despite early therapy, or by bacteria undetectable by conventional culture techniques, and may compromise male fertility.

Animals↗

PubMed1991

Comparison of immunofluorescence and culture for the detection of Actinomyces israelii in wearers of intra-uterine contraceptive devices.

A direct immunofluorescence (IF) method was compared with traditional culture methods for the detection of Actinomyces israelii in endocervical and intra-uterine-device (IUD) smears from 124 IUD wearers. Of 11 specimens that gave positive results by IF, only one was positive by culture. Of the 10 patients with positive IF specimens, three (30%) had signs and symptoms suggestive of pelvic infection and no other pathogen was detected. Direct IF of cervical smears offers a simple, relatively cheap method to screen IUD wearers for A. israelii. Clinical management of such cases is discussed.

Actinomyces↗

PubMed1991

Clinical, microbiological and immunological characteristics of subjects with "refractory" periodontal disease.

The aim of the present study was to analyze the relationship between attachment loss and clinical, microbiological, and immunological parameters in a group of 21 human subjects exhibiting poor response to previous periodontal therapy. All had been treated with periodontal surgery, tetracycline, and subsequent maintenance recalls to periodontists who, upon detection of disease progression, referred the subjects to our clinic. In our clinic, each subject received oral hygiene instruction and a thorough subgingival scaling and root planing utilizing as many appointments as necessary. Clinical indices, including gingival index, bleeding on probing, suppuration, plaque index, pocket depth, and duplicate measurements of attachment level from an acrylic stent, were collected at monthly intervals. Probing measurements were performed using the Florida Probe. When significant attachment loss (0.8-1.2 mm) was detected in at least 1 site, a bacterial sample was taken from that site and from a comparably deep, but non-progressing, control site. Microbial samples were enumerated by darkfield microscopy, on selective and non-selective media, and by predominant cultivable technique. Blood samples were also collected to determine antibody levels against potential pathogens. There was no difference in the amount of plaque present in sites gaining or losing attachment, but losing sites exhibited more bleeding and suppuration. 20 of the 21 subjects were tested; of these, 17 exhibited elevated serum antibody against one or more of the following microorganisms: Actinobacillus actinomycetemcomitans, Bacteroides, gingivalis, and Eikenella corrodens. However, few, if any, of the "classical" pathogens were detected in the plaque samples obtained at the time progressive disease was diagnosed. The only exception was Streptococcus intermedius, which occurred in slightly higher numbers in active sites.

Actinobacillus↗

PubMed2006

Multiplex detection of Ehrlichia and Anaplasma pathogens in vertebrate and tick hosts by real-time RT-PCR.

Tick-borne rickettsial infections are responsible for many emerging diseases in humans and several vertebrates. These include human infections with Ehrlichia chaffeensis, Ehrlichia ewingii and Anaplasma phagocytophilum. As single or co-infections can result from a tick bite, the availability of a rapid, multiplex molecular test will be valuable for timely diagnosis and treatment. We recently described a muliplex-molecular test that can detect single or co-infections with up to five Ehrlichia and Anaplasma species. We reported that the test has the sensitivity to identify single infections in the canine host with E. chaffeensis, E. canis, E. ewingii, A. phagocytophilum, and A. platys and co-infection with E. canis and A. platys. In this study, ticks were collected from different parts of the state of Kansas during summer months of the year 2003 and tested for the presence of infection using the molecular test. The analysis revealed a minimum of 3.66% of the ticks to be positive for either E. chaffeensis or E. ewingii in A. americanum and Dermacenter species. This assay will be valuable in monitoring infections in dogs and ticks, and with minor modifications it can be used for diagnosing infections in people and other vertebrates.

Anaplasma↗

PubMed1999

Enteric infections and diarrhea in human immunodeficiency virus-infected persons: prospective community-based cohort study. Swiss HIV Cohort Study.

BACKGROUND: Persons infected with human immunodeficiency virus (HIV) are at increased risk for diarrhea and enteric infections. We studied (1) the epidemiology of enteric pathogens associated with diarrhea, (2) the diagnostic yield of stool examination and endoscopic evaluation, (3) risks to develop diarrhea, and (4) the impact of diarrhea on patients' survival. METHODS: A total of 1933 participants in the Swiss HIV Cohort Study were prospectively followed up for a median of 25.5 months. A total of 560 diarrheal episodes were evaluated by standardized stool examination. Endoscopic evaluation was performed in 25% of patients with chronic diarrhea. RESULTS: The incidence of diarrhea was 14.2 per 100 person-years (95% confidence interval, 13.0-15.4). Among patients with CD4 cell counts below 0.05 x 10(9)/L, the probability to develop diarrhea within 1, 2, and 3 years was 48.5%, 74.3%, and 95.6%, respectively. The risk to develop diarrhea was increased among patients with severe immunodeficiency, homosexual men, and patients taking antiretroviral therapy. Pneumocystis carinii chemoprophylaxis did not reduce the risk of diarrhea. Diarrhea was an independent negative predictor of survival. Enteric pathogens were detected in 16.5% of 212 acute diarrheal episodes and in 46% of 348 chronic diarrheal episodes. The sensitivity of histological and stool examination was similar except for the diagnosis of intestinal cytomegalovirus infection and leishmaniasis, which required invasive evaluation. CONCLUSIONS: Intestinal infections were diagnosed in less than 50% of chronic diarrheal episodes. The prevalence of enteric pathogens tended to decrease during the observation period, possibly because of improved antiretroviral therapy. Endoscopic evaluation did not improve the diagnostic yield compared with stool examination except for the diagnosis of cytomegalovirus enteritis and leishmaniasis.

AIDS-Related Opportunistic Infections↗

PubMed2004

Cytochemical labeling for fungal and host components in plant tissues inoculated with fungal wilt pathogens.

Antibodies to detect pectin in present investigations attached to distinct fibrils in vessel lumina. In carnation infected with an isolate of Fusarium oxysporum f.sp., labeling of pathogen cells also occurred; in a resistant cultivar (cv.), it was coincident with proximate pectin fibrils and linked to altered fungal walls, which was the opposite in the susceptible cv., indicating that hindrance of pathogen ability to degrade pectin may be related to resistance. Labeling of the fungus in culture was nil, except in media containing pectin, showing that pectin is not native to the pathogen. Labeling of fungal walls for cellulose in elm (inoculated with Ophiostoma novo-ulmi) and carnation also occurred, linked to adsorbed host wall components. The chitin probe often attached to dispersed matter, in vessel lumina, traceable to irregularly labeled fungal cells and host wall degradation products. With an anti-horseradish peroxidase probe, host and fungal walls were equally labeled, and with a glucosidase, differences of labeling between these walls were observed, depending on pH of the test solution. Fungal extracellular matter and filamentous structures, present in fungal walls, predominantly in another elm isolate (Phaeotheca dimorphospora), did not label with any of the probes used. However, in cultures of this fungus, extracellular material labeled, even at a distance from the colony margin, with an anti-fimbriae probe.

Cell Wall↗

PubMed2004

Spectrum and frequencies of mutations in the GJB2 (Cx26) gene among 156 Czech patients with pre-lingual deafness.

Mutations in the gene gap junction beta 2 (GJB2), the gene for the connexin 26, are the most common cause of pre-lingual deafness worldwide. The mutation 35delG within GJB2 is prevalent in Europe. To date, there are no data about GJB2 mutation spectrum and frequencies from the Czech population. We investigated and report here the spectrum and frequencies of mutations in the GJB2 gene among 156 unrelated, congenital deafness Czech patients. Allele-specific polymerase chain reaction, together with fluorescent fragment analysis, were used for the detection of the 35delG mutation. The entire coding region of the GJB2 was directly sequenced in all patients who were not homozygous for the 35delG. No pathogenic mutation was detected in 51.9% of patients. At least one pathogenic mutation was found in 48.1% of patients, and both pathogenic mutations were detected in 37.8% of patients. Single mutations in a heterozygous state were detected in 10.3% of patients. The mutation 35delG accounts for 82.8% of detected disease mutations, Trp24stop accounts for 9.7% of pathogenic alleles and was found in patients with gypsy heritage. Mutation 313del14 accounts for 3.7% of pathogenic alleles. The frequency of 35delG heterozygotes in the Czech Republic is 1 : 29.6. Testing for only the three most common mutations would detect over 96% of all pathogenic alleles in the Czech Republic.

Cohort Studies↗

PubMed2006

Characterization of patients presenting for treatment to a university refractory periodontal diseases unit: three case reports.

BACKGROUND: Of the many forms of periodontal disease, refractory periodontal diseases are the least characterized. They are defined as the continued degeneration of the periodontium despite adequate management. This has led to the suggestion that there may be a systemic component that is a contributing factor to the development of this condition. The objectives of this report were to follow the progression of clinical changes associated with periodontal disease over a number of years in this unique population and review various hematologic and microbiologic factors that may be contributing to the disease progression. METHODS: Three subjects were profiled. They were referred to the Refractory Periodontal Disease Unit at the University of Toronto by periodontists or general practitioners in the Southern Ontario region. Complete medical and dental histories were obtained along with baseline clinical measurements. Periodontal examinations were facilitated with the use of a computer-assisted periodontal probe. A microbiologic analysis using immunofluorescence techniques was able to detect Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, and Actinobacillus actinomycetemcomitans and spirochetes. A hematologic analysis, including a complete blood count (CBC), immune profile, and glycosylated hemoglobin assay, was also performed. RESULTS: The clinical presentation revealed that patients receiving adequate maintenance therapy and with good to excellent oral hygiene demonstrated sites with continual loss of attachment. Few periodontal pathogens were detected. However, the most significant finding appeared to be the report elevated levels of CD8+ cells within this group of patients compared to normal laboratory ranges. CONCLUSIONS: This report is an attempt at characterizing a unique population within the periodontal realm. The long-term monitoring of these patients allowed for an assessment of factors that may be involved in the continued decline of the periodontal health of these patients. Based on the immune profile, it is possible that a hyperresponsive state may be the primary feature of this population. Future assessments, including full-mouth interleukin (IL)-1 and matrix metalloproteinase (MMP)-8 levels, may assist in characterizing this population further, with the goal of producing markers that will assist clinicians in predicting treatment outcome.

Adult↗

PubMed2006

Persistence of Mycobacterium avium subsp. paratuberculosis and other zoonotic pathogens during simulated composting, manure packing, and liquid storage of dairy manure.

Livestock manures contain numerous microorganisms which can infect humans and/or animals, such as Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., and Mycobacterium avium subsp. paratuberculosis (Mycobacterium paratuberculosis). The effects of commonly used manure treatments on the persistence of these pathogens have rarely been compared. The objective of this study was to compare the persistence of artificially inoculated M. paratuberculosis, as well as other naturally occurring pathogens, during the treatment of dairy manure under conditions that simulate three commonly used manure management methods: thermophilic composting at 55 degrees C, manure packing at 25 degrees C (or low-temperature composting), and liquid lagoon storage. Straw and sawdust amendments used for composting and packing were also compared. Manure was obtained from a large Ohio free-stall dairy herd and was inoculated with M. paratuberculosis at 10(6) CFU/g in the final mixes. For compost and pack treatments, this manure was amended with sawdust or straw to provide an optimal moisture content (60%) for composting for 56 days. To simulate liquid storage, water was added to the manure (to simulate liquid flushing and storage) and the slurry was placed in triplicate covered 4-liter Erlenmeyer flasks, incubated under ambient conditions for 175 days. The treatments were sampled on days 0, 3, 7, 14, 28, and 56 for the detection of pathogens. The persistence of M. paratuberculosis was also assessed by a PCR hybridization assay. After 56 days of composting, from 45 to 60% of the carbon in the compost treatments was converted to CO2, while no significant change in carbon content was observed in the liquid slurry. Escherichia coli, Salmonella, and Listeria were all detected in the manure and all of the treatments on day 0. After 3 days of composting at 55 degrees C, none of these organisms were detectable. In liquid manure and pack treatments, some of these microorganisms were detectable up to 28 days. M. paratuberculosis was detected by standard culture only on day 0 in all the treatments, but was undetectable in any treatment at 3 and 7 days. On days 14, 28, and 56, M. paratuberculosis was detected in the liquid storage treatment but remained undetectable in the compost and pack treatments. However, M. paratuberculosis DNA was detectable through day 56 in all treatments and up to day 175 in liquid storage treatments. Taken together, the results indicate that high-temperature composting is more effective than pack storage or liquid storage of manure in reducing these pathogens in dairy manure. Therefore, thermophilic composting is recommended for treatment of manures destined for pathogen-sensitive environments such as those for vegetable production, residential gardening, or application to rapidly draining fields.

Animals↗

PubMedDate not supplied

Separation, identification and quantification of riboflavin and its photoproducts in blood products using high-performance liquid chromatography with fluorescence detection: a method to support pathogen reduction technology.

A medical device using riboflavin (RB) and light is being developed for the reduction of pathogens in platelet concentrates (MIRASOL pathogen reduction technology [PRT]). A high-performance liquid chromatography (HPLC) method for the quantification of RB and its main photoproduct, lumichrome (LC) in blood components has been developed and validated. In addition, the same method has been used to identify and quantify the presence of additional photoproducts-catabolites of RB. Levels of these agents before and after treatment as well as endogenous levels present in normal donor blood are reported using this analytical technique. The method allows for quantitative and qualitative analysis of RB and LC in blood components using HPLC-fluorescence detection, a Zorbax SB-CN (stable bond cyano) column and a methanol-water mobile phase. Quantitation and qualitative analysis of additional photoproducts of RB was also performed, but the method has not been validated for these other components. The method described has passed an 8 day validation and has been found to be adequate for its intended use. The range of the method for RB is 0.016-1.500 microM and for LC is 0.060-1.500 microM. The method detection limit for RB is 0.0006 microM and for LC is 0.012 microM. The acceptance criteria for repeatability were met; the relative standard deviation for RB was 0.64% and for LC was 0.76%. The acceptance criteria for bias were met with a 97% average recovery for RB and a 102% recovery for LC. Samples were centrifuged and diluted 1:50 with 0.9% saline before analysis. No protein precipitation or extraction was required. A mass balance of approximately 93.4-94.4% was achieved after exposure of products to UV light in the intended pathogen reduction treatment method. The method permitted the identification of photoproducts in blood that were both naturally occurring and produced after photolysis of blood samples treated with the PRT process. The identity of these photoproducts has been established using HPLC Tandem Mass Spectrometry (MS/MS) and UV spectroscopic methods and has been correlated with known metabolites and catabolites of RB. HPLC with fluorescence detection using a reverse phase cyano-column allows for accurate separation, identification and quantification of both RB and LC in blood products without the need for solvent extraction or protein precipitation. Additional photoproducts could also be identified and quantified using this method. The presence of these agents in normal, untreated blood suggests that their presence in blood is ubiquitous.

Blood Chemical Analysis↗

PubMed1996

Rapid antigen tests for Neisseria gonorrhoeae and Chlamydia trachomatis are not accurate for screening women with disturbed vaginal lactobacillary flora.

We studied the accuracy of the rapid antigen detection tests Gonozyme and Chlamydiazyme in high-risk women in an outpatient prenatal clinic, Kalafong University Hospital, Pretoria, South Africa. Women (n = 433) presenting with uneventful pregnancy (n = 324), unavoidable miscarriage (n = 41) or infertility of 1 year's duration (n = 68) had a Pap smear for lactobacillary grading and detection of pathogens like Candida albicans or Trichomonas vaginalis, a swab for culture of Neisseria gonorrhoeae, and a swab for Gonozyme, Chlamydiazyme and Chlamydia immunofluorescence collected from the endocervix. Specificities of both antigen tests were high, but sensitivities and positive predictive values were disappointingly low. Chlamydial antigen was recovered in only 37% of samples with positive immunofluorescence, gonococcal antigen was detected in only 50% of samples with positive culture for N. gonorrhoeae. Although prevalence of N. gonorrhoeae, Chlamydia trachomatis and Trichomonas vaginalis was higher in women with disturbed lactobacillary grades on the Pap smears, sensitivities of the antigen tests were lower in this group. We conclude that detection of endocervical antigens of C. trachomatis and N. gonorrhoeae lacked sensitivity in pregnant and infertile women living in an area with high prevalence of chlamydial cervicitis, gonorrhoea and Trichomonas vaginitis. Furthermore, the rapid antigen tests lack accuracy when the lactobacillary flora is disturbed and are, therefore, not suitable for detection of C. trachomatis or N. gonorrhoeae in pre-screened patients.

Animals↗