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PubMed1998

Detection and analysis of cytokine mRNA in tissues and cell lines.

Many normal and pathological processes have been shown to be influenced by the quantitative and qualitative nature of cytokine production. Cytokines have also been shown to be central in modulating host responses to invasion by pathogens. Therefore detection and analysis of cytokine production are critical in the understanding of host responses to a variety of immunological insults. The detection of cytokines by bioassay or immunoassay provides data from in vitro experiments and can be used to determine the circulating plasma levels of particular cytokines. However, many investigations require the detection of cytokines in small numbers of cells or directly from tissue samples. It is in this particular area that detection and analysis of cytokine gene expression by use of the sensitive and specific reverse transcription-polymerase chain reaction (RT-PCR) has application. The purpose of these protocols is to provide a practical guide to the detection of cytokine gene expression using RT-PCR techniques. Procedures for analysing mRNA expression in cell culture, cell suspensions and tissue samples will be described and pitfalls and caveats concerning the techniques will be discussed. These protocols should provide a starting point for the development of RT-PCR technology in laboratories with only limited molecular experience.

Cytokines↗

PubMedDate not supplied

Recent advances in diagnosis of sexually transmitted diseases.

In an effort to better diagnose, treat, and control sexually transmitted diseases (STD), a number of new diagnostic assays using molecular techniques have been developed. By incorporating molecular amplification, the sensitivity for detecting sexually transmitted infections has become markedly enhanced, and organisms that were difficult or impossible to cultivate, such as human papillomavirus (HPV) or Treponema pallidum can now be detected and monitored. By using polymerase chain reaction (PCR) or ligase chain reaction (LCR), the sensitivity of detecting some pathogens is comparable to, or in some cases better than, direct in vitro cultivation of the agent. DNA fingerprint analysis of amplified microbial DNA also has been effectively used for detailed study of the epidemiology and pathophysiology of sexually transmitted infections. In addition to direct detection, molecular techniques have been used to enhance serologic techniques by use of cloned proteins and recombinant antigens. These techniques have enabled investigators to differentiate infection caused by closely related pathogens, such as human immunodeficiency virus type 1 (HIV-1) and HIV-2 and human T-cell lymphotrophic virus type 1 (HTLV-1) and HTLV-2. As a consequence of these molecular tools, the diagnostic repertoire of the clinical laboratory for the diagnosis of STD will expand significantly, allowing investigators to better diagnose and more effectively control the spread of STD. However, with such new technology, new problems and challenges have arisen, such as the risk of sample contamination resulting in false-positive results, and the presence of inhibitors resulting in false-negative results.(ABSTRACT TRUNCATED AT 250 WORDS)

Diagnosis, Differential↗

PubMed2002

Whole-gene APC deletions cause classical familial adenomatous polyposis, but not attenuated polyposis or "multiple" colorectal adenomas.

Familial adenomatous polyposis (FAP) is a dominantly inherited colorectal tumor predisposition that results from germ-line mutations in the APC gene (chromosome 5q21). FAP shows substantial phenotypic variability: classical polyposis patients develop more than 100 colorectal adenomas, whereas those with attenuated polyposis (AAPC) have fewer than 100 adenomas. A further group of individuals, so-called "multiple" adenoma patients, have a phenotype like AAPC, with 3-99 polyps throughout the colorectum, but mostly have no demonstrable germ-line APC mutation. Routine mutation detection techniques fail to detect a pathogenic APC germ-line mutation in approximately 30% of patients with classical polyposis and 90% of those with AAPC/multiple adenomas. We have developed a real-time quantitative multiplex PCR assay to detect APC exon 14 deletions. When this technique was applied to a set of 60 classical polyposis and 143 AAPC/multiple adenoma patients with no apparent APC germ-line mutation, deletions were found exclusively in individuals with classical polyposis (7 of 60, 12%). Fine-mapping of the region suggested that the majority (6 of 7) of these deletions encompassed the entire APC locus, confirming that haploinsufficiency can result in a classical polyposis phenotype. Screening for germ-line deletions in APC mutation-negative individuals with classical polyposis seems warranted.

Adenoma↗

PubMed1998

Detection of Legionella by PCR in respiratory specimens using a commercially available kit.

Using polymerase chain reaction (PCR) for the detection of pathogens that are difficult to grow, such as Legionella species, may reduce difficulties encountered with culture and immunofluorescent staining. We evaluated a commercial PCR and hybridization kit, designed for environmental samples, for the detection of Legionella in respiratory specimens. Sixteen Legionella species cultures tested positive with the Perkin Elmer Legionella EnviroAmp Amplification and Detection kits (Perkin Elmer, Foster City, Calif). The assay detected as few as 100 colony-forming units per milliliter of spiked bronchoalveolar lavage (BAL) fluid, and no false-negative results were obtained. PCR inhibition by blood in the specimens was removed by washing pelleted specimens in sterile distilled water. Of 126 specimens screened with the kit, 1 induced sputum and 3 BAL specimens were positive by PCR. All 4 were validated as true-positive results by culture or serologic testing. The entire PCR and hybridization assay can be completed in less than 6 hours, whereas isolation and identification by culture requires up to 12 days, and serologic conversion may not be demonstrated for weeks. Molecular techniques based on direct extraction and amplification of DNA from respiratory specimens nay be useful for the timely diagnosis of legionellosis.

Bronchoalveolar Lavage Fluid↗

PubMed2002

The emerging role of atypical pathogens in community-acquired pneumonia.

Community-acquired pneumonia (CAP) constitutes a major cause of morbidity and mortality. Although Streptococcus pneumoniae remains the bacterium most commonly implicated in CAP, the atypical respiratory pathogens Mycoplasma pneumoniae, Legionella species, and Chlamydia pneumoniae are being isolated with increasing frequency Contrary to previous beliefs, these agents are capable of causing severe as well as mild-to-moderate illness. Moreover, they can affect all age groups. Indeed, atypical pathogens are implicated in up to 40% of CAP cases and commonly occur as copathogens in mixed-infection CAP, an etiology associated with particularly high mortality (up to 25%). Laboratory methods for detecting atypical pathogens are slow, and there is significant overlap between atypical and typical CAP manifestations. For these reasons, accurate prediction of etiology cannot be made purely on clinical or radiologic grounds. Consequently, empiric antimicrobial therapy for atypical pathogens (with agents such as macrolides, fluoroquinolones, in some cases tetracyclines, or the new ketolides) warrants careful consideration and now is recommended for the treatment of CAP.

Anti-Bacterial Agents↗

PubMed1996

Evaluation of polymerase chain reaction-restriction enzyme analysis of mycobacteria cultured in BACTEC 12B bottles.

Polymerase chain reaction with restriction enzyme analysis (PRA) was first tested on 15 reference strains and 50 subcultured clinical isolates of mycobacteria according to the reference algorithm by Telenti et al [1]. Next, we evaluated the application of this method to 108 isolates from liquid media (BACTEC 12B). Of them, 15 M. tuberculosis complex and 81 mycobacteria other than tuberculosis (MOTT) had comparable results with both PRA and the BACTEC 460 TB systems. However, seven M. tuberculosis complex and three potentially pathogenic MOTT were identified by PRA rather than the BACTEC TB system. PRA seems to be an efficient method for the identification of mycobacteria to the species level and a good aid to detect potentially pathogenic mycobacteria, especially in mixed mycobacterial cultures.

Humans↗

PubMed2006

Detection and isolation of highly pathogenic H5N1 avian influenza A viruses from blow flies collected in the vicinity of an infected poultry farm in Kyoto, Japan, 2004.

During the outbreak of highly pathogenic avian influenza that occurred in Tamba Town, Kyoto Prefecture in 2004, a total of 926 flies were collected from six sites within a radius of 2.3 km from the poultry farm. The H5 influenza A virus genes were detected from the intestinal organs, crop, and gut of the two blow fly species, Calliphora nigribarbis and Aldrichina grahami, by reverse transcription-polymerase chain reaction for the matrix protein (M) and hemagglutinin (HA) genes. The HA gene encoding multiple basic amino acids at the HA cleavage site indicated that this virus is a highly pathogenic strain. Based on the full-length sequences of the M, HA, and neuraminidase (NA) segments of virus isolates through embryonated chicken eggs, the virus from C. nigribarbis (A/blow fly/Kyoto/93/2004) was characterized as H5N1 subtype influenza A virus and shown to have > 99.9% identities in all three RNA segments to a strain from chickens (A/chicken/Kyoto/3/2004) and crows (A/crows/Kyoto/53/2004) derived during this outbreak period in Kyoto in 2004. Our results suggest it is possible that blow flies could become a mechanical transmitter of H5N1 influenza virus.

Amino Acid Sequence↗

PubMed2005

Citrus huanglongbing in São Paulo State, Brazil: PCR detection of the 'Candidatus' Liberibacter species associated with the disease.

Symptoms of huanglongbing (HLB), one of the most serious diseases of citrus in Asia and Africa, have been noticed in March 2004 in the Araraquara region of São Paulo State, Brazil. HLB has not been reported previously from America. The causal HLB bacteria, Candidatus Liberibacter africanus in Africa and Candidatus Liberibacter asiaticus in Asia, can be detected in symptomatic citrus leaves by PCR amplification of their 16S rDNA with previously described primers. When this technique was applied to 43 symptomatic leaf samples from the Araraquara region, all PCR reactions were negative. This suggested that a new pathogen, not detected by the above primers, could be involved in HLB in the State of São Paulo. Indeed, by using universal primers for amplification of bacterial 16S rDNA, a new liberibacter species, Candidatus Liberibacter americanus, has recently been identified. Specific primers for PCR amplification of the 16S rDNA of Ca. L. americanus have been selected. Using these primers, the new liberibacter could be detected in 214 symptomatic leaf samples tested. The leaves of two additional samples were infected with Candidatus Liberibacter asiaticus, and two further samples contained both Ca. L. americanus and Ca. L. asiaticus. The samples came from 47 farms in 35 municipalities. The psyllid vector of Ca. L. asiaticus, Diaphorina citri, is established in South, Central, and North America (Florida and Texas). Ca. L. americanus could be detected by PCR in several batches of D. citri psyllids collected on symptomatic sweet orange trees infected with Ca. L. americanus, strongly suggesting that D. citri is the vector of Ca. L. americanus. The results reported here confirm the presence of HLB in the State of São Paulo. Ca. L. americanus is the most widely distributed pathogen.

Base Sequence↗

PubMed1991

Identification of foodborne pathogens by nucleic acid hybridization.

Nucleic acid hybridization methods have been developed and used to identify microorganisms in foods. Tests performed on mixed cultures save the time required to establish pure cultures. Enterotoxigenic or invasive strains of foodborne bacterial pathogens are detected with probes that identify genes responsible for virulence. Hybridization tests signal the presence or absence of a particular strain or an entire genus and are especially well suited for screening foods for specific pathogens. With the colony hybridization assay format, foodborne bacteria harboring a specific gene can be enumerated. However, hybridization tests require the presence of 10(5) to 10(6) cells to yield a positive result, thereby limiting sensitivity and necessitating a time-consuming growth step. In vitro DNA amplification techniques increase the amount of DNA segments 10(5)-10(6)-fold in 2 to 3 h, thus enhancing test sensitivity.

Bacteria↗

PubMed2004

Microbial-gut interactions in health and disease. Epithelial cell responses.

Pathogenic bacteria use many strategies to secure their survival within the host. Enteropathogens exploit intestinal epithelial cells in many ways, including the manipulation of normal cellular functioning, or of cellular structural components, or by the induction of signalling pathways, such as the production of pro-inflammatory cytokines. However, the enterocyte warns the host of impending danger and, in turn, elicits a protective response. Pathogens are detected by epithelial cells owing to their vast array of surface antigens and secreted products. Epithelial cells have developed both extracellular and intracellular sensing proteins that function as a first line of defence against pathogens; this is followed by acquired immunity, namely IgA, which is used as reinforcement. Thus, in a game of constant attack and defence, the pathogen and the enterocyte aim to outsmart each other in an effort to survive.

Enterobacteriaceae↗

PubMed2000

Occurrence of pathogenic Yersinia enterocolitica in Norwegian pork products determined by a PCR method and a traditional culturing method.

From October 1997 to April 1998, a survey was conducted to assess the occurrence of pathogenic Yersinia enterocolitica in Norwegian pork products, using a traditional culturing method and a PCR assay. A total of 300 pork samples was examined. Five slaughterhouses in the Norwegian Meat Cooperative were represented with 249 samples and another 51 samples were obtained from retail outlets in the city of Oslo. Using the NMKL method, Y. enterocolitica 0:3 was isolated from six (2%) of the samples, while the PCR method indicated presence of pathogenic Y. enterocolitica in 50 (17%) of the samples. The results indicate that a reduction has occurred in the prevalence of pathogenic Y. enterocolitica in Norwegian pork products, as compared to a previous Norwegian study conducted in 1988-1989. The study also highlights the need for further development and improvement of methods applied for the detection of pathogenic Y. enterocolitica in foods.

Animals↗

PubMedDate not supplied

Seasonal variation in host susceptibility and cycles of certain infectious diseases.

Seasonal cycles of infectious diseases have been variously attributed to changes in atmospheric conditions, the prevalence or virulence of the pathogen, or the behavior of the host. Some observations about seasonality are difficult to reconcile with these explanations. These include the simultaneous appearance of outbreaks across widespread geographic regions of the same latitude; the detection of pathogens in the off-season without epidemic spread; and the consistency of seasonal changes, despite wide variations in weather and human behavior. In contrast, an increase in susceptibility of the host population, perhaps linked to the annual light/dark cycle and mediated by the pattern of melatonin secretion, might account for many heretofore unexplained features of infectious disease seasonality. Ample evidence indicates that photoperiod-driven physiologic changes are typical in mammalian species, including some in humans. If such physiologic changes underlie human resistance to infectious diseases for large portions of the year and the changes can be identified and modified, the therapeutic and preventive implications may be considerable.

Communicable Diseases↗

PubMed1980

Considerations in receiving and quarantining laboratory rodents.

The development of increased bacteriologic and viral screening has revealed evidence of exposure to possible pathogens in animals from even the best suppliers. Because the cost in money and space to quarantine all rodents in many types of research institutions is great, and because of an increased ability to detect possible pathogens, a dilemma exists. At the same time, regulations are being implemented which, in many cases, mandate a quarantine period. In all cases, however, the professional judgement of laboratory animal medicine specialists is increasingly needed to interpret the best course of action. A proper matching of animal quality with a realistic assessment of research requirements seems the best course, with quarantine implemented where truly appropriate.

Animals↗

PubMed2003

First detection of group C rotavirus in children with acute diarrhea in Spain.

Group C rotavirus causes sporadic cases and outbreaks of acute diarrhea in children and adults in many countries, but has never been detected among children in Spain. In a recently conducted surveillance study to screen fecal specimens for bacteria and viruses from a cohort of 822 young children who were treated for acute diarrhea in Madrid, no pathogens were detected in fecal specimens from 238 (29%) children. In this study, we examined 147 of those specimens for group C rotavirus by EIA and PCR and found 22 (15%) were positive. Our findings demonstrate that group C rotavirus is an important cause of childhood diarrhea in Spain.

Child↗

PubMed2003

Molecular cloning and characterization of a novel leptospiral lipoprotein with OmpA domain.

A novel antigenic protein of pathogenic Leptospira, Loa22, was identified by using the PhoA fusion method followed by immunoblotting with convalescent mouse sera. Loa22 was shown to be a lipoprotein having a C-terminal OmpA consensus domain. Loa22 was detected among pathogenic leptospires but not among non-pathogenic leptospires, suggesting the possible involvement of this protein in virulence. The results of three different experiments suggested that Loa22 is located in the outer membrane and a small portion is exposed on the cell surface. Thus, Loa22 may be a candidate for a novel vaccine against infection with pathogenic leptospires.

Amino Acid Sequence↗

PubMed1993

Detection of antibodies to a pathogenic mycoplasma in desert tortoises (Gopherus agassizii) with upper respiratory tract disease.

Mycoplasma agassizii (proposed species novum) is the etiologic agent of an upper respiratory tract disease in the desert tortoise (Gopherus agassizii), which is threatened in most of its range. An enzyme-linked immunosorbent assay (ELISA) for the detection of M. agassizii-specific antibodies in desert tortoises was developed with a monoclonal antibody with specificity for desert tortoise immunoglobulin light chain. Plasma samples from one group of tortoises were tested immediately before and 1 month after challenge either with nasal exudate containing M. agassizii or with a purified preparation of M. agassizii. Plasma samples from a second group of known healthy and sick tortoises were also tested. In the first group, the ELISA detected seroconversion in individual tortoises following challenge with M. agassizii. In the second group, ELISA results were positively correlated with the health status of the tortoises, as determined by clinical and pathologic findings. In addition, the ELISA revealed that tortoise antimycoplasma antibodies were specific for M. agassizii when samples were assayed against M. agassizii, M. pulmonis, M. testudinis, and M. gallisepticum antigens. The observed direct correlation between the presence of nasal mucosal lesions and M. agassizii-specific antibodies proved that the ELISA reliably diagnosed M. agassizii infection in desert tortoises and advocates its use for monitoring M. agassizii-induced upper respiratory tract disease in free-ranging desert tortoises.

Animals↗

PubMed1999

Atypical outbreak of caprine cryptosporidiosis in the Sultanate of Oman.

An outbreak of cryptosporidiosis occurred in goats ranging in age from two days to adult, on a well-managed closed farm. None of the other animals on the farm, including sheep, cows and buffalo, were affected. Morbidity approached 100 per cent in goats less than six months of age. Despite intensive supportive care, 238 goats died, ranging in age from two days to over one year. Cryptosporidia were detected in large numbers in the intestinal contents of dead animals and in faecal smears of animals with diarrhoea. Massive numbers of the organisms were also demonstrated histopathologically and by electronmicroscopy, and no other significant pathogens were detected. The outbreak was unique in terms of the extreme virulence of the organism, its apparent species-specificity, and the shedding of the organism by animals over four weeks of age.

Animals↗

PubMed2002

Periodontopathic bacteria in young healthy subjects of different ethnic backgrounds in Los Angeles.

BACKGROUND: The present study determined risk indicators for oral colonization by Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola in 150 children and adolescents, 4 to 16 years of age, living in Los Angeles, California. METHODS: Fifty Caucasians, 50 Hispanics, and 50 Asian-Americans completed a questionnaire on demographic characteristics. 16S rRNA-based polymerase chain reaction identification was employed to determine the presence of test bacteria in unstimulated saliva. Step-wise logistic regression analysis identified explanatory variables (risk indicators) accounting for the salivary presence of periodontopathic bacteria. RESULTS: A. actinomycetemcomitans occurred in 15%, P. gingivalis in 15%, B. forsythus in 14%, and T. denticola in 18% of all subjects. Two or more pathogens were detected in 20% of Hispanic subjects and in 12% of Asian-American subjects but not in any Caucasians (P = 0.0005, chi square test). However, no stable multivariate model including ethnicity was found for multiple pathogens. Risk for harboring any pathogens increased with the length of time lapse from last dental visit (odds ratio [OR], 4.46; 95% confidence interval [CI], 1.83 to 12.21), and decreased with higher education level of the mother (OR, 0.258; 95% CI, 0.052 to 0.875). Risk for harboring 2 or more periodontal pathogens decreased with the years the parents had resided in the United States (OR, 0.95; 95% CI, 0.901 to 0.992). Risk for harboring A. actinomycetemcomitans decreased as the number of years the parents had resided in the United States increased (OR, 0.91; 95% CI, 0.86 to 0.95), and decreased with higher income level of the father (OR, 0.201; 95% CI, 0.038 to 0.948). Girls were at higher risk for harboring P. gingivalis (OR, 2.55; 95% CI, 1.02 to 7.03), but at lower risk for carrying T. denticola (OR, 0.42; 95% CI, 0.17 to 0.98). CONCLUSIONS: This study showed that salivary occurrence of periodontopathic bacteria in young individuals was related to the length of time the parents had lived in the United States, education level of the mother, length of time since last dental visit, and gender, but apparently not to ethnicity per se.

Adolescent↗