Polymerase Chain Reaction Amplification Assay Research Articles

Plasmid DNA could be delivered into Eimeria maxima unsporulated oocyst with gene gun system

Eimerian coccidia are the most common parasitic organisms infecting chickens. The feasibility of genetic manipulation of these parasites via electroporation is proven, but this method is cumbersome and time consuming. Here we report our endeavour to develop a rapid and simple transfection method by gene gun. Tungsten particles coated with plasmid DNA encoding enhanced yellow fluorescent protein (EYFP) were used for the bombardment of Eimeria maxima unsporulated oocysts. Seven Mpa (1015 psi) helium pressure, 65 mm target distance and -0.098 Mpa (24.8″ Hg) chamber vacuum were the optimised parameters for bombardment. After sporulation, the bombarded oocysts were inoculated into chickens, and the progeny oocysts were checked under fluorescent microscope and subjected to genomic DNA extraction, which was used either for polymerase chain reaction (PCR) amplification or plasmid rescue assay. Although the expression of EYFP was not observed, the gene was amplified from both genomic DNA and the rescued plasmid, suggesting that the plasmid DNA existed in the form of episome. These results are encouraging for the genetic processing of the sporogony stage of eimerian parasites.

Comparison of a TaqMan real-time polymerase chain reaction assay with a loop-mediated isothermal amplification assay for detection of Gallid herpesvirus 1

A TaqMan real-time polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) assay were developed to detect Gallid herpesvirus 1 (GaHV-1, formerly Infectious laryngotracheitis virus). The standard curve of real-time PCR was established, and the sensitivity reached 10 copies/μl. In the current study, the conversion between viral titer and GaHV-1 genomic copy number was constructed. Six primers for LAMP assay amplified target gene at 65°C within 45 min, and the detection limit was 60 copies/μl. The 6 primers were highly specific, sensitive, and reproducible for detection of GaHV-1. Although the sensitivity of LAMP was lower than that of real-time PCR, LAMP was faster, less expensive, and did not require a thermocycler. The LAMP assay would be a viable alternative assay in diagnostic laboratories that do not employ real-time PCR technology.

Molecular detection and characterization of West Nile virus associated with multifocal retinitis in patients from southern India

In late 2009/early 2010, approximately 2000 people were affected by a mysterious viral outbreak in a southern district of Tamil Nadu; this particularly affected those living in coastal areas. Blood samples from affected patients were sent for clinical analysis to determine the actual cause of the illness, but reports were inconclusive. The present study describes the clinical observations and laboratory investigations involving molecular methods performed on 170 of the 2000 clinically suspected cases. These were patients who were admitted to Aravind Eye Hospital, Madurai, Tamil Nadu with ocular complications. Conventional reverse transcription polymerase chain reaction (RT-PCR), real-time RT-PCR, and reverse transcription loop-mediated isothermal gene amplification (RT-LAMP) assays were used to detect West Nile virus (WNV) infection. Further investigation of the genetic diversity of the WNV implicated in ocular complications was undertaken by sequence phylogeny. Out of 170 samples, 25 (15%) were positive for chikungunya IgM antibody, 10 (6%) for chikungunya antigen, and 30 (18%) were positive for dengue IgM antibody. The remaining 105 seronegative samples were further processed for WNV detection by IgM capture ELISA and molecular methods. Out of the 105 samples, 35 (33%) were positive for WNV IgM antibody, 15 (14%) were positive for WNV by RT-PCR, and 27 (26%) were found to be positive for WNV by both real-time RT-PCR and RT-LAMP assays. Comparative evaluation with acute-phase patient serum samples revealed 100% concordance between the real-time RT-PCR and RT-LAMP assays. These assays had an overall higher sensitivity than the conventional RT-PCR as they picked up 12 additional samples with a low copy number of template. Further genotyping through sequence phylogeny revealed that all the WNV isolates were grouped in lineage I. The association of West Nile virus with ocular infection in South India during an epidemic of mysterious fever in the first half of 2010 was clearly established through molecular approaches employing envelope gene-specific real-time RT-PCR and RT-LAMP assays followed by nucleotide sequencing.

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Samples from infected domestic pigs associated with an outbreak of African swine fever (ASF) in three districts of central Uganda in 2007 were confirmed as being infected with African swine fever virus (ASFV) using a P72 gene-based polymerase chain reaction amplification (PCR) assay combined with restriction analysis. None of the sera collected from pigs with clinical symptoms were positive using the OIE serological prescribed tests. However, seven haemadsorbing viruses were isolated in macrophage culture and genotyped by partial p72 and full length p54-gene sequencing. Four of these viruses were isolated directly from serum samples. All the viruses were classified within the domesticpig cycle-associated p72 and p54 genotype IX which also includes viruses responsible for ASF outbreaks in Kenya in 2006 and 2007 and Uganda in 2003. To define virus relationships at higher resolution, typing was performed by analysis of tetrameric amino acid repeat regions within the central variable region (CVR) of the B602L gene. Ugandan isolates sequences exhibited 100% identity to viruses isolated from outbreaks in Kenya in 2007. The identity was greater than the viruses obtained from an earlier outbreak in Kenya in 2006. This provides further evidence that genetically similar ASFV virus within p72 Genotype IX may be circulating between Kenya and Uganda.

Detection of Early and Single Infections of Schistosoma japonicum in the Intermediate Host Snail, Oncomelania hupensis, by PCR and Loop-Mediated Isothermal Amplification (LAMP) Assay

Polymerase chain reaction (PCR) with the specific primer set amplifying 28S ribosomal DNA (rDNA) of Schistosoma japonicum was able to detect genomic DNA of S. japonicum, but not S. mansoni, at 100 fg. This procedure enabled us to detect the DNA from a single miracidium and a snail infected with one miracidium at just 1 day after infection. We compared these results with those from loop-mediated isothermal amplification (LAMP) targeting 28S rDNA and found similar results. The LAMP could amplify the specific DNA from a group of 100 normal snails mixed with one infected snail A PCR screening of infected snails from endemic regions in Anhui Province revealed schistosomal DNA even in snails found negative by microscopy. PCR and LAMP show promise for monitoring the early infection rate in snails, and they may be useful for predicting the risk of infection in the endemic places.

Possible use of RNA isolate from inactivated vaccine for external positive control in reverse transcription-based detection of foot-and-mouth disease virus in bull semen

This study has demonstrated the novel use of inactivated and purified vaccine against FMD virus for detection and analysis. RNA isolate was efficiently generated from the vaccine for an external positive control for reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays. The target DNA fragment sequences from the 2B region and 3D RNA polymerase gene of the virus for RT-PCR and RT-LAMP respectively were successfully amplified using the RNA template. Laboratories lacking complex equipment may not be feasible to handle high-risk viruses for conventional methods such as the isolation and culture of live viruses. Here, with the use of these molecular tools, novel use of RNA isolate from inactivated, purified vaccine proved to be an effective external positive control for the assays. Therefore, with these methods, the derived RNA control template aids in a safe method for screening FMD virus for diagnostic laboratories. And by using the same technique, it is then possible to generate a standard for diagnosing any other infectious viral diseases.

FMR1 repeat sizes in the high end of the normal range: no significant association with spontaneous 46,XX primary ovarian insufficiency detected

OBJECTIVE: To determine if FMR1 CGG repeat sizes of 35 to 54 (high end of the normal range) are associated with spontaneous 46,XX primary ovarian insufficiency (sPOI). A prior study found such an association (Bretherick et al 2005), yet CGG repeat sizes in this range are not uncommon in controls. This raises significant issues around genetic counseling in women with sPOI and led us to examine our own data in this regard.DESIGN: Cross-sectional.MATERIALS AND METHODS: We recruited 252 women with sPOI between the ages of 18 and 42 years. Southern blot and polymerase chain reaction (PCR) amplification assays were used to determine the size and methylation status of the CGG repeats within the FMR1 gene. The Southern blot assay utilizes the DNA probe StB12.3 and an Eco R1/Nru 1 double digest (Mayo Medical Laboratories). We compared findings to a published healthy control reference group of 161 Canadian women (Bretherick et al 2005). Analysis: Fisher exact test, Spearman correlation, and Wilcoxon rank sum test.RESULTS: We found 45/504 (8.9%, 95% CI 6.6, 11.8) of alleles in the 35 to 54 CGG repeat range in women with sPOI. This was not statistically different from 21/322 (6.5%) in the reference control group (p=0.24). There was a negative correlation of borderline significance between the bi-allelic mean repeat size and age of diagnosis of sPOI (r=-0.12, p=0.051). No such correlation was found using the highest allele repeat size (r=-0.04, p=0.54). Women with primary amenorrhea (22/252, 8.7%) did not differ significantly from other women with sPOI with regard to bi-allelic mean repeat size or the highest allele repeat size (p=0.75 and p=0.33, respectively).CONCLUSIONS: Despite a larger sample size, we were unable to confirm a prior report of an association between FMR1 CGG repeat sizes of 35 to 54 (high end normal range) and sPOI. Further studies are indicated. OBJECTIVE: To determine if FMR1 CGG repeat sizes of 35 to 54 (high end of the normal range) are associated with spontaneous 46,XX primary ovarian insufficiency (sPOI). A prior study found such an association (Bretherick et al 2005), yet CGG repeat sizes in this range are not uncommon in controls. This raises significant issues around genetic counseling in women with sPOI and led us to examine our own data in this regard. DESIGN: Cross-sectional. MATERIALS AND METHODS: We recruited 252 women with sPOI between the ages of 18 and 42 years. Southern blot and polymerase chain reaction (PCR) amplification assays were used to determine the size and methylation status of the CGG repeats within the FMR1 gene. The Southern blot assay utilizes the DNA probe StB12.3 and an Eco R1/Nru 1 double digest (Mayo Medical Laboratories). We compared findings to a published healthy control reference group of 161 Canadian women (Bretherick et al 2005). Analysis: Fisher exact test, Spearman correlation, and Wilcoxon rank sum test. RESULTS: We found 45/504 (8.9%, 95% CI 6.6, 11.8) of alleles in the 35 to 54 CGG repeat range in women with sPOI. This was not statistically different from 21/322 (6.5%) in the reference control group (p=0.24). There was a negative correlation of borderline significance between the bi-allelic mean repeat size and age of diagnosis of sPOI (r=-0.12, p=0.051). No such correlation was found using the highest allele repeat size (r=-0.04, p=0.54). Women with primary amenorrhea (22/252, 8.7%) did not differ significantly from other women with sPOI with regard to bi-allelic mean repeat size or the highest allele repeat size (p=0.75 and p=0.33, respectively). CONCLUSIONS: Despite a larger sample size, we were unable to confirm a prior report of an association between FMR1 CGG repeat sizes of 35 to 54 (high end normal range) and sPOI. Further studies are indicated.

Rapid virological response in hepatitis C virus genotype 1 and early ribavirin exposure†

We read with great interest the article by Loustaud-Ratti et al., published in a recent issue of HEPATOLOGY.1 The authors concluded that ribavirin exposure at day 0 (D0), an early pharmacokinetic predictor, is significantly related to sustained virological response (SVR) to 48-week combination therapy with peginterferon alfa 2a and ribavirin, and amantadine which was added at week 12 and for 36 weeks. The authors also proposed a minimum area under the curve at hour 0-4 (AUC0-4h) threshold of 1755 μg.hour/L at D0 as a target for ribavirin dose adjustment. In their study, the authors defined rapid virological response (RVR) by either a nondetectable viral load or a viral load drop >2 log10 international units (IU)/mL at week 4, and defined early virological response (EVR) by a nondetectable viral load at week 12. These definitions are not in accordance with the general consensuses that at least a 2-log10 decline from baseline hepatitis C virus (HCV) RNA level is applied to EVR but not RVR.2-4 Although the authors used the quantitative real-time polymerase chain reaction assay HCV Ampliprep TaqMan to determine RVR at week 4, which was more sensitive (cut-off 15 IU/mL) than the Cobas Roche Amplicor polymerase chain reaction assay (cut-off 50 IU/mL), the reason why the authors chose special definitions of RVR and EVR and the impact on the interpretations of RVR and EVR should be elucidated. Meanwhile, the percentages of RVR were markedly different by the definition of the authors [50.0% (12/24)] and as defined by the general consensus [8.3% (2/24)]. With peginterferon alfa and ribavirin therapy, Ferenci et al. previously reported that about 10% of patients infected with HCV genotype 1 achieved RVR.5 Other studies have demonstrated that the percentage of RVR in patients with hepatitis C genotype 1 was 16%-26.7%.4, 6-8 We have recently reported a RVR rate of 43.5% (87/200) among Taiwanese patients infected with HCV genotype 1 in our randomized trial.9 The rates of RVR in patients with hepatitis C genotype 1 differ greatly from different areas. Although we applaud the study conducted by Loustaud-Ratti et al.,1 the predictive value of ribavirin exposure at D0 for SVR and the proposed target for ribavirin dose adjustment of minimum AUC0-4h threshold of 1755 μg.h/L need further confirmatory studies in different countries. Chia-Yen Dai* , Wan-Long Chuang* , Jee-Fu Huang ?, Ming-Yen Hsieh , Ming-Lung Yu* ?, * Faculty of Internal Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, Department of Occupational Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, ? Department of Internal Medicine, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung, Taiwan.

Human papillomavirus-associated increase in p16INK4A expression in penile lichen sclerosus and squamous cell carcinoma

BackgroundHuman papillomaviruses (HPVs) are sexually transmitted human carcinogens that may play a role in the oncogenesis of penile cancer.ObjectivesTo investigate the role of HPV infection and expression of the tumour suppressor protein p16INK4A in the pathogenesis of penile cancer.MethodsBy means of polymerase chain reaction amplification and reverse hybridization line probe assay to detect HPV infection, and immunohistochemical staining for p16INK4A and Ki67, we analysed 26 penile squamous cell carcinomas (SCCs) and 20 independent penile lichen sclerosus (LS) lesions from 46 patients.ResultsHPV DNA was found in 54% of penile SCCs and 33% of penile LS cases in single and multiple infections. High-risk HPV 16 was the predominant HPV type detected. No relationship between Ki67 expression and HPV infection was observed. Strong immunostaining for p16INK4A correlated with HPV 16/18 infection in both penile LS and penile SCC. In our penile SCC series the cancer margins were also associated with penile LS in 13 of 26 lesions, and HPV was detected in seven of the 13 SCC cases associated with LS and in six of the 11 SCC lesions not involving LS.ConclusionsOur study shows a high prevalence of HPV 16 and p16INK4A expression in penile lesions, consistent with an active role for HPV in interfering with the retinoblastoma pathway. High-risk HPV infection could be involved in the tumorigenic process in 50% of penile cancers, and the use of prophylactic HPV vaccines has the potential to prevent these cancers.

Molecular analysis of the CHD7 gene in CHARGE syndrome: identification of 22 novel mutations and evidence for a low contribution of large CHD7 deletions

PurposeAutosomal dominant CHARGE syndrome (OMIM no. 214800) is characterized by choanal atresia or cleft lip or palate, ocular colobomas, cardiovascular malformations, retardation of growth, ear anomalies, and deafness, and is caused by mutations in the CHD7 gene. Here, we describe the outcome of a molecular genetic analysis in 18 Finnish and 56 German patients referred for molecular confirmation of the clinical diagnosis of suspected CHARGE syndrome. MethodsQuantitative real-time polymerase chain reaction or multiplex ligation-dependent probe amplification assays did not reveal deletions in mutation negative cases, suggesting that larger CHD7 deletions are not a major cause of CHARGE syndrome. ResultsIn this group of 74 patients, we found mutations in 30 cases. 22 mutations were novel, including 11 frameshift, 5 nonsense, 3 splice-site, and 3 missense mutations. One de novo frameshift mutation was found in the last exon and is expected to result in a minimally shortened CHD7 polypeptide. Because the mutation is associated with a typical CHARGE syndrome phenotype, it may indicate the presence of an as yet unknown functional domain in the very carboxyterminal end of CHD7. ConclusionsOur mutation detection rate of 40.5% is reflective of screening an unselected sample population referred for CHD7 testing based on suspected clinical diagnosis of CHARGE syndrome and not for having met strict clinical criteria for this disorder.

Can fleas from dogs infected with canine visceral leishmaniasis transfer the infection to other mammals?

In order to investigate the possible role of dog fleas in the transmission of trypanosomatids, ectoparasites were removed from 59 dogs testing positive for canine zoonotic visceral leishmaniasis according to the indirect fluorescent antibody test (IFAT). Of the fleas collected, 4/207 (1.9%) showed the presence of promastigotes in smears stained by Giemsa, whilst 43/144 (29.9%) exhibited positive polymerase chain reaction (PCR) amplification assays for Leishmania DNA. Fleas (409) from 9 Leishmania chagasi-infected dogs, each hosting more than 20 fleas per animal, were macerated and administered by peritoneal injection or orally to 36 hamsters. After 6 months, the 30 surviving hamsters were sacrificed and liver and spleen fragments were removed for PCA assay and to produce imprint smears, whilst blood samples were subjected to IFAT assay. Sixteen hamsters tested positive for Leishmania infection, 14 on the basis of PCR amplification and four by IFAT assay (two animals testing positive in both assays). Of the infected hamsters, 11/16 (68.7%) had been infected peritoneally and 5/16 (31.2%) orally. The imprint smears for all animals were, however, negative. Since both PCR and IFAT could present cross-reactivity for Leishmania and Leptomonas, the possibility of oral transmission of L. chagasi by fleas cannot be proven unambiguously even though the hamsters developed infection.

Immunoassay and polymerase chain reaction techniques for detection of enterotoxigenic <i>Bacillus cereus</i>

To compare the Reverse Passive Latex Agglutination (RPLA) and Enzyme Linked Immunosorbent Assay (ELISA) techniques with a Polymerase Chain Reaction (PCR) for detection of enterotoxigenic Bacillus cereus. A cross-sectional study. The Department of Public Health, Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Nairobi. Forty seven Bacillus cereus strains previously isolated from foods. Detection of hemolysin BL, non-hemolytic enterotoxin, binding protein gene (hblA) of the hemolysin BL, and binding protein gene (nheA) of nonhemolytic enterotoxin. Twenty five (53.2%) of the isolates produced hemolysin BL, while 81% of them produced non-hemolytic enterotoxin. Thirty eight (38.3%) produced both hemolysin BL and non-hemolytic enterotoxin. A polymerase chain reaction amplification assay detected the presence of hblA gene in all hemolysin BL positive isolates and nheA gene in 91.5% of non-hemolytic enterotoxin positive isolates. There was a strong association between PCR test and RPLA test (Pearson's X2 = 12.65; p < 0.001) as well as between PCR test and visual immunoassay test (Pearson's chi-square X2 = 18.46: p < 0.01). Polymerase chain reaction amplification assay technique for detection of enterotoxigenicity of B. cereus compare well with the immunoassay tests. The technique is sensitive detecting even strains with silentgenes, and is rapid with the test complete within 24 hours.

HCV-Associated B Cell Clonalities in the Liver Do Not Carry the t(14;18) Chromosomal Translocation *

Infection with HCV can be associated with B-cell non-Hodgkin lymphoma. Polymerase chain reaction (PCR) amplification assays for Bcl-2/IgH rearrangement were performed on nucleic acids extracted from portal tract inflammatory infiltrates, isolated with laser capture microdissection (LCM), from liver biopsy sections of 16 hepatitis C virus (HCV)-infected patients with and without extrahepatic B cell-related disorders. Results were compared with total DNA extracted from core liver biopsy specimens and from peripheral blood mononuclear cells (PBMCs). We failed to demonstrate specific Bcl-2/IgH amplicons either in liver tissue or in PBMCs in all patients of the current series. Multiple PCR assays for variable diversity joining (VDJ) IgH gene rearrangements were also performed in the liver compartment. Selective amplification compatible with mono or oligoclonal B cell clonotypes was demonstrated in 80% (6/8) and 25% (2/8) of patients with and without clinical evidence of B-cell disorders. V(H)1 and V(H)3 were the most represented V(H) families. In situ expression of Bcl-2 protein was carried out by immunohistochemistry on liver biopsy sections. Bcl-2 protein was detected in 2 (12.5%) patients who did not associate extrahepatic disorders. In conclusion, current data support the concept that production of IgH gene rearrangements is not associated with Bcl-2/IgH chromosomal translocation in hepatic compartment. Liver overexpression of Bcl-2 protein may occur in at least a minor proportion of HCV-infected patients.

Systematic Review: Noninvasive Testing for Chlamydia trachomatis and Neisseria gonorrhoeae

Testing of urine samples is noninvasive and could overcome several barriers to screening for chlamydial and gonococcal infections, but most test samples are obtained directly from the cervix or urethra. To systematically review studies that assessed the sensitivity and specificity of nucleic acid amplification tests for Chlamydia trachomatis and Neisseria gonorrhoeae in urine specimens and to compare test characteristics according to type of assay, site of sample collection, presence of symptoms, disease prevalence, and characteristics of the reference standard. Relevant studies in all languages were identified by searching the MEDLINE database (January 1991 to December 2004) and by hand-searching the references of identified articles and relevant journals. Studies were selected that evaluated 1 of 3 commercially available nucleic acid amplification tests, included data from tests of both a urine sample and a traditional sample (obtained from the cervix or urethra), and used an appropriate reference standard. From 29 eligible studies, 2 investigators independently abstracted data on sample characteristics, reference standard, sensitivity, and specificity. Articles were assessed qualitatively and quantitatively. Summary estimates for men and women were calculated separately for chlamydial and gonococcal infections and were stratified by assay and presence of symptoms. The pooled study specificities of each of the 3 assays exceeded 97% when urine samples were tested, for both chlamydial infection and gonorrhea and in both men and women. The pooled study sensitivities for the polymerase chain reaction, transcription-mediated amplification, and strand displacement amplification assays, respectively, were 83.3%, 92.5%, and 79.9% for chlamydial infections in women; 84.0%, 87.7%, and 93.1% for chlamydial infections in men; and 55.6%, 91.3%, and 84.9% for gonococcal infections in women. The pooled specificity of polymerase chain reaction to gonococcal infections in men was 90.4%. In subgroup analyses, the sensitivity did not vary according to the prevalence of infection or the presence of symptoms but did vary according to the reference standard used. Few published studies present data on the transcription-mediated amplification or strand displacement amplification assays, and few studies report data from asymptomatic patients or low-prevalence groups. Results of nucleic acid amplification tests for C. trachomatis on urine samples are nearly identical to those obtained on samples collected directly from the cervix or urethra. Although all 3 assays can also be used to test for N. gonorrhoeae, the sensitivity of the polymerase chain reaction assay in women is too low to recommend its routine use to test for gonorrhea in urine specimens.

Glutathione S-Transferase P1 and Lung Function in Patients With α1-Antitrypsin Deficiency and COPD

The glutathione S-transferase P1 (GSTP1) gene is involved in detoxification of electrophilic substances of tobacco smoke. A polymorphism at nucleotide 315 of this gene alters its enzymatic activity. We analyzed the association between the variability in the GSTP1 gene and impairment in lung function in smokers with and without alpha(1)-antitrypsin (AAT) deficiency and COPD. The study population consisted of 99 patients with smoking-related COPD and 69 patients with AAT deficiency; 198 healthy volunteers provided the frequency of the different polymorphisms in the general population. GSTP1 genotyping was performed by a real-time polymerase chain reaction amplification assay. The frequency (0.28) of the 105Val polymorphism was identical in COPD patients and the general population. However, the frequency was significantly increased (0.44) in patients with AAT deficiency (odds ratio [OR], 2.09; 95% confidence interval [CI], 1.17 to 3.72 compared to control subjects; and OR, 2.41; 95% CI, 1.27 to 4.59 compared to COPD). FEV(1) percentage of predicted was significantly impaired in AAT-deficient carriers of 105Val. This effect was not observed in COPD patients. These findings suggest that the frequency of the GSTP1 105Val polymorphism is increased in patients with AAT deficiency. Globally, GSTP1 genotypes, age, and tobacco smoking explained 41% of total FEV(1) percentage of predicted variability in patients with AAT deficiency. The modulatory role of GSTP1 in lung disease has only been observed in smokers lacking AAT.

Environmental contamination with an epidemic strain of Pseudomonas aeruginosa in a Liverpool cystic fibrosis centre, and study of its survival on dry surfaces

We conducted an environmental survey in the Liverpool adult cystic fibrosis (CF) centre in order to determine the extent of environmental contamination with an epidemic strain of Pseudomonas aeruginosa that colonizes most CF patients in Liverpool, and to identify possible reservoirs and routes of cross-infection. In addition, we studied the survival of this strain on dry surfaces, compared with that of other CF P. aeruginosa strains, to explore factors that might contribute to its high transmissibility. Samples were collected from staff, patients and the environment (drains, bath tubs, showers, dry surfaces, respiratory equipment and air) in the inpatient ward and outpatient clinic. P. aeruginosa strains were tested using a new polymerase chain reaction amplification assay specific for the Liverpool epidemic strain (LES). LES was isolated from patients' hands, clothes and bed linen. Environmental contamination with LES was only detected in close proximity to colonized patients (external surfaces of their respiratory equipment, and spirometry machine tubing and chair) and was short-lived. No persistent environmental reservoirs were found. LES was detected in the majority of air samples from inside patients' rooms, the ward corridor and the outpatient clinic. Survival of LES on dry surfaces was significantly longer than that for some other strains tested, but not compared with other strains shown not to be transmissible. Improved environmental survival on its own, therefore, cannot explain the high transmissibility of this epidemic strain. Our study suggests that airborne dissemination plays a significant role in patient-to-patient spread of LES, and confirms the need to segregate those patients colonized by epidemic P. aeruginosa strains from all other CF patients.

Detection of &lt;I&gt;Tritrichomonas Foetus&lt;/I&gt; in Cattle Using a PCR Amplification System

A trichomoniasis polymerase chain reaction (PCR) assay was successfully established at the Central Veterinary Laboratory (CVL). Four preputial and 27 vaginal washings of bulls and cows obtained from communal cattle in Zimbabwe's Buhera and Wedza districts were tested using a PCR amplification assay as well as the traditional method of microscopy. Out of the 31 cattle tested, two bulls and three cows were positive on trichomoniasis PCR assay. The trichomoniasis microscopy method detected only one cow which was also positive with the PCR assay. The use of the PCR assay for routine diagnosis will greatly improve the CVL's capacity to accurately diagnose bovine trichomoniasis. The PCR tests on clinical samples have for the first time confirmed tritrichomoniasis infection in communal cattle using a highly sensitive and specific molecular based diagnostic test. Zimbabwe Veterinary Journal (2002) 33; 1-5

Spread of varicella-zoster virus DNA to the environment from varicella patients who were treated with oral acyclovir.

The purpose of the present study was to determine the degree of spread of varicella-zoster virus to the environment (VZV) from varicella patients who received oral acyclovir treatment. Over a period of 8 months, seven healthy children (two girls and five boys, 23-64 months of age) with varicella who visited Fujita Health University School of Medicine were treated with routine doses of oral acyclovir (ACV) for 5 days, commencing within 24 h after onset of the disease. Swab samples from the throats of patients and their family members as well as from air purifier filters in their houses were collected for 7 days as frequently as possible after starting treatment for the disease. The VZV DNA in the samples was identified by a sensitive polymerase chain reaction amplification assay. The VZV DNA was detected in 33-100% of throat swab samples from varicella patients by day 7 of the disease, in 17-32% of throat swab samples from family members by day 4 and in 29% of filters from air purifiers by day 3. The results suggest a broad spread of VZV, probably by the airborne route, from the patients with varicella even after the administration of oral ACV.

Mutations in the TP53 gene in human malignant melanomas derived from sun-exposed skin and unexposed mucosal membranes.

Mutations in the p53 tumour suppressor gene ( ) have been linked to several types of cancer. We therefore investigated whether such mutations occur in malignant melanomas and, if so, whether they are linked to ultraviolet (sun) light exposure. For the first time, mutations in mucosal membranes and adjacent tissues shielded from sunlight were compared with those in cutaneous melanomas from sun-exposed skin. Archival tissues were obtained from 35 patients with a primary melanoma taken from unexposed mucosal areas and from 34 patients with a primary melanoma located in chronically sun-exposed head and neck skin. was characterized by means of polymerase chain reaction amplification and single-strand conformation polymorphism assay followed by nucleotide sequencing. The results showed that 17.6% of the primary cutaneous and 28.6% of the primary mucosal melanomas had point mutations in. Among the cutaneous melanomas, one showed three mutations in exon 7, and one had two mutations in exon 5; the mutation was in the same allele in both cases. One mucosal melanoma had two mutations in exon 7, both in the same allele, and another had two mutations, one in exon 7 and one in intron 6, both in the same allele. C<--T mutations at dipyrimidine sites, considered fingerprints for ultraviolet light-induced mutations, were about equally distributed among patients with melanomas from chronically sun-exposed areas (six out of nine; 67%) and those with melanomas from unexposed mucosal areas and adjacent skin (eight out of 14; 57%). Our data, demonstrating the presence of such mutations even in melanomas from mucosal membranes, clearly suggest that factors other than, or additional to, ultraviolet radiation are operational in the induction of mutations in melanomas.

Circular viral DNA and anomalous junction sequence in PBMC of HIV-infected individuals with no detectable plasma HIV RNA.

Closed circular (cc) forms of extrachromosomal HIV DNA are detected in patients with high viral loads; however, it is unclear whether these forms remain if virus replication is suppressed to undetectable levels by combination antiretroviral therapy. A nested primer polymerase chain reaction amplification assay was used to detect the presence of ccDNA containing two long terminal repeat sequences (2-LTR) in PBMC of patients with low or undetectable plasma HIV RNA. Fifty percent of patients with plasma RNA levels <50 copies/ml of blood had detectable 2-LTR DNA. Sequencing of the products identified normal LTR--LTR junctions in the minority of cases with the majority containing anomalies including deletions and insertions. The persistence of HIV ccDNA in patients with no detectable plasma RNA could be consistent with ongoing de novo infection of dividing cells or with stability of this form of DNA in nondividing cells.