Nested Polymerase Chain Reaction Test Research Articles

Nested PCR Targeting the Flagellin Gene (fliC) in Urine Sample for Diagnosis of Typhoid Fever – An Internal Pilot Study

Abstract Introduction Typhoid fever remains a significant public health challenge, especially in developing countries. Diagnostic complexities arise from the disease’s non-specific clinical manifestations that overlap with other febrile illnesses. Hence, reliable laboratory testing is crucial. Nested polymerase chain reaction (PCR) stands out for its diagnostic performance, especially when utilizing urine due to its minimal PCR inhibitors and ease of collection. Aim The study aimed to test the performance of Salmonella enterica serotype Typhi (Salmonella Typhi) fliC gene specific nested PCR in urine for laboratory diagnosis of typhoid fever. Subjects and Methods This observational, cross-sectional study was conducted as an internal pilot study during the period from August 2021 to October 2022. It included 25 clinically diagnosed enteric fever patients, recruited from Al-Abasseya Fever Hospital, Cairo, Egypt, and 25 healthy individuals as controls, matched for age and sex. Nested PCR was performed to detect the fliC gene of Salmonella Typhi in urine samples from both patients and controls. Urine culture was conducted for subjects in both groups, while blood and stool cultures were exclusively performed for the patients' group. Results The sensitivity of the nested PCR test targeting the fliC gene in urine was 44%, with a specificity of 88%. The positive predictive value was 78.57%, the negative predictive value was 61.11%, the positive likelihood ratio was 3.7, and the negative likelihood ratio was 0.63. Blood culture isolation exhibited a very low positivity rate of 4%. Conclusion Nested PCR targeting the fliC gene in urine samples holds promise as a diagnostic tool for typhoid fever. Despite its moderate sensitivity, the relatively higher specificity suggests its potential utility in confirming typhoid fever cases. The test offers a valuable adjunctive tool alongside conventional diagnostic method. However, further research and validation may be necessary before widespread clinical application. The performance of the test needs to be evaluated in clinically diagnosed and laboratory confirmed cases of typhoid fever.

Improved Antibody Detection for Canine Leptospirosis: ELISAs Modified Using Local Leptospiral Serovar Isolates from Asymptomatic Dogs.

Leptospirosis is a zoonotic disease of significant concern for human and animal health, with domestic animals, including dogs, acting as reservoirs for human infection. Serology is widely used for leptospirosis diagnosis, even though the standard microscopic agglutination test (MAT) using a panel of serovars lacks specificity and can lead to detection limitations in certain regions. In this study, we aimed to develop an antibody detection tool for dogs using an indirect enzyme-linked immunosorbent assay (ELISA) with a set of local serovar isolates, including Paidjan, Dadas, and Mini, to enhance the accuracy of leptospirosis surveillance in our region. The specificity and sensitivity of various antigen preparations, namely leptospiral whole-cell protein (WCP), total membrane protein (TMP), and outer membrane protein (OMP), were assessed using sera from infected and non-infected dogs, as well as negative puppy sera. Leptospirosis diagnosis was supported using a genus-specific nested polymerase chain reaction test on all collected sera. Protein preparations were validated using SDS-PAGE and Western blotting analysis. In the results, the standard MAT failed to detect antibodies in any of the dogs confirmed as being infected using PCR and isolation, highlighting its limitations. In contrast, the OMP-based ELISAs using local isolates of Leptospira serovars gave positive results with sera from all infected dogs, and negative results with sera from all dogs from non-endemic areas. IgG titres of infected and unvaccinated dogs from endemically affected areas were significantly higher than those in non-endemic regions. Using the OMP-based IgG/ELISAs with the local serovar Dadas resulted in higher specificity and lower sensitivity than when using the WCP- and TMP-based IgG/ELISAs. Agreement analysis revealed fair and moderate concordance between OMP-based IgG/ELISAs and PCR results, whereas slight and fair agreement was observed between OMP-based ELISAs and the MAT. Overall, the modified OMP-based IgG/ELISAs, utilising relevant local serovar isolates from dogs, demonstrated improved accuracy in detecting leptospirosis in the study area, overcoming the limitations of the MAT. This study highlights the importance of identifying and incorporating these local circulating serovar isolates into serological techniques for leptospirosis diagnosis and surveillance.

Significance of nested PCR testing for the detection of low-density malaria infection amongst febrile patients from the Malaria Elimination Demonstration Project in Mandla, Madhya Pradesh, India

BackgroundLow-density malaria infections (LDMI) are defined as infections that are missed by the rapid diagnostic test (RDT) and/or microscopy which can lead to continued transmission and poses a challenge in malaria elimination efforts. This study was conducted to investigate the prevalence of LDMI in febrile cases using species-specific nested Polymerase Chain Reaction (PCR) tests in the Malaria Elimination Demonstration Project, where routine diagnosis was conducted using RDT.MethodsEvery 10th fever case from a cross-sectional community based fever surveillance was tested with RDT, microscopy and nested PCR. Parasite DNA was isolated from the filter paper using Chelex based method. Molecular diagnosis by nested PCR was performed targeting 18SrRNA gene for Plasmodium species.ResultsThe prevalence of malaria was 2.50% (436/17405) diagnosed by PCR, 1.13% (196/17405) by RDT, and 0.68% (118/ 17,405) by microscopy. Amongst 17,405 febrile samples, the prevalence of LDMI was 1.51% (263/17405) (95% CI 1.33–1.70), which were missed by conventional methods. Logistic regression analysis revealed that illness during summer season [OR = 1.90 (p < 0.05)] and cases screened within three days of febrile illness [OR = 5.27 (p < 0.001)] were the statistically significant predictors of LDMI.ConclusionThe prevalence of malaria among febrile cases using PCR was 2.50% (436/17405) as compared to 1.13% (196/17405) by RDT. Higher number of the LDMI cases were found in subjects with ≤ 3 days mean duration of reported fever, which was statistically significant (p < 0.001). This observation suggests that an early detection of malaria with a more sensitive diagnostic method or repeat testing of the all negative cases may be useful for curtailing malaria transmission. Therefore, malaria elimination programme would benefit from using more sensitive and specific diagnostic methods, such as PCR.

Usefulness of Nested PCR Assay for the Molecular Diagnosis of Human Rickettsial Infection: A Study in Bangladesh

Background: Rickettsial infections are re-emerging arthropods born worldwide zoonotic disease caused by Rickettsia, which is responsible for spotted fever and typhus fever. The diagnosis of a rickettsial illness is important for appropriate antibiotic treatment.&#x0D; Aims: The study aimed to determine the diagnostic accuracy and clinical usefulness of using nested polymerase chain reaction (PCR) by comparing nested PCR, ELISA, and Weil-Felix (WF) tests. &#x0D; Methodology: This was a prospective type of cross-sectional study. A total of 135 clinically suspected rickettsial infection cases were enrolled. Peripheral blood was taken to detect gltA, 17 kDa lipoprotein antigen gene (17 kDa), ompA, and ompB gene of Rickettsia by nested PCR. ELISA and Weil-Felix tests were done to compare with nested PCR.&#x0D; Results: Out of 135 cases, we detected Rickettsia in 70(51.85%) cases by nested PCR assay (p&lt;0.01), 33((24.4%) by Weil- Felix test, 34 (25.18%) by ELISA. Only 26.66% of cases were PCR positive, which were negative by both ELISA and Weil-Felix test. Fifteen (11.11%) cases were positive by all three tests. Among 70 PCR positive rickettsia cases most frequently detected gene was ompB 42(60%), followed by 17kDa 34(48.58%); gltA 21(30%), and ompA 3(4.28%). Multiple gene combinations (ompB, 17kDa and gltA) detected in 98.57 % cases.&#x0D; Conclusion: Nested PCR assays showed the highest rate of detection of rickettsia cases than ELISA and Weil-Felix test. Multiple gene combinations (ompB, 17kDa, and gltA) showed the highest positivity. Therefore, diagnosis of rickettsial infection can be confirmed by PCR assay, and clinicians can plan appropriate treatment for these patients.

Molecular and serological characterization of occult hepatitis B virus infection among patients with hemophilia.

The occult hepatitis B virus infection (OBI) is a health concern among high-risk groups and immunosuppressed individuals. There is still a paucity of data regarding the occult hepatitis B virus infection among hemophilic patients. With this in mind, we aimed to evaluate the molecular prevalence of OBI among clients with hemophilia. Totally, 87 hemophilic patients were selected to be studied. To detect OBI, nested polymerase chain reaction test was used to amplify HBV-S, X, and Core regions. Viral load was determined using an in-house real-time PCR assay. Finally, sequence of S gene was used for genotyping and analysis of mutations. The mean age of patients was 28.4 ± 5.3 years old, with 90.7% of whom were men. HBV-DNA was detected in eight subjects (9.3%). The rate of OBI was much higher in anti-HBs seronegative subjects than that in other patients (P = 0.019). All OBI cases had HBV genotype D, subgenotype D1. In addition, five out of eight cases (62.5%) showed detectable viral loads (a mean viral load of 4.5 × 10 2 copies/mL). sR73H, sI110L, sP120A, sP127T, sQ129H, sG130R, and sC137S were shown to be the most determinant escape mutation and OBI-relevant mutants. The rate of OBI among the studied population of hemophilia seems to be remarkable. Therefore, screening for OBI must be a routine practice in patients with hemophilia and also patients undergoing immunosuppressive treatments. Amino acid substitutions were observed in the major hydrophilic region. However further investigations are needed for analysis of exact function.

Prevalence Rate of Isolated Cryptosporidium Spp. of Broiler Chicken in Al-Qadisiyah Province

This study has been carried out to determined the prevalence rate of cryptosporidiosis in broiler flocks. 150 fecal samples from 30 broiler flocks (5 samples for each flock) in different areas of AL-Qadisiyah province were used. They were collected starting of January till November 2016 with two age groups of chickens, (10-15 and 30-35) days old. Samples were examined with Modified Ziel -Neelsen stain to detect the positive samples by microscopic examination. After that, a nested-PCR technique was performed on 60 samples. Results of microscopic examination showed that total infection rate of Cryptosporidium was 29.33% . The highest rates of infection were recorded in 10-15 days group and during spring season, infection rates reached were 35.71% and 34.28% respectively. The lowest rates were observed in 30-35 days group and during summer season were 23.75% and 20% respectively. No significant differences within percentage in both two age group or among percentage in seasons at level (P < 0.05) were recorded. According to Nested Polymerase Chain Reaction test, the results showed that overall percentage of infection is 20%. The highest rate of observed infection is 23.33% in 10-15 days aged group, while the lowest rate of infection is (16.67 %) in the other age. No significant differences was observed between percentage in ages. On the other hand, result of comparing nested-PCR test with microscopic examination shows that there are no significant differences between the percentage of both tests. In conclusion, cryptosporidiosis is widespread in broiler flocks in AL-Qadisiyah province, but there is no significant impact found concerning the relationship between infection rates and age of chickens or seasons of the year..

Prevalence Rate of Isolated Cryptosporidium Spp. of Broiler Chickenin Al-Qadisiyah Province

This study has been carried out to determined the prevalence rate of cryptosporidiosis in broiler flocks. 150 fecal samples from 30 broiler flocks (5 samples for each flock) in different areas of AL-Qadisiyah province were used. They were collected starting of January till November 2016 with two age groups of chickens, (10-15 and 30-35) days old. Samples were examined with Modified Ziel -Neelsen stain to detect the positive samples by microscopic examination. After that, a nested-PCR technique was performed on 60 samples. Results of microscopic examination showed that total infection rate of Cryptosporidium was29.33% . The highest rates of infection were recorded in 10-15 days group and during spring season, infection rates reached were 35.71% and 34.28% respectively. The lowest rates were observed in 30-35 days group and during summer season were 23.75% and 20% respectively. No significant differences within percentage in both two age group or among percentage in seasonsat level (P &lt; 0.05) were recorded. According to Nested Polymerase Chain Reaction test, the results showed that overall percentage of infection is20%. The highest rate of observed infection is 23.33% in 10-15 days aged group, while the lowest rate of infection is (16.67 %) in the other age. No significant differences was observed between percentage in ages. On the other hand, result of comparing nested-PCR test with microscopic examination shows that there are no significant differences between the percentage of both tests.&#x0D; In conclusion, cryptosporidiosis is widespread in broiler flocks in AL-Qadisiyah province, but there is no significant impact found concerning the relationship between infection rates and age of chickens or seasons of the year

Clinical and laboratory features of PCR-confirmed periocular tuberculosis in China

Experts lack knowledge of periocular tuberculosis (TB) in China. Nested polymerase chain reaction (PCR) shows advantages in diagnosis of extrapulmonary TB. Our study aims to explore the clinical and laboratory features of PCR-confirmed periocular TB. We retrospectively reviewed medical records of presumptive periocular TB and performed nested PCR test to confirm diagnosis. Nine cases were recruited. Clinical symptoms were chronic and insidious. Eight cases achieved favorable visual acuity, while one underwent enucleation due to fungal-TB panophthalmitis. Sensitivity of caseous necrosis, acid-fast bacilli (AFB) staining and interferon γ release assay (T-SPOT) test are 33.3%, 44.4% and 85.7% respectively. Low lymphocyte percentage (P=0.019) and high monocyte-lymphocyte ratio (P=0.042) positively correlate with AFB staining. Male gender (P=0.048) and Langhans giant cell (P=0.048) positively correlate with caseous necrosis. To conclude, traditional TB ancillary tests are not as sensitive as nested PCR technique. Several factors facilitate diagnosis including male gender, decreased lymphocytes, and typical Langhans giant cells.

Serological diagnosis and molecular characterization of Leptospira spp. in the blood and urine of bovine females from refrigerated slaughterhouses

Leptospirosis is an important socioeconomic disease in humans, as well as in domestic and wild animals, being caused by Leptospira spp. Bovine animals are considered reservoirs of this disease, because they intermittently disseminate the bacteria into the environment through their urine. In this way, the cattle an important source of Leptospira infection. The objective of this study was to detect Leptospira spp. antibodies and DNA in bovine females from two refrigerated slaughterhouses in the microregion of Umuarama, Paraná, Brazil. In particular, blood and urine samples from 52 crossbred bovine females older than 36 months from the two slaughterhouses were used. The microscopic agglutination test (MAT) was used to detect leptospiral antibodies, and the polymerase chain reaction (PCR) and subsequent sequencing were used to detect Leptospira DNA. The MAT yielded 22 (42.3%) serum samples considered reagent, while the nested PCR test resulted in one amplified sample (1.9%) of 289 bp. This single sample was then amplified again using primers for the SecY gene (549 bp). Sequencing of this gene characterized the bacteria as L. borgpetersenii that were similar to the serovar Hardjo of the genotype Hardjobovis. This is the first molecular confirmation of Hardjobovis-like L. borgpetersenii in the urine of crossbred bovine females older than 36 months from slaughterhouses in the microregion of Umuarama. This study’s results show that it is important to combine serological and molecular diagnosis in the detection of Leptospira spp. Therefore, both methods were used to improve our understanding of the epidemiology of this disease in bovine animals from the microregion of Umuarama. In addition, the analysis informed the subsequent adoption of preventive measures and educational One Health actions to prevent economic losses related to the herd, as well as social losses related to workers and the environment.

Caprine lentivirus in sheep milk and semen

ABSTRACT With the objective of detecting the presence of caprine lentivirus (CLV) in ewe milk and in ram semen, ten matrixes and four reproducers experimentally infected with CLV were used. Samples of ewe milk were collected during the four months of lactation, five collections per animal, totaling 50 samples. Regarding the rams, eight semen collections were made per animal, during one year of experimentation, totaling 32 samples. The milk and semen samples were submitted to DNA extraction and the nested polymerase chain reaction test (nPCR) to detect CLV proviral DNA. Eight (16%) of the milk samples were positive in nPCR originating from two ewes. Only one (3.12%) semen sample was positive. The amplification products were sequenced, and were confirmed to be a CLV genomic sequence. Thus, the presence of CLV proviral DNA in sheep milk and semen was demonstrated, confirming the feasibility of infection between species, and alerting to the risk of spreading infections.

Do blood parasites infect Magellanic penguins (Spheniscus magellanicus) in the wild? Prospective investigation and climatogeographic considerations.

Magellanic penguins (Spheniscus magellanicus) are native to Argentina, Chile and the Falkland Islands. Magellanic penguins are highly susceptible to blood parasites such as the mosquito-borne Plasmodium spp., which have been documented causing high morbidity and mortality in zoos and rehabilitation centres. However, to date no blood parasites have been detected in wild Magellanic penguins, and it is not clear whether this is reflective of their true absence or is instead related to an insufficiency in sampling effort or a failure of the diagnostic methods. We examined blood smears of 284 Magellanic penguins from the Argentinean coast and tested their blood samples with nested polymerase chain reaction tests targeting Haemoproteus, Plasmodium, Leucocytozoon and Babesia. No blood parasites were detected. Analysing the sampling effort of previous studies and the climatogeography of the region, we found there is strong basis to conclude that haemosporidians do not infect wild Magellanic penguins on the Argentinean coast. However, at present it is not possible to determine whether such parasites occur on the Chilean coast and at the Falkland Islands. Furthermore, it is troubling that the northward distribution expansion of Magellanic penguins and the poleward distribution shift of vectors may lead to novel opportunities for the transmission of blood parasites.

Análisis molecular de un fragmento del gen env del virus de leucosis bovina, por PCR anidada en vacas lecheras de Pasto, Nariño

Introducción: El diagnóstico de rutina para la leucosis bovina se realiza con pruebas serológicas indirectas, aunque se recomienda usar la reacción en cadena de la polimerasa (PCR), que permite un diagnóstico confiable en fases iniciales y en animales jóvenes. Además, mediante la amplificación del ADN se pueden secuenciar fragmentos del virus, que permite identificar los genotipos presentes y construir árboles filogenéticos. Objetivo: Analizar un fragmento del gen env del virus de leucosis bovina (VLB) aislado de animales positivos a ELISA indirecta en fincas lecheras del municipio de Pasto, Nariño. Materiales y métodos: Una vez establecida la presencia del VLB en 48 animales mayores de dos años en siete fincas lecheras del municipio de Pasto mediante la prueba de ELISA indirecta, se realizó una prueba de PCR anidada para confirmar el diagnóstico y secuenciar un fragmento del gen env en los animales positivos y sus hijas. Las secuencias obtenidas se compararon con los siete genotipos descritos en el mundo mediante el programa MEGA 6. Resultados: Las secuencias de los fragmentos comparados no difieren de las descritas; pero permiten su agrupación en diferentes clústeres de acuerdo con su similitud. Los genotipos encontrados en estas fincas corresponden al genotipo 1 y 2 descritos en la literatura. Conclusiones: En las fincas con reposición propia y adecuadas medidas de bioseguridad solo se encontró un genotipo y en las fincas con menor control en las prácticas de manejo y de reposición se encontraron los dos genotipos descritos para la región.

Therapeutic Assessment of Chloroquine-Primaquine Combined Regimen in Adult Cohort of Plasmodium vivax Malaria from Primary Care Centres in Southwestern India.

BackgroundSeveral reports of chloroquine treatment failure and resistance in Plasmodium vivax malaria from Southeast Asian countries have been published. Present study was undertaken to assess the efficacy of chloroquine-primaquine (CQ-PQ) combined regimen for the treatment of P. vivax malaria patients who were catered by the selected primary health centres (PHCs) of Udupi taluk, Udupi district, Karnataka, India.MethodFive PHCs were selected within Udupi taluk based on probability proportional to size. In-vivo therapeutic efficacy assessment of CQ (1500 mg over three days) plus PQ (210 mg over 14 days) regimen was carried out in accordance with the World Health Organization’s protocol of 28 days follow-up among microscopically diagnosed monoinfection P. vivax cohort.ResultsIn total, 161 participants were recruited in the study of which, 155 (96.3%) participants completed till day 28 follow-up, fully complied with the treatment regimen and showed adequate clinical and parasitological response. Loss to follow up was noted with 5 (3.1%) participants and non-compliance with treatment regimen occurred with one participant (0.6%). Glucose-6-phosphate dehydrogenase deficiency (G6PDd, <30% of normal mean activity) was noted among 5 (3.1%) participants and one of them did develop PQ induced dark-brown urination which subsided after PQ discontinuation. G6PDd patients were treated with PQ 45 mg/week for eight weeks while PQ was discontinued in one case with G6PD 1.4 U/g Hb due to complaint of reddish-brown coloured urine by 48 hours of PQ initiation. Nested polymerase chain reaction test revealed 45 (28%) cases as mixed (vivax and falciparum) malaria.ConclusionsThe CQ-PQ combined regimen remains outstandingly effective to treat uncomplicated P. vivax malaria in Udupi taluk and thus it should continue as first line regimen. For all P. vivax cases, G6PD screening before PQ administration must be mandatory and made available in all PHCs.

Therapeutic assessment of chloroquine-primaquine combined regimen in adult cohort of Plasmodium vivax malaria from a tertiary care hospital in southwestern India.

BackgroundOf late there have been accounts of therapeutic failure and chloroquine resistance in Plasmodium vivax malaria especially from Southeast Asian regions. The present study was conducted to assess the therapeutic efficacy of chloroquine–primaquine (CQ–PQ) combined regimen in a cohort of uncomplicated P. vivax mono-infection.MethodsA tertiary care hospital-based prospective study was conducted among adult cohort with mono-infection P. vivax malaria as per the World Health Organization’s protocol of in vivo assessment of anti-malarial therapeutic efficacy. Participants were treated with CQ 25 mg/kg body weight divided over 3 days and PQ 0.25 mg/kg body weight daily for 2 weeks.ResultsOf a total of 125 participants recruited, 122 (97.6%) completed day 28 follow up, three (2.4%) participants were lost to follow-up. Eight patients (6.4%) were ascertained to have mixed P. vivax and Plasmodium falciparum infection by nested polymerase chain reaction test. The majority of subjects (56.8%, 71/125) became aparasitaemic on day 2 followed by 35.2% (44/125) on day 3, and 8% (10/125) on day 7, and remained so thereafter. Overall only one therapeutic failure (0.8%, 1/125) occurred on day 3 due to persistence of fever and parasitaemia.ConclusionsCQ–PQ combined regimen remains outstandingly effective for uncomplicated P. vivax malaria and should be retained as treatment of choice in the study region. One case of treatment failure indicates possible resistance which warrants constant vigilance and periodic surveillance.

Evaluation of Immunohistochemical Expression of p16 and Presence of Human Papillomavirus in Oral and Oropharyngeal Carcinoma

Carcinogenesis concerns several changes that eventually result in the inactivation of tumor suppressor genes and activation of protooncogenes, leading to loss of cell cycle control. Inactivation of p16 seems to be an early event in this process and occurs in approximately 80% of squamous cell carcinoma cases. The aims of this study were to evaluate the immunohistochemical expression of p16 protein in oral and oropharyngeal squamous cell carcinoma cases, with both the tumoral area itself and its surgical margin being analyzed (dysplastic areas and histologically normal epithelium adjacent to carcinoma), and to verify the presence of human papillomavirus (HPV) and its relation to p16 expression. Paraffin-embedded biopsy tissues from 26 patients, 13 with oral squamous cell carcinoma and 13 with oropharyngeal squamous cell carcinoma, comprised the analyzed samples. To detect HPV, a nested polymerase chain reaction test using PGMY 09/11 and GP5*/GP6* primers and visualization of the product on a 2% agarose gel was performed. Demographic data were obtained from medical records. The results showed low expression of p16 in the tumor area (38.46%), compared with surgical margins in the histologically normal epithelium (84.6%) and dysplastic areas (57.7%). These findings indicate the inactivation of p16 in the process of malignant transformation. The association described in the literature between expression of p16 and presence of HPV could not be verified in this study, because none of the cases was HPV positive.

Detection and identification of mycobacteria in fixed stained smears and formalin-fixed paraffin-embedded tissues using PCR

To determine the feasibility of using polymerase chain reaction to amplify DNA from methanol-fixed, Romanowsky-stained and Ziehl-Neelsen-stained smears to confirm the presence of mycobacteria. Tissue was obtained from 10 archival slides and 27 slides from a prospective series of consecutive cases. Phosphate buffered saline (500 μL) was pipetted onto a stained smear (on a glass slide) using a disposable filtered pipette tip. The material adherent to the slide was scraped from its surface and drawn up into the saline. Routine DNA extraction and purification was carried out before nested polymerase chain reaction testing targeting the 16S-23S internal transcribed spacer region or a TaqMan real-time polymerase chain reaction. The real-time polymerase chain reaction was also used on thick sections cut from formalin-fixed paraffin-embedded tissue blocks from 24 canine leproid granulomas. Mycobacterial DNA was detected in 34 of 37 slides. Polymerase chain reaction products could not be amplified from three archived smears stained using the Ziehl-Neelsen acid-fast method, probably because its harsher fixation damaged the DNA. With the nested polymerase chain reaction, species identification using internal transcribed spacer sequence analysis was achieved in all instances, diagnosing a wide range of mycobacteria. The real-time polymerase chain reaction detected Mycobacterium sp. CLG DNA within all 24 formalin-fixed paraffin-embedded specimens tested. This technique should provide a non-invasive and cost-effective means of diagnosing mycobacterial infections.

Absence ofHelicobacter pyloriin healthy laryngeal mucosa

To evaluate the presence of Helicobacter pylori in healthy laryngeal mucosa. Prospective analysis of Helicobacter pylori colonisation in healthy laryngeal mucosa, using the 13C urea breath test and polymerase chain reaction analysis. Twenty randomly chosen men (28-78 years) without laryngeal pathology or gastrointestinal disease were investigated. All subjects were scheduled for elective operative procedures, under general, endotracheal anaesthesia. Cytobrush samples were taken for Helicobacter pylori DNA detection. Nested polymerase chain reaction testing was performed on DNA solutions using two primer pairs from the urease A gene of the Helicobacter pylori genome. The 13C urea breath test was performed on two exhalation samples. Eight (40 per cent) of the subjects were positive for urease on urea breath testing; none were positive for Helicobacter pylori DNA on polymerase chain reaction testing. Based on these results, we do not consider Helicobacter pylori to be a normal constituent of healthy laryngeal microflora.

Clinical usefulness of the nested polymerase chain reaction in the diagnosis of extrapulmonary tuberculosis

To evaluate the effectiveness of nested polymerase chain reaction (PCR) for diagnosis of extrapulmonary tuberculosis (ETB), as well as the impact of PCR results on clinical management. We conducted a study of nested PCR tests in 45 patients and a review of patient hospital files, calculating sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). PCR was positive in 51% of cases; PCR sensitivity for diagnosing TB was 86%, specificity was 79%, PPV was 76%, and NPV was 88%. When solely analyzing urine samples, sensitivity and NPV increased to 100%. PCR exerted an influence on management in 27% of patients. PCR for rapid diagnosis of extrapulmonary TB has an adequate effect, which improves when performed on urine. The results of PCR exerted an acceptable impact on the clinical management of these patients.

Association among Results of Serum ELISA, Faecal Culture and Nested PCR on Milk, Blood and Faeces for the Detection of Paratuberculosis in Dairy Cows

Paratuberculosis is a chronic, infectious disease of ruminants that entails a serious concern for the cattle industry. One of the main issues relates to the efficiency of diagnosis of subclinically infected animals. The objective of this field study was to analyse the association among results of a serum enzyme-linked immunosorbent assays (ELISA), faecal culture and nested PCR tests on milk, blood and faeces for Mycobacterium avium subsp. paratuberculosis detection in dairy cows. Faeces, blood and milk samples were collected from 328 lactating dairy cows in four known infected herds. Results were analysed to determine associations and levels of agreement between pairs of tests. A total of 61 animals (18.6%) tested positive when all the tests were interpreted in parallel. The agreement between results in different pairs of tests was poor, slight and fair in two, five and three of the 10 possible combinations respectively. Faecal culture and faecal polymerase chain reaction (PCR) resulted in the highest kappa coefficient (0.39; fair agreement), with the lowest agreement being for ELISA and blood PCR (-0.036; poor agreement). Fisher's exact test resulted in statistically significant associations (P < or = 0.05) between the following test pairs: ELISA : faecal culture; ELISA : faecal PCR; milk PCR : faecal PCR, blood PCR : faecal PCR and faecal culture : faecal PCR. Enzyme-linked immunosorbent assays showed the highest complementary sensitivity values for all the possible two-test combinations, followed by faecal PCR. The combined use of ELISA and faecal PCR has the potential to increase the overall sensitivity for the diagnosis of paratuberculosis infection.

Detection of Cyprinid herpesvirus-3 (CyHV-3) DNA in infected fish tissues by nested polymerase chain reaction

Diagnostic tests with high analytical sensitivity are required to detect Cyprinid herpesvirus-3 (CyHV-3) in carriers that have been implicated in the dissemination of this important disease of koi and common carp Cyprinus carpio. A nested polymerase chain reaction (PCR) test was developed to detect CyHV-3 DNA in tissues of infected fish. The 2-round PCR amplified 529 and 379 bp segments of the CyHV-3 gene coding for the major capsid protein using specific external and internal primers, respectively. The PCR test did not amplify genomic DNA from either Cyprinid herpesvirus-1 (CyHV-1) or Cyprinid herpesvirus-2 (CyHV-2). The nested PCR test detected CyHV-3 DNA at a 10-fold lower concentration than a routinely used, 1-round test. Hence, the new method should provide more sensitive diagnosis of CyHV-3 infection among koi and carp populations.