Polymerase Chain Reaction In Samples Research Articles

In vivo structural and cellular remodeling of engineered bone–ligament–bone constructs used for anterior cruciate ligament reconstruction in sheep

ABSTRACTAnterior cruciate ligament (ACL) ruptures rank among the most prevalent and costly sports-related injuries. Current tendon grafts used for ACL reconstruction are limited by suboptimal biomechanical properties. We have addressed these issues by engineering multiphasic bone–ligament–bone (BLB) constructs that develop structural and mechanical properties similar to native ACL. The purpose of this study was to examine the acute remodeling process that occurs as the BLB grafts advance toward the adult ligament phenotype in vivo. Thus, we implanted BLB constructs fabricated from male cells into female host sheep and allowed 3, 7, 14, or 28 days (n = 4 at each time point) for recovery. To address whether or not graft-derived cells were even necessary, a subset of BLB constructs (n = 3) were acellularized, implanted, and allowed 28 days for recovery. At each recovery time point, the following histological analyses were performed: picrosirius red staining to assess collagen alignment and immunohistochemistry to assess both graft development and host immune response. Polymerase chain reaction (PCR) analysis, performed on every explanted BLB, was used to detect the presence of graft-derived male cells remaining in the constructs and/or migration into surrounding host tissue. The analysis of the PCR and histology samples revealed a rapid migration of host-derived macrophages and neutrophils into the graft at 3 days, followed by increased collagen density and alignment, vascularization, innervation, and near complete repopulation of the graft with host cells within 28 days. This study provides a greater understanding of the processes of ligament regeneration in our BLB constructs as they remodel toward the adult ligament phenotype.

A bimetallic nanocomposite modified genosensor for recognition and determination of thalassemia gene

The main roles of DNA in the cells are to maintain and properly express genetic information. It is important to have analytical methods capable of fast and sensitive detection of DNA damage. DNA hybridization sensors are well suited for diagnostics and other purposes, including determination of bacteria and viruses. Beta thalassemias (βth) are due to mutations in the β-globin gene. In this study, an electrochemical biosensor which detects the sequences related to the β-globin gene issued from real samples amplified by polymerase chain reaction (PCR) is described for the first time. The biosensor relies on the immobilization of 20-mer single stranded oligonucleotide (probe) related to βth sequence on the carbon paste electrode (CPE) modified by 15% silver (Ag) and platinum (Pt) nanoparticles to prepare the bimetallic nanocomposite electrode and hybridization of this oligonucleotide with its complementary sequence (target). The extent of hybridization between the probe and target sequences was shown by using linear sweep voltammetry (LSV) with methylene blue (MB) as hybridization indicator. The selectivity of sensor was investigated using PCR samples containing non-complementary oligonucleotides. The detection limit of biosensor was calculated about 470.0pg/μL.

Detection of Hepatitis E Virus Genotype 1 Among Blood Donors From Southwest of Iran.

BackgroundInfection with hepatitis E virus (HEV) is endemic in developing countries and reveals significant regional differences. Several studies have reported virus transmission via blood transfusion. To date, however, no cases of HEV RNA detection in blood donors have been reported from Iran.ObjectivesThe aim of this study was to determine the presence of HEV RNA in plasma samples of blood donors referred to a blood transfusion center in Shiraz in the southwest of Iran. The HEV genotypes were also investigated using nucleotide sequencing.Patients and MethodsBlood samples were collected from 700 blood donors who were referred to Fars blood transfusion organization from January to March 2014. Plasma samples were screened for the presence of HEV IgG and IgM antibodies by standard enzyme immunoassay. Samples seroreactive to anti-HEV were further tested for the presence of HEV RNA using nested polymerase chain reaction (PCR) with universal primers for detection of all four HEV genotypes. Positive PCR samples were then subjected to DNA sequencing for further analysis.ResultsFifty (50, 7.1%) out of 700 plasma samples tested positive for anti-HEV antibodies. HEV RNA was detected in 7/50 (12%) of the antibody-positive samples, the majority of which were IgM positive. Sequence analysis of seven isolates of the HEV RNA ORF 2 gene region revealed > 80% similarity with genotype 1.ConclusionsThe analysis indicates that the HEV isolated from blood donors in the southwest of Iran belongs to genotype 1. However, more samples from other geographic regions of Iran are needed to confirm these findings. Because transmission of HEV by administration of blood or blood components is likely to occur, it may be sensible to screen donor blood for HEV to eliminate transfusion-transmitted HEV infection when the recipient is immunocompromised.

Differential expression of circulating microRNAs in blood and haematoma samples from patients with intracerebral haemorrhage.

ObjectiveTo measure the differential expression of microRNAs (miRNAs) in peripheral blood samples from patients with intracerebral haemorrhage (ICH) and to measure the levels of hsa-miR-21-5p in peripheral blood and haematoma samples from patients with ICH.MethodsThis case–control study enrolled individuals with ICH in the putamen treated by craniotomy and age- and sex-matched healthy control subjects. Serum miRNA expression profiles were determined in the patient and control groups using miRNA polymerase chain reaction (PCR) arrays. The ICH-related miRNA hsa-miR-21-5p was selected and its differential expression was assessed in peripheral blood and haematoma specimens from patients with ICH compared with peripheral blood samples controls using real-time PCR.ResultsSeven patients and five control subjects were included in the miRNA expression profile analysis; and 31 patients and 22 control subjects provided samples for the real-time PCR of hsa-miR-21-5p expression. A total of 59 miRNAs were significantly downregulated in patients with ICH. Relative hsa-miR-21-5p levels of 0.43 and 0.31 for peripheral blood and haematoma samples, respectively, were obtained in the patient group compared with the control subjects.ConclusionHsa-miR-21-5p levels were significantly reduced in both peripheral blood and haematoma samples in patients with ICH.

Early detection of novel Leishmania species DNA in the saliva of two HIV-infected patients.

BackgroundLeishmaniasis caused by two new species of Leishmania; L. siamensis and L. martiniquensis have been recently described in Thailand. The disease has mainly been documented in AIDS patients from southern Thailand. In this study, polymerase chain reaction (PCR) was used to determine HIV-Leishmania co-infection in southern Thailand.MethodsOne ml of saliva and 3 ml of EDTA blood were collected from HIV-infected patients for PCR detection of Leishmania DNA, cloning and sequencing. The positive PCR samples were then cultured on Schneider’s insect medium.ResultsThree out of 316 saliva samples collected from HIV-infected patients were found to be positive for Leishmania DNA (0.95 %). Among the positive samples, one patient was observed with disseminated cutaneous lesions and also tested positive via saliva, whole blood and buffy coat in PCR. The second case presenting with nodular lesions also gave a positive saliva test via PCR two months prior to buffy coat. This diagnosis was confirmed by microscopic examination and a culture of biopsy samples from a nodule. The last case was an asymptomatic Leishmania infection which tested PCR positive only in saliva with a consecutive sample collection conducted for three months.ConclusionsThe prevalence of Leishmania infection in HIV infected patients within this study is 0.95 %. Leishmania DNA was detected in saliva by PCR prior to blood and buffy coat of two HIV infected patients. Early detection of Leishmania DNA in saliva would be beneficial for the follow up of asymptomatic Leishmania infected patients, the early treatment of leishmaniasis and for surveillance survey purpose. However, full evaluation of sensitivity and specificity of this technique with a large cohort of patients is required before deployment.

Label-free Electrostatic Detection of DNA Amplification by PCR Using Capacitive Field-effect Devices

A capacitive field-effect EIS (electrolyte-insulator-semiconductor) sensor modified with a positively charged weak polyelectrolyte of poly(allylamine hydrochloride) (PAH)/single-stranded probe DNA (ssDNA) bilayer has been used for a label-free electrostatic detection of pathogen-specific DNA amplification via polymerase chain reaction (PCR). The sensor is able to distinguish between positive and negative PCR solutions, to detect the existence of target DNA amplicons in PCR samples and thus, can be used as tool for a quick verification of DNA amplification and the successful PCR process.

Isolation of Canine Parvovirus-2 in A-72 Cell Line

Objectives: Isolation of Canine parvovirus -2 in A-72 cell line. Material and method: In the present study, out of 20 clinical samples (faecal swab), five samples were subjected to CPV-2 isolation in A-72 cell linebased on positive confirmation in both polymerase chain reaction (PCR) and haemagglutination (HA) test. Result: Out of five confirmed PCR and HA positive faecal sample only in one A-72 cell line flask cytopathic effect was seen in mild form. Discussion: A-72 cell line permit the faster growth of CPV-2 than other cell line as pronounced CPE was seen. However four PCR and HA positive faecal samples were failed to grow in A-72 cell line might be due to lack of sufficient virus titre as 8–10 days of post-infection specific anti-CPV antibodies formed in the intestinal lumen frequently entrap many of the virions thus preventing the virus adsorption to cells. Conclusion:Although CPV-2 was isolated in one of the infected A-72 cell line flask but visual confirmation by CPE was poor. Therefore, it is recommended that Fluorescent antibody test (FAT) is only way for proper visualisation of CPV-2 induced CPE in any suitable cell line

Accuracy of mucocutaneous leishmaniasis diagnosis using polymerase chain reaction: systematic literature review and meta-analysis.

The diagnosis of mucocutaneous leishmaniasis (MCL) is hampered by the absence of a gold standard. An accurate diagnosis is essential because of the high toxicity of the medications for the disease. This study aimed to assess the ability of polymerase chain reaction (PCR) to identify MCL and to compare these results with clinical research recently published by the authors. A systematic literature review based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses: the PRISMA Statement was performed using comprehensive search criteria and communication with the authors. A meta-analysis considering the estimates of the univariate and bivariate models was performed. Specificity near 100% was common among the papers. The primary reason for accuracy differences was sensitivity. The meta-analysis, which was only possible for PCR samples of lesion fragments, revealed a sensitivity of 71% [95% confidence interval (CI) = 0.59; 0.81] and a specificity of 93% (95% CI = 0.83; 0.98) in the bivariate model. The search for measures that could increase the sensitivity of PCR should be encouraged. The quality of the collected material and the optimisation of the amplification of genetic material should be prioritised.

What is the burden of submicroscopic malaria in pregnancy in central India?

Background:Conventional microscopy underestimates the burden of malarial infection when compared with molecular diagnosis using polymerase chain reaction (PCR)-based methods. Lower density parasitemias serve as a reservoir for infection. We evaluated the prevalence of submicroscopic infections in an area of unstable malarial transmission in India and determined whether these infections negatively impacted maternal or fetal outcomes.Methods:This cross-sectional study (2007–2008) was undertaken in two districts of Chhattisgarh, recruiting women from both antenatal clinics (ANCs) and delivery units (DUs). For ANC/DU subjects, peripheral/placental blood, respectively, was obtained for conventional microscopy and collected onto filter paper for PCR analysis.Results:There were 3425 pregnant women, including 2477 ANC subjects and 948 DU subjects who had both microscopic and PCR samples available. Polymerase chain reaction detected significantly more Plasmodium infections than traditional light microscopy both from peripheral (3·4 vs 1·2%; OR 2·9, 95% confidence intervals (CIs) 1·9–4·5) and placental (4·2 vs 1·7%; OR 2·5, 95% CIs 1·4–4·8) blood samples. Submicroscopic infections were not associated with anemia or severe maternal anemia among ANC or DU participants and were not associated with low birth weight (LBW) among DU participants. In contrast, microscopically detected infections were associated with severe anemia and LBW.Conclusions:In this area of unstable malarial transmission from India, submicroscopic infections did not identify a set of pregnant women at increased risk for anemia or LBW. Until PCR techniques become much less expensive and available as a point of care test for the field setting, its use will be limited for malarial detection.

Microscopy for Acid Fast Bacilli: A Useful but Neglected Tool in Routine Laboratory Diagnosis of Buruli Ulcer

Background: Laboratory diagnosis of Buruli ulcer disease has become vital with the introduction of antibiotic treatment. Polymerase chain reaction (PCR) for the IS2404 repeat sequence of Mycobacterium ulcerans is the gold standard for laboratory diagnosis. This is expensive and only carried out in reference laboratories in endemic countries in Africa. In order to improve the efficiency of diagnosis at the point of care and reduce the total cost of patient management, we decided to evaluate Ziehl-Neelsen (ZN) staining for acid-fast bacilli (AFB) as an inexpensive diagnostic tool. Methods: Two smears directly prepared at the point of care were examined under oil-immersion microscopy after ZN staining for AFB and compared the results to PCR samples from the same patients. Results: Good quality smears were obtained from all subjects and our results showed that when a second smear was added the sensitivity of microscopy for AFB was increased from 52 to 55% for FNA samples and from 51 to 57 % for swabs. Conclusion: If PCR were to be omitted in all patients with suspected Buruli ulcer disease when AFB were detected it would result in a considerable saving.

Community Analysis of Ammonia Oxidizing Bacteria through Molecular Genetics in Activated Sludge of Effluent Treatment Plant

We investigated the communities of Ammonia-Oxidizing Bacteria (AOB) in activated sludge using Polymerase Chain Reaction (PCR) followed by Terminal Restriction Fragment Length Polymorphism (T-RFLP), cloning, and sequencing of the alpha-subunit of the ammonia monooxygenase gene (amoA). In this study the techniques of specific amplification of ammonia oxidiser 16S rDNA fragments by PCR, separation of mixed PCR samples by Denaturing Gradient Gel Electrophoresis (DGGE), and band identification by specific hybridization with oligonucleotide probes were combined to allow for the comparison of the community composition of multiple samples over space and time. DGGE bands of interest were also excised for DNA isolation, reamplification, sequence determination and phylogenetic analysis. We compared monthly samples by the emergent macrophyte Glyceria maxima to determine the seasonal effects that the plant roots and the oxygen availability might have on the β-subgroup ammoniaoxidiser populations present. Similarly, five soil or sediment samples, varying in oxygen availability, from different locations were compared. Although the presence of two previously defined Nitrosospira sequence clusters could be differentially detected in the samples examined, there was no evidence for a particular group which was specific to periodically anoxic environments.

Comparison of primer pairs: Greater degeneracy improves small ruminant lentivirus (SRLVs) detection by seminested PCR

The identification of small ruminant lentivirus (SRLV) infected animals is crucial to obtain success through eradication, control and monitoring programs worldwide. Several authors have demonstrated that serology alone is not sufficient to detect SRLV infection. Polymerase chain reaction (PCR) is suggested as the choice test to identify seronegative infected animals. The aim of this study was to evaluate and compare degenerate primer pairs in order to detect a great number of virus variants by a seminested PCR. Based on already described primers and the alignment of gag nucleotide sequences from databases, degenerate primers were designed. Increasing the degree of degeneracy has improved the detection of the provirus in ovine and caprine genomic DNA samples. The number of positive PCR samples was twice higher when using the more degenerate primers in the established experimental conditions. Thus, we propose the use of this seminested PCR as a diagnostic tool, in combination with serology, especially in the situations where the circulating virus variants are unknown.

High prevalence of large trematode eggs in schoolchildren in Cambodia

Large trematode eggs (LTE) resembling Fasciola spp. eggs were reportedly found in the stools of schoolchildren in Kandal province, Cambodia. This study aimed to assess the prevalence of LTE in the stools of children attending the affected school, identify potential risk factors for infection and ascertain the trematode species. We performed a cross-sectional study involving an in-depth questionnaire administered to schoolchildren at the affected school, and examined cattle droppings in the surrounding area and the livers of slaughtered cattle. Three stool samples were examined per child, using Kato–Katz and formalin–ether concentration techniques. In addition, blood serum enzyme-linked immunosorbent assay (ELISA) and coprological polymerase chain reaction (PCR) was conducted for species clarification. Cattle droppings were examined by cup sedimentation and coprological ELISA. LTE were observed in the stools of 106 schoolchildren (46.5%). Two blood serum samples from schoolchildren were positive for Fasciola hepatica in a first ELISA but were negative in a confirmation immunofluorescence antibody test. Out of 221 PCR samples, only one tested positive for Fasciola spp. and none for Fasciolopsis buski. The consumption of raw aquatic plants (odds ratio (OR)=3.3) and fermented fish sauce (OR=2.1) were significantly associated with LTE in the stool. Fasciola spp. flukes were observed in 18.3% of 191 cattle livers. The prevalence of fascioliasis in cattle droppings was 88.8%. The low prevalence of schoolchildren that tested positive for Fasciola spp. with specific molecular diagnostics and who had no diagnostic evidence of F. buski strongly indicates that the majority of microscopically observed LTE are from Echinostoma spp. Fasciola spp. transmission from cattle to human is possible and public health services need to be alerted accordingly.

Detection of deoxyribonucleic acid (DNA) targets using polymerase chain reaction (PCR) and paper surface-enhanced Raman spectroscopy (SERS) chromatography.

Surface-enhanced Raman spectroscopy (SERS) enables multiplex detection of analytes using simple, portable equipment consisting of a single excitation source and detector. Thus, in theory, SERS is ideally suited to replace fluorescence in assays that screen for numerous deoxyribonucleic acid (DNA) targets, but in practice, SERS-based assays have suffered from complexity and elaborate processing steps. Here, we report an assay in which a simple inkjet-fabricated plasmonic paper device enables SERS-based detection of multiple DNA targets within a single polymerase chain reaction (PCR). In prior work, we demonstrated the principles of chromatographic separation and SERS-based detection on inkjet-fabricated plasmonic paper. The present work extends that capability for post-PCR gene sequence detection. In this design, hydrolysis DNA probes with 5' Raman labels are utilized; if the target is present, the probe is hydrolyzed during PCR, freeing the reporter. After applying the PCR sample to a paper SERS device, an on-device chromatographic separation and concentration is conducted to discriminate between hydrolyzed and intact probes. SERS is then used to detect the reporter released by the hydrolyzed probes. This simple separation and detection on paper eliminates the need for complex sample processing steps. In this work, we simultaneously detect the methicillin-resistant Staphylococcus aureus genes mecA and femB to illustrate the concept. We envision that this approach could contribute to the development of multiplex DNA diagnostic tests enabling screening for several target sequences within a single reaction, which is necessary for cases in which sample volume and resources are limited.

Detecção de Brucella abortus em tecidos bovinos utilizando ensaios de PCR e qPCR¹

Objetivou-se no presente estudo avaliar as técnicas reação em cadeia da polimerase (PCR) e PCR em Tempo Real (qPCR) para detectar Brucella abortus, a partir de tecidos bovinos com lesões sugestivas de brucelose. Para isto, 21 fragmentos de tecidos bovinos coletados em abatedouros de Mato Grosso do Sul foram processados e submetidos ao cultivo microbiológico e extração do DNA genômico para realização das reações de PCR e qPCR. No cultivo microbiológico, oito amostras apresentaram crescimento bacteriano e cinco foram confirmadas como B. abortus por PCR. Diretamente das amostras de tecido, DNA do gênero Brucella (oligonucleotídeos IS711) foi detectado em 13 (61,9%) amostras de tecido e 17 (81%) amostras de homogeneizado. Já com os oligonucleotídeos espécie-específicos BruAb2_0168F e BruAb2_0168R, 14 (66%) amostras de tecido e 18 (85,7%) amostras de homogeneizado foram amplificadas. Seis amostras positivas na PCR espécie-específica foram sequenciadas e o best hit na análise BLASTn foi B. abortus. Na qPCR, 21 (100%) amostras de tecidos e 19 (90,5%) amostras de homogeneizado foram positivas para B. abortus. Dez amostras de DNA de sangue bovino de rebanho certificado livre foram utilizadas como controle negativo nas análises de PCR e qPCR utilizando-se os oligonucleotídeos BruAb2_0168F e BruAb2_0168R. Na PCR nenhuma amostra amplificou, enquanto que na qPCR 2 (20%) amplificaram. Conclui-se que as duas técnicas detectam a presença de B. abortus diretamente de tecidos e homogeneizados, porém a qPCR apresentou maior sensibilidade. Os resultados obtidos indicam que a qPCR pode representar uma alternativa rápida e precisa para a detecção de B. abortus diretamente de tecidos, e ser utilizada em programas de vigilância sanitária, por apresentar sensibilidade e especificidade satisfatórias.

Urine real-time polymerase chain reaction detection for children virus pneumonia with acute human cytomegalovirus infection

BackgroundHuman cytomegalovirus (HCMV) is an important pathogen of viral pneumonia in children. The diagnosis of acute HCMV infection is complicated and difficult.MethodsClinical and laboratory data of 6063 hospitalized children with respiratory infection and 509 with respiratory virus infection alone were retrospectively analyzed. Urine and respiratory specimens of 186 hospitalized children with pneumonia were also prospectively collected. Real-time polymerase chain reaction (PCR) and a chemiluminescent assay were used to detect HCMV DNA copy number, the pp65 gene, and HCMV IgM.ResultsThe patients with respiratory virus infection alone and those with pulmonary HCMV infection (n = 422) were mostly children aged <6 months old (82.91%, 422/509). The accuracy of urine HCMV DNA (82.32%) was higher than that of HCMV IgM (67.78%), indicating that PCR of urine samples is suitable for determining pediatric acute pulmonary HCMV infection. There was no significant difference in detecting HCMV DNA or the pp65 gene between urinary and respiratory specimens (P > 0.05) in 186 pediatric pneumonia cases. The accuracy of the pp65 gene measured in urine for determining acute pulmonary HCMV infection was the highest (93.01%).ConclusionsOur study shows a novel method for investigating acute pulmonary HCMV infection in children by using real-time PCR and non-invasive samples. This study also highlights the superiority and potential use of the pp65 gene as an important target for the diagnosis of acute pulmonary HCMV infection.

Pilot study to evaluate the role of Mycoplasma species in cat bite abscesses.

Mycoplasma species are common inhabitants of the feline oral cavity, and so likely contaminate many cat bite abscesses. The objectives of this study were to determine whether Mycoplasma species are common contaminants of cat bite abscesses and whether they are are associated with β-lactam-resistant clinical disease. Twenty-six privately owned cats with clinical evidence of an abscess suspected to be from a cat bite were included in the study. Samples from each cat were evaluated by aerobic and anaerobic culture, as well as Mycoplasma species culture and polymerase chain reaction (PCR). All cats were initially treated with appropriate wound management and were administered an antibiotic of the β-lactam class (amoxicillin, amoxicillin clavulanate or cefovecin sodium). Mycoplasma species DNA was amplified by PCR from 4/26 samples (15.4%); one of these cases was concurrently culture positive. Adequate DNA for sequencing was present for 2/4 positive PCR samples; one was most homologous with Mycoplasma felis, and the other was most homologous with Mycoplasma equigenitalium and Mycoplasma elephantis. Of the 26 cats, 25 responded to the initial treatment by day 7. The cat that failed initial treatment was positive for M equigenitalium or M elephantis DNA on days 0 and 12, and ultimately responded to administration of enrofloxacin and clindamycin. The results suggest that while Mycoplasma species can contaminate cat bite abscesses, routine wound management and β-lactam antibiotic therapy is adequate for treatment in most cases of abscess. However, as Mycoplasma species infections do not respond to β-lactam class antibiotic therapy, these organisms should be on the differential list for cats with abscesses that fail treatment with this antibiotic class.

A case of zoster sine herpete presenting with thoracic radicular pain diagnosed by polymerase chain reaction in skin exudate

Varicella zoster virus (VZV) can cause radicular pain in the absence of skin lesions; such cases are referred to as zoster sine herpete (ZSH) and are usually diagnosed by using serological assays or polymerase chain reaction (PCR). An effort is underway to detect VZV DNA in novel specimens rather than conventional samples (e.g., blood or cerebrospinal fluid) for PCR. There are two reports that PCR analysis in the exudate of the auricular skin can be a useful diagnostic tool for the diagnosis of ZSH in patients presenting with cranial nerve paralysis without herpetic eruptions. Here, we report a case of ZSH diagnosed by using PCR analysis of skin exudates in a patient who developed thoracic radicular pain. This is believed to be the first case of ZSH diagnosed using PCR analysis of skin exudate in a patient in whom the cranial nerve was not involved.

Mycobacterium leprae is identified in the oral mucosa from paucibacillary and multibacillary leprosy patients

In leprosy, the nasal mucosa is considered as the principal route of transmission for the bacillus Mycobacterium leprae. The objective of this study was to identify M. leprae in the oral mucosa of 50 untreated leprosy patients, including 21 paucibacillary (PB) and 29 multibacillary (MB) patients, using immunohistochemistry (IHC), with antibodies against bacillus Calmette-Guérin (BCG) and phenolic glycolipid antigen-1 (PGL-1), and polymerase chain reaction (PCR), with MntH-specific primers for M. leprae, and to compare the results. The material was represented by 163 paraffin blocks containing biopsy samples obtained from clinically normal sites (including the tongue, buccal mucosa and soft palate) and visible lesions anywhere in the oral mucosa. All patients and 158 available samples were included for IHC study. Among the 161 available samples for PCR, 110 had viable DNA. There was viable DNA in at least one area of the oral mucosa for 47 patients. M. leprae was detected in 70% and 78% of patients using IHC and PCR, respectively, and in 94% of the patients by at least one of the two diagnostic methods. There were no differences in detection of M. leprae between MB and PB patients. Similar results were obtained using anti-BCG and anti-PGL-1 antibodies, and immunoreactivity occurred predominantly on free-living bacteria on the epithelial surface, with a predilection for the tongue. Conversely, there was no area of predilection according to the PCR results. M. leprae is present in the oral mucosa at a high frequency, implicating this site as a potential means of leprosy transmission.

Comparitive study on the efficacy of ELISA and IS900 PCR for the diagnosis of Paratuberculosis in goats

Paratuberculosis, one of the chronic granulomatous enteritis that predominantly affects ruminants worldwide, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It is most efficiently diagnosed by MAP from faeces by Polymerase Chain Reaction (PCR). Serological tests like Enzyme Linked Immuno Sorbent Assay (ELISA) also provides a rapid and cost-effective alternative diagnostic tool. Present study was carried out to directly evaluate the sensitivity and specificity of ELISA (ID vet innovative diagnostics; France) using IS900 PCR as a gold standard. Serum and faecal samples were collected from 180 adult goats of either sex, from Malappuram and Thrissur districts of Kerala with unknown paratuberculosis status. Faecal samples were processed for direct IS900 PCR and serum samples were tested for MAP antibodies using Indirect ELISA kit. IS900 PCR detected 38 out of 180 confirmed to be shedding MAP. ELISA detected 22 out of 180 animals as positive. Overall, ELISA was 50 % sensitive and 97.9 % specific in comparison to IS900 PCR. The IS900 PCR outperformed ELISA in detecting animals potentially infected with MAP and is more sensitive than ELISA at detecting animals suspected of paratuberculosis. But, for early diagnosis of paratuberculosis in goats, ELISA can be done as easy and rapid farm level identification and IS900 PCR as individual confirmatory test.