Use Of Polymerase Chain Reaction Research Articles

An alternative approach for evaluating the phenotypic virulence factors of pathogenic Escherichia coli

Escherichia coli is a recognized zoonotic food-borne pathogen; however, the use of polymerase chain reaction (PCR) in the underdeveloped countries to differentiate pathogenic from non-pathogenic E. coli is a problematic issue. Our grail was to assess the phenotypic virulence markers motility, hemolysin, congo red agar, embryo lethality assay and serum resistance for pathogenic E. coli (PEC) correlated to PCR tests which is currently used world-wide to evaluate the PEC. The 448 strains of Escherichia coli that were isolated from different sources, were characterized for phenotypic virulence factors such as motility, hemolysin, Congo red binding, Embryo Lethality assay (ELA) and serum resistance, as well as antibiotic susceptibility using disc diffusion method to 23 antibiotics. Results exhibited 100% motility and Congo red binding, 97.1% for hemolysin production and 90.2% in the ELA. As a result, we were able to hypothetically conclude that the aforementioned virulence markers are plain, straightforward, economical, rapid, more dynamic, uncomplicated methodology, duplicatable and cost next to nothing when compared to the molecular PCR. Their implementation in a diagnostic microbiology laboratory for vetting is a rewarding task in the underdeveloped countries. It augments endeavors to minimize the use of PCR in our investigations especially during epidemiological and outbreak investigations of PEC.

Erysipelothrix rhusiopathiae bloodstream infection – A 22‐year experience at Mayo Clinic, Minnesota

Erysipelothrix rhusiopathiae is a facultatively anaerobic Gram-positive bacillus found mostly in swine, fish and sheep. E.rhusiopathiae classically causes cutaneous eruptions in butchers, fish handlers and veterinarians. Based solely on case reports, 90% of E.rhusiopathiae bloodstream infections (BSI) have been associated with infective endocarditis (IE). To assess the true frequency of IE in E.rhusiopathiae BSI as well as other clinical associations, we performed a retrospective cohort analysis of E.rhusiopathiae BSI at Mayo Clinic. This is a single-centre, retrospective study conducted between 1/1/1994 and 20/6/2016 at Mayo Clinic in Rochester, MN. Medical records were reviewed for demographics, E.rhusiopathiae BSI, anti-microbial susceptibilities, incidence of IE, patient comorbidities, intensive care unit (ICU) admission and duration of antibiotics. Five cases of E.rhusiopathiae BSI were identified. Risk factors included animal exposures, immunosuppression, diabetes and kidney disease. All cases involved penicillin-sensitive strains and high-grade BSI. Four cases showed no signs of IE on transesophageal echocardiogram. All patients recovered fully with intravenous antibiotics. Our retrospective review illustrates that E.rhusiopathiae can cause invasive BSI in the absence of IE and that the previously reported 90% association between BSI and IE may be overestimated due to reporting bias. E.rhusiopathiae should be suspected in any patient with Gram-positive bacilli in blood cultures and the aforementioned risk factors. A limitation of our study was the low sample size, and future studies may involve multicentre collaborations and the use of polymerase chain reaction (PCR) or serologic testing to increase the number of diagnoses..

Treatment of Infected Women of Childbearing Age Prevents Congenital Trypanosoma cruzi Infection by Eliminating the Parasitemia Detected by PCR.

We evaluated the effectiveness of treating women of childbearing age with benznidazole to prevent congenital Chagas disease (CCD), as well as the usefulness of polymerase chain reaction (PCR) as a tool to predict the risk of transmission. Prospective study involving 144 T. cruzi seropositive pregnant women. The parasitological status was studied by PCR in 159 pregnancies, 38 of which involved a cohort of previously treated mothers. One hundred sixty children were examined by PCR and serologically studied at 0-6, 9 and 12 months and annually after treatment. PCR was seen to be useful for predicting the risk of congenital transmission: 18.8% of mothers with a positive PCR result transmitted the infection (16 infected children out of 85 pregnancies). No infected infants were detected among 74 pregnancies when PCR was negative. Of the treated mothers, 92.1% had negative PCR results, compared with 32.2% of untreated mothers. No infected infants were detected from previously treated mothers, compared with 13.2% among untreated mothers (P = .019; χ2). All infants treated before the first year of life were cured. Treating infected women of childbearing age prevents congenital Chagas disease. Polymerase chain reaction screening of T. cruzi-infected pregnant women is a useful tool for predicting the risk of congenital transmission.

Evaluation of the utility of conventional polymerase chain reaction for detection and species differentiation in human hookworm infections.

Background:Human hookworm infection is caused mainly by Necator americanus and Ancylostoma duodenale. Among the zoonotic hookworm species, only Ancylostoma ceylanicum causes potent human infections where dogs and cats act as reservoir of infection. Hence, species differentiation is imperative because the eradication of both anthroponotic and zoonotic hookworm depends on the concurrent human and animal health programs, hygienic practices, and mass drug administration for humans and dogs.Objective:This study was performed to evaluate the utility of polymerase chain reaction (PCR) for detection of hookworm infections.Materials and Methods:A total of 209 stool samples were collected and subjected to stool microscopy, Kato-Katz method to identify the intensity of the infection, coproculture for L3 larval identification and species differentiation and semi-nested PCR with sequencing.Results:The prevalence of hookworm was estimated as 7.6%. Highest hookworm prevalence was seen in 20–30 years of age group. Majority of the infections were mild intensity infections. Sensitivity of stool microscopy was found to be 81.2% and the specificity was 100%. Sensitivity of Kato-Katz method was 87.5% and specificity was 100%. True positivity by agar plate culture was 83.3% and false positivity rate was 16.6%.Conclusion:Stool microscopy is the major mode of detection, but it has a higher false negative rate. Coproculture is time-consuming and needs the expertise to differentiate the species. On the other hand, PCR is known to be a sensitive, specific, and a reliable investigative tool which can help in diagnosis as well as in species differentiation.

Chapter 9 - Preimplantation genetics and other reproductive options in Huntington disease

Preimplantation genetic diagnosis (PGD) is a form of prenatal diagnosis applied to potential parents with known carrier status of a genetic disease, such as Huntington disease. It employs the use of polymerase chain reaction to amplify single cells from early embryos obtained with in vitro fertilization (IVF) techniques. PGD allows the couple the chance to have a pregnancy and livebirth child without Huntington disease, although there are some risks and expenses related to the procedures. Success of the procedure may be greater than standard IVF because the patients are not infertility patients, but are undergoing the procedure to avoid passing a highly deleterious disease gene to offspring. Recent advances in sequencing may allow for higher success rates as the chromosomally abnormal embryos will be identified more easily and the embryos with the highest chance of survival will be transferred.

Molecular genetic and morphological features of malignant neoplasms of the stomach and colon with tissue eosinophilia

Aim. To study molecular genetic and morphological features of neoplasms in stomach and colon cancer associated with tissue eosinophilia. 
 Methods. Study material was the tissue samples of malignant neoplasms of the stomach and colon obtained from the surgical procedures. Study groups were identified depending on the presence or absence of eosinophilic infiltration of the tumour tissue as well as on neoplasm localization. Medical records and outpatient patient cards were analyzed, presence of matastases in regional lymph nodes and grade of differentiation of malignant cells were evaluated. In tumour tissue expression of p53 and p21 proteins was evaluated by means of immunohistochemical technique. To study the distribution of polymorphic variants of р53 (G215C) and р21 (A1026G) genes, deoxyribonucleic acid extraction was performed from formalin-fixed and paraffin-embedded tissue samples obtained from the resection margins. Polymorphism genotyping of deoxyribonucleic acid samples was performed by restriction fragment length polymorphism analysis of amplification products with the use of polymerase chain reaction of specific genome regions with further visualization with ultraviolet light. 
 Results. In patients with colon cancer associated with tissue eosinophilia tumour cells were shown to have higher grade of differentiation than in patients without eosinophilia. In stomach cancer associated with eosinophilic infiltration low expression of mutant p53 protein was revealed significantly more frequently as opposed to cancer without eosinophilia. Also in patients with stomach and colon cancer, association of tissue eosinophilia with carriage of G allele of р53 (G215C) gene polymorphism was found. 
 Conclusion. Tissue eosinophilia in stomach and colon cancer is associated with favourable morphological cancer characteristics, low expression of mutant p53 protein and carriage of G allele of р53 (G215C) polymorphism.

Dual mutations and complex mutations in metastatic nonsmall cell lung cancer: A single-institution experience from South India.

Wide use and incorporation of newer diagnostic tools in the management of metastatic nonsmall cell lung cancer (NSCLC) has helped in achieving the goal of personalized treatment of this disease. With the wide use of polymerase chain reaction and fluorescence in situ hybridization increasing number of patients are being diagnosed with dual and complex mutations posing a new challenge in the management of patient with metastatic NSCLC. In this article, we would like to bring forth six such cases and the varied responses to the current available treatment in patients with complex and dual mutations. The appropriate management in these groups of patients is yet to be standardized.

Impact of the Use of Polymerase Chain Reaction for the Diagnosis and Management of Pneumocystis Jirovecii Pneumoniae in a Retrospective Cohort of Patients with Lymphoid Malignancies

Introduction: Pneumocystis jirovecii (Pj) is an opportunistic fungus and a common cause of pneumonia (PCP) in immunocompromised patient. The gold standard for the diagnosis is the direct exam by microscopy. Patients with hematological malignancies may have a small burden of Pj with a negative direct exam. A polymerase chain reaction assay (PCR) can be used for the diagnosis. Real-time qPCR methods have a good sensitivity (97-100%) and specificity (90-94%) for the PCP detection whereas direct examination has lower sensitivity. Currently, the diagnostic impact of a positive PCR with a negative direct examination for patients with lymphoid malignancies suspected of PCP is not well known.Methods: We retrospectively analysed in this study all patients followed in the Department of Hematology of the Leon Berard Cancer Center (Lyon, France) between January 2003 and March 2014 who developed PCP. In at risk patients, PCP was considered when clinical signs and typical radiological features (e.g. ground glass opacities). Diagnosis was confirmed by the microscopic observation of Pj cysts and/or trophic forms on the bronchoalveolar lavage fluid (Group 1, gold standard) or a positive PCR with negative direct examination (Group 2).Results: PCP was diagnosed in 68 patients with lymphoid malignancies (76% non-Hodgkin lymphoma, 15% Hodgkin lymphoma, 7% chronic lymphocytic leukemia and 2% myeloma). PCP was diagnosed during first-line therapy for 42 patients (62%). Seven breakthrough infections (10%) under sulfamethoxazole and trimethoprim (S/T) prophylaxis were observed. PCP was confirmed in 27 patients (40%) by a positive direct exam (Group 1) and 41 patients (60%) by PCR only (Group 2). The clinical and biological characteristics at diagnosis of PCP were not significantly different between Group 1 and 2 (fever, cough, lymphocyte count, neutrophil count, hemoglobin level, C-reactive protein level, lactate deshydogenase level). However, Group 1 presented more frequently an oxygen saturation of less than 92% (88.9% vs. 56.1%, P=0.007) and dyspnea (88.9% vs. 65.9%, P=0.045) as compared to Group 2. The CT-scan showed bilateral ground-glass opacities in 74% and 71% of Group 1 and 2, respectively. Median time between the hematological diagnosis (3.02 months vs. 4.7 months, P=0.54) or the last chemotherapy (20.5 days vs. 21 days, P=0.62) and PCP diagnosis were similar in both groups. The median time between the first day of hospitalization and the initiation of curative treatment by S/T was similar in the two groups (6 days [0-30] vs. 7 days [0-33], P=0.71) but Group 1 received corticosteroids more frequently as compared to Group 2 (74% vs. 49%, P=0.047). We observed a similar rate of co-infections between the two groups (Group 1, 44% vs. Group 2, 39%, P=0.80). After the initiation of S/T, the median time to fever resolution (2 days [1-21] vs. 1 days [0-8], P=0.006) and to normal oxygen saturation (6 days [2-28] vs. 3 days [0-11], P=0.002) was longer for Group 1 as compared to Group 2. Hence, the median time between S/T initiation and hospital discharge was longer for Group 1 (16 days [4-53] vs. 10 days [2-44], P=0.02). The rate of hospitalization in intensive care unit was higher for Group 1 than for Group 2 (59% vs. 24%, P=0.005). In Group 1 and 2, 5 (18.5%) and 3 (7.3%) patients died from PCP, respectively (P=0.25).Conclusions: In our series, 60% of immunocompromised patients with lymphoid malignancies were diagnosed with PCP on the basis of a positive PCR and a negative direct examination. The presentation and clinical course of these patients are less severe than patients with a positive direct exam, possibly in relation with a lower fungal load. Quick improvement of clinical parameters was observed after curative S/T. These results suggest to promptly initiate a specific treatment for PCP in patients with a positive PCR and a negative direct examination. DisclosuresBlay:MDS: Research Funding; Lilly: Research Funding; Bayer: Consultancy, Research Funding; Pharmamar: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; F. Hoffmann-La Roche: Consultancy, Research Funding.

Molecular Evidence of Malaria and Zoonotic Diseases Among Rapid Diagnostic Test-Negative Febrile Patients in Low-Transmission Season, Mali.

From November to December 2012 in Sélingué-Mali, blood samples from 88 febrile patients who tested negative by malaria Paracheck® rapid diagnostic tests (RDTs) were used to assess the presence of sub-RDT Plasmodium falciparum as well as Borrelia, Coxiella burnetii, and Babesia applying molecular tools. Plasmodium sp. was present among 57 (60.2%) of the 88 malaria RDT-negative patients, whereas the prevalence of Borrelia, C. burnetii, and Babesia were 3.4% (N = 3), 1.1% (N = 1), and 0.0%, respectively. The additional diagnostic use of polymerase chain reaction (PCR) identified a high proportion of Plasmodium sp.-positive samples and although this may be a concern for malaria control, the respective PCR-identified malaria infections were less likely responsible for the observed fevers given the low parasite density. Also, the low infection levels of Borrelia and C. burnetii and lack of Babesia among the febrile patients call for further studies to assess the causes of fever among malaria RDT-negative patients in Sélingué.

Taq1B Polymorphism of Cholesteryl Ester Transfer Protein (CETP) and Its Effects on the Serum Lipid Levels in Metabolic Syndrome Patients.

The metabolic syndrome (MetS) is one of the most important risk factors for type 2 diabetes and cardiovascular disease. This syndrome is characterized by abdominal obesity, hypertension, insulin resistance, and dyslipidemia. The plasma origin of Cholesteryl ester transfer protein (CETP) is responsible for transferring cholesterol esters from high-density lipoprotein particles to apolipoprotein B containing lipoproteins compartment. We conducted this study to investigate the association between CETP gene Taq1B (rs708272) polymorphism in the metabolic syndrome among Iranian subjects. A sample size of 200 patients diagnosed with MetS together with 200 healthy donors as control were enrolled in this study. The investigation of polymorphism was performed by the use of polymerase chain reaction and restriction fragment length polymorphism analysis. To determine the relationship between polymorphism and lipid profile, we measured lipids and CETP concentration in metabolic syndrome and control subjects. Genotype distribution and allelic frequencies of polymorphism were determined and compared in both groups. Our findings showed that all clinical and biochemical characteristics in patients differed from the control group. The results showed that genotype and allele frequency of the Taq1B polymorphism was not significantly different between two groups. Instinctively, CETP was significantly higher in metabolic syndrome (1.64±0.32µg/ml) than in control (1.53±0.34µg/ml). A low level of CETP was found in blood of B2B2 typified genotype. In spite of Taq1B polymorphism on ester transfer protein concentration, no direct correlation was found between this polymorphism and metabolic syndrome.

Herpes zoster und postzosterische Neuralgie

Herpes zoster develops by endogenous reactivation of varizella zoster virus (VZV). Incidence increases with age. Females are more frequently affected than males. The reactivation rate in seropositive individuals is about 20 %. After ashort prodromal stage, herpetiform-grouped vesicles appear in segmental arrangement. Pain and paresthesia are typical zoster symptoms. Complications like bacterial superinfections, vasculopathy, paresis, and oculopathy may occur. During pregnancy herpes zoster is athreat for mother and child. Among elderly patients, cardiovascular risk is increased during the first week of herpes zoster infection. Postherpetic neuropathy is feared. Diagnosis can be made clinically and by the use of polymerase chain reaction. First-line treatment is systemic antiviral drug therapy with either acyclovir or brivudine. Adjuvant therapies consist of pain management and topical treatment.

PCR targeting IS6110 in diagnosing tuberculosis in children in comparison to MGIT culture

BackgroundDiagnosis of tuberculosis (TB) in children is difficult in children especially in extrapulmonary tuberculosis (EPTB). This study was conducted to evaluate the use of polymerase chain reaction (PCR) targeting IS6110 in the diagnosis of TB in children with pulmonary TB and EPTB and also to compare its performance with MGIT 960 culture and conventional microscopy. MethodsA total of 142 cases (50 pulmonary, 92 extrapulmonary) of suspected TB patients <15 years of age were included in the study. The clinical specimens obtained from these cases were subjected to Ziehl–Neelsen staining (ZN), MGIT 960 TB culture and PCR targeting insertion sequence IS6110. Sensitivity and specificity of PCR were calculated in pulmonary and extrapulmonary specimens. The results were compared to MGIT culture. ResultsPCR targeting IS6110 sequence had sensitivity of 69.01% in various clinical specimens which was significantly more than MGIT culture showing a sensitivity of 47.41% (p<0.05). Sensitivity of PCR IS6110 in extrapulmonary specimens was 65.21% which was lower than sensitivity in pulmonary specimens (76%) but was not statistically significant (p>0.05). ConclusionsDiagnostic efficacy of PCR IS6110 in pulmonary and extrapulmonary TB cases was similar. PCR using IS6110 primer had significantly better efficiency than MGIT culture in diagnosing TB in children.

La pneumocystose au cours de l’infection à VIH

Pneumocystosis is an opportunistic disease caused by invasion of unicellular fungus Pneumocystic jirovecii which is responsible for febrile pneumonia among patients with cellular immunodeficiency especially those HIV infected. Despite the decreasing of its incidence due to the introduction of antiretroviral therapy, as well as anti-Pneumocystis prophylaxis among these patients, Pneumocystis pneumonia remains the first AIDS-defining event and a leading cause of mortality among HIV-infected patients. The usual radiological presentation is that of diffuse interstitial pneumonia. The diagnosis is confirmed by the detection of trophozoides and/or cysts P.jirovecii in bronchoalveolar lavage (BAL) samples using several staining techniques. The use of polymerase chain reaction in the BAL samples in conjunction with standard immunofluorescent or colorimetric tests have allowed for more has allowed for more rapid and accurate diagnosis. The standard regimen of treatment is the association of trimethoprim-sulfamethoxazole which has been utilized as an effective treatment with a favourable recovery. Early HIV diagnosis and antiretroviral therapy should reduce the incidence of this dreaded disease.

Primary subacute hematogenous osteomyelitis in children: a clearer bacteriological etiology.

BackgroundThis study aimed to describe the spectrum of pediatric primary subacute hematogenous osteomyelitis (PSAHO) and to investigate its bacterial etiology.MethodsSixty-five consecutive cases of PSAHO admitted to our institution over a 16-year period (2000–2015) were retrospectively reviewed to assess their laboratory and radiographic imaging features, as well as their bacteriological etiology.ResultsOn evaluation, white blood cell count and C-reactive protein were normal in 53 (81.5 %) and 34 cases (52.3 %), respectively, whereas the erythrocyte sedimentation rate was superior to 20 mm/h in 44 cases (72.1 %). Blood cultures failed to identify the pathogen in all but one patient, and classic bone sample cultures only managed to isolate the pathogen in five cases (11.6 %). Use of polymerase chain reaction (PCR) assays on bone aspirates or blood allowed the causative microorganism to be isolated in a further 22 cases. Using classic cultures and PCR assays together resulted in pathogen detection in 27 cases (62.8 % of the children bacteriologically investigated), with Kingella kingae being the most frequently reported microorganism.ConclusionsTwo distinct forms of PSAHO should be distinguished on the basis of age of patients and bacteriological etiology. The infantile form affects children aged between 6 months and 4 years and is predominantly due to K. kingae. The juvenile form involves children aged >4 years and Staphylococcus aureus appears to be the main bacteriological etiology. Appropriate nucleic amplification assays drastically improve the detection rate of the microorganisms responsible for PSAHO.Level of evidence: Case series, level IV.

A comparative survey of Gopherus polyphemus hemoparasites in four different South Florida habitats

Abstract The gopher tortoise (Gopherus polyphemus) is a keystone species, for its burrows house more than 300 species of animals. Habitat destruction and its consequent fragmentation have led to a decline in populations within southeastern ranges of the United States. Haemogregarina (intracellular protozoan parasites) have previously been identified in the blood of gopher tortoises. High levels of blood parasitaemia have shown to be a potential indication of stress resulting from overcrowding. The goals of this study are: (1) to characterize hemoparasite species through the use of Polymerase Chain Reaction (PCR) from tortoise blood samples and the ticks attached to tortoises and (2) to determine if free ranging gopher tortoises that live in poorly maintained habitats exhibit higher prevalence and parasitaemia levels within their blood when compared to tortoises inhabiting better maintained sites with prescribed fires. Research goals will be examined at four sites: Blazing Star Preserve (BSP), Pine Jog Preserve (PJP), Florida Atlantic University Preserve (FAUP), and Johnathan Dickinson State Park (JDSP), to determine both hemoparasite prevalence and parasitaemia levels. PJP and JDSP are sites currently practicing fire management and herbicides, whereas FAUP utilizes mechanical and chemical management while BSP is not managed. We hypothesize that parasitaemia will be higher in poorly managed sites as a result of crowding, when compared to well managed sites. This study will benefit current conservation and management practices for gopher tortoises in South Florida and help provide a baseline study for reptile hemoparasites.

A Compendium for Mycoplasma pneumoniae.

Historically, atypical pneumonia was a term used to describe an unusual presentation of pneumonia. Currently, it is used to describe the multitude of symptoms juxtaposing the classic symptoms found in cases of pneumococcal pneumonia. Specifically, atypical pneumonia is a syndrome resulting from a relatively common group of pathogens including Chlamydophila sp., and Mycoplasma pneumoniae. The incidence of M. pneumoniae pneumonia in adults is less than the burden experienced by children. Transmission rates among families indicate children may act as a reservoir and maintain contagiousness over a long period of time ranging from months to years. In adults, M. pneumoniae typically produces a mild, “walking” pneumonia and is considered to be one of the causes of persistent cough in patients. M. pneumoniae has also been shown to trigger the exacerbation of other lung diseases. It has been repeatedly detected in patients with bronchitis, asthma, chronic obstructive pulmonary disorder, and cystic fibrosis. Recent advances in technology allow for the rapid diagnosis of M. pneumoniae through the use of polymerase chain reaction or rapid antigen tests. With this, more effort has been afforded to identify the causative etiologic agent in all cases of pneumonia. However, previous practices, including the overprescribing of macrolide treatment in China and Japan, have created increased incidence of macrolide-resistant M. pneumoniae. Reports from these countries indicate that >85% of M. pneumoniae pneumonia pediatric cases are macrolide-resistant. Despite its extensively studied past, the smallest bacterial species still inspires some of the largest questions. The developments in microbiology, diagnostic features and techniques, epidemiology, treatment and vaccines, and upper respiratory conditions associated with M. pneumoniae in adult populations are included within this review.

Determination of Leishmania species by PCR-RFLP in the smear samples taken from the lesions of cutaneous leishmaniasis cases

The forms of the disease caused by Leishmania species in Turkey as well as in Aegean region are cutaneous and visceral leishmaniasis (CL and VL, respectively), and the agent of CL is commonly L.tropica. However, L.infantum was also reported as being CL agent recently. Direct microscopic examination, serological tests and culture are the conventional methods used for the diagnosis of CL. Since the specificities of these methods are high their sensitivities are variable and identification at species level is not possible. Recently, the use of polymerase chain reaction (PCR)-based molecular methods enabled the rapid and reliable diagnosis and species identification. The aim of this study was to investigate the performance of PCR-restriction fragment length polymorphism (RFLP) method both for the detection and identification of Leishmania species simultaneously in CL patients. A total of 30 smear samples that were positive for Leishmania amastigotes with microscopic examination, obtained from CL-suspected cases admitted to Adnan Menderes University Medical School Hospital, Parasitology Laboratory (located at Aydin, in the Aegean region of Turkey) between 2012-2014 period were included in the study. Ten samples taken from the skin lesions caused by Staphylococcus aureus (n= 5) and Candida albicans (n= 5) were also included as negative controls. DNA extractions from the smears were performed by the use of a commercial kit (Macherey-Nagel NucleoSpin Tissue® Kit, Germany). DNA isolation was also performed from L.major, L.infantum and L.tropica promastigotes that were grown in culture as positive controls. In PCR method LITSR and L5.8S primers targeting to ITS (internal transcribed spacer)-1 region were used. In RFLP method, the amplified PCR products were cleaved by BsuRI (HaeIII) restriction enzyme for the species identification. As a result, restriction profiles of all samples (n= 30) were in accordance with L.tropica restriction profile. No band was observed in the control samples (n= 10). The data of this study showed that the most common CL agent in Aydin is L.tropica. In conclusion, ITS-1 PCR-RFLP method may be used directly as a single routine procedure for both the detection and identification of Leishmania species in the clinical samples of CL patients, in laboratories with adequate facilities.

Utility of Polymerase chain reaction in diagnosis of Acanthamoeba and Microsporidial keratitis

Acanthamoeba and Microsporidia are two opportunistic parasitic organisms which are now increasingly recognized as significant emerging cause of microbial keratitis. The reason for this rise in numbers could possibly be because of increasing awareness of the condition, gradual rise in the use of contact lens and improvement in the diagnostic modalities. Early and accurate diagnosis is the most vital step in managing Acanthamoeba and Microsporidial keratitis as prognosis is directly related to timely diagnosis. Current methods of diagnosing these parasites depends mainly upon their morphologic demonstration in the clinical specimen by microscopy. Culture which is the gold standard in diagnosis of Acanthamoeba keratitis is time consuming and requires advanced laboratory set up because of the special growth requirements International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 5 Number 2(2016) pp. 854-860 Journal homepage: http://www.ijcmas.com

Predominant Bacteria Detected from the Middle Ear Fluid of Children Experiencing Otitis Media: A Systematic Review.

BackgroundOtitis media (OM) is amongst the most common childhood diseases and is associated with multiple microbial pathogens within the middle ear. Global and temporal monitoring of predominant bacterial pathogens is important to inform new treatment strategies, vaccine development and to monitor the impact of vaccine implementation to improve progress toward global OM prevention.MethodsA systematic review of published reports of microbiology of acute otitis media (AOM) and otitis media with effusion (OME) from January, 1970 to August 2014, was performed using PubMed databases.ResultsThis review confirmed that Streptococcus pneumoniae and Haemophilus influenzae, remain the predominant bacterial pathogens, with S. pneumoniae the predominant bacterium in the majority reports from AOM patients. In contrast, H. influenzae was the predominant bacterium for patients experiencing chronic OME, recurrent AOM and AOM with treatment failure. This result was consistent, even where improved detection sensitivity from the use of polymerase chain reaction (PCR) rather than bacterial culture was conducted. On average, PCR analyses increased the frequency of detection of S. pneumoniae and H. influenzae 3.2 fold compared to culture, whilst Moraxella catarrhalis was 4.5 times more frequently identified by PCR. Molecular methods can also improve monitoring of regional changes in the serotypes and identification frequency of S. pneumoniae and H. influenzae over time or after vaccine implementation, such as after introduction of the 7-valent pneumococcal conjugate vaccine.ConclusionsGlobally, S. pneumoniae and H. influenzae remain the predominant otopathogens associated with OM as identified through bacterial culture; however, molecular methods continue to improve the frequency and accuracy of detection of individual serotypes. Ongoing monitoring with appropriate detection methods for OM pathogens can support development of improved vaccines to provide protection from the complex combination of otopathogens within the middle ear, ultimately aiming to reduce the risk of chronic and recurrent OM in vulnerable populations.

The Clinical Usefulness of Polymerase Chain Reaction as a Supplemental Diagnostic Tool in the Evaluation and the Treatment of Children With Septic Arthritis.

Culture-negative septic arthritis occurs frequently in children. The supplemental use of polymerase chain reaction (PCR) techniques improves the detection of bacteria in the joint fluid. This study evaluates the clinical utility of PCR at a tertiary pediatric medical center. Children with septic arthritis were studied prospectively from 2012 to 2014. Culture results and clinical infection parameters were recorded. PCR was performed whenever sufficient fluid was available from the joint aspiration. A statistical comparison was made for the rates of identification of the causative organism by these methods. A subgroup analysis was performed to assess the correspondence of clinical and laboratory parameters with the results of joint fluid culture and PCR. Ninety-nine children with septic arthritis were enrolled consecutively. A broad range of parameter results was identified among these children with an average of 3.6 of 6 parameters per child that met thresholds of infection. Joint fluid cultures were positive in 34 of 97 (35.1%) children from whom they were sent. Among the 68 children from whom the material was sent for PCR, the result was positive in 32 (47.1%). The combination of blood culture, joint fluid culture, and PCR resulted in bacterial detection in 49 of 97 (50.5%) children. PCR improved the rate of detection of Kingella kingae markedly when compared with joint fluid culture. PCR results were available at an average of 14.6 days after the acquisition of joint fluid. 16S PCR results were reported at an average of 17.5 days, whereas Kingella PCR took 5.1 days. PCR provides supplemental information for diagnostic confirmation through an increased rate of detection of bacteria. The timing of results and the inability to provide antibiotic sensitivity are factors that limit its clinical usefulness currently.