Arbitrarily primed polymerase chain reaction for fingerprinting the genotype identification of mutans streptococci in children with Down syndrome

This study investigated the possible intrafamilial similarity of mutans streptococcal strains in some families with a child with Down syndrome using chromosomal DNA fingerprinting. The isolates were genotyped using arbitrarily primed polymerase chain reaction with the OPA 02 and OPA 03 primers. The results showed that five children with Down syndrome harbored mutans streptococci genotypes different from those of their mothers. A matching of genotypes was observed within the control pair (mother/child without Down syndrome). After six months, new samples were collected from all participants. Analysis showed that samples from children with Down syndrome were colonized by a new strain of Streptococcus mutans that did not match the previously collected one. The results suggest the S. mutans indigenous bacteria change more than once in children with Down syndrome.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb)gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Presumptive Identification of the Fungal Communities in Cystic Fibrosis Patients' Sputum Using Amplicon Length Heterogeneity Polymerase Chain Reaction

This thesis would not have been possible without the aid of my family, friends, laboratory members, and professors. First and foremost, I would like to thank Dr. Kalai Mathee for allowing me to enter her lab in August 2007 and enabling to embark on this journey. This experience has transformed me into more mature scientist, teaching me how to ask the right questions and the process needed to solve them. I would also like to acknowledge Dr. Lisa Schneper. She has helped me throughout the whole process, by graciously giving me input at every step of the way. I would like to express gratitude to Dr. Jennifer Richards for all her input in writing the thesis. She has been a great teacher and being in her class has been a pleasure. Moreover, I would like to thank all the committee members for their constructive criticism throughout the process. When I entered the lab in August, there was one person who literally was by my side, Melissa Doud. Without your input and guidance I would not have even been able to do these experiments. I would also like to thank you and Dr. Light for allowing me to meet some cystic fibrosis patients. It has allowed me to put a face on the disease, and help the patients' fight. For a period before I had entered the lab, Ms. Doud had an apprentice, who started the fungal aspect of the project, Caroline Veronese. Her initial work has enabled me to prefect the protocols and complete the ITS 1 region.One very unique aspect about Dr. Mathee's lab is the camaraderie. I would like to thank all the lab members for the good times in and out of the lab. These individuals have been able to make smile and laugh in parties and lab meetings. I would like to individually thank Balachandar Dananjeyan, Deepak Balasubramanian, and V arinderpal Singh Pannu for all the PCR help and Natalie Maricic for the laughs and being a great classmate. Last, but not least, I would like to acknowledge my family and friends for their support and keeping me sane: Cecilia, my mother, Mohammad, my father, Amir, my older brother, Billal, my younger brother, Ouday Akkari and Stephanie De Bedout, my best friends.

The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis : C. parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis . In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

Sensitive and specific detection of Clavibacter xyli subsp. xyli, causal agent of ratoon stunting disease of sugarcane, with a polymerase chain reaction-based assay

A sensitive, specific polymerase chain reaction-based assay was developed for the detection of the causal agent of ratoon stunting disease of sugarcane, Clavibacter xyli subsp. xyli. This assay uses oligonucleotide primers derived from the internal transcribed spacer region between the 16S and 23S rRNA genes of the bacterial rRNA operon. The assay is specific for C. xyli subsp. xyli and does not produce an amplification product from the template of the closely related bacterium C. xyli subsp. cynodontis, nor from other bacterial species. The assay was successfully applied to the detection of C. xyli subsp. xyli in fibrovascular fluid extracted from sugarcane and was sensitive to approximately 22 cells per PCR assay. A multiplex PCR test was also developed which identified and differentiated C. xyli subsp. xyli and C. xyli subsp. cynodontis in a single PCR assay.

Immunohistochemistry and polymerase chain reaction on paraffin-embedded material improve the diagnosis of cutaneous leishmaniasis in the Amazon region

Background Recently, there has been an increase in the incidence of cutaneous leishmaniasis (CL), which represents an important health problem. This increase may be related to the epidemiologic expansion of the infective agent and the increase in tourism in tropical areas. The difficulty in clinical diagnosis, mainly in areas in which CL is not the first consideration of local physicians, has intensified efforts to describe diagnostic tests, which should be specific, sensitive, and practical. Amongst the new tests described are those including nucleic acid amplification (polymerase chain reaction, PCR) and immunohistochemistry (IHC). Methods In this study, we evaluated the sensitivity of a PCR based on small subunit (SSU) ribosomal DNA, in comparison with IHC using Leishmania spp. antibodies, in biopsies embedded in paraffin. Result The results indicated a total sensitivity of 96% (90.9% with PCR and 68.8% with IHC), showing the possibility of using paraffin-embedded biopsies to diagnose CL. Conclusion We propose the use of the two tests together as a routine protocol for diagnosis. This would require the provision of local medical services to perform molecular biology techniques and adequate Leishmania antibodies.

Dual candidemia detected by nested polymerase chain reaction in two critically ill children

The use of improved microbiological procedures associated with molecular techniques has increased the identification of Candida bloodstream infections, even if the isolation of more than one species by culture methods remains uncommon. We report the cases of two children presenting with severe gastrointestinal disorders and other risk factors that contribute to Candida infections. In the first patient, C. albicans DNA was initially detected by a nested-amplification and C. tropicalis was found later during hospitalization, while blood cultures were persistently negative. In the second child, there was amplification of C. albicans and C. glabrata DNA in the same samples, but blood cultures yielded only C. albicans. Both patients received antifungal therapy but had unfavorable outcomes. These two cases illustrate that PCR was more successful than culture methods in detecting Candida in the bloodstream of high risk children, and was also able to detect the presence of more than one species in the same patient that might impact therapy when the fungi are resistant to azole compounds.

Polymerase chain reaction compared to other laboratory findings and to clinical evaluation in the diagnosis of cutaneous tuberculosis and atypical mycobacteria skin infection

Cutaneous tuberculosis has re-emerged in the last 15 years together with the higher incidence of pulmonary tuberculosis and multidrug resistance. The choice for a single diagnostic tool among the many available today is a challenge. Our objective was to compare polymerase chain reaction (PCR) with other exams in the diagnosis of cutaneous tuberculosis and atypical mycobacteria skin infection. PCR and a set of five different exams were performed in 32 patients (34 samples of paraffin-embedded tissue) evaluated for 3 years in a university hospital, considering the response to mycobacterial infection treatment as a positive case. PCR was the most sensitive (88%) and specific (83%) exam. Culture, immunohistochemistry and acid-fast bacilli were not in agreement with clinical response to treatment. Although PCR is a useful tool, careful clinical exam is still the gold standard for the evaluation and treatment of cutaneous tuberculosis and mycobacteria skin infection.

Advantages and Pitfalls of the Polymerase Chain Reaction in the Diagnosis of Esophageal Ulcers in AIDS Patients

HIV-1-infected patients frequently have opportunistic esophageal infections which, when associated with severe immunodeficiency, can be attributed to unusual pathogens. The clinical presentation of several esophageal diseases is similar and the best method for a specific diagnosis of these patients has not been well defined. To evaluate the role of the polymerase chain reaction (PCR) in the etiologic definition of esophageal ulcers in HIV-1-infected patients, 96 esophageal biopsies from 79 HIV-1-infected patients were processed by PCR using specific primers for cytomegalovirus (CMV), herpes virus (HSV), human papilloma virus (HPV), HIV-1, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Treponema pallidum, and Haemophilus ducreyi. The PCR results were compared to the histopathologic results. Seventy-nine patients were studied (mean age: 34 years; 62% men; median CD4 + T cell = 103.59 cells/mu l (range 1-795.2 cells/mu l). The most common endoscopic findings were as follows: esophageal candidiasis (37.1%), esophageal ulcers (24.7%), esophagitis (11.2%), and lugol-negative areas (10.1%). The histopathologic findings in the esophageal ulcers (22 biopsies) were non-specific inflammation (31.8%), HSV (36.4%), Candida (13.6%), CMV (13.6%), or HPV disease (4.5%). In the esophageal ulcer biopsies, the PCR results were negative in 27.6% of cases, and positive for HIV (65.5%), CMV (31%), HPV (20.7%), HSV (10.3%), and H. ducreyi (6.9%). The histopathologic examination did not identify a pathogen or identified only Candida in 15 biopsies of esophageal ulcers. PCR was positive in ten (66.7%) and negative in five (33.3%) of these biopsies (idiopathic ulcers). PCR detected: HIV (53.3%), CMV (20%), HPV (13.3%), and H. ducreyi (6,7%). PCR detected more etiologic agents in esophageal ulcers than histopathology and was able to detect unusual pathogens. On the other hand, sometimes more than one pathogen was detected in the esophageal ulcers, making it difficult to reach an accurate diagnosis. This finding indicates the need for more studies to evaluate the benefit of this method in the routine evaluation of esophageal ulcer biopsies in HIV-1-infected patients.

Detection of dengue viral RNA in Aedes aegypti (Diptera : Culicidae) exposed to sticky lures using reverse-transcriptase polymerase chain reaction

Active surveillance for dengue (DEN) virus infected mosquitoes can be an effective way to predict the risk of dengue infection in a given area. However, doing so may pose logistical problems if mosquitoes must be kept alive or frozen fresh to detect DEN virus. In an attempt to simplify mosquito processing, we evaluated the usefulness of a sticky lure and a seminested reverse-transcriptase polymerase chain reaction assay (RT-PCR) for detecting DEN virus RNA under laboratory conditions using experimentally infected Aedes aegypti (L.) mosquitoes. In the first experiment, 40 male mosquitoes were inoculated with 0.13 mul of a 10(4) pfu/ml DEN-2 stock solution. After a 7-d incubation period, the mosquitoes were applied to the sticky lure and kept at room temperatures of 23-30 degreesC. Following 7,10,14, and 28 d application, 10 mosquitoes each were removed from the lure pooled and assayed for virus. DEN virus nucleic acid was clearly detectable in all pools up to 28 d after death. A second study evaluated sensitivity and specificity using one, two, and five DEN-infected mosquitoes removed after 7, 10, 14, 21 and 30 d application and tested by RT-PCR. All four DEN serotypes were individually inoculated in mosquitoes and evaluated using the same procedures as experiment 1. The four serotypes were detectable in as few as one mosquito 30 d after application to the lure with no evidence of cross-reactivity. The combination of sticky lures and RT-PCR show promise for mosquito and dengue virus surveillance and warrant further evaluation.

Development and evaluation of a species diagnostic polymerase chain reaction-restriction fragment-length polymorphism procedure for cryptic members of the Culex sitiens (Diptera : Culicidae) subgroup in Australia and the southwest Pacific

Members of the Culex sitiens subgroup are important vectors of arboviruses, including Japanese encephalitis virus, Murray Valley encephalitis virus and Ross River virus. Of the eight described species, Cx. annulirostris Skuse, Cx. sitiens Wiedemann, and Cx. palpalis Taylor appear to be the most abundant and widespread throughout northern Australia and Papua New Guinea (PNG). Recent investigations using allozymes have shown this subgroup to contain cryptic species that possess overlapping adult morphology. We report the development of a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) procedure that reliably separates these three species. This procedure utilizes the sequence variation in the ribosomal DNA ITS1 and demonstrates species-specific PCR-RFLP profiles from both colony and field collected material. Assessment of the consistency of this procedure was undertaken on mosquitoes sampled from a wide geographic area including Australia, PNG, and the Solomon Islands. Overlapping adult morphology was observed for Cx. annulirostris and Cx. palpalis in both northern Queensland and PNG and for all three species at one site in northwest Queensland.

Single step polymerase chain reaction (PCR) for the diagnosis of the Leishmania (Viannia) subgenus

In Brazil, the main etiologic agent of Leishmaniasis that frequently presents with mucosal involvement belongs to the Viannia subgenus. The therapeutic conduct in this disease depends on the parasitological diagnosis, and classical methods are restricted in identifying the agent. In this paper we describe a polymerase chain reaction (PCR), which uses primers designed from mini-exons repetitive sequences. The PCR amplifies a 177bp fragment that can distinguish (Viannia) from (Leishmania) subgenus. This test could be a useful diagnostic tool.

USE OF THE POLYMERASE CHAIN REACTION FOR THE DIAGNOSIS OF ASYMPTOMATIC Leishmania INFECTION IN A VISCERAL LEISHMANIASIS-ENDEMIC AREA

The diagnosis of asymptomatic infection with Leishmania (Leishmania) chagasi has become more important over recent years. Expansion of visceral leishmaniasis might be associated with other routes of transmission such as transfusion, congenital or even vector transmission, and subjects with asymptomatic infection are potential reservoirs. Moreover, the identification of infection may contribute to the management of patients with immunosuppressive conditions (HIV, transplants, use of immunomodulators) and to the assessment of the effectiveness of control measures. In this study, 149 subjects living in a visceral leishmaniasis endemic area were evaluated clinically and submitted to genus-specific polymerase chain reaction (PCR), serological testing, and the Montenegro skin test. Forty-nine (32.9%) of the subjects had a positive PCR result and none of them developed the disease within a follow-up period of three years. No association was observed between the results of PCR, serological and skin tests. A positive PCR result in subjects from the endemic area did not indicate a risk of progression to visceral leishmaniasis and was not associated with a positive result in the serological tests.

DETECTION OF Leishmania (Viannia) IN Nyssomyia neivai AND Nyssomyia whitmani BY MULTIPLEX POLYMERASE CHAIN REACTION, IN SOUTHERN BRAZIL

Sandflies transmit pathogens of leishmaniasis. The natural infection of sandflies by Leishmania (Viannia) was assessed in municipalities, in the state of Paraná, in Southern Brazil. Sandflies were collected with Falcão and Shannon traps. After dissection in search of flagellates in digestive tubes and identification of the species, female sandflies were submitted to the Multiplex Polymerase Chain Reaction (multiplex PCR) for detection of the fragment of the kDNA of Leishmania (Viannia) and the fragment from the IVS6 cacophony gene region of the phlebotomine insects. The analysis was performed in pools containing seven to 12 guts from females of the same species. A total of 510 female sandflies were analyzed, including nine Migonemyia migonei, 17 Pintomyia fischeri, 216 Nyssomyia neivai, and 268 Nyssomyia whitmani. Although none of the females was found naturally infected by flagellates through dissection, the fragment of DNA from Leishmania (Viannia) was shown by multiplex PCR in one sample of Ny. neivai (0.46%) and three samples of Ny. whitmani (1.12%). It was concluded that Ny. neivai and Ny. whitmani are susceptible to Leishmania infection, and that multiplex PCR can be used in epidemiological studies to detect the natural infection of the sandfly vector, because of its sensitivity, specificity and feasibility.