Usefulness of qualitative polymerase chain reaction for Trypanosoma cruzi DNA in endomyocardial biopsy specimens of chagasic heart transplant patients

BACKGROUND: Chagas` disease reactivation (CDR) after heart transplantation is characterized by relapse of the infectious disease, with direct detection of Trypanosoma cruzi parasites in blood, cerebrospinal fluid, or tissues. CDR affecting the myocardium induces lymphocytic myocarditis and should be distinguished from acute cellular rejection in endomyocardial biopsy (EMB) specimens. METHODS: We performed retrospectively qualitative polymerase chain reaction for T cruzi DNA using 2 sets of primers targeting nuclear DNA (nDNA) or kinetoplast DNA (kDNA) in 61 EMB specimens of 11 chagasic heart transplant recipients who presented with CDR. Thirty-five EMB specimens were obtained up to 6 months before (pre-CDR group) and 26 up to 2 years after the diagnosis of CDR. The control group consisted of 6 chagasic heart transplant recipients with 18 EMB specimens who never experienced CDR. RESULTS: Amplification of kDNA occurred in 8 of 35 (22.9%) EMB specimens of the pre-CDR group, in 5 of 18(27.8%) of the control group, and in 17 of 26(65.4%) EMB specimens obtained after the successful treatment of CDR. Amplification of nDNA occurred in 3 of 35 (8.6%) EMB specimens of the pre-CDR group, 0 of 18 (0%) of the control group, and 6 of 26 (23.1%) EMB specimens obtained after the successful treatment of CDR. CONCLUSIONS: Amplification of kDNA in EMB specimens is not specific for the diagnosis of CDR, occurring also in patients with no evidence of CDR (control group). However, amplification of nDNA occurred in a few EMB specimens obtained before CDR, but in none of the control group specimens. Qualitative PCR for T cruzi DNA in EMB specimens should not be used as a criterion for cure of CDR because it can persist positive despite favorable clinical evolution of the patients. J Heart Lung Transplant 2011;30:799-804 (C) 2011 International Society for Heart and Lung Transplantation. All rights reserved.

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 Leptospira spp. in semen and vaginal fluids of goats and sheep by polymerase chain reaction

Thirteen goat herds and seven sheep flocks in the state of Rio de Janeiro, Brazil were screened for leptospirosis. From the three herds and three flocks with greatest seroreactivity, 19 goats (16 females and three bucks) and 40 sheep (26 ewes and 14 rams) that were seropositive (specific anti-Leptospira titres >= 400, based on a microscopic agglutination test), were selected for more detailed studies. From those animals, samples of vaginal fluids or semen were collected for bacteriological and molecular assays. For both species of animals, the most prevalent reactions were to serovars Hardjo, Shermani, and Grippotyphosa. Although leptospires were detected by darkfield microscopy in three vaginal fluid samples (from two goats and one ewe), pure isolates were not obtained by bacteriological culture of vaginal fluids or semen. However, seven vaginal fluid samples (from four goats and three ewes) and six semen samples (all from rams) were positive on polymerase chain reaction (PCR). Based on these findings, in addition to analogous findings in cattle, we inferred that there is potential for venereal transmission of leptospirosis in small ruminants. (c) 2008 Elsevier Inc. All rights reserved.

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.

Polymerase chain reaction tests for the identification of Ross River, Kunjin and Murray Valley encephalitis virus infections in horses

Objective To develop and validate specific, sensitive and rapid diagnostic tests using RT-PCR for the detection of Ross River virus (RRV), Kunjin virus (KV) and Murray Valley encephalitis virus (MVEV) infections in horses. Methods Primer sets based on nucleotide sequence encoding the envelope glycoprotein E2 of RRV and on the nonstructural protein 5 (NS5) of KV and MVEV were designed and used in single round PCRs to test for the respective viruses in infected cell cultures and, in the case of RRV, in samples of horse blood and synovial fluid. Results The primer pairs designed for each of the three viruses amplified a product of expected size from prototype viruses that were grown in cell culture. The identity of each of the products was confirmed by nucleotide sequencing indicating that in the context used the RT-PCRs were specific. RRV was detected in serums from 8 horses for which there were clinical signs consistent with RRV infection such that an acute-phase serum sample was taken and submitted for RRV serology testing. The RRV RT-PCR was analytically sensitive in that it was estimated to detect as little as 50 TCID50 of RRV per mL of serum and was specific in that the primer pairs did not amplify other products from the 8 serum samples. The RRV primers also detected virus in three independent mosquito pools known to contain RRV by virus isolation in cell culture. Samples from horses suspected to be infected with KV and MVEV were not available. Conclusion Despite much anecdotal and serological evidence for infection of horses with RRV actual infection and associated clinical disease are infrequently confirmed. The availability of a specific and analytically sensitive RT-PCR for the detection of RRV provides additional opportunities to confirm the presence of this virus in clinical samples. The RTPCR primers for the diagnosis of KV and MVEV infections were shown to be specific for cell culture grown viruses but the further validation of these tests requires the availability of appropriate clinical samples from infected horses.

Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR) method

We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination

Detection of mycoplasmas in urethral swabs from HIV-1 infected patients and control individuals using culture techniques and polymerase chain reaction

The objective of the present study was to determine the prevalence of certain mycoplasma species, i.e., Mycoplasma hominis, Ureaplasma urealyticum and Mycoplasma penetrans, in urethral swabs from HIV-1 infected patients compared to swabs from a control group. Mycoplasmas were detected by routine culture techniques and by the Polymerase Chain Reaction (PCR) technique, using 16SrRNA generic primers of conserved region and Mycoplasma penetrans specific primers. The positivity rates obtained with the two methods were comparable. Nevertheless, PCR was more sensitive, while the culture techniques allowed the quantification of the isolates. The results showed no significant difference (p < 0.05) in positivity rates between the methods used for mycoplasma detection.

NEW PRIMERS FOR DETECTION OF Leishmania infantum USING POLYMERASE CHAIN REACTION

SUMMARY Leishmania infantum causes visceral leishmaniasis (VL) in the New World. The diagnosis of VL is confirmed by parasitological and serological tests, which are not always sensitive or specific. Our aim was to design new primers to perform a Polymerase Chain Reaction (PCR) for detecting L. infantum. Sequences of the minicircle kinetoplast DNA (kDNA) were obtained from GenBank, and the FLC2/RLC2 primers were designed. Samples of DNA from L. infantum, Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis, Leishmania major and Trypanosoma cruzi were used to standardize the PCR. PCR with FLC2/RLC2 primers amplified a fragment of 230 bp and the detection limit was 0.2 fg of L. infantum DNA. Of the parasite species assayed, only L. infantum DNA was amplified. After sequencing, the fragment was aligned to GenBank sequences, and showed (99%) homology with L. infantum. In the analysis of blood samples and lesion biopsy from a dog clinically suspected to have VL, the PCR detected DNA from L. infantum. In biopsy lesions from humans and dogs with cutaneous leishmaniasis, the PCR was negative. The PCR with FLC2/RLC2 primers showed high sensitivity and specificity, and constitutes a promising technique for the diagnosis of VL.

Polymerase chain reaction and restriction fragment length polymorphism analysis of the ITS2 region for differatiation of Brazilian Biomphalaria intermediate hosts of the Schistosoma mansoni

We sequenced the internal transcribed spacer 2 of the ribosomal DNA (ITS2-DNAr) from the three Schistosoma mansoni intermediate hosts in Brazil: Biomphalaria glabrata, Biomphalaria tenagophila and Biomphalaria straminea. Analysis of a restriction map from those sequences allowed us to select putative restriction enzymes able to identify the snail species under study. Four restriction enzymes were used and HpaII provided simple species-specific profiles easily visualized in polyacrylamide gels. The use of ITS2 is advantageous as it provides a small fragment of 460 bp which may be easily amplified by PCR. In the current work, we showed that the amplification of ITS2-DNAr together with HpaII enzyme restriction is an auxiliary molecular tool for the morphological identification of such snails as well as for taxonomic and phylogenetic studies of neotropical planorbids.

Polymerase chain reaction and nested-PCR approaches for detecting Cryptosporidium in water catchments of water treatment plants in Curitiba, State of Paraná, Brazil

Introduction Cryptosporidium is an important protozoan cause of waterborne disease worldwide of concern to public health authorities. To prevent outbreaks of cryptosporidiosis, the monitoring of this parasite in drinking water is necessary. In the present work, the polymerase chain reaction (PCR) and nested-PCR techniques were used to detect Cryptosporidium in raw water from catchment points of four water treatment plants (WTP) in Curitiba, Paraná, Brazil. Methods First, DNA extraction techniques were tested in samples containing decreasing amount of oocysts in reagent water, and PCR and nested-PCR with specific primers for 18SSU rDNA of Cryptosporidium were conducted to determine their sensitivity. In reagent water, a commercial extraction kit provided the best analytical sensitivity, and PCR and nested-PCR allowed the detection of five and two oocysts, respectively, with the primers XIAOR/XIAOF and XIAO1F/XIAO2R. Results In the spiking experiments, only the PCR with the primers AWA995F/AWA1206R was successful at detecting concentrations of 0.1 oocysts/mL. Two catchments samples of raw water and/or water sludge from four WTPs were contaminated with Cryptosporidium. Conclusions The application of the techniques to monitor Cryptosporidium in water and detect contamination in water catchments of WTPs in Curitiba are discussed in the present work.

Identification of species related to Anopheles (Nyssorhynchus) albitarsis by random amplified polymorphic DNA-Polymerase chain reaction (Diptera: Culicidae)

Species-specific Random Amplified Polymorphic DNA-Polymerase chain Reaction (RAPD-PCR) markers were used to identify four species related to Anopheles (Nyssorhynchus) albitarsis Lynch-Arribàlzaga from 12 sites in Brazil and 4 in Venezuela. In a previous study (Wilkerson et al. 1995), which included sites in Paraguay and Argentina, these four species were designated "A", "B", "C" and "D". It was hypothesized that species A is An. (Nys.) albitarsis, species B is undescribed, species C is An. (Nys) marajoara Galvão and Damasceno and species D is An. (Nys.) deaneorum Rosa-Freitas. Species D, previously characterized by RAPD-PCR from a small sample from northern Argentina and southern Brazil, is reported here from the type locality of An. (Nys.) deaneorum, Guajará-Mirim, state of Rondônia, Brazil. Species C and D were found by RAPD-PCR to be sympatric at Costa Marques, state of Rondônia, Brazil. Species A and C have yet to be encountered at the same locality. The RAPD markers for species C were found to be conserved over 4,620 km; from Iguape, state of São Paulo, Brazil to rio Socuavo, state of Zulia, Venezuela. RAPD-PCR was determined to be an effective means for the identification of unknown species within this species complex.

Quantitative real-time polymerase chain reaction for detection of adenovirus after T cell-replete hematopoietic cell transplantation: viral load as a marker for invasive disease.

BACKGROUND: The value of adenovirus plasma DNA detection as an indicator for adenovirus disease is unknown in the context of T cell-replete hematopoietic cell transplantation, of which adenovirus disease is an uncommon but serious complication. METHODS: Three groups of 62 T cell-replete hematopoietic cell transplant recipients were selected and tested for adenovirus in plasma by polymerase chain reaction. RESULTS: Adenovirus was detected in 21 (87.5%) of 24 patients with proven adenovirus disease (group 1), in 4 (21%) of 19 patients who shed adenovirus (group 2), and in 1 (10.5%) of 19 uninfected control patients. The maximum viral load was significantly higher in group 1 (median maximum viral load, 6.3x10(6) copies/mL; range, 0 to 1.0x10(9) copies/mL) than in group 2 (median maximum viral load, 0 copies/mL; range, 0 to 1.7x10(8) copies/mL; P<.001) and in group 3 (median maximum viral load, 0 copies/mL; range 0-40 copies/mL; P<.001). All patients in group 2 who developed adenoviremia had symptoms compatible with adenovirus disease (i.e., possible disease). A minimal plasma viral load of 10(3) copies/mL was detected in all patients with proven or possible disease. Adenoviremia was detectable at a median of 19.5 days (range, 8-48 days) and 24 days (range, 9-41 days) before death for patients with proven and possible adenovirus disease, respectively. CONCLUSION: Sustained or high-level adenoviremia appears to be a specific and sensitive indicator of adenovirus disease after T cell-replete hematopoietic cell transplantation. In the context of low prevalence of adenovirus disease, the use of polymerase chain reaction of plasma specimens to detect virus might be a valuable tool to identify and treat patients at risk for viral invasive disease.

A low stringency polymerase chain reaction approach to the identification of Biomphalaria glabrata and B. tenagophila, intermediate snail hosts of Schistosoma mansoni in Brazil

The low stringency-polymerase chain reaction (LS-PCR) with a pair of specific primers for the amplification of the 18S rRNA gene was evaluated as a means of differentiating between the two Schistosoma mansoni intermediate host species in Brazil: Biomphalaria glabrata and B. tenagophila. Individual snails obtained from different states of Brazil were used and the amplification patterns obtained showed a high degree of genetic variability in these species. Nevertheless, 4 and 3 clearly defined specific diagnostic bands was observed in individuals from B. glabrata and B. tenagophila respectively. The detection of snail specific diagnostic bands suggests the possibility of reliable species differentiation at the DNA level using LS-PCR.

Analysis of Respiratory Syncytial Virus in Clinical Samples by Reverse Transcriptase-Polymerase Chain Reaction Restriction Mapping

The aim of this study was to develop a polymerase chain reaction (PCR) for the detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respiratory ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100% sensitivity and 80% specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed