Real-time Polymerase Chain Reaction Targeting Research Articles

Ameliorative effects of propolis and wheat germ oil on acute toxoplasmosis in experimentally infected mice are associated with reduction in parasite burden and restoration of histopathological changes in the brain, uterus, and kidney.

Toxoplasmosis continues to be a prevalent parasitic zoonosis with a global distribution. This disease is caused by an intracellular parasite known as Toxoplasma gondii, and the development of effective novel drug targets to combat it is imperative. There is limited information available on the potential advantages of wheat germ oil (WGO) and propolis, both individually and in combination, against the acute phase of toxoplasmosis. In this study, acute toxoplasmosis was induced in Swiss albino mice, followed by the treatment of infected animals with WGO and propolis, either separately or in combination. After 10 days of experimental infection and treatment, mice from all groups were sacrificed, and their brains, uteri, and kidneys were excised for histopathological assessment. Additionally, the average parasite load in the brain was determined through parasitological assessment, and quantification of the parasite was performed using Real-Time Polymerase Chain Reaction targeting gene amplification. Remarkably, the study found that treating infected animals with wheat germ oil and propolis significantly reduced the parasite load compared to the control group that was infected but not treated. Moreover, the group treated with a combination of wheat germ oil and propolis exhibited a markedly greater reduction in parasitic load compared to the other groups. Similarly, the combination treatment effectively restored the histopathological changes observed in the brain, uterus, and kidney, and the scoring of these reported lesions confirmed these findings. In summary, the present results reveal intriguing insights into the potential therapeutic benefits of wheat germ oil and propolis in the treatment of acute toxoplasmosis.

Molecular Surveillance of Yersinia pestis from Stray Dogs and Cats and their Fleas in Algiers

In recent years, plague has re-emerged in several countries around the world and remains endemic in some regions. In a natural environment, and in contact with rodents and their fleas, stray carnivores are most at risk of catching the disease and maintaining the spread of the bacillus. The objective of this study is to demonstrate the presence or absence of Yersinia pestis in stray dogs and cats in the Algiers region by molecular methods and thus contribute their role in epidemiology of this disease. Molecular research of Yersinia pestis has also been conducted on fleas from these carnivores. Preliminary identification of ectoparasites to genus and species level was performed. Real-time polymerase chain reaction targeting Yersinia pestis pla gene was used to survey the plague agent in fleas and carnivores captured as stray animals in Algiers (Algeria). Positive qPCR results were tested by PCR sequencing using glpD gene. Among 327 fleas captured from 107 dogs and 365 fleas from 140 cats, prevalence of Ctenocephalides felis was higher in cats (86,96%), whereas that of Ctenocephalides canis and Xenopsylla cheopis was higher in dogs (90,57% and 92,63% respectively). While internal and external PCR positive controls were positive, none of the 107 dogs spleen and 140 cat spleens and none of the 256 analyzed fleas were positive for Y. pestis. These results suggest that stray cats and dogs are unlikely sources for plague in Algeria contrary to that has been reported in other plague-endemic countries. This observation illustrates that plague epidemiological chain varies from one region to another one.

Comparison between Two Molecular Techniques: Nested and Real-Time Polymerase Chain Reaction Targeting 100-kDa Hc Protein for Detection of Histoplasma capsulatum in Environmental Samples.

ABSTRACT.Histoplasmosis, one of the most frequent endemic mycoses in the Americas, is caused by the inhalation of airborne conidia of Histoplasma capsulatum. Better understanding of the distribution of this fungus in the environment is important for the development of appropriate public health measures to prevent human infections. Previously, we used Hc100 nested polymerase chain reaction (PCR) to identify H. capsulatum DNA in 10% of environmental samples in Colombia. Here, we validate a 100-kDa real-time PCR assay for the detection of this fungus in the environment. Using this method, we identified H. capsulatum DNA in 80% of samples of raw organic materials, such as chicken manure, soil from caves, and bird and bat guano, as well as in 62% of samples of organic fertilizer that underwent the composting process. We demonstrated that 100-KDa real-time PCR is a useful tool for environmental surveillance that can be used to identify the potential reservoirs of H. capsulatum and to prevent outbreaks, especially in people with the higher risk of exposure, such as spelunkers, farmers, poultry manure collectors, and anyone who handle organic fertilizers or bat and bird excreta.

Systematic evaluation of TaqMan real-time polymerase chain reaction assays targeting the dsb and gltA loci of Ehrlichia canis in recombinant plasmids and naturally infected dogs.

Background and Aim:Because of the diversity of local genotypes of Ehrlichia canis, genes targeted by TaqMan real-time polymerase chain reaction (RT-PCR) assays should be systematically evaluated. This study evaluated the amplification efficiency, linearity, precision, and sensitivity of two TaqMan RT-PCR assays targeting the dsb and gltA loci of E. canis in recombinant plasmids and naturally infected dogs.Materials and Methods:Thirty blood samples were collected from dogs showing clinical signs of canine monocytic ehrlichiosis at the Kasetsart University Veterinary Teaching Hospital, Bangkok, Thailand. The dsb and gltA genes were amplified by conventional PCRs (cPCRs) on the blood samples and were then sequenced. Meanwhile, RT-PCR was used to detect dsb and gltA genes in 10-fold dilutions of the recombinant plasmids.Results:Both dsb and gltA were amplified with a high degree of linearity (R2≥0.975 and 0.993, respectively) in all dilutions, although the mean percentage of relative standard deviation of gltA was lower, but the difference was non-significant. The detection limits of RT-PCR and cPCR were 10−7 and 10−6, respectively, for both loci. RT-PCR targeting dsb (22/30; 73.3%) and gltA (15/30; 50%) yielded a number of positive results that did not differ significantly (p=0.06). The RT-PCR positive results of the dsb gene (22/30) differed significantly from that of cPCR (11/30) (p=0.004). In contrast, the RT-PCR positive results of the gltA gene (15/30) did not differ significantly from that of cPCR (12/30) (p=0.43). The mean Ct value (30.2) based on dsb RT-PCR of 22 positive cases was higher than that of gltA RT-PCR (Ct=27.4) on 15 positive cases. The Ct values from dsb RT-PCR were >30 in all seven discordant samples that were not detected by the gltA RT-PCR.Conclusion:RT-PCR targeting the dsb gene was more sensitive for detecting E. canis in naturally infected dogs. This study suggested that TaqMan RT-PCR of the dsb gene should be selected for E. canis research in this region.

An automated real‐time PCR assay for synovial fluid improves the preoperative etiological diagnosis of periprosthetic joint infection and septic arthritis

Synovial fluid is important for the preoperative etiological diagnosis of suspected periprosthetic joint infection (PJI) or septic arthritis (SA). GENECUBE, an automated real-time polymerase chain reaction (PCR) assay, was used to detect bacterial mecA (methicillin resistance) and was compared with microbiological cultures for preoperatively diagnosing PJI and SA in 74 patients suspected of these infections and thus earmarked for surgery. PJI and SA were diagnosed in 21 and 6 cases, respectively, using modified ICM 2018 diagnostic criteria. Microbiological cultures determined methicillin-resistant staphylococcus (MRS) as the causative organism in six samples, which were all positive in the GENECUBE assay. Significantly also, the GENECUBE assay detected six MRS infections in culture-negative but infection-diagnosed patients, and in one inconclusive case, suggesting a higher sensitivity of this assay. Compared with microbiological culture, the sensitivity and specificity of the GENECUBE assay for mecAwas 100% and 92.2%, respectively. However, GENECUBE also produced invalid results in three cases, suggesting possible PCR inhibitors in the synovial fluid samples. We additionally validated the accuracy of pan-bacterial real-time PCR targeting 16S rRNA and other tests. Pan-bacterial real-time PCR was as effective as preoperative bacterial culture testing, although the α-defensin assay had the highest sensitivity at 100%. Hence, fully automated real-time PCR targeting of the bacterial mecA gene improves the etiological diagnosis of PJI and SA by reducing the testing time and lowering the false-positive detection rates. A screening approach for α-defensin followed by bacterial mecA gene testing in synovial fluids is therefore a more efficient method of preoperatively diagnosing PJI and SA.

Evaluation of real time polymerase chain reaction targeting mpb64 gene for diagnosis of extrapulmonary tuberculosis

BackgroundPaucibacillary nature of extrapulmonary tuberculosis (EPTB) has paved way for molecular methods increasingly being used for diagnosis. We undertook a study for evaluation of sensitivity and specificity of real-time polymerase chain reaction (RT-PCR) targeting mpb64 gene for diagnosis of EPTB. MethodsA total of 152 clinical samples from suspected cases of EPTB were included in this study. All samples were extracted using spin column based commercial DNA extraction kit and were subjected to RT-PCR targeting mpb64 and IS6110. Smear and culture was also done for samples whenever quantity was sufficient. Cytology report was noted from hospital information system. Receiver operating characteristic (ROC) curve analysis was done for determining cut-off Ct value for mpb64 RT-PCR. Melt curve analysis was done for samples whose cycle threshold (Ct) value was more than 37. The sensitivity and specificity of the mpb64 RT-PCR was calculated using a composite gold standard i.e., positive for one or more of the following: microscopy (including fine needle aspiration cytology (FNAC), acid-fast bacilli positivity), culture and IS6110 RT-PCR. ResultsOut of the 152 samples, 72 (47.4%) were positive for tuberculosis by composite gold standard. Samples consisted of ascitic fluid (12), CSF (35), pus (23), lymph node aspirate (35), pleural fluid (37), synovial fluid (4), urine (1), pericardial fluid (1) and tissue bits (4). Microscopy (AFB smear including lymph node aspirate) was done for 124 samples of which 43 (34.7%) were positive. Culture results were available for 79 samples, 25 (31.6%) of which were positive and 42 (27.6%) of the 152 samples were positive by IS6110 PCR. Based on ROC and melt curve analysis, mpb64 RT-PCR was able to detect 38 (52.8%) of the 72 positive samples. In comparison to IS6110 RT PCR, 4 additional cases were detected by mpb64 RT-PCR. Compared to composite gold standard mpb64 showed overall sensitivity of 52.8%. ConclusionThe mpb64 RT-PCR is highly specific or MTB and can be used as a supplemental test for diagnosis of EPTB along with other diagnostic tests. However the overall sensitivity of mpb64 RT-PCR is too low to be used as an independent test for diagnosis of EPTB. Combining the results of IS6110 RT PCR and mpb64 RT PCR improved the overall sensitivity and hence mpb64 can be used as an additional target for diagnosis of EPTB.

Comparison of an in house and a commercial real-time polymerase chain reaction targeting Toxoplasma gondii RE gene using various samples collected from patients in Turkey

BackgroundToxoplasma gondii is an opportunistic protozoan parasite that can infect all warm-blooded animals including humans and cause serious clinical manifestations. Toxoplasmosis can be diagnosed using histological, serological, and molecular methods. In this study, we aimed to detect T. gondii RE gene in various human samples by in house and commercial real time polymerase chain reactions.MethodsA total of 38 suspected cases of toxoplasmosis [peripheral blood (n:12), amnion fluid (n:11), tissue (n:9), cerebrospinal fluid (n:5), and intraocular fluid (n:1)] were included to the study. An in house and a commercial RT-PCR were applied to investigate the T. gondii RE gene in these samples.ResultsThe compatibility rate of the two tests was 94.7% (37/38). When the commercial RT-PCR kit was taken as reference, the sensitivity and specificity of in house RT-PCR test was 87.5 and 100%. When the in house RT-PCR test was taken as reference, the commercial RT-PCR kit has 100% sensitivity and 96.8% specificity. Incompatibility was detected in only in a buffy coat sample with high protein content.ConclusionsBoth the commercial and in house RT-PCR tests can be used to investigate T. gondii RE gene in various clinical specimens with their high sensitivity and specificity. In house RT-PCR assay can be favorable due to cost savings compared to using the commercial test.

Comparison of Nested Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction Targeting 47kDa Gene for the Diagnosis of Scrub Typhus

AbstractIntroduction: Scrub typhus is a zoonotic infection caused by Orientia tsutsugamushi which is transmitted by Leptotrombidium mites. The disease manifests as a mild-to-severe illness with non-specific clinical symptoms. Rapid diagnosis and prompt treatment are essential for patient management. Both serological and molecular methods are used for the diagnosis of scrub typhus. The present study assessed the usefulness of detection of the gene encoding the 47kDa outer-membrane protein (OMP) for the laboratory diagnosis of scrub typhus. Materials and Methods: Nested polymerase chain reaction (nPCR) and real-time PCR targeting 47 kDa OMP antigen gene of O. tsutsugamushi were performed on ethylenediaminetetraacetic acid blood samples. Results: Six of the 103 (5.8%) patients showed the presence of 47kDa gene by nPCR. Seventy of 103 (67.9%) cases showed the presence of 47kDa gene by qPCR. Among the 70 positive cases, the majority of them were females (40/70, 57.1%). The highest number of positive cases was observed during October–February. Conclusion: Real-time PCR targeting O. tsutsugamushi-specific 47-kDa gene is more sensitive than nPCR and may be the assay of choice for the detection of the organism in patients with suspected scrub typhus.

Detection of Bartonella quintana in African body and head lice.

Currently, the body louse is the only recognized vector of Bartonella quintana, an organism that causes trench fever. In this work, we investigated the prevalence of this bacterium in human lice in different African countries. We tested 616 head lice and 424 body lice from nine African countries using real-time polymerase chain reaction targeting intergenic spacer region 2 and specific B. quintana genes. Overall, B. quintana DNA was found in 54% and 2% of body and head lice, respectively. Our results also show that there are more body lice positive for B. quintana in poor countries, which was determined by the gross domestic product, than in wealthy areas (228/403 versus 0/21, P < 0.001). A similar finding was obtained for head lice (8/226 versus 2/390, P = 0.007). Our findings suggest that head lice in Africa may be infected by B. quintana when patients live in poor economic conditions and are also exposed to body lice.

Molecular Diagnostic Tools for the Detection of Nodal Micrometastases in Prostate Cancer Patients Undergoing Radical Prostatectomy with Extended Pelvic Lymph Node Dissection: A Prospective Study

Routine pathological examination can miss micro-metastatic tumor foci in the lymph nodes (LN) of patients with prostate cancer (PCa) that undergo radical prostatectomy and pelvic lymph node dissection (PLND). The aim of the present prospective study was to evaluate the impact of micrometastases assessed by serial section (SS), immunohistochemistry (IHC), and Real-time Polymerase Chain Reaction (RT-PCR) in patients undergoing radical prostatectomy with extended PLND. 32 consecutive patients who underwent radical prostatectomy with extended PLND (obturator, internal/external and distal 2 cm common iliac lymph-nodes (LN)) for intermediate (clinical T1c-T2 and PSA:10-20 ng/mL and clinical Gleason Score = 7) or high (clinical stage T3 or PSA>20 or clinical Gleason Score = 8-10) PCa were enrolled. The nodes were processed by the one uropathologist, both according to the routine pathological examination (analysis of the central section for 4 mm nodes or every 2 mm for LN>4 mm), which served as comparative method, both according to SS, IHC with antibodies against PSA and broad-spectrum Cytokeratins (BSCK), and quantitative RT-PCR targeting PSA, PSMA (PS Membrane Antigen), and Glucuronidase-S-Beta (GUSB) mRNA, that are over-expressed in prostatic cancer cells. A total of 628 LN were analyzed, with a mean number of LN removed of 19.6 (SD = 7.2). Applying the routine pathological examination, 10 (31.2%) patients and 23 (3.9%) LN resulted positive for nodal involvement, with mean positive LN of 2.2 (SD = 1.4). After applying the SS and the molecular method of analysis (IHC and RT-PCR), micrometastases were found in 7 LN (SS showed micrometastases in 3 of them, IHC in 6 of them and RT-PCR in 7 of them); a total of 3 (9.3%) node-negative patients showed micrometastases at routine pathological examination (in 2 patients with RT-PCR and in 1 with IHC). The significance of micrometastases in PCa and the potential therapeutic role of PLND is not yet clarified, but the molecular analysis of the LN can detect a significant percentage of patients who harbor micro-metastatic PCa missed at routine pathological examination, and can enhance the accuracy of lymphadenectomy as a staging method.

An advanced PCR method for the specific detection of viable total coliform bacteria in pasteurized milk

Pasteurized milk is a complex food that contains various inhibitors of polymerase chain reaction (PCR) and may contain a large number of dead bacteria, depending on the milking conditions and environment. Ethidium monoazide bromide (EMA)-PCR is occasionally used to distinguish between viable and dead bacteria in foods other than pasteurized milk. EMA is a DNA-intercalating dye that selectively permeates the compromised cell membranes of dead bacteria and cleaves DNA. Usually, EMA-PCR techniques reduce the detection of dead bacteria by up to 3.5 logs compared with techniques that do not use EMA. However, this difference may still be insufficient to suppress the amplification of DNA from dead Gram-negative bacteria (e.g., total coliform bacteria) if they are present in pasteurized milk in large numbers. Thus, false positives may result. We developed a new method that uses real-time PCR targeting of a long DNA template (16S-23S rRNA gene, principally 2,451bp) following EMA treatment to completely suppress the amplification of DNA of up to 7logs (10(7)cells) of dead total coliforms. Furthermore, we found that a low dose of proteinase K (25U/ml) removed PCR inhibitors and simultaneously increased the signal from viable coliform bacteria. In conclusion, our simple protocol specifically detects viable total coliforms in pasteurized milk at an initial count of ≥1colony forming unit (CFU)/2.22ml within 7.5h of total testing time. This detection limit for viable cells complies with the requirements for the analysis of total coliforms in pasteurized milk set by the Japanese Sanitation Act (which specifies <1CFU/2.22ml).

Diagnosis of Bacteremia in Febrile Neutropenic Episodes in Children With Cancer

Bacterial isolation using conventional microbiologic techniques rarely surpasses 25% in children with clinical and laboratory findings indicative of an invasive bacterial infection. The aim of this study was to determine the role of real-time polymerase chain reaction (RT-PCR) from whole blood samples compared with automated blood cultures (BC) in detection of relevant microorganisms causing bacteremia in episodes of high-risk febrile neutropenia (HRFN) in children with cancer. Children presenting with HRFN at 6 hospitals in Santiago, Chile, were invited to participate. Blood samples were obtained at admission for BC, and at admission and 24 hours for RT-PCR targeting DNA of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa causing bacteremia in children with HRFN. A total of 177 HRFN episodes were evaluated from May 2009 to August 2010, of which 29 (16.3%) had positive BC, 9 (5%) positive for 1 of the 3 selected bacterial species: 5 for E. coli, 3 for S. aureus, and 1 for P. aeruginosa. RT-PCR detected 39 bacteria in 36 episodes (20%): 14 E. coli, 20 S. aureus, and 5 P. aeruginosa. The sensitivity, specificity, and positive and negative predictive values of RT-PCR compared with BC were 56%, 80%, 13%, and 97%. The final clinical diagnosis was compatible with an invasive bacterial infection in 30/36 (83%) RT-PCR-positive episodes. In our series, RT-PCR significantly improved detection of the most relevant bacteria associated with HRFN episodes. Large number of patients and close clinical monitoring, in addition to improved RT-PCR techniques will be required to fully recommend RT-PCR-based diagnosis for the routine workup of children with cancer, fever, and neutropenia.

Low-Frequency K103N Strengthens the Impact of Transmitted Drug Resistance on Virologic Responses to First-Line Efavirenz or Nevirapine-Based Highly Active Antiretroviral Therapy

There are conflicting data on the impact of low-frequency transmitted drug-resistant mutants on responses to first-line highly active antiretroviral therapy (HAART). Patients started nevirapine or efavirenz with two or more nucleoside/nucleotide reverse transcriptase inhibitors in 1998-2007 without a prior resistance test at a median 1.0 (interquartile range, 0.0-3.4) year after diagnosis and with a median 218 (interquartile range, 131-296) CD4 cells/mm3, and had at least 24 weeks of follow up. Pre-HAART plasma samples were tested retrospectively by bulk genotyping and sensitive real-time polymerase chain reaction targeting reverse transcriptase K65R, K103N, Y181C, M184V, and G190A (interpretative cutoff 0.3%-0.9%). Among 93 patients, seven of 18 who experienced virologic failure and zero of 75 who maintained virologic suppression showed pre-HAART resistance, including three with high-frequency mutations detectable by bulk genotyping (two K103N, one G190A) and four with low-frequency K103N detectable only by polymerase chain reaction. Detection of either bulk (P = 0.006) or low-frequency (P = 0.001) resistance was significantly associated with the odds of virologic failure; combining the two markedly increased the strength of the association (P < 0.0001). At failure, the pre-HAART mutations were detected by bulk genotyping in five of seven patients alongside additional reverse transcriptase mutations. Low-frequency K103N mutants were as prevalent as bulk-detectable variants before starting HAART. Both high- and low-frequency mutants were significantly associated with virologic failure.