Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia

In this study, we have established and evaluated a genus-specific polymerase chain reaction (PCR) and species-specific nested PCRs for the detection of Candida species in blood samples of neutropenic mice and patients suspected of candidemia. DNA segments of the gene encoding cytochrome P450 L1A1 were targeted for amplification by using genus and species-specific primers. As compared to the genus-specific PCR, the species-specific nested PCRs improved the sensitivity by 10 times with the detection limit < 10 yeast cells. Of the 18 blood samples tested daily over a period of 8 days following Candida albicans infection in neutropenic mice, four samples were positive by genus-specific PCR and 11 were positive by species-specific nested PCR. The PCR results were correlated with culture findings obtained on blood samples. Two of the three blood culture-positive samples were positive by genus-specific PCR and all the three with species-specific nested PCR. Among 15 mice, which were negative by blood culture but had C. albicans isolated from visceral organs, 2 and 8 mice yielded positive results by genus-specific PCR and species-specific nested PCR, respectively. Consistent with the results of the animal study, species-specific nested PCR yielded much higher positivity as compared to culture (52.2% versus 21.2%) in patients suspected for candidemia. Moreover, 8 specimens which were negative for Candida by genus-specific PCR became positive by species-specific nested PCR. No correlation was apparent between PCR positivity and Candida antigen titers. The results suggest that nested PCR is a sensitive technique for the detection of Candida species from blood samples, and thus it may have application in the diagnosis of suspected cases of candidemia and candidiasis.     

Comparison of different PCR methods for detection of Brucella spp. in human blood samples

For detection of Brucella species by PCR four DNA extraction methods and four targets were compared using pure culture of Brucella melitensis and the best conditions were applied in clinical samples. It was found that the MagNA Pure LC method was the most efficient and sensitive method showing a positive PCR reaction with DNA extracted from as low as 25 and 100 CFU suspended in one ml blood and one ml water, respectively. Detection of Brucella spp. by conventional PCR was investigated using four different targets. The results indicated that The B4-B5 amplification method was the most sensitive one as it could amplify DNA extracted from as a low as 25 and 100 CFU/ml suspended in one ml water and blood, respectively. Furthermore real-time PCR was able to detect Brucella using DNA extracted from as low as 50 CFU/ml blood and 15 CFU/ml water, respectively. The best and optimum detection conditions were applied to the clinical samples. Evaluation of conventional PCR assays on blood specimens confirmed 72% of the results obtained by conventional blood culture methods with a specificity of 95%, while serum samples had a sensitivity of 54% and specificity of 100%. Real-time PCR was generally found to be more sensitive and specific for detecting Brucella spp. in blood and serum samples compared to conventional PCR. The real-time PCR done on blood specimens confirmed 77.5% of the results obtained by conventional blood culture methods with specificity of 100%, while 60% of serum samples were found to be positive with specificity of 100%. These results suggest that serum and blood analysis by conventional and real time PCR is a convenient and safe method for rapid and accurate diagnosis of brucellosis.     

Serological status for N. caninum, bovine viral diarrhea virus, and infectious bovine rhinotracheitis virus at pregnancy testing and reproductive performance in beef herds

Neospora caninum, infectious bovine rhinotracheitis (IBR), and bovine viral diarrhea virus (BVDV) are important differentials for the diagnosis of infectious reproductive loss in beef herds. The objective of this study was to describe the serological status of both pregnant and non-pregnant beef cows from herds with varying levels of reproductive success. The study provided an opportunity to examine whether there were any associations between serological status for BVDV, IBR, and N. caninum and pregnancy status, as well as the subsequent risk of abortion, or stillbirth. Samples were collected from 2516 cows and heifers from 66 herds; 31 herds where the proportion pregnant was <90% and 35 randomly selected herds where the proportion pregnant was ≥90%. Of these samples 5.9% were positive for antibodies to N. caninum, 20.4% had titres >1:80 to IBR, 91.8% had titres ≥1:256 to BVDV type 1, and 23.9% had titres ≥1:256 to BVDV type 2. N. caninum antibody concentration was associated with an increased individual animal risk of non-pregnancy (OR_log S/P, 1.9; 95% CI, 1.2–2.9) and abortion (OR_pos/neg, 2.8; 95% CI, 1.1–7.5). The proportion of animals at pregnancy testing with antibodies to BVDV type 2 above 1:3000 (OR_{10% change in prevalence}, 2.3; 95% CI, 1.5–3.5) was also associated with an increased risk of abortion. No other measures of antibody status were associated with reduced reproductive performance in this group of herds. Antibodies to Mycobacterium avium spp.paratuberculosis were also measured; 0.7% of samples were positive (sample to positive (S/P) >0.25) and 3.6% were suspicious (S/P, 0.10–0.25).     

Development of a semi-nested PCR based method for sensitive detection of tumorigenic Agrobacterium in soil

AIMS: To develop a specific, sensitive and rapid PCR-based method for detection of tumorigenic Agrobacterium in soil. METHODS AND RESULTS: Three newly designed primers complementary to tms2 gene amplified DNA of only the tumor-inducing agrobacteria of 113 strains tested, resulting in 617 bp and 458 bp products in the first and second rounds of semi-nested PCR respectively. As optimized method of pre-incubation of soil suspensions on selective medium, DNA isolation and two-round semi-nested PCR enabled detection of 1-2 bacterial cells in 1 g of soil. Using this method tumour-inducing Agrobacterium was detected in 67 of 69 samples of naturally infested soil originating from the field, where plants with crown gall symptoms occurred. The pathogen was detected only in two samples of 15 tested, collected from a nursery where crown gall symptoms were not observed. CONCLUSIONS: The semi-nested PCR-based method allowed for sensitive and rapid detection of tumorigenic agrobacteria in soil. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is proposed for testing of soil in fields intended for nursery production of fruit trees, roses or other plants susceptible to crown gall, as well as a tool for ecological studies.     

Detection of toxigenic Vibrio cholerae from environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure

A pit-stop semi-nested PCR assay for the detection of toxigenic Vibrio cholerae in environmental water samples was developed and its performance evaluated. The PCR technique amplifies sequences within the cholera toxin operon specific for toxigenic V. cholerae. The PCR procedure coupled with an enrichment culture detected as few as four V. cholerae organisms in pure culture. Treated sewage, surface, ground and drinking water samples were seeded with V. cholerae and following enrichment, a detection limit of as few as 1 V. cholerae cfu ml(-1) was obtained with amplification reactions from crude bacterial lysates. The proposed method, which includes a combination of enrichment, rapid sample preparation and a pit-stop semi-nested PCR, could be applicable in the rapid detection of toxigenic V. cholerae in environmental water samples.     

Influence of storage temperature on infectious hematopoietic necrosis virus detection by cell culture isolation and RT-PCR methods

The detection of infectious hematopoietic necrosis virus (IHNV) in infected rainbow trout Oncorhynchus mykiss and in cell culture supernatants stored under different conditions was studied. IHNV-positive fish visceral organ homogenates and cell culture supernatants were incubated at 4 and 25 degrees C. Virus titre was measured by virus isolation on epithelioma papulosum cyprini (EPC) cells and the IHNV RNA was detected by RT-PCR and semi-nested RT-PCR. The influence of repeated freezing and thawing on the virus isolation from organ homogenates and from cell culture supernatants was studied as well. It was possible to isolate the virus from IHNV-positive organ material during the 3 d of incubation at 4 degrees C but, only on the first day of incubation at 25 degrees C. Viral RNA could be amplified during the incubation period of 35 d at 4 degrees C but only during 8 d of incubation at 25 degrees C. In IHNV-infected cell culture supernatant stored at 4 degrees C, it was possible to detect virus for 36 and 16 d in supernatant stored at 25 degrees C. Viral RNA could be followed by using molecular methods during the entire experimental period of 123 d. Each cycle of freezing and thawing of samples resulted in a reduction of IHNV titre in the suspension of visceral organs, while the virus titre in cell culture supernatant remained almost the same following 33 freezing-thawing cycles. The present results show that rapid laboratory processing and storage of potentially virus-containing tissue samples as well as the use of different detection methods are very important for efficient IHNV diagnosis.     

Semi-nested polymerase chain reaction for detection of toxigenic <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from environmental water samples

A rapid and sensitive direct cell semi-nested PCR assay was developed for the detection of viable toxigenic V. cholerae in environmental water samples. The semi-nested PCR assay amplified cholera toxin (ctxA2B) gene present in the toxigenic V. cholerae. The detection sensitivity of direct cell semi-nested PCR was 2 × 10³ CFU of V. cholerae whereas direct cell single-step PCR could detect 2 × 10⁴ CFU of V. cholerae. The performance of the assay was evaluated using environmental water samples after spiking with known number of Vibrio cholerae O1. The spiked water samples were filtered through a 0.22 micrometer membrane and the bacteria retained on filters were enriched in alkaline peptone water and then used directly in the PCR assay. The semi-nested PCR procedure coupled with enrichment could detect less than 1 CFU/ml in ground water and sea water whereas 2 CFU/ml and 20 CFU/ml could be detected in pond water and tap water, respectively. The proposed method is simple, faster than the conventional detection assays and can be used for screening of drinking water or environmental water samples for the presence of toxigenic V. cholerae.     

Population genetic structure among populations of three predaceous nabid species: Nabis alternatus Parshley, Nabis roseipennis Reuter and Nabis americoferous Carayon (Hemiptera: Nabidae)

An electrophoretic survey of 28 presumptive loci distributed among 15 enzyme systems was conducted to determine the genetic structure among local populations of three predaceous nabid species: Nabis alternatus, N. roseipennis and N.americoferous in human-disturbed habitat. For all three species, F_IS values indicated a deficiency of heterozygotes at most loci in most populations. The amount of genetic differentiation based on the standardized variance, F_ST when combined across all poulations was 0.100 for N. alternatus, 0.189 for N. roseipennis, and 0.335 for N. americoferous. When local populations were grouped according to a set of defined geographical limits, based on the naturally occurring spatial arrangement of the sampled localities, some unexpected patterns of genetic variation were revealed. The degree of genetic differentiation detected among local populations of nabids is discussed in light of the frequent disruptions and ecological changes in their ephemeral habitats.     

Pediatric visceral leishmaniasis diagnosis in Tunisia: comparative study between optimised PCR assays and parasitological methods

There has been a steady increase of visceral leishmaniasis during the past 20 years in Tunisia. In this study, we assess the value of two optimised PCR versus those of classical methods for the diagnosis of human visceral leishmaniasis. 106 samples were collected from 53 cases of pediatric visceral leishmaniasis. Peripheral blood and bone marrow samples were analysed both by parasitological methods (direct examination, leukocytoconcentration (LCC) and culture) and by PCR methods with two primer pair (R221/R332 and Lei 70L/Lei 70R). We diagnosed visceral leishmaniasis in all patients: 44 cases were diagnosed by culture (83%), 42 by direct examination of bone marrow (79%), 17 by LCC (32%), and 53 positive cases with both PCR assays (R221/R332 and/or Lei 70L/Lei 70R) (100 %). Regarding each PCR assay, for blood samples, the difference between the sensitivities of PCR Lei 70L/Lei 70R (86,8%) and PCR R221/R332 (17 %) is statistically significant with p-value 0.025. For bone marrow, the sensitivities of the two PCR methods were respectively 96,2% (Lei 70L/Lei 70R) and 75,5% (R221/R332). On the whole, PCR Lei 70L/Lei 70R was more effective than PCR R221/R332 and conventional methods for the two biological samples. Moreover, the requirement of less invasive sample using blood has the advantage of being repeatable for screening and for post therapeutic monitoring.     

大熊猫轮状病毒PCR检测方法的建立及应用

轮状病毒是威胁大熊猫健康的主要病原微生物之一。为了对大熊猫轮状病毒进行快速、方便且准确地检测,研发适合于基层饲养单位和保护区的检测方法是非常有必要的。本研究通过合成大熊猫轮状病毒Vp7基因序列,构建了PUC-VP7的重组质粒,并将其作为阳性对照对大熊猫轮状病毒样本进行PCR检测和分析。结果表明,在进行PCR扩增分析时,该质粒和病毒cDNA二者均在340 bp处出现了特异性条带。此外,对收集到的45份大熊猫粪便样本进行轮状病毒抗原检测时,其中2份样品在340 bp处出现条带,该基因片段与大熊猫轮状病毒CH-1株的相似性为99.89%。本研究构建的PUC-VP7质粒不但可以作为大熊猫轮状病毒PCR检测中的阳性质控品,而且还能有效地促进该PCR病毒检测技术在基层饲养单位和保护区的推广和应用。     

Sequencing a specific kinetoplast DNA fragment of Leishmania donovani for polymerase chain reaction amplification in diagnosis of leishmaniasis in bone marrow and blood samples

A set of oligonucleotide primers I and II was developed by analyzing the specificity of a cloned kinetoplast DNA (kDNA) fragment of Leishmania donovani and sequencing the fragment. Polymerase chain reaction (PCR) was conducted with the primers to amplify a minicircle kDNA fragment (297 bp) to detect L. donovani in the bone marrow (22 samples), whole blood (16 samples), and serum (17 samples) of 22 patients with visceral leishmaniasis. All of 22 patients were diagnosed by microscopic identification. Control samples of bone marrow, whole blood, and serum were obtained from patients with leukemia and from healthy volunteers. In addition, 12 dogs were infected with L. donovani promastigotes for the PCR test. The total number of patients positive by PCR testing was 95.5% (21/22), with 91.0% (20/22) from the bone marrow, 68.8% (11/16) from the blood, and 29.4% (5/17) from the sera. Similar results were obtained in infected dogs. No amplification products were seen in control samples from humans or dogs. Our results suggest that PCR may be useful in detecting kDNA in the bone marrow and blood of patients with visceral leishmaniasis.     

A survey of malarial infection in endemic areas of Savannakhet province, Lao PDR and comparative diagnostic efficiencies of Giemsa staining, acridine orange staining, and semi-nested multiplex PCR

Malaria remains one of the most important parasitic diseases in Lao PDR, especially in forested rural areas. Knowing the rate of infection using highly sensitive and specific methods, and the factors related to malarial infection, may be helpful in reducing the infection and mortality rates. We aimed to study the malarial infection rate by comparing three detection methods, i.e., Giemsa staining, acridine orange (AO) staining and semi-nested multiplex PCR. The study also included some factors related to malarial infection in the endemic areas of Savannakhet province, Lao PDR. The respective malarial infection rates by Giemsa staining, AO staining and semi-nested multiplex PCR in Houy Jang vs. Keng Thong villages were 13.1 vs. 20.8, 16.2 vs. 25.4 and 20.8 vs. 30.8%. The infection rate among children not over 10 years of age was higher than infection rate among the older ages (p = 0.002, Z-test for two proportions). The higher infection rates by semi-nested multiplex PCR over Giemsa and AO staining suggest the existence of many subclinical cases with low level parasitemia, undetected by microscopic techniques. We found no mixed infections using Giemsa or AO staining, but using semi-nested multiplex PCR we found 1.2% (3/260) mixed P. falciparum and P. vivax infections, suggesting that semi-nested multiplex PCR is suitable for detecting malarial infection from endemic areas whose cases may have low parasitemia and/or mixed infection. The factors significantly related to malarial infection from 260 questionnaires were: (1) children and young adults, (2) not having lived in the area more than 5 years, and (3) not using a mosquito net over the bed, indicating an increased risk of new residents of contracting malaria and a need to promote bed nets.     

Survey of Theileria parasites of sheep in eastern Turkey using polymerase chain reaction

This study provides the first molecular data on infection Theileria of sheep in Turkey. A total of 218 blood samples were collected from sheep in five distinct geographical locations of eastern Turkey. Theileria piroplasm DNAs, extracted from sheep blood, were subjected to the polymerase chain reaction (PCR) procedures, using specific primers for Theileria spp. and Theileria lestoquardi. Blood smears were examined for Theileria piroplasms by microscopic observation. In PCR analysis, the expected amplicons were obtained from 90 (41.2%) blood samples with a molecular size of 1098 bp for Theileria spp. whereas none were amplified by Theileria lestoquardi-specific primers. Piroplasms of Theileria spp. were detected in 34 (15.5%) of the samples.     

Detection and quantitative measurement of transforming growth factor-beta1 (TGF-beta1) gene expression using a semi-nested competitive PCR assay

A polymerase chain reaction (PCR) technique was optimized for detection and quantification of very low concentrations (down to a few molecules) of transforming growth factor beta1 (TGF-beta1) mRNA. The strategy involved a combination of a competitive PCR assay and a semi-nested PCR. In the present study, the semi-nested PCR technique was tested in several rat organs containing different concentrations of target mRNA. A control fragment for TGF-beta1 was used to correct for differences in amplification of various cDNA samples. TGF-beta1 mRNA levels were also corrected according to the abundance of the "housekeeping" gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the same samples. The differences of sensitivity among the standard (one-step) and semi-nested (two-step) competitive PCR assays for the detection of TGF-beta1 are discussed. In conclusion, the semi-nested PCR protocol provides greatly enhanced sensitivity over standard PCR analysis. It is a reproducible and very specific method for quantification of only a few molecules of TGF-beta1 mRNA in a background of non-target molecules.     

Comparison of culture and PCR for the detection of Brucella melitensis in blood and lymphoid tissues of serologically positive and negative slaughtered sheep

Aims: To compare the culture and PCR methods for detection of Brucella melitensis in blood and lymphoid tissue samples obtained from slaughtered sheep (n = 162) testing positive/negative in serological tests (Rose Bengal test and serum agglutination test). Methods and Results: Of 162 sheep examined, 45 were positive and 117 negative in serological tests. A PCR assay based on a pair of Br. melitensis‐specific primers was used to detect DNA in blood and lymphoid tissue. Brucella melitensis was isolated from 1·2% (2/162) and 17·2% (28/162) of the blood and lymphoid tissue samples respectively. Positive PCR products with a molecular size of 731 bp were obtained from 27·7% (45/162) of blood and 29·0% (47/162) of lymphoid tissue samples. Conclusions: The species‐specific PCR assay detected a higher number of Br. melitensis DNA both from serologically positive (P < 0·01 in blood PCR, P < 0·001 in tissue PCR) and serologically negative (P < 0·001 in both blood PCR and tissue PCR) sheep compared with classical bacteriological culture methods. Significance and Impact of the Study: The results emphasize the importance of using more than one type of diagnostic technique for the detection of animals positive for brucellosis, especially with epidemiological purposes.     

Development and Evaluation of a Blood Culture PCR Assay for Rapid Detection of Salmonella Paratyphi A in Clinical Samples

Background

Enteric fever remains an important cause of morbidity in many low-income countries and Salmonella Paratyphi A has emerged as the aetiological agent in an increasing proportion of cases. Lack of adequate diagnostics hinders early diagnosis and prompt treatment of both typhoid and paratyphoid but development of assays to identify paratyphoid has been particularly neglected. Here we describe the development of a rapid and sensitive blood culture PCR method for detection of Salmonella Paratyphi A from blood, potentially allowing for appropriate diagnosis and antimicrobial treatment to be initiated on the same day.

Methods

Venous blood samples from volunteers experimentally challenged orally with Salmonella Paratyphi A, who subsequently developed paratyphoid, were taken on the day of diagnosis; 10 ml for quantitative blood culture and automated blood culture, and 5 ml for blood culture PCR. In the latter assay, bacteria were grown in tryptone soy broth containing 2.4% ox bile and micrococcal nuclease for 5 hours (37°C) before bacterial DNA was isolated for PCR detection targeting the fliC-a gene of Salmonella Paratyphi A.

Results

An optimized broth containing 2.4% ox bile and micrococcal nuclease, as well as a PCR test was developed for a blood culture PCR assay of Salmonella Paratyphi A. The volunteers diagnosed with paratyphoid had a median bacterial burden of 1 (range 0.1–6.9) CFU/ml blood. All the blood culture PCR positive cases where a positive bacterial growth was shown by quantitative blood culture had a bacterial burden of ≥ 0.3 CFU/ ml blood. The blood culture PCR assay identified an equal number of positive cases as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood), but utilized only half the volume of specimens.

Conclusions

The blood culture PCR method for detection of Salmonella Paratyphi A can be completed within 9 hours and offers the potential for same-day diagnosis of enteric fever. Using 5 ml blood, it exhibited a lower limit of detection equal to 0.3 CFU/ml blood, and it performed at least as well as automated blood culture at higher bacterial loads (≥0.3 CFU/ml blood) of clinical specimens despite using half the volume of blood. The findings warrant its further study in endemic populations with a potential use as a novel diagnostic which fills the present gap of paratyphoid diagnostics.     

Detection of the protozoan Neospora caninum Using in situ Polymerase Chain Reaction

A new method to detect the protozoan Neospora caninum using indirect in situ polymerase chain reaction (PCR) is described. In situ PCR combines the advantages of the extraordinarily high sensitivity and specificity of PCR and the in situ representation of immunohistochemical methods. We describe an indirect in situ PCR, whereby the amplified products were detected using a primed in situ (PRINS) reaction with hapten-labeled nucleotides and visualized using fluorochrome-labeled antibodies. This technique was carried out in both infected cell cultures and formalin fixed, paraffin embedded tissues. Clear signals were obtained in the N. caninum positive samples using in situ PCR, whereas control slides with Toxoplasma gondii infected tissues always yielded negative results.     

Detection of Wuchereria bancrofti DNA in paired serum and urine samples using polymerase chain reaction-based systems

The Global Program for the Elimination of Lymphatic Filariasis (GPELF) aims to eliminate this disease by the year 2020. However, the development of more specific and sensitive tests is important for the success of the GPELF. The present study aimed to standardise polymerase chain reaction (PCR)-based systems for the diagnosis of filariasis in serum and urine. Twenty paired biological urine and serum samples from individuals already known to be positive for Wuchereria bancrofti were collected during the day. Conventional PCR and semi-nested PCR assays were optimised. The detection limit of the technique for purified W. bancrofti DNA extracted from adult worms was 10 fg for the internal systems (WbF/Wb2) and 0.1 fg by using semi-nested PCR. The specificity of the primers was confirmed experimentally by amplification of 1 ng of purified genomic DNA from other species of parasites. Evaluation of the paired urine and serum samples by the semi-nested PCR technique indicated only two of the 20 tested individuals were positive, whereas the simple internal PCR system (WbF/Wb2), which has highly promising performance, revealed that all the patients were positive using both samples. This study successfully demonstrated the possibility of using the PCR technique on urine for the diagnosis of W. bancrofti infection.     

聚合酶链式反应检测结核杆菌的研究

以人型结核杆菌基因组DNA为模板,合成二段引物各20个碱基进行聚合酶链式反应(PCR)。经琼脂糖凝胶电泳证实,获得一条245bp扩增带。PCR检测的敏感性染色体基因组DNA为1pg,菌悬液为13个活菌／ml。在特异性试验中,人型结核杆趋,牛型结核杆菌、BCG可见此扩增带。被试的其它14种扰酸菌以及变铅青链霉菌、大肠杆菌质粒Puc19、星状诺卡氏菌、红球菌均未见该扩增带。54例肺结核痰标本3种方法检查的阳性率分别为：萋尼氏抗酸染色16．7％,培养法14．8％,PCR 37．0％。前2种检查方法分别与PCR比较,经统计学处理均有显著性差异(P<0．01)。12例非结核性肺部疾患痰标本抗酸染色和PCR均为阴性。结果表明,PCR技术是快速、敏感、特异诊断结核病的方法。     

人结核分枝杆菌特异性核酸探针制备及结核病PCR检测技术的初步临床应用

采用人结核分枝杆菌(Mycobacterium tuberculosis TB)染色体DNA为模板,选择位于插入片段IS6110中884～865和568～588碱基对处的两个片段为引物,扩增出317bp的特异性片段．将其克隆进pUCl9载体。酶切图谱分析和DNA序列测定证实为目的片段。该片段经DIG标记,分别与11种分枝杆菌DNA进行Southern杂交,结果证明只与人型复合分枝杆菌发生杂交反应。利用该对引物建立的PcR检测拄术对74份结核病痰液标本进行检测,并与临床细菌快速培养结果相比较,发现48份临床阳性均为PcR阳性,在26份临床阴性标本中亦发现11份PCR检测阳性。将标本PCR产物与克隆探针进行杂交,显示两者结果完全一致。说明PCR检测体系结果可靠,其灵敏度明显高于目前临床所采用的方法,可作为一种常规技术用于结核病的临床检测。