Evaluation of a new assay for HBV DNA quantitation in patients with chronic hepatitis B

Background: The Amplicor™ HBV Monitor Test for quantitative determination of serum hepatitis B virus (HBV) DNA has recently been introduced. This assay is based on PCR and a non-radioactive hybridization and detection system on microwell plates.Objective: The performance of the Amplicor™ HBV Monitor Test was evaluated in a routine diagnostic laboratory. The Amplicor™ HBV Monoitor assay was compared to the Digene Hybrid Capture™ System HBV DNA assay for the quantitation of HBV in patient sera.Study design: Sensitivity and reproducibility were determined with 10-fold dilution series of two Eurohep HBV reference plasma specimens. Furthermore, 196 sera from 14 children with chronic HBV infection and interferon therapy were tested with both assays.Results: The detection limit was found to be 10³ copies/ml with the Amplicor™ PCR assay compared to 10⁶ to 10⁷ copies/ml with the Digene™ hybridization assay. Both assays were quasi-linear over the measurable ranges. The new PCR assay proved to be very reliable. With the Amplicor™ PCR assay, 26.2% of the HBV DNA-positive clinical samples were found between 10³ and 10⁷ copies/ml and all of them tested below the detection limit with the hybridization assay.Conclusion: The Amplicor™ HBV Monitor Test shows excellent sensitivity and provides a valuable tool for the detection of HBV DNA in serum. It can be used for recognizing those patients who might benefit from antiviral therapy, for evaluation of the efficacy of anti-HBV therapy, and for validation of blood products.     

A nested PCR assay to detect DNA in sera for the diagnosis of deep-seated trichosporonosis

Deep-seated trichosporonosis caused by Trichosporon asahii has a high mortality rate and a very poor prognosis. New species-specific oligonucleotide primers for T. asahii were developed from a sequence analysis of rRNA genes that included the internal transcribed spacer regions. A nested PCR assay with specific primers was used to examine 11 serum samples from 7 patients, who were diagnosed with deep-seated trichosporonosis histologically at autopsy. In addition, Trichosporon cell wall polysaccharide (PS) was detected by a latex agglutination (LA) test. Of 11 samples, seven had a positive LA test, and T. asahii DNA was also detected with the nested PCR assay. Of the four samples in which PS antigen was not detected, the nested PCR of two samples was positive. Our new nested PCR assay may be used as an adjunct to conventional methods for diagnosing T. asahii infection.     

Clinicopathologic features and genotyping of patients with chronic HBV infection in the Upper Egypt

The aim of this study was to determine the clinicopathologic features and Hepatitis B virus genotypes in HBV-infected patients in the Upper Egypt. Eighty-three HBsAg-positive patients (28 carriers, 14 with chronic hepatitis, 32 with liver cirrhosis and 9 with hepatocellular carcinoma) were enrolled. Blood was collected and serum samples obtained were screened for Hepatitis markers genotyping was conducted for 6 HBV genotypes (A through F) using a method for genotyping HBV by primer specific polymerase chain reaction. Genotype D was the only genotype detected in different clinical forms of chronic HBV infection (carriers, chronic hepatitis, cirrhosis and hepatocellular carcinoma) and, in all patients who had elevated or normal alanine aminotransferase levels and in all ages. HBeAg was absent in 78 patients suggesting the presence of pre-core or core mutations. Positive correlation was found among serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), histological activity index and grade of hepatitis. This study provides the first indication about the clinicopathologic features of HBV-infected patients in the Upper Egypt. It also reports the predominance of genotype D in this region.     

In vitro investigation of HBV clinical isolates from Chinese patients reveals that genotype C isolates possess higher infectivity than genotype B isolates

Hepatitis B virus (HBV) genotype B and C are two major genotypes that are prevalent in Asia and differ in natural history and disease progression. The impact of HBV genotypes on viral replication and protein expression has been explored by the transfection of hepatoma cells with replication-competent HBV DNA, which mimics the later stages of the viral life cycle. However, the influence of HBV genotypes on the early events of viral infection remains undetermined, mainly due to the difficulties in obtaining sufficient infectious viral particles for infection assays. Here, we report that a high-titer HBV inoculum can be generated from the transient transfection-based cell model after optimizing transfection conditions and modifying the HBV-expressing construct. By performing in vitro infection assays using transiently transfected derived viruses, we found that clinical genotype C isolates possessed higher infectivity than genotype B isolates. Moreover, we identified a naturally occurring mutation sL21S in small hepatitis B surface protein, which markedly decreased the infectivity of HBV genotype C isolates, but not that of genotype B isolates. In summary, using infectious viral particles provided by the optimized transient transfection-based cell model, we have been able to investigate a wide range of HBV variants on viral infectivity, which may contribute to our understanding of the reasons for different clinical outcomes in HBV infections and the development of therapeutic drugs targeting the early stages of HBV life cycle.     

A nested real-time PCR assay has an increased sensitivity suitable for detection of viruses in aerosol studies

Aims:  Influenza is commonly spread by infectious aerosols; however, detection of viruses in aerosols is not sensitive enough to confirm the characteristics of virus aerosols. The aim of this study was to develop an assay for respiratory viruses sufficiently sensitive to be used in epidemiological studies.
Method:  A two-step, nested real-time PCR assay was developed for MS2 bacteriophage, and for influenza A and B, parainfluenza 1 and human respiratory syncytial virus. Outer primer pairs were designed to nest each existing real-time PCR assay. The sensitivities of the nested real-time PCR assays were compared to those of existing real-time PCR assays. Both assays were applied in an aerosol study to compare their detection limits in air samples.
Conclusions:  The nested real-time PCR assays were found to be several logs more sensitive than the real-time PCR assays, with lower levels of virus detected at lower Ct values. The nested real-time PCR assay successfully detected MS2 in air samples, whereas the real-time assay did not.
Significance and Impact of the Study:  The sensitive assays for respiratory viruses will permit further research using air samples from naturally generated virus aerosols. This will inform current knowledge regarding the risks associated with the spread of viruses through aerosol transmission.     

A real-time PCR assay for the rapid determination of 16S rRNA genotype in Vibrio vulnificus

In a terminal restriction fragment polymorphism (T-RFLP) study, we recently reported a significant association between the type B 16S rRNA gene and clinical strains of Vibrio vulnificus associated with the consumption of raw oysters. In the present study we describe a real-time PCR assay for the rapid determination of the 16S rRNA type of V. vulnificus isolates. This assay was used to reexamine the 16S rRNA gene type in the strains studied previously by T-RFLP and additional isolates from selected sources. Analyses revealed that 15 of the strains (10 environmental and 5 clinical) previously found to be 16S rRNA type A actually appear to possess both the type A and B genes. The presence of both alleles was confirmed by cloning and sequencing both gene types from one strain. To our knowledge, this is the first report of 16S rRNA sequence heterogeneity within individual strains of V. vulnificus. The findings confirm the T-RFLP data that 16S rRNA type may be a useful marker for determining the clinical significance of V. vulnificus in disease in humans and cultured eels. The real-time PCR assay is much more rapid and less resource-intensive than T-RFLP, and should facilitate further study of the occurrence and distribution of the 16S rRNA genotypes of V. vulnificus. These studies should provide more definitive estimates of the risks associated with this organism and may lead to a better understanding of its virulence mechanism(s).     

A rapid infection assay for Armillaria and real-time PCR quantitation of the fungal biomass in planta

Slow and unreliable infection in the greenhouse has been a barrier to research on Armillaria root disease. The existing infection assay takes 7–18 months for detectable infection, during which time the inoculum often dies, resulting in unequal challenge among plants. Because symptom expression and mortality are rare, presence or absence of infection, determined by culturing, is the only datum derived from the existing infection assay. This limits both routine comparisons of strain virulence and complex investigations of pathogenesis, neither of which have been done for Armillaria mellea. We tested a new infection assay, in which grape rootstocks growing in tissue culture medium are inoculated, and compared to rootstocks previously characterized from the existing infection assay as tolerant (Freedom) or susceptible (3309C). Culture media of 25 plants per rootstock was inoculated and five plants per rootstock were harvested 0, 2, 4, 6, and 8 weeks postinoculation; the experiment was completed twice. Confocal microscopy and quantitative PCR (Q-PCR) were used to quantify infection. Roots were treated with WGA-AlexaFluor488, hyphae and roots were scanned on green and red channels on a confocal microscope, and percent root colonization was quantified. A fungal gene (EF1α) was determined to have a single copy in A. mellea, and both EF1α and a single-copy grape gene (UFGT) were amplified by Q-PCR; fungal DNA: plant DNA served as a measure of fungal biomass. Armillaria was detected by culture, microscopy, and Q-PCR starting 2 weeks postinoculation from all inoculated plants, demonstrating that the new infection assay is rapid and plants do not escape infection. Our findings of higher percent root colonization (as measured by microscopy) of 3309C than Freedom at all harvests (P < 0.0001), consistently higher fungal biomass (as measured by Q-PCR) of 3309 than Freedom, and a significant positive correlation between percent root colonization and fungal biomass (P = 0.01) suggests that the quantitative methods of our new assay give similar results to the qualitative method of the existing infection assay.     

重庆地区乙肝血清特殊模式患者HBV基因分型及临床自然病程分析

目的了解重庆地区乙肝病毒（HBV）血清学标志物为特殊模式的HBV感染患者病毒基因型的分布情况,分析其临床特征及自然病程。方法从1000例HBV感染者中检测到48例乙肝病毒血清学标志物为特殊模式的患者（HBsAg与抗一HBs同时阳性,HBeAg与抗一HBe同时阳性）。采用巢式聚合酶链式反应（nPCR）对特殊模式患者的HBV进行基因分型,同时对两组特殊模式患者的临床资料和HBV感染的自然史进行分析。结果48例乙肝病毒血清学标志物为特殊模式的HBV感染者中,36例患者HBsAg与抗-HBs同时阳性,12例患者HBeAg与抗-HBe同时阳性。HBeAg＋／抗-HBe＋患者组的年龄较HBsAg＋／抗-HBs＋患者组的小（P〈0．05）。HBsAg＋／抗-HBs＋患者中,3例（8．3％）为B2亚型,12例（33．3％）为c2亚型,21例（58．4％）未分型;HBeAg＋／抗-HBe＋患者中,8例（66．7％）为B2亚型,1例（8．3％）为c2亚型,3例（25．0％）未分型,两组在HBV基因型的分布上差异具有统计学意义（Y2=17．44,P〈0．05）。在HBsAg＋／抗-HBs＋患者中,2例（4．2％）处于免疫清除期,14例（29．2％）处于低复制期,7例（14．6％）处于再活动期。HBeAg＋／抗-HBe＋患者中,5例（10．4％）处于免疫清除期。两组在HBV感染的自然病程中的分布差异具有统计学意义（X2=18．26,P〈0．05）。结论重庆地区乙肝病毒血清学标志物为特殊模式的慢性HBV感染者中,HBeAg与抗-HBe同时阳性的HBV感染者中B2亚型为优势基因型;HBsAg与抗-HBs同时阳性的HBV感染者中,HBV基因型以C2亚型为主。     

PCR for diagnosis of male Trichomonas vaginalis infection with chronic prostatitis and urethritis

The aim of this study was to assess the usefulness of PCR for diagnosis of Trichomonas vaginalis infection among male patients with chronic recurrent prostatitis and urethritis. Between June 2001 and December 2003, a total of 33 patients visited the Department of Urology, Hanyang University Guri Hospital and were examined for T. vaginalis infection by PCR and culture in TYM medium. For the PCR, we used primers based on a repetitive sequence cloned from T. vaginalis (TV-E650). Voided bladder urine (VB1 and VB3) was sampled from 33 men with symptoms of lower urinary tract infection (urethral charge, residual urine sensation, and frequency). Culture failed to detect any T. vaginalis infection whereas PCR identified 7 cases of trichomoniasis (21.2%). Five of the 7 cases had been diagnosed with prostatitis and 2 with urethritis. PCR for the 5 prostatitis cases yielded a positive 330 bp band from bothVB1 and VB3, whereas positive results were only obtained from VB1 for the 2 urethritis patients. We showed that the PCR method could detect T. vaginalis when there was only 1 T. vaginalis cell per PCR mixture. Our results strongly support the usefulness of PCR on urine samples for detecting T. vaginalis in chronic prostatitis and urethritis patients.     

A fast and efficient method for screening of nested deletions using PCR

Summary A procedure is presented for using PCR to screen individual colonies, representing clones obtained after controlled unidirectional exonuclease III digestion, for the sizes of the individual inserts. Many clones can be screened at one time since the plasmid templates used for amplification need not be purified but are prepared from individual colonies by a simple procedure that takes less than 30 minutes.     

A specific PCR assay for the diagnosis of Clonorchis sinensis infection in humans, cats and fishes

Clonorchiasis caused by Clonorchis sinensis is a fish-borne parasitic disease which is endemic in a number of countries. Using the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of C. sinensis as genetic markers, a pair of C. sinensis-specific primers was designed and used to establish a specific PCR assay for the diagnosis of C. sinensis infection in humans, cats and fish. This approach allowed the specific identification of C. sinensis after optimizing amplification conditions, with no amplicons being amplified from related heterogeneous DNA samples, and sequencing of amplicons confirmed the identity of the sequences amplified. The detection limit of this assay was 1.03 pg of adult C. sinensis, 1.1 metacercariae per gram of fish filet, and a single egg in human and cat feces. The PCR assay should provide a useful tool for the diagnosis and molecular epidemiological investigation of clonorchiasis in humans and animals.     

Serum microRNAs as biomarkers for hepatocellular carcinoma in Chinese patients with chronic hepatitis B virus infection

Background

MicroRNAs (miRNAs) have been shown to anticipate great cancer diagnostic potential. Recently, circulating miRNAs have been reported as promising biomarkers for various pathologic conditions. The objective of this study was to investigate the potential of serum miRNAs as novel biomarkers for hepatocellular carcinoma (HCC).

Methodology/Principal Findings

This study was divided into four phases: (I) Ten candidate serum miRNAs were detected by using real-time RT-PCR, corresponding 10 HCC patients with chronic hepatitis B virus (HBV) infection and 10 age- and sex-matched healthy subjects. (II) Marker validation by real-time RT-PCR on HBV patients with (n = 48) or without HCC (n = 48), and healthy subjects (n = 24). (III) Marker detection by real-time RT-PCR in sera from another 14 HCC patients before and 1 month after surgical resection. (IV) We examined the correlation between the expressions of candidate serum miRNAs with clinical parameters of HCC patients. Although miR-222, miR-223 or miR-21 were significantly up- or down-regulated between HCC patients and healthy controls, no significant difference was observed in the levels of these miRNAs between HBV patients without and with HCC. MiR-122 in serum was significantly higher in HCC patients than healthy controls (p<0.001). More importantly, it was found that the levels of miR-122 were significantly reduced in the post-operative serum samples when compared to the pre-operative samples. Although serum miR-122 was also elevated in HBV patients with HCC comparing with those without HCC, the difference was at the border line (p = 0.043).

Conclusions/Significance

Our results suggest that serum miR-122 might serve as a novel and potential noninvasive biomarker for detection of HCC in healthy subjects, moreover, it might serve as a novel biomarker for liver injury but not specifically for detection of HCC in chronic HBV infection patients.     

A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.)

A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V‐C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA‐A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl₂, Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low‐cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V‐C.

Significance and Impact of the Study

Incidence of phytoplasma diseases is increasing worldwide and particularly in the tropical and subtropical world. Co‐infection of phytoplasma and virus(s) is also common. Therefore, use of single primer PCR in detecting these pathogens would require more time and effort, whereas multiplex PCR involving several pairs of primers saves time and reduces cost. In this study, we have developed a multiplex nested PCR assay that provides more sensitive and specific detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma in white jute simultaneously. It is the first report of simultaneous detection of CoGMV and a phytoplasma in Corchorus capsularis by multiplex nested PCR.     

Performance of nested multiplex PCR assay targeting MTP40 and IS6110 gene sequences for the diagnosis of tubercular lymphadenitis

The conventional methods for diagnosis of tubercular lymphadenitis (TBLN) such as - fine needle aspiration cytology, Ziehl-Neelsen staining and culture have limitations of low sensitivity and/or specificity. So, it becomes essential to develop a rapid, sensitive, and specific method for an early diagnosis of TBLN. Therefore, the present study was conducted to evaluate nested multiplex polymerase chain reaction (nMPCR) targeting MTP40 and IS6110 gene sequences of Mycobacterium tuberculosis and Mycobacterium tuberculosis complex, respectively in 48 successive patients of TBLN and 20 random patients with non-tubercular lymph node lesions. Out of the 48 cases of TBLN, 14 (29.2%) were found to be positive by Ziehl-Neelsen staining, 15 (31.2%) were positive by culture and 43 (89.6%) cases were positive after first round of PCR while 48 (100%) cases were positive by nMPCR assay. The sensitivity and specificity of nMPCR was found to be 100% for the diagnosis of TBLN. The results thus obtained indicate that nMPCR assay is a highly sensitive and specific tool for the diagnosis of TBLN.     

乙型肝炎病毒树鼩体内慢性感染模型的研究历程和展望

乙型肝炎病毒(HBV)感染是全球和我国的重要健康问题,建立方便有效的HBV感染动物体内模型是开展HBV相关基础和临床研究的重要前提。该实验室地处乙型肝炎和肝癌高发流行的广西地区,自建立以来一直以肝癌的病因、发病机制和化学预防作为主要研究方向,于20世纪80年代初开始树鼩体内感染HBV模型方面的研究并持续至今。该文对此研究历程作一概述,并重点介绍关于用新生树鼩慢性感染HBV的新进展。     

In situ detection of the Clostridium botulinum type C1 toxin gene in wetland sediments with a nested PCR assay.

A nested PCR was developed for detection of the Clostridium botulinum type C1 toxin gene in sediments collected from wetlands where avian botulism outbreaks had or had not occurred. The C1 toxin gene was detected in 16 of 18 sites, demonstrating both the ubiquitous distribution of C. botulinum type C in wetland sediments and the sensitivity of the detection assay.     

Circulating circRNA predicting the occurrence of hepatocellular carcinoma in patients with HBV infection

A microarray‐based high‐throughput screening of human circulating circular RNA (circRNA) was applied with five patients newly diagnosed with hepatocellular carcinoma (HCC), five patients with HBV‐positive chronic hepatitis (CH) and five healthy controls (NC) enrolled. The plasma of HCC patients after hepatectomy was also collected. After multiple staged validation, we obtained five circRNAs as candidate. Based on the stratified risk score analysis, three increased circRNAs including circ_0009582, circ_0037120 and circ_0140117 were confirmed as candidate circulating fingerprints for distinguishing HCC from CH or NC group. With the combination of AFP, higher sensitivity and specificity were further guaranteed, suggesting that circ_0009582, circ_0037120 and circ_0140117 may serve as potential biomarkers for predicting the occurrence of HCC in patients with HBV infection.     

A new LAMP-based assay for the molecular diagnosis of toxoplasmosis: comparison with a proficient PCR assay

Toxoplasmosis is generally a benign infection caused by the protozoan parasite Toxoplasma gondii but can have severe consequences in fetuses of mothers infected during pregnancy (congenital toxoplasmosis) and immunocompromised individuals. PCR-based diagnostic tests have become crucial for its diagnosis. However, this molecular diagnosis essentially relies upon laboratory-developed methods and suffers from a lack of standardization, leading to great variation in methods and performance among laboratories. With the need for accreditation of clinical microbiological laboratories, the use of commercial PCR kits has become an attractive alternative; but thorough evaluation of newly commercialized kits by proficient groups is necessary before any recommendation can be made to parasitology laboratories by health authorities or learned societies. Here, we compared the performance of an original commercial method, the Iam TOXO Q-LAMP (DiaSorin®), using Loop-mediated isothermal amplification (LAMP) technology, with our reference laboratory-developed method using real-time PCR. The kit was first tested using amniotic fluid (AF) and plasma samples (either negative or spiked with live T. gondii tachyzoites at different concentrations (from 7 to 10⁵?tachyzoites/mL)). It was then assessed using a cohort of 11 AF, five placental and 32 blood clinical samples preserved at ?20?°C. For the processing of placental/blood samples, a pretreatment step was used, which did not strictly follow the manufacturer’s recommendations. The practical ease of use and compliance with good laboratory practices were also evaluated. Although the LAMP assay was less sensitive than the laboratory-developed method at very low parasite concentrations (0.1?T. gondii genome equivalents/mL), the two methods yielded identical results qualitatively and, in some instances, quantitatively, particularly for AF samples.