Virus morphology as an aid for rapid diagnosis.

Standard methods of virus diagnosis may take many days to complete. As antiviral drugs are being used with more effectiveness, it becomes more important to develop rapid diagnostic methods. It takes only a few minutes to prepare and examine a specimen for electron microscopy (EM), using the negative staining technique. Viruses in the specimen can readily be identified by their morphology. In order to be detected by EM there must be at least 10(7) virus particles per milliliter of sample. This concentration is frequently found in certain types of specimens. The sensitivity of EM is increased 100-fold if homologous antibody is used to aggregate the virus. Visualization of virus-antibody aggregates forms the basis for serotyping by immunoelectron microscopy (IEM).     

Comparison of Three Methods Used to Isolate Dengue Virus Type 2

During the 1969 dengue epidemic in Puerto Rico, human sera and Aedes aegypti mosquitoes were collected for virus isolation and identification. Three methods of isolation were used and compared. In the first method, we inoculated newborn mice by the intracranial route, noted any signs of illness, and serially passed specimens in mice until virus was isolated. In the second method, we inoculated tube cultures of LLC-MK(2) cells, noted any cytopathic effect (CPE), and assayed fluids for virus by plaque formation in LLC-MK(2) cell monolayers. The third method was different from the second only in that the original specimens were first inoculated into fluid cultures of Singh's A. albopictus cells. No significant CPE was seen in LLC-MK(2) cultures; however, distinct syncytial CPE was observed in A. albopictus cells. About the same number of virus isolates were made in each isolation system. Virus isolates from both sera and mosquitoes were identified as dengue type 2 by a plaque-reduction neutralization test in LLC-MK(2) cells. The utility of the three methods, individually or in combination, is discussed and related to diagnostic and epidemic situations.     

Rapid viral diagnosis: role of electron microscopy

Starting in the 1960, electron microscopy (EM) became widely applied also in viral diagnosis. During the 1970th and 80th, many new agents were characterized from diagnostic cell cultures and clinical specimens. The wide introduction of ELISA- and PCR-techniques as well as cost-arguments recently reduced the role of EM in routine viral diagnosis. Compared to other diagnostic techniques, however, EM excells by speed and "open view", i.e. by the ability to detect also the "un-expected" without the need for specific reagents. As shown in 2003 by the elucidation of the SARS pandemia and the human monkeypox outbreak in US, EM is well suited as a safe, front-line diagnostic method in infectious diseases emergencies and/or in possible bioterrorist attacks.     

Electron microscopy: essentials for viral structure, morphogenesis and rapid diagnosis

Electron microscopy(EM) should be used in the front line for detection of agents in emergencies and bioterrorism,on accounts of its speed and accuracy.However,the number of EM diagnostic laboratories has decreased considerably and an increasing number of people encounter difficulties with EM results.Therefore,the research on viral structure and morphology has become important in EM diagnostic practice.EM has several technological advantages,and should be a fundamental tool in clinical diagnosis of viruses,particularly when agents are unknown or unsuspected.In this article,we review the historical contribution of EM to virology,and its use in virus differentiation,localization of specific virus antigens,virus-cell interaction,and viral morphogenesis.It is essential that EM investigations are based on clinical and comprehensive pathogenesis data from light or confocal microscopy.Furthermore,avoidance of artifacts or false results is necessary to exploit fully the advantages while minimizing its limitations.     

The use of biomagnetic separation to recover DNA suitable for PCR from Claviceps species

DNA analysis of agriculturally important fungi using polymerase chain reaction (PCR)-based methods is becoming routine in research and for diagnostic purposes. Rapid, small-scale DNA isolation methods that take advantage of the sensitivity, speed and automation potential of PCR technology are needed for timely analysis of important plant pathogens. DNA isolated from Claviceps africana (causal agent of ergot of sorghum) using several standard DNA extraction protocols was found to be unamplifiable using PCR. The standard methods apparently failed to separate DNA from substances inhibitory to the Taq polymerase enzyme. We obtained DNA amenable to PCR analysis using a novel method involving magnetic beads and high salt extraction buffer. The biomagnetic purification method allowed us to obtain reliable PCR amplification of the internal transcribed spacer (ITS) regions of rDNA of Claviceps africana, making genetic comparisons possible.     

An Occurrence of Diarrheal Cases Associated with Group C Rotavirus in Adults

Six of the 23 college students who joined a group trip in February of 1991 developed acute nonbacterial gastroenteritis with severe diarrhea. The causal agent was identified as group C rotaviruses by electron microscopy (EM), immune-EM (IEM) and the molecular examinations including polyacrylamide gel electrophoresis (PAGE) and polymerase chain reaction (PCR) on virus particles detected in the extract of watery fecal specimens of the patients. The patients positive for virus isolation showed significant increase in IEM antibody to the isolated virus in their paired sera. These findings suggest that the group C rotavirus is an important etiological agent of diarrhea and may also cause serious food-borne diarrheal disease in adults.     

PCR Amplification and Species Determination of Microsporidia in Formalin-Fixed Feces after Immunomagnetic Separation

The term microsporidia is used to describe several species of opportunistic protozoan parasites. Encephalitozoon intestinalis and Enterocytozoon bieneusi have been found in stools of more than 40% of AIDS patients with diarrhea. Diagnosis of infection with these small protozoans has been difficult, and until recently their occurrence has not been well documented. Formalin is widely used to preserve clinical specimens, but due to the nature of the fixation process, subsequent analysis, especially analysis by the PCR, is difficult. This study evaluated methods used to prepare formalin-fixed fecal specimens for PCR amplification of microsporidial DNA. Two methods were devised to allow PCR detection and subsequent identification of microsporidia in formalin-fixed fecal specimens to the species level. One method involved immunomagnetic separation to concentrate microsporidial spores from fecal specimens. In the second method Chelex resin (Bio-Rad, Hercules, Calif.) was used to remove inhibitory substances, followed by a DNA concentration step. Both methods resulted in reproducible, confirmed detection of microsporidia in formalinized fecal specimens and subsequent species determination by PCR sequencing. The detection sensitivity was two in vitro culture-derived spores (Encephalitozoon intestinalis) for the direct PCR. The reproducible detection sensitivity for DNA amplification from formalin-fixed fecal samples was 200 spores for either the Chelex method or the immunomagnetic bead separation method. Thus, we developed two methods for rapid, inexpensive detection of microsporidial spores in formalin-fixed fecal specimens.     

The role of IgG avidity in diagnosis of cytomegalovirus infection in newborns and infants

To evaluate the value of IgG avidity in diagnosis of congenital cytomegalovirus (CMV) infection in newborns and infants we collected serum samples from 40 infants under 12 months of age with suspected congenital CMV infection. Sera were tested for IgM, IgG and IgG avidity. For 25 of them, virus isolation and/or polymerase chain reaction (PCR) on urine specimens were performed. Thirteen (32.5%) patients showed the presence of CMV IgM antibodies, 3 (7.5%) had equivocal IgM result, and 24 (60.0%) patients had IgG antibodies only. Using IgG avidity, CMV infection (low avidity index-AI) was documented in 61.5% IgM positive and 54.2% IgM negative patients. Eight of nine (88.8%) IgM positive patients were positive either on virus isolation or PCR. In IgM negative patients, 46.6% urine cultures were positive for CMV and 66.6% were PCR positive. According to age, IgG avidity demonstrated acute/recent primary CMV infection in 58.8% patients younger than three months compared with 91.7% and 81.8% in 3-6 and 6-12 months old babies, respectively. In conclusion, IgG avidity is useful in diagnosis of CMV infection either in IgM positive or IgM negative children older than 3 months of age. In infants less than 3 months, transplacentally derived maternal IgG antibodies of high avidity influence on the IgG avidity result. In these children, CMV infection should be confirmed by direct virologic methods such as virus isolation or PCR.     

A five-minute DNA extraction method for expedited detection of Phytophthora ramorum following prescreening using Phytophthora spp. lateral flow devices

In a direct comparison with established methods for Phytophthora ramorum detection (isolation followed by morphological identification, or conventional DNA extraction followed by TaqMan real-time PCR) a rapid, simplified detection method in which membranes of lateral flow devices (LFDs) are added directly to TaqMan real-time PCR reactions was used to test 202 plant samples collected by plant health inspectors in the field. P. ramorum prevalence within the 202 samples was approximately 40% according to routine testing by isolation or TaqMan real-time PCR. The diagnostic sensitivity and specificity of the rapid detection method were 96.3% and 91.2%, respectively. This method can be used in conjunction with Phytophthora spp. lateral flow devices to reduce the number of samples requiring testing using more laborious conventional methods. The effect of combining prescreening for Phytophthora spp. with P. ramorum-specific tests is discussed in terms of the positive and negative predictive values of species-specific detection when testing samples collected in different inspection scenarios.     

成人腹泻轮状病毒ELISA方法的建立和应用

本文通过特异性试验、阻断试验、交叉试验、敏感度试验和重复性试验,建立了成人腹泻轮状病毒一酶联免疫吸附试验法(ADRV—ELISA)。应用此法检测了全国20多个省区202份病人腹泻标本,检出率为91％。采用本ELISA、核酸电泳、电镜三种方法对48份病人腹泻标本进行了双盲法检测比较,结果三种方法的阳性检出率分别为100％、85.4％、56.25％(P<0.05)。实验结果表明,本ELISA应用于检测成人腹泻轮状病毒(ADRV),具有敏感度高。特异性强等优点。     

Negative staining in the detection of viruses in clinical specimens

Viruses have unique morphology and are therefore good candidates for negative staining. Negative staining with phosphotungstic acid (PTA) or uranyl acetate has facilitated the detection of many viruses in clinical specimens. Enhancement procedures have included the use of centrifugation and agar diffusion for concentrating virus particles, the use of solid phase capture reagents to trap virus particles and the use of secondary antibodies and electron dense markers to help visualize them. Techniques currently in use and employing negative staining include direct EM, immune electron microscopy (IEM), solid phase immune electron microscopy (SPIEM), colloidal gold-labeled protein A (PAG), solid phase IEM employing a second decorator antibody (SPIEMDAT), and solid phase IEM using colloided gold-labeled secondary antibodies (SPEIMDAGT). IEM methods assist with the detection of small viruses or viruses present in low numbers while PAG offers increased sensitivity over direct EM and IEM. In our experience the serum-in-agar (SIA) method is the most sensitive of the PAG IEM techniques for detection of rotavirus particles in clinical specimens. SPIEMDAT enhances the detection of small viruses which are often missed by other techniques due to background staining in specimens. SPEIMDAGT employing colloidal gold-labeled secondary antibody has increased sensitivity and offers the advantage of detecting viral antigen when whole virus particles are not visible. IEM techniques have recently been used for typing viruses using either monospecific antisera or monoclonal antibodies and colloidal gold-labeled secondary antibody.     

生殖器疱疹病毒抗原的两种检测结果比较分析

比较分析生殖器疱疹病毒抗原的两种检测方法,找出更为准确、简便、经济、实用的检测方法,分别利用疱疹病毒抗原酶免疫(ELISA)法和荧光定量PCR(FQ-PCR)法检测南方医科大学珠江医院门诊患者538例。酶免疫法检出率为27.5%,略低于荧光定量PCR法的28.3%,经SPSS12.0分析,两种方法检测结果无显著差异。酶免疫法检测生殖器疱疹病毒抗原具有操作简便、快捷、经济等特点,而敏感性和特异性均无显著差别,是一种更适合临床的检验方法,更具有值得推广使用的价值。     

禽流感H5、H7、H9亚型多重实时荧光RT-PCR检测方法的建立

为了对致病性强、危害性大的H5、H7、H9亚型禽流感病毒进行同时集成化快速检测,通过对GenBank已报道的禽流感病毒的HA基因进行序列分析比较,设计了H5、H7、H9 3个亚型的特异性引物和分别用3个荧光基团标记的Taqman MGB核酸探针。将各个亚型引物与探针优化组合,筛选出能够同时检测禽流感病毒H5、H7、H9 3个亚型、且对Ct值和扩增效率影响不大的3组引物和探针,建立了三重实时荧光RT-PCR方法。该方法特异性好,在我们检测的样品中,没有发现假阳性和假阴性现象。同时敏感性高,检测禽流感病毒H5、H7、H9亚型的敏感性分别达到1 0001、000、500个模板拷贝数;此外抗干扰能力强,对禽流感H5、H7、H9 3个亚型的不同模板浓度进行组合,仍可有效地同时检测3个病毒亚型。所建立的方法对保存的89个禽流感病毒样品进行检测,结果与经典检测方法(病毒分离鉴定、HA、HI)的符合率达100%。用上述建立的方法与鸡胚分离法同时对新鲜采集的4 000多份临床样品进行检测,两种方法的检测结果符合率为100%。     

Viral Population Estimation Using Pyrosequencing

The diversity of virus populations within single infected hosts presents a major difficulty for the natural immune response as well as for vaccine design and antiviral drug therapy. Recently developed pyrophosphate-based sequencing technologies (pyrosequencing) can be used for quantifying this diversity by ultra-deep sequencing of virus samples. We present computational methods for the analysis of such sequence data and apply these techniques to pyrosequencing data obtained from HIV populations within patients harboring drug-resistant virus strains. Our main result is the estimation of the population structure of the sample from the pyrosequencing reads. This inference is based on a statistical approach to error correction, followed by a combinatorial algorithm for constructing a minimal set of haplotypes that explain the data. Using this set of explaining haplotypes, we apply a statistical model to infer the frequencies of the haplotypes in the population via an expectation–maximization (EM) algorithm. We demonstrate that pyrosequencing reads allow for effective population reconstruction by extensive simulations and by comparison to 165 sequences obtained directly from clonal sequencing of four independent, diverse HIV populations. Thus, pyrosequencing can be used for cost-effective estimation of the structure of virus populations, promising new insights into viral evolutionary dynamics and disease control strategies.     

Viral population estimation using pyrosequencing

The diversity of virus populations within single infected hosts presents a major difficulty for the natural immune response as well as for vaccine design and antiviral drug therapy. Recently developed pyrophosphate-based sequencing technologies (pyrosequencing) can be used for quantifying this diversity by ultra-deep sequencing of virus samples. We present computational methods for the analysis of such sequence data and apply these techniques to pyrosequencing data obtained from HIV populations within patients harboring drug-resistant virus strains. Our main result is the estimation of the population structure of the sample from the pyrosequencing reads. This inference is based on a statistical approach to error correction, followed by a combinatorial algorithm for constructing a minimal set of haplotypes that explain the data. Using this set of explaining haplotypes, we apply a statistical model to infer the frequencies of the haplotypes in the population via an expectation-maximization (EM) algorithm. We demonstrate that pyrosequencing reads allow for effective population reconstruction by extensive simulations and by comparison to 165 sequences obtained directly from clonal sequencing of four independent, diverse HIV populations. Thus, pyrosequencing can be used for cost-effective estimation of the structure of virus populations, promising new insights into viral evolutionary dynamics and disease control strategies.     

Diagnosis of Xanthomonas axonopodis pv. citri, causal agent of citrus canker, in commercial fruits by isolation and PCR-based methods

AIMS: To show the results of the detection of an EU quarantine organism, Xanthomonas axonopodis pv. citri (Xac), in citrus fruits imported from countries where this bacterium is present, using an integrated approach that includes isolation, pathogenicity assays and molecular techniques. METHODS AND RESULTS: Citrus fruits with canker-like symptoms, exported to Spain from South American countries were analysed by several methods. Bacterial isolation, three conventional polymerase chain reaction (PCR) protocols, and real-time PCR with SYBR Green or a TaqMan probe, were compared. Canker-like lesions were disrupted in PBS buffer, and the extract used for bacterial isolation and DNA extraction followed by PCR amplification. Canker lesions, identified by PCR, showed viable bacteria in eleven of fifteen fruit samples. In 16 out of 130 lesions analysed from these samples, Xac was isolated, and pathogenicity on grapefruit leaves confirmed. By real-time PCR, using SYBR green or a Taqman probe, Xac was detected in 58 and 80 lesions respectively. By conventional PCR the bacterium was detected in 39-52 lesions depending on the protocol employed. CONCLUSIONS: An integrated approach for reliable detection of Xac in lesions of fruit samples, employing several techniques and with real-time PCR using a TaqMan probe as the fastest and most sensitive screening method, has been established and validated and is proposed as a useful tool for the analysis of Xac on fresh fruits. SIGNIFICANCE AND IMPACT OF THE STUDY: This work faces up to the real threat of the importation of citrus fruits that can harbour quarantine bacteria and will be useful in diagnostic laboratories for the analysis of commercial fresh fruits from countries where citrus canker is present.     

An effective method for isolation of DNA from pig faeces and comparison of five different methods

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and beta-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp DNA Stool Mini Kit.     

A novel simple one-step single-tube RT-duplex PCR method with an internal control for detection of bovine viral diarrhoea virus in bulk milk, blood, and follicular fluid samples.

A simple one-step single-tube RT-PCR method was developed for detection of bovine viral diarrhea virus (BVDV) in bulk milk, blood and follicular fluid samples. A set of universal primers (UTR DL1/4) was designed for RT-PCR to detect BVDV type I and II viruses simultaneously and was tested for efficacy in comparison to published primers for two type strains, 42 field isolates, and 95 diagnostic samples. The sensitivity (100%) and specificity (96.2%) of the RT-PCR assay, with the universal primers for 95 diagnostic samples, were equal to those of virus isolation. An internal control targeting a bovine actin gene was also included in the same reaction tube to monitor RNA preparation and RT-PCR procedure. A total of 632 specimens (378 bulk milk, 140 blood, and 112 follicular samples) were tested in the year 2000 by the RT-duplex PCR assay in parallel with virus isolation. The one-step single-tube RT-duplex PCR with the universal primers UTR DL1/4 was sensitive, specific, less complicated and cost effective for detection of BVDV in various types of specimens.     

A real-time PCR antibiogram for drug-resistant sepsis

Current molecular diagnostic techniques for susceptibility testing of septicemia rely on genotyping for the presence of known resistance cassettes. This technique is intrinsically vulnerable due to the inability to detect newly emergent resistance genes. Traditional phenotypic susceptibility testing has always been a superior method to assay for resistance; however, relying on the multi-day growth period to determine which antimicrobial to administer jeopardizes patient survival. These factors have resulted in the widespread and deleterious use of broad-spectrum antimicrobials. The real-time PCR antibiogram, described herein, combines universal phenotypic susceptibility testing with the rapid diagnostic capabilities of PCR. We have developed a procedure that determines susceptibility by monitoring pathogenic load with the highly conserved 16S rRNA gene in blood samples exposed to different antimicrobial drugs. The optimized protocol removes heme and human background DNA from blood, which allows standard real-time PCR detection systems to be employed with high sensitivity (<100 CFU/mL). Three strains of E. coli, two of which were antimicrobial resistant, were spiked into whole blood and exposed to three different antibiotics. After real-time PCR-based determination of pathogenic load, a ΔC(t)<3.0 between untreated and treated samples was found to indicate antimicrobial resistance (P<0.01). Minimum inhibitory concentration was determined for susceptible bacteria and pan-bacterial detection was demonstrated with 3 gram-negative and 2 gram-positive bacteria. Species identification was performed via analysis of the hypervariable amplicons. In summary, we have developed a universal diagnostic phenotyping technique that assays for the susceptibility of drug-resistant septicemia with the speed of PCR. The real-time PCR antibiogram achieves detection, susceptibility testing, minimum inhibitory concentration determination, and identification in less than 24 hours.