Laboratory diagnosis of toxoplasmosis using polymerase chain reaction

Polymorphism of clinical manifestations in Toxoplasma infection and variegated disease patterns virtually rule out the diagnosis based solely on clinical symptoms, which makes modern laboratory tests particularly important. Amplification test system based on the polymerase chain reaction has been developed for the diagnosis of Toxoplasma infection. Computer analysis of nucleotide sequence of Toxoplasma gondii surface antigen gene P30 was analyzed, which helped choose and synthesize specific oligonucleotide primers. A method for biological material processing was selected, allowing sufficient DNA output. Optimal conditions for amplification reaction, ensuring absolute specificity and high (10-100 cells/sample) sensitivity, were determined.     

A comparative evaluation of dot immunobinding assay (Dot-Iba) and polymerase chain reaction (PCR) for the laboratory diagnosis of tuberculous meningitis

The results of a Dot immunobinding assay (Dot Iba) for the detection of mycobacterial antigen in the cerebrospinal fluid (CSF) of 45 patients with tuberculous meningitis (TBM) were compared with the results of a polymerase chain reaction (PCR) for the detection of Mycobacterium tuberculosis. In eight patients with culture proven TBM, Dot-Iba gave positive results, while PCR yielded positive results only in six patients. The overall sensitivities of Dot-Iba and PCR in 37 patients with culture negative (probable) TBM were 75.67% and 40.5% respectively. Dot-Iba, in contrast to PCR is a rapid and relatively easier method. More importantly, Dot-Iba is suitable for the routine application for the laboratory diagnosis of TBM and therefore best suited to laboratories in the developing world.     

Bordetella pertussis detection by spectrofluorometry using polymerase chain reaction (PCR) and a molecular beacon probe

Bordetella pertussis was detected by spectrofluorometry following PCR incorporating a molecular beacon probe in the reaction. A DNA fragment from the tandem repeat sequence region (IS 481) of the genome of B. pertussis was amplified in presence of the probe complementary to an internal segment of the amplified DNA fragment. Fluorescein (FAM) and DABCYL were used as the fluorophore and quencher in the probe. The probe was characterized for its signal to noise ratio by homogeneous solution hybridization with a complementary oligonucleotide. Measurement of fluorescent signal at the emission maxima of FAM, immediately after a PCR was used to detect the B. pertussis target, with no additional steps. Presence of B. pertussis in a sample was also examined by agarose gel electrophoresis of the PCR product. A serial diluted stock of B. pertussis (ATCC strain #9797) and fourteen clinical isolates of B. pertussis were examined. The sensitivity of detection by fluorescent measurement was found to be at least in the range of 0.01-0.1 CFU per 10 microl of the sample and was equal to or better than that detected by agarose gel analysis.     

Specific diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in dried blood spots by a polymerase chain reaction (PCR) assay detecting a point-mutation (G985) in the MCAD gene.

The discovery of a point-mutation, adenine-to-guanine, at position 985 in the gene coding for MCAD (G985), gave the basis for an easy and specific polymerase chain reaction test. We tested the specificity of such a PCR based assay and detected correctly G985 and A985 in sequence verified cDNA clones. We showed that the G985 mutation is present in genomic DNA from 48 of 50 patients with confirmed MCAD deficiency, originating from various European countries, Australia and the USA. On the basis of this high frequency of the G985 mutation among patients, we improved and optimized the assay with respect to reliability and convenience for routine diagnostic and screening purposes. As little as 2 microliters blood from filter-paper blood-spots (Guthrie spots) is sufficient for the test.     

Single tube multiplex real-time PCR for the rapid detection of herpesvirus infections of the central nervous system

Human herpesvirus infection of immunocompromised hosts may lead to central nervous system (CNS) infection and diseases. In this study, a single tube multiplex real-time PCR was developed for the detection of five herpesviruses (HSV-1, HSV-2, VZV, EBV and CMV) in clinical cerebrospinal fluid (CSF) specimens. Two primer pairs specific for the herpesvirus polymerase gene and five hybridization probe pairs for the specific identification of the herpesvirus types were used in a LightCycler multiplex real-time PCR. A singleplex real-time PCR was first optimized and then applied to the multiplex real-time PCR. The singleplex and multiplex real-time PCRs showed no cross-reactivity. The sensitivity of the singleplex real-time PCR was 1 copy per reaction for each herpesvirus, while that of the multiplex real-time PCR was 1 copy per reaction for HSV-1 and VZV and 10 copies per reaction for HSV-2, EBV and CMV. Intra and inter-assay variations of the single tube multiplex assay were in the range of 0.02%–3.67% and 0.79%–4.35%, respectively. The assay was evaluated by testing 62 clinical CSF samples and was found to have equivalent sensitivity, specificity and agreement as the routine real-time PCR, but reducing time, cost and amount of used sample.     

巢式聚合酶链式反应检测布鲁氏菌核酸DNA方法的建立

目的建立一种具有灵敏性高,特异性强的巢式聚合酶链式反应(PCR)方法检测血液标本中布鲁氏菌核酸DNA。方法使用细菌基因组提取试剂盒提取纯菌核酸DNA;使用血液等组织基因组核酸DNA提取试剂盒提取血液标本核酸DNA,对提取的核酸DNA先行常规PCR预扩增,以扩增的PCR产物为模板进行荧光定量PCR第二次扩增(即巢式PCR)。对纯菌提取的核酸DNA进行灵敏度和和特异性测试,构建巢式PCR的Ct值与核酸DNA拷贝数之关系曲线;检测临床血液标本核酸DNA,同时比较常规两种PCR方法检测结果。结果常规PCR检测的灵敏度为512个核酸DNA拷贝数;巢氏PCR检测有效范围为921.6 ng/μl^6.8 fg/μl,对应的Ct值为12.04~37.50,其指数关系为:y=(e-0.695x)×1012;R2=0.9986,巢式PCR扩增效率为2.28×109倍,检测限为2个布鲁氏核酸DNA拷贝数。巢式PCR的灵敏度为91.67%,特异度为93.10%,阳性预测值为91.67%,阴性预测值为93.10%。对一起羊养殖场采集的25份血液标本应用巢式PCR方法检测,结果阳性率为92.00%(23/25);27份健康人群血液标本没有检测出(无Ct值)。结论巢式PCR具有较好的灵敏性和特异性,特别适合于血液标本布鲁氏菌核酸DNA的检测。     

Detection of central nervous system leukemia in children with acute lymphoblastic leukemia by real-time polymerase chain reaction

Accurate detection of central nervous system (CNS) involvement in children with newly diagnosed acute lymphoblastic leukemia (ALL) could have profound prognostic and therapeutic implications. We examined various cerebrospinal fluid (CSF) preservation methods to yield adequate DNA stability for polymerase chain reaction (PCR) analysis and developed a quantitative real-time PCR assay to detect occult CNS leukemia. Sixty CSF specimens were maintained in several storage conditions for varying amounts of time, and we found that preserving CSF in 1:1 serum-free RPMI tissue culture medium offers the best stability of DNA for PCR analysis. Sixty CSF samples (30 at diagnosis and 30 at the end of induction therapy) from 30 children with ALL were tested for CNS leukemic involvement by real-time PCR using patient-specific antigen receptor gene rearrangement primers. Six of thirty patient diagnosis samples were PCR-positive at levels ranging from 0.5 to 66% leukemic blasts in the CSF. Four of these patients had no clinical or cytomorphological evidence of CNS leukemia involvement at that time. All 30 CSF samples drawn at the end of induction therapy were PCR-negative. The data indicate that real-time PCR analysis of CSF is an excellent tool to assess occult CNS leukemia involvement in patients with ALL and can possibly be used to refine CNS status classification.     

中枢神经系统神经母细胞瘤的CT、MRI诊断(附6例报告)

目的分析中枢神经系统神经母细胞瘤的影像学特点,探讨CT及MRI对该病的诊断价值。材料与方法对6例经手术病理证实的中枢神经系统神经母细胞的影像资料进行回顾性分析。结果男5例,女1例,年龄3~24岁,平均13.8岁。幕上3例,分别位于颞极区1例,颞枕叶及顶叶各1例;幕下四脑室内和桥脑各1例,另1例跨小脑天幕。肿块呈囊实性2例,实性为主4例。MRI表现肿瘤实性部分呈等或稍长T1、T2信号,FLAIR序列呈等、稍高信号;囊性部分呈长T1、长T2信号,FLAIR序列呈低信号;增强扫描后,2例强化不明显,4例肿瘤实性部分呈明显强化,肿块边界较清楚,瘤周水肿较轻。CT表现肿瘤实性部分呈等、稍高密度,其内可见钙化,囊性部分呈低密度。结论中枢神经系统神经母细胞瘤影像表现有一定的特点,根据肿瘤部位、边界、信号(或密度)、水肿、增强等特点有助于该病的诊断和鉴别诊断。     

Diagnosis of human parechovirus infections of the central nervous system with a commercial real-time reverse transcription-polymerase chain reaction kit and direct genotyping of cerebrospinal fluid specimens

We screened 100 cerebrospinal fluid specimens for the human parechoviruses (HPeV) genome with the commercial parechovirus r-gene™ kit, which allows results to be available in a clinically relevant time frame. HPeV infection was diagnosed in 4 infants (< 4 months) and all genotyped viruses were HPeV3.     

Imaging aspects of pyogenic infections of the central nervous system

Although pyogenic infections of the central nervous system are not a frequent group of diseases, their morbidity and mortally are very high. For this reason they require prompt diagnosis and treatment to avoid several complications that can lead to an undesired outcome. In this article, we review the imaging findings of these infections according to the anatomic site, their complications, and their differential diagnosis. Special attention is given to the different techniques of magnetic resonance imaging like perfusion, spectroscopy, and diffusion, for each specific situation such as meningitis, abscess, ventriculitis, purulent extra axial collections, and vascular complications.     

Ataxia in Listeria monocytogenes infections of the central nervous system

From 1976 to 1981 Listeria monocytogenes was second only to Neisseria meningitidis as the cause of bacterial infections of the central nervous system in adults at our hospital. None of the patients with Listeria infection was immunosuppressed or had an underlying malignancy. Ataxia was an initial feature in five of the eight patients, and in three of them it persisted beyond their discharge from the hospital. Ataxia was not a feature of the clinical picture of 14 other adult patients with meningococcal and pneumococcal meningitis. Our data indicate that L monocytogenes should be suspected as the etiologic agent in an adult with ataxia and infection of the central nervous system.     

Laboratory diagnosis of chancroid using species-specific primers from Haemophilus ducreyi groEL and the polymerase chain reaction

To enhance laboratory identification of Haemophilus ducreyi, the causative agent of the genital ulcer disease chancroid, a polymerase chain reaction (PCR) assay was developed using target DNA sequences from the essential H. ducreyi gene, groEL. Positive reactions were obtained in this PCR assay with 139 isolates of H. ducreyi from patients in worldwide locations from the 1940s to the 1990s. In contrast, 24 other bacterial species were negative. When genital ulcer specimens from 162 African patients with clinically diagnosed chancroid were evaluated, 66 were culture positive. The sensitivity of PCR as compared with culture was 89% (59 of 66), and specificity was 79% (76 of 96). However, representative samples of the 20 culture-negative, PCR-positive specimens were confirmed as positive by a second PCR assay using different H. ducreyi-specific primers. Thus, combined results of culture and PCR detected H. ducreyi in 86 specimens, with resolved sensitivities of 92% (79 of 86) for PCR, and 77% (66 of 86) for culture. These results suggest that PCR assays for H. ducreyi have great potential for augmenting or replacing problematic cultural techniques.     

Evaluation of a real-time polymerase chain reaction assay for the laboratory diagnosis of giardiasis

A real-time polymerase chain reaction (PCR) assay was evaluated in comparison with the combination of conventional methods (microscopic examination and antigen detection assay) during the period 2006 to 2008 on 771 fecal samples belonging to 386 patients to assess its usefulness for an accurate laboratory diagnosis of giardiasis. The real-time PCR assay detected Giardia intestinalis DNA in 195 samples (106 patients), including 26 samples (21 patients) negative by the conventional assays. Among the 21 patients, in 8 cases, giardiasis was previously diagnosed also by conventional methods in additional samples of the same patients, whereas in 13, it would have been undiagnosed if real-time PCR assay was not used. The real-time PCR assay demonstrated a detection limit of 2 cysts per reaction and 100% specificity and sensitivity compared to conventional methods. A genotype analysis targeting the β-giardin gene allowed to identify 53 samples (23 patients) containing genotype A and 59 samples (45 patients) containing genotype B.     

应用巢式PCR检测实验小鼠组织中申克孢子丝菌

目的研究实验小鼠申克孢子丝菌感染的分子生物学鉴定方法,为建立检测人申克孢子丝菌感染的快速、敏感、特异方法提供依据。方法建立申克孢子丝菌感染小鼠模型,应用合成的申克孢子丝菌种特异性引物ITS3-SSP进行巢式PCR扩增小鼠皮损组织内核糖体DNA的ITS2靶序列,将检测结果与标准形态学鉴定结果对比,观察种特异性引物PCR扩增结果的准确程度、敏感性和特异性。结果组织学检查可以从11只感染小鼠尾组织中见到染成紫红色的圆形、卵圆形孢子;第一循环PCR检测实验小鼠申克孢子丝菌,只有3只小鼠呈阳性;而巢式PCR检测申克孢子丝菌特异性DNA,11只实验小鼠中9只呈阳性;而3只对照组小鼠无一呈阳性。结论本实验结果说明巢式PCR结合种特异性引物ITS3-SSP可以有效检出实验小鼠皮损中的申克孢子丝菌,具有一定的敏感性和特异性,尤其是对于组织学检查和真菌培养阴性的标本,本方法是一个很有潜力的替代方法。     

套式聚合酶链反应单管法检测单纯疱疹病毒

应用聚合酶链反应（ＰＣＲ）方法、套式ＰＣＲ双管法及套式ＰＣＲ单管法分别检测１３０份散发性脑炎患者脑脊液（ＣＳＦ）中单纯疱疹病毒（ＨＳＶ）的ＤＮＡ，阳性率依次为１８．５％（２４／１３０）、２９．２％（３８／１３０）、２９．２％（３８／１３０）；６０份非中枢神经系统感染者ＣＳＦ标本的检测阳性率分别为０、３．３％（２／６０）、０。结果显示：套式ＰＣＲ虽然提高了检测的敏感性，但假阳性也大大增加。改良的套式ＰＣＲ单管法则大大减少了污染机会，使操作更方便、省时，适于临床推广应用。