Early diagnosis of central nervous system aspergillosis using polymerase chain reaction, latex agglutination test, and enzyme-linked immunosorbent assay

To investigate the usefulness of examination of cerebrospinal fluids (CSF) in the diagnosis of central nervous system (CNS) aspergillosis, we examined five patients with either brain abscesses or cerebral infarctions and 11 control patients. CSF samples were subjected to enzyme-linked immunosorbent assay (EIA), latex agglutination test (LA) and polymerase chain reaction (PCR). Cultures of CSF samples were negative in all the patients, but PCR, EIA and LA were positive in five, four and four patients with CNS aspergillosis, respectively. None of these tests were positive in the control patients. CSF examination may be beneficial in the diagnosis of CNS aspergillosis.     

Latex based, rapid and easy assay for human leptospirosis in a single test format

Leptospirosis is an often severe disease which requires prompt treatment. Laboratory testing is required to reach a valid diagnosis. An agglutination assay for the detection of Leptospira-specific antibodies consisting of individually wrapped agglutination cards containing a stable, dried detection reagent is evaluated. The assay is simply performed by suspending the dried reagent with a drop of serum. The result is obtained within 30 s. The sensitivity of the assay varied with the stage of the disease and was 72.3% for samples collected during the first 10 days of the illness and 88.2% for samples collected at a later stage. The specificity was 93.9% and 89.8%, respectively. These characteristics make the test ideal for use in areas where the disease is common and where laboratory support is not routinely available.     

Field evaluation of a one-step dipstick assay for the diagnosis of human leptospirosis in the Seychelles

OBJECTIVE AND METHOD: To compare the response of a dipstick assay (DSA) detecting Leptospira-specific immunoglobulin M (IgM) antibodies with that of an enzyme-linked immunosorbent assay (ELISA), an indirect haemagglutination assay (IHA), the microagglutination test (MAT) and a polymerase chain reaction assay (PCR) in patients with leptospirosis confirmed by MAT alone or by MAT and/or PCR (MAT/PCR). RESULT: In 75 patients with acute leptospirosis diagnosed by MAT (respectively, 90 patients diagnosed by MAT/PCR), the response in paired early and convalescent sera was positive in 78.9% (67.9%) by DSA, 76.0% (67.8%) by ELISA, 58.7% (55.6%) by IHA, 44.0% (53.3%) by PCR, and 100% (90.0%) by MAT. In early serum only, the response in patients diagnosed by MAT (respectively by MAT/PCR) was positive in 36.0% (38.9%) by DSA, 36.0% (37.8%) by ELISA, 14.7% (18.9%) by IHA, 39.2% (48.3%) by PCR, and 53.3% (58.9%) by MAT titre > or =1:100. DSA detected the main serogroups implicated in human leptospirosis in Seychelles and demonstrated sensitivity comparable to ELISA. In 124 single sera from control subjects without overt disease, the response was positive in 4.8% by DSA, 3.2% by ELISA, 3.2% by IHA, 13.8% by PCR, 37.9% by MAT titre > or =1:100, and 2.4% by MAT titre > or =1:800, giving evidence of the frequency of both past and current subclinical infection in Seychelles and that DSA was less sensitive than MAT to detect moderate levels of leptospiral antibodies. CONCLUSION: DSA is a simple and reproducible assay well adapted to field conditions and could usefully contribute to the evaluation of leptospirosis in areas devoid of serological laboratory facilities.     

Outbreak of leptospirosis in New Caledonia: diagnosis issues and burden of disease

A leptospirosis epidemic affected New Caledonia during the first semester of 2008. A total of 135 cases were diagnosed with a relatively low fatality rate of 3.7%. Heavy rainfalls, related to La Niña, favoured this epidemic. The PCR, routinely used, confirmed 54% of the cases, and the microagglutination test 56%. Epidemiological and economical data on this epidemic are presented and discussed.     

International multi-centre evaluation of a dipstick assay for human leptospirosis

A dipstick assay for the detection of Leptospira-specific immunoglobulin M (IgM) antibodies in human sera was evaluated in 27 laboratories in 23 countries. 873 serum samples from 711 patients including 329 laboratory-confirmed leptospirosis case patients, 239 noncase patients and 69 patients with viral infections causing heamorrhagic fever were tested. Relative to the results of the reference leptospirosis test, the sensitivity of the dipstick assay was 84.5% for serum samples collected during the first 10 days of the disease and 92.1% for serum samples collected 10-30 days after the onset of disease. The specificity was 87.5% and 94.4%, respectively. Similar to viral haemorrhagic fevers, leptospirosis may cause bleeding. A small number of serum samples from patients with haemorrhagic viral infections gave a weak (1 +) stain. All other samples were negative. In conclusion, the dipstick assay is sensitive and specific and reacts well with serum samples from patients infected with a range of leptospiral strains. It is also easy to use and does not require special equipment or refrigeration. Therefore the assay is ideal for use in developing countries and rural settings.     

聚合酶链反应在诊断结核性胸腔积液中的应用

应用聚合酶链反应（ＰＣＲ）技术对６２例胸水进行结核菌ＤＮＡ检则，并同时与胸水涂片及培养结果进行比较。结果显示：３４例结核性胸水涂片抗酸染色、结核菌培养和ｐＣＲ检测阳性率分别为５．９％、８．８％和５２．９％，后者显著高于前二者（Ｐ均＜０．０１）。２８例非结核性胸水涂片和培养结果均呈阴性，但ＰＣＲ有４例（１４．３％）出现阳性。结果表明ＰＣＲ直接检测胸水中结核菌显示出快速、敏感和高效等优点。同时还对影响ＰＣＲ检测结核菌的某些因素作了分析。     

极速实时荧光聚合酶链反应与常规方法对新型布尼亚病毒检测的评价

目的探讨极速实时荧光聚合酶链反应(polymerase chain reaction,PCR)、实时荧光PCR、酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)和胶体金免疫层析法(gold immunochromatography assay,GICA)4种方法检测新型布尼亚病毒的特异度和灵敏度,为发热伴血小板减少综合征的早期诊断提供依据。方法采集2017年6月1日至9月30日山东大学附属济南市传染病医院86例临床诊断为发热伴血小板减少综合征患者的血清样本,分别应用极速实时荧光PCR、实时荧光PCR、ELISA和GICA 4种方法进行检测。统计学分析采用χ^2检验。结果86份患者血清标本中,极速实时荧光PCR、实时荧光PCR、IgM-ELISA、IgG-ELISA、IgM-GICA、IgG-GICA的新型布尼亚病毒阳性分别为82份(95.34%)、79份(91.86%)、41份(47.67%)、8份(9.3%)、19份(22.09%)和3份(3.49%)。极速实时荧光PCR特异度为100%,灵敏度达到1×103拷贝/mL,3次重复扩增试验显示其Ct值变异系数均<2%。在发热伴血小板减少综合征进展的1期、2期、3期病程中,极速实时荧光PCR的阳性检出率为41份(97.62%)、34份(94.44%)、7份(87.50%),实时荧光PCR的阳性检出率为39份(92.86%)、33份(91.67%)、7份(87.50%),在1期和2期两个病程,极速实时荧光PCR阳性检出率略高;IgM-ELISA阳性检出率从1期(28.57%)到3期(87.50%)显著增高,2期、3期与1期相比,差异均有统计学意义(χ^2=8.347、7.561,均P<0.01);IgM-GICA的阳性检出率从1期(14.29%)到2期(33.33%)也有增高,差异有统计学意义(χ^2=3.962,P<0.05),但与其他方法相比,其检出率偏低。1期,实时荧光PCR阳性检出率显著高于ELISA(IgM和IgG)和GICA(IgM和IgG),差异均有统计学意义(χ^2=33.740、55.080、49.010、64.340,均P<0.01)。2期,实时荧光PCR的阳性检出率高于ELISA(IgM和IgG)和GICA(IgM和IgG),差异均有统计学意义(χ^2=7.700、46.720、23.700、50.630,均P<0.01)。3期,极速实时荧光PCR、实时荧光PCR和IgM-ELISA表现出同样高的阳性检出率,远高于IgG-ELISA和GICA(IgM和IgG)。实时荧光PCR阳性检出率和IgG-ELISA、IgM-GICA、IgG-GICA之间差异均有统计学意义(均χ^2=6.250,P<0.05)。结论极速实时荧光PCR在新型布尼亚病毒的早期检测中有更高的灵敏度和特异度,且重复性好、稳定度高,与传统实时荧光PCR相比大大缩短了扩增时间,对发热伴血小板减少综合征的早期快速诊断具有重要价值。     

Polymerase chain reaction diagnosis of Helicobacter pylori in gastroduodenal diseases: Comparison with culture and histopathological examinations

Helicobacter pylori has been associated with a variety of upper gastrointestinal diseases. Histopathological examination and culture are considered to be the more specific tests in the diagnosis of H. pylori infection. In the present study, we evaluated the efficiency of a polymerase chain reaction (PCR) assay of the H. pylori urease A gene as a procedure in the diagnosis of gastric H. pylori infection in various gastroduodenal diseases. Biopsy specimens were obtained from the antral mucosa of 83 patients during endoscopic examination and were submitted to three tests for the detection of H. pylori infection. The detection rates of H. pylori using PCR, histopathological examination and culture were 84, 77 and 63%, respectively. When the infection was defined, by the agreement of culture and histopathological examination or by positive culture, the PCR assay had a sensitivity of 98.1% and a specificity of 84.6%. When the infection was defined by a positive result of either two of the three tests or by positive culture, the PCR assay had a sensitivity of 98.6% and a specificity of 85.7%. We conclude that the PCR assay is a valuable test for the diagnosis of H. pylori infection in gastroduodenal diseases.     

结核抗体检测联合结核感染T细胞斑点试验的临床诊断价值

目的 探讨结核抗体检测联合结核感染T细胞斑点试验(T-SPOT.TB)在结核病诊断中的应用价值。方法 选择2016年1月1日至2017年4月12日就诊于北京老年医院的961例结核病患者[结核病组;包括364例菌阳肺结核患者(通过细菌学诊断确诊)和597例菌阴肺结核患者(通过临床进行诊断)]和非结核病的1046例患者(对照组)。所有患者同时使用上海奥普生物医药有限公司及新加坡MP生物医学亚太私人有限公司生产的结核抗体金标试剂(分别为TB-DOT、ASSURE TB试剂盒)检测血清中的结核抗体;961例结核病组中的574例及1046例对照组中的664例患者同时使用T-SPOT.TB试剂盒和2种结核抗体金标试剂进行检测。对两组患者采用3种实验室技术进行检测或联合检测(包括串联与并联),对检测结果的敏感度、特异度进行统计学分析。结果 TB-DOT和ASSURE TB检测结核病组和对照组患者的敏感度和特异度分别为52.86%(508/961)、74.95%(784/1046)和45.99%(442/961)、76.00%(795/1046)。TB-DOT 和ASSURE TB对菌阳和菌阴肺结核患者检测的敏感度分别为64.84%(236/364)、62.09%(226/364)和45.56%(272/597)、36.18%(216/597);菌阳肺结核患者TB-DOT和ASSURE TB检测的敏感度均高于菌阴肺结核患者,差异均有统计学意义(χ ²值分别为60.99、33.69,P值均<0.05)。结核病组和对照组患者在2种结核抗体金标试剂和T-SPOT.TB试剂盒的串联联合检测中,敏感度为24.04%(138/574),但特异度达到89.91%(597/664);并联联合检测中,特异度为21.23%(141/664),但敏感度达到89.55%(514/574)。结核病组和对照组在ASSURE TB、TB-DOT和T-SPOT.TB 3种检测方法中,均为阳性和均为阴性两种谱型的构成比分别为24.04%(138/574)、10.09%(67/664)和10.45%(60/574)、21.23%(141/664),差异均有统计学意义(χ ²值分别为43.37和26.32,P值均<0.05)。 结论 两种结核抗体与T-SPOT.TB行串联联合检测能够提高检测特异度,并联联合检测能够提高检测敏感度;串联联合检测时,3种检测方法结果均为阳性和均为阴性两种谱型在临床上对结核病血清学诊断具有辅助诊断价值。     

丙型肝炎抗体的检测与HCVRNA定量及ALT的关系探讨

目的比较不同实验诊断方法在丙型肝炎(丙肝)抗体检测中的应用价值,并探讨HCV RNA定量与ALT指标之间的关系。方法对105例增强化学发光法(CIA)检测丙肝抗体初筛结果呈阳性(S/Co>1)的标本,分别采用酶联免疫吸附试验(ELISA)和荧光定量聚合酶链反应(RFQ-RT-PCR)进行检测,并对可疑结果(①CIA检测结果S/Co在1～8之间的标本,②ELISA检测结果呈阴性的标本)采用HCV RIBA3.0进行确认;应用OLYMPUS AU-5400全自动生化仪及其生化检测试剂检测全部标本ALT指标。结果ELISA及RFQ-RT-PCR方法检测样本阳性率分别为93.33%和70.48%;对可疑标本经HCV RIBA3.0确认试验显示,2例结果呈阳性(分别为NS3、HCV-C和NS3、NS4阳性),1例为阴性,4例仍为可疑(其中1例HCV RNA>103copies/ml);HCV RNA含量呈阳性的样本中,ALT异常率与HCV RNA含量间呈正相关(r=0.96,P<0.01),而ALT数值的变化与HCV RNA含量并无相关性(r=0.19,P>0.05)。结论第三代ELISA诊断试剂盒检测丙肝抗体同样具有较高的灵敏度,2种检测方法均可能存在假阳性或假阴性情况;RFQ-RT-PCR检测结果阳性率低于CIA与ELISA检测结果;在临床诊断中丙肝抗体的检测应与荧光定量聚合酶链反应一起使用,以提高临床丙肝诊断的准确率。     

Detection of immunoglobulin gene rearrangement in acute and chronic lymphoid leukemias of B-cell lineage by polymerase chain reaction gene amplification

Polymerase chain reaction (PCR) was used to amplify the DNA fragments of the complementarity determining region 3 of the immunoglobulin (Ig) gene heavy chain from the leukemic cell specimens of patients with acute and chronic lymphoid leukemias of B-cell lineage. Two different pairs of primers were tested. Fourteen of the 17 (82%) cases of acute lymphoblastic leukemia (ALL), and all 15 cases (100%) of B-cell chronic lymphocytic leukemia, who had rearrangement of the Ig gene heavy chain by Southern analysis, were positive by PCR with either one or both pairs of primers. This technique was able to detect leukemic cells at the level of 0.1%. Applying it to study the remission marrow specimens following induction chemotherapy was more useful than morphology alone in predicting early relapse of the leukemia.     

双重聚合酶链反应法早期诊断军团菌肺炎的初步研究

目的探讨双重聚合酶链反应（DPCR）法检测痰及支气管肺泡灌洗液（BALF）中军团菌DNA在早期诊断军团菌肺炎的意义。方法用DPCR对军团菌肺炎组（15例）和普通肺炎组（31例）患者病程早期留取的痰及BALF进行军团菌DNA检测。用军团菌16SrRNA基因和mip基因序列引物,可扩增386bp16SrRNA基因片段和206bpmip基因片段。通过模拟标本来检测DPCR方法用于临床标本时的灵敏度。结果军团菌肺炎组患者痰、BALF的DPCR结果均为阳性,其中13例患者留取的23份痰、7份BALF标本386bp16SrRNA基因片段和206bpmip基因片段的扩增结果均阳性,余2例患者留取的2份痰和1份BALF标本单一386bp基因片段扩增阳性。DPCR结果与血清军团菌抗体结果相符。普通肺炎组患者痰和BALF的DPCR结果均为阴性。同一患者留取的多份标本呈现相同的DPCR结果。模拟痰和BALF的DPCR最低检出限均为1×103cfu/ml。结论采用DPCR法检测痰和BALF中的军团菌DNA,具有较好的敏感性、特异性和稳定性,在临床早期诊断军团菌肺炎方面具有一定的价值。     

Use of polymerase chain reaction in the diagnosis of abdominal tuberculosis

BACKGROUND: Early diagnosis and prompt treatment of abdominal tuberculosis is vitally important as it greatly reduces disease and treatment related morbidity and even mortality in extreme cases. A polymerase chain reaction (PCR) test was evaluated for its feasibility as a diagnostic tool in abdominal tuberculosis (TB) in the Indian scenario. METHODS: PCR for the identification of M. tuberculosis amplified a 340 bp nucleotide sequence located within the 38 kDa protein gene of M. tuberculosis. Tissues for processing were obtained from patients suspected to have abdominal TB. These were from various sources such as abdominal lymph nodes, segments of intestine and bowel obtained at various times and in different ways such as laparoscopy, colectomy, bowel and lymph node resection. Fifty such patients had their tissues sent for PCR. RESULTS: PCR results were compared with histopathology (HP). Of the 50 samples, 31 were positive for abdominal TB by HP whereas 30 were positive by PCR. Twenty-four of these were positive for both HP and PCR while of the seven samples positive for HP, five were negative and two gave inhibition by PCR. Six samples negative by HP were positive by PCR. CONCLUSION: This study demonstrates that PCR can be used as an effective tool to diagnose abdominal TB.     

Field evaluation of rapid diagnostic tests for meningococcal meningitis in Niger

Objective  To evaluate dipstick rapid diagnostic tests (RDTs) for meningococcal meningitis in basic health facilities.
Methods  Health facility staff received a one-day training. During the meningitis season, they performed RDTs on cerebrospinal fluid (CSF) specimens from suspected cases of meningitis. A frozen aliquot of CSF was later tested using polymerase chain reaction (PCR) to establish the reference diagnosis. RDTs used in health facilities were archived to allow checking the concordance between reported diagnosis and observed results. Reported diagnosis was also compared to PCR diagnosis. A second RDT was performed on each CSF specimen at the reference laboratory.
Results  Using RDTs, health facilities reported 382 negative results (73.9%), 114 NmA (22.1%), 12 NmW135 (2.3%) and nine uninterpretable results (1.7%), the latter corresponding to the misuse of a reagent by three agents. The agreement between reported diagnosis and archived dipsticks was excellent (kappa = 0.98). The agreement between PCR diagnosis and reported RDTs results was strong (kappa = 0.82). In health facilities, the sensitivity of RDTs for N. meningitidis A was Se = 0.91. The kappa coefficient measuring the agreement between RDTs operated in the reference laboratory and RDTs operated in health facilities was κ = 0.78.
Conclusion  We confirmed that dipstick RDTs to identify N. meningitidis serogroups A, C, W135 and Y can be reliably operated by non-specialized staff in basic health facilities. RDTs proved very useful to recommend vaccination in NmA epidemics, and also to avoid vaccination in epidemics due to serogroups not included in vaccines (NmX).     

Evaluation of serological tests for diagnosis of Bordetella pertussis infection in adolescents and adults

Background and objective: Bacterial agglutination antibodies against Bordetella pertussis, Yamaguchi and Tohama strains, are frequently measured for serodiagnosis of pertussis infection in Japan. To determine the serological criteria, the comparative titres of bacterial agglutination antibody and anti‐pertussis toxin (PT) antibody were evaluated. Methods: Antibody titres were analysed in 36 definitive (fourfold increase in agglutination antibody) and 137 presumptive (high titre of single‐antibody) cases of B. pertussis infection among adolescents and adults, and in a control group of 318 healthy volunteers. Results: When a single Yamaguchi agglutinin titre of ≥1:1280 (> three SD above the geometric mean for the control group) was taken as diagnostic, the sensitivity and specificity at 4–5 weeks after onset of cough were 58% and 98%, respectively. Using this criterion, the clinical findings in presumptive cases were almost identical to those in definitive cases. When the two tests were compared using 318 control sera, there was no association between the Tohama agglutinin titre and the anti‐PT antibody titre, whereas a weak association between the Yamaguchi agglutinin titre and the anti‐PT antibody titre was observed. When the numbers of pertussis cases with high antibody titres in the two tests were compared, 60% of cases with a Yamaguchi agglutinin titre of ≥1:1280 showed an anti‐PT antibody titre of ≥100 EU/mL. Conclusions: These results indicate that the bacterial agglutination test is a method with low sensitivity and specificity for the diagnosis of B. pertussis infection. Therefore, to yield an accurate diagnosis, anti‐PT antibody levels should be measured instead of bacterial agglutination antibody.     

Polymerase chain reaction (PCR) is highly sensitive for diagnosis of mucosal leishmaniasis

We evaluated the use of polymerase chain reaction (PCR) for diagnosis of mucosal leishmaniasis (ML) in an endemic area in Acre, Brazil, where Leishmania braziliensis is present. Leishmania DNA was detected 34 of 35 cases, yielding a positivity rate of 97.1%, which was higher than the positivity rates for all of the other diagnostic methods studied, namely Montenegro skin test (MST), anti-Leishmania serological testing and microscopic examination of lesion biopsy specimens. These findings have led us to propose guidelines for the diagnosis of ML that use PCR as the principal method of parasitological confirmation of cases.     

聚合酶链反应芯片筛选5-氟尿嘧啶对乳鼠心肌细胞心脏毒性差异表达基因的作用

目的应用聚合酶链反应（polymerase chain reaction,PCR）芯片技术观察5-氟尿嘧啶（5-fluorouracil,5-FU）对乳鼠心肌细胞心脏毒性相关基因表达的调控作用。方法利用差速贴壁法分离培养乳鼠心肌细胞．分为对照组和5一FU刺激组。提取并纯化细胞RNA,紫外分光光度计检测RNA提取物浓度、变性琼脂糖凝胶电泳检测其纯度及完整性。选择包含84个已知心脏毒性相关基因的PCR芯片,采用比较阈值（△△Ct）法分析两组基因的表达差异。以表达差异（即上调或下调）大于2倍的基因为有意义的差异基因。结果共有48条基因出现差异表达,其中5条基因表达上调,43条基因表达下调。结论利用PCR芯片技术筛选相关基因,为深入阐明5-FU心脏毒性的作用机制提供了新思路。     

Comparative evaluation of parasitology and serological tests in the diagnosis of visceral leishmaniasis in India: a phase III diagnostic accuracy study

In this phase III trial for diagnostics for visceral leishmaniasis (VL) in India, we compared parasitological diagnosis with several serological tests: direct agglutination test (freeze dried; DAT-FD), rK-39 strip test, rK-26 strip test and a latex agglutination test for antigen detection in urine (KAtex) in 452 subjects from the endemic regions of Bihar, India. The subjects were segregated into four categories: 230 confirmed patients, 52 probable cases, 70 non-cases and 100 healthy endemic controls. The first two groups were used for estimating sensitivity, the latter two for specificity. Sensitivity of DAT-FD was 98.9%, rK-39: 98.9%, KAtex: 67.0% and rK-26: 21.3%. Sensitivity of DAT-FD on blood taken on filter paper (DAT-FDF) was 99.3%, which was comparable with that using serum. Specificity of serological tests was comparable and high (DAT-FD and DAT-FDF: 94%, rK-39 strip test: 97%, KAtex: 99% and rK-26 strip test: 100%). The classical 'gold standard' parasitological demonstration in splenic smear performed poorly as it missed 18.4% of cases that benefited from VL treatment. Reproducibility of the serological tests between field and central laboratories was excellent (kappa = 1.0, 0.99, 0.96 and 0.94 respectively for microscopy, DAT-FD, rK-39 strip test and rK-26 strip test). A high degree of agreement was observed between DAT-FD and rK-39 strip test (kappa = 0.986). Although DAT-FD and rK-39 strip test were highly sensitive with excellent specificity, the ease of use of the latter makes it most suitable for the diagnosis of VL in the field conditions.