多聚酶链反应和病毒分离检测巨细胞病毒的比较研究

９２名住院患儿病毒分离，常规ＰＣＲ，热启动ＰＣＲ，碘化钠抽提结合热启动ＰＣＲ的ＣＭＶ检出率分别为６１％，２８％，３９％和４８％，与病毒分离比较，常规ＰＣＲ，热启动ＰＣＲ，碘化钠抽提结合热启动ＰＣＲ检测极低拷贝数ＣＭＶＤＮＡ的敏感性分别为８％，３６％和６４％。从而显示碘化钠抽提结合加热启动ＰＣＲ，敏感性高，简便快速，４ｈ内可判定结果，颇具临床实用价值。     

广东地区人微小病毒B19母婴传播及异常胎儿和新生儿人微小病毒B19感染的研究

目的 为研究人微小病毒Ｂ１９在广东地区母婴及异常胎儿和新生儿中的感染状况。方法 应用ＰＣＲ方法检测７００份孕妇外周血和新生儿脐血,ＰＣＲ结合限制性内切酶分析以及原位杂交检测２４份异常胎儿和新生儿组织。结果 孕妇和胎儿血清中Ｂ１９病毒的检出率分别为１．１４％（４／３５０）和０．２８％（１／３５０）;１７例异常胎儿和７例新生儿中,分别有５份胎儿和３份新生儿组织ＰＣＲ结果阳性。ＰＣＲ产物经限制性内切酶Ｈ     

性病衣原体三种检测方法的比较研究

目的通过实验探讨一种简便、快速、准确而又经济的性病衣原体检测方法。方法对所选择的临床标本同时用ＭｃＣｏｙ细胞分离培养、ＭｃＡｂ荧光染色和ＰＣＲ法进行沙眼衣原体的检测，并用统计学方法分析结果。结果４４份临床标本中有３０份用３种方法检测均为阴性，７份为阳性，余７份结果不尽相同。三种方法检出率依次为２０．４５％、２５．００％、２９．５５％，用多组配对计数资料的ｘ２检验表明差别有显著意义。以培养法为标准，ＭｃＡｂ荧光法的符合率（９０．９１％）高于ＰＣＲ法（８６．３６％），而阳性率低于ＰＣＲ法。结论ＭｃＡｂ荧光法和ＰＣＲ法可为一种简便、快速而经济的判断衣原体感染的方法，但在进行临床诊断时，宜以一种以上方法同时检测，并结合临床表现进行评判为宜。     

聚合酶链反应检测间日疟原虫的现场应用研究

为探讨聚合酶链反应（ＰＣＲ）检测间日疟原虫（Ｐ．ｖ．）的现场应用价值。根据Ｐ．ｖ．红内期ＳＳｕｒＲＮＡ基因序列合成１对寡核苷酸引物建立检测Ｐ．ｖ．的ＰＣＲ技术，扩增产物片段为３４１ｂｐ，同时采用ＰＣＲ法和镜检法对８份临床疑为Ｐ．ｖ．感染患者的血液标本进行检测。用ＰＣＲ法检测阳性者６９份，镜检法检测阳性者５９份，ＰＣＲ检出率（８８．５％）明显高于镜检法检出率（７５．６％），两者符合率为８７．２％。结     

聚合酶链反应检测间日疟原虫的现场应用研究

为探讨聚合酶链反应（ＰＣＲ）检测间日疟原虫（Ｐ．ｖ．）的现场应用价值。根据Ｐ．ｖ．红内期ＳＳｕｒＲＮＡ基因序列合成１对寡核苷酸引物建立检测Ｐ．ｖ．的ＰＣＲ技术，扩增产物片段为３４１ｂｐ，同时采用ＰＣＲ法和镜检法对７８份临床疑为Ｐ．ｖ．感染患者的血液标本进行检测。用ＰＣＲ法检测阳性者６９份，镜检法检测阳性者５９份，ＰＣＲ检出率（８８．５％）明显高于镜检法检出率（７５．６％），两者符合率为８７．２％。结果表明ＰＣＲ检测Ｐ．ｖ．具有简便、快速、特异性强、敏感性高等优点，可作为Ｐ．ｖ．诊断和流行病学调查的重要手段     

核酸体外扩增技术诊断骨结核的临床研究

目的探讨核酸体外扩增（ＰＣＲ）技术在骨结核诊断中的价值。方法对６０例骨结核标本与２０例非骨结核标本分别应用ＰＣＲ、抗酸染色镜检及分离培养法进行结核分支杆菌检测。同时，分析了影响ＰＣＲ结果的有关因素与相应处理措施。结果６０例骨结核标本中三种方法的阳性检出率分别为：ＰＣＲ法８３％，镜检法３％，培养法７％。经统计学处理，Ｐ＜０．００５，ＰＣＲ法与镜检及培养法对结核分支杆菌的阳性检出率比较具有显著性差异，ＰＣＲ法明显优于镜检及培养法。２０例非骨结核标本镜检及培养法均阴性，ＰＣＲ法阳性率１０％。盲法结核分支杆菌和对照菌ＰＣＲ检测结果表明，ＰＣＲ的特异性为１００％。ＰＣＲ扩增整个过程自动化控制，可在数小时内完成。结论ＰＣＲ技术是一种快速、敏感、特异与简便的骨结核标本结核分支杆菌检测方法，对骨结核的诊断与鉴别诊断具有重要价值。     

改良式聚合酶链反应检测幽门螺杆菌

为建立一种检测幽门螺杆菌感染的最佳方法，用尿苷酶抗污染聚合酶链反应（改良式ＰＣＲ）检测幽门螺杆菌，并与组织病理学检查、尿素酶试验、酶联免疫吸附试验及细菌培养相比较。共９５例病人５种方法的检出率分别为８３．１％，７８．９％，７０．５％，６６．３％，４２．１％。２项检查阳性即判断为阳性，否则为阴性。９５例病人中有７９例阳性，１６例阴性。ＰＣＲ方法的敏感性、特异性、阳性预测值及阴性预测值分别为：９７．５％，８７．５％，９７．５％，８７．５％，明显优于其它检查方法。改良后的ＰＣＲ方法为其从单纯的科研方法转为临床应用作了尝试。     

一例普通转状病毒腹泻患儿血中轮状病毒的检出及测序

目的 探讨普通轮状病毒（ＲＶ）腹泻患儿病毒血症出现情况。方法 套式－逆转录ＰＣＲ检测血中ＲＶ，阳性ＰＣＲ产物测序。结果 ６０例ＲＶ腹泻患儿中，检出１例病毒血症阳性者，检出率为１．６７％，测序证实为ＲＶ序列，未见明显变异。结论 普通ＲＶ腹泻患儿也可发生病毒血症。     

病毒性心肌炎心内膜心肌活检石蜡包埋标本中巨细胞病毒基因的原位多聚酶链反应

对２５例病毒性心肌炎（ＶＭＣ）心内膜心肌活检（ＥＭＢ）石蜡包埋标本，用原位多聚酶链反应（ＩＳＰＣＲ）方法进行了巨细胞病毒（ＣＭＶ）的原位检测，心肌细胞内阳性检出率为４４％，明显高于对照组（Ｐ＜０．０１），但病毒基因与心肌病理学改变的关系尚不能确定。提示在心肌内ＣＭＶ基因检测中，ＩＳＰＣＲ是一种迅速、敏感和特异的方法，与原位杂交方法相比，其阳性检出率虽略有提高，但差异不具有统计学意义。     

幽门螺杆菌flaA基因的变异

目的鞭毛是幽门螺杆菌（Ｈｐ）的重要侵袭因子,本研究拟检测Ｈｐ鞭毛素Ａ基因的变异性,进而研究其与Ｈｐ致病性的关系．方法我们对５２例胃粘膜活检标本和１８株Ｈｐ临床分离株进行Ｈｐ特异的１６ＳｒＲＮＡ基因和鞭毛素Ａ基因ＰＣＲ扩增和ＰＣＲＲＦＬＰ及ＰＣＲＲＦＬＰＳＳＣＰ分析．结果对４３例Ｈｐ（＋）的ｆｌａＡ基因ＰＣＲＲＦＬＰ（ＨｉｎｄⅢ）大约可分７个型（变异检出率为１６３％）,而ＰＣＲＲＦＬＰＳＳＣＰ可分３７个型（变异检出率为８６０％）,两者有非常显著性差异．结论ｆｌａＡ基因变异性极大,ＰＣＲＲＦＬＰＳＳＣＰ优于ＰＣＲＲＦＬＰ,是检测其变异的有效方法．     

Use of dengue NS1 antigen for early diagnosis of dengue virus infection

Accurate and timely diagnosis of dengue virus is important for early detection of dengue virus infection. In this study, the usefulness of the dengue NS1 antigen test was evaluated as a routine, rapid diagnostic test for dengue virus infection. A total of 208 sera from patients suspected of having dengue virus infection were collected and tested for dengue antibody, dengue genome and dengue NS1 antigen. Dengue antibody test, dengue PCR test and dengue antigen test were able to detect dengue virus infection from Days 1 to 8 in 72.8, 52.8 and 44.0% of samples, respectively. Of the 208 sera tested, 69.2% (144/208) of the acute sera were positive for dengue virus infection based on IgM antibody, IgG antibody, NS1 antigen and PCR tests. Thirty-two point two percent of the samples (67/208) were found positive for dengue NS1 antigen, 38.5% (80/208) were PCR positive, 40.9% (85/208) were IgM positive and 36.1% (75/208) were IgG positive for dengue virus. The results reveal the detection rate of dengue virus infection was similar for PCR and dengue antibody (65.9%) and for NS1 antigen and dengue antibody (62.0%) combinations. Therefore, the dengue NS1 antigen test can be used to complement the current antibody test used in peripheral laboratories. Thus, the combination of the NS1 antigen and antibody tests could increase the diagnostic efficiency for early diagnosis of dengue infection.     

Dengue virus infections in the first 2 years of life and the kinetics of transplacentally transferred dengue neutralizing antibodies in thai children

BACKGROUND: Understanding dengue virus infection in children and the kinetics of maternal dengue neutralizing antibodies is essential for effective dengue immunization of children in endemic areas. METHODS: Serum samples from 219 mother-child pairs and 140 children at 3, 6, 9, 12, 18, and 24 months of age from Bangkok, Thailand, were tested for serotype-specific dengue antibodies. Febrile episodes in the children were recorded. RESULTS: Antibodies were found in 97% of cord serum samples and disappeared in 27%, 80%, and 95% of the children by the age of 6, 9, and 12 months, respectively. Geometric mean titers (GMTs) of the antibodies to 4 dengue serotypes decreased to 5.4-15.5 in 6-month-old infants. Eleven of 12 children acquired dengue virus infection at 6 months of age and beyond; 1 had the infection at 3 months of age. Two exhibited undifferentiated febrile illnesses, and 10 had subclinical infections. CONCLUSIONS: Evidence of dengue virus infection and very low GMTs against all dengue serotypes in children at 6 months of age and beyond was demonstrated. There was no evidence that maternal antibodies were harmful to infants. Dengue virus infection rates increase from 12 months of age onward. These data provide information for supporting the optimal age at vaccination.     

Secondary dengue virus type 4 infections in Vietnam

This study was designated to describe clinical and biological features of patients with a suspected diagnosis of dengue fever/dengue hemorrhagic fever during an outbreak in Central Vietnam. One hundred and twenty-five consecutive patients hospitalized at Khanh Hoa and Binh Thuan Provincial hospitals between November 2001 and January 2002 with a diagnosis of suspected dengue infection were included in the present study.Viruses were isolated in C6/36 and VERO E6 cell cultures or detected by RT-PCR. A hemagglutination-inhibition test (HI) was done on each paired sera using dengue antigens type 1-4, Japanese encephalitis (JE) virus antigen, Chickungunya virus antigen and Sindbis virus antigen. Anti-dengue and anti-JE virus IgM were measured by a capture enzyme-linked immunosorbent assay (MAC-ELISA). Anti-dengue and anti-JE virus IgG were measured by an ELISA test. Dengue viruses were isolated in cell culture and/or detected by RT-PCR in 20.8% of blood samples. DEN-4 and DEN-2 serotypes were found in 18.4% and 2.4% of the patients, respectively. A total of 86.4% of individuals had a diagnosis of acute dengue fever by using the HI test and/or dengue virus-specific IgM capture-ELISA and/or virus isolation and/or RT-PCR. The prevalence of primary and secondary acute dengue infection was 4% and 78.4%, respectively. Anti-dengue IgG ELISA test was positive in 88.8% of the patients. In 5 cases (4%), Japanese encephalitis virus infection was positive by serology but the cell culture was negative. No Chickungunya virus or Sindbis virus infection was detected by the HI test. In patients with acute dengue virus infection, the most common presenting symptom was headache, followed by conjunctivitis, petechial rash, muscle and joint pain, nausea and abdominal pain. Four percent of hospitalized patients were classified as dengue hemorrhagic fever. The clinical presentation and blood cell counts were similar between patients hospitalized with acute dengue fever and patients with other febrile illnesses.     

Expression of the dengue virus type 2 NS5 protein in a CD4(+) T cell line inhibits HIV replication

Human immunodeficiency virus (HIV) load is suppressed during dengue virus infection. The NS5A phosphoprotein of GB virus C (a related flavivirus) inhibits HIV replication in vitro. To determine whether the dengue virus NS5 protein inhibits HIV replication, CD4(+) T cell lines expressing this protein were generated. HIV replication in dengue virus NS5-expressing cells decreased by >90% compared with control cells ([Formula: see text]), and this was mediated in part by decreased HIV coreceptor (CXCR4) expression and increased production of SDF-1. Thus, the dengue virus NS5 protein inhibits HIV replication in vitro, potentially explaining the reduction in HIV load observed during acute dengue virus infection.     

Virologic and serologic surveillance for dengue fever in Jeddah, Saudi Arabia, 1994-1999.

Dengue fever infection was first documented in Jeddah, Saudi Arabia, by virus isolation of dengue type 2 virus in 1994 at the virology laboratory of Dr. Soliman Fakeeh Hospital. Dengue virus surveillance was established after that time. Blood samples were collected from 985 patients (710 male patients and 275 female patients) with suspected cases of dengue from February 1994 to December 1999. Dengue virus isolates were obtained in 207 patients (21%; 162 male patients and 45 female patients). Dengue type 2 was the predominant serotype (138 of 207 isolates, 66.7%), followed by dengue type 1 with (56 of 207 isolates, 27%) and dengue type 3 (13 of 207 isolates, 6.3%). The largest number of isolates (186 of 207 isolates, 90%) was in 1994, a year during which there was a dengue epidemic. In the next 5 years, 1995-1999, only 21 isolates (10%) were isolated. Immunoglobulin M capture enzyme-linked immunosorbent assay was positive in 160 acute samples; 52 of them were from virus culture-positive cases and 108 (11%) from culture-negative cases. The total number of cases diagnosed by both methods was 315 (32%). The prevalence of dengue immunoglobulin G antibodies, as assessed on the basis of immunofluorescent assay, hemagglutination inhibition titers > or = 1/20, or both, in the acute samples was 314 (32%) of 985, indicating past Flavivirus infection. Two patients died, one man with dengue hemorrhagic fever and one woman with dengue shock syndrome. Both fatal dengue cases were due to infection with type 2 virus. All other cases were simple dengue fever. To our knowledge, this is the first report confirming the circulation of 3 dengue serotypes in Jeddah.     

Vertical transmission of dengue viruses by mosquitoes of the Aedes scutellaris group

Seventeen strains of mosquitoes belonging to 12 species in the Aedes scutellaris subgroup were tested for an ability to transmit one or more dengue virus serotype(s) vertically. Strains of virus employed for dengue types 1, 2, 3, and 4 were from Fiji, Bangkok, Burma, and Medan, respectively. After parental females were infected by intrathoracic inoculation, F1 larval and pupal progeny were tested for the presence of virus by inoculating aliquots of triturated suspensions into Toxorhynchites amboinensis mosquitoes. Dengue type 1 was transmitted vertically by 11 strains of mosquitoes representing 8 species with the highest filial infection rates observed for Ae. cooki (1.2%). Vertical transmission of the other dengue virus serotypes was observed for fewer species of mosquitoes, however the filial infection rates of those demonstrating vertical transmission were between 1%-2% for types 2 and 3, and about 0.5% for type 4. Tests with the progeny of individual Ae. cooki and Ae. polynesiensis infected with dengue virus types 1 and 3, respectively, showed that approximately greater than or equal to 50% of the parental females transmitted virus to their progeny. Highest filial infection rates were 6.7% for Ae. cooki and 4.6% for Ae. polynesiensis.     

Variation among geographic strains of Aedes albopictus in susceptibility to infection with dengue viruses.

The comparative susceptibility to dengue virus infection of 13 geographic strains of Aedes albopictus was studied by feeding the mosquitoes on a virus-erythrocyte-sugar suspension. Significant variation in susceptibility for each of the four dengue serotypes was observed among the geographic strains. Mosquito strains which were more susceptible to infection with one dengue serotype also were more susceptible to the other dengue serotypes. There was a direct relationship between the amount of virus ingested and the infection rate in a given mosquito strain. A 100-fold difference in oral ID50 was noted between the most and the least susceptible strains. Crossing experiments between susceptible and resistant mosquito strains produced hybrid progeny with intermediate susceptibility. Susceptibility to infection by dengue 2 virus was decreased by selective inbreeding in one strain from 74% to 13% in two generations. Further selection, however, failed to produce a completely resistant line. It appeared that the actual "barrier" to infection was in the mosquito midgut.     

Molecular characterization and clinical evaluation of dengue outbreak in 2002 in Bangladesh

During the febrile illness epidemic in Bangladesh in 2002, 58 people died out of the 6,132 affected. Two hundred hospitalized patients were analyzed clinically, serologically and virologically to determine the features of this dengue infection. Among the 10- to 70-year-old age group of the 200 clinically suspected dengue patients, 100 (50%) were confirmed as dengue cases by virus isolation and dengue IgM-capture ELISA. Of the 100 dengue-confirmed cases, the mean age was 29.0 (+/-12.4). The possible dengue secondary infection rate determined by Flavivirus IgG-indirect ELISA was 78% in 2002. Eight dengue virus strains were isolated, representing the first dengue virus isolation in the country, and all of the strains were dengue virus type-3 (DEN-3). Sequence data for the envelope gene of the DEN-3 Bangladeshi isolates were used in a phylogenetic comparison with DEN-3 from other countries. A phylogenetic analysis revealed that all 8 strains of DEN-3 were clustered within a well-supported independent sub-cluster of genotype II and were closely related to the Thai isolates from the 1990s. Therefore, it is likely that the currently circulating DEN-3 viruses entered Bangladesh from neighboring countries.     

The effect of dengue virus infection on the clinical sequelae of Japanese encephalitis: a one year follow-up study in Thailand.

In order to determine if prior dengue virus infection reduces the severity of Japanese encephalitis (JE), we examined 127 patients hospitalized during the 1970 JE epidemic in the Chiangmai and Lampang Valleys of northern Thailand. Patients were studied during the first 30 days after onset of JE; 120 of these patients were examined one year later for residual neurologic sequelae. About 21% of patients had serological evidence of a prior dengue virus infection. Morbidity and mortality in patients with and without prior dengue virus experience were compared. These comparisons were made within two age groups to exclude differences due to age alone;     

Emerging viral pathogens in long-term expatriates (II): dengue virus

Dengue virus infections have been well known for many years; still dengue virus is regarded as an ‘emerging’ pathogen, as the disease profile is changing. Its geographical range and oveall incidence, and the incidence of the associated complications, dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS), are on the increse. Modern-day travel and increasing urbanization seem to be the main contributing factors. In order to estimate the risk of infection during long-term stays in dengue-endemic countries, we tested sera obtained from 323 development aid workers and their family members who had spent on average 9.8 years in dengue-endemic regions for the presence of dengue virus antibodies. Dengue virus antibody screening was done by a commercially available immunofluorescence test (IF). Reactive samples were re-tested by an in-house IF and also tested for cross-reactivity to yellow fever virus using yellow fever IF and neutralization test (NT). Evaluation of the results revealed that the screening test has a specificity of at least 63.2%. In 12 of 19 initially positive cases crossreacting antibodies against yellow fever virus could be ruled out. Three cases remained indeterminable, whereas four of the reactive and 10 (out of 12) of the borderline reactive cases showed crossreactivity with yellow fever virus, probably due to previous vaceination. We found seroprevalence rates of 4.3% with no significant differences related to gender or area of upbringing. Scroprevalence rates were evaluated according to region of suspected or confirmed infection. In two cases the dengue infection had taken a classical clinical course; in another three cases an extraordinary febrile illness was reported in the history. None of the other seropositive individuals had a history of an illness possibly attributable to dengue virus infection. Our results show that there definitely is a risk for long-term expatriates to acquire (mostly non- or oligo-symptomatic) dengue infection, which might be important especially in the light of the supposed aetiology of DHF or DSS as a secondary infection with another dengue virus serotype.