Rapid diagnosis of cytomegalovirus infections by direct immunoperoxidase staining with human monoclonal antibody against an immediate-early antigen.

Direct immunoperoxidase technique using a horseradish peroxidase (HRP)-conjugated Fab' fragment of human monoclonal antibody (humab C7), designated HRP-C7, was evaluated as a rapid diagnosis of cytomegalovirus (CMV) infection. A total of 138 clinical specimens consisting of 124 urine samples and 14 oral swabs were examined for CMV by the direct HRP-C7 staining in comparison with conventional virus isolation. The number of CMV-positive samples by each method was 40 (29.0%) for the former and 37 (26.8%) for the latter, respectively. By HRP-C7 staining, CMV was identifiable within 24 hr after inoculation. By conventional isolation method, an average of 10.3 days had passed before cytopathic effect characteristic of CMV appeared in the cell culture. Some false-positive and false-negative cases were discussed in relation to toxicity of urine samples, storage of the samples, and amount of CMV in the sample. The sensitivity and specificity of HRP-C7 method against conventional isolation method were 89.2% and 93.1%, respectively. Thus, HRP-C7 staining is useful for a rapid diagnosis of CMV infections.     

Direct diagnostic methods for the demonstration of cytomegaloviruses in the blood

CMV can be detected in blood with two techniques: isolation of CMV from the buffy-coat on human embryonic fibroblasts; CMV genome detection by DNA hybridization technique. Isolation of CMV from blood plated on embryonic fibroblasts necessitates a delay of 10-30 days. Using monoclonal antibodies we have developed a 96 hours test to diagnose viremia. Fibroblasts grown on coverslip in tubes are inoculated with buffy-coat. Indirect immunofluorescence test is performed using the monoclonal antibodies E 13, 48 and 96 hours post-infection. DNA-DNA hybridization technique necessitates purification of CMV DNA. CMV DNA is then labelled with 32 P by nick-translation. This labelled DNA is used as probe for detection of CMV genome in DNA extracted from leukocytes. The hybridization is virus-specific. Cytomegaloviremia correlates with active infection and is not indicate of the carrier state of blood donors. CMV genome research in blood can potentially identify infective donors. Further studies are needed to correlate CMV genome detection in blood with presence of CMV antibodies.     

Replication footprint analysis of cucumber mosaic virus electroporated into tomato protoplasts

Total RNA extracted from cucumber mosaic virus (CMV) strains WT, with its associated satellite CARNA 5 (CMV-associated RNA 5), was successfully electroporated into isolated tomato protoplasts. At various time intervals samples were extracted for total nucleic acids and analyzed by semidenaturing polyacrylamide gel electrophoresis (PAGE). Sequence-specific hybridization probes were used for the detection of viral and satellite RNAs following Northern transfer. The resulting PAGE patterns and/or autoradiographs depict the proportional presence of viral and satellite RNAs in the extracts over time and have been referred to as "replication footprint profiles" (RFPs) of specific CMV/CARNA 5 combinations. The effective isolation and infection of tomato protoplasts, combined with the ability to follow virus/satellite titers during the infection by RFP analysis, yield results similar to those of infected plants and reduces experiments of 21 or more days in whole plants to less than 72 h in protoplasts.     

Genetic analysis of a cytomegalovirus-like agent isolated from human brain.

An unusual cytomegalovirus (CMV, strain Colburn) isolated from brain biopsy of a boy with clinical encephalopathy was studied for genetic relatedness to human and simian CMV. Cross-examination of the purified viral DNA by DNA-DNA reassociation kinetics analyses showed more than 90% homology between Colburn virus and simian CMV (strain GR2757) and a lack of detectable homology between Colburn virus and human CMV (strains AD-169 and TW-87). Restriction endonuclease analysis of Colburn DNA showed some similarity of the DNA fragment pattern with that of simian CMV DNA, although the DNA fragment patterns were not identical, and showed no similarity to that of human CMV DNA. The molecular size and density of viral DNA were close to those of simian CMV DNA. The antigenic study, as performed by complement fixation and neutralization tests, showed strong cross-reactivity of Colburn virus to simian GR2757 virus. One-way cross-reaction of Colburn virus to several human CMV isolates (AD-169, Davis, and Town) was detected by complement fixation; this one-way cross-reaction was not obvious in a plaque neutralization test. It was concluded that Colburn is a simian CMV-related virus.     

Detection of the markers of herpes simplex virus and cytomegalovirus in newborns and infants

A total of 111 children suspected for herpesvirus infection were examined. In blood and urine samples the infectious activity of herpes simplex virus (HSV) and cytomegalovirus (CMV) was detected by the rapid culture method (RCM) and the presence of virus DNA--by the polymerase chain reaction (PCR). HSV and/or CMV were detected by two laboratory methods in 57 examined children (51%). Of these, in 18 children (16.2%) both HSV and CMV were detected. The coincidence of the results of the detection of HSV and CMV by these two methods was observed in 72.4% and 75.2% of cases respectively. The comparative analysis of the detection of anti-CMV IgG and IgM was made with the use of commercial test systems produced bythe following manufacturers: "Vector-Best" and "Bioservice" (Russia), "HUMAN" and "Boehringer" (Germany). The effective detection of both anti-CMV (IgG and IgM) was ensured by the test systems "Boehringer". The test system "Vector-Best" for anti-CMV IgG proved to be not inferior as regards sensitivity and specificity. The German test systems demonstrated the highest specificity in the detection of low-avid antibodies. The data obtained in this study indicate that the detection rate of HSV and CMV markers in newborns and infants suspected for herpesvirus infection was, on the average, 20 - 40%. Reliable diagnostics in newborns and infants is possible only in the presence of the combination of at least 2 serological tests (the determination of antivirus IgM and IgG avidity) and 2 methods for the detection of direct herpesvirus markers (PCR and RCM).     

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.     

Rapid diagnosis of cytomegalovirus in immunocompromised patients using monoclonal antibodies that detect early viral antigens

A technique was applied to detect early fluorescent antigens (DEFA) of cytomegalovirus (CMV) using the E13 monoclonal antibodies in 52 immuno-compromised patients hospitalized in the Nephrology Institute of Havana. Of the 75 urine or blood (buffy coat) samples taken, 15 were found positive to CMV. Using classical diploid human fibroblast isolation technique, 12 CMV strains were isolation of previously detected positive samples by DEFA. In addition, CMV was isolated from one sample reported to be negative by DEFA. A coincidence of 80% was found between both techniques. With the ELISA test, all the sample studied have IgG antibodies to CMV.     

Study by in situ hybridization of the prevalence of herpes virus as cofactors of the tumoral development of papillomatous lesions of the anogenital region in seropositive and seronegative men against HIV]

Infection with specific types of HPV has emerged as necessary but not sufficient factor in the neoplastic transformation of anogenital condylomas. Some viruses (HIV, Herpes viridae: HSV, CMV, EBV) might act as cofactors in the neoplastic changes and cancer. To study the prevalence of these viral pathogens in anogenital lesions, biopsies were obtained from HIV seropositive or seronegative men and tested using in situ hybridization technique. Infection by "high risk" HPV, HSV and CMV are facilitated in patients immunocompromised by HIV. Presence of CMV is more frequent in high risk HPV-induced lesions than in low risk HPV lesions.     

Immunological relationships among transaldolases in the genus Bifidobacterium

Antisera were prepared against electrophoretically homogeneous transaldolase (dihydroxyacetone transferase, E.C. 2.2.1.2.) of Bifidobacterium thermophilum (B. ruminale) RU326 (ATCC 25866), B. cuniculi RA93 (ATCC 27916) and B. minimum (homology group) F392 (ATCC 27538).Crude extracts of eighty six strains previously assigned to twenty one species of the genus Bifidobacterium on the basis of deoxyribonucleic acid (DNA) homology (DNA-DNA hybridization), were compared by double diffusion tests on Ouchterlony plates. Eight groups of identical antigenic specificity were recognized. By analysis of the spur formation, the groups of identical specificity were arranged in preliminary sequences of decreasing similarity to each of the three homologous transaldolases used as reference point. The relationships between immunological data and the genetic similarity among the species of the genus measured by means of DNA-DNA hybridization were discussed together with some relevant points of bifidal ecology.This investigation was supported by a research grant of Consiglio Nazionale delle Ricerche, Roma.     

Relationship between genome similarity and DNA-DNA hybridization among closely related bacteria

DNA-DNA hybridization has been established as an important technology in bacterial species taxonomy and phylogenetic analysis. In this study, we analyzed how the efficiency with which the genomic DNA from one species hybridizes to the genomic DNA of another species (DNA-DNA hybridization) in microarray analysis relates to the similarity between two genomes. We found that the predicted DNA-DNA hybridization based on genome sequence similarity correlated well with the experimentally determined microarray hybridization. Between closely related strains, significant numbers of highly divergent genes (<55% identity) and/or the accumulation of mismatches between conserved genes lowered the DNA-DNA hybridization signal, and this reduced the hybridization signals to below 70% for even bacterial strains with over 97% 16S rRNA gene identity. In addition, our results also suggest that a DNA-DNA hybridization signal intensity of over 40% indicates that two genomes at least shared 30% conserved genes (>60% gene identity). This study may expand our knowledge of DNA-DNA hybridization based on genomic sequence similarity comparison and further provide insights for bacterial phylogeny analyses.     

人巨细胞病毒的生物素DNA探针的制备和应用

本研究用克隆的HCMV AD169株DNA片段,制备了生物素标记的DNA探针,建立了检测临床脐带血、尿标本中HCMV DNA的核酸探针杂交方法。该探针可测出100pg同源DNA,不与人胚肺细胞、Hep-2细胞DNA以及其他疱疹病毒的DNA发生反应。用核酸杂交方法检测了30份脐带血标本,有11例阳性,阳性率为33％。10例孕妇尿标本中,3例阳性,阳性率为30％。检测结果表明：我们建立的生物素标记的HCMV DNA探针的点杂交法,具有高度的特异性、敏感性,比分离病毒法更迅速,可用于HCMV感染的临床标本的病毒核酸检测。     

Prediction of Maternal Cytomegalovirus Serostatus in Early Pregnancy: A Retrospective Analysis in Western Europe

Background

Cytomegalovirus (CMV) is the most prevalent congenital viral infection and thus places an enormous disease burden on newborn infants. Seroprevalence of maternal antibodies to CMV due to CMV exposure prior to pregnancy is currently the most important protective factor against congenital CMV disease. The aim of this study was to identify potential predictors, and to develop and evaluate a risk-predicting model for the maternal CMV serostatus in early pregnancy.

Methods

Maternal and paternal background information, as well as maternal CMV serostatus in early pregnancy from 882 pregnant women were analyzed. Women were divided into two groups based on their CMV serostatus, and were compared using univariate analysis. To predict serostatus based on epidemiological baseline characteristics, a multiple logistic regression model was calculated using stepwise model selection. Sensitivity and specificity were analyzed using ROC curves. A nomogram based on the model was developed.

Results

646 women were CMV seropositive (73.2%), and 236 were seronegative (26.8%). The groups differed significantly with respect to maternal age (p = 0.006), gravidity (p<0.001), parity (p<0.001), use of assisted reproduction techniques (p = 0.018), maternal and paternal migration background (p<0.001), and maternal and paternal education level (p<0.001). ROC evaluation of the selected prediction model revealed an area under the curve of 0.83 (95%CI: 0.8–0.86), yielding sensitivity and specificity values of 0.69 and 0.86, respectively.

Conclusion

We identified predictors of maternal CMV serostatus in early pregnancy and developed a risk-predicting model based on baseline epidemiological characteristics. Our findings provide easy accessible information that can influence the counseling of pregnant woman in terms of their CMV-associated risk.     

基因芯片技术检测重要人兽共患病病毒方法的建立

为了建立能对25种重要人兽共患病病毒进行筛查及鉴定用的基因芯片技术,本实验首先设计针对每种病毒的寡核苷酸探针并进行探针特异性的生物信息学验证.然后探索病毒核酸随机扩增方法,优化杂交动力学条件,建立本芯片标准的数据处理分析方法.最后用细胞培养的病毒和模拟临床标本验证芯片的敏感性与特异性．结果表明,锚定随机PCR扩增法适合于本芯片病毒核酸的扩增;芯片杂交前用0.25% NaBH4进行封闭,最优杂交条件为51 ℃,2 h及50%甲酰胺浓度;芯片具有较好的敏感性及检测特异性．初步结果表明,本实验所建立的基因芯片技术可应用于对25种重要人兽共患病病毒进行筛查及鉴定．     

Heat denaturation increases the sensitivity of the cytomegalovirus loop‐mediated isothermal amplification method

A new protocol for CMV LAMP with an additional heat denaturation step was developed. While the sensitivity of the original CMV LAMP method was 500 copies/tube, sensitivity was increased by up to 100 copies/tube by additional heat denaturation. CMV DNA was detected in 103 of 350 samples (29.4%) by the original CMV LAMP procedure and 148 of 350 samples (42.3%) by the new CMV LAMP protocol. When the pp65 antigenemia assay was used as the standard method, the sensitivity, specificity, PPV, and NPV of the new protocol were 92.9%, 77.7%, 62.2%, and 96.5%, respectively.     

Predictive Value of Proteinuria in Adult Dengue Severity

Background

Dengue is an important viral infection with different presentations. Predicting disease severity is important in triaging patients requiring hospital care. We aim to study the value of proteinuria in predicting the development of dengue hemorrhagic fever (DHF), utility of urine dipstick test as a rapid prognostic tool.

Methodology and principal findings

Adult patients with undifferentiated fever (n = 293) were prospectively enrolled at the Infectious Disease Research Clinic at Tan Tock Seng Hospital, Singapore from January to August 2012. Dengue infection was confirmed in 168 (57%) by dengue RT-PCR or NS1 antigen detection. Dengue cases had median fever duration of 6 days at enrolment. DHF was diagnosed in 34 cases according to the WHO 1997 guideline. Dengue fever (DF) patients were predominantly younger and were mostly seen in the outpatient setting with higher platelet level. Compared to DF, DHF cases had significantly higher peak urine protein creatinine ratio (UPCR) during clinical course (26 vs. 40 mg/mmol; p<0.001). We obtained a UPCR cut-off value of 29 mg/mmol based on maximum AUC in ROC curves of peak UPCR for DF versus DHF, corresponding to 76% sensitivity and 60% specificity. Multivariate analysis with other readily available clinical and laboratory variables increased the AUC to 0.91 with 92% sensitivity and 80% specificity. Neither urine dipstick at initial presentation nor peak urine dipstick value during the entire illness was able to discriminate between DF and DHF.

Conclusions

Proteinuria measured by a laboratory-based UPCR test may be sensitive and specific in prognosticating adult dengue patients.     

Distribution and Determinants of Cytomegalovirus Induced End Organ Disease/s among People Living with HIV/AIDS in a Poor Resource Setting: Observation from India

BackgroundIn India, despite well-established anti-retroviral treatment programs, Cytomegalovirus (CMV) infection-related end-organ diseases (EODs) still remain a major concern resulting in exacerbation of morbidity and mortality among HIV/AIDS patients. A prospective study was designed to understand the distribution and prognosis of CMV associated EODs and to determine a standardized cut-off value for serum CMV viral load associated with the development of EODs amongst HIV/AIDS subjects.MethodsIn a cohort of 400 late-diagnosed HAART naïve HIV/AIDS subjects attending anti-retroviral centers of Kolkata during 2008–2014, the median duration of follow-up was 560 days, and at least 3 visits subsequent to the baseline were mandatory for eligibility. HIV-1 and CMV viral load were estimated by performing Real-Time Polymerase Chain Reactions (PCR).ResultsAmong subjects, 40.5% (162/400) had CMV EODs which were more common at lower CD4 counts. Poor prognosis and higher death rate were associated with a low CD4 count and increased HIV-1 and CMV viral loads. Subjects having higher CD4 count responded better to therapy [for CD4 = 60–100: Risk Ratio:RR = 1.48 (95% Confidence Interval: 95%CI = 1.18–1.82) and for CD4 = 30–59: RR = 1.64 (95%CI = 1.18–2.27)]. The cut off value of the serum CMV viral load (expressed as log10DNA/ml serum) associated with the development of EODs and disseminated CMV EODs was determined as 5.4 (p<0.0001) and 6.4 (p<0.0001) respectively. These cut offs were found to have satisfactorily high sensitivity, specificity, positive and negative predictive values.ConclusionPrognosis of CMV EOD was poor as indicated by higher death rates among subjects with lower CD4 count, and specific cut-off values were found to have useful potential for identification and treatment of CMV infected HIV/AIDS patients in due time to avoid CMV EODs among HIV/AIDS subjects. Targeted intervention programs seemed to be required urgently to make these cut-offs operational in order to minimize the burden of CMV EOD in this vulnerable population.     

用核酸杂交法检查北京地区幼儿园正常儿童尿中人巨细胞病毒DNA

以克隆的人巨细胞病毒(Human Cytomegalovirus,简称HCMV)DNA片段做探针,用核酸杂交方法检查了50例北京地区幼儿园正常儿童的尿标本。查出HCMV DMA阳性者24例,占48％。在两个群体和两个年龄组之间,阳性检出率有显著性差异。利用探针可查出同源DNA5～10pg,而不与其它病毒或细胞DNA杂交。标记不同的探针,使用不同的固定方法,其敏感性不同。此法可在分子水平上查出HCMV基因,比分离病毒更迅速敏感。     

Detection of human cytomegalovirus DNA and viral antigens in tissues of different manifestations of CMV infection

Biopsy and autopsy specimens from 22 patients with cytomegalovirus (CMV) infections were investigated by means of in situ hybridization (ISH) to detect viral DNA and by immunohistochemistry (IHC) to visualize viral proteins. Both methods proved to be valuable tools for histopathology. ISH sometimes recognized cells that did not show typical CMV inclusions. An antiserum against the full spectrum of viral proteins (non-infectious enveloped particles) detected most cytomegalic cells in disseminated and organ-limited infections. An antiserum against a recombinant polypeptide (XP1) was particularly useful in connatal CMV infections and organ-limited infections. We have demonstrated that IHC and ISH studies in parallel are the best approach to the detection of CMV infections in pathological specimens.     

Advances in the Diagnosis of Human Opisthorchiasis: Development of Opisthorchis viverrini Antigen Detection in Urine

Background

Many strategies to control opisthorchiasis have been employed in Thailand, but not in the other neighbouring countries. Specific control methods include mass drug administration (MDA) and health education to reduce raw fish consumption. These control efforts have greatly shifted the epidemiology of Opisthorchis viverrini (OV) infection over the last decade from presenting as densely concentrated "heavy" infections in single villages to widespread "light" OV infections distributed over wide geographical areas. Currently, the "gold standard" detection method for OV infection is formalin ethyl-acetate concentration technique (FECT), which has limited diagnostic sensitivity and diagnostic specificity for light OV infections, with OV eggs often confused with eggs of minute intestinal flukes (MIFs) in feces. In this study, we developed and evaluated the diagnostic performance of a monoclonal antibody-based enzyme-linked immunosorbent assay for the measurement of OV excretory-secretory (ES) antigens in urine (urine OV-ES assay) for the diagnosis of opisthorchiasis compared to the gold standard detection FECT method.

Methodology

We tested several methods for pre-treating urine samples prior to testing the diagnostic performance of the urine OV-ES assay. Using trichloroacetic acid (TCA) pre-treated urine, we compared detection and quantification of OV infection using the urine OV-ES assay versus FECT in OV-endemic areas in Northeastern Thailand. Receiver operating characteristic (ROC) curves were used to determine the diagnostic sensitivity and specificity of the urine OV-ES assay using TCA pre-treated urine, and to establish diagnostic positivity thresholds. The Positive Predictive Value as well as the likelihood of obtaining a positive test result (LR+) or a negative test result (LR-) were calculated for the established diagnostic positivity threshold. Diagnostic risks (Odds Ratios) were estimated using logistic regression.

Results

When urine samples were pre-treated with TCA prior to use in the urine OV-ES assay, the analytical sensitivity was significantly improved. Using TCA pre-treatment of urine, the urine OV-ES assay had a limit of detection (LoD) of 39 ng/ml compared to the LoD of 52 ng/mL reported for coprological antigen detection methods. Similarly, the urine OV-ES assay correlated significantly with intensity of OV infection as measured by FECT. The urine OV-ES assay was also able to detect 28 individuals as positive from the 63 (44.4%) individuals previously determined to be negative using FECT. The likelihood of a positive diagnosis of OV infection by urine OV-ES assay increased significantly with the intensity of OV infection as determined by FECT. With reference to FECT, the sensitivity and specificity of the urine OV-ES assay was 81% and 70%, respectively.

Conclusion

The detection of OV-infection by the urine OV-ES assay showed much greater diagnostic sensitivity and diagnostic specificity than the current "gold standard" FECT method for the detection and quantification of OV infection. Due to its ease-of-use, and noninvasive sample collection (urine), the urine OV-ES assay offers the potential to revolutionize the diagnosis of liver fluke infection and provide an effective tool for control and elimination of these tumorigenic parasites.