Rapid Detection of Oculopathogenic Adenovirus in Conjunctivitis

Ocular adenovirus (Ad) infections occur throughout the world in both sporadic and epidemic forms. Accurate laboratory diagnosis of Ad in conjunctival samples is always valuable. The present study was carried out to explore the presence of Ad as a causative agent in clinically suspected viral conjunctivitis and to compare the performance of conventional virus isolation on cell cultures, direct detection of Ad antigens in conjunctival cells by a direct fluorescence assay, Ad DNA detection by polymerase chain reaction (PCR), and specific IgM measurement by ELISA. Samples included scrapes from conjunctiva. Scrapes were subjected to study by direct immunofluorescence stain, culture on the Hep-2 cell line, and PCR for Ad detection. Blood samples were also taken and subjected to study for specific anti-Ad IgM determination. The culture for Ad was positive in 77.8%, direct antigen detection by fluorescent stain was positive in 72.2%, PCR was positive in 83.3%, and serology was positive in 88.9% of patients. Both determination of antibody IgM and PCR correctly identified a larger group of patients compared to cell culture. The most sensitive and specific method for diagnosis of Ad compared to culture was PCR (100%), followed by IgM detection (92.9%) then direct antigen detection by fluorescent stain (85.8%). From this study, we conclude that Ad is a common pathogen in sporadic cases of conjunctivitis. Screening of adenoviral conjunctivitis is possible by using specific IgM due to its high sensitivity. A confirmatory test can be done by PCR for diagnosis of Ad, as it is a rapid, specific, and accurate method.     

Trichomonas vaginalis detection using real-time TaqMan PCR

1978 women and 93 men, all suspected of having a Trichomonas vaginalis infection, were tested for the presence of T. vaginalis by real-time PCR using the T. vaginalis-specific 2-kb repeated sequence, and by direct microscopy and culture. 40 samples were positive by T. vaginalis real-time PCR and 27 were positive by wet mount microscopy, either direct or after culture. All samples positive by direct microscopy of culture were also positive by real-time PCR. Of the 13 samples which were real-time PCR positive but negative by direct microscopy and culture 11 were confirmed by another T. vaginalis real-time PCR based on the beta tubulin gene. Only 2 samples (0.1%) showed inhibition in the PCR. The prevalence of T. vaginalis infection in the female patients was 1.8%. The sensitivity, specificity, positive and negative predictive values of the real-time PCR were 100%, 99.9%, 95% and 100%, respectively. The same test characteristics for the combined conventional T. vaginalis detection methods (microscopy+culture) were 71%, 100%, 100% and 99%, respectively. Therefore, real-time PCR is the method of choice for the diagnosis of T. vaginalis infection.     

Detection of antibodies to herpes simplex virus type 2 with a mammalian cell line expressing glycoprotein gG-2

The gene (US4) coding for herpes simplex virus type 2 (HSV-2) glycoprotein G (gG-2) was cloned and constitutively expressed in Chinese hamster ovary (CHO) cells. The expression vector containing the dihydrofolate reductase (dhfr) gene, and the HSV-2 US4 gene under the control of the Simian virus 40 early promoter (SV40 EP), was transfected into dhfr-deficient CHO cells. The transfected cells were selected and amplified using methotrexate (MTX). To demonstrate that the gG-2 produced in these transformed cells had antigenic determinants in common with the native glycoprotein, CHO cells expressing gG-2 were used in an immunofluorescent assay (IFA) for the detection of HSV-2 type-specific antibodies in human serum samples. Seven of eight serum samples from adults with prior episodes of culture proven HSV-2 infections were found to be positive by the IFA method whereas none of seven serum samples from young children with culture documented HSV-1 infections were positive by IFA. Thus the recombinant CHO : gG-2 cells have diagnostic utility in an HSV-2 specific serologic assay.     

Tegument-specific, virus-reactive CD4 T cells localize to the cornea in herpes simplex virus interstitial keratitis in humans

Herpes stromal keratitis (HSK) is a prevalent and frequently vision-threatening disease associated with herpes simplex virus type 1 (HSV-1) infection. In mice, HSK progression occurs after viral clearance and requires T cells and neutrophils. One model implicates Th1-like CD4 T cells with cross-reactivity between the HSV-1 protein UL6 and a corneal autoantigen. HSK can be prevented by establishing specific immunological tolerance. However, HSK can also occur in T-cell receptor-transgenic X SCID mice lacking HSV-specific T cells. To study the pathogenesis of HSK in the natural host species, we measured local HSV-specific T-cell responses in HSK corneas removed at transplant surgery (n = 5) or control corneas (n = 2). HSV-1 DNA was detected by PCR in two specimens. HSV-specific CD4 T cells were enriched in three of the five HSK specimens and were not detectable in the control specimens. Reactivity with peptide epitopes within the tegument proteins UL21 and UL49 was documented. Responses to HSV-1 UL6 were not detected. Diverse HLA DR and DP alleles restricted these local responses. Most clones secreted gamma interferon, but not interleukin-5, in response to antigen. HSV-specific CD8 cells were also recovered. Some clones had cytotoxic-T-lymphocyte activity. The diverse specificities and HLA-restricting alleles of local virus-specific T cells in HSK are consistent with their contribution to HSK by a proinflammatory effect.     

血中检测SARS冠状病毒N蛋白在SARS实验室早期诊断中的作用

为明确严重急性呼吸综合症(SARS)冠状病毒N蛋白在SARS实验室早期诊断中的作用,通过微量中和试验及酶联免疫方法、间接免疫荧光法检测疑似病人恢复期血清(大于28天)中SARS-IgG抗体,确诊SARS患者。同时收集发病不同时期SARS及普通发热病人血清,利用酶联免疫方法检测SARS-CoVN蛋白,并与荧光定量PCR早期诊断方法相比较。共检测：广州地区2003年12月～2004．年1月新发4例确诊SARS患者不同时期的血液和咽漱液标本;恢复期血清SARS-CoV中和抗体阳性病人不同时期的血清46份;广州地区2003年1月～4月临床确诊SARS患者159例的血清和56例疑似患者血清;非SARS普通发热病人血清97份;正常人体检血清100份。结果：4例新发SARS患者的不同时期标本中,3例患者急性期血均检出N蛋白,优于常用的荧光定量PCR检测方法。46份SARS-CoV中和抗体阳性的血清标本,N蛋白检出率为100％。159例临床确诊病例中,发病早期5天以内SARS-CoVN蛋白的检出率为92．3％,随后呈现逐步下降的趋势,在发病第18天仍可检出。56例临床疑似患者发病早期也有23．2％检出率。而97例普通发热病人及100份正常人血清中均未能检测出SARS-CoVN蛋白。表明在血清中检测SARS冠状病毒N蛋白的方法敏感性和特异性都好,对SARS实验室早期诊断具有重要作用。     

Detection of typhoid fever by Widal and indirect fluorescent antibody (IFA) tests. A comparative study

Widal test is a conventional method for the detection of typhoid fever. However, it takes 18-24 hours to complete the test. In the present study indirect fluorescent antibody test has been compared with the Widal test using single serum specimens and was found to be rapid, sensitive and specific. Serum specimens from 41 culture proven cases of typhoid fever, 14 clinically suspected cases and 22 normal individuals were collected. Whereas Widal test detected 63.41% positive cases, IFA test detected 87.80% from among culturally proven typhoid cases. Among the clinically suspected cases of typhoid fever, IFA test detected 85.71% (28.57 + 57.14%) while Widal test detected only 57.13% (35.71 + 21.42%) positive cases out of above 14 cases.     

Diagnosis of Genital Herpes by Polymerase Chain Reaction Amplification

A new polymerase chain reaction (PCR) method employing type-specific primers and probes was applied to 114 clinical specimens obtained from 58 female patients with genital lesions or who had a history of genital herpes. Ten and 15 specimens, respectively, were positive for herpes simplex virus (HSV)-1 and HSV-2 by cell culture. All of 10 culture-confirmed HSV-1 cases and 11 of 15 (73%) culture-confirmed HSV-2 cases were identified by PCR. Although there were several cases with discrepancy between cell culture and PCR for HSV-2, the results suggest that this PCR procedure could be applied to clinical specimens from the female genital tract.     

B7 costimulation molecules encoded by replication-defective, vhs-deficient HSV-1 improve vaccine-induced protection against corneal disease

Herpes simplex virus 1 (HSV-1) causes herpes stromal keratitis (HSK), a sight-threatening disease of the cornea for which no vaccine exists. A replication-defective, HSV-1 prototype vaccine bearing deletions in the genes encoding ICP8 and the virion host shutoff (vhs) protein reduces HSV-1 replication and disease in a mouse model of HSK. Here we demonstrate that combining deletion of ICP8 and vhs with virus-based expression of B7 costimulation molecules created a vaccine strain that enhanced T cell responses to HSV-1 compared with the ICP8⁻vhs⁻ parental strain, and reduced the incidence of keratitis and acute infection of the nervous system after corneal challenge. Post-challenge T cell infiltration of the trigeminal ganglia and antigen-specific recall responses in local lymph nodes correlated with protection. Thus, B7 costimulation molecules expressed from the genome of a replication-defective, ICP8⁻vhs⁻ virus enhance vaccine efficacy by further reducing HSK.     

Development and evaluation of a PCR method for diagnosis of Salmonella enteric fever, based on DNA sequences of the hilA gene

Typically, diagnosis of enteric fever due to Salmonella spp. is by bacterial isolation from blood culture; however, the blood culture method is slow, not always available, and not informative in patients with antibiotic treatment. Salmonella spp. uses the hilA gene (component of the pathogenicity island I) to invade epithelial cells and produce infection. Using the hilA gene sequence a PCR test was designed to detect Salmonella in blood samples. The sensitivity (S), specificity (SP), positive predictive value (PPV) and negative predictive value (NPV) of the PCR method were obtained by testing the blood samples from 34 patients with suspected of enteric fever. Presence of S. typhi was confirmed by blood culture. Blood samples were also tested from 35 patients with infections due to other non-Salmonella pathogens, again corroborated by blood culture (Klebsiella pneumoniae, 9; Serratia marcescens, 5; Escherichia coli, 4; Pseudomonas aeruginosa, 9; Providencia alcalifaciens, 4; Enterobacter cloacae, 4). Control samples were obtained from 150 healthy volunteers. The S, SP, PPV and NPV for the PCR method were all 100%. The lowest number of colony forming units/ml detected by PCR in blood samples was 10.     

Etiologic Agents of Central Nervous System Infections among Febrile Hospitalized Patients in the Country of Georgia

Objectives

There is a large spectrum of viral, bacterial, fungal, and prion pathogens that cause central nervous system (CNS) infections. As such, identification of the etiological agent requires multiple laboratory tests and accurate diagnosis requires clinical and epidemiological information. This hospital-based study aimed to determine the main causes of acute meningitis and encephalitis and enhance laboratory capacity for CNS infection diagnosis.

Methods

Children and adults patients clinically diagnosed with meningitis or encephalitis were enrolled at four reference health centers. Cerebrospinal fluid (CSF) was collected for bacterial culture, and in-house and multiplex RT-PCR testing was conducted for herpes simplex virus (HSV) types 1 and 2, mumps virus, enterovirus, varicella zoster virus (VZV), Streptococcus pneumoniae, HiB and Neisseria meningitidis.

Results

Out of 140 enrolled patients, the mean age was 23.9 years, and 58% were children. Bacterial or viral etiologies were determined in 51% of patients. Five Streptococcus pneumoniae cultures were isolated from CSF. Based on in-house PCR analysis, 25 patients were positive for S. pneumoniae, 6 for N. meningitidis, and 1 for H. influenzae. Viral multiplex PCR identified infections with enterovirus (n = 26), VZV (n = 4), and HSV-1 (n = 2). No patient was positive for mumps or HSV-2.

Conclusions

Study findings indicate that S. pneumoniae and enteroviruses are the main etiologies in this patient cohort. The utility of molecular diagnostics for pathogen identification combined with the knowledge provided by the investigation may improve health outcomes of CNS infection cases in Georgia.     

Diagnostics for herpes simplex virus: is PCR the new gold standard?

Herpes simplex virus (HSV) is one of the most common, yet frequently overlooked, sexually transmitted infections. Since the type of HSV infection affects prognosis and subsequent counseling, type-specific testing to distinguish HSV-1 from HSV-2 is recommended. Although PCR has been the diagnostic standard for HSV infections of the central nervous system, until now viral culture has been the test of choice for HSV genital infection. However, HSV PCR, with its consistently and substantially higher rate of HSV detection, will likely replace viral culture as the gold standard for the diagnosis of genital herpes in people with active mucocutaneous lesions, regardless of anatomic location or viral type. Alternatively, type-specific serologic tests based on glycoprotein G should be the test of choice to establish the diagnosis of HSV infection when no active lesion is present. Given the difficulty in making the clinical diagnosis of HSV, the growing worldwide prevalence of genital herpes and the availability of effective antiviral therapy, there is an increased demand for rapid, accurate laboratory diagnosis of patients with HSV.     

Diagnostic value of viral culture, polymerase chain reaction and western blot for HIV-1 infection in 218 infants born to HIV-infected mothers and examined at different ages.

In a prospective longitudinal 10-year (1988 to 1998) study, 308 sequential blood samples from 218 infants born to HIV-1 seropositive women were examined by blood culture, polymerase chain reaction (PCR) and Western Blot (WB) for HIV-1 infection within the first month of life (no. 47 specimens), at 2-6 (no. 125), 7-18 (no. 80), and > 18 (no. 56) months after birth. Clinical status at follow-up after the initial diagnosis of HIV infection was also evaluated. Vertically transmitted HIV infection was diagnosed in 45 children (24 children were diagnosed before 18 months of age), whereas 173 were found to be uninfected (transmission rate 20.6%). Sensitivities of viral culture, PCR and WB were 95.2%, 97.8%, 94.4%, and specificities were 99.5%, 97.6% and 20.7%, respectively. Thus, cumulative positive predictive values (PPV) of blood culture, PCR and WB were 97.5%, 88.2% and 23.4%, while negative predictive values (NPV) were 99.0%, 99.6% and 100.0%, respectively. In view of defining the optimal time of sampling for a correct diagnosis of HIV infection, a PPV of 100.0% was achieved earlier by viral culture (2-6 months of age) than by PCR (7-18 months of age). Meanwhile, a NPV of 100% was obtained earlier by PCR (within the first month of age) than by viral culture (2-6 months). These results indicate that a combination test strategy requiring two blood samples analyzed by viral culture and PCR may confirm or exclude HIV perinatal infection within the first 2 months of life rather than being delayed to later times. Clinical follow-up was performed in 35 children, of whom 7 developed a rapidly progressive disease, 23 showed a slow progression, while 5 children are still younger than 5 years and do not present severe clinical symptoms.     

Genital co-infection with herpes simplex viruses type 1 and 2: comparison of real-time PCR assay and traditional viral isolation methods

We report the clinical case of a genital outbreak with both Herpes Simplex Type 1 (HSV-1) and Herpes Simplex Type 2 (HSV-2) during pregnancy. Herpes was presumptively identified by clinical presentation of lesion and Tzanck smear while serotypes were identified by cell culture and polymerase chain reaction (PCR). This case report highlights the need for increased surveillance of both serotypes in genital infection of pregnant women for effective disease management and reduced risk of transmission. Increasing rates of genital infection with HSV-1, the possibility of genital co-infection with HSV-1 and HSV-2 and the non-specificity and lack of sensitivity of traditional viral isolation methods may lead to under-diagnosis of genital HSV-1 infections unless molecular diagnostic methods, such as polymerase chain reaction (PCR) are routinely deployed in the clinical setting.     

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. Of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 x 10(0) cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations.     

B7-H1 (CD274) inhibits the development of herpetic stromal keratitis (HSK)

The co-signaling molecule B7-H1 (CD274) functions as both a co-inhibitor through programmed death-1 (PD-1) receptor and a co-stimulator via an as-yet-unidentified receptor on T cells. We investigated the physiological role of endogenous B7-H1 in the pathogenesis of herpetic stromal keratitis (HSK) caused by herpes simplex virus type 1 (HSV-1). Following HSV-1 infection of the cornea of mice, B7-H1 expression was up-regulated in the CD11b+ macrophage population in the draining lymph nodes (dLN) and in the inflamed cornea. In addition, HSV-1 infection significantly increased PD-1 expression on CD4+ T cells in the dLN and inflamed cornea. The administration of antagonistic B7-H1 monoclonal antibody resulted in the proliferation of HSV-specific CD4+ T cells that secreted interferon (INF)-gamma, and inhibited the apoptosis of HSV-specific CD4+ T cells, which exaggerated HSK. These results strongly suggest that the B7-H1 may be involved in suppression of the development of HSK.     

Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia

In this study, we have established and evaluated a genus-specific polymerase chain reaction (PCR) and species-specific nested PCRs for the detection of Candida species in blood samples of neutropenic mice and patients suspected of candidemia. DNA segments of the gene encoding cytochrome P450 L1A1 were targeted for amplification by using genus and species-specific primers. As compared to the genus-specific PCR, the species-specific nested PCRs improved the sensitivity by 10 times with the detection limit < 10 yeast cells. Of the 18 blood samples tested daily over a period of 8 days following Candida albicans infection in neutropenic mice, four samples were positive by genus-specific PCR and 11 were positive by species-specific nested PCR. The PCR results were correlated with culture findings obtained on blood samples. Two of the three blood culture-positive samples were positive by genus-specific PCR and all the three with species-specific nested PCR. Among 15 mice, which were negative by blood culture but had C. albicans isolated from visceral organs, 2 and 8 mice yielded positive results by genus-specific PCR and species-specific nested PCR, respectively. Consistent with the results of the animal study, species-specific nested PCR yielded much higher positivity as compared to culture (52.2% versus 21.2%) in patients suspected for candidemia. Moreover, 8 specimens which were negative for Candida by genus-specific PCR became positive by species-specific nested PCR. No correlation was apparent between PCR positivity and Candida antigen titers. The results suggest that nested PCR is a sensitive technique for the detection of Candida species from blood samples, and thus it may have application in the diagnosis of suspected cases of candidemia and candidiasis.     

Culture and PCR analysis of joint fluid in the diagnosis of prosthetic joint infection

This prospective study compared PCR and culture techniques in the diagnosis of prosthetic joint infection (PJI). We obtained joint fluid samples (JFS; n=115) from patients who had failed total joint arthroplasty between January 2003 and June 2005; 49 were positive for PJI according to established strict criteria. JFS were analyzed by PCR (n=35; control n=66) or culture (n=46, control n=48). PCR was positive in 71% of PJI cases, resulting in sensitivity, specificity, accuracy, positive predictive value, negative predictive value, and likelihood ratio for positive results as follows: 0.71; 0.97; 0.88; 0.93; 0.87 and 23.6, respectively. Culture was positive in 44% of PJI samples. Corresponding statistics were 0.44; 0.94; 0.69; 0.87; 0.63 and 7.0, respectively. Significantly higher sensitivity, accuracy and negative predictive values were calculated for PCR versus culture, and there was 83% concordance between the results of intraoperative culture and PCR detection of causative bacteria. Therefore, we conclude that PCR analysis of synovial fluid increases the utility of pre-operative aspiration for patients who require revision total joint surgery.     

MCP-1 derived from stromal keratocyte induces corneal infiltration of CD4+ T cells in herpetic stromal keratitis

Herpetic stromal keratitis (HSK) is an inflammatory disorder induced by HSV-1 infection and characterized by T cell-dependent destruction of corneal tissues. It is not known what triggers CD4(+) T cell migration into the stroma of HSV-1-infected corneas. The keratocyte is a fibroblast-like cell that can function as an antigen-presenting cell in the mouse cornea by expressing MHC class II and costimulatory molecules after HSV-1 infection. We hypothesized that chemokines produced by stromal keratocytes are involved in CD4(+) T cell infiltration into the cornea. We found that keratocytes produce several cytokines and chemokines, including MCP-1, RANTES, and T cell activation (TCA)-3. HSV-1 infection increased the production of MCP-1 and RANTES by keratocytes, and these acted as chemoattractants for HSV-1-primed CD4(+) T cells expressing CCR2 and CCR5. Expression of MCP-1 in the corneal stroma was confirmed in vivo. Finally, when HSV-1-primed CD4(+) T cells were adoptively transferred into wild type and MCP-1-deficient mice that had been sublethally irradiated to minimize chemokine production from immune cells, infiltration of CD4(+) T cells was markedly reduced in the MCP-1-deficient mice, suggesting that it is the MCP-1 from HSV-1-infected keratocytes that attracts CD4(+) T cells into the cornea.