Detection and typing of human herpesvirus 6 by molecular methods in specimens from patients diagnosed with encephalitis or meningitis

Human herpesvirus 6 (HHV-6) was detected in specimens from patients hospitalized with symptoms of encephalitis or meningitis. A real-time PCR assay was developed which has a linear dynamic range of 5 to 5 x 10(6) copies of HHV-6 and a sensitivity of five gene copies per reaction. While the assay detects both subtypes, HHV-6A and HHV-6B, it is specific and does not cross-react with a selected specificity panel. A total of 1,482 patient specimens, which were collected between 2003 and 2007, were tested; 26 specimens from 24 patients were found to be positive for HHV-6 by real-time PCR. The HHV-6 detection rate in this population was therefore 1.75%. The majority of the specimens tested (>95%) were cerebrospinal fluid (CSF) specimens. We were able to type 20 of the 26 positive specimens by conventional PCR and sequence analysis; all were HHV-6B. Forty-two percent of the patients were 3 years of age or younger, which may indicate a primary infection in these patients. Given the ages of the remaining patients (from 4 to 81 years), their infections were most probably due to virus reactivations. Where information was available, symptoms of patients included fever (71%), altered mental status (67%), and abnormal CSF profile (75%). Fifty percent of patients of 3 years of age or younger suffered from seizures. The detection of HHV-6 in specimens from patients diagnosed with encephalitis or meningitis, in the absence of a positive PCR result for other agents, strongly suggests a role for HHV-6 in the pathogenesis of these central nervous system diseases.     

Rapid and sensitive detection of enteroviruses in specimens from patients with aseptic meningitis.

A 5-h PCR assay (Amplicor enterovirus test) was compared with viral culture for the detection of enteroviruses in cerebrospinal fluid. Of the cerebrospinal fluid specimens collected during a summer outbreak of aseptic meningitis, 34% were positive by viral culture whereas 66% were positive by the Amplicor PCR, suggesting that this technique improves the diagnosis of enteroviral meningitis.     

PCR-RFLP based molecular typing of enteroviruses isolated from patients with aseptic meningitis in Korea

Summary.  We have evaluated PCR–RFLP as a practical method for rapid typing of enteroviruses causing aseptic meningitis in Korea. Through blind examination of 80 clinical isolates from patients with aseptic meningitis, we have compared the results of conventional serotyping with PCR–RFLP based genotyping, which was developed for this study. Among the 80 case isolates, which had been previously typed by routine neutralization test, only 42 cases (52.5%) were matched with typing by PCR–RFLP. The result clearly demonstrated that the enterovirus serotype does not coincide with the genotype. Therefore, the classification of enteroviruses by genotyping with PCR–RFLP, although rapid and simple, may be complicated by regional or seasonal differences. However, the PCR–RFLP method developed in this study is applicable to the epidemiological study of enteroviruses when regional or seasonal differences exist, and is useful in identifying the source of an infection. Received August 6, 2001; accepted April 15, 2002 Published online July 10, 2002     

Detection of microsporidial spores in fecal specimens from patients diagnosed with cryptosporidiosis.

Patients infected with Cryptosporidium parvum may have concurrent infections with microsporidia. Two modified trichrome stains and a polyclonal indirect fluorescent-antibody procedure were used for the detection of microsporidia; the Merifluor Cryptosporidium-Giardia monoclonal direct immunofluorescence detection kit was used for the detection of C. parvum. Formalinized stool specimens from 60 immunocompromised patients strongly suspected of having or previously diagnosed with cryptosporidiosis or microsporidiosis were examined. All patients were positive for one or both parasites, 18 (30%) with C. parvum only, 25 (42%) with microsporidia only, and 17 (28%) with both C. parvum and microsporidia. These findings emphasize the importance of considering both organisms as potential causative agents of diarrhea in compromised patients.     

无菌性脑膜炎和脑炎病人脑脊液肠道病毒RNA的检测及其临床意义

目的 检测肠道病毒（EV）在中枢神经系统感染中的致病情况,探讨检测EV感染的方法。方法 就用逆转录－聚合酶链反应（RT－PCR0和病毒培技术检测46例无菌性脑膜炎及脑炎病人脑脊液（CSF）标本。结果 RT－PCR方法敏感特异;46例无菌性脑膜炎和脑炎急性期CSF标本中,31例EV阳性（67．4％）,14例病毒培养阳性（26．1％）。统计结果显示,RT－PCR敏感性明显高于病毒培养。结论 EV是引起无菌性脑膜炎和脑炎的重要病原;RT－PCR快速敏感特异,简单易行,易于推广,是诊断EV感染的有效方法。     

Molecular characterization of enteroviruses isolated from patients with aseptic meningitis in Korea, 2005

Summary. Enteroviruses are the most common causative agents of aseptic meningitis. In 2005, an outbreak of aseptic meningitis occurred in Seoul, Korea, which appeared to be caused by certain enterovirus strains. This study analyzed the clinical isolates from 24 different aseptic meningitis patients at our center from January to September 2005. Serotyping using type-specific antisera, RT-PCR, and direct sequencing of the 5′ noncoding and VP1 regions were carried out. The serotyping study identified 15 coxsackievirus B5 (CBV5), 4 coxsackievirus B1 (CBV1), 1 coxsackievirus B3 (CBV3) and 4 echovirus 9 (ECV9). Phylogenic analysis of the VP1 region revealed CBV5 to be the most predominant strain (15 strains). Twelve of these strains were grouped together with recent strains isolated in China and France. The 12 CBV5 strains contained a novel amino acid change in the BC-loop (the 90th and 91st, AY → GN) compared to the reference strain (1954/UK/85). The amino acid changes in the BC-loop in some recent CBV5 strains tend to be located in the C-terminal part, and these changes suggest some antigenic significance. First two authors contributed equally to this work.     

Molecular typing and epidemiology of enteroviruses identified from an outbreak of aseptic meningitis in Belgium during the summer of 2000

Non-polio enteroviruses are the most common cause of aseptic meningitis worldwide. From May to September 2000, a major outbreak of aseptic meningitis occurred in Belgium. Cerebrospinal fluid samples (CSF) of 122 patients were found to contain enterovirus RNA using diagnostic RT-PCR that targeted a 231-bp gene fragment in the 5' noncoding region. In addition, a molecular typing method was developed based on RT-nested PCR and sequencing directly from CSF(a) 358-bp fragment in the aminoterminal part of the VP1 capsid protein. To identify the enterovirus type, nucleotide sequences of the VP1 amplicons were compared to all the enterovirus VP1 sequences available in GenBank. Echovirus 30 (31.2%), echovirus 13 (23.8%), and echovirus 6 (20.5%) were identified most frequently during the epidemic. Coxsackievirus B5 was present in 15.6% of the samples, and could be subdivided in two distinct epidemic clusters, coxsackievirus B5a (10.7%) and B5b (4.9%). Other enteroviruses encountered were echovirus 16 (5.7%), echovirus 18 (1.6%), coxsackievirus B4 (0.8%) and echovirus 7 (0.8%). The high prevalence of echovirus 13, considered previously a rare serotype, indicates it is an emerging epidemic type. To verify the typing results and to explore further the intratypical genetic variation, phylogenetic analysis was carried out. Geographical clustering of most of the strains within each type and subtype could be observed. The RT-nested PCR strategy, carried out directly on clinical samples, is a simple and rapid method for adequate molecular typing of the Group B enteroviruses causing aseptic meningitis.     

Molecular identification of enteroviruses associated with aseptic meningitis in children from India

We identified and characterized enteroviruses associated with aseptic meningitis in children between April 2009 and March 2010. Enterovirus RNA was detected in 51 (45.5 %) of 112 CSF samples. Molecular typing by RT-PCR and sequencing of a partial VP1 region revealed the predominance of echovirus (ECV) 32 (n = 20), followed by ECV 11 (n = 10), ECV 13 and ECV 14 (n = 5 each), coxsackievirus (CV) B3 and CV B6 (n = 3 each), CV A2, CV A10 and ECV 30 (n = 1 each). Phylogenetic analysis of ECV 32 showed 0 to 4 % sequence divergence among strains of the present study and 20-23 % from the prototype Puerto Rico strain at the nucleotide level. This is the first report of ECV 32 associated with an aseptic meningitis epidemic and identification of seven different enterovirus serotypes (CV A2, CV A10, CV B3, CV B6, ECV 13, ECV 14 and ECV 32) in meningitis cases from India.     

Detection of the Candida antigen mannan in cerebrospinal fluid specimens from patients suspected of having Candida meningitis

Cerebrospinal fluid samples from five patients from which Candida cells were cultured were tested for the presence of mannan. Samples from four patients categorized as having proven candidosis reacted positively. Samples from the remaining patient and from patients with other central nervous system infections were negative. Detection of mannan may be valuable in the diagnosis of Candida meningitis.     

Detection of enteroviruses from clinical specimens by spin amplification shell vial culture and monoclonal antibody assay.

Conventional tube cell culture was compared with a 72-h, spin-amplified shell vial indirect immunofluorescence assay for the detection of enterovirus from clinical specimens. The sensitivity for the shell vial assay after resolution of discrepant results were 93 and 100%, respectively. The shell vial assay detected 93% of the positive cultures within 72 h of incubation while conventional tube culture detected only 51% of the positive cultures within the same time interval. The data suggest that a spin-amplified shell vial indirect immunofluorescence assay may be useful for the detection of enterovirus from clinical specimens.     

Detection of enteroviruses by reverse-transcriptase polymerase chain reaction in cell culture negative stool specimens of patients with acute flaccid paralysis

The aim of this study was the detection of a 114 base pairs amplicon in 5' non-translated region of enterovirus genome in stool specimens of patients with acute flaccid paralysis (AFP) which were negative on cell culture. One hundred and twenty stool specimens were collected from AFP cases and tested with cell culture (RD, L20B and Hep2 cell lines). RT-PCR was carried out for the specimens with negative cell culture result. A 10% raise in enterovirus detection was observed with RT-PCR. This increased sensitivity can improve the detection of enterovirus serotypes which grow poorly in cell culture, and can thus alter significantly the medical care of patients with acute flaccid paralysis.     

Detection of precytopathic effect of enteroviruses in clinical specimens by centrifugation-enhanced antigen detection

Rapid enterovirus detection is important for decisions about antibiotic administration and length of hospital stay. The efficacy of rapid antigen detection-cell culture amplification (Ag-CCA) was evaluated with monoclonal antibodies (MAbs) 5-D8/1 (DAKO) and Pan-Enterovirus clone 2E11 (Chemicon) with 10 poliovirus, echovirus, and coxsackievirus type A and B stock isolates and College of American Pathologists check samples. By using Ag-CCA technology, MAb 2E11 was more sensitive than 5-D8/1 at detecting a greater number of stock isolates at or past tube (cytopathic effect [CPE]) culture (TC) end points. The efficacy of Ag-CCA in the clinical setting was subsequently confirmed with 273 consecutively freshly collected nasopharyngeal aspirate or swab specimens, rectal swab, and cerebrospinal fluid specimens during the 1999 enterovirus season. All specimens were tested by Ag-CCA in parallel with rhesus monkey kidney (RhMk), MRC-5, and A549 conventional TCs. Approximately 60% of field specimens were additionally tested with Hep-2 and HNK conventional TCs. Sixty-two percent of the clinical specimens tested were Ag-CCA positive after 48 h. Among 51 isolates, the mean time to CPE or culture confirmation was 5.5 days (range, 2 to 18 days). After 48 h, Ag-CCA achieved sensitivity, specificity, and positive and negative predictive values of 62, 100, 100, and 93%, respectively. During the same period, TC-CPE displayed test parameters of 12, 100, 100, and 85%, respectively. After 5 days, the sensitivity and specificity of Ag-CCA increased to 92 and 98%, respectively. Within the same period, isolation attained sensitivity and specificity of 52 and 100%, respectively. Although Ag-CCA displayed slightly reduced sensitivity and reduced specificity compared with conventional cell culture after 14 days, the markedly superior 48-h enterovirus Ag-CCA detection rate supports incorporation of this assay into the routine clinical setting.     

Detection of cancer cells in effusions from patients diagnosed with gynaecological malignancies

The detection of malignant cells in pleural, peritoneal, and pericardial fluids of cancer patients marks the presence of metastatic disease and is associated with a grave prognosis. We evaluated five epithelial markers for the detection of cancer cells in 94 fresh pleural, peritoneal and pericardial effusions. Eighty-four of the samples were regarded as adequate for analysis after evaluation of cytological smears, including 61 samples from patients known to have gynaecological neoplasms. The other 23 samples were from patients with various non-gynaecological malignancies or tumours of unknown origin. Our control cases were 10 fallopian tubes not affected by any malignancy and 12 malignant mesotheliomas. Cell blocks from all cases were stained for CA-125, BerEP4, carcinoembryonic antigen (CEA), BG8 (Lewis Y blood antigen), and B72.3 (TAG-72). Fifty-one of 84 cases were diagnosed as malignant or suggestive of malignancy in cytological smears and/or cell block sections. However, staining for epithelial markers highlighted the presence of malignant cells in 7 additional cases. When membrane staining was evaluated, the sensitivity of the markers studied in detecting malignant cells was as follows: CA-125: 88%, BerEP4: 78%, CEA: 26%, BG8: 86%, B72.3: 79%. Membrane positivity for CEA, B72.3 and BerEP4 was not detected in reactive mesothelial cells. However, membranous staining in mesothelial cells was evident in 13% and 31% of cases with the use of BG8 and CA-125, respectively. Weak cytoplasmic staining for CEA was observed in mesothelial cells in 2 cases. When Ber-EP4, B72.3, and BG8 staining results in cancer cells were combined, the following sensitivity levels were observed: BG8+B72.3: 91%; BG8+Ber-EP4: 90%; B72.3+Ber-EP4: 93%; BG8+ Ber-EP4+B72.3: 95%. The detection of malignant cells in effusions is facilitated by the use of immunocytochemistry using a wide panel of antibodies. BerEP4 and B72.3 appear to be the best markers when both sensitivity and specificity are considered, followed by BG8, while CEA and CA-125 have a limited role in the detection of metastases from gynaecological tumours owing to the low sensitivity of the former and the low specificity of the latter. Analysis of all staining results should be based on a thorough morphological examination. Received: 14 December 1998 / Accepted: 23 February 1999     

Usefulness of the FilmArray meningitis/encephalitis (M/E) panel for the diagnosis of infectious meningitis and encephalitis in Taiwan

Background/purposeEarly recognition of causative pathogens is critical for the appropriate management of central nervous system infection and improved outcomes. The BioFire^® FilmArray^® Meningitis/Encephalitis Panel (BioFire^® ME Panel, BioFire Diagnostics) is the first U.S. Food and Drug Administration (FDA)-approved multiplex PCR assay that allows the rapid detection of 14 pathogens, including bacteria (n = 6), viruses (n = 7), and fungi (n = 1), from cerebrospinal fluid (CSF). The performance of the panel is expected to be dependent on the epidemiology of M/E in different geographical regions.MethodsIn this preliminary study, we used the BioFire^® ME Panel in 42 subjects who presented to the emergency department with symptoms of M/E in our hospital. The results were compared to conventional culture, antigen detection, PCR, and various laboratory findings.ResultsThe panel detected six positive samples, of which five were viral and one bacterial. We observed an overall agreement rate of 88% between the BioFire^® ME Panel results and the conventional methods. There were no false-positive findings, but five discordant results were observed for enterovirus, herpes simplex virus type 1, Escherichia coli, and Cryptococcus species.ConclusionsThe BioFire^® ME Panel performed equivalently to the traditional PCR methods for virus detection, and better than bacterial cultures. This revolutionary system represents a paradigm shift in the diagnosis of M/E and may aid in the rapid identification of community-acquired M/E. However, the usefulness of this tool is limited in regions with a high prevalence of infectious M/E caused by microorganisms not included in the panel.     

Molecular typing of Beta-hemolytic streptococci from two patients with lower-limb cellulitis: identical isolates from toe web and blood specimens

Intertriginous toe webs harboring cellulitis-causing bacteria constitute a risk factor for lower-limb cellulitis. Molecular typing of Streptococcus pyogenes and S. dysgalactiae subsp. equisimilis isolates from blood and toe webs of two cellulitis patients revealed identical strains for each species. This finding supports the role of toe webs as a potential site of entry for cellulitis pathogens.     

结核性脑膜炎患者的经颅多普勒超声和脑电图检测

目的：探讨经颅多普勒超声（ＴＣＤ）和脑电图（ＥＥＧ）在结核性脑膜炎中的应用价值。方法：对８０例结核性脑膜炎进行ＴＣ Ｄ和ＥＥＧ检测与分析。结果：治疗前８６％的ＥＥＧ和８１％的ＴＣ Ｄ均显示异常。治疗后除个别病例外，随着临床症状好转和痊愈，ＴＣＤ和 ＥＦＧ也好转或恢复正常。结论：用 ＴＣＤ和ＥＥＧ观察结核性脑膜炎的演变过程对估计预后有重要意义。