Karen A. McCaustland

Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA.

ABSTRACT The accurate diagnosis of pneumococcal disease has frequently been hampered not only by the difficulties in obtaining isolates of the organism from patient specimens but also by the misidentification of pneumococcus-like viridans group streptococci (P-LVS) as Streptococcus pneumoniae. This is especially critical when the specimen comes from the respiratory tract. In this study, three novel real-time PCR assays designed for the detection of specific sequence regions of the lytA, ply, and psaA genes were developed (lytA-CDC, ply-CDC, and psaA, respectively). These assays showed high sensitivity (<10 copies for lytA-CDC and ply-CDC and an approximately twofold less sensitivity for psaA). Two additional real-time PCR assays for lytA and ply described previously for pneumococcal DNA detection were also evaluated. A panel of isolates consisting of 67 S. pneumoniae isolates (44 different serotypes and 3 nonencapsulated S. pneumoniae isolates from conjunctivitis outbreaks) and 104 nonpneumococcal isolates was used. The 67 S. pneumoniae isolates were reactive in all five assays. The new real-time detection assays targeting the lytA and psaA genes were the most specific for the detection of isolates confirmed to be S. pneumoniae, with lytA-CDC showing the greatest specificity. Both ply PCRs were positive for all isolates of S. pseudopneumoniae, along with 13 other isolates of other P-LVS isolates confirmed to be non-S. pneumoniae by DNA-DNA reassociation. Thus, the use of the ply gene for the detection of pneumococci can lead to false-positive reactions in the presence of P-LVS. The five assays were applied to 15 culture-positive cerebrospinal fluid specimens with 100% sensitivity; and serum and ear fluid specimens were also evaluated. Both the lytA-CDC and psaA assays, particularly the lytA-CDC assay, have improved specificities compared with those of currently available assays and should therefore be considered the assays of choice for the detection of pneumococcal DNA, particularly when upper respiratory P-LVS might be present in the clinical specimen.

Use of TaqMan Real-Time Reverse Transcription-PCR for Rapid Detection, Quantification, and Typing of Norovirus

ABSTRACT Noroviruses (NoVs) are the most commonly identified cause of outbreaks and sporadic cases of acute gastroenteritis. We evaluated and optimized NoV-specific TaqMan real-time reverse transcription (RT)-PCR assays for the rapid detection and typing of NoV strains belonging to genogroups GI and GII and adapted them to the LightCycler platform. We expanded the detection ability of the assays by developing an assay that detects the GIV NoV strain. The assays were validated with 92 clinical samples and 33 water samples from confirmed NoV outbreaks and suspected NoV contamination cases. The assays detected NoV RNA in all of the clinical specimens previously confirmed positive by conventional RT-PCR and sequencing. Additionally, the TaqMan assays successfully detected NoV RNA in water samples containing low viral concentrations and inhibitors of RT and/or PCR, whereas the conventional method with region B primers required dilution of the inhibitors. By means of serially diluted NoV T7 RNA transcripts, a potential detection limit of <10 transcript copies per reaction mixture was observed with the GII assay and a potential detection limit of <100 transcript copies per reaction mixture was observed with the GI assay. These results and the ability to detect virus in water that was negative by RT-PCR demonstrate the higher sensitivity of the TaqMan assay compared with that of a conventional RT-PCR assay. The TaqMan methods dramatically decrease the turnaround time by eliminating post-PCR processing. These assays have proven useful in assisting scientists in public health and diagnostic laboratories report findings quickly to outbreak management teams.

Detection of pathogenic Leptospira spp. through TaqMan polymerase chain reaction targeting the LipL32 gene

Rapid diagnosis of leptospirosis, through culture and/or serology, can be difficult without proper expertise and is often delayed because of the length of time required to obtain results. In this study, we developed a real-time polymerase chain reaction (PCR) assay using a TaqMan probe targeting lipL32, which is present only in pathogenic Leptospira spp. Using Leptospira interrogans serovar Icterohaemorrhagiae DNA, the lower limit of detection was found to be 20 genomic equivalents/reaction with a 95% cutoff value. The assay detected pathogenic Leptospira strains, but not intermediately pathogenic or nonpathogenic strains. When testing the assay on spiked clinical specimens, whole blood and plasma were better specimens for detecting the same initial number of leptospires compared with serum from clotted and centrifuged blood. Leptospira spiked at the same concentration was better detected in centrifuged urine. This real-time PCR assay with high specificity and sensitivity may prove to be a rapid method for diagnosing acute leptospirosis.

Aetiological Agent of Enterically Transmitted Non-A, Non-B Hepatitis

Virus-like particles (VLPs) with a mean diameter of 32 nm were recovered from the stools of three acute phase cases of enterically transmitted non-A, non-B hepatitis (ET-NANBH) occurring in the Soviet Union, North Africa and North America. VLPs from two of these cases were studied in detail and were shown to react specifically with antibody in acute phase sera obtained from other cases of ET-NANBH in Asia, the Soviet Union, North Africa and North America. Partially purified VLPs were found to sediment at 183S in sucrose gradients and to cross-react with antibody in acute phase sera from geographically isolated cases of ET-NANBH. The latter virus preparations were also used to document the seroconversion of experimentally ET-NANBH-infected cynomolgus macaques to 32 nm VLPs. Our findings indicate that one virus or class of viruses is responsible for the majority of ET-NANBH.

Real-time reverse transcription-polymerase chain reaction assay for SARS-associated coronavirus.

A real-time reverse transcription–polymerase chain reaction (RT-PCR) assay was developed to rapidly detect the severe acute respiratory syndrome–associated coronavirus (SARS-CoV). The assay, based on multiple primer and probe sets located in different regions of the SARS-CoV genome, could discriminate SARS-CoV from other human and animal coronaviruses with a potential detection limit of <10 genomic copies per reaction. The real-time RT-PCR assay was more sensitive than a conventional RT-PCR assay or culture isolation and proved suitable to detect SARS-CoV in clinical specimens. Application of this assay will aid in diagnosing SARS-CoV infection.

The sequence of hepatitis E virus isolated directly from a single source during an outbreak in China

In this study an IgM antibody-mediated antigen-capture procedure for direct extraction of hepatitis E virus (HEV) RNA from clinical specimens was developed and used with an efficient method for generating viral cDNA that was subsequently sequenced using the dideoxy chain termination method. This is the first time the complete HEV genome has been isolated directly from a single human clinical specimen obtained during an outbreak of enterically transmitted non-A, non-B hepatitis. When the Chinese-derived sequence was compared with the original isolate of Burmese HEV from an experimentally infected cynomolgus macaque, the homology between the two sequences was 94% and 98.5% at the nucleotide and amino acid levels, respectively. The methods we developed for generating and sequencing genomic HEV cDNA dramatically improved the efficiency of cloning the viral genome and should be helpful for continued analysis of this virus as well as other RNA viruses that have proven to be difficult to clone and sequence directly.

Infectivity of Hepatitis C Virus in Plasma After Drying and Storing at Room Temperature

OBJECTIVE To determine effect of environmental exposure on the survival and infectivity of hepatitis C virus (HCV). METHODS Three aliquots of chimpanzee plasma containing HCV and proven infectious HCV inoculum were dried and stored at room temperature, 1 aliquot for 16 hours, 1 for 4 days, and 1 for 7 days. A chimpanzee (CH247) was sequentially inoculated intravenously with each of these experimental inocula, beginning with the material stored for 7 days. Each inoculation was separated by at least 18 weeks of follow-up to monitor for infection. The concentration of HCV RNA was measured and quasi species were sequenced for each experimental inoculum and in serum samples from CH247. RESULTS Evidence of HCV infection developed in CH247 only after inoculation with the material stored for 16 hours. No infection occurred after inoculation with the material stored for 7 days or 4 days. Compared with the original infectious chimpanzee plasma, the concentration of HCV RNA was 1 log lower in all 3 experimental inocula. The same predominant sequences were found in similar proportions in the original chimpanzee plasma and in the experimental inocula, as well as in serum samples from CH247. CONCLUSION HCV in plasma can survive drying and environmental exposure to room temperature for at least 16 hours, which supports the results of recent epidemiologic investigations that implicated blood-contaminated inanimate surfaces, objects, and/or devices as reservoirs for patient-to-patient transmission of HCV. Healthcare professionals in all settings should review their aseptic techniques and infection control practices to ensure that they are being performed in a manner that prevents cross-contamination from such reservoirs.

A new cluster of hepatitis A infection in hemophiliacs traced to a contaminated plasma pool.

Recently, several clusters of hepatitis A have been observed among hemophiliacs linked to factor VIII concentrates treated for virus inactivation solely with the solvent/detergent (S/D) method, a procedure that does not affect nonenveloped viruses such as the hepatitis A virus (HAV). A new outbreak of hepatitis A in six hemophiliacs treated with the same lot of a factor VIII preparation occurred recently in Germany. The objective of the study was to clarify whether these diseases were caused by the administration of the S/D‐treated plasma product, rather than a community‐acquired infection. Polymerase chain reactions designed to detect HAV nucleic acid have been carried out in the implicated factor VIII lots, in the corresponding plasma pools, and in serum samples of four out of six infected individuals. The nucleic acid sequences were determined in samples that resulted in positive amplification products. HAV sequences were found in one of the two plasma pools used for manufacture of the incriminated product, in the incriminated lot itself, and in all recipient sera tested so far, although the latter were collected up to 7 weeks after the onset of jaundice. The sequences obtained were completely identical, revealing a unique HAV strain of genotype IA. This study provides conclusive evidence that hepatitis A can be transmitted by factor VIII concentrates treated solely by the S/D procedure for virus inactivation. This inactivation method is not effective against nonenveloped viruses. Since a number of hepatitis A transmission episodes have been described with such preparations during the past 10 years, their continued use seems to be questionable unless additional virus removal or inactivation steps are introduced to prevent the transmission of nonenveloped viruses. Molecular approaches again proved to be reliable tools for elucidating the chain of virus transmission. J. Med. Virol. 57:91–99, 1999.

Posttransfusion non-A, non-B hepatitis in chimpanzees

Posttransfusion non-A, non-B hepatitis associated with the formation of hepatocyte cytoplasmic tubules was experimentally transmitted to chimpanzees by intravenous inoculation of a proven-infectious plasma that had been pelleted and microfiltrated, or purified by a combination of pelleting and rate-zonal banding. The results of these studies indicate that a factor VIII-derived non-A, non-B tubule-forming agent will pass through an 80-nm membrane filter and that it can be recovered from infected plasma by use of a purification procedure that assumes the non-A, non-B tubule-forming agent is a small, enveloped virus. Our findings, in combination with the known sensitivity of the non-A, non-B tubule-forming agent to chloroform and its apparent lack of nucleic acid homology with hepatitis B virus, further suggest that at least one etiologic agent of human posttransfusion non-A, non-B hepatitis may be a small, enveloped RNA virus.

Enhancement of detection and quantification of rotavirus in stool using a modified real‐time RT‐PCR assay

A sensitive and specific real‐time RT‐PCR assay to detect rotavirus in stool samples was optimized and validated using a wide range of rotavirus genotypes. The target of the original TaqMan® assay is an 87 bp fragment of the highly conserved non‐structural protein 3 (NSP3) gene. Here we modified the original assay by introducing degeneracy into the forward primer to account for sequence variation between rotavirus genotypes, added four nucleotides at the 3′ end of the reverse primer to reduce its stability, and modified the probe label. Amplification and detection conditions were optimized using purified dsRNA from two cultivated strains. The limit of detection of the modified assay was calculated to be approximately 44 genome copies per reaction. To validate the reactivity of the assay, 103 archived RNAs that had been extracted from stools and genotyped during routine U.S. surveillance were tested. Samples were selected to represent both rare and common genotypes that have been detected in U.S. children. Nine genotypes known to be circulating in the United States were detected by the real‐time assay demonstrating broad reactivity. In addition, other enteric viruses were not detected demonstrating that the assay is specific for rotavirus and does not cross‐react with other viruses potentially present in stool samples. This real‐time assay is an important addition to the arsenal of molecular tools available to quickly identify rotavirus in stool samples during routine surveillance. J. Med. Virol. 80:1489–1496, 2008.