Use of a rapid test of pneumococcal colonization density to diagnose pneumococcal pneumonia.

BACKGROUND: There is major need for a more sensitive assay for the diagnosis of pneumococcal community-acquired pneumonia (CAP). We hypothesized that pneumococcal nasopharyngeal (NP) proliferation may lead to microaspiration followed by pneumonia. We therefore tested a quantitative lytA real-time polymerase chain reaction (rtPCR) on NP swab samples from patients with pneumonia and controls. METHODS: In the absence of a sensitive reference standard, a composite diagnostic standard for pneumococcal pneumonia was considered positive in South African human immunodeficiency virus (HIV)-infected adults hospitalized with radiographically confirmed CAP, if blood culture, induced good-quality sputum culture, Gram stain, or urinary Binax demonstrated pneumococci. Results of quantitative lytA rtPCR in NP swab samples were compared with quantitative colony counts in patients with CAP and 300 HIV-infected asymptomatic controls. RESULTS: Pneumococci were the leading pathogen identified in 76 of 280 patients with CAP (27.1%) using the composite diagnostic standard. NP colonization density measured by lytA rtPCR correlated with quantitative cultures (r = 0.67; P < .001). The mean lytA rtPCR copy number in patients with pneumococcal pneumonia was 6.0 log(10) copies/mL, compared with patients with CAP outside the composite standard (2.7 log(10) copies/mL; P < .001) and asymptomatic controls (0.8 log(10) copies/mL; P < .001). A lytA rtPCR density ≥8000 copies/mL had a sensitivity of 82.2% and a specificity of 92.0% for distinguishing pneumococcal CAP from asymptomatic colonization. The proportion of CAP cases attributable to pneumococcus increased from 27.1% to 52.5% using that cutoff. CONCLUSIONS: A rapid molecular assay of NP pneumococcal density performed on an easily available specimen may significantly increase pneumococcal pneumonia diagnoses in adults.

New method for titration of virus infectivity by immunostaining.

We have developed a new method for titration of viruses utilizing automated microtiter technology. Compared to existing methods such as plaque assay or hemagglutination titration of influenza virus, the new method offers distinct advantages in terms of time and effort. In addition because multiple replicates can easily be employed accuracy can be increased.

Use of the polymerase chain reaction to screen cell banks for retroviruses.

The polymerase chain reaction (PCR) may be used to increase the sensitivity for detecting nucleic acids of specific adventitious agents. PCR can be used in combination with other assays such as infectivity or co-cultivation to increase the sensitivity of detecting adventitious agents or to identify a particular adventitious agent present in the test sample. In addition, PCR is useful for confirming the results of other assays such as reverse transcriptase or electron microscopy. Sources of the test sample, passage history and raw materials added to the test sample are considered when deciding upon the appropriate screening procedures and the adventitious agents for which to screen. In developing a PCR assay, the experiments should be designed to address specificity, sensitivity and reproducibility. Assay development issues include design of primers for the particular retroviruses in question, determination of quantity of sample to test, potential levels of interference, and appropriate positive and negative controls. Validation of a PCR assay is necessary to prove reproducibility for detecting the retrovirus. Data are presented on the qualification of a primate master cell bank to assure the absence of simian immunodeficiency viruses.

Relation between the levels of mRNA abundance and kinetics of protein synthesis in pseudorabies virus-infected cells.

Virus proteins synthesized by pseudorabies virus-infected cells can be classified into five groups on the basis of the kinetics of their synthesis at various stages of the infective process; virus mRNAs can similarly be classified into four groups. To determine whether the kinetics of synthesis of specific proteins are determined solely by the level of abundance in the cells of their mRNAs, we have compared at various times after infection the relative synthesis of these proteins with the relative abundance of their mRNAs. We have focused on two proteins: the 142K major capsid protein, an early-late protein, and the 136K major DNA binding protein, an early protein. The mRNAs encoding these proteins were identified. The relative abundances of these mRNAs in the cytoplasms of the infected cells were found to be the same as those associated with the polysome fractions. The relative amount of the proteins synthesized by the infected cells at a given stage of the infective process closely reflected the relative amount of the mRNA encoding these proteins that was present in the cells at that stage of the infective process. Most virus mRNA species that are present in the cytoplasm of infected cells were represented on polysomes to approximately an equal extent. Some RNA species were, however, significantly underrepresented under certain conditions in the polysomal fractions. We conclude that whereas the amount of many virus proteins synthesized by the infected cells is determined mainly by the level of the abundance of their mRNAs, additional controls operate in the cells that determine the relative rates of synthesis of some other virus proteins.

The large late virus transcripts synthesized in herpesvirus suis (pseudorabies) virus-infected cells are not precursors of mRNA.

The sizes of early and late pseudorabies virus transcripts were compared to those of early and late mRNA. While the early primary transcripts were of approximately the same size as early mRNA, a large proportion of late primary transcripts was much larger than late mRNA. Furthermore, most early transcripts were transported efficiently to the cytoplasm and were relatively stable. In contrast, a large proportion of the late transcripts were retained in the nucleus and turned over rapidly. Specific retention in the nucleus of transcripts originating from some regions of the genome could be detected. These were, however, not preferentially degraded; degradation of transcripts originating from all regions of the genome, including those from which late mRNA originates, occurred. Experiments designed to determine whether part of the large transcripts are processed into mRNA revealed that most of the large late transcripts synthesized by the infected cells bear no precursor relationship to mRNA. Thus, during late phase of infection, most regions of the genome are abundantly transcribed as large RNA molecules; these are not destined to be processed into mRNA.

RNA from an immediate early region of the type 1 herpes simplex virus genome is present in the trigeminal ganglia of latently infected mice.

Transcription of the type 1 herpes simplex virus (HSV-1) genome in trigeminal ganglia of latently infected mice was studied using in situ hybridization. Probes representative of each temporal gene class were used to determine the regions of the genome that encode the transcripts present in latently infected cells. Probes encoding HSV-1 sequences of the five immediate early genes and representative early (thymidine kinase), early-late (major capsid protein), and late (glycoprotein C) genes were used in these experiments. Of the probes tested, only those encoding the immediate early gene product infected-cell polypeptide (ICP) 0 hybridized to RNA in latently infected tissues. Probes containing the other immediate early genes (ICP4, ICP22, ICP27, and ICP47) and the representative early, early-late, and late genes did not hybridize. Two probes covering approximately equal to 30% of the HSV-1 genome and encoding over 20 early and late transcripts also did not hybridize to RNA in latently infected tissues. These results, with probes spanning greater than 60% of the HSV-1 genome, suggest that transcription of the HSV-1 genome is restricted to one region in latently infected mouse trigeminal ganglia.

Equalization of the inverted repeat sequences of the pseudorabies virus genome by intermolecular recombination.

During a study designed to identify changes in the genomes that are observed in mutant populations of pseudorabies virions, a thymidine kinase-defective population of virions which contains genomes that possess inverted repeated sequences of unequal sizes has been identified. This population of virions has been used to ascertain the mechanism and rates of equalization of the repeats. Results showed that when the virions were passed in cell cultures at high multiplicities of infection (either once or several times), one-half of the molecules had repeats of equal size and one-half had repeats of unequal size. This result is to be expected if conditions allowing a high degree of intermolecular recombination involving exchange of repeats exist. The process of intermolecular exchange of inverted repeats is rapid and the DNA molecules were at equilibrium by the time a virion had produced a small plaque. If the virions were passed in cell culture at low multiplicities of infection, they acquired genomes with equal-size repeats. This is probably due to segregation of the virions which had acquired genomes with equal repeats by recombination; in the absence of coinfection of the cells with virions containing genomes with different repeat sizes, heterogeneity of the repeats could not be generated. The fact that virions containing genomes with equal repeats are usually isolated from nature can thus be attributed to equalization of the repeats which results from intermolecular recombination, followed by segregation. A special mechanism ensuring equalization of repeats, such as one repeat acting as a template for the synthesis (or repair) of the other, need therefore not be invoked.

Human herpes virus infections and Alzheimer's disease.

Herpes viruses cause acute and chronic diseases of the peripheral and central nervous systems and have been implicated in the aetiology of Alzheimer's disease (AD). In this investigation the relevance of human herpes virus infection to AD was assessed by in situ hybridization. The abundant latency associated transcript(s) of herpes simplex virus type 1 (HSV-1) were detected at a significantly higher incidence in the trigeminal ganglia of individuals with AD than in controls. But we could find no evidence of viral RNA in the central nervous system (CNS), looking specifically in the hippocampal cortex of demented individuals with extensive neuropathological changes of AD. These studies solve one problem in testing the viral hypothesis of causation, i.e. the sensitivity of the methods used in the search for latent infection. But the central issue remains unresolved because of necessity, only the end stage of a prolonged pathophysiological process has been examined. Our conclusions are qualified accordingly.

Latent herpes simplex virus type 1 transcripts in peripheral and central nervous system tissues of mice map to similar regions of the viral genome.

Herpes simplex virus type 1 (HSV-1) DNA and RNA have been detected in peripheral nervous system (PNS) and central nervous system (CNS) tissues of latently infected mice. However, explant methods are successful in reactivating HSV-1 only from latently infected PNS tissues. In this report, latent herpesvirus infections in mouse PNS and CNS tissues were compared by in situ hybridization to determine whether the difference in reactivation was at the level of the virus or the host tissue. It was demonstrated that the HSV-1 transcripts present during latency in the mouse PNS and CNS originated from the same region of the genome, the repeats which bracket the long unique sequence. Therefore, the difference in reactivation with PNS and CNS tissues cannot be accounted for by differences in the extent of the HSV-1 genome transcribed during herpesvirus latency. Latent HSV-1 RNA was detected in the trigeminal ganglia (PNS) and the trigeminal system in the CNS from the mesencephalon to the spinal cord as well as other regions of the CNS not noted previously. Latent HSV-1 RNA was found predominantly in neurons but also in a small number of cells which could not be identified as neuronal cells. It is suggested that host differences in CNS and PNS tissues, rather than differences in latent virus transcription, may be important determinants in the HSV-1 reactivation process in explanted tissues.

Poliomyelitis in intraspinally inoculated poliovirus receptor transgenic mice.

Mice transgenic with the human poliovirus receptor gene develop clinical signs and neuropathology similar to those of human poliomyelitis when neurovirulent polioviruses are inoculated into the central nervous system (CNS). Factors contributing to disease severity and the frequencies of paralysis and mortality include the poliovirus strain, dose, and gender of the mouse inoculated. The more neurovirulent the virus, as defined by monkey challenge results, the higher the rate of paralysis, mortality, and severity of disease. Also, the time to disease onset is shorter for more neurovirulent viruses. Male mice are more susceptible to polioviruses than females. TGM-PRG-3 mice have a 10-fold higher transgene copy number and produce 3-fold more receptor RNA and protein levels in the CNS than TGM-PRG-1 mice. CNS inoculations with type III polioviruses differing in relative neurovirulence show that these mouse lines are similar in disease frequency and severity, demonstrating that differences in receptor gene dosage and concomitant receptor abundance do not affect susceptibility to infection. However, there is a difference in the rate of accumulation of clinical signs. The time to onset of disease is shorter for TGM-PRG-3 than TGM-PRG-1 mice. Thus, receptor dosage affects the rate of appearance of poliomyelitis in these mice.

Characterization of mouse lines transgenic with the human poliovirus receptor gene.

Two mouse lines transgenic with the human poliovirus receptor gene (PVR), TGM-PRG-1 and TGM-PRG-3, were characterized to determine whether transgene copy number and PVR expression levels influence susceptibility to poliovirus. The mouse lines have been bred for more than 10 generations and the transgene was stably transmitted to progeny as determined by Southern blot hybridization and restriction fragment length polymorphism. The transgene copy number is 10 in the TGM-PRG-3 mouse line and one in the TGM-PRG-1 mouse line. Abundance of PVR RNA is on average three-fold higher in TGM-PRG-3 relative to TGM-PRG-1 tissues, and the abundance of the receptor molecule is three-fold higher in TGM-PRG-3 central nervous system tissues compared to TGM-PRG-1 tissues as determined by Western blot analysis. When TGM-PRG-1 and TGM-PRG-3 mice were inoculated intracranially with a neurovirulent type III poliovirus strain, they developed clinical symptoms and CNS lesions characteristic of human poliomyelitis. These results indicate that the PVR gene is expressed as a functional receptor in the CNS of both mouse lines rendering the mice susceptible to poliovirus infection. Even though the two mouse lines have different copy numbers of the transgene and different levels of PVR RNA and protein, they are similar in their susceptibility to poliovirus.