Comparison of droplet digital PCR to real-time PCR for quantitative detection of cytomegalovirus.

Quantitative real-time PCR (QRT-PCR) has been widely implemented for clinical viral load testing, but a lack of standardization and relatively poor precision have hindered its usefulness. Digital PCR offers highly precise, direct quantification without requiring a calibration curve. Performance characteristics of real-time PCR were compared to those of droplet digital PCR (ddPCR) for cytomegalovirus (CMV) load testing. Tenfold serial dilutions of the World Health Organization (WHO) and the National Institute of Standards and Technology (NIST) CMV quantitative standards were tested, together with the AcroMetrix CMV tc panel (Life Technologies, Carlsbad, CA) and 50 human plasma specimens. Each method was evaluated using all three standards for quantitative linearity, lower limit of detection (LOD), and accuracy. Quantitative correlation, mean viral load, and variability were compared. Real-time PCR showed somewhat higher sensitivity than ddPCR (LODs, 3 log(10) versus 4 log(10) copies/ml and IU/ml for NIST and WHO standards, respectively). Both methods showed a high degree of linearity and quantitative correlation for standards (R(2) ≥ 0.98 in each of 6 regression models) and clinical samples (R(2) = 0.93) across their detectable ranges. For higher concentrations, ddPCR showed less variability than QRT-PCR for the WHO standards and AcroMetrix standards (P < 0.05). QRT-PCR showed less variability and greater sensitivity than did ddPCR in clinical samples. Both digital and real-time PCR provide accurate CMV load data over a wide linear dynamic range. Digital PCR may provide an opportunity to reduce the quantitative variability currently seen using real-time PCR, but methods need to be further optimized to match the sensitivity of real-time PCR.

Effect of HSV-2 suppressive therapy on genital tract HIV-1 RNA shedding among women on HAART: a pilot randomized controlled trial.

BACKGROUND: The role of suppressive HSV therapy in women coinfected with HSV-2 and HIV-1 taking highly active antiretroviral therapy (HAART) is unclear. METHODS: 60 women with HIV-1/HSV-2 coinfection on HAART with plasma HIV-1 viral load (PVL) ≤75 copies/mL were randomized to receive acyclovir (N = 30) or no acyclovir (N = 30). PVL, genital tract (GT) HIV-1, and GT HSV were measured every 4 weeks for one year. RESULTS: Detection of GT HIV-1 was not significantly different in the two arms (OR 1.23, P = 0.67), although this pilot study was underpowered to detect this difference. When PVL was undetectable, the odds of detecting GT HIV were 0.4 times smaller in the acyclovir arm than in the control arm, though this was not statistically significant (P = 0.07). The odds of detecting GT HSV DNA in women receiving acyclovir were significantly lower than in women in the control group, OR 0.38, P < 0.05. CONCLUSIONS: Chronic suppressive therapy with acyclovir in HIV-1/HSV-2-positive women on HAART significantly reduces asymptomatic GT HSV shedding, though not GT HIV shedding or PVL. PVL was strongly associated with GT HIV shedding, reinforcing the importance of HAART in decreasing HIV sexual transmission.

Real-time PCR improves detection of Trichomonas vaginalis infection compared with culture using self-collected vaginal swabs.

OBJECTIVE: To compare a real-time polymerase chain reaction (PCR) assay with broth culture for the detection of Trichomonas vaginalis using self-collected vaginal swabs. METHODS: Self-collected vaginal swabs were obtained from adolescent and young adult African-American women participating in HIV-1 prevention programs. T. vaginalis culture was performed using the InPouch TV System. Samples for the real-time PCR assay were collected using the BDProbeTec ET Culturette Direct Dry Swab system and tested in a laboratory-developed assay which targeted a repeated sequence of the genome. Discrepant samples that were culture negative and positive in the real-time PCR assay were tested in a confirmatory PCR which targeted a different region of the T. vaginalis genome, the18S ribosomal DNA gene. RESULTS: Of the 524 specimens tested by both culture and real-time PCR, 36 were culture positive and 54 were positive in the real-time PCR assay; 16 of the 18 discrepant specimens were also positive in the confirmatory PCR assay. Using a modified gold standard of positive by culture or positive in both PCR assays, the sensitivity of the real-time PCR assay was 100% and the specificity was 99.6%, whereas culture had a sensitivity of 69.2% and a specificity of 100%. CONCLUSIONS: The real-time PCR assay was sensitive and specific for the detection of T. vaginalis DNA from self-collected vaginal swab specimens. The ability to use the BDProbeTec dry swab system for the real-time PCR testing allowed for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and T. vaginalis from a single specimen.

Human immunodeficiency virus in plasma and cervicovaginal secretions in Filipino women.

This study examined 30 HIV-infected women in Manila to assess the relationship between cervicovaginal and plasma HIV-1 viral load. An interview and gynaecologic examination was conducted and cervicovaginal lavage (CVL) and venous blood specimens were collected. HIV-1 RNA was detected in plasma samples of 24 patients (80%) and in CVL samples of 18 women (60%); 16 patients (53%) had detectable levels in both. CVL HIV-1 RNA was detectable in 75% of women (6/8) with plasma viral loads between 10,000 and 100,000 copies/mL and in 77% of women (10/13) with plasma viral loads higher than 100,000 copies/mL (P =0.0086). Among women with CD4 cell counts of less than 200, 200-500, and greater than 500/mm(3), CVL HIV-1 RNA was detected in 73%, 69%, and 17% of women, respectively (P =0.1428). HIV-1 RNA shedding in the genital tract was significantly associated with plasma viral load.

Generation and maintenance of tandemly repeated extrachromosomal plasmid DNA in Chlamydomonas chloroplasts.

Unusual chloroplast transformants of Chlamydomonas reinhardtii that contain 2000 copies of a mutant version of the chloroplast atpB gene, maintained as an extrachromosomal tandem repeat, have recently been described. In this paper studies have been undertaken to (i) address possible mechanisms for generating and maintaining the amplified DNA and (ii) determine whether it is possible to use chloroplast gene amplification to overexpress chloroplast or foreign genes. Data presented here indicate that high copy number transformants harbor characteristic rearrangements in both copies of the chloroplast genome large inverted repeat. These rearrangements appear to be a consequence of, or required for, maintenance of the amplified DNA. In an attempt to mimic the apparently autonomous replication of extrachromosomal DNA in the chloroplast, transformation was carried out with a plasmid that lacked homology with the chloroplast genome or with the same plasmid carrying a putative chloroplast DNA replication origin (oriA). Transformants were recovered only with the plasmid containing oriA, and all transformants contained an integrated plasmid copy at oriA, suggesting that establishment or maintenance of the extrachromosomal tandem repeat requires conditions that were not replicated in this experiment. To determine whether other genes could be maintained at high copy number in the chloroplast, plasmids carrying the wild-type atpB gene or the bacterial aadA gene were introduced into a high copy number transformant. Surprisingly, the copy number of the plasmid tandem repeat declined rapidly after the secondary transformation events, even when strong selective pressure for the introduced gene was applied. Thus, chloroplast transformation can either create or destabilize high copy number tandem repeats.

Evaluating HIV prevention community planning.

To improve human immunodeficiency virus (HIV) prevention efforts nationwide, the Centers for Disease Control and Prevention (CDC) has funded HIV Prevention Community Planning, an initiative that promotes parity, representativeness, and inclusion of community and the application of scientific principles for decision making. This initiative was welcomed enthusiastically in New York City, an AIDS epicenter with limited prevention resources. In the first year of implementation, the New York City Department of Health (NYCDOH) supported a comprehensive evaluation of the planning initiative. This article reviews the evaluation design, including its process and outcome components, the study findings, and its strengths and limitations. It provides overall guidance for those who are assessing prevention planning in HIV as well as other health areas.

Effect of promoter-leader sequences on transient expression of reporter gene chimeras biolistically transferred into sugarbeet (Beta vulgaris) suspension cells.

Chimeric constructs consisting of the gus coding region fused downstream of promoterun-translated leader sequences from the tobacco osmotin and PR-S genes, the potato proteinase inhibitor 2 gene (pin2), and the cauliflower mosaic virus (CaMV) 35S promoter were biolistically transferred into sugarbeet suspension cells. Each construct was expressed in recipient cells at 6 h after bombardment with maximum levels observed between 12 and 48 h. Expression of the PR-S construct mimicked the time-course expression of the constitutively expressed 35S construct but reached levels almost 50% higher. The pin2-promoter construct was ultimately expressed at levels similar to that of PR-S. Expression of the osmotin promoter-leader construct was highest, reaching levels approximately 2.5-fold higher than those of the 35S construct.

A novel anther-expressed adh-homologous gene in Lycopersicon esculentum.

Two novel tandemly-oriented open reading frames (ORFs) with homology to alcohol dehydrogenase (ADH) were isolated from tomato. The predicted amino acid composition for each of the two tandem adh genes indicates the presence of 22 and 21, respectively, of 22 amino acids conserved in ADH proteins from plants and animals. However, comparison to known plant adh genes reveals a significantly lower similarity indicating that they belong to a novel class of ADHs. According to mapping data, the adh-homologous ORFs do not represent either of the previously studied adh1 or adh2 genes of tomato. The tandem genes, termed adh3a and adh3b, mapped to a distal region of the long arm of chromosome 4, unlike adh1, which maps closer to the centromere. Adh3a and adh3b have over 90% similarity to each other at the nucleotide and putative peptide levels. The adh3a gene has ten exons and nine introns with the transcription initiation site 57 bp upstream of the translation start. A putative TATA box and polyadenylation site have been identified. Adh3a is transcribed and, according to cDNA sequence analysis, fully processed in the late stages of anther development. According to transformation analysis, tissue-specific regulatory elements reside within the -448 to +724 region. The termination codon of adh3a is separated from the putative adh3b translation start site by 789 bp of intervening sequence. The 5' untranscribed sequences of each gene contain a stretch of 68 bp with 78% similarity. Within this stretch are sequences which are homologous to sequences found in anaerobically-induced or pollen-expressed genes from various plant species.

AT-rich promoter elements of soybean heat shock gene Gmhsp17.5E bind two distinct sets of nuclear proteins in vitro.

A 33 bp double-stranded oligonucleotide homologous to two AT-rich sequences located upstream (-907 to -889 and -843 to -826) to the start of transcription of heat shock gene Gmhsp17.5E of soybean stimulated transcription when placed 5' to a truncated (-140) maize Adh1 promoter. The chimeric promoter was assayed in vivo utilizing anaerobically stressed sunflower tumors transformed by a pTi-based vector of Agrobacterium tumefaciens. Nuclear proteins extracted from soybean plumules were shown to bind double-stranded oligonucleotides homologous to AT-rich sequences in the 5' flanking regions of soybean beta-conglycinin, lectin, leghemoglobin and heat shock genes. These proteins were also shown to bind AT-rich probes homologous to homeobox protein binding sites from the Antennapedia and engrailed/fushi tarazu genes of Drosophila. Binding activity specific for AT-rich sequences showed a wide distribution among various plant organs and species. Preliminary characterization indicated that two sets of nuclear proteins from soybean bind AT-rich DNA sequences: a diverse high-molecular-weight (ca. 46-69 kDa) group, and a low-molecular-weight (23 and 32 kDa) group of proteins. The latter meets the operational criteria for high-mobility group proteins (HMGs).

Protection against feline infectious peritonitis by intranasal inoculation of a temperature-sensitive FIPV vaccine.

Cats vaccinated intranasally (i.n.) with a temperature sensitive feline infectious peritonitis virus (ts-FIPV) vaccine were protected against an FIP-inducing challenge. Seventeen of 20 vaccinated cats (85%) survived a rigorous virulent FIPV challenge that caused FIP in 12 of 12 non-vaccinated cats (100%), 10 (83%) of which died. Intranasal vaccination stimulated serum IgG and serum and salivary IgA antibody responses (measured by ELISA), FIPV-neutralizing antibody (VN), and a cell-mediated immune (CMI) response as measured by lymphocyte proliferation. The serum antibody response to vaccination was not associated with protection. In fact, the IgG, IgA and VN titres were much higher in control cats than in vaccinated cats following challenge suggesting an immune-mediated pathogenesis. In contrast, stimulation of a mucosal IgA response to vaccination was related to protection. The in vitro proliferation of peripheral blood lymphocytes in response to virulent FIPV was observed in vaccinated cats, in vaccinated and challenged cats but not in non-vaccinated challenged cats.

Characterization of an attenuated temperature sensitive feline infectious peritonitis vaccine virus.

Intranasal administration of a ts-FIPV vaccine protected cats against two rigorous challenges of immunity. Investigations showed that ts-FIP viral RNA synthesis was normal at 39 degrees C and structural proteins were synthesized, but not expressed at the cell surface. Lack of surface expression combined with decreased virus titer indicate that, although structural viral proteins were initially synthesized, they were not packaged into intact virions at the nonpermissive temperature. The ts-FIP vaccine virus was shown to replicate exclusively in the upper respiratory tract, where lower temperatures allow maturation of the virus. Viral proteins expressed on cells in the upper respiratory tract probably stimulate the development of local IgA and CMI responses and a systemic CMI response which in turn may stop the dissemination of virulent FIPV if it crosses the mucosal barrier. Investigations are ongoing to study the protective mechanism of ts-FIPV induced immunity.

Characterization of a temperature sensitive feline infectious peritonitis coronavirus.

The characteristics of a temperature sensitive feline infectious peritonitis virus (TS-FIPV) were examined. TS-FIPV, unlike its parent strain, DF2 wild type FIPV (WT-FIPV), propagated at 31 degrees C (permissive temperature) but not at 39 degrees C (nonpermissive temperature). This temperature preference of TS-FIPV was also demonstrated in cats by the ability of the virus to replicate only at the lower temperature in the upper respiratory tract and not at systemic sites where higher temperatures (38-39 degrees C) prevail. Viral structural proteins and RNA were synthesized at 39 degrees C but some undefined maturational defect prevented the formation of infectious TS-FIPV at its nonpermissive temperature. TS-FIPV was more thermolabile than WT-FIPV which indicated alterations in the structural proteins of TS-FIPV, and a difference in the envelope protein of the two viruses was revealed by Western blot analysis. Plaque assay characterization showed that TS-FIPV produced small plaques in comparison to the large plaques of WT-FIPV. These unique characteristics possessed by TS-FIPV may account for its nonvirulent nature and ability to stimulate protective immune responses in cats.

Identification of viral antigens that induce antibody responses on exposure to coronaviruses.

Various techniques were used to look for protective, non-cross-reactive antibodies in the sera of cats exposed to virulent feline infectious peritonitis virus (FIPV). Antibodies reactive with feline enteric coronavirus (FECV) from FIPV-exposed cats were adsorbed by several passages over an FECV-Sepharose column. In an ELISA against FECV and FIPV, the activity against both viruses was removed at the same rate; thus, no FIPV-specific antibodies could be identified. By gel electrophoresis-derived ELISA, the responses of cats surviving FIPV exposure were compared with those of cats succumbing to FIPV exposure to determine whether survival could be correlated with an antibody response against a particular virus protein. Results indicated that both groups responded in the same way to the matrix envelope protein and nucleocapsid proteins. Even though the response to peplomer in each group was weak, the survivor group responded better to this protein. Furthermore, the response of this group to the peplomer protein had the highest correlation with virus neutralization titer.

Comparison of serologic assays for measurement of antibody response to coronavirus in cats.

Serologic virus neutralization tests, indirect immunofluorescence tests, and ELISA, using tissue culture-adapted feline infectious peritonitis virus (FIPV) or feline enteric coronavirus (FECV) were compared for their ability to distinguish specific virus exposure in cats. Sera of specific-pathogen-free cats inoculated with virulent or modified FIPV or FECV were used to compare the sensitivity and specificity of the homologous assays to a heterologous assay that measures antibody reactivity with transmissible gastroenteritis virus of swine. The geometric means of the serologic titers in FIPV and FECV assays were higher for FIPV- or FECV-infected specific-pathogen-free cats than the geometric means of the transmissible gastroenteritis virus assays for most groups. None of the assays was specific enough to discern the virus to which a cat had been exposed. However, the FIPV virus neutralization test appeared to be more sensitive for detection of an early response to FIPV infection than did the FIPV immunofluorescence test or FIPV-ELISA.

A survey of radionuclide contents and radon emanation rates in building materials used in the U.S.

Building materials commonly used in the U.S. were surveyed for both radionuclide content and radon emanation rate. Particular emphasis was placed on concrete, for which samples from ten major metropolitan areas were measured. Gamma-ray spectroscopy techniques, as well as direct radon emanation measurements, were employed in this study. The results of this survey suggest that these materials are not the primary source of radon in typical U.S. houses.

Radon concentrations and infiltration rates measured in conventional and energy-efficient houses.

To elucidate any connection between high radon concentrations and low-infiltration houses, we have concurrently measured the 222Rn concentration and the infiltration rate in U.S. houses. Three housing surveys have been undertaken: one in "energy-efficient" houses located throughout the U.S. and two in "conventional" houses in the San Francisco area and in Maryland. In each of the groups surveyed, no clear correlation was observed between 222Rn concentrations and infiltration rate, although each parameter varied over a wide range. Infiltration rates for the entire sample, numbering 98 houses, ranged between 0.02 and 1.6 air changes per hr, and 222Rn concentrations ranged from 0.1 to 27 pCi/l. It appears that the major cause of the observed differences in 222Rn concentration is variation from one house to another in the rate at which 222Rn enters houses from its sources.

Cross-protection among feline caliciviruses.

Each of five groups of specific-pathogen-free and conventionally reared cats was infected with a different strain of feline calicivirus. Two of the strains were pathogenic, producing characteristically fever, depression, loss of appetite, buccal ulceration, and occasionally increased ocular and nasal secretion. Two of the other strains were midly pathogenic and associated with fever or buccal ulceration or both; the fifth strain was nonpathogenic. The two pathogenic strains plus three others shown also to be pathogenic were used 3 months after the initial infection to challenge the cats in rearranged groupings. Of the 28 conventional cats challenged six (21.4%) showed at least a febrile response, although none of the 30 specific-pathogen-free cats showed any clinical signs. After challenge, virus was recovered from throat swabs of 37 or the 58 cats (63.8%) including the six which showed symptoms, but the duration of the excretion of virus was significantly less than that seen with the initial infection. The homologous and heterotypic antibody responses correlated well with the clinical protection, or lack of it, seen on challenge. The results provide further evidence for significant cross-relationships between feline caliciviruses.