René Boom

Detection and quantitation of human cytomegalovirus DNA in faeces

The development and performance of a robust and sensitive PCR assay are described for the detection and quantitation of human cytomegalovirus DNA in human faecal specimens. In this assay, CMV DNA was purified by an optimised DNA extraction protocol together with internal control DNA that monitored both DNA extraction efficiency and PCR efficiency. The lower detection limit of the assay was reached at about 100 CMV particles per ml of (25-50%) faecal suspension. CMV DNA could be quantitated in the range of about 300-100000 molecules per ml of faecal suspension. CMV DNA loads obtained in clinical faeces specimens suggest that the assay can be used to monitor the efficacy of antiviral treatment. Reconstruction experiments that monitored the efficiency of DNA extraction of a preliminary DNA extraction protocol, showed low DNA yields for 9% of the specimens (n = 78). In all cases, low DNA extraction efficiency seemed to be due to a component present in faeces that prevented DNA binding to silica particles, presumably by competitive binding. Choosing the right ratio of silica particles to faeces specimen solved this problem. Similarly, reconstruction experiments showed that the strong PCR inhibition that was observed in 8% of the specimens could effectively be relieved by the inclusion of alpha-casein in the PCR mixtures.

Human cytomegalovirus DNA in plasma and serum specimens of renal transplant recipients is highly fragmented.

ABSTRACT Quantitation of cytomegalovirus (CMV) DNA in plasma and serum by PCR is increasingly used to identify patients at risk for developing CMV disease and to monitor the efficacy of antiviral therapy. Although CMV DNA levels are generally interpreted as viral loads, the exact nature of the viral DNA in these specimens is unknown. We studied the state of CMV DNA in plasma and serum specimens obtained from three renal transplant recipients at peak viral DNA levels during primary CMV infection. For this purpose, DNA isolated from these specimens was fractionated by size, and CMV DNA levels in the resulting DNA fractions were measured by quantitative PCR targeted at large (578-bp) and small (134-bp) amplicons. These experiments showed that the molecular sizes of DNA fragments from which CMV DNA is amplified were small (<2,000 bp), indicating that CMV DNA in plasma and serum is highly fragmented. Furthermore, CMV DNA levels were consistently higher when targeted at the smaller amplicon, providing additional evidence for the fragmentation of viral DNA. In conclusion, the first results with three patients have shown that CMV DNA in plasma and serum is highly fragmented and does not necessarily reflect the amount of infectious virus. These observations have potential consequences for understanding CMV pathogenesis and interpreting CMV DNA levels in individual patient management.

Reduced PCR Sensitivity Due to Impaired DNA Recovery with the MagNA Pure LC Total Nucleic Acid Isolation Kit

ABSTRACT The increasing demand for molecular diagnostics in clinical microbiology laboratories necessitates automated sample processing. In the present study, we evaluated the performance of the MagNA Pure LC total nucleic acid isolation kit (M extraction) in comparison with the manual method (Si extraction) according to Boom et al. (R. Boom, C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. Wertheim-van Dillen, and J. van der Noordaa, J. Clin. Microbiol. 28:495-503, 1990) for the detection of viral DNA by competitive quantitative PCR. Reconstruction experiments with HindIII-digested phage lambda DNA and HaeIII-digested φX174 DNA showed that the recovery of DNA from phosphate-buffered saline, cerebrospinal fluid, EDTA-anticoagulated plasma, and EDTA-anticoagulated whole blood by M extraction is, on average, 6.6-fold lower compared to Si extraction. PCR signals of spiked PCR control DNAs for Epstein-Barr virus and varicella-zoster virus were also between 1.9- and 14.2-fold lower after M extraction compared to Si extraction, also suggesting impaired DNA recovery. M extraction of spiked cytomegalovirus strain AD 169 in whole blood showed a 5- to 10-fold reduction in PCR sensitivity compared to Si extraction. This reduction of PCR sensitivity was also observed when clinical whole blood samples were processed by M extraction. Before implementing M extraction, the clinical consequences of the reduced recovery should first be considered, especially when maximal sensitivity is required.

Molecular diagnosis of visceral herpes zoster

Patients with disseminated herpes zoster may present with severe abdominal pain that results from visceral involvement of varicella-zoster-virus infection. In the absence of cutaneous eruptions of herpes zoster, visceral herpes zoster is extremely difficult to diagnose. This diagnostic difficulty has the potential to cause devastating delays in treatment. We report a case series of four patients with visceral herpes zoster in whom large concentrations of DNA from varicella zoster virus were detectable in blood by PCR before signs of infection appeared on the skin, thus enabling early diagnosis and treatment.

Heat-shock induction of the human immunodeficiency virus long terminal repeat.

Rat cell lines were established in which the bacterial chloramphenicol acetyltransferase (CAT) gene under control of the human immunodeficiency virus (HIV) long terminal repeat (LTR) was stably integrated. The cell lines showed a repressed phenotype for CAT expression, but could be induced for it by inhibition of protein synthesis, as well as by heat-shock and chemical inducers of the cellular stress response, such as sodium arsenite, 8-hydroxyquinoline and the heavy metals cadmium and copper. A decameric sequence present in the NF-kB binding sites in the HIV LTR (GGGACTTTCC) resembles the cellular heat-shock core sequence and may therefore be involved in the heat-shock response.

Detection and Identification of Enterocytozoon bieneusi and Encephalitozoon Species in Stool and Urine Specimens by PCR and Differential Hybridization

ABSTRACT Several species of microsporidia can cause disease in humans in both immunocompromised and immunocompetent individuals. Enterocytozoon bieneusi and Encephalitozoon intestinalis are most commonly associated with chronic diarrhea. All Encephalitozoon species, including E. intestinalis, E. hellem, and E. cuniculi, also cause disseminated infections. As distinctive treatment options are available for the different genera, identification is clinically important. We evaluated a PCR with primers directed to a conserved region of the small subunit rRNA gene of microsporidia. Hybridization with a generic microsporidium probe and specific probes for each of the four different species was used for identification. Probes were labeled with ruthenium and detected by electrochemiluminescence. The sensitivity of the assay was tested with plasmids containing the region of interest from each of the four different species and Vittaforma corneae as a control. In addition, the assay was tested with feces spiked with cultured spores from each of the three Encephalitozoon species and V. corneae. An analytical sensitivity of 3.5 × 102 to 3.5 × 103 spores per g of feces, corresponding to 17 to 170 gene copies per PCR, was found, which is several orders of magnitude more sensitive than microscopy after Uvitex 2B fluorescent staining. Stool samples from 22 microscopically diagnosed patients and from 61 uninfected controls were evaluated, showing a sensitivity of at least 95% and a specificity of 100% compared to microscopy. The method was further tested by spiking urine samples with spores of the different Encephalitozoon species.

Induction of gene expression under human cytomegalovirus immediate early enhancer-promoter control by inhibition of protein synthesis is cell cycle-dependent.

In this paper we describe stably transfected rat cell lines which harbour either the human cytomegalovirus (HCMV) immediate early (IE) gene encoding the 72K IE nuclear antigen (IEA) or the bacterial chloramphenicol acetyltransferase (CAT) gene both under transcriptional control of the HCMV IE enhancer-promoter (-484 to -19 relative to the IE cap site, +1). In these cell lines IE gene or CAT gene expression is repressed but can be induced by heat-shock, by sodium arsenite and by inhibitors of protein synthesis such as cycloheximide (CH). In addition, we present evidence suggesting that CH-mediated activation is cell cycle-dependent. Thus CH-mediated induction of the 72K IEA as well as CAT gene expression was impaired and accumulation of mRNAs did not occur when cellular DNA synthesis was inhibited. Activation of IE genes by CH occurred almost exclusively in those cells which were in S-phase. In contrast, activation of gene expression by sodium arsenite occurred independently of cellular DNA synthesis and was not restricted to cells in S-phase. The data are consistent with, but not proof of, the hypothesis that the activation of IE transcription, brought about by inhibition of protein synthesis, resulted from a disturbed chromatin conformation due to DNA synthesis continuing in the absence of a supply of chromatin-organizing proteins. The possible relevance of these observations with regard to HCMV latency and reactivation is discussed.

Human immunodeficiency virus type 1 RNA populations in faeces with higher homology to intestinal populations than to blood populations

To determine whether human immunodeficiency virus type 1 (HIV-1) in faeces is representative of the HIV-1 population in intestinal tissue, we studied HIV-1 V3 variation in faeces, intestinal biopsies and serum from two individuals. Phylogenic analysis of HIV-1 V3-coding RNA in faeces from one individual showed three distinct genotypes. Viruses belonging to all three genotypes were also present in sigmoidal tissue and in serum. Jejunal tissue contained two of these three genotypes. Analysis of the V3-coding RNA in faeces of the other individual showed five distinct genotypes. One of these genotypes was present in all specimens from this individual. Besides this shared genotype, jejunal tissue and serum contained sequences belonging to one other genotype. In addition, one of the other three V3 variants was detected in sigmoidal tissue. For both persons the shared HIV-1 RNA genotypes in faeces and serum displayed a distinctly different frequency distribution. In one individual, the genotype which was detected in a majority of the clones in faeces (59%) and as a minority in serum (11%), was the most abundant genotype in jejunal and sigmoidal tissue (61% and 80%, respectively). For the other individual the genotype that was present in faeces in a significant number of clones (43%) was detected in serum as a minority (8%), whereas this genotype composed 47% of the clones isolated from jejunal tissue. Taken together these data suggest that faeces contain HIV-1 sequences that are derived from local HIV-1 replication in intestinal tissue.

Fractionation of Nucleic Acids into Single-Stranded and Double-Stranded Forms

We describe a rapid and efficient procedure for the fractionation of mixtures of nucleic acids (NA) into double-stranded (ds) and single-stranded (ss) forms regardless of the nature of the nucleic acid (DNA or RNA). The procedure is based on the differential binding of dsand ss-NA forms to silica particles in different lysis/binding buffers which have in common that they contain a high concentration of the chaotropic agent guanidinium thiocyanate (GuSCN). Previously we reported on a procedure (protocol Y) for the routine purification of total NA from clinical specimens (1). The method is based on the lysing and nuclease-inactivating properties of the chaotropic agent GuSCN together with the NA-binding properties of silica particles (or diatoms) in the presence of this agent. Comparison of different GuSCN-containing lysis/binding buffers with respect to the binding of different NA-types to silica particles revealed that only ds-forms were bound when using lysis/binding buffer L11 (see below) whereas both dsand ss-forms were bound in lysis/binding buffer L6 (1). This observation formed the basis for the development of a procedure (protocol R) for the fractionation of mixtures of ssand ds-NA. The procedure is summarised in Figure 1. A 50 μl specimen (containing a mixture of NA-types in TE buffer) was added to a mixture of 900 μl lysis/binding buffer L11 and 40 μl size-fractionated silica particles (SC) in an Eppendorf tube and subsequently homogenized by vortexing. After a 10 min binding step at room temperature, the tube was centrifuged (2 min at ∼12 000 g) which resulted in a silica/ds-NA pellet (‘initial silica pellet’) and a supernatant containing ss-NA. To recover ss-NA forms (protocol R-sup) 900 μl of the supernatant were added to a mixture of 400 μl binding buffer L10 (see below) and 40 μl SC. Thereafter the ss-NA was bound during a 10 min binding step at room temperature. The tube was subsequently centrifuged (15 s at ∼12 000 g), and the supernatant discarded (by suction). The resulting pellet was subsequently washed twice with 1 ml of washing buffer L2 (1), twice with 1 ml ethanol 70% (vol/vol) and once with 1 ml acetone. The silica pellet was dried (10 min at 56 C with open lid in an Eppendorf heating block) and eluted (10 min at 56 C; closed lid) in 50 μl TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0). After centrifugation (2 min at ∼12 000 g) the supernatant contained the ss-NA fraction. To recover ds-NA forms (protocol R-pellet) from the initial silica-pellet, the remaining supernatant was discarded, and the silica pellet was washed twice with 1 ml lysis/binding buffer L11 to remove unbound ss-NA. The resulting silica pellet Figure 1. Outline of protocol R. Recovery of ds-NA takes place from the initial pellet (R-pellet), recovery of ss-NA takes place from the initial supernatant (R-sup). L11, L10, L6 and L2 are GuSCN containing buffers; SC is silica particle suspension. For details see text.

Resistance to methylation de novo of the human cytomegalovirus immediate early enhancer in a model for virus latency and reactivation in vitro

Rat-9G cells carry several stably integrated copies of the major immediate early (IE) transcription unit of the human cytomegalovirus (HCMV). In these cells IE expression is repressed but inducible. In this report we describe the DNA methylation status of HpaII, HhaI and AhaII sites within the IE gene, determined at different passage levels. Most, if not all, of the resident IE genes were progressively methylated in a similar fashion. This resulted in DNA methylation patterns in which sites surrounding the IE upstream region were preferentially methylated to a high degree. In contrast, sites within the 19 bp IE enhancer elements were markedly under-methylated. This particular DNA methylation pattern probably resulted from differences in DNA methylation rates, sites within the IE enhancer being methylated at only a very low rate. Methylation of the IE genes did not affect their inducibility, which might be related to the very low methylation level of the IE enhancer.