The epidemiology of tuberculosis among asylum seekers in The Netherlands: implications for screening.

OBJECTIVE: To identify low-risk groups among asylum seekers in the Netherlands that may be excluded from tuberculosis (TB) screening at entry or during follow-up. METHODS: A retrospective cohort study of medical records of asylum seekers entering the country between January 1994 and March 1997. RESULTS: Medical records were available for 46,424 of the 96,000 asylum seekers (48%) in this period. One hundred and three pulmonary TB cases were diagnosed at entry (prevalence 222/100,000). Risk factors were age >11 years, history of imprisonment and country of origin at war or with TB incidence >100/100,000. During a mean follow-up period of 10 months, 51 pulmonary TB cases were diagnosed (incidence 134/100,000 person-years). Risk factors were age >11 years, old lesions on entry X-ray, and country of origin whose asylum seekers had a prevalence of TB at entry >200/100,000. CONCLUSIONS: We conclude that 1) those with abnormal X-ray at entry should receive preventive therapy after exclusion of active TB, or undergo intensive follow-up, 2) periodic screening is not indicated for immigrants from countries whose asylum seekers have a low prevalence of pulmonary TB at entry, and 3) children <12 years can be excluded from screening.

Tuberculosis screening among immigrants in The Netherlands: what is its contribution to public health?

Understanding the epidemiology of tuberculosis in migrant communities and designing adequate and comprehensive control strategies is a major challenge facing public health authorities in many low-prevalence countries. In The Netherlands, screening immigrants from tuberculosis high prevalence countries has been conducted since 1966. In this paper, we review risk factors for tuberculosis in migrant populations, the public health importance of tuberculosis and the current screening policy in The Netherlands. TB treatment outcome in migrant populations and operational considerations that ought to be taken into account to optimize current screening practices are also reviewed. The article recommends the setting-up of an information system to evaluate the effectiveness of screening immigrants in The Netherlands, and adjustment of screening policies where needed.

Rapid and sensitive detection of Chlamydia trachomatis using a ligatable binary RNA probe and Q beta replicase.

A simple assay format was developed for the direct detection of C. trachomatis rRNA utilizing ligation of recombinant MDV-1 probe RNA fragments hybridized to 23S rRNA after capture and release from a solid support. Assay background (equivalent to 10(4) targets) was suppressed by blocking sequences in the 5' MDV reporter probe fragment complementary to the 3' fragment by prehybridization of a DNA oligonucleotide. A pair of reporter fragments bearing a deletion within the region, obtained by a hydrid-selection-amplification protocol, yielded a low level of assay background which was reduced to < 2% with a blocker directed against the remaining pairing sequence. This probe set showed a sensitivity of 10(3) molecules of 23S rRNA (> 95% responding) and could detect a single elementary body (EB) of Chlamydia trachomatis or 1-10 EB added to a clinical matrix of pooled negative human cervical swab samples. The time of first appearance of amplification products by real-time fluorescence detection showed a linear response to log increases in the target level over a 10(5)-fold range, permitting the determination of target level within an order of magnitude. The assay showed approximately 10(9)-fold discrimination over Chlamydia pneumonae (TWAR) rRNA. High levels of cultured C. albicans, E. coli, S. aureus, or N. gonorrhoeae had no detectable effect on assay background or the ability to detect a single elementary body.

Single molecule detection of RNA reporter probes by amplification with Q beta replicase.

The addition of target-specific probe sequences within MDV RNA, an otherwise efficient template for Q beta replicase, generally resulted in RNA molecules with inferior replication properties, including reduced replication rates and poor sensitivities. We have discovered that the replication characteristics of MDV RNA molecules with internally placed probe sequences are dramatically affected by short RNA sequences (spacer elements) at the 5' and 3' ends of the probe sequence. For a given probe sequence, the sequences of the flanking spacer elements effected replication sensitivity by six orders of magnitude and replication rate by three fold. By taking advantage of spacer elements, internal MDV probes were developed that permitted the reproducible, real time, fluorescence detection of a single RNA molecule in less than 25 min through amplification with Q beta replicase. RNA structural analysis of such probes suggested that the spacer elements functioned by allowing the RNA to fold in a way which substantially maintained the tertiary structure of the MDV domain. MDV reporter probes with suitable replication properties were obtained from libraries of RNA molecules in which the probe sequence was flanked by many different spacer elements (generated by random nucleotide synthesis). We demonstrated that this is a general method for developing RNA reporter molecules which are rapidly and reproducibly amplified by Q beta replicase, even from a single molecule.

Real-time fluorescence detection of RNA amplified by Q beta replicase.

Amplification of RNA probes by Q beta replicase can be used to detect a wide range of analytes with a potential sensitivity of a single molecule. A system has been developed in which Q beta amplification of midivariant-(MDV)-based RNA is measured in real time by fluorescence. This was accomplished by including a fluorescent intercalating dye, propidium iodide, in the reactions and monitoring the fluorescence change using a custom fluorometer. The time at which fluorescence is detectable above background is referred to as the "response time" and is calculated using curve-fitting algorithms. A response time is inversely and linearly proportional to the logarithm of the number of template RNA molecules which initiated the reaction. Therefore, this system permits an unknown amount of input RNA probe to be quantified through 11 orders of magnitude when compared to a standard curve. Under the described conditions with MDV RNA, the response time occurs when about 3 x 10(11) RNA molecules are synthesized and occurs within the exponential phase of the reaction, before the number of active enzyme molecules are saturated with RNA templates. This system has been used to determine the replication properties of MDV RNA reporter molecules bearing specific probe sequences and to develop hybridization assays for the clinical diagnostic field.

Evolution of host cell RNA into efficient template RNA by Q beta replicase: the origin of RNA in untemplated reactions.

Q beta replicase can replicate a single molecule of certain species of RNA to 10(14) copies in minutes. This replication ability has been used for in vitro studies of molecular evolution and is currently being utilized as a method of amplifying RNAs that contain probe sequences. It has been observed that Q beta replicase can produce replicatable RNA even in the absence of exogenously added template RNA. The origin of this RNA has been ascribed either to contamination with replicatable RNA or to an ability of Q beta replicase to synthesize RNA de novo from the nucleotides present in the reaction. Technologies that employ Q beta replicase require a thorough understanding of the conditions that lead to this so-called spontaneous RNA production. We have created an expression system and purification method with which we produce gram quantities of highly purified Q beta replicase, and we have identified reaction conditions that prevent the amplification of RNA in assays that do not contain added RNA. However, when these reaction conditions are relaxed, spontaneous RNA replication is seen in up to 100% of the assays. To understand the origin of this RNA, we have cloned several spontaneously produced RNAs. Sequence analysis of one of these RNAs shows that it arose by the evolution of Escherichia coli tRNA into a replicatable template and not by de novo synthesis from nucleoside triphosphates in the reaction.

Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction.

We applied the polymerase chain reaction to detection of the pathogenic protozoan Toxoplasma gondii based on our identification of a 35-fold-repetitive gene (the B1 gene) as a target. Using this procedure, we were able to amplify and detect the DNA of a single organism directly from a crude cell lysate. This level of sensitivity also allowed us to detect the B1 gene from purified DNA samples containing as few as 10 parasites in the presence of 100,000 human leukocytes. This is representative of the maximal cellular infiltration (10(5)/ml) in 1 ml of cerebrospinal fluid obtained from patients with toxoplasmic encephalitis. The B1 gene is present and conserved in all six T. gondii strains tested to date, including two isolates from patients with acquired immunodeficiency syndrome. No signal was detected by using this assay and DNAs from a variety of other organisms, including several which might be found in the central nervous system of an immunocompromised host. This combination of sensitivity and specificity should make detection of the B1 gene based on polymerase chain reaction amplification a very useful method for diagnosis of toxoplasmosis both in immunocompromised hosts and in congenitally infected fetuses.

Cloning of cDNAs encoding a 28 kilodalton antigen of Toxoplasma gondii.

By screening cDNA libraries in lambda gt11 with antibodies raised against the previously described protective F3G3 antigen of Toxoplasma gondii, and subsequently screening with nucleic acid probes, we have isolated cDNA clones that encode a 28 kDa antigen of T. gondii that is likely one of the two antigenic components of the F3G3 antigen. The gene apparently exists as a single copy in the tachyzoite haploid genome of the three strains of T. gondii examined. Northern blot analyses revealed that the cDNAs hybridize with a major T. gondii RNA species of 1.1 kb. Together the cDNAs encompass 1051 bp of cDNA sequence containing an open reading frame with the capacity to encode a 28 kDa protein. Antibodies that were affinity purified using recombinant fusion proteins produced by two of the clones reacted on protein blots of whole T. gondii lysate with a single antigen having an apparent molecular mass of 28 kDa. Both recombinant fusion proteins reacted with IgG antibodies in sera of mice and humans infected with T. gondii and therefore might be useful for the development of diagnostic assays for T. gondii infection.

Molecular analysis of the gene encoding the major surface antigen of Toxoplasma gondii.

The complete sequence of P30, the major surface Ag of the protozoan parasite, Toxoplasma gondii, has been deduced through the cloning and analysis of its gene. Using polyclonal serum specific for P30, we have isolated a P30 cDNA clone from a lambda gt11 cDNA expression library derived from tachyzoites of T. gondii (RH strain). This clone produces a beta-galactosidase fusion protein which reacts with several anti-P30 mAb. In addition, polyclonal anti-serum raised to the fusion protein reacts with purified P30 protein and exclusively with P30 in a whole cell lysate of T. gondii. This cDNA clone was used to isolate near full-length cDNA molecules and a cosmid clone containing the P30 gene. Sequence analysis of the cDNA reveals a single open reading frame with coding capacity for 34.7 kDa of primary translation product (consistent with the apparent Mr of P30 on SDS-acrylamide gels) including a presumptive hydrophobic signal sequence. The P30 primary translation product also has a carboxy-terminal hydrophobic tail which is predictive of a posttranslational cleavage and modification with a glycolipid anchor. We have identified the apparent 5' and 3' ends of the P30 mRNA transcript which is extremely abundant, 1500 nucleotides in length, and polyadenylated. The P30 gene is single copy and contains no introns.

Introduction of superhelical turns into DNA by adenoviral core proteins and chromatin assembly factors.

The interaction in vitro between adenoviral histone-like proteins and DNA in the presence of chromatin assembly factors was investigated. Viral core protein VII or its precursor pVII was incubated with DNA in the presence of an extract of HeLa cell chromatin, which mediates nucleosome assembly from histones and DNA. We have demonstrated that either protein can introduce superhelical turns into relaxed closed-circular DNA and that the presence of chromatin extract is necessary for the supertwisting effect. A greater density of superhelical turns was produced by pVII than by VII, but neither protein-DNA interaction resulted in the "physiological" amount of supertwisting produced by histones. The inhibition of histone-induced supercoiling by both proteins and the protection of turns in supertwisted starting material are also described. The nucleosome assembly factor, nucleoplasmin, fails to mediate the introduction of superhelical turns by VII or pVII.