The emergence of epidemic multiple-drug-resistant (MDR) strains of Mycobacterium tuberculosis in conjunction with an increase in the number of reported cases of tuberculosis (TB) represents a major public health problem. In light of a recent outbreak of MDR M. tuberculosis at our center, we began the development of a polymerase chain reaction (PCR) assay for the rapid diagnosis of pulmonary TB using two sets of primers, one based on the IS6110 repeated sequence of M. tuberculosis and the other based on the protein antigen b (PAB). Reaction conditions were first optimized as to the appropriate extraction protocol and the concentrations of primer pairs, nucleotides, and MgCl2. Following a preliminary evaluation of the assay with clinical specimens, extraction and amplification procedures were further modified. PAB and IS6110 primers detected between 2 and 23 and 0.023 and 0.23 CFU of M. tuberculosis, respectively, in pooled, M. tuberculosis-negative sputa by our optimized PCR assay. After routine processing for mycobacteria, 734 specimens were subsequently amplified. DNA for amplification was obtained by boiling and beating the sediments with Tween 20. For each reaction, DNA (10 ,ul) was added to an amplification mixture containing 12 pmol of IS6110 primers, 20 pmol of PAB primers, 2 mM MgCl2, 200 JLM nucleotides, and 2.5 U of Taq polymerase and the mixture was then amplified for 40 cycles. The sensitivity and specificity of our PCR assay were 87.2 and 97.7%, respectively. We were unable to interpret the results for seven specimens (1%). In our experience, PCR proved to be a useful rapid diagnostic test for TB in a clinical setting and a valuable epidemiological tool for determining exposure groups in the hospital setting. Our findings also underscore the need for the systematic optimization of PCR assay conditions.

We have isolated and characterised the gene encoding the glycolytic enzyme enolase (2-phospho-D-glycerate hydrolase) from the human malaria parasite Plasmodium falciparum. This was achieved using a combination of cDNA sequencing and inverse-PCR techniques. The gene maps to chromosome 10 of the parasite. We have also mapped two further glycolytic enzyme genes, glyceraldehyde-3-phosphate dehydrogenase and triose-phosphate isomerase, to chromosome 14. The enolase gene encodes a protein of 446 amino acids (48.7 kDa), and all amino acid residues implicated in substratekofactor binding and catalysis are conserved in the malarial enolase molecule. The predicted protein sequence displays approximately 60-70% identity to enolase molecules of other eukaryotes, the closest relationship with its homologues seen amongst the seven fully described glycolytic pathway enzymes of R falciparum. Of particular significance in this well conserved molecule is a characteristic 5-amino-acid insertion sequence that is identical in position and virtually identical in primary structure to that which is otherwise found uniquely in plant enolase proteins. This pentapeptide, together with other features of the plasmodia1 sequence, points to a common ancestry with photosynthetic organisms at the level of a protein-encoding nuclear gene, thus extending earlier analyses of nuclear small-subunit ribosomal RNA genes, and of an extrachromosomal circular 35-kb DNA element found in I? falciparum, which have also indicated such a relationship.The glycolytic pathway has a number of features which suggest that it is particularly important to the blood-stage forms of the human malaria parasite Plasmodium falciparum, and thus make it an attractive candidate for experimental investigation. The level of glycolysis of parasite-infected cells is about 100-times greater than that observed in uninfected cells [l, 21 and can result in clinical hypoglycaemia as the infection progresses. ATP generation during this process is

Interfering substances have been reported to inhibit PCR assays for the direct detection of Mycobacterium tuberculosis in clinical specimens. Using an internal control, we determined that 52% of respiratory specimens interfered with our PCR assay. On the basis of these findings, we tried to circumvent the problem by simply diluting prepared sediments. With sediment from a routinely processed sputum known to be inhibitory to PCR, one aliquot was prepared in a routine manner for PCR. Remaining sediment was diluted in phosphate-buffered saline, Middlebrook 7H10 broth, or BACTEC 12B broth; an internal control was added to all reaction mixtures and controls. Internal control was detected only in the sample diluted with BACTEC 12B medium. Components of the BACTEC 12B medium including PANTA reagent (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin), reconstituting fluid, 0.2% glycerol, 0.05% Tween 80, and 0.05% bovine serum albumin (BSA) were tested in a similar manner. Only 0.05% BSA resulted in amplification of the internal control DNA. Varying concentrations of BSA were added to 11 aliquots of a respiratory sediment known to be inhibitory to the PCR. Internal control was detected in all reaction mixtures containing 0.00038 to 0.1% BSA.To determine the ability of BSA to override inhibition, respiratory specimens were run in triplicate: undiluted, diluted 1:2 with BACTEC 12B medium, or diluted with 0.026% BSA. For 21 of 22 inhibitory specimens, BSA was able to override the presence of interfering substances. These data suggest that the presence of BSA in a PCR assay is critical for the direct detection of M. tuberculosis in respiratory specimens.

Serology for parvovirus B19 has been hampered by limited availability of antigen which has often had to be isolated from viraemic blood donations. We have determined the sequence of the genome of one such isolate (Stu). It is 99% similar to the sequences of two other isolates (Wi and Au) except at the far 5'-end, where it is more similar to the terminus of another isolate (Ala/Alb). Recombinant nonstructural protein, NS, was constructed. Antibodies to NS, as well as to the capsid proteins, VP1/2, were detected in patients with B19 infection.

Introduction of PCR to directly detect Mycobacterium tuberculosis in clinical specimens has shown promise; however, interfering substances in clinical material have contributed to lowered assay sensitivities. We evaluated the ability of a PCR assay to detect M. tuberculosis in BACTEC 12B broth cultures. Clinical specimens were processed and inoculated into BACTEC 12B vials. Evaluation was approached in two phases, starting with an initial evaluation in which an aliquot of 12B broth was removed when the growth index (GI) was .10 and stored at 4°C until assayed by PCR. Of the 290 specimens initially assayed, 129 were culture negative for mycobacteria as well as PCR negative for M. tuberculosis. Except for one, cultures (n = 102) which grew mycobacteria other than M. tuberculosis were all PCR negative. The remaining 59 broths were all culture and PCR positive for M. tuberculosis; 39%o (n = 23) of these cultures when assayed by PCR had GIs of s50. Following initial evaluation, 200 12B BACTEC vials with GIs of :10 were assayed in a similar manner except that specimens were amplified twice weekly to determine PCR's impact on the length of time to identification of M. tuberculosis as compared with standard laboratory practices. Utilization of PCR resulted in a mean time to detection of M. tuberculosis of 14 days, compared with 29 days by using commercially available nucleic acid probes to identify M. tuberculosis complex from growth of BACTEC 12B subcultures on solid media. In light of an overall sensitivity and specificity of 100 and 99.7%, respectively, coupled with the ability to identify M. tuberculosis days or weeks before other methods can be applied, we conclude that PCR might prove to be a rapid alternative for identification of M. tuberculosis in culture and allow for earlier setup of susceptibility testing.

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A dot blot hybridization assay for parvovirus B19 diagnosis was developed by using a PCR-generated probe, digoxigenin labelling, and chemiluminescence detection. Different labelling techniques and hybridization solutions were evaluated. From this analysis a protocol was devised for routine diagnostic use. The protocol enabled 1 pg of B19 DNA to be detected. The results of applying this method to 8,369 diagnostic samples collected during 1994 and 1995 are given.

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