S.B. Svenson

PCR is, in principle, a simple and rapid test for use in the detection of Mycobacterium tuberculosis. However, virtually no data are available on the reliability and reproducibility of the method. In order to assess the validity of PCR for the detection of mycobacteria in clinical samples, seven laboratories participated in a blinded study of 200 sputum, saliva, and water samples containing either known numbers of Mycobacterium bovis BCG cells or no added organisms. Each laboratory used its own protocol for pretreatment, DNA extraction, and detection of the amplification product. Insertion sequence IS6110 was the target for DNA amplification. Several participating laboratories reported high levels of false-positive PCR results, with rates ranging from 3 to 20% and with one extreme value of 77%. The levels of sensitivity also ranged widely among the different participants. A positive PCR result was reported for 2 to 90% of the samples with 10(3) mycobacteria. Although most participants did include control tests to check the sensitivity and specificity of the PCR, the sequence of operations from sample pretreatment to purification of DNA from bacteria was not always monitored adequately. During these procedures cross-contaminating DNA was introduced and/or bacterial DNA was lost. The results of the study show that the implementation of an effective system for monitoring sensitivity and specificity is required before the PCR can be used reliably in the diagnosis of tuberculosis.

A total of 237 Escherichia coli strains isolated from the urine of patients with various forms of urinary tract infection or from feces of healthy children were analyzed for O group, possession of K1 capsule, type 1 fimbriae, P fimbriae, X adhesin, and production of hemolysin. Some of the strains were also analyzed for K and H antigens, outer membrane protein pattern, and plasmid content. P fimbriation, hemolysin production, and certain O groups were found to be significantly correlated with pyelonephritogenicity. Possession of type 1 fimbriae or of K1 capsule or plasmid content did not significantly correlate with virulence. Outer membrane protein patterns in 139 strains of the more common O groups were analyzed. Only one to three patterns, which varied between serotypes, were usually found within any one O group. Distinctive groups (clones) were found when the strains were grouped according to complete serotype, fimbriation, hemolysin production, and outer membrane protein pattern; also, the mean number of plasmids was typical of the strains in a given clone. Seven clones associated with pyelonephritis were found; together they accounted for 57% of the O serotypable strains from the pyelonephritis patients. The seven clones were P fimbriated but differed in their serotypes as follows: O1:K1:H7, O4:K12:H1, O4:K12:H5, O6:K2:H1, O16:K1:H6, or O18ac:K5:H7. All O1:K1:H7 strains observed fell into two clones according to the presence or absence of type 1 fimbriae and hemolysin production. One clone associated with cystitis was also found; this consisted of O6:K13:H1 strains lacking P fimbriae. Not a single representative of these eight clones was found among the fecal strains from the healthy children. They are proposed to represent virulent clones with special ability to cause human urinary tract infection.

Two Salmonella typhi mutants, 541Ty (Vi+) and 543Ty (Vi-), auxotrophic for p-aminobenzoate and adenine, were evaluated as live oral vaccines. 33 volunteers ingested single doses of 10(8), 10(9), or 10(10) vaccine organisms, while four others received two 2 X 10(9) organism doses 4 d apart. No adverse reactions were observed. Vaccine was recovered from coprocultures of 29 of 37 vaccinees (78%) and from duodenal string cultures of two; repeated blood cultures were negative. The humoral antibody response to S. typhi O, H, Vi, and lysate antigens in serum and intestinal fluid was meager. In contrast, all vaccinees manifested cell-mediated immune responses. After vaccination, 69% of vaccinees overall and 89% of recipients of doses greater than or equal to 10(9) responded to S. typhi particulate or purified O polysaccharide antigens in lymphocyte replication studies but not to antigens of other Salmonella or Escherichia coli. All individuals, postvaccination, demonstrated a significant plasma-dependent mononuclear cell inhibition of wild S. typhi.

Lipoarabinomannan (LAM) is a major structural carbohydrate antigen of the outer surface of Mycobacterium tuberculosis. High antibody titres against LAM are often seen in active tuberculosis (TB). The role of such LAM-specific antibodies in the immune response against TB is unknown. Here we have investigated a monoclonal antibody (MoAb) SMITB14 of IgG1 subclass and its corresponding F(ab')(2) fragment directed against LAM from M. tuberculosis strain H37Rv. MoAb SMITB14 was shown by immunofluorescence to bind to whole cells of the clinical isolate M. tuberculosis strain Harlingen as well as to M. tuberculosis H37Rv. The binding of MoAb SMITB14 to LAM was inhibited by arabinomannan (AM) and oligosaccharides (5.2 kDa) derived from LAM, showing that the MoAb binds specifically to the AM carbohydrate portion of LAM. In passive protection experiments BALB/c mice were infected intravenously with M. tuberculosis Harlingen. MoAb SMITB14 was added intravenously either prior to, or together with, the bacteria. The antibody proved to be protective against the M. tuberculosis infection in terms of a dose-dependent reduction in bacterial load in spleens and lungs, reduced weight loss and, most importantly, increased long-term survival.