The application of genetic techniques in community health surveillance
English: The study was designed to investigate the application of a range of genetic methods for the detection and monitoring of bacterial pathogens responsible for key Free State community health issues. The rapid detection and differentiation of potentially pathogenic organisms from water sources is vital for the safety and the state of health of many. Conventional culture methods can be complex and time-consuming, whereas detection by the polymerase chain reaction (PCR) is rapid but could be impaired due to regional strain sequence variations and detection of dead cells. Neisseria gonorrhoeae is treated syndromically in South Africa. To ensure continued efficacy of antibiotics, resistance development and plasmid content (β-Iactamase type or tetM-conjugative type) are important factors to be monitored. The study of pulmonary tuberculosis, which has reemerged as a significant problem in the developed as well as the developing world, is greatly benefited by genetic techniques. DNA fingerprinting is a powerful method and may be used in the context of Mycobacterium tuberculosis surveillance for determining transmission versus reactivation rates and for following patient compliance. The first-line antibiotics employed against M. tuberculosis in the Free State are isoniazid (INH), rifampin (RIF), ethambutol and pyrazinamide. Resistance to rifampin is known to arise as a result of missense and other mutations occurring in a discrete 23 amino acid region (69 bp) of the rpoB gene. Detection of such mutations can be performed by PCR-based methods. The objectives of the study were as follows: (1) surveillance of community and environmentally acquired infections including waterborne pathogens (conventional and PCR detection techniques), N. gonorrhoeae (randomly amplified polymorphic DNA [RAPD] and plasmid analysis) and M. tuberculosis (genomic fingerprinting); (2) to determine antibiotic susceptibilities of N. gonorrhoeae and M. tuberculosis; (3) to investigate the acquisition and dissemination of tetracycline resistance in N. gonorrhoeae and the development of rifampin antibiotic resistance in M. tuberculosis (rpoB gene sequencing). One hundred and five water samples (shaken and brushed from containers, sewage effluent and river water) were collected during March - May 1999. The detection of waterborne pathogens Escherichia coli, Shigella sp., Salmonella sp. and Listeria monocytogenes was performed by the widely versatile PCR technique. The primer sets were designed to detect the verotoxin genes of Enterohaemorrhagic E. coli (EHEC), the invasive plasmid antigen gene of Shigella sp. and Enteroinvasive E. coli and the enterotoxin gene of Salmonella sp. A final primer set was used to amplify the listeriolysin 0 gene of Listeria monocytogenes. Where possible the suitability of primers against local clinical strains was shown to be successful. Selective and enrichment media was employed to provide presumptive confirmation of the detection of the pathogens by PCR. PCR detection revealed four cytotoxic E. coli, seven ipaH Shigella sp., ten enterotoxin Salmonella species, and thirteen listeriolysin Listeria monocytogenes strains in the waters examined. Culture confirmed only a single Salmonella sp. This indicated a higher potential for rapid detection (compared with conventional culture methods) of waterborne pathogens by PCR especially when the bacteria could have entered a non-culturable but viable state. The problem of residual DNA from non-viable bacteria being detected by PCR is still a setback to this particular genetic technique. The detection of four verotoxin containing EHEC, followed by the inability to confirm the E. coli serovar 0157:H7 (culture, immunomagnetic separation and latex agglutination) emphasises the dangers in concentrating efforts to detect only one specific serovar when screening water samples. The N. gonorrhoeae investigated were isolated from the Bloemfontein community during 1993-1997. To overcome the problems and difficulties in speciating and strain typing Neisseria for epidemiological surveillance, RAPD surveillance analysis was performed. The primer used had been shown to exhibit excellent discriminatory power for the differentiation of N. meningitidis strains. The results (significantly enhanced by RAPD analysis beads) showed that this analysis can be used to augment auxotype/serovar typing of N. gonorrhoeae populations. With observed shifts in clinical isolation sites of Neisseria species, the RAPD technique has potential use for taxonomic studies of Neisseria. Investigations into tetracycline resistance development in N. gonorrhoeae were performed by amplification of tetM genes by PCR. The PCR products were digested with Hpall and the fragments separated on agarase gels. Plasmid analysis was performed using a plasmid Miniprep DNA purification system. TetM-conjugative and conjugative plasmids were restricted with enzymes Bgll, Smal and Hincll and fragments separated on agarase gels. The conjugative (24.5 MOa) plasmid was present in 29/102 (28.4%) strains while the tetM-conjugative (25.2 MOa) plasmid was present in 48/102 (47%) strains. The Bloemfontein N. gonorrhoeae strains carried both African and Asian β-Iactamase plasmids. Seventy percent of strains showed increased tetracycline resistance (≥ 2 µg/ml) while 42% of strains exhibited high-level (16-128 µg/ml) resistance. The restriction of tetM-conjugative and conjugative plasmids isolated in 1996 revealed different profiles to those previously described showing that these plasmid types are continuing to evolve. Amplification of a fragment of the tetM gene provided a simple and quick method for predicting high-level tetracycline resistance. On restricting the 43 high-level tetracycline-resistant strains (MICs ≥ 16 µg/ml) all were found to contain the American-type tetM gene and 25.2 MDa plasmids were demonstrated. The establishment of tetM-conjugative plasmids containing the American-type tetM gene is increasing, 2% in 1994 to 47% in 1997. Three hundred and thirteen sputum samples were collected from the Rocklands community in Bloemfontein. Subsequent sputum samples were collected to monitor community response to reassessment and to ensure eradication. Detection of M. tuberculosis (MTB) was accomplished by Ziehl-Neelsen (ZN) staining and conventional culture on L6wenstein-Jensen (LJ) agar slopes. Thirty three sputum samples were ZN positive, with LJ detecting an additional 7 M. tuberculosis isolates. Discrepancies in ZN and LJ results were confirmed by amplifying a 123 bp fragment of the IS6110. PCR also indicated the need for additional diagnostic methods as 11% of isolates were not detected by ZN or LJ. The BACTEC system was used for confirmation as well as for susceptibility testing. Only 63% of persons receiving treatment returned after 1-3 months indicating possible non-compliance. A single patient (old case) had a maintained ZN positive result for 6 months with full susceptibility to all antibiotics tested. The standard method of fingerprinting involved Pvull restriction endonuclease digestion of genomic DNA followed by Southern blotting and probing for IS6110 elements. The fingerprinting of 50 INH -and/or RIF-resistant strains from 1997 revealed 32 diverse profiles. Non-adherence and the emergence of resistant clonal groups were evident. Five clonally related clusters were evident that were either localised or had disseminated to different districts in the Free State. Of 26 person's initial samples (ZN+/LJ+) investigated in 1998, 25 diverse fingerprint profiles were found. Fingerprinting of 11 rifampicin-resistant strains (1998) showed the emergence of many diverse resistant strain types. The possible spread of TB in a hospital ward was revealed through shared fingerprint profiles of two samples. The monitoring of rifampin resistance through sequencing of a key region 157 bp of the rpoB gene was performed. Previously reported mutation sites were evident in the study; 516, 526, 531 and 533. The two local 1997 clonal groups (identified by fingerprint profile) did not share mutated rpoB alleles. This could possibly be explained by clonally related susceptible strains independently developing sub-clones bearing distinct rpoB alleles. Inaccuracies in susceptibility testing were evident as a Bloemfontein strain reported to be rifampin-susceptible presented with a variant rpoB allele. From 13 MTB (new cases, 1998) screened for the rpoB gene and subsequently sequenced it was found that two ZN/LJ positive samples had missense mutation at positions 516 and 526. A reduced outcome would result with these patients emphasising the need for accurate susceptibility testing to be conducted earlier than presently stipulated. Eleven rifampin-resistant strains (1998) revealed only one strain without rpoB gene mutations in the 157 bp region examined. The same mutated codon was evident with two strains (with shared fingerprint profile from same hospital ward) again strongly implying dissemination of a strain type between patients. A family community from a semi-rural area (Bainsvlei) situated 15 km from Bloemfontein was investigated. The Bainsvlei family member's samples from 1995, 1997 and 1998 revealed the same fingerprint profile (shared by other family members in 1995) and same mutated rpoB codon indicating the persistence of a rifampin/isoniazid-resistant strain. Subsequent information on the brother's past MTB infections and treatment showed that a possible reinfection of a multiplyresistant strain could have occurred. The situation has not been fully resolved due to lack of community involvement and funding. Genetic techniques investigating infectious diseases in the community setting certainly provides required rapid results and epidemiological information essential for the future success of infection control programmes.