The application of genetic techniques in community health surveillance

[摘要] English: The study was designed to investigate the application of a range of geneticmethods for the detection and monitoring of bacterial pathogens responsible forkey Free State community health issues. The rapid detection and differentiationof potentially pathogenic organisms from water sources is vital for the safety andthe state of health of many. Conventional culture methods can be complex andtime-consuming, whereas detection by the polymerase chain reaction (PCR) israpid but could be impaired due to regional strain sequence variations anddetection of dead cells. Neisseria gonorrhoeae is treated syndromically in SouthAfrica. To ensure continued efficacy of antibiotics, resistance development andplasmid content (β-Iactamase type or tetM-conjugative type) are importantfactors to be monitored. The study of pulmonary tuberculosis, which has reemergedas a significant problem in the developed as well as the developingworld, is greatly benefited by genetic techniques. DNA fingerprinting is a powerfulmethod and may be used in the context of Mycobacterium tuberculosissurveillance for determining transmission versus reactivation rates and forfollowing patient compliance. The first-line antibiotics employed against M.tuberculosis in the Free State are isoniazid (INH), rifampin (RIF), ethambutol andpyrazinamide. Resistance to rifampin is known to arise as a result of missenseand other mutations occurring in a discrete 23 amino acid region (69 bp) of therpoB gene. Detection of such mutations can be performed by PCR-basedmethods.The objectives of the study were as follows: (1) surveillance of community andenvironmentally acquired infections including waterborne pathogens(conventional and PCR detection techniques), N. gonorrhoeae (randomlyamplified 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 anddissemination of tetracycline resistance in N. gonorrhoeae and the developmentof 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. Thedetection of waterborne pathogens Escherichia coli, Shigella sp., Salmonella sp.and Listeria monocytogenes was performed by the widely versatile PCRtechnique. The primer sets were designed to detect the verotoxin genes ofEnterohaemorrhagic E. coli (EHEC), the invasive plasmid antigen gene ofShigella sp. and Enteroinvasive E. coli and the enterotoxin gene of Salmonellasp. A final primer set was used to amplify the listeriolysin 0 gene of Listeriamonocytogenes. Where possible the suitability of primers against local clinicalstrains was shown to be successful. Selective and enrichment media wasemployed to provide presumptive confirmation of the detection of the pathogensby PCR. PCR detection revealed four cytotoxic E. coli, seven ipaH Shigella sp.,ten enterotoxin Salmonella species, and thirteen listeriolysin Listeriamonocytogenes strains in the waters examined. Culture confirmed only a singleSalmonella sp. This indicated a higher potential for rapid detection (comparedwith conventional culture methods) of waterborne pathogens by PCR especiallywhen the bacteria could have entered a non-culturable but viable state. Theproblem of residual DNA from non-viable bacteria being detected by PCR is stilla setback to this particular genetic technique. The detection of four verotoxincontaining EHEC, followed by the inability to confirm the E. coli serovar 0157:H7(culture, immunomagnetic separation and latex agglutination) emphasises thedangers in concentrating efforts to detect only one specific serovar whenscreening water samples.The N. gonorrhoeae investigated were isolated from the Bloemfontein communityduring 1993-1997. To overcome the problems and difficulties in speciating andstrain typing Neisseria for epidemiological surveillance, RAPD surveillanceanalysis was performed. The primer used had been shown to exhibit excellentdiscriminatory power for the differentiation of N. meningitidis strains. The results(significantly enhanced by RAPD analysis beads) showed that this analysis canbe used to augment auxotype/serovar typing of N. gonorrhoeae populations.With observed shifts in clinical isolation sites of Neisseria species, the RAPDtechnique has potential use for taxonomic studies of Neisseria. Investigationsinto tetracycline resistance development in N. gonorrhoeae were performed byamplification of tetM genes by PCR. The PCR products were digested with Hpalland the fragments separated on agarase gels. Plasmid analysis was performedusing a plasmid Miniprep DNA purification system. TetM-conjugative andconjugative plasmids were restricted with enzymes Bgll, Smal and Hincll andfragments separated on agarase gels. The conjugative (24.5 MOa) plasmid waspresent in 29/102 (28.4%) strains while the tetM-conjugative (25.2 MOa) plasmidwas present in 48/102 (47%) strains. The Bloemfontein N. gonorrhoeae strainscarried both African and Asian β-Iactamase plasmids. Seventy percent of strainsshowed increased tetracycline resistance (�?2 µg/ml) while 42% of strainsexhibited high-level (16-128 µg/ml) resistance. The restriction of tetM-conjugativeand conjugative plasmids isolated in 1996 revealed different profiles to thosepreviously described showing that these plasmid types are continuing to evolve.Amplification of a fragment of the tetM gene provided a simple and quick methodfor predicting high-level tetracycline resistance. On restricting the 43 high-leveltetracycline-resistant strains (MICs �?16 µg/ml) all were found to contain theAmerican-type tetM gene and 25.2 MDa plasmids were demonstrated. Theestablishment of tetM-conjugative plasmids containing the American-type tetMgene is increasing, 2% in 1994 to 47% in 1997.Three hundred and thirteen sputum samples were collected from the Rocklandscommunity in Bloemfontein. Subsequent sputum samples were collected tomonitor 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. Thirtythree sputum samples were ZN positive, with LJ detecting an additional 7 M.tuberculosis isolates. Discrepancies in ZN and LJ results were confirmed byamplifying a 123 bp fragment of the IS6110. PCR also indicated the need foradditional diagnostic methods as 11% of isolates were not detected by ZN or LJ.The BACTEC system was used for confirmation as well as for susceptibilitytesting. Only 63% of persons receiving treatment returned after 1-3 monthsindicating possible non-compliance. A single patient (old case) had a maintainedZN positive result for 6 months with full susceptibility to all antibiotics tested. Thestandard method of fingerprinting involved Pvull restriction endonucleasedigestion of genomic DNA followed by Southern blotting and probing for IS6110elements. The fingerprinting of 50 INH -and/or RIF-resistant strains from 1997revealed 32 diverse profiles. Non-adherence and the emergence of resistantclonal groups were evident. Five clonally related clusters were evident that wereeither localised or had disseminated to different districts in the Free State. Of 26person's initial samples (ZN+/LJ+) investigated in 1998, 25 diverse fingerprintprofiles were found. Fingerprinting of 11 rifampicin-resistant strains (1998)showed the emergence of many diverse resistant strain types. The possiblespread of TB in a hospital ward was revealed through shared fingerprint profilesof two samples. The monitoring of rifampin resistance through sequencing of akey region 157 bp of the rpoB gene was performed. Previously reported mutationsites were evident in the study; 516, 526, 531 and 533. The two local 1997 clonalgroups (identified by fingerprint profile) did not share mutated rpoB alleles. Thiscould possibly be explained by clonally related susceptible strains independentlydeveloping sub-clones bearing distinct rpoB alleles. Inaccuracies in susceptibilitytesting were evident as a Bloemfontein strain reported to be rifampin-susceptiblepresented with a variant rpoB allele. From 13 MTB (new cases, 1998) screenedfor the rpoB gene and subsequently sequenced it was found that two ZN/LJpositive samples had missense mutation at positions 516 and 526. A reducedoutcome would result with these patients emphasising the need for accuratesusceptibility testing to be conducted earlier than presently stipulated. Elevenrifampin-resistant strains (1998) revealed only one strain without rpoB genemutations in the 157 bp region examined. The same mutated codon was evidentwith two strains (with shared fingerprint profile from same hospital ward) againstrongly implying dissemination of a strain type between patients. A familycommunity from a semi-rural area (Bainsvlei) situated 15 km from Bloemfonteinwas investigated. The Bainsvlei family member's samples from 1995, 1997 and1998 revealed the same fingerprint profile (shared by other family members in1995) and same mutated rpoB codon indicating the persistence of arifampin/isoniazid-resistant strain. Subsequent information on the brother's pastMTB infections and treatment showed that a possible reinfection of a multiplyresistantstrain could have occurred. The situation has not been fully resolveddue to lack of community involvement and funding.Genetic techniques investigating infectious diseases in the community settingcertainly provides required rapid results and epidemiological informationessential for the future success of infection control programmes.

[发布机构] University of the Free State