The application of genetic techniques in community health surveillance
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Date
1999-12
Authors
Stanley, Kim
Journal Title
Journal ISSN
Volume Title
Publisher
University of the Free State
Abstract
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.
Description
Keywords
Diagnostic microbiology, Communicable diseases -- Genetics, Dissertation (M.Sc. (Medical Sciences))--University of the Free State, 1999