The two MLVA schemes evaluated in our study were selected based on the availability of published MLVA schemes at the time of the study design, their abilities to type human B. melitensis isolates, and the availability of the resources, technology, and technical expertise required to perform the techniques. As mentioned above, the gel-based MLVA assay was carried out in Cairo, Egypt, and data from the automated MLVA system were collected at the CDC in Atlanta, GA. Thus far, there has been no study demonstrating the typing abilities of different MLVA systems for a common collection of Brucella isolates. The objective of our study was to compare the resolving powers of two different MLVA techniques on a common collection of 101 clinical B. melitensis isolates from the Middle East and to provide an assessment of the limitations, advantages, and field applications of the two MLVA assays for genotyping B. melitensis isolates.
The MLVA techniques compared in this study were developed for different purposes, which are reflected in the unique combination of tandem-repeat markers that makes up each scheme. Originally intended for forensic purposes, the MLVA-15NAU assay contains 12 markers that have small repeat units less than or equal to 8 bp; 8 of these markers have diversity indexes (DIs) greater than 0.75 in our study. The MLVA-15IGM assay, developed for epidemiological typing, contains two panels: a panel of eight conserved markers with larger repeat units (>8 bp) (panel 1), exhibiting DIs less than 0.40 in the strains we examined, and a panel of seven more-diverse loci (panel 2) with small repeat units (<8 bp).
Overall, the VNTR markers in the MLVA-15NAU
assay presented a broader number of alleles and higher DIs than the markers examined by the MLVA-15IGM
scheme. Six of the 15 VNTR loci in the MLVA-15NAU
scheme exhibited DIs of >0.80 for the 101 B. melitensis
isolates examined, with allele numbers ranging from 9 to 20 (Table ). The Bruce VNTR 21 and Bruce VNTR 29 markers display very low genetic DIs of 0.06 and 0.02, respectively, while the Bruce VNTR 7, Bruce VNTR 14, and Bruce VNTR 25 loci were monomorphic in the strains we examined. The invariance we observed at these loci is most likely due to the localized geographic origins of our isolates and represents a level of strain-to-strain conservation within a geographic area that can be better evaluated in a larger population of isolates from similar locations. The monomorphic nature of the Bruce VNTR 7 and Bruce VNTR 25 loci in the Middle Eastern strains could represent geographically specific alleles, since these markers have been reported to exhibit higher DIs in other B. melitensis
strains, from the United States (n
= 85) (16
). The Bruce VNTR 14 locus is consistently monomorphic in our strains and in other B. melitensis
strains examined by Huynh et al. (16
) and could be examined as a species-specific marker. Bruce VNTR 02, 20, 28, 31, and 33 were initially difficult to amplify in certain isolates. However, all VNTR loci were successfully amplified in all isolates except for Bruce VNTR 33 in isolate E5 and Bruce VNTR 02 in isolate E53.
Only one of the 15 VNTR markers from the MLVA-15IGM assay had a DI of >0.80 in the 101 strains we examined. The Bruce 04, 07, 09, 16, and 18 VNTR markers exhibit the highest DIs, ranging from 0.64 to 0.82, among the 15 markers (Table ). The remaining 10 tandem-repeat markers ranged in diversity from 0.18 to 0.37. The Bruce 11 VNTR marker is the only monomorphic marker in the Middle Eastern strains we examined. Two of the 101 B. melitensis isolates (E21 and E26) exhibited null alleles at the Bruce 09 locus; however, all other markers were successfully amplified in all isolates.
The two MLVA schemes evaluated in this study were able to correlate similar genetic relationships among the 101 B. melitensis strains we examined. The MLVA-15NAU scheme, exhibiting greater DIs, resolved 101 unique genotypes, while the MLVA-15IGM assay discriminated 99 genotypes. Dendrograms generated by UPGMA clustering analysis (Fig. and ) show similar groupings of B. melitensis strains by the MLVA methods, such as the clustering of Qatar isolates Q1, Q3, and Q5 with each other and the genetic similarity of Q6, Q7, and Q13 strains to the Egyptian genotypes. Also, the Libyan strain consistently clusters with the Egyptian genotypes by both MLVA methods. Because none of the strains we examined were epidemiologically linked, since they were sporadic cases from various regions of Egypt and Qatar, we did not expect to see a great deal of homogeneity by either MLVA method. However, by the MLVA-15IGM scheme, two strains, E41 and E42, which were isolated in the same hospital in Sohag, Egypt, 6 weeks apart from each other, had identical genotypes. Two other strains, E61 and E62, isolated 2 years apart from each other from another hospital, in Assiut, Egypt, also had identical genotypes. The same strains (E41 and E42; E61 and 62) did not have identical genotypes by the MLVANAU assay but diverged by a mutation at one or both of the hypervariable markers VNTR 01 and VNTR 33. By both MLVA schemes, it is possible to see some strains originating from the same hospital location or the same region (see Table for regions) within Egypt that have very similar but not identical genotypes, which is expected for nonoutbreak strains.
The MLVA schemes evaluated in this study have different logistical and technical requirements. The gel-based MLVA method requires more time for typing from start to finish, requires less technical expertise, and involves less-expensive materials, although it does require BioNumerics software, which is quite costly and requires technical training. The use of multiple individual PCRs and agarose gels for typing one strain is cumbersome and requires considerable subjectivity in the stages of analysis of the raw data. The automated system is much faster and has a high throughput capacity; however, the instrumentation and proprietary reagents make this method more expensive. The use of an automated sequencer and the genetic analysis software require a skilled technician; however, the data output is more reliable, and once properly optimized, the method is very reproducible.
Both MLVA schemes examined in this study can be used to epidemiologically type human B. melitensis
strains and can be used for high- and low-resolution typing by the inclusion and exclusion of highly variable markers. We evaluated the two MLVA schemes and compared their abilities to resolve relationships between geographically localized strains from the Middle East, where B. melitensis
is the primary etiologic agent of human brucellosis. Other studies have looked at globally diverse and localized Brucella
strains using the MLVAIGM
). However, it would be interesting to demonstrate the capability of the MLVANAU
method for differentiating global genotypes in Brucella
species. Although we currently utilize the MLVA-15NAU
assay for genotyping Brucella
isolates in our reference lab at the CDC and prefer the automated platform for its reproducibility, the MLVA-15IGM
scheme is used widely in the European community and collaborating laboratories. The comparable geographic and in-country resolution of the two MLVA systems, targeting different VNTR regions, validates the utility of a combination analysis system, such as MLVA, for genotyping genetically monomorphic bacteria. With the increasing use of multiple MLVA systems for typing Brucella
species and the increasing need to share data, it would be effective to investigate the discriminatory value of all the currently utilized VNTR markers in these combination systems by using statistical methods such as principal-component analysis in order to establish one streamlined MLVA system that serves the global need for an epidemiological genotyping tool to improve brucellosis surveillance.