|Home | About | Journals | Submit | Contact Us | Français|
The multiple-locus variable-number repeat analysis of 90 human Brucella melitensis isolates from a large urban area in central Peru revealed variations at 4 (Bruce07, Bruce09, Bruce18, and Bruce42) out of 16 loci investigated, of which 1 (Bruce42) also is used for species identification. Ten genotypes were identified, separated by the number of Bruce42 repeats into two groups that may have distinct phenotypic characteristics. Whereas genotypes with five or six Bruce42 repeats were cultured mainly from adult patients, genotypes with three Bruce42 repeats were isolated from children and young adolescents as well as from adults. In addition, the isolates with three Bruce42 repeats were obtained more often from patients with splenomegaly (P = 0.02) or hepatomegaly (P = 0.006). An annual variation in the diversity of genotypes was observed, possibly reflecting changes in sources of fresh dairy products, supply routes to city shops and markets, and the movement of infected dairy goat herds.
Brucellosis is endemic in Peru and continues to be transmitted to humans. The number of reported cases of human brucellosis has decreased in recent years, presumably due to ongoing vaccination campaigns of goats and the promotion of the pasteurization of dairy products (33, 34). Between 10 and 25% of the human cases are reported from Callao, the harbor city of the capital Lima (22). Human brucellosis is a nonspecific but serious febrile illness without distinctive signs or characteristic symptoms that can affect multiple organ systems and cause various debilitating complications (19, 40). Treatment consists of a combination of antibiotics (17), most commonly doxycycline in combination with an aminoglycoside, rifampin (rifampicin), or both.
Brucellosis is caused by infection with bacteria of the genus Brucella. Several species are pathogenic to humans, most notably Brucella melitensis, B. abortus, and B. suis (32). In Peru, human brucellosis is caused almost exclusively by infection with B. melitensis, a species that principally infects goats and sheep. Based on their agglutinating properties with specific antisera, B. melitensis can be differentiated into three biovars, biotypes 1, 2, and 3, of which biotype 1 is known to be present in Peru (12, 14, 26). Recently, a highly discriminatory method for the genotyping of Brucella known as multiple-locus variable-number repeat analysis (MLVA) has become available (25). This method makes use of various loci on the Brucella genome that are composed of repeats of short nucleotide sequences. These tandem-repeat units tend to occur in various numbers, and various alleles can be observed in different species and isolates. The recently published MLVA-16 assay, developed for the genotyping of Brucella, makes use of eight minisatellite loci for species identification, supplemented with a selection of eight more polymorphic microsatellite loci for the further characterization and differentiation of isolates (2, 25). Whereas the MLVA-16 assay can be used for the biovar classification of B. abortus and B. suis, no correlation between biovar and genotype has been observed for B. melitensis (2, 25).
The MLVA-16 typing of animal and human Brucella isolates has revealed that clusters of individual genotypes within a species may show a distinct geographic distribution. For instance, human isolates of B. melitensis from Europe and North Africa can be divided according to their geographic origin into a west and an east Mediterranean cluster. Within the west Mediterranean cluster (which includes isolates from France, Switzerland, Tunisia, and Algeria), a clearly separate cluster originating from Italy can be identified (2). Genotypes are relatively stable, and isolates with identical MLVA patterns have been obtained from the same geographic area during a time span of almost three decades (2). A considerable number of distinct B. melitensis genotypes already have been identified (2). MLVA typing additionally has some practical clinical applications, such as tracing sources of infections and discriminating relapse from reinfection (2, 28, 37).
Recently, we showed that B. melitensis biotype 1 from Peru is clearly distinct from the east and west Mediterranean groups, forming a Latin American cluster that is most closely related to two previously genotyped isolates from Mexico (37). Here, we investigate the genotypes of a series of consecutive human Brucella isolates cultured from patients hospitalized at a major hospital of Callao.
Ethical approval was obtained from the ethical committees of the U.S. Naval Medical Research Center and from the Hospital Nacional Daniel Alcides Carrión. Written informed consent was obtained from all patients, their parents, or their guardians.
All patients hospitalized between November 2003 and April 2007 at the Hospital Nacional Daniel Alcides Carrión in Callao with a Brucella-positive blood culture were entered in the study. All patients were Rose Bengal positive with reactive serum agglutination tests (median titer, 1:400; range, 1:50 to 1:12,800). During this 3.5-year period, 91 isolates were cultured from 89 patients. Two patients experienced second disease episodes, and two isolates each were obtained from these patients. Patients were classified according to the duration of illness as acute (<6 months of illness), subacute (6 to 12 months of illness), and chronic (>12 months of illness).
Genotyping was performed using MLVA-16 panel 1 for species identification (Bruce06, Bruce08, Bruce11, Bruce12, Bruce42, Bruce43, Bruce45, and Bruce55) and MLVA-16 panels 2A (Bruce18, Bruce19, and Bruce21) and 2B (Bruce04, Bruce07, Bruce09, Bruce16, and Bruce30) for further subspecies differentiation (25). Primer pairs and PCR conditions were as described by Le Flèche and coworkers (25). PCR products were separated by electrophoresis on 1.5 to 3% agarose gels. For each run, DNA from the B. melitensis 16M reference strain was included. In anticipation of the expected tandem-repeat unit length, a 100- or 25-bp molecular marker ladder (hyper ladder no. IV and V; Bioline, Berlin, Germany) was used. Ethidium bromide-stained gels were visualized by UV light and graphically evaluated (GeneGenius; Syngene, Cambridge, United Kingdom). Genotyping was performed for all but one isolate. The MLVA-16 patterns from the Peruvian isolates were compared to the public database Brucella2007 (http://mlva.u-psud.fr) to identify the most closely related isolates. The cluster analysis was performed by the unweighted-pair group method with an arithmetic mean (UPGMA) algorithm, and a rooted tree was generated. In this study, we define a genotype as an isolate with a distinct MLVA-16 pattern. The distance between two genotypes is defined as the minimum number of changes in the number of repeats of any locus that converts one genotype to the other.
Chi-square analysis was used to correlate patient characteristics with genotype.
The mean age of the patients was 32 years (range, 1 to 92), and the ratio of males (n = 35) to females (n = 54) was 0.65 (Table (Table1).1). Male patients (mean age, 34.3 years; range, 1 to 92) were slightly older than female patients (mean age, 26.7 years; range, 5 to 81). Most (74.7%) patients presented with acute disease. All patients presented with one or more significant signs and/or symptoms, of which fever, malaise, sweats, headache, hyporexia, shaking chills, and arthralgia each were observed in more than 50% of the patients. None of the patients presented with focal disease or complications requiring intervention other than antibiotic treatment. Most (78.7%) patients came from Callao, with other patients coming from different districts of greater Lima. Almost all (92.1%) patients reported the consumption of unpasteurized dairy products. As expected for patients from an urban area, contact with livestock was rare.
A total of 90 isolates could be typed by MLVA-16, and patterns obtained for all 90 isolates were consistent with B. melitensis. The results revealed that the panel 1 locus Bruce42, the panel 2A locus Bruce18, and the panel 2B loci Bruce07 and Bruce09 were polymorphic (Table (Table2).2). Isolates with three, five, and six repeats were detected for panel 1 locus Bruce42, isolates with seven and eight repeats for panel 2A locus Bruce18, isolates with five and six repeats for panel 2B locus Bruce07, and isolates with four, five, six, seven, and eight repeats for panel 2B locus Bruce09. All isolates were monomorphic for the panel 1 loci Bruce06 (3 repeats), Bruce08 (4 repeats), Bruce11 (2 repeats), Bruce12 (13 repeats), Bruce43 (2 repeats), Bruce45 (3 repeats), and Bruce55 (3 repeats), as well as for the panel 2 loci Bruce04 (2 repeats), Bruce16 (4 repeats), Bruce19 (18 repeats), Bruce21 (6 repeats), and Bruce30 (4 repeats). In total, three genotypes with a single isolate each and seven genotypes comprising 2 to 50 isolates each could be distinguished. Four genotypes together constituted 90% of the isolates: genotype G1 with 19 isolates, G2 with 50 isolates, and genotypes G3 and G8 with six isolates each. Genotypes G1, G4, and G8 were identical to 3 out of 15 previously described human B. melitensis genotypes isolated in 2000 and 2001 from Peruvians also residing in Callao and Lima (36). All other genotypes found in this study differed from the 15 previously characterized Peruvian B. melitensis genotypes, which were polymorphic for Bruce07, Bruce09, Bruce16, and Bruce42 (Table (Table2).2). The MLVA-16 results are illustrated in Fig. Fig.1,1, showing the length of the PCR products for the four polymorphic loci Bruce07, Bruce09, Bruce18, and Bruce42 for each of the 10 genotypes of Brucella isolates obtained during the period 2003 to 2007.
Of all published non-Peruvian isolates characterized by MLVA-16 genotyping, the Brucella isolate key bru128 showed the closest relationship, with a maximum distance of 2 to 4 from the 10 Peruvian genotypes isolated in the period of 2003 to 2007 (37). According to database information, bru128 is a B. melitensis biovar 1 isolate from Mexico. Furthermore, genotype G5 closely matched with the Brucella isolate key bru651 (distance of 4), a B. melitensis biovar 1 isolate from Spain. The relationship of these 10 Peruvian genotypes with bru128 is depicted in the dendrogram presented in Fig. Fig.2A2A.
According to the dendrogram, the 10 genotypes separate into two branches, with branch 1 including genotypes G1 (type strain 1), G4 (type strain 8), G7 (type strain 35), G8 (type strain 59), G9 (type strain 90), and G10 (type strain 87), and branch 2 including genotypes G2 (type strain 2 strains), G3 (type strain 7), G5 (type strain 20), and G6 (type strain 28). Isolates with a genotype grouped in branch 2 were isolated throughout the study period, whereas isolates comprising branch 1 were isolated mainly in the period from 2005 to 2007 (Table (Table3).3). No differences were observed between the spectrum of genotypes isolated from males and females, and the ratio of branch 1 and branch 2 genotypes was similar for the two sexes (Table (Table4).4). Consistently with the larger number of patients from Callao, the diversity of genotypes derived from patients from this city was higher than those from patients from Lima (Table (Table5).5). Interestingly, a clear difference was observed between the spectrum of genotypes isolated from children and young adolescents (age group, ≤20 years) and those isolated from adults (Table (Table6).6). Very few branch 1 genotypes were obtained for children and adolescents compared to findings for adults. Twenty-six branch 1 and 34 branch 2 genotypes were isolated from adult patients (age, ≥20 years), whereas 4 branch 1 and 26 branch 2 genotypes were isolated from children and adolescents. The difference was significant (relative risk, 3.25; 95% confidence interval, 1.25 to 8.46; P = 0.005).
Branch 1 genotypes differ from branch 2 genotypes in the number of Bruce42 repeats. Branch 1 genotypes are characterized by the presence of five or six (one isolate) Bruce42 repeats, whereas branch 2 genotypes have three Bruce42 repeats (Table (Table2).2). The pattern of panel 1 loci repeats for the genotype with five Bruce42 repeats (e.g., 3 Bruce06, 4 Bruce08, 2 Bruce11, 13 Bruce12, 5 Bruce42, 2 Bruce43, 3 Bruce45, and 3 Bruce55 repeats) was determined previously for a B. melitensis biovar 1 (BCNN 96 137b; bru146) isolate cultured from a patient from Argentina (2). B. melitensis genotypes with three or six Bruce42 repeats and with a pattern of panel 1 locus alleles similar to that of the Peruvian isolates have not been described for isolates from other countries.
We did not observe a relationship between the consumption of specific dairy products and genotype (data not shown). Also, we did not find a relationship between genotype and specific symptoms and signs, except that a branch 2 genotype was isolated from 92.9% of the patients presenting with splenomegaly, whereas the branch 2 genotype was isolated from 62.0% of the patients with no evidence of splenomegaly (P = 0.02). Additionally, a branch 2 genotype was isolated from 85.0% of the patients with hepatomegaly and 47.2% of the patients without evidence of hepatomegaly (P = 0.006). The response to treatment was not recorded in the patient database, hence we could not determine a relationship between genotype and recovery after treatment.
The genotypes of isolates obtained from one patient during the subacute phase and after a relapse were identical (genotype G2). In another case, two genotype G2 isolates were cultured from a single patient's blood during the acute and subacute phases of a single illness episode. In a third case, genotype G3 was cultured from the blood of two patients living at the same address and hospitalized on the same day. These two patients recalled that they had consumed fresh cheese and eaten papa a la Huancaína, a popular local dish prepared with potatoes and fresh cheese. No evidence for a common source of infection was obtained for any of the other patients.
A dendrogram constructed for all 22 B. melitensis genotypes currently identified in Peru is presented in Fig. Fig.2B.2B. This dendrogram shows a more complex structure, which is related to the relatively high degree of variation in the MLVA-16 panel 2B loci Bruce07, Bruce09, and Bruce16 observed in these genotypes.
Consistently with our previous molecular studies describing the genotypes of 24 human Brucella isolates from Peru isolated in 2000 and 2001 (37), all 90 isolates obtained between 2003 and 2007 were identified by MLVA-16 typing as B. melitensis and showed the closest homology to a B. melitensis biovar 1 isolate (bru128) from Mexico. Essentially all previously described Brucella isolates from Peru were determined to be B. melitensis biovar 1 (12, 14, 26). Classical biovar typing, however, was not performed for the current isolates, and MLVA-16 typing does not allow biovar classification (2, 25). The homology of the Peruvian isolates with the B. melitensis biovar 1 isolate from Mexico further supports our notion that the majority of the B. melitensis strains circulating in Peru form a Latin American cluster distinct from the west and east Mediterranean clusters (37). The similarity of two Peruvian isolates to an isolate originating from Spain may indicate that either the source of infection of the latter case came from Peru or that Brucella recently has been reimported into Peru. This could have occurred with the import of replacement animals for herds sacrificed in the course of the brucellosis control program. If so, this illustrates a risk of global trade and the transboundary transport of livestock. Replacement animals recently imported into Peru include Anglo-Nubians, a breed developed in the United Kingdom, Alpine and Saanen breeds of Swiss origin and imported from Chile, and Murcina and Granadina breeds from Spain (11 and M. Vargas, personal communication). Also, semen from the Murcina and Granadina breeds has been imported during the last decade (O. Arroyo, personal communication). Historically, goats and other livestock animals were introduced into Latin America as early as 1493 with the second journey of Columbus to the Antilles (35). These goats were of Spanish origin and have developed over time into the Creole goats that are kept by most farmers. Genetic analysis has indicated the phylogenetic relationship of Creole goats from Peru with certain goat breeds from Spain and with Creole goats from other Latin American countries (15). Brucella may have evolved with these Creole goats, thereby separating from the present day west and east Mediterranean clusters of B. melitensis genotypes and forming a distinct cluster of Latin American B. melitensis genotypes (37). However, brucellosis is common in camels and theoretically already may have been present in indigenous Peruvian camelids (including the llama, vicuña, and alpaca) at the time of the Spanish conquest (1, 39). Marine mammals may be infected with B. pinnipedialis, but this species is clearly distinct from B. melitensis (25). Marine mammals are abundantly present in the Pacific Ocean along the Peruvian coast, and the infection of Peruvians with B. pinnipedialis has been reported, but the transmission of this pathogen to humans is very rare (21, 38). Most Brucella patients in Peru belong to the lower socioeconomic classes, and infection during international travel or through the consumption of imported food is highly unlikely.
Using the same typing method as the one we have used, Kattar and coworkers identified a series of unique B. melitensis isolates in patients from Lebanon that were closely related to isolates from neighboring countries (23). The genotypic variation of these Lebanese isolates was limited, with most of the variation occurring in panel 2B loci, and it was postulated that these isolates had evolved from indigenous strains with a common ancestor. The genotypic variation in the Peruvian isolates is similarly restricted, with variation occurring in one panel 1 locus (Bruce42), one panel 2A locus (Bruce18), and three panel 2B loci (Bruce07, Bruce09, and Bruce16), indicating that these Latin American isolates have evolved from a small number of ancestors. Genotypic variation revealed by MLVA-16 typing also was observed for Rev-1 isolates, providing an example of the evolution of Brucella (18).
Currently, a total of 22 genotypes have been identified among 114 human Peruvian B. melitensis isolates. Most likely, changes in the repeat number of the more heterogenous panel 2B loci in particular occurred on different occasions. Thus, identical or similar panel 2B loci patterns may have developed independently in strains with dissimilar panel 1 and/or panel 2A locus patterns. Therefore, the genetic relationship between the 22 genotypes as shown in the dendrograms does not reveal their phylogenetic relationship. In our hypothesis, the different genotypes have developed by microevolution from a small number of ancestors characterized by differences in the panel 1 Bruce42 locus. Further, it is possible that the genotype (G5) that is similar to that of the isolate from Spain has evolved separately in Peru.
Brucella species strains showing a specific MLVA pattern may circulate in a geographic area and may be transmitted to the human population over many decades (2). Our results show that the spectrum of strains isolated from patients from central Peru changes over time. Of the 15 B. melitensis genotypes isolated in 2000 and 2001, only 3 were identified among the 10 genotypes isolated between 2003 and 2007 (37). Also, the percentage of isolates with five or six Bruce42 repeats (branch 1) and with three Bruce42 repeats (branch 2) has varied considerably over the years. The majority (83.3%) of the isolates obtained in 2000 and 2001 had five Bruce42 repeats and just 16.7% had three Bruce42 repeats, but almost all (92.9%) isolates obtained in 2003 and 2004 had three Bruce42 repeats and 7.1% had five Bruce42 repeats. A further change in the prevalence of these two groups was observed for the period of 2005 to 2007, by which time 45.2% of the isolates had five or six Bruce42 repeats, and 54.8% had three Bruce42 repeats.
Although the 114 isolates presented here and in our previous study (37) were obtained from blood cultures from patients hospitalized at three different hospitals, these three hospitals serve patients from the same districts of Callao and Lima, with most patients coming from specific districts within Callao. Most likely, this variation over time in the spectrum of genotypes is the result of movements of migratory herds and perhaps changes in sources and supply routes of dairy products. The major route of infection with Brucella in cities in Peru is through the ingestion of contaminated dairy products, the origin of which is unclear. Contaminated dairy products may be obtained from remote farms, from migratory goat herds in the Andean highlands that have not yet been reached by the vaccination programs, or from farms with goat herds that have been partially vaccinated but still include infected animals. Thus, herds from different areas may be infected with different genotypes. Shops also may obtain their products from multiple vendors and areas depending on available supplies. There is no evidence to suggest that shops and markets in Callao obtain their dairy products from areas clearly distinct from those in Lima; Lima and Callao form one large metropolis. The migration of herds makes the epidemiology and control of brucellosis more difficult. The identification of the supply routes may help to trace the sources of infection, and these sources then may be confirmed by the genotyping of isolates obtained from milk samples or other animal materials. Changes in the supply and marketing of dairy products originating from goat herds infected with different Brucella genotypes may cause a change in the predominant genotype isolated from patients. Alternatively, an outbreak of brucellosis in certain herds may have caused a predominance of human infections with a certain genotype during a particular period. Finally, the ongoing vaccination of goats may influence the transmission of Brucella genotypes to the human population as well.
More than half of the Brucella patients in Peru are females. We speculated earlier that Peruvian women, who do most of the grocery shopping, have an increased risk of infection due to sampling different products while shopping (29). Women also may become exposed to the pathogen while preparing meals. This is typical for urban exposure. Studies in rural, pastoral communities in the Balkans (4, 6) and rural Spain (36) show a much higher rate of disease, or at least seroprevalence, in men in herding communities.
One of the major challenges in brucellosis research is to delineate differences in pathogenicity and host susceptibility. A growing number of studies have shown a relationship between host susceptibility and the gene polymorphism of various human genes, such as the transforming growth factor β1 gene, major histocompatibility complex class I genes, and certain interleukin genes (7, 8, 10). Here, we provide evidence that susceptibility to different B. melitensis biovar 1 genotypes is age related and that specific Brucella genotypes are more frequently isolated from Brucella patients presenting with specific clinical manifestations, such as splenomegaly and hepatomegaly. The genotypes G1, G4, G7, G8, G9, and G10, comprising branch 1 of the dendrogram drawn for the Peruvian isolates, were isolated almost exclusively from adult patients, and a relatively high proportion of the patients infected with the four genotypes G2, G3, G5, and G6 comprising branch 2 presented with splenomegaly and/or hepatomegaly. Studies of mice may help to elucidate differences in organ tropism and pathological effects (30).
The 10 genotypes comprising branch 1 and 2 genotypes showed variation at 4 (Bruce07, Bruce09, Bruce18, and Bruce42) of the 16 tandem-repeat loci investigated, of which variation in one (Bruce42) locus separated branch 1 from branch 2. Bruce42 is used for species identification in combination with other MLVA-16 panel 1 loci and is considered relatively invariable. The number of Bruce42 alleles for a panel of 21 reference strains, including B. melitensis biotypes 1 to 3, B. abortus biotypes 1 to 6 and 9, B. suis biotypes 1 to 5, B. ovis, B. canis, B. neotomae, B. pinnipedialis, and B. ceti was five with either one, two, three, four, or six repeats (25). Three Bruce42 repeats were found for B. pinnipedialis, B. ceti, and B. suis biotypes 3 and 4, and six Bruce24 repeats were found for B. suis biotype 2. Branch 1 of the Peruvian isolates is characterized by the presence of five or six Bruce42 repeats and branch 2 isolates by three Bruce42 repeats. A B. melitensis isolate with three Bruce42 repeats was isolated from 26 (86.7%) of the 30 children and adolescent patients and from 34 (56.7%) of the 60 adult patients. From the other patients, an isolate with five or six Bruce42 repeats was obtained. In our previous study, all patients were 19 years or older (mean, 30 years of age), and 20 of the 24 isolates had five Bruce42 repeats (37). The other four (16.7%) isolates had three Bruce42 repeats. These results suggest that the number of Bruce42 repeats mark a phenotypical characteristic that relates to age-dependent host susceptibility. Differences in the mode of exposure need to be considered. Occupational exposure is rare in patients residing in Lima or Callao, and food-borne transmission is the most common mechanism for both adults and children. This seems to speak against the route of infection being the principle discriminating factor. The possibility that adult patients had acquired brucellosis from a common point source (for instance, from a meal served during a wedding ceremony) is considered highly unlikely. In Peru, brucellosis is a reportable disease, and each case requires detailed epidemiological investigations. We have not found any such demographic relationship between patients, excepting only two patients living at the same address. Furthermore, most patients in this study came from Callao, a large urban area with almost 800,000 inhabitants, or from Lima, a city with almost 8.5 million inhabitants. A more extensive analysis of the genomes of branch 1 and 2 genotypes may help to identify the bacterial factors that determine differences in host susceptibility. Several bacterial factors are involved in the virulence and pathogenicity of Brucella, including the lipopolysaccharide, the type IV secretion system, and the BvrR/BvrS two-component signaling system (24, 31). However, genetic differences that result in a modification of pathogenicity have not yet been identified. In B. melitensis, Bruce42 is located within the 5′ region of the tRNA modification GTPase gene, which stretches from nucleotide 5526 to nucleotide 6854 of chromosome I of B. melitensis strain 16M (25). The outer membrane proteins thought to be involved in virulence are encoded by genes of this chromosome (13) and are among others involved in cell adhesion and cell entry. It is possible that branch 1 and 2 isolates differ in a mutation in one of the outer membrane proteins, causing a critical change in a functional activity. Most, but not all, of the other virulence-associated genes also are located on chromosome I; the genes encoding the type IV secretion system are located on chromosome II (13).
As noted before, the clinical signs and symptoms of brucellosis include such nonspecific features such as fever, malaise, headache, and sweats (19). A B. melitensis genotype belonging to branch 2 was isolated from most of the brucellosis patients presenting with hepatomegaly and/or splenomegaly. As branch 2 isolates were obtained from 66.7% of all Brucella patients, further studies will be needed to demonstrate a correlation of disease severity or of specific disease presentations with the genotype of the infecting strain. (It should be noted that hepatosplenomegaly was assessed clinically, and radiographic confirmation with sonography or computed tomography was not obtained.)
With regard to data analysis, we consider it unlikely that an age-related difference in the diagnosis led to a selection bias, as the percentage of adults and children in whom splenomegaly or hepatomegaly was observed was similar. Nevertheless, we are aware that there is intrinsic bias in the study due to the nature of the data at hand, which was not obtained through a structured or planned survey, and of the consequences that could result from this for the interpretation of the findings. Therefore, the confirmation of our findings should come from future investigations into the genotypic diversity of human B. melitensis isolates between patient groups. Because Brucella is a fastidious organism that is more easily cultured from the blood of acute or subacute patients than from patients with more persistent disease (15), this led to the inclusion of patients with mostly early-stage disease. However, this is unlikely to have resulted in the selection of a specific group of genotypes, unless one assumes that a separate group of genotypes causes predominantly persistent disease and that some genotypes are more easily isolated by culture than others.
The recurrence of brucellosis is a major problem in Peru and worldwide and may be due to either relapse or reinfection (27). In relapsing patients, the same genotype can be expected to be isolated during both the first episode and relapse (3). Conversely, the isolation of a different genotype from a patient with a repeat episode of brucellosis would be consistent with reinfection. The limited variation among genotypic characteristics in a given patient population, however, makes it more difficult to discriminate between relapse and reinfection in a patient experiencing two or more disease episodes. Further studies that investigate the inclusion of other more highly variable tandem-repeat loci or the use of other potentially more discriminatory genotyping methods, such as single-nucleotide polymorphism analysis, could help to discriminate in such cases between these two options (9, 16, 20). By using MLVA genotyping for the same panel of loci, we previously demonstrated probable reinfection in three out of three brucellosis patients from Peru with recurrent disease (37). The diversity of Brucella genotypes isolated from that particular group of patients hospitalized in 2000 and 2001 was larger than that among the isolates obtained from the patients hospitalized in the period of 2003 to 2007. Finally, the isolation of the less frequently observed B. melitensis genotype G3 from two patients living at the same address and hospitalized on the same day supports a common source of infection in these two patients.
The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, or the U.S. government.
E.H., B.E., and R.M. are U.S. military service members working at the U.S. Naval Medical Research Center Detachment in Lima, Peru. This work was prepared as part of their official duties. Title 17 U.S.C. § 105 provides that copyright protection under this title is not available for any work of the U.S. Government. Title 17 U.S.C. § 101 defines U.S. Government work as work prepared by a military service member or employee of the U.S. government as part of that person's official duties.
Published ahead of print on 5 August 2009.