What is Brucellosis? Symptoms, diagnosis, treatment
BRUCELOSIS: CURRENT ASPECTS OF MAIN INTEREST
Javier Ariza Cardinal
Service Infectious Diseases, CSU de Bellvitge
EPIDEMIOLOGY
Brucellosis has been well known in recent decades as a zoonosis of worldwide distribution. The disease has been eradicated in many countries of the developed world, but the infection by Brucella melitensis is still endemic in the Mediterranean area, the Middle East, the Arabian Peninsula, the Indian subcontinent and Latin America.
In Spain, the great majority of isolates from patients with brucellosis have corresponded to B. melitensis. The human disease has been associated with direct contact with livestock, typical of some professions such as shepherds, cattle ranchers, slaughterers, etc., but also in large part with the consumption of uncontrolled dairy foods, predominant in rural areas, but also existing in the urban and its surroundings. The rates corresponding to the compulsory declaration registration have shown a progressive decrease in the last 15 years, coinciding with intense vaccination and intervention campaigns on animal brucellosis in many Spanish regions.
While the maximum rate was reached in 1984, with 22.33 cases per 100,000 inhabitants, the last records were 3.9 in the years 1998 and 1999, and 2.8 cases per 100,000 inhabitants in
2000.
ETIOPATOGENIA
The genus Brucella includes a homogeneous group of intracellular facultative microorganisms belonging to the α-2 subdivision of the Proteobacteria. Due to the similarity of the DNA of the different varieties, it has been proposed to group them in a single species, B. melitensis. However, for practical and epidemiological reasons the distinction of the 6 main types is maintained: B. melitensis, Brucella abortus, Brucella suis, Brucella canis, Brucella. ovis and Brucella neotomae.
The microorganism lacks plasmids as an expression of its adaptation to a stable intracellular medium, free of bacterial competition. Its particular intracellular parisitism is related to some structural differences of its outer membrane in comparison with other gram-negative bacteria. In its intracellular location, it is resistant to the action of polycations and to the oxygen-dependent killing systems of phagocytes. It uses the autophagosome pathway to evade phagolysosome fusion and replicate inside the cells of the phagocytic mononuclear system. This capacity for intracellular survival determines the clinical pattern characteristic of brucellosis, the undulating course of the disease, its tendency to relapse and evolve into chronic forms. Cellular immunity is the main defense mechanism, through macrophage activation and its capacity to eradicate intracellular bacteria by the action of some cytokines (interferon-γ, tumor necrosis factor α and interleukin-12) produced by sensitized lymphocytes. Coinciding with the development of cellular immunity also appears a delayed hypersensitivity that seems to be important in the pathogenesis of the disease. However, the role of humoral immunity in defense mechanisms is undoubtedly significant. CLINICAL MANIFESTATIONS
The clinical behavior of brucellosis is well known and has been the object of multiple descriptions. The infection can affect any system or tissue of the organism.
The disease due to B. melitensis is the most aggressive and that caused by B. suis has the greatest capacity to produce abscess. The use of antibiotic therapy and the greater health development can modify the clinical presentation referred to in the classic descriptions and the symptomatology may differ depending on the social environment in which it is developed. The involvement of the organs of the mononuclear phagocytic system, hepatosplenomegaly and lymphadenopathy and of the ostearticular is especially characteristic. The development of focalities of the disease occurs in more than 30% of cases, a frequency that is fundamentally related to the time of evolution of the disease before starting antibiotic therapy. The observation of a patient with spondylitis, sacroiliitis, polyarthritis or tenosynovitis, orchiepididymitis and lymphocytic meningitis should raise the differential diagnosis of brucellosis in our environment. The observation of abscessed focal forms, such as reactivation of an old brucellosis, considered very rare in our days, could be proportionally more frequent in the coming years if the cases of brucellosis of recent acquisition continue to decrease and can pose diagnostic problems to unfamiliar doctors. with the disease.
DIAGNOSIS
Bacteriological
diagnosis The definitive diagnosis of the disease requires the isolation of Brucella, which is usually done in blood cultures.Castañeda's two-phase medium has been very effective and the
standard system recognized for many years. In general terms, 75-80% of infections due to B. melitensis and about 50% of
those produced by B. abortus occur with a positive blood culture .
The new systems of automated blood cultures, of the Bactec 9200 type or similar, have proved to be extraordinarily effective. Its detection capacity is probably greater than the classic Castañeda method and the other described systems, but the most significant thing is the precocity of this detection, which usually occurs between 3 and 5 days. Using this methodology, the microorganism is recovered during the first week of incubation in more than 95% of the cases in which Brucella can be isolated, which guarantees its isolation even in cases without clinical suspicion of the disease. The routine blind subculture at 7 days is not necessary and should be reserved for those cases with a high suspicion of brucellosis, especially if there has been previous antibiotherapy.
The isolation of Brucella in other samples is less frequent. The microorganism grows well in the usual media, and in a few days, when joint fluid is cultured, pus from abscesses, cerebrospinal fluid (CSF) or other tissue samples, although the frequency of recovery in these cases is usually around 30%. Also in this type of samples, the Bactec 9200 system increases the profitability of the crop significantly compared to conventional methods.
In recent years, the group of Colmenero et al. has observed very satisfactory results with the use of a polymerase chain reaction (PCR) technique in peripheral blood of patients with brucellosis, for the diagnosis of the initial phase of the disease and for the identification of relapses (sensitivity 100% , specificity 98.5%). However, these results have not been confirmed by other authors and the interference of some blood elements has been considered a limiting factor in the sensitivity of the test. Recently, Zerva et al. obtained good results when performing the test on serum samples (sensitivity 94%, specificity 100%). Therefore, PCR can be very useful for the evolutionary follow-up of patients who present a complicated clinical course, but for this it will be necessary that other groups corroborate their effectiveness and that the most appropriate methodology be precisely defined. The application of PCR in different clinical samples other than blood, (joint fluid, CSF, abscess pus, etc.), whose cultures are often negative in the usual media seems highly recommended in those laboratories that have the infrastructure necessary for its realization.
Serological diagnosis Serological
tests are of great importance in the diagnosis of brucellosis. Most of them detect antibodies against lipopolysaccharide (LPS) of the outer membrane. Its main limitation is its inability to differentiate with sufficient sensitivity and specificity between active and cured infection, since antibodies usually persist for a prolonged period after clinical recovery.
The most widely used classical tests, with which a large majority of patients with brucellosis can be managed, are the Wright seroagglutination test and the Rose Bengal agglutination test (Huddleson, Hudleson, Huddlesson, Hudlesson reaction). detect the presence of binding antibodies, and the Coombs test to quantify non-binding antibodies. The negativity of these three tests practically excludes the diagnosis of brucellosis. The adequate assessment of its results requires the use of a quality antigen and well standardized, since otherwise it is a frequent cause of unreliable titles and diagnostic confusions. Classically, a title of 1 / 80-1 / 160 has been considered a cut-off point indicative of disease if it is accompanied by a suggestive or compatible clinical picture. However, any positive titre should be carefully evaluated in the light of the clinical data, before being discarded as not significant.
The Rose Bengal test is a very effective rapid agglutination test, which was initially used as a screening test to allow a diagnostic approach in a few minutes. However, the accumulated practical experience has given it a prominence that goes beyond that corresponding to a simple screening test. The acid medium in which the test is carried out considerably favors the expression of the binder component of the antibodies. Its sensitivity and specificity for identifying anti-Brucella agglutinating antibodies is very high, so that only exceptionally it is negative in the acute phase of the infection and very infrequently in the evolved or chronic phases of the disease.
The Coombs test for the detection of non-binding antibodies of IgG and IgA type has been shown to be extremely reliable in clinical practice over the years and provides, in 48 hours, very useful complementary information. The modification of the method introduced by Otero et al, carrying out the test in plaque instead of tube, simplifies it remarkably and makes it possible to recommend its use in the usual way to patients with brucellosis. In the vast majority of cases, the titles obtained are superior to those of agglutination, one or two dilutions in the first phases of the disease, but several times more as the evolution time is prolonged.
Recently, a new immunocapture-agglutination test called Brucellacapt has been incorporated in our country for the detection of total antibodies against Brucella. The test uses a lamellae with a series of wells containing antihuman immunoglobulins. Several works by Spanish authors have shown a specificity similar to the Coombs test, but with greater sensitivity. Probably, one of the main reasons for the sensitivity of the test is that its realization occurs at an acid pH, which greatly favors the agglutination of the antibodies. Titres ≥1 / 320 are considered significant. This results,
For about 15 years, the enzyme immunoassay (ELISA) method has been used for the determination of specific immunoglobulins against Brucella LPS, showing an extraordinary sensitivity and specificity. The existing studies on the ELISA IgG, IgM and IgA and their correlation with the classic serological tests have allowed us to know in detail the evolutionary course of the specific immunoglobulins in the different phases of the disease and their different role in the results of the tests of agglutination and Coombs. The determination of IgM antibodies has a very high correlation with seroagglutination, especially in the first months of evolution. After these first, there is a usual decrease in the ELISA-IgM titers, independently of the clinical evolution of the patient. The immunoglobulins IgG and IgA also have a binding component, but their relative role in the agglutination tests is less important than that of the IgM in the first months of the disease. Its correlation with seroagglutination increases markedly over time and becomes very noticeable after the first three months; The role of these immunoglobulins in agglutination tests is all the more important the longer the evolutionary period considered. IgG and IgA have a very notable component as non-binding antibodies, which are the ones detected by the Coombs test. Thus, the IgA ELISA test, and especially the ELISA IgG determination, show a high correlation with the Coombs test. The detailed and reliable information provided by the ELISA methods, together with the possibility of being introduced on a large scale without great technical difficulties and for an acceptable cost, led us to think that the IgM-ELISA would replace the agglutination test and the IgG ELISA. to the Coombs test in the usual diagnosis of brucellosis. However, in these years, the widespread and habitual use of this test has met with the lack of adequate standardization of commercial systems. In fact, laboratories that use this method often provide confusing results that are difficult to interpret. Thus, its generalized use is not advisable as long as a well-established standardization does not occur.
To differentiate whether the antibodies are of the IgM or IgG type in the seroagglutination test and thus have an information regarding the evolution time of the disease, the seroagglutination test has been used classically after a subsequent treatment of the sample with 2-mercaptoethanol. (2ME) or ditrioteitol (DTT), which detects only IgG antibodies because those of the IgM class are inactivated. The information provided by this test is useful, but much less accurate than that obtained with the ELISA method, since it has been shown that it can also inactivate IgA in addition to IgM. Recently, a very simple and fast colorimetric test has been designed, the dipstick test that identifies the IgM by means of a nitrocellulose ribbon impregnated with anti-human IgM monoclonal antibodies.
In patients suffering a relapse of the disease, a new increase in IgG and IgA, but not IgM, in the evolutionary course of immunoglobulins, is observed very well objectified by the ELISA method. Among the classic tests, this turn is detected much better with the Coombs test than with the agglutination test. The data provided recently seem to indicate that Brucellacapt, whose titles seem to have more pronounced variations, could better detect these oscillations related to the relapse of brucellosis.
The interpretation of serology in relation to the chronic evolution of the disease is an unresolved problem. The classic presumption of the American authors that the agglutination test with 2ME was a good marker of evolution to chronicity is not currently supported, in light of the data previously commented. In this sense, it must be taken into account that serological persistence is more frequent in patients who have started from very high titers at the beginning of the disease and in those who have had focality of the infection even though they have followed a satisfactory evolution. From a practical point of view, any serological title should be compared with the previous titles if they were available; an increase in IgG or IgA antibodies, or the persistence of these titres in patients with a compatible clinical situation, should suggest the activity of the infection. In this sense, it is worth remembering that the comparison of two serological titles to each other requires their realization in a couplet and that a variation is only considered to be valuable if it is greater than a dilution.
The tests that determine antibodies against the cytoplasmic proteins of Brucella (counter-immunoelectrophoresis and more recently the ELISA method) have also been shown to be very sensitive and specific in the diagnosis of brucellosis, although its use has been much less widespread. These antibodies appear later than the anti-LPS and their evolution is further interfered by the course of antibiotic treatment. Therefore, it has been reported that these antibodies may be a marker of activity more sensitive than anti-LPS.
In our opinion, brucellosis that has a low level serological response and especially if it is of IgG and IgA type, with absence of IgM, could correspond to forms of reactivation or not recent acquisition, after a previous asymptomatic or unnoticed infection; In this regard it should be remembered that, in some patients, the incubation time of the disease may be several months.
TREATMENT
The treatment of brucellosis has not undergone significant variations in recent years. In its intracellular location the microorganism finds a way to evade the antimicrobial action, so that it has not required to develop resistance mechanisms against the antibiotics used for decades to treat the disease. The problem lies in the difficulty to achieve the intracellular eradication of the microorganism, so that no single antibiotic achieves it, and this has led to the necessary use of various combinations with synergistic or additive effect, administered for several weeks, to reduce as much as possible the appearance of relapses. When these occur, the bacterium maintains an antibiotic sensitivity identical to that of the initial episode, so a similar antibiotic regimen can be used for its treatment.
Tetracyclines are the most effective antibiotics in the treatment of brucellosis and should be the basis of any therapeutic combination. Aminoglycosides, despite their poor intracellular penetration, show a synergistic effect with tetracyclines.The combination of doxycycline, at a dose of 100 mg / 12 h orally for 6 weeks, and an aminoglycoside by intramuscular route for 2 weeks is the most active, being considered as the classic treatment and choice of the disease, with a of relapses of around 5%. Although the classically used aminoglycoside has been streptomycin (1 g / day, 750 mg / day in patients older than 50 years), the use of gentamicin in monodose 4 mg / kg is currently recommended, since it has greater activity in vitro and less toxicity, but the period of 2 weeks must be maintained. The oral combination of doxycycline (100 mg / 12 h) and rifampicin (15 mg / kg / day, usually 900 mg / day in a morning fasting dose), both for 45 days, is the best alternative to classical treatment. Due to its tolerance and comfort it has more acceptance, but it is accompanied by a higher percentage of relapses, around 15%, which
It has special importance in the complicated forms of the disease. Fluoroquinolones, cotrimoxazole and azithromycin have given poor results in experimental studies and in the treatment of human disease.
The combinations of doxycycline or rifampin with quinolones or cotrimoxazole have been used by some authors, but their results have not been sufficiently contrasted and these guidelines should be considered as secondary alternatives. For brucellosis of the pregnant woman, a rifampin monotherapy regimen for 8 weeks could be a reasonable option.Infantile brucellosis in children over 7 years of age should be managed like adult brucellosis. In the case of young children, rifampin or cotrimoxazole is recommended for 4-6 weeks, plus gentamicin for 7-10 days; The best alternative to this regimen is the oral combination of rifampin and cotrimoxazole for 6 weeks.
The majority of laboratory accidents consisting of punctures do not require the preventive administration of antibiotics.However, if the accident involves the conjunctival route, the use of doxycycline is recommended; Although the recommended time is a matter of controversy, a guideline of 7-10 days is probably adequate.
The treatment of many localized forms of brucellosis does not require modifications with respect to the general recommendations mentioned. However, in the case of spondylitis, some of osteoarthritis or orchiepidimitis and suppurative forms of the disease, which are accompanied by a higher rate of therapeutic failure, it is recommended to prolong the treatment with doxycycline for at least 8 weeks; Surgical drainage is not usually necessary in many of these patients. For cases of endocarditis, the triple combination of doxycycline, rifampin and aminoglycosides is recommended to optimize bactericidal activity; gentamicin should be prolonged for 3 weeks and doxycycline and rifampicin for at least 8 weeks. The criteria used for valve replacement are the same as for other infective endocarditis, although this is often required in the case of brucellosis. The treatment of neurobrucellosis encounters the difficulty of achieving high levels of antibiotics in the CSF; The addition of rifampicin to classical treatment and the prolongation of antibiotic therapy according to clinical response is recommended.
BIBLIOGRAPHY
ARIZA J, PELICER T, PALLARÉS R, FOZ A, GUDIOL F. Specific antibody profile in human brucelosis. Clin Infect Dis 1992; 14: 131-140.
ARIZA J, GUDIOL F, PALLARÉS R, et al. Treatment of human brucellosis with doxycycline plus rifampin or doxycycline plus streptomycin, a randomized, double-blind study. Ann Intern Med 1992; 117:25-30.
ARIZA J. Brucellosis: an update. The perspective from the Mediterranean basin. Rev Med Microbiol 1999; 10:125-135.
COLMENERO JD, REGUERA JM, MARTOS F, et al.. Complications associated with Brucella melitensis infection: a study of 530 cases. Medicine 1996; 75:195-211.
DIAZ R, MORIYÓN I. Laboratory techniques in the diagnosis of human brucellosis. En: Young EJ, Corbel MJ (eds). Brucellosis: Clinical and laboratory aspects. Boca Ratón: CRS Press, 1992. MORATA P, QUEIPO-ORTUÑO MI, REGUERA JM, GARCÍA-ORDOÑEZ MA, PICHARDO C, COLMENERO JD. Post-treatment follow-up of brucellosis by PCR assay. J Clin Microbiol
1999; 37: 4163-4166.
ORDUÑA A, ALMARAZ A, PRADO A, et al. Evaluation of an immunocapture-agglutination test (Brucellacapt) for the serodiagnosis of human brucellosis. J Clin Microbiol 2000;
38:4000-4005.
SOLERA J, LOZANO E, MARTÍNEZ-ALFARO E, ESPINOSA A, CASTILLEJOS ML, ABAD L. Brucella spondylitis: review of 35 cases and literature survey. Clin Infect Dis 1999; 29: 1440-1449
.
YAGUPSKY P. Detection of Brucella in blood cultures. J Clin Microbiol 1999; 37:3437-3442.
YOUNG EJ. An overview of human brucellosis. Clin Infect Dis 1995; 21:283-290.
Javier Ariza Cardinal
Service Infectious Diseases, CSU de Bellvitge
EPIDEMIOLOGY
Brucellosis has been well known in recent decades as a zoonosis of worldwide distribution. The disease has been eradicated in many countries of the developed world, but the infection by Brucella melitensis is still endemic in the Mediterranean area, the Middle East, the Arabian Peninsula, the Indian subcontinent and Latin America.
In Spain, the great majority of isolates from patients with brucellosis have corresponded to B. melitensis. The human disease has been associated with direct contact with livestock, typical of some professions such as shepherds, cattle ranchers, slaughterers, etc., but also in large part with the consumption of uncontrolled dairy foods, predominant in rural areas, but also existing in the urban and its surroundings. The rates corresponding to the compulsory declaration registration have shown a progressive decrease in the last 15 years, coinciding with intense vaccination and intervention campaigns on animal brucellosis in many Spanish regions.
While the maximum rate was reached in 1984, with 22.33 cases per 100,000 inhabitants, the last records were 3.9 in the years 1998 and 1999, and 2.8 cases per 100,000 inhabitants in
2000.
ETIOPATOGENIA
The genus Brucella includes a homogeneous group of intracellular facultative microorganisms belonging to the α-2 subdivision of the Proteobacteria. Due to the similarity of the DNA of the different varieties, it has been proposed to group them in a single species, B. melitensis. However, for practical and epidemiological reasons the distinction of the 6 main types is maintained: B. melitensis, Brucella abortus, Brucella suis, Brucella canis, Brucella. ovis and Brucella neotomae.
The microorganism lacks plasmids as an expression of its adaptation to a stable intracellular medium, free of bacterial competition. Its particular intracellular parisitism is related to some structural differences of its outer membrane in comparison with other gram-negative bacteria. In its intracellular location, it is resistant to the action of polycations and to the oxygen-dependent killing systems of phagocytes. It uses the autophagosome pathway to evade phagolysosome fusion and replicate inside the cells of the phagocytic mononuclear system. This capacity for intracellular survival determines the clinical pattern characteristic of brucellosis, the undulating course of the disease, its tendency to relapse and evolve into chronic forms. Cellular immunity is the main defense mechanism, through macrophage activation and its capacity to eradicate intracellular bacteria by the action of some cytokines (interferon-γ, tumor necrosis factor α and interleukin-12) produced by sensitized lymphocytes. Coinciding with the development of cellular immunity also appears a delayed hypersensitivity that seems to be important in the pathogenesis of the disease. However, the role of humoral immunity in defense mechanisms is undoubtedly significant. CLINICAL MANIFESTATIONS
The clinical behavior of brucellosis is well known and has been the object of multiple descriptions. The infection can affect any system or tissue of the organism.
The disease due to B. melitensis is the most aggressive and that caused by B. suis has the greatest capacity to produce abscess. The use of antibiotic therapy and the greater health development can modify the clinical presentation referred to in the classic descriptions and the symptomatology may differ depending on the social environment in which it is developed. The involvement of the organs of the mononuclear phagocytic system, hepatosplenomegaly and lymphadenopathy and of the ostearticular is especially characteristic. The development of focalities of the disease occurs in more than 30% of cases, a frequency that is fundamentally related to the time of evolution of the disease before starting antibiotic therapy. The observation of a patient with spondylitis, sacroiliitis, polyarthritis or tenosynovitis, orchiepididymitis and lymphocytic meningitis should raise the differential diagnosis of brucellosis in our environment. The observation of abscessed focal forms, such as reactivation of an old brucellosis, considered very rare in our days, could be proportionally more frequent in the coming years if the cases of brucellosis of recent acquisition continue to decrease and can pose diagnostic problems to unfamiliar doctors. with the disease.
DIAGNOSIS
Bacteriological
diagnosis The definitive diagnosis of the disease requires the isolation of Brucella, which is usually done in blood cultures.Castañeda's two-phase medium has been very effective and the
standard system recognized for many years. In general terms, 75-80% of infections due to B. melitensis and about 50% of
those produced by B. abortus occur with a positive blood culture .
The new systems of automated blood cultures, of the Bactec 9200 type or similar, have proved to be extraordinarily effective. Its detection capacity is probably greater than the classic Castañeda method and the other described systems, but the most significant thing is the precocity of this detection, which usually occurs between 3 and 5 days. Using this methodology, the microorganism is recovered during the first week of incubation in more than 95% of the cases in which Brucella can be isolated, which guarantees its isolation even in cases without clinical suspicion of the disease. The routine blind subculture at 7 days is not necessary and should be reserved for those cases with a high suspicion of brucellosis, especially if there has been previous antibiotherapy.
The isolation of Brucella in other samples is less frequent. The microorganism grows well in the usual media, and in a few days, when joint fluid is cultured, pus from abscesses, cerebrospinal fluid (CSF) or other tissue samples, although the frequency of recovery in these cases is usually around 30%. Also in this type of samples, the Bactec 9200 system increases the profitability of the crop significantly compared to conventional methods.
In recent years, the group of Colmenero et al. has observed very satisfactory results with the use of a polymerase chain reaction (PCR) technique in peripheral blood of patients with brucellosis, for the diagnosis of the initial phase of the disease and for the identification of relapses (sensitivity 100% , specificity 98.5%). However, these results have not been confirmed by other authors and the interference of some blood elements has been considered a limiting factor in the sensitivity of the test. Recently, Zerva et al. obtained good results when performing the test on serum samples (sensitivity 94%, specificity 100%). Therefore, PCR can be very useful for the evolutionary follow-up of patients who present a complicated clinical course, but for this it will be necessary that other groups corroborate their effectiveness and that the most appropriate methodology be precisely defined. The application of PCR in different clinical samples other than blood, (joint fluid, CSF, abscess pus, etc.), whose cultures are often negative in the usual media seems highly recommended in those laboratories that have the infrastructure necessary for its realization.
Serological diagnosis Serological
tests are of great importance in the diagnosis of brucellosis. Most of them detect antibodies against lipopolysaccharide (LPS) of the outer membrane. Its main limitation is its inability to differentiate with sufficient sensitivity and specificity between active and cured infection, since antibodies usually persist for a prolonged period after clinical recovery.
The most widely used classical tests, with which a large majority of patients with brucellosis can be managed, are the Wright seroagglutination test and the Rose Bengal agglutination test (Huddleson, Hudleson, Huddlesson, Hudlesson reaction). detect the presence of binding antibodies, and the Coombs test to quantify non-binding antibodies. The negativity of these three tests practically excludes the diagnosis of brucellosis. The adequate assessment of its results requires the use of a quality antigen and well standardized, since otherwise it is a frequent cause of unreliable titles and diagnostic confusions. Classically, a title of 1 / 80-1 / 160 has been considered a cut-off point indicative of disease if it is accompanied by a suggestive or compatible clinical picture. However, any positive titre should be carefully evaluated in the light of the clinical data, before being discarded as not significant.
The Rose Bengal test is a very effective rapid agglutination test, which was initially used as a screening test to allow a diagnostic approach in a few minutes. However, the accumulated practical experience has given it a prominence that goes beyond that corresponding to a simple screening test. The acid medium in which the test is carried out considerably favors the expression of the binder component of the antibodies. Its sensitivity and specificity for identifying anti-Brucella agglutinating antibodies is very high, so that only exceptionally it is negative in the acute phase of the infection and very infrequently in the evolved or chronic phases of the disease.
The Coombs test for the detection of non-binding antibodies of IgG and IgA type has been shown to be extremely reliable in clinical practice over the years and provides, in 48 hours, very useful complementary information. The modification of the method introduced by Otero et al, carrying out the test in plaque instead of tube, simplifies it remarkably and makes it possible to recommend its use in the usual way to patients with brucellosis. In the vast majority of cases, the titles obtained are superior to those of agglutination, one or two dilutions in the first phases of the disease, but several times more as the evolution time is prolonged.
Recently, a new immunocapture-agglutination test called Brucellacapt has been incorporated in our country for the detection of total antibodies against Brucella. The test uses a lamellae with a series of wells containing antihuman immunoglobulins. Several works by Spanish authors have shown a specificity similar to the Coombs test, but with greater sensitivity. Probably, one of the main reasons for the sensitivity of the test is that its realization occurs at an acid pH, which greatly favors the agglutination of the antibodies. Titres ≥1 / 320 are considered significant. This results,
For about 15 years, the enzyme immunoassay (ELISA) method has been used for the determination of specific immunoglobulins against Brucella LPS, showing an extraordinary sensitivity and specificity. The existing studies on the ELISA IgG, IgM and IgA and their correlation with the classic serological tests have allowed us to know in detail the evolutionary course of the specific immunoglobulins in the different phases of the disease and their different role in the results of the tests of agglutination and Coombs. The determination of IgM antibodies has a very high correlation with seroagglutination, especially in the first months of evolution. After these first, there is a usual decrease in the ELISA-IgM titers, independently of the clinical evolution of the patient. The immunoglobulins IgG and IgA also have a binding component, but their relative role in the agglutination tests is less important than that of the IgM in the first months of the disease. Its correlation with seroagglutination increases markedly over time and becomes very noticeable after the first three months; The role of these immunoglobulins in agglutination tests is all the more important the longer the evolutionary period considered. IgG and IgA have a very notable component as non-binding antibodies, which are the ones detected by the Coombs test. Thus, the IgA ELISA test, and especially the ELISA IgG determination, show a high correlation with the Coombs test. The detailed and reliable information provided by the ELISA methods, together with the possibility of being introduced on a large scale without great technical difficulties and for an acceptable cost, led us to think that the IgM-ELISA would replace the agglutination test and the IgG ELISA. to the Coombs test in the usual diagnosis of brucellosis. However, in these years, the widespread and habitual use of this test has met with the lack of adequate standardization of commercial systems. In fact, laboratories that use this method often provide confusing results that are difficult to interpret. Thus, its generalized use is not advisable as long as a well-established standardization does not occur.
To differentiate whether the antibodies are of the IgM or IgG type in the seroagglutination test and thus have an information regarding the evolution time of the disease, the seroagglutination test has been used classically after a subsequent treatment of the sample with 2-mercaptoethanol. (2ME) or ditrioteitol (DTT), which detects only IgG antibodies because those of the IgM class are inactivated. The information provided by this test is useful, but much less accurate than that obtained with the ELISA method, since it has been shown that it can also inactivate IgA in addition to IgM. Recently, a very simple and fast colorimetric test has been designed, the dipstick test that identifies the IgM by means of a nitrocellulose ribbon impregnated with anti-human IgM monoclonal antibodies.
In patients suffering a relapse of the disease, a new increase in IgG and IgA, but not IgM, in the evolutionary course of immunoglobulins, is observed very well objectified by the ELISA method. Among the classic tests, this turn is detected much better with the Coombs test than with the agglutination test. The data provided recently seem to indicate that Brucellacapt, whose titles seem to have more pronounced variations, could better detect these oscillations related to the relapse of brucellosis.
The interpretation of serology in relation to the chronic evolution of the disease is an unresolved problem. The classic presumption of the American authors that the agglutination test with 2ME was a good marker of evolution to chronicity is not currently supported, in light of the data previously commented. In this sense, it must be taken into account that serological persistence is more frequent in patients who have started from very high titers at the beginning of the disease and in those who have had focality of the infection even though they have followed a satisfactory evolution. From a practical point of view, any serological title should be compared with the previous titles if they were available; an increase in IgG or IgA antibodies, or the persistence of these titres in patients with a compatible clinical situation, should suggest the activity of the infection. In this sense, it is worth remembering that the comparison of two serological titles to each other requires their realization in a couplet and that a variation is only considered to be valuable if it is greater than a dilution.
The tests that determine antibodies against the cytoplasmic proteins of Brucella (counter-immunoelectrophoresis and more recently the ELISA method) have also been shown to be very sensitive and specific in the diagnosis of brucellosis, although its use has been much less widespread. These antibodies appear later than the anti-LPS and their evolution is further interfered by the course of antibiotic treatment. Therefore, it has been reported that these antibodies may be a marker of activity more sensitive than anti-LPS.
In our opinion, brucellosis that has a low level serological response and especially if it is of IgG and IgA type, with absence of IgM, could correspond to forms of reactivation or not recent acquisition, after a previous asymptomatic or unnoticed infection; In this regard it should be remembered that, in some patients, the incubation time of the disease may be several months.
TREATMENT
The treatment of brucellosis has not undergone significant variations in recent years. In its intracellular location the microorganism finds a way to evade the antimicrobial action, so that it has not required to develop resistance mechanisms against the antibiotics used for decades to treat the disease. The problem lies in the difficulty to achieve the intracellular eradication of the microorganism, so that no single antibiotic achieves it, and this has led to the necessary use of various combinations with synergistic or additive effect, administered for several weeks, to reduce as much as possible the appearance of relapses. When these occur, the bacterium maintains an antibiotic sensitivity identical to that of the initial episode, so a similar antibiotic regimen can be used for its treatment.
Tetracyclines are the most effective antibiotics in the treatment of brucellosis and should be the basis of any therapeutic combination. Aminoglycosides, despite their poor intracellular penetration, show a synergistic effect with tetracyclines.The combination of doxycycline, at a dose of 100 mg / 12 h orally for 6 weeks, and an aminoglycoside by intramuscular route for 2 weeks is the most active, being considered as the classic treatment and choice of the disease, with a of relapses of around 5%. Although the classically used aminoglycoside has been streptomycin (1 g / day, 750 mg / day in patients older than 50 years), the use of gentamicin in monodose 4 mg / kg is currently recommended, since it has greater activity in vitro and less toxicity, but the period of 2 weeks must be maintained. The oral combination of doxycycline (100 mg / 12 h) and rifampicin (15 mg / kg / day, usually 900 mg / day in a morning fasting dose), both for 45 days, is the best alternative to classical treatment. Due to its tolerance and comfort it has more acceptance, but it is accompanied by a higher percentage of relapses, around 15%, which
It has special importance in the complicated forms of the disease. Fluoroquinolones, cotrimoxazole and azithromycin have given poor results in experimental studies and in the treatment of human disease.
The combinations of doxycycline or rifampin with quinolones or cotrimoxazole have been used by some authors, but their results have not been sufficiently contrasted and these guidelines should be considered as secondary alternatives. For brucellosis of the pregnant woman, a rifampin monotherapy regimen for 8 weeks could be a reasonable option.Infantile brucellosis in children over 7 years of age should be managed like adult brucellosis. In the case of young children, rifampin or cotrimoxazole is recommended for 4-6 weeks, plus gentamicin for 7-10 days; The best alternative to this regimen is the oral combination of rifampin and cotrimoxazole for 6 weeks.
The majority of laboratory accidents consisting of punctures do not require the preventive administration of antibiotics.However, if the accident involves the conjunctival route, the use of doxycycline is recommended; Although the recommended time is a matter of controversy, a guideline of 7-10 days is probably adequate.
The treatment of many localized forms of brucellosis does not require modifications with respect to the general recommendations mentioned. However, in the case of spondylitis, some of osteoarthritis or orchiepidimitis and suppurative forms of the disease, which are accompanied by a higher rate of therapeutic failure, it is recommended to prolong the treatment with doxycycline for at least 8 weeks; Surgical drainage is not usually necessary in many of these patients. For cases of endocarditis, the triple combination of doxycycline, rifampin and aminoglycosides is recommended to optimize bactericidal activity; gentamicin should be prolonged for 3 weeks and doxycycline and rifampicin for at least 8 weeks. The criteria used for valve replacement are the same as for other infective endocarditis, although this is often required in the case of brucellosis. The treatment of neurobrucellosis encounters the difficulty of achieving high levels of antibiotics in the CSF; The addition of rifampicin to classical treatment and the prolongation of antibiotic therapy according to clinical response is recommended.
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