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0International Journal of Pediatrics and Adolescent Medicine (2014) 1, 40-46
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CLINICAL PRACTICE GUIDELINES
Brucellosis in children: Prevention, diagnosis and management guidelines for general pediatricians endorsed by the Saudi Pediatric Infectious Diseases Society (SPIDS)
Mohammad A. Alshaalan a *, Sulaiman A. Alalola a,
Maha A. Almuneef ab, Esam A. Albanyan a, Hanan H. Balkhy a,
Dayel A. AlShahrani a, Sameera AlJohani c
a Pediatric Infectious Diseases, King Abdulaziz Medical City, Riyadh, Saudi Arabia b ISPCAN, Arab Professionals Society for Prevention of Violence Against Children (Arab-ISPCAN), King Abdulaziz Medical City, NGHA, Saudi Arabia
c Microbiology, Department of Pathology and Laboratory Medicine, King Abdulaziz Medical City, Riyadh, Saudi Arabia
Received 19 August 2013; accepted 29 November 2013 Available online 5 October 2014
KEYWORDS
Brucellosis;
Children;
Diagnosis;
Treatment;
Prevention
Abstract In Saudi Arabia, brucellosis is an endemic zoonotic disease. Although it is believed that children are not commonly involved, a number of reports from endemic areas exhibited a high percentage of pediatric patients (20—30% of affected patients). Clinical manifestations of childhood brucellosis are varied and range from minimal symptoms to extreme morbidity and occasional fatality. Asymptomatic infections are also not uncommon. The fact that brucellosis is endemic in the Kingdom became clear in the early 1980s. Several reasons have been considered, but the most prominent of them is the increase in the importation of animals from areas where brucellosis is endemic, especially some African countries. Consumption of raw milk and, to a lesser extent, contact with infected animals or their products are the primary routes of infection. The consumption of fresh, unpasteurized milk from camels is a traditional practice, and people believe that boiling the milk removes nutritional value.
Copyright © 2014, King Faisal Specialist Hospital & Research Centre (General Organization), Saudi Arabia. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
* Corresponding author. Tel.: +966 8011111x11630, +966 0505209532 (mobile); fax: +966 8011111x11641. E-mail addresses: shaalanm1@ngha.med.sa (M.A. Alshaalan), alolas@ngha.med.sa (S.A. Alalola), MuneefM@NGHA.MED.SA (M.A. Almuneef), banyane@ngha.med.sa (E.A. Albanyan), daalshahrani@kfmc.med.sa (D.A. AlShahrani).
Peer review under responsibility of King Faisal Specialist Hospital & Research Centre (General Organization), Saudi Arabia.
http://dx.doi.org/10.1016/j.ijpam.2014.09.004
2352-6467/Copyright © 2014, King Faisal Specialist Hospital & Research Centre (General Organization), Saudi Arabia. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
1. Purpose of the guidelines
1. To increase the awareness of pediatricians and other child care providers, such as family medicine and general practitioners, about brucellosis, its varied clinical presentations and its impact on child health.
2. To provide insight to the epidemiology of brucellosis in Saudi Arabia, the modes of transmission and preventive measures.
3. To set a standard scheme for the diagnosis, antimicrobial therapy, management and follow up of childhood brucellosis.
2. Epidemiology
Brucellosis is endemic in Saudi Arabia with a reported incidence of 18/100,000 population/year, as reported by the MOH in 2011. An overall seroprevalence rate of 15% was found in the Saudi population, and the seroprevalence in children aged 0—14 years was 10% [1].
The reason for the high prevalence of brucellosis in Saudi Arabia is attributed, but not limited, to the following:
1. The nomadic life style including animal raising, especially of sheep and camels.
2. The traditional belief of the great benefit of ingesting raw milk, especially camel milk.
3. The high rate of animals imported from Africa where the disease is endemic, with a lag in compliance with national and international policies of animals screening and quarantine rules. In Saudi Arabia, the prevalence of brucella among different animals is high; 8% in camels, 18.7% in cattle, 6.5% in sheep and 9.7% in goats [2].
4. The mixing of different animal herds together, such as raising sheep and cattle together.
5. The low levels of public awareness about the seriousness of brucellosis as a human disease.
6. The resistance to slaughtering infected animals.
3. Transmission
Brucella spp. are small, gram negative, non-motile, non-spore-forming, rod shaped (coccobacilli) bacterial organisms. It is a zoonotic disease caused by the ingestion of raw unpasteurized milkfrom infected animalsorclose contact with their secretions. There are different animal reservoirs for different Brucella spp. that are known to cause human disease:
Other species include Brucella ovis, Brucella neotomae, Brucella microti and marine Brucella species (Brucella pinnipediae and Brucella cetaceae); however, only the marine species have been reported to rarely cause human disease [3].
In Saudi Arabia and most of the neighboring countries, the most prevalent species is Brucella melitensis causing 70—90% of brucellosis cases. Brucella abortus is the second most common disease causing pathogen. The other species rarely cause brucellosis.
Transmission of the organism to humans occurs as a result of:
1.The consumption of unpasteurized raw milk or other dairy products, especially soft cheese, butter and cream. Hard cheese, sour milk and yogurt are unlikely to transmit the disease because of the propionic and lactic fermentation.
2. Direct contact with the secretions of infected animals or their products, such as the placenta or aborted materials.
3. The air borne transmission of aerosolized materials through open wounds or mucus membranes either in the animal areas or in the laboratory when dealing with blood and other infected fluid cultures. It has been found that direct contact with soil, animal feces and dust contaminated with Brucella is associated with a higher risk of infection [4].
4. Vertical transmission, sexual transmission and breast milk transmission are anecdotally reported routes of infection.
4. Clinical presentation
Most infected children have acute and sub-acute presentation of 2—4 weeks of symptomatology [5,6]. Brucellosis symptomatology is very variable, in part because of the variable pathogenicity of different strains. It is known that B. abortus causes milder disease with either mild symptomatology or focal lesions. However, infection with B. melitensis is usually associated with a high rate of bacteremia, short incubation periods, and noticeable symptoms [3,6,7]. Almost any organ can be affected and varied complications are reported. Almost 76% of affected patients present within two weeks of the onset of their symptoms [4]. Fever and arthritis are the most common presenting signs. Monoarthritis was more common than polyarthritis. This may create confusion with pyogenic arthritis in children; therefore, in a community where brucella is common, awareness about this entity should prompt the investigation of this disease, and physicians should have a high index of suspicion for Brucella arthritis. The most commonly affected joints were the hip and knee. Unlike in adults, the sacroiliac joint and the axial skeleton were rarely affected [4]. Brucella osteomyelitis is rare in children. Studies showed that only l—2% of children with brucellosis has osteomyelitis [5,6,8]. Neurobrucellosis is rare in children and it has been reported in only 0.5—1% of children with brucellosis. The clinical presentation of neurobrucellosis is variable with meningitis and
Species Animal Host Virulence
Brucella Goats, sheep, camels ++++
melitensis
Brucella Cows, other bovidae animals +++
abortus and camels
Brucella canis Dogs +
Brucella suis Pigs +
meningoencephalitis being the most common. Other presentations of neurobrucellosis include polyradiculopathy, myelitis and psychiatric disorders, mainly depression. Rare CNS manifestations include brain abscess, hydrocephalus, pesudotumor cerebri, Guillain Barre syndrome, cranial nerve palsy, cerebral venous sinus thrombosis, subdural and epidural collection and stroke [9,10,11]. Clinical manifestations of neurobrucellosis range from subtle manifestation, such as fever and malaise, to frank meningitis. Other manifestations include cranial nerve neuropathy, hearing loss, visual disturbances, chronic vomiting, altered senso-rium and coma and various neurological focal motor deficits. Recovery is typical and sequelae are rare. Classic general symptoms include the following:
• Headache
• Myalgia/bone pain
• Anorexia/weight loss
• Sweats
• Malodorous perspiration
• Depression, or mood disorders
• Fever
• Arthralgia/arthritis
The main signs are:
• Fever
• Arthritis
• Hepatomegaly
• Splenomegaly
• Neck stiffness
• Miscellaneous (skin rash, cervical nodes, drowsiness, periorbital swelling, ataxia)
Other rare manifestations include:
■ CNS meningitis, encephalitis, meningoencephalitis, brain abscess, and Guillain Barr syndrome
■ Lung pneumonia
■ Cardiac endocarditis and myocarditis
■ Liver transaminasemia and abscess
■ Spleen abscess
■ Genitourinary epedidemo-orchitis and nephritis
■ Eye uveitis
■ Thyroid abscess
■ Spine epidural abscess
■ Hematologic immune thrombocytopenic purpura and hemophagocytic syndrome
4.2. Culture
Although positive culture should be the gold standard for diagnosis, its yield remains suboptimal. The culture yield is greater in the first two weeks of illness and as the duration of illness increases the yield decreases. In a patient with fever and chills and short duration of illness, the culture yield is up to 90%; however, as the disease duration increases the yield drops to 30%. Most studies have shown a 40—70% yield. Rapid diagnosis of brucella using the new third generation continuous monitoring blood culture systems, such as non-radiometric BACTEC or BACT/ALERT, has shown a better and faster recovery with all positive cultures occurring by day 5—7 of incubation; therefore, extending the blood culture incubation to 4 weeks for improving the yield of culture is rarely required [12,13].
4.3. Specimen collection and requisition
Blood is collected aseptically by venipuncture. The volume withdrawn at a single venipuncture (3—5 ml and 10 ml from older children) and inoculated into a pediatric vial. For the diagnosis of bloodstream infection, two "sets" drawn 20 min apart are normally recommended. In infants and children, partly because the intensity of bacteremia is greater and partly due to physiological reasons, 3.0 ml in the specially formatted pediatric vial is acceptable. Inoculated vials should be transported promptly to the laboratory and held at room temperature until they are entered into the BacT—Alert system. Vials must be incubated up to 5 days routinely, in cases with a high index of suspicion along with a negative initial blood culture, the incubation period can be increased up to 21 days.
4.4. Species identification
• Gram stain: Brucella are Gram-negative coccobacilli that strongly retain the crystal violet and resist counter-staining. It may be necessary to counterstain for 1 —3 min.
• For any blood culture that flags positive and is indicative of Brucella species, 0.5 ml of the blood culture fluid is to be aspirated from the bottle and inoculated onto a urea slant. This tube is sealed with parafilm and incubated in CO2 for 24 h. These slants must be checked for a positive reaction every 2 h and the length of time taken to become positive is documented on the electronic worksheet. Brucella is usually oxidase positive, catalase positive and urease positive.
4.1. Diagnosis
Laboratory diagnosis of brucellosis relies on:
1. Positive blood or other sterile body fluid culture, such as synovial fluid, CSF, and plural fluid, for brucella species.
2. Or, a positive brucella serology test of 1:>160, using the Standard Agglutination Test (SAT) for patients presenting with symptoms suggestive of brucellosis. For the purpose of screening and in the absence of clinical indicators of active brucellosis, a titer of 1:320 or higher is more specific for the presence of the disease.
4.5. Brucella susceptibility testing
Brucella susceptibility testing is performed by E-test for the following antibiotics: tigecycline, SXT, rifampin, tetracy-cline, streptomycin and ciprofloxacin. The interpretation is according to the CLSI guidelines as well as published references. Some discrepancy exists between in vitro susceptibility results and in vivo outcomes. This precludes the exact extrapolation of in vitro results to in vivo application. However, every attempt should be made to follow susceptibility data in the choice of antimicrobial regimen for the treatment of brucellosis [13,14,15].
5. Serology
Because of the lower yield of culture, serological tests remain the best diagnostic modality available. There are multiple serological tests for brucella including the standard agglutination test (SAT), the enzyme linked immunosorbent assay (ELISA), indirect Coombs, brucellacapt, indirect fluorescent antibody and immunochromatogrphic lateral flow [13,14]. The most commonly used tests are SAT and ELISA.
5.1. Standard agglutination test (SAT)
SAT is the most commonly used and most standardized test. It is based on measuring an agglutination titer of different serum dilutions against a standardized concentration of whole brucella cell suspension. It is usually measured by doubling the serum dilution from 1:20 up to 1:20,480. A positive titer is 1:160 or more. In endemic areas, there may be a persistent and continuing exposure to the source of infection, and therefore there may be a persistent low titer in the range of 1:80—1:160 in the absence of true infection. It was found that 2.9 and 2.5% of the healthy Saudi population have a titer of 1:160 and 1:320, respectively [1]. Our study showed that 92% of children with acute brucellosis have a titer of 1:320 or more [6]. Brucella antibodies can persist for a long period after acute infection with a median time to serological cure of 18 months. Among cured patients, 29% continued to have a titer of 1:320 or higher 2 years or more after infection [16,17]. Therefore, the interpretation of serology in endemic areas should be correlated to the clinical presentation. As such, for screening purposes and in the absence of suggestive symptomatology, a titer of 1:320 or more should be taken as the cutoff level for positivity. Coupling SAT with the 2-mercaptoethanol agglutination test is useful in differentiating acute from chronic brucellosis. The 2-mercaptoethanol agglutination test elutes IgM out leaving IgG. Elevated IgG titers indicate an acute disease. A negative 2-mercaptoethanol test after therapy indicates a good response to treatment.
There are a few limitations to the SAT including:
• The inability to diagnose B. canis
• The appearance of cross-reactions of IgM with Francisella tularensis, Escherichia coli O116 and O157, Salmonella urbana, Yersinia enterocolitica O: 9, Vibrio cholerae, Xanthomonas maltophilia, and Afipia clevelandensis
• The SAT may be negative in presence of disease in rare occasions
Lack of seroconversion can be attributed to:
• The performance of the SAT early in the course of infection
• The presence of blocking antibodies
• The prozone phenomenon (i.e. the inhibition of agglutination at low dilutions due to an excess of antibodies or to nonspecific serum factors)
5.2. Enzyme linked immunosorbent assay (ELISA)
The ELISA is performed using 96-well microtiter plates that are precoated with a standardized antigen, usually purified lipopolysaccharide. It has the advantage of measuring different classes of reactive antibodies including IgG, IgA and IgM. Thus, it has greater ability to differentiate between acute infection and relapsing infection. Various studies showed different results regarding the sensitivity of the ELISA. In general, the sensitivity ranges from 60 to 98%. In acute brucellosis, the sensitivity and specificity of ELISA for IgG were found to be 45 and 97%, respectively, and were 79 and 100% for IgM, respectively [18]. Another study found that the sensitivity and specificity of ELISA for IgG were 84 and 100%, respectively, and 60 and 100% for IgM, respectively [19]. The sensitivity and specificity of combined of ELISA for IgG and ELISA for IgM are comparable to that of the SAT [14]. Therefore, the SAT remained the preferred test of choice for acute brucellosis. However, for follow up and for the diagnosis of a brucella relapse ELISA is better as it gives a separate titer for IgG, IgM and IgA and thus can be used to assess response to therapy and relapse. The ELISA is the best for detecting brucella antibody in the cerebrospinal fluid in cases of neurobrucellosis [14].
5.3. PCR-based diagnosis
Various PCR tests are being studied for diagnosing brucellosis both at the genus and species levels. These tests have shown promising results regarding their sensitivity, specificity and clinical applicability. They have advantages over the gold standard of culture isolation in that they are easy to perform, require a short period of time and avoid the risk of laboratory acquired infection. Once validated, these diagnostic tests will be the future diagnostic modality of choice [20,21].
6. Laboratory investigation
All patients suspected of having brucella should have the following tests done:
1. Complete blood count and differentiation:
The result will be normal in most cases; however, in some patients variable affection of different cell lines may be noted such as leukopenia, anemia, thrombocy-topenia or a combination of some or all of them.
2. Erythrocyte sedimentation rate:
As an acute reactant marker, it will be raised but usually of modest value ranging from 20 to 80 mm/h.
3. Liver function test:
A mild to moderate elevation of transaminases can be found in some cases. Increased bilirubin is rare but it can occur.
4. Renal function test:
Mostly normal; however, in rare cases, glomerulone-phritis may occur with variable elevations of creatinine and BUN.
5. Blood culture
Table 1 Drugs used for brucellosis and their dosages.
Drug Dosage
Rifampicin 20 mg/kg/day in two divided doses (max.
600 mg)
Doxycycline 5 mg/kg/day in two divided doses (max.
200 mg) (only for children more than 8
year of age)
TMP/SMX 10 mg of trimethoprim/kg/day (max.
480 mg)
Gentamicin 5—7.5 mg/kg/day IM or IV either as a
single dose or three divided doses
Streptomycin 15 mg/kg IM or IV once daily (max. 1 g/
day) (only for children more than 8 year
of age)
Ciprofloxacin 30 mg/kg/day in two divided doses (max.
1.5 g)
6. Other sterile body fluid, tissue or bone marrow culture as indicated.
7. Brucella serology
7. Management[4,9,10,11,21,22,23]
Management of brucellosis relies on adherence to the following criteria:
1. Using an antibiotic that has the ability to act intracel-lularly and in acidic media.
2. Using combined therapy.
3. Using antimicrobials for a prolonged duration according
to the system involved.
There is a limited number of antibiotics that can be used for the treatment of brucella infections including doxycy-cline, rifampicin, trimethoprime—sulfamethoxazole, streptomycin, gentamicin and ciprofloxacin (see Table 1).
■ A common combination for children yielding successful
results is as follows:
• Rifampicin and TMP/SMX for children below 8 years of age.
• Doxycycline and TMP/SMX or rifampicin for children older than 8 years of age. This combination has been shown to have the highest success rate and should be used in children above 8 years to avoid the staining of the teeth in younger children.
• In serious infections, such as neurobrucellosis and endocarditis, three to five drugs need to be used for a longer period of time, usually for three to 12 months (see Table 2).
• Gentamicin for 7 days or streptomycin for 14 days can be used for patients requiring hospitalization.
• The use of streptomycin has been associated with a lesser degree of relapse but is not significantly superior.
8. Monitoring the response to therapy 8.1. Clinical response
Most patients respond promptly to therapy. Additionally, most patients with acute brucellosis without neuro-brucellosis or brucella endocarditis can be managed as
Table 2 Regimes for different manifestations of brucellosis.
Disease
Therapy
Children < 8 years
Children > 8 year
Comment and duration of therapy
Common diseases: Acute brucellosis, brucella arthritis, brucella osteomyelitis, brucella bacteremia Serious illness
Brucella endocarditis
Neurobrucellosis
Rifampicin and septra OR rifampicin for 45 days and gentamicin for 7 days
Rifampicin, septra, and ciprofloxacin
Rifampicin, septra, and ciprofloxacin
Doxycycline and rifampicin OR Doxycycline for 45 days and streptomycin for 14 days OR Doxycycline for 45 days and gentamicin for 7 days Doxycycline, septra, and rifampicin
Doxycycline, septra, and rifampicin
Hospitalized patients add gentamicin for 5—7days Duration of therapy 6 weeks
Gentamicin for the initial two weeks
Surgical intervention is indicated
Duration of therapy is 3—9 months
Gentamicin for the initial two weeks
Ceftriaxone has shown some efficacy and it is usually used in the initial therapy for 2—4 weeks
Duration of therapy is 3—6 months up to one year in complicated cases
outpatients even if they are bacteremic. They usually show improvement within 3—7 days after starting therapy. All patients who are started on therapy for brucellosis should be followed closely in the clinic to monitor the persistence of the response and compliance to therapy. In patients who have CBC laboratory abnormalities, such as a positive blood culture and/or liver enzymes, they should have their tests repeated one week after starting therapy. Such patients usually normalize their laboratory abnormalities by then. If culture yields a positive result, attention needs to be paid to the susceptibility pattern although most brucella isolates remain sensitive to the first line antibiotics.
8.2. Serology response
Brucella titers decline slowly and may remain moderately high for months. Therefore, there is no need to repeat a titer early in the course of therapy. One serology titer should be repeated by the end of therapy to evaluate the trend and demonstrate a decrease in the titer. However, even if the titer remains high or decrease only slightly this does not mean that the patient did not respond as some patients may maintain high titers for a prolonged period of time. Almuneef et al. demonstrated that a titer over 320 can be sustained for up to 18 months in 25% of patients after resolution of the infection [16,17].
8.3. Relapse
Among treated patients, 3—9% will have a relapse or reinfection. Most relapses occur in the first year following therapy. If an affected patient begins to have symptoms, serology and blood culture should be repeated. Sites that may be affected such as the CNS or heart should be examined fully. The titer usually will be raised. However, obtaining an estimation of the IgG and IgM levels separately is optimal. This can be obtained by the ELISA or SAT with 2-ME. If the IgG level is high, relapse is indicated. Once relapse or reinfection occurs, referral to a pediatric infectious disease clinic is warranted for diagnosis confirmation and further management.
8.4. Prevention
1. Increase public awareness about the endemicity of brucellosis in Saudi Arabia and the importance of avoiding all risk factors that could lead to acquiring this infection. This entails stressing the importance of avoiding drinking raw milk or its products, and avoiding contact with sick animals or their products such as aborted fetuses.
2. Animal owners should be aware that brucellosis is prevalent among animals and thus regular checkups of animals are required.
3. Avoid mixing different herds of animals together as this practice facilitates the transmission of disease among animals.
4. The government should stress the screening of animals, the vaccination of seronegative animals and slaughtering diseased ones.
5. A collaborative team to implement a brucella control program should be arranged and maintained among the concerned government sectors including the Ministry of Health, the Ministry of Agriculture, the Custom Department and the Municipal Department.
6. Screening the family members of patients with acute brucellosis in endemic areas is strongly recommended to enhance the detection rate, to initiate early treatment and to reduce complications.
Where resources are limited, the screening of family members could be limited to the symptomatic and to children [24]. In these cases, the cutoff limit for a positive titer is 1:320.
Conflict of interest
There is no conflict of interest.
References
[1] Al-Sekait MA. Seroepidemiology survey of brucellosis antibodies in Saudi Arabia. Ann Saudi Med 1999 May-Jun;19(3): 219-22.
[2] Gul ST, Khan A. Epidemiology and epizootology of brucellosis: a review. Pak Vet J 2007;27(3):145-51.
[3] Zhen Q, Lu Y, Yuan X, Qiu Y, Xu J, Li W, et al. Asymptomatic brucellosis infection in humans: implications for diagnosis and prevention. Clin Microbiol Infect 2013 Apr 23.
[4] Pappas G, Akritidis N, Bosilkovski M, Tsianos E. Brucellosis. N Engl J Med 2005 Jun 2;352(22):2325-36.
[5] al-Eissa Y, al-Zamil F, al-Mugeiren M, al-Rasheed S, al-Sanie A, al-Mazyad A. Childhood brucellosis: a deceptive infectious disease. Scand J Infect Dis 1991;23(2):129-33.
[6] Shaalan MA, Memish ZA, Mahmoud SA, Alomari A, Khan MY, Almuneef M, et al. Brucellosis in children: clinical observations in 115 cases. Int J Infect Dis 2002 Sep;6(3):182-6.
[7] Roushan MR, Amiri MJ. Update on childhood brucellosis. Recent Pat Antiinfect Drug Discov 2013 Apr;8(1):42-6.
[8] Sharda DC, Lubani M. A study of brucellosis in childhood. Clin Pediatr 1986:25492-5.
[9] Turel O, Sanli K, Hatipoglu N, Aydogmus C, Hatipoglu H, Siraneci R. Acute meningoencephalitis due to Brucella: case report and review of neurobrucellosis in children. Turk J pediatr 2010;52:426-9.
[10] Haji-Abdolbagi M, Rasooli-Nejad M, Jafari S, Hasibi M, Soudbakhsh A. Clinical and laboratory findings in neurobrucellosis: review of 31 cases. Arch Iran Med 2008;11:21-5.
[11] Altas M, Evirgen O, Arica V, Tutanc M. Pediatric neuro-brucellosis associated with hydrocephalus. J Pediatr Neurosci 2010;5:144-6.
[12] Bannatyne RM, Jackson MC, Memish Z. Rapid diagnosis of Brucella bacteremia by using the BACTEC 9240 system. J Clin Microbiol 1997 0ct;35(10):2673-4.
[13] Al Dahouk S, Sprague LD, Neubauer H. New developments in the diagnostic procedures for zoonotic brucellosis in humans. Rev Sci Tech 2013 Apr;32(1):177-88.
[14] Araj GF. Update on laboratory diagnosis of human brucellosis. Int J Antimicrob Agents 2010 Nov;36(Suppl. 1):S12-7.
[15] Constantine MV, Vangelis E, Evdokia V, Chryssanthy P. Brucellosis in humans: why is it so elusive? Rev Med Micro 2009;20:63-73.
[16] Almuneef M, Memish ZA. Prevalence of Brucella antibodies after acute brucellosis. J Chemother 2003 Apr;15(2):148-51.
[17] Almuneef M, Memish ZA. Persistence of Brucella antibodies after successful treatment of acute brucellosis in an area of endemicity. J Clin Microbiol 2002 Jun;40(6):2313.
[18] Memish ZA, Almuneef M, Mah MW, Qassem LA, Osoba AO. Comparison of the brucella standard agglutination test with the ELISA IgG and IgM in patients with Brucella bacteremia. Diagn Microbiol Infect Dis 2002 Oct;44(2):129-32.
[19] Gomez MC, Nieto JA, Rosa C, Geijo P, Escribano MA, Munoz A, et al. Evaluation of seven tests for diagnosis of human brucellosis in an area where the disease is endemic. Clin Vaccine Immunol 2008 Jun;15(6):1031-3.
[20] Surucuoglu S, El S, Ural S, Gazi H, Kurutepe S, Taskiran P, et al. Evaluation of real-time PCR method for rapid diagnosis of brucellosis with different clinical manifestations. Pol J Microbiol 2009;58(1):15-9.
[21] Solera J. Update on brucellosis: therapeutic challenges. Int J Antimicrob Agents 2010 Nov;36(Suppl. 1):S18—20.
[22] Ariza J, Bosilkovski M, Cascio A, Colmenero JD, Corbel MJ, Falagas ME, et al., International Society of Chemotherapy; Institute of Continuing Medical Education of Ioannina. Perspectives for the treatment of brucellosis in the 21st century: the Ioannina recommendations. PLoS Med 2007 Dec;4(12): e317.
[23] Soli's Garcia del Pozo J, Solera J. Systematic review and meta-analysis of randomized clinical trials in the treatment of human brucellosis. PLoS One 2012;7(2):e32090.
[24] Alsubaie S, Almuneef M, Alshaalan M, Balkhy H, Albanyan E, Alola S, et al. Acute brucellosis in Saudi families: relationship between brucella serology and clinical symptoms. Int J Infect Dis 2005 Jul;9(4):218—24.
