Brucellosis is one of the most common endemic zoonotic diseases worldwide. It is caused by gram-negative coccobacilli belonging to the genus Brucella . Brucellamelitensis and Brucellaabortus are the most important Brucella spp., primarily infecting small ruminants (sheep and goats) and cattle, respectively . Humans are considered incidental hosts that can be infected through contact with animals and animal products . Acute infection manifests as a disabling flu-like syndrome with nonspecific clinical signs, including an undulating fever, sweating, chills, myalgia, arthralgia, and fatigue . Single-agent therapy with drugs such as cotrimoxazole, doxycycline, rifampicin, and ciprofloxacin has been abandoned owing to the high relapse rates, and a combination of two drug regimens is preferred . Multiple drug combinations are used in brucellosis treatment. The combination of doxycycline plus rifampicin for 6 weeks is the most used regimen. Other alternatives include a combination of doxycycline plus trimethoprim-sulfamethoxazole, or a fluoroquinolone plus rifampicin . Failure of treatment and relapse after treatment is common, varying from 5 to 15% in mild cases. In the last decade, resistance to Brucella treatment has emerged in brucellosis-endemic regions worldwide (such as Malaysia, Egypt, Qatar, and China) . Shevtsov et al.  reported in their study that resistance to rifampicin is prevalent in Kazakhstan, and about half of the isolates were rifampicin resistant (including the intermediate resistance). Elbehiry et al.  also observed resistances to ampicillin, ampicillin-sulbactam, and rifampicin in Saudi Arabia. Rifampicin is one of the cornerstones in the treatment of tuberculosis. Moreover, With the emergence of multiresistant strains of mycobacteria, there is an increased need for rifampicin-free regimens in the treatment of brucellosis. Streptomycin is effective in the treatment of brucellosis but still carries the risk of nephrotoxicity and ototoxicity. In addition, parenteral intake is a challenge in some village areas where health facilities are deficient. The combination of doxycycline with trimethoprim-sulfamethoxazole is a possible solution for these obstacles .
In this study, we aimed to compare the efficacy of two drug regimens used for the treatment of brucellosis: doxycycline plus trimethoprim-sulfamethoxazole (TMX-Doxy) versus doxycycline plus rifampicin (RIF-Doxy) in Egyptian patients.
Patients and methods
Patient’s selection and study settings
This is a prospective study that was carried out in the Department of Tropical Medicine and Gastroenterology, Assiut University, between June 2020 and January 2022. A total of 100 consecutive patients with brucellosis who attended our outpatient clinic and inpatient ward were enrolled in our study. Exclusion criteria were complicated localized brucellosis (spondylitis, endocarditis, and meningoencephalitis), age less than 18 years, pregnancy, receiving antibiotics for more than 1 week before enrollment, and having COVID-19.
For all enrolled patients, the following were done: complete history taking and physical examination, standard tube agglutination test (STAT), complete blood picture, liver enzymes, blood glucose level, erythrocyte sedimentation rate (ESR), C-reactive protein. History taking, clinical examination, and all of these investigations were done before starting treatment, 2, 4 weeks, at the end of treatment, 3 and 6 months after the end of treatment. STAT was done at baseline, at the end of treatment, 3 months after, and 6 months after the end of treatment. Diagnosis was based on a positive STAT result more than or equal to 1/160 association with compatible clinical findings . STAT test was done by the same laboratory clinician. Plain antigen of B. abortus S99 (IVRI, Izatnagar) was used. Two-fold serial dilutions (1 : 80 to 1 : 1280) of the sera were prepared in phenol saline, and an equal quantity (0.5 ml) of antigen was added to each tube. All of the tubes were incubated at 37°C for 24 h. The results were compared with the antigen control tube showing 50% agglutination. A titer of 1 : 160 or above was considered positive. Brucellosis was classified into acute (if the duration of illness is <2 months) and chronic (if the duration of illness is more than 2 months) . Half of the enrolled patients randomly received doxycycline plus trimethoprim-sulfamethoxazole, whereas the other half received doxycycline plus rifampicin. The two regimens were given for 6 weeks. Randomization was done by writing 100 cards. Overall, 50 of them were assigned to the first group that received the TMX-Doxy combination, whereas the other 50 were assigned to the group that received the RIF-Doxy combination. The cards were placed in sealed envelopes, and all were mixed. The patient or the treating doctor chose one of the cards to put the patient in the group that was drawn blindly. The dosage of drugs was doxycycline (100 mg twice a day), trimethoprim-sulfamethoxazole (160 mg of trimethoprim plus 800 mg of sulfamethoxazole twice a day), and rifampicin (600 mg per day) .
Study definitions 
Successful treatment was defined by subsiding fever, back pain, and sweating with or without decline in STAT titer.
Relapse was diagnosed when the clinical manifestations reappeared and titer of STAT re-increase again after its reduction.
Therapeutic failure was defined by persistence of symptoms (fever, sweating or back pain) at the end of treatment with persistently high STAT titer.
The data in the two groups were collected and analyzed using the Statistical Package for the Social Sciences (version 21; SPSS Inc., Chicago, Illinois, USA). Data were presented as numbers, percentages, means, and SDs. The χ2 test was used to compare the therapeutic effect of two regimens. Overall, 95% confidence intervals were calculated when appropriate. Difference with a P value of less than 0.05 was considered significant.
Ethical consideration and funding
The study protocol was approved by the ethics committee of the Faculty of Medicine, Assiut University (IRB No. 17300850), and was adherent to the ethical guidelines of the 1975 Declaration of Helsinki. Written informed consent was obtained from each participant. The study was registered at pactr.samrc.ac.za (PACTR202210778524094).
Basic clinical and imaging characteristics
Table 1 shows the clinical data and abdominal ultrasound findings. A total of 100 patients with acute and chronic brucellosis were involved in our study. Overall, 50 patients, with a mean age of 39.3±15.23 years, was treated with TMX-Doxy group, and 50 patients, with a mean age of 38.1±14.8 years, were treated with rifampicin plus doxycycline RIF-Doxy group. Male sex was predominant in both groups (71 males versus 29 females). The most observed symptom was fever, followed by sweating. Acute brucellosis was diagnosed in 77 patients, whereas chronic brucellosis was diagnosed in 23 patients in both groups.
Table 2 shows the laboratory investigations of the patients. Leukocytosis was observed in 13 (13%) patients of all patients in the two groups, whereas leukopenia was observed in three (3%) of the 100 patients. Anemia was present in 18 (18%) patients in both groups. Thrombocytosis was recorded in three (3%) patients only. Erythrocyte sedimentation rate was raised in 19 (19%) patients and C-reactive protein was positive in 68 (68%) patients. STAT was positive with variable titers. The most observed titers were 1/160 and 1/320. For five of the 100 patients, higher dilutions were requested when the initial STAT was negative but still clinically suspected cases to avoid the prozone phenomenon and the test was positive with 1/1280 dilution.
Outcomes of treatment
Successful treatment (disappearance of symptoms and no relapse within 6 months of treatment) was recorded in 45 (90%) patients of the TMX-Doxy group, whereas it was seen in 47 (94%) patients in the RIF-Doxy group, with no statistically significant difference between the two groups. Failure of treatment was observed in two (4%) patients in the TMX-Doxy group and one (2%) patient in the RIF-Doxy group. Relapse of brucellosis was recorded in three (6%) cases treated in the TMX-Doxy group, whereas two (4%) cases relapsed treated in the RIF-Doxy group. Table 3 shows the outcomes of treatment.
As shown in Table 4, some adverse effects of the used drugs were reported. All of them were mild and self-limited and did not necessitate stoppage of therapy. Diarrhea, mild hypersensitivity (mild itching and skin rash), and gastrointestinal upset were the most observed adverse effects. Liver injury with mildly raised liver enzymes was detected in only one (2%) patient in the TMX-Doxy group and three (3%) patients in the RIF-Doxy group. Follow-up liver enzymes were normal, and treatment was continued.
Brucellosis is still a challenging infectious intracellular zoonotic disease that has both domestic animal and human burden with significant economic losses. Brucellosis is one of the group B notifiable diseases in Egypt. Nevertheless, 50–60% of patients are managed in the primary care private sector . Many trials were done to study monotherapy with ciprofloxacin for the treatment of brucellosis, but relapse rate was high in all of these studies [14,15]. Therefore, combination therapy is now recommended for at least 6 weeks, with rifampicin-based combination antibiotics are still the most recommended and most used regimens [15,16].
Communities, like Egypt, with high prevalence of tuberculosis suffer from emerging of resistant strains of tuberculosis, may be partly due to overuse of rifampicin in the treatment of brucellosis, especially in villages where both brucellosis and tuberculosis are prevalent [17,18,19]. Other alternative regimens have been suggested and studied for the treatment of brucellosis to avoid using rifampicin and reserve it for tuberculosis. One of the suggested regimens was TMX-Doxy Combination for 6 weeks. Few previous studies have evaluated this regimen [20,21]. In our study, we aimed to compare two regimens in the treatment: TMX-Doxy in comparison with rifampicin plus doxycycline.
Our results showed that the TMX-Doxy combination is highly effective in the treatment of brucellosis with a success rate of 90%. This is comparable to the success rate of the other regimen (RIF-Doxy combination), which has a 94% success rate in our study.
These results confirm the high efficacy of both TMX-Doxy and RIF-Doxy combinations. In contrast with our results, Alavi et al.  reported a higher efficacy of the TMX-Doxy group over the RIF-Doxy combination (92.15 vs. 78.43%) with significant differences between the two regimens.
In another older study, Roushan et al.  reported that the efficacy of TMX-Doxy combination was 84.3%, which was higher than the TMX-RIF combination (73.6%).
Regarding failure of therapy, in our study, we found only two (4%) cases in the TMX-Doxy group versus only one (2%) case in the RIF-Doxy group. This is comparable to the results of Alavi and Rajabzadeh , who reported a 1.94% failure rate in the TMX-Doxy group.
In contrast to our study, Roushan et al.  reported a higher failure rate of treatment with the TMX-Doxy combination (7.1%). This may be explained by the larger sample size in their study.
Regarding relapse of brucellosis after treatment, we diagnosed three (6%) patients in the TMX-Doxy group and two (4%) patients in the RIF-Doxy group. Alavi and Rajabzadeh  found slightly higher rate of relapse (5.88% in the TMX-Doxy group and 11.76% in the RIF-Doxy group). The same results were obtained in the study by Roushan et al. , who reported an 8.6% relapse rate in the TMX-Doxy group.
The reported adverse effects of the used drugs in our study were all mild and self-limited. None of them necessitated stoppage of treatment. Gastrointestinal upset, diarrhea, mild skin allergy, and mild liver injury were observed after 2 weeks of treatment and improved at the second follow-up after 1 month of treatment.
In areas where brucellosis is endemic, tuberculosis may also be common. Trials to avoid use of rifampicin and use of other drug combinations should be made to decrease the rate of emerging resistant strains of TB. In our study, we found that the combination of TMX-Doxy was highly effective and safe with low rate of relapse and failure of treatment. It is tolerable with no serious adverse effects.
In conclusion, both regimens (TMX-Doxy and RIF-Doxy) are effective in treating brucellosis. Both regimens are also safe with no major adverse effects encountered. We recommend the TMX-Doxy combination as an effective and safe alternative combination to the RIF-Doxy combination to save rifampicin for patients with tuberculosis to decrease the incidence of drug-resistant mycobacteria.
In our study, the sample size was relatively small, so the number of cases that failed to response or relapsed was too small to compare in the acute or chronic stages. Therefore, we recommend a larger study to assess the risk factors for failure of treatment or relapse.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
Author’s contributions: Waleed Attia Hassan conceived and designed the experiments. Waleed Attia Hassan, Muhammad Abdel-Gawad, and Ahmed Shawkat performed the experiments. Waleed Attia Hassan and Muhammad Abdel-Gawad performed the data analysis, data accuracy, data validation, statistical analysis, investigation, and supervision. Waleed Attia Hassan and Ahmed Shawkat drafted the manuscript. Waleed Attia Hassan and Muhammad Abdel-Gawad wrote, reviewed, and edited the article. All authors have read and agreed to the published version of the manuscript.
1. Godfroid J, Cloeckaert A, Liautard JP, Kohler S, Fretin D, Walravens K, et al From the discovery of the Malta fever’s agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis Vet Res. 2005;36:313–326
2. Poester FP, Samartino LE, Santos RL. Pathogenesis and pathobiology of brucellosis in livestock Rev Sci Tech. 2013;32:105–115
3. Dean AS, Crump L, Greter H, Schelling E, Zinsstag J. Global burden of human brucellosis: a systematic review of disease frequency PLoS Negl Trop Dis.. 2012;6:e1865
4. Montejo JM, Alberola I, Glez-Zarate P, Alvarez A, Alonso J, Canovas A, Aguirre C. Open, randomized therapeutic trial of six antimicrobial regimens in the treatment of human brucellosis Clin Infect Dis.. 1993;16:671–676
5. Al-Tawfiq JA, Memish ZA. Antibiotic susceptibility and treatment of brucellosis Recent Pat Antiinfect Drug Discov. 2013;8:51–54
6. Pozo JSGD, Solera JBaddour MM. Treatment of human brucellosis — review of evidence from clinical trials Updates on brucellosis. 2015 London IntechOpen
7. Shevtsov A, Syzdykov M, Kuznetsov A, Shustov A, Shevtsova E, Berdimuratova K, et al Antimicrobial susceptibility of Brucella melitensis
in Kazakhstan Antimicrob Resist Infect Control. 2017;6:130
8. Elbehiry A, Aldubaib M, Al Rugaie O, Marzouk E, Abaalkhail M, Moussa I, et al Proteomics-based screening and antibiotic resistance assessment of clinical and sub-clinical Brucella species: an evolution of brucellosis infection control PLoS ONE. 2022;17:e0262551
9. Seung KJ, Keshavjee S, Rich ML. Multidrug-resistant tuberculosis and extensively drug-resistant tuberculosis Cold Spring Harb Perspect Med.. 2015;5:a017863
10. Franco MP, Mulder M, Gilman RH, Smits HL. Human brucellosis Lancet Infect Dis.. 2007;7:775–786
11. Salata RA, Ravdin JIMandell GL, Douglas RG, Bennett JE. Brucella species (brucellosis) Principles and practices of infectious diseases. 19852nd ed. New York John Wiley & Sons, Inc:1283–1289
12. Corbel MJ Brucellosis in humans and animals. 2006 World Health Organization
13. Crump JA, Youssef FG, Luby SP, Wasfy MO, Rangel JM, Taalat M, et al Estimating the incidence of typhoid fever and other febrile illnesses in developing countries Emerg Infect Dis.. 2003;9:539–544
14. Doganay M, Aygen B. Use of Ciprofloxacin in treatment of brucellosis Eur J Clin Microbiol Infect Dis. 1992;11:74–75
15. Lang R, Raz R, Sacks T, Shapiro M. Failure of prolonged treatment with ciprofloxacin in acute brucellosis J Antimicrob Chemother. 1990;6:841–846
16. Young JEMandell GL, Dolin R, Benett JE. Brucella species Principles and practice of infectious diseases. 1995 New York Churchill Livingstone:2053–2057
17. Gadallah MA, Mokhtar A, Rady M, El-Moghazy E, Fawzy M, Kandil SK. Prognostic factors of treatment among patients with multidrug-resistant tuberculosis in Egypt J Formos Med Assoc. 2016;115:997–1003
18. World Health Organization. . Global tuberculosis report 2019 World Health Organization Document 2019. WHO/CDS/TB/2019.15:1-283. Google Scholar.
19. Abdel Dayem AM, Sharkawy SH, Mohammed Fathy RM, Hassanin OM, Ali A. Detection of drug-resistant mycobacterium tuberculosis using Geno Type MTBDRplus assay in smear-positive cases Egypt J Chest Dis Tuberc. 2019;68:284–289
20. Alavi SM, Rajabzadeh AR. Comparison of two chemotherapy regimen: doxycycline-rifampicin and doxycycline cotrimoxazol in the brucellosis patients Ahvaz, Iran, 2004–2006 Pak J Med Sci. 2007;23:6
21. Roushan MR, Gangi SM, Ahmadi SA. Comparison of the efficacy of two months of treatment with co-trimoxazole plus doxycycline vs. co-trimoxazole plus rifampin in brucellosis Swiss Med Wkly.. 2004;134:564–568