Babesia species (Babesiosis)
Authors: Edouard Vannier, Ph.D., Peter J. Krause, M.D.
Parasitology
Babesiosis is an emerging tick-borne zoonotic disease caused by intraerythrocytic protozoa of the genus Babesia and transmitted by hard-bodied (Ixodid) ticks. Several species are known to cause disease in humans.
Epidemiology
Babesia microti infection is endemic in the northeastern and midwestern United States (12, 36, 38, 42). Babesia duncani infection has been reported in Washington State and California (7, 27). MO-1 was isolated from a resident of Missouri and is closely related to Babesia divergens, a species known to cause human disease in Europe (3, 12, 14). The yearly recurrence of human B. microti infection in endemic regions of the United States contrasts with the sporadic occurrence of the disease in Europe, Africa, Asia, and South America (3, 12, 36, 37, 38). Because Ixodes scapularis (also known as Ixodes dammini) is the vector for B. microti, and for the causative agents of Lyme disease and of human granulocytic anaplasmosis, coinfection may occur (17). Babesiosis is occasionally transmitted through blood transfusion and on rare occasions, perinatally (8, 11, 23, 44).
Clinical Manifestations
Babesia infection may be associated with no symptoms or may cause an illness that ranges in severity from mild to fulminant, sometimes resulting in death (2, 12, 17, 22, 35, 42). The replication of the parasite may cause severe hemolytic anemia with numerous clinical manifestations including high fever, chills, sweats, headache, and arthralgias. Complications include pulmonary edema, hypotension, disseminated intravascular coagulation, congestive heart failure, splenic rupture, and renal failure. The incubation period typically is one to six weeks when the infection is tick-transmitted and up to nine weeks in transfusion transmitted cases (11, 44). Patients at increased risk of severe babesiosis include those who have malignancy, lack a spleen, are infected with HIV, are receiving immunosuppressive drugs, or are beyond the age of 50 years (1, 22, 24, 36, 38). Such patients may experience a prolonged and recurrent illness despite standard anti-babesial therapy (22). Based upon a case review of 139 patients hospitalized with babesiosis, White noted that male gender, alkaline phosphatase >125 U/L, and WBC>5 x 109/L are indicators of severe outcome (42).
Babesiosis should be suspected in any patient with unexplained febrile illness who has recently lived in or traveled to an endemic region during the months of May through September, with or without a history of tick bite. Recipients of blood products are also at increased risk when blood donors live in or travel to endemic areas.
Laboratory Diagnosis
During the acute phase of babesiosis, definitive laboratory diagnosis can be made by identification of the causative agent on Giemsa-stained thin blood smears (5, 36, 38). Support of a specific diagnosis can be made by serological evaluation. The most common serologic test is the indirect immunofluorescent assay (IFA) (18, 20, 38). Patients with a Babesia antibody titer of 1:64 or higher are considered to be seropositive whereas those with a titer of 1:1024 or greater usually are actively or recently infected (18, 38). Cross-reactivity with other Babesia species andPlasmodium species can occur. In cases that are difficult to diagnose by smear and serology, detection of Babesia can be accomplished by amplification of babesial DNA using polymerase chain reaction or by inoculation of patient blood into a hamster or gerbil (28, 38).
Pathogenesis
Babesia species are intraerythrocytic protozoa that multiply by asexual budding within the erythrocyte to produce 2 to 4 daughter cells or merozoites. The egress of merozoites into the circulation is accompanied by erythrocyte lysis. Erythrocyte lysis is responsible for many of the clinical manifestations of the disease (12, 36, 38). Ischemia and necrosis may result from obstruction of blood vessels by erythrocytes parasitized with certain Babesia spp. Cytokines are thought to be of central importance for protection against the infection, and for the development of symptoms and complications (21, 38). The spleen also plays an important role in the protection against babesiosis by clearing parasitized erythrocytes. Activation of complement, fibronectin, and kallikreins may be responsible for hypotension and vascular congestion associated with the disease (12, 36, 38).
SUSCEPTIBILITY IN VITRO AND IN VIVO
In vitro susceptibility testing has been reported using B. divergens cultures. Microbial growth was measured by parasite number on Giemsa-stained thin blood smears and by parasite incorporation of 3H hypoxanthine (3). Susceptibility testing has not been developed for B. microti.
Various animal models used to test the safety and efficacy of anti-babesial compounds generally show that a combination of two antiprotozoal drugs is more effective than a single drug in eradicating infection. Early studies of 20 antiprotozoal agents in a Mongolian jird model demonstrated that pentamidine and diminazene were the most effective agents tested (26, 32). The combination of clindamycin and quinine, one of the current standard regimens for human babesiosis, effectively eradicates B. microti infection in hamsters (31, 40). Quinine is inactive againstB. divergens (3, 25) and atovaquone often fails to eradicate B. microti infection in hamsters, even at high doses (13, 15). While atovaquone and clindamycin also fail to completely clear Babesia microti infection in the gerbil model of infection, the combination is more effective than atovaquone alone in delaying recrudescence of infection (13). Similarly, the combination of quinine and azithromycin is more effective in reducing B. microti parasitemia in hamsters than either drug alone (40). Atovaquone monotherapy leads to the development of drug resistance but not when combined with azithromycin (43). Although antiprotozoal combinations such as quinine and pyrimethamine sometimes are ineffective in eradicating Babesia infection, animal models have consistently demonstrated the superiority of combination drugs for the treatment of babesiosis.
ANTIPARASITIC THERAPY
Antiparasitic Agents of Choice
The combination of atovaquone and azithromycin is the treatment of choice for immunocompetent patients experiencing mild to moderate babesiosis while clindamycin and quinine should be used for more severe infections (Table 1).
Mild to Moderate Babesiosis
In the first prospective, randomized trial of anti-babesial therapy in people, atovaquone and azithromycin was compared with clindamycin and quinine for treatment of adults experiencing mild to moderate B. microti infection (16). The atovaquone (750 mg every 12 hours) and azithromycin (500 mg on day 1, then 250 mg per day thereafter) combination was found to be as effective in clearing parasitemia and resolving symptoms as the clindamycin (600 mg every 8 hours) and quinine (650 mg every 8 hours) combination. Both drug combinations were given by mouth over 7 days. After three months, there was no evidence of piroplasms or amplifiable B. microti DNA in either group. Significantly fewer adverse effects were associated with the atovaquone and azithromycin combination. Three-fourths of patients receiving clindamycin and quinine experienced adverse drug reactions and a third were forced to decrease the dose or to discontinue the medication. Adverse effects of therapy include tinnitus, decreased hearing, GI symptoms (anorexia, vomiting, and diarrhea), headache, visual disturbances, rash, and vertigo. By contrast, only fifteen percent in the atovaquone and azithromycin group experienced symptoms consistent with adverse drug reaction and only one patient (2%) discontinued medication. The combination of atovaquone and azithromycin is suggested as the therapy of choice in immunocompetent adult patients experiencing mild or moderate babesial symptoms and in those who cannot tolerate clindamycin and quinine (Table 1) (16, 45).
Severe Infection
Clindamycin (administered intravenously) and oral quinine should be given to patients who experience severe babesial illness with high parasitemia (>10%), significant hemolysis, or renal, hepatic, or pulmonary compromise (Table 1). When necessary, oral quinine may be replaced with intravenous quinidine. The combination of clindamycin and quinine was the first successful antimicrobial therapy used to treat B. microtiinfection (44).
Exchange transfusion should be considered in patients experiencing severe babesiosis (1, 4, 24, 29, 45). Partial or complete red blood cell exchange (whole blood or packed red blood cells) or plasmapheresis are rapid and reliable methods for removing parasite-infected red blood cells and vasoactive elements such as cytokines and thromboplastic substances that may contribute to renal failure and disseminated intravascular coagulation (1, 4, 24). Due to the risks associated with multiple blood exposures, these techniques should not be considered as routine therapy but only used for those who are severely ill (9).
Immunosuppressed Hosts
Data from a recent study of highly immunocompromised patients experiencing recurrent symptomatic babesiosis despite a variety of anti-babesial therapeutic combinations indicate that while no drug combination is clearly superior in treating such patients, cure requires anti-babesial treatment for at least six weeks, including two weeks after babesia are no longer detected on blood smear (22). Higher doses of azithromycin (600 to 1,000 mg per day) in combination with atovaquone (750 mg twice a day) have been used successfully to treat immunosuppressed patients experiencing babesiosis (41). Recent data suggest that prolonged (longer than four weeks) atovaquone and azithromycin therapy may result in the development of resistance to this combination.
Alternative Therapy
The combination of pentamidine (240 mg IV/day) and cotrimoxazole (3 grams/ day) was found to be moderately effective in clearing parasitemia and resolving symptoms in a mild case of B. divergens infection (30). Potential adverse reactions to pentamidine (pain at the site of injection, formation of sterile abscess and nephrotoxicity) limit the use of this drug. The combination of azithromycin and quinine was used successfully in two patients who had not improved with clindamycin and quinine (33, 34). An AIDS patient was successfully treated with high doses of clindamycin and azithromycin in combination with doxycycline after he became allergic to quinine (10). High-level parasitemia eventually decreased in another AIDS patient after exchange transfusion and atovaquone-proguanil was given along with azithromycin, atovaquone, clindamycin and quinine. In this patient, parasitemia recurred but was eventually cleared with a monotherapy of atovaquone-proguanil (39). A variety of other medications have been used in highly immunocompromised hosts but none has been shown to be clearly superior to current standard therapy (22).
ENDPOINTS FOR MONITORING THERAPY
Patients with babesiosis should be closely monitored during therapy. In most cases, improvement will occur within a day or two after antiprotozoal therapy is begun. In a recent study, symptoms completely resolved in all subjects three months after clindamycin and quinine or atovaquone and azithromycin therapy was completed (16). In severely ill patients, the hematocrit and percentage of parasitized erythrocytes should be monitored daily or every other day until the patient has improved and the parasitemia has decreased to less than 5 percent. Some patients may have persistence of low-grade parasitemia for months after infection with or without antimicrobial therapy (19, 22). When parasitemia is difficult to detect on blood smear, the more sensitive polymerase chain reaction for amplifying parasite DNA should be used (28). We suggest consideration of retreatment of patients if they show evidence of parasitemia by analysis of thin blood smear or PCR for more than 3 months after initial therapy or if the parasite has been detected for more than 3 months in asymptomatic individuals (45). Physicians also should consider the possibility of coinfection with Lyme disease or human granulocytic anaplasmosis or both, in patients who experience especially severe or persistent symptoms despite appropriate antibabesial therapy. Such patients may benefit from the addition of doxycycline therapy because neither clindamycin and quinine nor atovaquone and azithromycin are effective for the treatment of Lyme disease or human granulocytic anaplasmosis.
VACCINES
Vaccines have successfully been developed to prevent babesiosis caused by B. divergens or B. bovis in cattle. No vaccine is currently available for the prevention of human babesiosis (38).
PREVENTION
Prevention of babesiosis can be accomplished by avoiding areas from May through September where ticks, deer, and mice are known to thrive (36). It is especially important for those at increased risk in endemic areas, such as asplenic individuals, to avoid tall grass and brush where ticks may abound. Use of clothing that covers the lower part of the body and that is sprayed or impregnated with diethyltoluamide (DEET), dimethyl phthalate or permethrin (Permanone®) is recommended for those who travel in the foliage of endemic areas. DEET has been shown to be more effective than other repellents against ticks, but the risk of adverse effects is greater. Topically applied DEET is absorbed into the systemic circulation and as much as 10-15% of each dose can be found in the urine (6). Dermatological effects such as bullous eruptions and urticaria have been documented. Systemic effects such as toxic encephalopathy, anaphylaxis, and grand mal seizures have been noted with higher DEET concentrations (6). Finally, DEET ingestion may be fatal to both children and adults (6). Thus, while DEET can help to prevent tick bites, care must be taken with repeated exposure to high concentrations. A search for ticks on people and pets should be carried out and the ticks removed as soon as possible. The latter is best accomplished with tweezers by grasping the mouthparts without squeezing the body of the tick. Prospective blood donors in endemic areas are not allowed to donate if they have a history of babesiosis (23). Laboratory screening of blood donors for asymptomatic babesial infection has been proposed but has yet to be implemented.
REFERENCES
1. Bonoan JT, Johnson DH, Cunha BA. Life-threatening babesiosis in an asplenic patient treated with exchange transfusion, azithromycin, and atovaquone. Heart and Lung 1998; 27: 424-8. [PubMed]
2. Benach JL, Habicht GS. Clinical characteristics of human babesiosis. J Infect Dis 1981; 144; 481. [PubMed]
3. Brasseur P, Lecoublet S, Kapel N, Favennec L, Ballet JJ. Quinine in the treatment of Babesia divergens infections in humans. Euro J Clin Microbiol Infect Dis 1996; 15: 840-1. [PubMed]
4. Cahill KM, Benach JL, Reich LM, Bilmes E, Zins JH, Siegel FP, Hochweis S. Red cell exchange: treatment of babesiosis in a splenectomized patient. Transfusion 1981; 21: 193-8. [PubMe
5. Carr JM, Emery S, Stone BF, Tulin L. Babesiosis. Diagnostic pitfalls. Am J Clin Pathol 1991; 95: 774-7. [PubMed]
6. Centers for Disease Control: Seizures temporally associated with use of DEET insect repellant-New York and Connecticut. M. M. W. R. 1989; 38: 678-80. [PubMed]
7. Conrad PA, Kjemtrup AM, Carreno RA, et al. Description of Babesia duncani n.sp. (Apicomplexa: Babesiidae) from humans and its differentiation from other piroplasms. Int J Parasitol 2006; 36(7):779-89. [PubMed]
8. Esernio-Jenssen D, Scimeca PG, Benach JL, Tenenbaum MJ. Transplacental/ perinatal babesiosis. J Pediatr 1987; 110: 570-2. [PubMed]
9. Evenson DA, Perry E, Kloster B, Hurley R, Stroncek DF. Therapeutic apheresis for babesiosis. J Clin Apheresis 1998; 13: 32-6. [PubMed]
10. Falagas ME, Klempner MS. Babesiosis in patients with AIDS: a chronic infection presenting as fever of unknown origin. Clin Infect Dis 1996;22:809-12. [PubMed]
11. Gerber MA, Shapiro E, Krause PJ, Cable R, Badon SJ, Ryan R. The risk of acquiring Lyme disease or babesiosis from a blood transfusion. J Infect Dis 1994; 170: 231-4. [PubMed]
12. Gorenflot A, Moubri K, Precigout E, Carcy B, Schetters TP. Human Babesiosis. Ann Trop Med Parasitol 1998; 92: 489-501. [PubMed]
13. Gray JS, Pudney M. Activity of atovaquone against Babesia microti in the Mongolian gerbil, Meriones unguiculatus. J Parasitol 1999; 85: 723-8. [PubMed]
14. Herwaldt B, Persing DH, Precigout EA, Goff WL, Mathiesen DA, Taylor PW, Eberhard ML, Gorenflot AF. A fatal case of babesiosis in Missouri: identification of another piroplasm that infects humans. Ann Intern Med 1996; 124: 643-50. [PubMed]
15. Hughes WT, Oz HS. Successful prevention and treatment of babesiosis with atovaquone. J Infect Dis 1995; 172: 1042-6. [PubMed]
16. Krause PJ, Lepore T, Sikand VK, Gadbaw J Jr, Burke G, Telford SR 3rd, Brassard P, Pearl D, Azlanzadeh J, Christianson D, McGrath D, Spielman A. Atovaquone and azithromycin for the treatment of babesiosis. N Engl J Med 2000; 16; 343: 1454-8. [PubMed]
17. Krause PJ, McKay K, Thompson CA, Sikand VK, Lentz R, Lepore T, Closter L, Christianson D, Telford SR, Persing D, Radolf, JD Spielman A and the Deer-associated infection study group. Disease-specific diagnosis of coinfecting tick-borne zoonoses: Babesiosis, human granulocytic ehrlichiosis and Lyme disease. Clin Inf Dis 2002;34:1184-1191. [PubMed]
18. Krause PJ, Telford SR 3rd, Ryan R, Conrad PA, Conrad M, Wilson M, Thomford JW, Spielman A: Diagnosis of Babesiosis: Evaluation of a serologic test for the detection of Babesia microti antibody. J Infect Dis 1994; 169: 923-6. [PubMed]
19. Krause PJ, Spielman A , Telford SR 3rd, Sikand VK, McKay K, Christianson D, Pollack RJ, Brassard P, Magera J, Ryan R, Persing DH. Persistent parasitemia following acute babesiosis. New Engl J Med 1998; 339: 160-5.[PubMed]
20. Krause PJ, Ryan R, Telford S, Persing D, Spielman A. Efficacy of an IgM serodiagnostic test for the rapid diagnosis of acute babesiosis. J Clin Microbiol 1996; 34: 2014-16. [PubMed]
21. Krause PJ, Daily J, Telford SR, Vannier E, Lantos P, Spielman A. Shared features in the pathobiology of babesiosis and malaria. Trends Parasitol, 2007;23:605-610. [PubMed]
22. Krause PJ, Gewurz BE, Hill D, Marty FM, Vannier E, Foppa IM, Furman RR, Neuhaus E, Skowron G, Gupta S, McCalla C, Pesanti EL, Young M, Heiman D, Hsue G, Gelfand JA, Wormser GP, Dickason J, Bia FJ, Hartman B, Telford SR, Christianson D, Dardick K, Coleman M, Girotto JE, Spielman A. Persistent and relapsing babesiosis in immunocompromised patients. Clin Infect Dis 2008, 46:370-6. [PubMed]
23. Leiby DA. Babesiosis and blood transfusion: flying under the radar. Vox Sang 2006; 90:157-65. [PubMed]
24. Machtinger L, Telford SR III, Inducil C, Klapper E, Pepkowitz SH, Gold nger D. Treatment of babesiosis by red blood cell exchange in an HIV-positive, splenectomized patient. J Clin Apheresis 1993; 8: 78-81. [PubMed]
25. Marcus LC, Mabray CJ, Sturgis GH. Babesia microti infection in the hamster: failure of quinine and pyrimethamine in chemotherapeutic trials. Am J Trop Med Hyg 1984; 33: 21-3. [PubMed]
26. Marley SE, Eberhard ML, Steurer FJ, Ellis WL, McGreevy PB, Ruebush TK II. Evaluation of selected antiprotozoal drugs in theBabesia microti hamster model. Antimicrob Agents Chemother 1997; 41: 91-4. [PubMed]
27. Persing D, Herwaldt BL, Glaser C, Lane RS, Thomford JW, Mathiesen D, Krause PJ, Phillip DF, Conrad PA. Infection with aBabesia-like organism in the western United States. New Engl J Med 1995; 332: 298-303. [PubMed]
28. Persing DH, Mathiesen D, Marshall WF, et al. Detection of Babesia microti by polymerase chain reaction. J Clin Microbiol 1992; 30: 2097-103. [PubMed]
29. Powell VI and Grima K. Exchange transfusion for malaria and babesia infection. Trans Med Rev 2002; 16: 239-250. [PubMed]
30. Raoult D, Soulayrol L, Toga B, Dumon H, Casanova P. Babesiosis, pentamidine, and cotrimoxazole. Ann Intern Med 1987; 107: 944 (letter to the editor). [PubMed]
31. Rowin KS, Tanowitz HB, Witter M. Therapy of experimental babesiosis. Ann Intern Med 1982; 97: 556-8. [PubMed]
32. Ruebush TK II, Contacos PG, Steck EA. Chemotherapy of Babesia microti infections in Mongolian jirds. Antimicrob Agents Chemother 1980; 18: 289-91. [PubMed]
33. Shih CM, Wang CC. Ability of azithromycin in combination with quinine for the elimination of Babesial infection in humans. Am J Trop Med Hyg 1998; 59: 509-12. [PubMed]
34. Shih CM, Wang CC. Response to babesiosis to a combined regimen of quinine and azithromycin. Trans Royal Society Trop Med Hyg 1997; 91: 214-215. [PubMed]
35. Sun T, Tenenbaum MJ, Greenspan J, Teichberg S, Wang RT, Degnan T, Kaplan MH. . Morphologic and clinical observations in human infection with Babesia microti . J Infect Dis 1983; 148: 239-48. [PubMed]
36. Telford SR, Goren ot A, Brasseur P, Spielman A: Babesial infections in man and wildlife. In: Kreier JP, Baker JR, editors. Parasitic protozoa, Vol 5. 2nd ed. New York: Academic Press, 1993. p. 1-47.
37. Uhnoo I, Cars O, Christensson D, Nystrom-Rosander C. First documented case of human babesiosis in Sweden. Scand J Infect Dis 1992; 24: 541-7. [PubMed]
38. Vannier E, Gewurz B, Krause PJ. Human babesiosis. Infect Dis Clin N Amer 2008;22:469-88. [PubMed]
39. Vyas JM, Telford SR, Robbins GK. Treatment of refractory Babesia microti infection with atovaquone-proguanil in an HIV-infected patient: case report. Clin Infect Dis 2007; 45:1588-90. [PubMed]
40. Weiss LM, Wittner M, Wasserman S, Oz HS, Retsema J, Tanowitz HB. Efficacy of azithromycin for treating Babesia microtiinfection in the hamster model. J Infect Dis 1993; 168:1289-92. [PubMed]
41. Weiss LM, Wittner M, Tanowitz HB. The treatment of babesiosis. New Engl J Med ( Letter) 2001; 344: 773. [PubMed]
42. White DJ, Talarico J, Chang HG, Birkhead GS, Heimberger T, Morse DL. Human babesiosis in New York State: Review of 139 hospitalized cases and analysis of prognostic factors. Arch Intern Med 1998, 158 (19):2149-54. [PubMed]
43. Wittner M, Lederman J, Tanowitz HB, Rosenbaum GS, Weiss LM. Atovaquone in the treatment of Babesia microti infections in hamsters. Am J Trop Med Hyg 1996; 55: 219-22. [PubMed]
44. Wittner M, Rowin KS, Tanowitz HB, Hobbs JF, Saltzman S, Wenz B, Hirsch R, Chisholm E, Healy GR. Successful chemotherapy of transfusion babesiosis. Ann Intern Med 1982, 96: 601-4. [PubMed]
45. Wormser GP, Dattwyler RJ, Shapiro ED, Halperin JJ, Steere AC, Klempner MS, Krause PJ, Bakken JS, Strle F, Stanek G, Bockenstedt L, Fish D, Dumler JS, Nadelman RB. The Clinical Assessment, Treatment, and Prevention of Lyme Disease, Human Granulocytic Anaplasmosis, and Babesiosis. Clinical Practice Guidelines by the Infectious Disease Society of America. Clin Infect Dis 2006;43:1089-134.
Tables
Table 1: ANTI-BABESIA THERAPY
| TREATMENT | DOSE | FREQUENCY |
|---|---|---|
| Mild to moderate B. microti disease | ||
| Atovaquone and Azithromycin | ||
| Atovaquone | Adult: 750 mg | Every 12 hours |
| Child: 20 mg/kg (maximum 750 mg/dose) | Every 12 hours | |
| Azithromycin | Adult: 500 to 1000 mg 250 to 1000 mg Child: 10 mg/kg (maximum 500 mg/dose) 5 mg/kg (maximum 250 mg/dose) | On day 1 On subsequent days On day 1 On subsequent days |
| Clindamycin and Quinine | ||
| Clindamycin | Adult: 600 mg Child: 7-10 mg/kg (maximum 600 mg/dose) | Every 8 hours Every 6-8 hours |
| Quinine | Adult: 650 mg Child: 8 mg/kg (maximum 650 mg/dose) | Every 6-8 hours Every 8 hours |
| Severe B. microti disease, B. divergens and B. duncani disease | ||
| Clindamycin and quinine | ||
| Clindamycin | Intravenous administration Adult: 300-600 mg Child: 7-10 mg/kg (maximum 600 mg/dose) | Every 6 hours Every 6-8 hours |
| Quinine | Adult: 650 mg Child: 8 mg/kg (maximum 650 mg/dose) | Every 6-8 hours Every 8 hours |
- All antibiotics are administered by mouth unless otherwise specified. All doses administered for 7 to 10 days except for persistent relapsing infection (see text).
- Exchange transfusion should be performed in all B. divergens cases and considered for use in severe cases caused by other Babesia spp.
What's New
Wormser GP, et al. Emergence of Resistance to Azithromycin-Atovaquone in Immunocompromised Patients with Babesia microti Infection. Clin Infect Dis. 2010 Feb 1;50:381-6.
Guided Medline Search For:
Review Articles
Vannier E, et al. Human Babesiosis. N Engl J Med 2012;366:2397-407.
Blevins SM, et al. Blood smear analysis in babesiosis, ehrlichiosis, relapsing fever, malaria, and Chagas disease. Cleveland Clinic Journal of Medicine 2008;75:521-530.
Adhikari P, Mietzner T. Cell Mediated Immunity.