Cytomegalovirus retinitis in hospitalized people living with HIV in the late antiretroviral therapy era in São Paulo,Brazil

Abstract

Background

There is scarce information on AIDS-related cytomegalovirus (CMV) retinitis in middle-income countries. The objectives of this study were to identify the prevalence of active CMV retinitis in severely immunosuppressed people living with HIV (PLWHIV) and to describe its main features.

Methods

This retrospective cohort study was carried out at a tertiary center in São Paulo, Brazil. We included hospitalized adults PLWHIV with CD4 count ≤100 cells/μL, ≥ one quantitation of CMV DNA in plasma, and indirect ophthalmoscopy evaluation.

Results

Thirty-eight (21.6%) of 176 participants had at least an ophthalmoscopy diagnosis and only 3 (1.7%) individuals presented active CMV retinitis. All these participants were male, and retinitis was asymptomatic in 2 cases. Two participants had extraocular end-organ CMV disease and detectable CMV DNA in plasma.

Conclusions

These results show a low prevalence of active CMV retinitis in the evaluated population. However, 2 of 3 participants had asymptomatic active CMV retinitis and a fifth of participants had at least one ophthalmoscopy diagnosis, suggesting the need for routine ophthalmologic evaluation in hospitalized severely immunosuppressed PLWHIV. The profile of participants with active CMV retinitis was similar to that described in the pre-ART era and quantitation of CMV DNA in plasma was variable.

Keywords

cytomegalovirus retinitis epidemiology highly active antiretroviral therapy acquired immunodeficiency syndrome Brazil

Introduction

Acute cytomegalovirus (CMV) infection is common worldwide, is usually asymptomatic, and often remains latent in immunocompetent hosts. Chronic CMV infection can reactivate and cause end-organ disease (EOD) and death in immunosuppressed hosts.^1,2

Retinitis has been described as the most frequent CMV EOD and occurs in severely immunosuppressed patients living with human immunodeficiency virus (PLWHIV).^2,3 Retinal lesions occur by hematogenous dissemination and retinitis can be asymptomatic or oligosymptomatic (when lesions are peripheral), and symptomatic (when lesions are in macula or optic nerves).^3,4

Before the combined antiretroviral therapy (ART) era, approximately one-third of severely immunosuppressed PLWHIV had CMV retinitis in high-income countries.⁵ In this scenario, the lesions tended to progress, despite anti-CMV treatment,² and disseminated CMV disease was a common cause of morbidity and mortality in this set of patients.^1,6

After the introduction of ART, studies from high-income countries reported a dramatic reduction in the incidence of CMV retinitis, as well as the presence of inactive retinal lesions^6–8 due to effective immune reconstitution.^4,9 However, active CMV retinitis continues to occur in subgroups of severely immunosuppressed PLWHIV, due to a variety of reasons, including late HIV diagnosis, nonadherence to ART, and antiretroviral resistance.

The burden of CMV retinitis in the late ART era is not fully known in low- and middle-income countries, where retinal examination is rarely performed routinely, and diagnosis is often delayed, following vision damage or even blindness. Thus, CMV retinitis poses a serious challenge in resource-limited settings, as this opportunistic disease has historically been neglected, largely undiagnosed and untreated.²

The objectives of the present study are to identify the prevalence of active CMV retinitis in severely immunosuppressed PLWHIV and describe the main epidemiological and clinical characteristics of these individuals in São Paulo, Brazil.

Methods

A retrospective cohort study was carried out at the Instituto de Infectologia Emílio Ribas (IIER), a tertiary health care center, located in São Paulo, Brazil. Patients were consecutively admitted from January 1 to 31 December 2019.

The inclusion criteria of the study were patients with age ≥18 years, diagnosis of HIV infection, CD4 count ≤100 cells/μL, results of quantitative CMV DNA in plasma, and indirect ophthalmoscopy evaluation. The exclusion criteria were patients using ganciclovir and/or foscarnet 30 days before hospital admission for any indication, and no access to or absence of medical records.

CMV retinitis was diagnosed by experienced ophthalmologists and classified into active or inactive disease by indirect fundoscopy with fully dilated pupils. Photographic registers were not available.

In routine clinical conditions at IIER, indirect ophthalmoscopy evaluation and quantitation of CMV DNA in plasma are usually requested in patients with CD4 count ≤100 cells/μL. The management of the patients evaluated in this study followed the individual decisions of the attending physicians, including the use of anti-CMV therapy in cases with CMV viremia but no CMV EOD.

Patients were selected from a list of hospital admissions prepared by the Medical Archives Section of IIER. Then, we reviewed the electronic charts of the patients and other sources of laboratory (SISCEL and Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC-FMUSP) or therapeutic (SICLOM and TB WEB) information. Quantitation of CMV DNA in plasma was performed at HC-FMUSP using the Abbott RealTime CMV assay (linear range: 31.2–156.000.000 UI/mL). All data were registered in a standardized electronic form. Continuous variables were described with medians and interquartile ranges (IQRs). Categorical variables were described as frequency and percentages. This study was designed and reported using STROBE guidelines.¹⁰ This report is a subanalysis of a study approved by the Scientific Division (Protocol No. 38/2020) and the Ethics Committee of the IIER (CAAE 38976520.1.0000.0061).

Results

Initially, we identified 833 PLWHIV admitted to IIER during the study period: 14 cases were under 18 years old; 78 cases had no CD4 count; 397 cases had a CD4 count >100 cells/μL; 72 cases had no quantitation of CMV DNA in plasma; 72 cases had no ophthalmologic evaluation; and in 24 cases, the medical records were not found. Thus, 176 patients were included.

Table 1 presents the baseline characteristics of the 176 patients included in this study. Median (range) age was 37 (19–68) years and 136 (77.8%) of patients were males. Median time of HIV diagnosis (range) was 7 (0–35) years and 131 (74.4%) patients had received ART. One-hundred sixty-five (98%) of 168 patients with serum anti-CMV IgG test showed positive results and 111 (63%) patients had CMV DNA detected in plasma with median CMV viral load (range) of 396 (32–969 535) IU/mL.

Table 1.

Main characteristics of 176 severely immunosuppressed people living with HIV with ophthalmoscopy evaluation at the Instituto de Infectologia Emilio Ribas, São Paulo, Brazil, between 1 January 2019 and 31 December 2019.

Characteristics	n (%)
Age, years, median (range)	37 (19–68)
Male, n (%)	136 (77.8)
HIV diagnosis, years, median (range)	7 (0–35)
CD4⁺ count, median (range), cell/μL	17 (1–99)
Prior opportunistic infection, n (%), n = 158	79 (50)
Prior or current use of cART, n (%)	131 (74.4)
Undetectable HIV viral load, n (%)	18 (10.2)
CD4⁺ count, cells/μL, median (range)	25 (1–100)
HIV viral load, log/μL, median (range), n = 173	4.92 (0–6.9)
Positive anti-CMV IgG antibodies, n (%), n=168	165 (98)
CMV DNA detection in plasma, n (%)	111 (63.1)

Notes. cART: combined antiretroviral therapy. CMV: cytomegalovirus.

Six (3.4%) cases had CMV retinitis, and 3 (1.7%) of these 6 had active CMV retinitis. Table 2 presents the ophthalmologic diagnoses of the 176 patients of this study. Thirty-eight (21.6%) patients had at least one ophthalmoscopy diagnosis. The 3 patients with active CMV retinitis were males, had a median age (range) of 40 (37–42) years, median (range), HIV diagnosis of 11 (4–15) years, and none were being treated with ART. Two patients had no visual complaints and no extraocular end-organ CMV disease. The median (range) CD4 count was 12 (2–51) cells/μL; the 3 patients had positive serum anti-CMV IgG and 2 patients had CMV DNA detected in plasma. The 3 patients received anti-CMV therapy and were discharged to home. Table 3 shows the main characteristics of the 3 patients with active CMV retinitis.

Table 2.

Ophthalmoscopy diagnoses in 176 severely immunosuppressed people living with HIV at the Instituto de Infectologia Emilio Ribas, São Paulo, Brazil, between 1 January 2019 and 31 December 2019.

Ophtalmoscopy diagnoses	n (%)
Active CMV retinitis	3 (1.7)
HIV microangiopathy	11 (6.3)
Retinal scare^a	10 (5.7)
Glaucoma	4 (2.3)
Thrombocytopenic retinal hemorrhages	4 (2.3)
Intracranial hypertension papilledema	2 (1.1)
Myopia	2 (1.1)
Mycobacterium tuberculosis IRIS	1 (0.6)
Retinal dystrophy	1 (0.6)
Normal findings	138 (78.4)
Total^b	177

Notes. CMV: Cytomegalovirus. IRIS: immune reconstitution inflammatory syndrome.

^aCMV retinitis, n = 3; toxoplasmosis, n = 1; trauma, n = 1; retinal detachment, n = 1; unknown cause, n = 4.

^bOne participant had myopia and glaucoma.

Table 3.

Details of the three people living with HIV and active cytomegalovirus retinitis admitted at Instituto de Infectologia Emilio Ribas, São Paulo, Brazil, between 1 January 2019 and 31 December 2019.

Case	Sex, age (years)	HIV diagnosis (years)	Prior opportunistic	CD4⁺ count (cell/μl)/Plasma HIV VL (copies/mL)	Visual complaint	Indirect ophtalmoscopy	Plasma CMV VL (copies/ml)*	Extra-ocular CMV end-organ disease	Other diagnosis	Anti-CMV therapy	ART	Outcome (in hospital/6 months)
1	Male, 37	4	Yes	51/886.663	Right amaurosis	Right CMV retinitis	396^a	Not	Pulmonary tuberculosis	Ganciclovir (8 days)	None	Left against medical advice/Death
2	Male, 40	11	No	2/177	Not	Left CMV Retinitis	124.509^b	Encephalitis, polyradiculopathy	Oral candidíasis	Ganciclovir (33 days) +foscarnet (26 days)	TDF+3TC+ATV/r	Discharged home/unknown
3	Male, 42	15	Yes	12/1.337.101	Not	Right CMV retinitis	Undetectable^b	Polyradiculopathy	Disseminated histoplasmosis	Ganciclovir (42 days)	DRV/r + DTG	Discharged home/death

Notes. CMV: cytomegalovirus. VL: viral load. ART: combined antiretroviral therapy.

*Undetectable: <31.2 UI/mL.

^aSample collected on day 4 of the anti-CMV therapy.

^bSample collected before starting of the anti-CMV therapy.

Discussion

The present study identified a low prevalence (1.7%) of active CMV retinitis among PLWHIV at risk of having CMV EOD, since they were hospitalized with severe immunosuppression (CD4 count ≤100 cells/mm³), showed a high rate of prior cytomegalic infection (98% with serum anti-CMV IgG antibodies) and most of them had CMV viremia (63% with detection of CMV DNA in plasma).

Cytomegalovirus infection and CMV retinitis has followed the history of acquired immunodeficiency syndrome (AIDS) since the initial reports of the disease.^11,12 CMV disease of an organ other than liver, spleen, or lymph nodes were included early as an AIDS-defining condition in the classification of the US Centers for Disease Control and Prevention.¹³ Retinitis was the most common clinical presentation of CMV EOD (80%) in severely immunosuppressed PLWHIV.^1,14,15 However, autopsy studies showed that CMV retinitis is a component of systemic CMV disease due to the concomitant involvement of extraocular organs.^16,17

Before the introduction of ART, about 20–30% of severely immunosuppressed PLWHIV developed CMV retinitis in high-income countries.^18,19 Following the availability of ART and improvements in early diagnosis of treatment of HIV infection, the incidence of CMV retinitis in the United States declined by over 95%. CMV retinitis has been neglected in low- and middle-income countries and reported prevalence is scarce and variable.²⁰ Studies of CMV retinitis in the pre-ART era reported 20–30% of CMV retinitis in severely immunosuppressed PLWHIV in Brazil,^21,22 12%–30% in Asia,^2,23,24 and <5% in Africa.^25,26 Following the availability of ART, information on CMV retinitis continues to be scarce in low- and middle-income countries. Of 65 studies (n = 20 280 PLWHIV), only 5 studies (n = 2836 patients) were from Latin America.²⁰ Low prevalence of CMV retinitis was reported in Latin America (<5% in Brazil and Peru)^20,27 and in Africa (<3%)²⁴ in the ART era. In contrast, maintenance of high prevalence has been reported in Asia (∼15%).^9,20,28,29 These results are affected by multiple factors, but some individual variables may be more relevant depending on the epidemiological scenario. For example, free and universal access to ART in most countries in Latin America suggests a protective strategy to reduce the prevalence of CMV retinitis;⁸ the low prevalence of CMV retinitis in Africa suggests that PLWHIV either die from other diseases before they enter the stage of severe immunosuppression or their survival at this stage is very short;²⁶ and the persistent high prevalence of CMV retinitis in Asia suggests the possible influence of genetic factors.^30,31

Patients of this study with active CMV retinitis were a median age of 40 years and all were males, consistent with other studies.^9,15,17,29 There are no conclusive data indicating increased risk of CMV retinitis in males, and this finding may only reflect the epidemiological trends. Patients with CMV retinitis had a median CD4 count of only 12 cells/μL, median time of HIV diagnosis of 11 years, and had received ART. Thus, these patients showed a demographic and laboratory profile similar to that described in the pre-ART era.¹⁶ This fact demonstrates the need to improve retention and re-engagement strategies in the care of PLWHIV.

Median survival of PLWHIV with CMV retinitis was approximately 10 months in the pre-ART era.³² However, PLWHIV with CMV retinitis remain at increased risk of mortality in the ART era.³³ The 3 patients with active CMV retinitis of this study were discharged from the hospital, but 2 died within 6 months and the other was lost to follow-up. This result confirms the poor outcome of a vulnerable subset of PLWHIV in recent years.

Acute CMV infection is probably related to socioeconomic and environmental factors.³⁴ Consequently, serum prevalence of CMV is high in low- and middle-income countries, including Brazil, as shown in this study, in which 98% of patients had positive anti-CMV IgG antibodies. The high CMV viremia prevalence observed in this study (63%) was expected considering the profile of the patients included. CMV DNA detection is not currently recommended for diagnosis of CMV EOD because of its poor positive predictive value in people with advanced HIV disease.³⁵ In addition, CMV DNA detection in plasma or whole blood is often present in CMV EOD and a negative plasma result does not rule out this outcome.³⁵ This spectrum of results were observed in the patients with active CMV retinitis of the present study.

Cytomegalovirus EOD is best prevented by using ART to maintain CD4⁺ count >100 cells/mm³ and to suppress the HIV viral load.^3,6 Nowadays, the value of performing periodic ophthalmology examinations in PLWHIV without visual abnormalities and CD4⁺ counts ≤50–100 cells/mm³ is unknown in high-income countries, mainly due to the significant decline in the incidence of CMV retinitis in the ART era.³⁴ This recommendation probably cannot be extrapolated to most low- and middle-income countries due to the higher proportion of PLWHIV subgroups with severe immunosuppression and the relevant proportion of asymptomatic CMV retinitis. We recommend that every patient with a CD4⁺ count <100 cells/mm³ should undergo a baseline ophthalmologic examination. This procedure allows identifying a broad spectrum of diseases in severely immunosuppressed individuals, as shown in this study, where one in five patients had at least one ophthalmoscopy diagnosis.

This study has potential limitations. First, this was a retrospective study carried out in a single urban center from a middle-income country and only hospitalized patients were included. Therefore, we should be cautious about extrapolating from these results. In this study, ophthalmological evaluations were performed by experienced specialists, and CMV DNA detection in plasma was carried out, which are resources that are usually unavailable in resource-limited settings and which reinforce the reliability of our results. Second, the sample size was not calculated, and we used a convenience sample. However, we included consecutive cases admitted within a period of one year. The low prevalence CMV retinitis limited further analysis of association, but it reflects the current epidemiological scenario at least in hospitalized patients. Third, a relevant number of severely immunosuppressed patients were excluded because they did not undergo ophthalmologic evaluation. This result demonstrates the loss of opportunities for routine ophthalmologic evaluation in high-risk hosts and indicates the need to reinforce its importance. Finally, the ophthalmologic diagnosis was neither detailed (i.e., localization of the retinal lesions) nor photographically documented, a fact that reflects the clinical practice in our hospital.

In conclusion, we identified a low prevalence of active CMV retinitis in severely immunosuppressed PLWHIV admitted in a tertiary referral center from a middle-income country. Two of three patients with active CMV retinitis had no ophthalmologic symptoms and one of five patients of this study had at least one ophthalmoscopic diagnosis, justifying the recommendation of routine ocular evaluation in severely immunosuppressed PLWHIV. The profile of patients with active CMV retinitis was similar to that described in the pre-ART; all of them received anti-CMV therapy, and CMV DNA detection in plasma was variable.

Footnotes

Acknowledgements

We would like to thank the patients and their relatives.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Giuliane Bogoni

Rodovaldo M Lucas Júnior

Gustavo Arthur Reis Schneider

Nidyanara F Castanheira de Souza

Maria K Carvalho

Rosa Maria Marcusso

Jose E Vidal

References

Jabs

. Cytomegalovirus retinitis and the acquired immunodeficiency syndrome-bench to bedside: LXVII Edward Jackson Memorial Lecture. Am J Ophthalmol 2011; 151(2): 198–216.

Heiden

Ford

Wilson

, et al. Cytomegalovirus retinitis: the neglected disease of the AIDS pandemic. Plos Med 2007; 4(12): 1845–1852.

Jabs

Van Natta

Kempen

, et al. Characteristics of patients with cytomegalovirus retinitis in the era of highly active antiretroviral therapy. Am J Ophthalmol 2002; 133(1): 48–61.

Liu

Chen

Kamphaengkham

, et al. Diagnostic utility of ocular symptoms and vision for cytomegalovirus retinitis. PLoS One 2016; 11(10): e0165564.

Holbrook

Jabs

Weinberg

, et al. Visual loss in patients with cytomegalovirus retinitis and acquired immunodeficiency syndrome before widespread availability of highly active antiretroviral therapy. Arch Ophthalmol 2003; 121(1): 99–107.

Yust

Fox

Burke

, et al. Retinal and extraocular cytomegalovirus end-organ disease in HIV-infected patients in Europe: a EuroSIDA study, 1994-2001. Eur J Clin Microbiol Infect Dis 2004; 23(7): 550–559.

Deayton

Wilson

Sabin

, et al. Changes in the natural history of cytomegalovirus retinitis following the introduction of highly active antiretroviral therapy. AIDS 2000; 14(9): 1163–1170.

Schwarcz

Chen

Vittinghoff

, et al. Declining incidence of AIDS-defining opportunistic illnesses: results from 16 years of population-based AIDS surveillance. AIDS 2013; 27(4): 597–605.

Colby

Teoh

, et al. Prevalence and predictors of cytomegalovirus retinitis in HIV-infected patients with low CD4 lymphocyte counts in Vietnam. Int J STD AIDS 2014; 25(7): 516–522.

10.

von Elm

Altman

Egger

, et al. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. Lancet 2007; 370(2596): 1453–1457.

11.

Holland

Gottlieb

Yee

, et al. Ocular disorders associated with a new severe acquired cellular immunodeficiency syndrome. Am J Ophthalmol 1982; 93(4): 393–402.

12.

Centers for Disease Control (CDC) . Pneumocystis Pneumonia--Los Angeles [internet]. MMWR Morb Mortal Wkly Rep 1981.

13.

Centers for Disease Control (CDC) . Classification system for human T-lymphotropic virus type III/lymphadenopathy-associated virus infections [internet]. MMWR Morb Mortal Wkly Rep 1986.

14.

Gallant

Moore

Richman

, et al. Incidence and natural history of cytomegalovirus disease in patients with advanced human immunodeficiency virus disease treated with zidovudine. The Zidovudine Epidemiology Study Group. J Infect Dis 1992; 166(6): 1223–1227.

15.

Jabs

Green

Fox

, et al. Ocular manifestations of acquired immune deficiency syndrome. Ophthalmology 1989; 96(7): 1092–1099.

16.

Holland

Vaudaux

Jeng

, et al. UCLA CMV Retinitis Study Group. Characteristics of untreated AIDS-related cytomegalovirus retinitis. I. Findings before the era of highly active antiretroviral therapy (1988 to 1994). Am J Ophthalmol 2008; 145(1): 5–11.

17.

Holland

Vaudaux

Jeng

, et al. Characteristics of untreated AIDS-related cytomegalovirus retinitis. I. Findings before the era of highly active antiretroviral therapy (1988 to 1994). Am J Ophthalmol 2008; 145(1): 5–11.

18.

Jabs

Van Natta

Holbrook

, et al. Longitudinal study of the ocular complications of AIDS: 1. Ocular diagnoses at enrollment. Ophthalmology 2007; 114(4): 780–786.

19.

Sugar

Jabs

Ahuja

, et al. Studies of the ocular complications of AIDS research group. Incidence of cytomegalovirus retinitis in the era of highly active antiretroviral therapy. Am J Ophthalmol 2012; 153(6): 1016–1024.

20.

Ford

Shubber

Saranchuk

, et al. Burden of HIV-related cytomegalovirus retinitis in resource-limited settings: a systematic review. Clin Infect Dis 2013; 57(9): 1351–1361.

21.

Arruda

Muccioli

Belfort

. Achados Achados Oftalmológicos em Infectados Pelo HIV na Era Pós-Haart e Comparação Com Série de Pacientes Acientes Avaliados Aliados no Período Pré-Haart. Rev Assoc Med Bras 2004; 50(2): 148–152.

22.

Matos

KTF

Santos

MCM

Muccioli

. Manifestações oculares do paciente infectado pelo HIV atendido no Departamento de Oftalmologia da Universidade Federal de São Paulo. Rev Assoc Med Bras 1999; 45(4): 323–326.

23.

Gharai

Venkatesh

Garg

, et al. Ophthalmic manifestations of HIV infections in India in the era of HAART: analysis of 100 consecutive patients evaluated at a tertiary eye care center in India. Ophthalmic Epidemiol 2008; 15(4): 264–271.

24.

Pathai

Gilbert

Weiss

, et al. Differing spectrum of HIV-associated ophthalmic disease among patients starting antiretroviral therapy in India and South Africa. Trop Med Int Health 2011; 16(3): 356–359.

25.

Jaffar

Ariyoshi

Frith

, et al. Retinal manifestations of HIV-1 and HIV-2 infections among hospital patients in The Gambia, west Africa. Trop Med Int Health 1999; 4(7): 487–492.

26.

Kestelyn

. The epidemiology of CMV retinitis in Africa. Ocul Immunol Inflamm 1999; 7(3–4): 173–177.

27.

Telles

Fernandes

Barros

, et al. Neurological manifestations in people living with HIV/AIDS in the late cART era: a prospective observational study at a tertiary healthcare center in São Paulo, Brazil. HIV Res Clin Pract 2021; 22(4): 87–95.

28.

Jirawison

Yen

Leenasirimakul

, et al. Telemedicine screening for cytomegalovirus retinitis at the point of care for human immunodeficiency virus infection. JAMA Ophthalmol 2015; 133(2): 198–205.

29.

Tang

Sun

, et al. Clinical features of cytomegalovirus retinitis in HIV infected patients. Front Cel Infect Microbiol 2020; 10: 136.

30.

Sezgin

Jabs

Hendrickson

, et al. Effect of host genetics on the development of cytomegalovirus retinitis in patients with AIDS. J Infect Dis 2010; 202(4): 606–613.

31.

Sezgin

van Natta

Ahuja

, et al. Studies of the ocular complications of AIDS research group. Association of host genetic risk factors with the course of cytomegalovirus retinitis in patients infected with human immunodeficiency virus. Am J Ophthalmol 2011; 151(6): 999–1006.

32.

Hoover

Peng

Saah

, et al. Occurrence of cytomegalovirus retinitis after human immunodeficiency virus immunosuppression. Arch Ophthalmol 1996; 114(7): 821–827.

33.

Jabs

Ahuja

Van Natta

, et al. Course of cytomegalovirus retinitis in the era of highly active antiretroviral therapy: five-year outcomes. Ophthalmology 2010; 117(11): 2152–2161.

34.

de Jong

Galasso

Gazzard

, et al. Summary of the II international symposium on cytomegalovirus. Antivir Res 1998; 39(3): 141–162.

35.

Jabs

Martin

Forman

, et al. Cytomegalovirus (CMV) blood DNA load, CMV retinitis progression, and occurrence of resistant CMV in patients with CMV retinitis. J Infect Dis 2005; 192(4): 640–649.