Study design
In the prospective ENDOCOVID study, monitoring will be carried out at admission (baseline), weekly, at discharge and, a follow-up 4 weeks post-discharge (if possible) (Figs. 1 and 2). The four- week post-discharge collection may be hampered by unpredictable lockdowns; therefore, this measurement may not be carried out in all the participants (that is why it is indicated as “when possible”). Measurements will include health questionnaires, anthropometric measurements, a cardiovascular physical examination, blood collection, Ultrasound-based vascular and endothelial measurements, and fundus imaging for retinal blood vessel analysis (Fig. 2). Results obtained at admission (baseline), weekly, at discharge and, 4 weeks post-discharge (if possible) (Fig. 1) will be compared. ENDOCOVID (protocol version 1 from August 13, 2020) has received ethics approval from Walter Sisulu University, South Africa (EC_202011_006 and 053/2020), as well as University of Illorin Teaching Hospital, Nigeria (NHREC/02/05/2010) and Lagos State University Teaching Hospital, Nigeria (NHREC04/04/2008). Protocol modifiers will be submitted to the respective ethics committees as well as to the clinical trial registration. Verbal and written informed consent will be obtained by the study clinician from each participant prior to being included into the study.
The study was registered under clinicaltrials.gov: NCT04709302, 13 January 2021, https://clinicaltrials.gov/show/NCT04709302.
Participant recruitment and study groups
Inclusion criteria
Participantsof the ENDOCOVID study population are patients with COVID-19, who at the same time are infected with HIV (with or without ART), or are HIV negative. Some may be admitted in the ICU. Participants will be recruited by qualified and trained researchers at hospitals based on pre-determined inclusion criteria. For inclusion into the study, participants must be positive for SARS-CoV-2 RNA and 18 years or older. Participants will be excluded from the study if they are less than 3 months post-partum and those with other co-infections than HIV such as tuberculosis (Fig. 2). Participants fulfilling the criteria will be invited into the study and asked to provide written informed consent.
Presence of SARS-CoV-2 RNA will be determined by real-time PCR. Subsequently, potential participants with unknown HIV status will be tested for HIV-infection via a rapid HIV test. A negative HIV test will be used to assign the participants to the HIV-negative group and a positive HIV test to the HIV-positive group, which is further split into two sub-groups according to the ART status of participants. Firstly, participants without ART as the “HIV-positive ART naïve” group. However, it has to be taken into consideration that, the majority of participants in this sub-group will start with ART, soon after recruitment. The third group are patients diagnosed as “HIV-positive on ART”. Therefore, we can evaluate cardiovascular risk and early endothelial changes in both the untreated and treated groups. SARS-CoV-2 RNA, CD4+ T-cell count, and HIV RNA viral load of the HIV-positive participants will be determined at admission (baseline), weekly, at discharge and, 4 weeks post-discharge (if possible) (Figs. 1 and 2). Participants are also required to refrain from smoking, drinking coffee or alcohol, or doing exercise for 4 h prior to the study. The menstrual phase of each female will be recorded.
Assessments: clinical and laboratory investigations
Health questionnaire, cardiovascular measurements, blood chemistry
General information will be obtained from a health questionnaire which include details about cardiovascular health including family and personal history of CVD, medication, smoking, and alcohol consumption (for detailed information, please find the full questionnaire in the supplementary material). Additionally, personal data about participants and their demographic characteristics based on the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) will be collected. Aspects related to the usage of medication, presence of co-infections and HIV disease characteristics (first diagnosis and duration of HIV infection, past and current regimens of ART treatment, viral load, nadir/current CD4+ T-cell count) will also be recorded [31]. Cardiovascular risk factors will be screened through analyses of (i) Lipid profile, (ii) glucose, (iii) glycated hemoglobin (HbA1c), (v) liver function enzymes, and (vi) high sensitivity hs-CRP, (vii) heart rate, (viii) blood pressure and (ix) body anthropometrics [32]. Together with evaluation of SARS-CoV-2 and HIV infection, these measurments in the participants will allow identification of common cardiovascular risk factors: smoking, alcohol consumption, overweight/obesity, hypertension, diabetes mellitus, dyslipidemia, renal impairment, inflammation, and liver disease.
Anthropometric assessment
Anthropometric measurements include body mass (BM), body height (h), sitting height (s), and the circumferences of waist and hip. Measurement of height will be performed by a Seca 217 portable stadiometer (Seca, Hamburg, Germany) and measurement of BM (in underwear) will be performed using a Seca 862 electronic scale (Seca, Hamburg, Germany). Body mass index (BMI) is computed as kg/m2 [33]. Waist circumference will be measured at the mid-point between the lower costal border, 10th rib, and the iliac crest, using a Lufkin W606P flexible steel tape (Apex Tool Group, Sparks Glencoe, MD, USA). Anthropometric measurements will be carried out according to the International Society for the Advancement of Kinanthropometry [34].
Subcutaneous Apidose tissue measurement to assess cardio-metabolic risk
Ultrasound is the most exact technique for thickness measurements of subcutaneous adipose tissue (SAT) layers. This method was recently standardised using eight sites to display SAT and distinguish between fat and present fibrous structures (the trunk (three), the arms (two), and the legs (three)). For calculating mean SAT thickness, it is necessary to carry out repeated series measurements of randomly selected sites of these eight standardized sites [35,36,37]. Advantages of using this method are that it avoids compression artifacts, distinguishes between fat tissue and neighbouring structures, is not invasive, does not use ionizing radiation, and is easily applicable in the field. The usage of this recent method in overweight and obese groups has recently been reported [36]. Ultrasound imaging is based on the pulse-echo technique whereby a series of ultrasound pulses (each several wavelengths long) is directed into the tissues. While ultrasound systems for diagnostics purposes utilize 1540 ms-1 for distance determination in soft tissues, in adipose tissues 1450 ms-1 [35] is used for measurement of thickness. To minimize fat compression errors, the ultrasound probe is normally placed on the measuring site without any pressure and on a thick layer (5 mm) of ultrasound gel between the probe and the skin. Ultrasound measurements will be performed according to the standardized ultrasound measurement approach [35].
Flow mediated dilation (FMD)
FMD is non-invasive technique, ideal for evaluating vascular endothelial functions. Measurement of vascular endothelial functions by FMD has been validated from a prognostic point of view, as well in successful monitoring of disease progression/regression in clinical studies [38,39,40,41]. We have also used it previously to assess the cardiovascular risk in PLHIV/AIDS in SSA in the ongoing EndoAfrica Study [39].
The FMD procedure is performed as described in detail in the EndoAfrica study [39]. Briefly, images of the right radial artery are obtained in B-mode with an Esaote MyLabTMFive US and 12 MHz linear probe (Esaote, Italy). Doppler mode is used to visualise and locate the radial artery with pulse repetition frequency (PRF) set at 6.7 kHz. In pulse wave mode, the angle of insonation is set to + 60°. Radial artery diameter and shear rate measures are recorded continuously with FMD Studio and Cardiovascular Suite version 2.8.1 software (Quipu, Italy), which makes use of automatic edge detection technology. The measurements begin with 1 min baseline recording (rest phase). The blood pressure cuff is then inflated to 50 mmHg above the systolic blood pressure value and it remains at this level for 5 min (ischaemic phase). After 5 min, the cuff is deflated leading to hyperaemia and increased shear rate inducing endothelium-dependent dilatation of the radial artery (post-ischemic recovery phase). The software calculates baseline, and maximum artery diameter, recovery artery diameter, FMD % (difference between maximum, and baseline diameter expressed as %), baseline shear rate and maximum shear rate.
Carotid intima-media thickness (IMT)
Carotid IMT is an indirect sonographic assessment of the degree of atheromatous vascular diseases. IMT is a biomarker of arterial wall thickening and therefore also atherosclerosis [42,43,44,45]. Enhancements in IMT associate with prevalent and incident cardiovascular morbidity and mortality, including coronary heart disease [45]. The thickness of the media and the intima of the vessels’ change following many conditions and it can be easily and reliably assessed with ultrasound using the B-mode of the common carotid arteries.
IMT measurements will be performed according to pre-established protocols [46]. Briefly, the participants are laid in supine position and the head tilted 30° to the left to evaluate of the right carotid artery, and 30° to the right for evaluation of the left carotid artery. After visualisation of the carotid artery bifurcation in B-mode, IMT measurements will be carried out on the posterior carotid wall on the right and on the left side, 1 cm from the carotid bifurcation. The measured segment must be free of atherosclerotic plaques and the lumen-intima and media-adventitia interfaces must be clearly defined. At least five sagittal measurements on each side are needed for obtaining the average IMT value. Carotid artery IMT average values greater than 1 mm are considered abnormal. The evaluation of the carotid IMT is carried out using Quality Intima-Media Thickness (QIMT) software. Th QIMT software enables automatic measurements of the intima-media thickness and calculates and records the median ± standard deviation IMT values during several cardiac cycles [45].
Pulse wave velocity (PWV)
PWV, a marker of aortic stiffness [47], is most commonly measured as the time it takes a pulse wave to travel from the carotid to the femoral arteries divided by the distance multiplied by 0.8. PWV can be measured by several devices. The non-invasive Vicorder device has been shown to have good reproducibility [48] - even when the assessor has limited experience in its usage [49] - and the results obtained reflect those obtained via invasive central blood pressure measurements [50] and those of SphygmoCor device [48]. Based on the criteria of the ARTERY Society Recommendations [51], the accuracy of PWV recorded by the Vicorder device has been described as “excellent” [52]. Vicorder measurements are performed by trained researchers. Patients lie in a supine position on a bed with the head raised to approximately 30°. PWV is measured by a cuff on the right carotid and the right thigh. The distance between the carotid and femoral arteries is recorded by measuring the length between the suprasternal notch and the mid-point of the thigh cuff. Measurements continue until pressure waveforms over the carotid and thigh are clear and reproducible. Subsequently, all tracings are reviewed, and, if necessary, repeated. Selected are only data that show clear pressure waveform upstrokes.
Retinal vessel analyses
Retinal imaging analysis is a procedure which enables us to visualize the microcirculation in the eye consisting of blood vessels that are less than ~ 150 μm and include the smallest resistance vessels. They form an important part of our circulatory system and assessment of these retinal microvasculature can provide an indication of cardiovascular health [40, 53]. Several studies have reported that there is a connection between changes in retinal microvasculature and coronary heart disease [54]. The ratio between the diameter of the retinal arteries and veins (A/V) reflects the predisposition and/or association with hypertension and atherosclerosis [55]. Quantitative features from the retinal images are therefore useful for identifying microvascular changes, which can be used as a predictive tool for assessment of the risk of developing cardiovascular diseases [56, 57].
In our study, a non-mydriatic, hand-held, portable digital retinal camera (Optomed Aurora, Optomed Oy, Oulu, Finland) will be used for collecting retinal images. Dimensions of vessels and microvascular state will be analyzed offline with the semi-automated MONA REVA software (VITO, Belgium). Analysis of retinal images will be performed by a trained person in this field. In order to prevent bias, the participant characteristics will not be provided to the person analyzing the images. The vessel widths and pattern features of both the right and left eye will be performed. The width is calculated from the largest six arterioles and venules which cross a zone of 0.5–1.0 disc diameter from the optic disc margin. Final calculations related to the average diameter of arterioles and venules in the retina are presented as central retinal arteriolar equivalent (CRAE) and central retinal venular equivalent (CRVE), respectively [58]. However, for analysis of retinal microvasculature pattern features such as tortuosity, fractal analysis, and lacunarity, it is necessary to measure all blood vessels crossing a zone of 2x disc diameter from the optic disc margin. A summary of the many features of the retina that can be characterized and calculated is provided by Prabhakara and colleagues [59].
Sample collection for SARS-Cov-2 RNA detection
Nasopharynegal swab will be used to obtain nasopharyngeal sample for the qualitative detection of SARS-Cov-2 RNA using real time PCR. Plain urine container will be used to obtain urine from participants for the detection of urine albumin and creatinine. Two collection tubes will be used for blood obtained from the ante-cubital vein: One 9-mL plain tube and another 6-mL EDTA tube. Some blood will then be dispensed into EDTA container for determination of CD4+ T cells and full blood count and viral load, while approx. 7 mls will then be dispensed into plain containers allowed to clot at room temperature, spun at 2000 g for 10 min; the serum will then be separated and cryopreserved at − 80 degree Celsius for analysis.
Serum biomarker analyses
Blood samples will be collected at admission (baseline), weekly, at discharge and, 4 weeks post-discharge (if possible), which is described in detail elsewhere [60] (Fig. 1). A number of plasmatic pro-inflammatory markers that play a role in endothelial dysfunction will be measured to supplement the endothelial function assessments done on the participants. Levels of serum cytokine and inflammatory markers, such as VCAM- 1, ICAM-1, TNF-α, TNF-β, and NF-κB will be measured at the same time for each participant using Luminex technology (Luminex Bio-Plex 200 system) [61, 62]. Markers of endothelial function such as ADMA will be measured using commercially available ELISA kits. Measurement of effects of COVID-19 on coagulation will be carried out via coagulation tests, calibrated automated thrombogram, thromboelastometry, impedance aggregometry, and markers of thrombin formation. These measurements are important as an increased level of D-dimer over 2500 μg/L is seen, for example, in all cases of pulmonary embolism [63]. Hence, patients with high levels of D-dimer will undergo CT angiography to visualize any accompanying thrombosis of segmental pulmonary arteries [63]. Screening for cardiovascular risk factors will be performed by measurements of (i) Lipid profile, (ii) glucose, (iii) HbA1c, (v) liver function enzymes, and (vi) high sensitivity, hs-CRP.
Data collection, management and statistical analysis
Data from the questionnaires, clinical evaluations, and biochemical analyses will be gathered and managed by Research Electronic Data Capture (REDCap) tools hosted at the Walter Sisulu University [64] (Fig. 1).
Statistical analyses will be performed in collaboration with the Biostatistics Unit in the Centre for Evidence-based Health Care, Walter Sisulu University. Descriptive statistical models (ANOVA, Chi Square) will be used for inter-group comparisons, and associations between independent and dependent variables will be tested by multivariate regression analysis models, taking into account confounding variables such as age, gender, ethnicity, smoking, alcohol consumption, medication, and BMI. For long term changes in the same individual, a repeated measures model approach will be engaged.
Strijdom et al., in their discussion of the EndoAfrica study design, suggested that sample sizes of up to 300 participants are sufficient to secure statistical power [39]. Fischer’s formula was also used for calculation of sample size: (n) = Z2P (1-P)D2. Since there are no data on the number of people with coinfection of HIV and SARS-CoV-2, the sample size was calculated using the prevalence rate of PLHIV of 4.9% in SSA (UNAIDS report, 2019) [65]. To achieve statistical significance, 94 HIV-infected persons are required. Since we have 3 groups, we will require a total of (94 × 3) = 282 participants. Due to 20–30% drop out rate, 20 more persons in each group will be added to this number, so that the final number of participants required in this project has similar participant numbers that were recruited in the EndoAfrica study (282 + 60 = 342 at each recruiting center). Demographic and background data (e.g., smoking and drinking status, BMI) will also be collected and included in the linear regression models as putative confounders [66].
