Is laboratory screening prior to antiretroviral treatment useful in Johannesburg, South Africa? Baseline findings of a clinical trial.

BACKGROUND: Screening for renal, hepatic and haematological disorders complicates the initiation of current first-line antiretroviral therapy (ART). Each additional test done adds substantial costs, both through direct laboratory expenses, but also by increasing the burden on health workers and patients. Evaluating the prevalence of clinically relevant abnormalities in different population groups could guide decisions about what tests to recommend in national guidelines, or in local adaptations of these. METHODS: As part of enrolment procedures in a clinical trial, 771 HIV-positive adults, predominantly from inner-city primary health care clinics, underwent laboratory screening prior to ART. Participants had to be eligible for ART, based on the then CD4 eligibility threshold of 350 cells/µL, antiretroviral naïve and have no symptoms of peripheral neuropathy. RESULTS: Participants were mostly female (57%) and a mean 34 years old. Creatinine clearance rates were almost all above 50 mL/min (99%), although 5% had microalbuminuria. Hepatitis B antigenaemia was common (8% of participants), of whom 40% had a raised AST/ALT, though only 2 had transaminase levels above 200 IU/L. Only 2% of participants had severe anaemia (haemoglobin <8 g/dl) and 1% neutropaenia (neutrophils <0.75 × 10^9/L). Costs per case detected of hepatitis B infection was USD135, but more than USD800 for a raised creatinine. CONCLUSIONS: Hepatitis B continues to be a common co-infection in HIV-infected adults, and adds complexity to management of ART switches involving tenofovir. Routine renal and haematological screening prior to ART detected few abnormalities. The use of these screening tests should be assessed among patients with higher CD4 counts, who may even have fewer abnormalities. Formal evaluation of cost-effectiveness of laboratory screening prior to ART is warranted.

Assessment of T-dependent and T-independent immune responses in cattle using a B cell ELISPOT assay.

Understanding the mechanisms that maintain protective antibody levels after immunisation is important for vaccine design. In this study, we have determined the kinetics of plasma and memory B cells detectable in the blood of cattle immunised with model T-dependent or T-independent antigens. Immunisation with the T-D antigen resulted in an expansion of TNP-specific plasma cells post-TNP primary and booster immunisations, which was associated with increased titres of TNP-specific IgG antibodies. Although no TNP-specific memory B cells were detected in the T-D group following the primary immunisation, we detected an increase in the number of TNP-specific memory B cells post-TNP boost. In contrast, no TNP-specific plasma or memory B cells were detected after primary or secondary immunisation with the T-I antigen. We then investigated if immunisation with a third party antigen (tetanus toxin fragment C, TTC) would result in a bystander stimulation and increase the number of TNP-specific plasma and memory B cells in the T-D and/or T-I group. TTC immunisation in the T-D group resulted in a small increase in the number of TNP-specific plasma cells post-TTC primary immunisation and boost, and in an increase in the number of TNP-specific memory B cells post-TTC boost. This bystander effect was not observed in the animals previously immunised with the T-I antigen. In conclusion, the present study characterised for the first time the B cell response in cattle to immunisation with T-D and T-I antigens and showed that bystander stimulation of an established T-D B cell memory response may occur in cattle.

Masked hypertension unfavourably affects haemostasis parameters.

OBJECTIVE. Recent evidence demonstrates that masked hypertension (MH) is a significant predictor of cardiovascular disease. The aim of our study was to examine the impact of MH on haemostasis parameters and to compare the findings to those of healthy normotensives matched for age, sex, body mass index and the rest of risk factors. DESIGN AND METHOD. 130 (60 male, 70 female) healthy subjects mean age 45 ± 12 years who had clinic blood pressure < 140/90 mmHg were studied. The whole study population underwent 24-h ambulatory blood pressure monitoring (ABPM). According to the ABPM recordings, 24 individuals (eight males, 16 females) had MH (daytime systolic blood pressure ≥ 135 mmHg or daytime diastolic blood pressure ≥ 85 mmHg - group A) and the remaining 106 subjects (52 males, 54 females) had normal ABPM recordings - group B. Fibrinogen, thrombomodulin ™, the antigens of plasminogen activator inhibitor 1 (PAI-1Ag) and tissue plasminogen activator (tPA-Ag) were determined in the two groups. Results. The PAI-1 Ag, tPA-Ag, fibrinogen and TM levels were significantly higher in the masked hypertensive group than to normotensive control group. CONCLUSIONS. Our findings suggest that subjects with MH have significantly higher fibrinogen, TM, PAI-1Ag and tPA-Ag plasma levels compared with normotensives. This observation may have prognostic significance for future cardiovascular events in subjects with MH and needs further investigation.

A quantitative assessment of primary and secondary immune responses in cattle using a B cell ELISPOT assay.

The aim of the study was to build a comprehensive picture of the appearance in the blood stream of Ag-specific plasma cells and memory B cells in the bovine model. For this purpose, we have developed a method allowing the detection and quantification of both cell types within individual calves immunised with ovalbumin. During the primary response, we detected a burst of ovalbumin-specific plasma cells at days 6 and 7 post-immunisation, which was followed by the production of specific Ab, whereas a gradual increase of memory B cells was only detected from day 15. As expected, a boost immunisation performed 7 weeks later induced a quicker and stronger secondary response. Indeed, a burst of plasma cells was detected in the blood at days 3 and 4, which was followed by a strong increase in Ab titres. Furthermore, a burst of memory B cells, and not a gradual increase, was detected at days 5 and 6 post-boost immunisation. Importantly, we showed a strong correlation between the anti-ovalbumin-specific IgG titres detected 5 months after secondary immunisation and the plasma cell numbers detected in the blood at the peak response after secondary immunisation. The detection and quantification of plasma cells following an mmunisation/vaccination strategy could constitute a very effective means for predicting the magnitude and longevity of an Ab response.

Tests for the measurement of factor VII-activating protease (FSAP) activity and antigen levels in citrated plasma, their correlation to PCR testing, and utility for the detection of the Marburg I-polymorphism of FSAP.

BACKGROUND: The single nucleotide Marburg I (MRI) polymorphism of the factor VII-activating protease (FSAP) gene, the prourokinase-activating activity of FSAP, and antigen levels of FSAP in plasma have been associated with incidence and progression of carotid stenosis and venous thromboembolism. However, more information on the extent of these associations, potential further ones, and respective clinical utilities remain to be determined. At present, testing is performed mainly by PCR assays based on probes or SYBR Green I. Some studies include testing for antigen levels of total FSAP and its ability to activate prourokinase. To test large cohorts, it is beneficial to rely on assays that are cost-effective, reliable, easy to use, rapid to perform, and that may eventually be automated. In addition, it appears advantageous to use functional tests or tests that determine antigen levels as they may relate more closely to the phenotype than the genotype does. METHODS: Tests for the measurements of antigen levels of FSAP and its prourokinase-activating activity were improved and performance characteristics assessed. To determine the FSAP genotypes, an amplification created restriction site (ACRS) PCR test was developed. RESULTS: Key performance characteristics of the FSAP activity and antigen tests were as follows: measuring range: 350-1400 mPEU/mL and 1.8-120 ng/mL, total coefficients of variation (CV): 5%-20% and 5%-14%, within-run CV: 4%-11% and 2.3%-12%, and run-to-run CV: 2%-17% and 4.3%-8.3%, respectively. The ratio of the activity and antigen level of FSAP correctly identified the FSAP genotypes of 126 samples tested. CONCLUSIONS: The ACRS PCR test is useful for laboratories that do not have the equipment to perform probe or SYBR Green I based real-time PCR. Furthermore, the tests developed for the determination of FSAP activity and antigen levels are convenient for determining clinical correlations, even for large population studies. The ratio of activity and antigen level of FSAP appears to be a promising and efficient alternative to molecular diagnostic techniques to detect the MRI polymorphism of FSAP.

Assessment of KL-6 as a tumor marker in patients with hepatocellular carcinoma.

AIM: To investigate the clinical significance of KL-6 as a tumor marker of HCC in two different ethnic groups with chronic liver disease consecutively encountered at outpatient clinics. METHODS: Serum KL-6 was measured by the sandwich enzyme immunoassay method using the KL-6 antibody (Ab) as both the capture and tracer Ab according to the manufacturer's instructions (Eisai, Tokyo, Japan). Assessment of alpha fetoprotein (AFP) and protein induced vitamin K deficiency or absence (PIVKA-II) was performed in both groups using commercially available kits. RESULTS: A significantly higher mean serum KL-6 (556+/-467 U/L) was found in HCC in comparison with non-HCC groups either with (391+/-176 U/L; P<0.001) or without (361+/-161 U/L; P<0.001) liver cirrhosis (LC). Serum KL-6 level did not correlate with either AFP or PIVKA-II serU/Levels. Using receiver operating curve analysis for KL-6 as a predictor for HCC showed that the area under the curve was 0.574 (95%CI = 0.50-0.64) and the KL-6 level that gave the best sensitivity (61%) was found to be 334 U/L but according to the manufacturer's instructions; a cut-off point of 500 U/L was used that showed the highest specificity (80%) in comparison with AFP and PIVKA-II (78% vs 72% respectively). Combining the values of the three markers improved specificity of AFP for HCC diagnosis from 78% for AFP alone; 93% for AFP plus PIVKA-II to 99% for both plus KL-6 value (P<0.001). Mean serum alkaline phosphatase level was significantly higher in KL-6 positive (564+/-475) in comparison with KL-6 negative (505+/-469) HCC patients (P = 0.021), but such a difference was not found among non-HCC corresponding groups. CONCLUSION: KL-6 is suggested as a tumor for HCC. Its positivity may reflect HCC-associated cholestasis and/or local tumor invasion.

[Assessment of serum markers KL-6 and SP-D for interstitial pneumonia associated with connective tissue diseases].

There are a lot of difficulties in the estimation of interstitial pneumonia and subsequent pulmonary fibrosis associated with connective tissue diseases. Recently, serum KL-6 (KL-6) and serum surfactant protein D (SP-D) have been reported to be useful to estimate the severity of interstitial pneumonia. We investigated the usefulness of these serum markers comparing to the spirometric parameters in patients with interstitial pneumonia associated with connective tissue diseases. We found significant inverse correlation between KL-6 and spirometric % VC. Furthermore, KL-6 was more significantly inverse-related with %DLco. On the other hand, we found neither correlation between SP-D and %VC, nor between SP-D and %DLco, suggesting SP-D level seems to be not affected by the degree of pulmonary fibrosis itself. These results indicate that KL-6 is useful to estimate the severity of pulmonary fibrosis more precisely than SP-D in patients with interstitial pneumonia associated with connective tissue diseases.

Tissue polypeptide-specific antigen in pediatric patients: assessment of normal values.

Measurement of serum concentrations of tissue polypeptide-specific Antigen (TPS) has been demonstrated to the useful in diagnosis and monitoring of adult epithelial tumors. So far, no data have been available on normal or pathologic TPS values in children. Therefore, the present study was designed to evaluate the normal values of TPS in childhood. Using a commercial enzyme linked immunosorbent assay (ELISA) kit, serum TPS was determined in 361 healthy children. Median (M) TPS was found to be 107 U/l at birth (n = 124). By the end of the first week, the value rose to M = 150 U/l (n = 68) and then continuously decreased with age (1 week-1 year, n = 45, M = 88 U/l; 1-7 years, n = 75, M = 51 U/l) until reaching the adult level (8-18 years, n = 49, M = 34 U/l). Additionally, the serum TPS values of 45 mothers right after delivery (M = 161 U/l) were assessed, and there was no correlation to the marker levels determined in the cord blood of their children. The age-dependent distribution of serum TPS in healthy children must be taken into account in the clinical application of this tumor marker.

Comparison of serum 7S fragment of type IV collagen and serum central triple-helix of type IV collagen for assessment of liver fibrosis in patients with chronic viral liver disease.

BACKGROUND/AIMS: A competitive radioimmunoassay for serum 7S fragment of type IV collagen (7S collagen) using a polyclonal antibody against 7S collagen and a sandwich enzyme immunoassay for serum central triple-helix of type IV collagen (IV collagen) using two monoclonal antibodies against the pepsin-solubilized type IV collagen may be used as diagnostic aids for liver fibrosis in clinical medicine. We compared the clinical usefulness for assessing liver fibrosis of serum 7S collagen and IV collagen tests in chronic viral liver diseases, and also examined the elution pattern of 7S collagen- and IV collagen-related antigens in serum by gel filtration analysis. METHODS: Serum 7S collagen and IV collagen levels were assayed in 151 patients with chronic viral liver disease and 30 healthy control subjects. RESULTS: Gel filtration on the Sephacryl S400HR column revealed that the 7S collagen antigenicity in serum was heterogeneous, whereas the IV collagen antigen in serum was uniform in size. Serum levels of 7S collagen and IV collagen showed increases closely correlated with the severity of liver disease. The abnormal percentage of 7S collagen in three patient groups was similar to that of IV collagen in the corresponding groups. Serum 7S collagen and IV collagen levels were strongly correlated with the histological degree of liver fibrosis; the correlation coefficients were r = +0.675 for 7S collagen and r = +0.665 for IV collagen. When we assessed the ability of each test to detect cirrhosis with a receiver operating curve, the serum 7S collagen test was a slightly better marker than the serum IV collagen test. For the detection of cirrhosis, serum 7S collagen was 83% sensitive and 88% specific at a cutoff value of 9 ng/ml, and serum IV collagen was 80% sensitive and 81% specific at a cutoff value of 160 ng/ml. CONCLUSIONS: These findings suggested that serum 7S collagen and IV collagen tests are similarly useful for assessing liver fibrosis in patients with chronic viral liver disease, although the former is slightly better for diagnosing cirrhosis than the latter.

Von Willebrand factor antigen in assessment of vasculitis in patients with connective tissue diseases.

Von Willebrand factor antigen (vWF:Ag) is synthesized and secreted by endothelial cells. In the present study we tried to assess the relationship between plasma level of vWF:Ag and vascular damage in patients with vasculitis. The study was carried out on 59 patients with connective tissue diseases. Vasculitis was diagnosed by biopsies of the skin. The patients with vasculitis had a significantly elevated level of vWF:Ag; however, no significant correlation between the amount of plasma vWF:Ag and the degree of vasculitis was found. The obtained results show that the plasma level of vWF:Ag may reflect the presence of vascular, especially endothelial, damage in patients with connective tissue diseases.