BD FACSCount Research Articles

Cryptococcal antigenemia among HIV seropositive patients accessing care in antiretroviral therapy (ART) clinics in Calabar, South Southern Nigeria

Background: Cryptococcus neoformans infection is a life threatening disease especially when associated with immunosupression like HIV/AIDS. The greatest burden of disease occurs in sub-Saharan Africa, where mortality is estimated to be 17%. The finding of cryptococcal antigen in the blood represents a condition of systemic invasion with the fungus. Clinical manifestation of infection with Cryptococcus neoformans in AIDS patients is generally more evident at CD4 cells ≤100 cells/µl. This study was carried out to determine cryptococcal antigenemia among HIV seropositive patients accessing care in ART clinics as patients are not screened routinely despite reports of relatively high co-infection rates with HIV. Methods: This prospective cross-sectional study was carried out on ART-treated and ART-naive HIV positive adult patients attending ART clinics in two tertiary hospitals in Calabar, Nigeria. The sero-prevalence of cryptococcal antigen among the patients was determined using the cryptococcal antigen latex agglutination system (CALAS) (Wampole Laboratories, USA), according to the manufacturer’s instruction. The CD4 count levels of each patient were determined by flow cytometry using the fluorescent activated cell sorter BD FACS Count system (Becton Dickinson). Results: Out of the 272 HIV positive subjects enrolled in the study, 116(42.6%) were ARV-naive and 156(57.4%) were ARV-treated patients. A 5.1% cryptococcal antigenemia prevalence was established in the study. Infection rates were higher among subjects receiving ART 11/156 (7.1%) than in ART-naive subjects 3/116 (2.6%). Infection rates 5(35.7%) peaked at age 25-34 years. The mean CD4 counts of subjects with cryptococcal infection were 100.7±67.8 cells/µl, with a minimum CD4 count of 13.0. All the infections occurred among subjects with CD4 counts ≤200 cells/µl of blood. There was a statistically significant effect of cryptococcal antigenemia on the CD4 counts of the subjects (t=3.7, p=0.002). Conclusion: This study reveals that cryptococcal antigenemia is a health problem among HIV/AIDS patients in our locality. Cryptococcal antigenaemia seem to be more common among HIV patients on ART. The CD4 count levels among the ART treated subjects could have been boosted by administration of ART

Multisite evaluation of point of care CD4 testing in Papua New Guinea.

Laboratory-based CD4 monitoring of HIV patients presents challenges in resource limited settings (RLS) including frequent machine breakdown, poor engineering support and limited cold chain and specimen transport logistics. This study assessed the performance of two CD4 tests designed for use in RLS; the Dynal assay and the Alere PIMA test (PIMA). Accuracy of Dynal and PIMA using venous blood was assessed in a centralised laboratory by comparison to BD FACSCount (BD FACS). Dynal had a mean bias of −50.35 cells/µl (r2 = 0.973, p<0.0001, n = 101) and PIMA −22.43 cells/µl (r2 = 0.964, p<0.0001, n = 139) compared to BD FACS. Similar results were observed for PIMA operated by clinicians in one urban (n = 117) and two rural clinics (n = 98). Using internal control beads, PIMA precision was 10.34% CV (low bead mean 214.24 cells/µl) and 8.29% (high bead mean 920.73 cells/µl) and similar %CV results were observed external quality assurance (EQA) and replicate patient samples. Dynal did not perform using EQA and no internal controls are supplied by the manufacturer, however duplicate testing of samples resulted in r2 = 0.961, p<0.0001, mean bias = −1.44 cells/µl. Using the cut-off of 350 cells/µl compared to BD FACS, PIMA had a sensitivity of 88.85% and specificity of 98.71% and Dynal 88.61% and 100%. A total of 0.44% (2/452) of patient samples were misclassified as “no treat” and 7.30% (33/452) “treat” using PIMA whereas with Dynal 8.91% (9/101) as “treat” and 0% as “no treat”. In our setting PIMA was found to be accurate, precise and user-friendly in both laboratory and clinic settings. Dynal performed well in initial centralized laboratory evaluation, however lacks requisite quality control measures, and was technically more difficult to use, making it less suitable for use at lower tiered laboratories.

Population-based survey of HIV sero-status and vertical transmission among naive pregnant women in Sokoto, Nigeria

Background: This study aims at investigating the seroprevalence of HIV infection among status naive pregnant women and probable vertical transmission in Sokoto, Nigeria.Materials and Methods: This cross-sectional study examined 13,026 apparently healthy pregnant women aged between 14 and 45 years and 312 mother-baby pairs in 4 different hospital settings in Sokoto State, North West, Nigeria between March, 2011 and February, 2013. The babies were aged between 8 and 16 months. HIV screening was performed using qualitative rapid tests and ELISA and HIV-DNA polymerase chain reaction (PCR) techniques. Measurement of CD4+ T-lymphocytes was carried out by the BD FACScount System. All seropositive pregnant women were immediately placed on triple antiretroviral therapy (ART) throughout the duration of the pregnancy and beyond.Results: An overall 2.4% prevalence of HIV-1 infection among the pregnant women and 20.5% incident of mother-to-child transmission were found. Of the seropositive pregnant women, 75.0% were full-time house wives, 13.8% and 11.2% were traders and civil servants respectively; of which, 70.2% were within the ages of 14 and 27 years (youthful predominance). Pearson’s χ2analysis did not show any statistically significant difference in the Mean values in the 4 health facilities (χ2 =2.084, df=3, P-value=0.555). Similarly, no significant difference in HIV seropositivity in the demographic data of the pregnant women were observed (P&gt;0.05). Infection was recorded in all age groups but there was no statistical significance between age groups and infection (P = 0.833). Of the 64 seropositive babies, 62 (92.5%) contracted HIV from antiretroviral therapy non-adherence mothers (χ2 =271.457, df=1, P&lt;0.01), OR=1506.6 (95%CI=285.5-7950.4). Conclusion: This study found high prevalence of vertical transmission due to ART non-adherence. Intervention initiatives should, therefore, focus seriously on ART non-adherence. DOI: http://dx.doi.org/10.3126/ajms.v6i3.11530Asian Journal of Medical Sciences Vol.6(3) 2015 49-57

Technical performance evaluation of the MyT4 point of care technology for CD4+ T cell enumeration.

ObjectiveThough absolute CD4+ T cell enumeration is the primary gateway to antiretroviral therapy initiation for HIV-positive patients in all developing countries, patient access to this critical diagnostic test is relatively poor. We technically evaluated the performance of a newly developed point-of-care CD4+ T cell technology, the MyT4, compared with conventional CD4+ T cell testing technologies.DesignOver 250 HIV-positive patients were consecutively enrolled and their blood tested on the MyT4, BD FACSCalibur, and BD FACSCount.ResultsCompared with the BD FACSCount, the MyT4 had an r2 of 0.7269 and a mean bias of −23.37 cells/µl. Compared with the BD FACSCalibur, the MyT4 had an r2 of 0.5825 and a mean bias of −46.58 cells/µl. Kenya currently uses a CD4+ T cell test threshold of 350 cells/µl to determine patient eligibility for antiretroviral therapy. At this threshold, the MyT4 had a sensitivity of 95.3% (95% CI: 88.4–98.7%) and a specificity of 87.9% (95% CI: 82.3–92.3%) compared with the BD FACSCount and sensitivity and specificity of 88.2% (95% CI: 79.4–94.2%) and 84.2% (95% CI: 78.2–89.2%), respectively, compared with the BD FACSCalibur. Finally, the MyT4 had a coefficient of variation of 12.80% compared with 14.03% for the BD FACSCalibur.ConclusionsWe conclude that the MyT4 performed well at the current 350 cells/µl ART initiation eligibility threshold when used by lower cadres of health care facility staff in rural clinics compared to conventional CD4+ T cell technologies.

Determination of hematological and immunological parameters among HIV positive patients taking highly active antiretroviral treatment and treatment naïve in the antiretroviral therapy clinic of Gondar University Hospital, Gondar, Northwest Ethiopia: a comparative cross-sectional study

BackgroundAnemia, leucopenia and thrombocytopenia are the commonest hematological abnormalities resulting from human immunodeficiency virus infection. The use of antiretroviral drugs could positively or negatively affect these disorders. Thus a specific diagnosis and a determination of hematological and immunological parameters are required for initiating and monitoring early treatment to avert disease progression. Therefore, this study aimed to compare hematological and immunological parameters in HIV positive patients taking antiretroviral therapy and those treatment naïve patients in Gondar University Hospital.MethodsA comparative cross-sectional study was conducted on a total of 290 HIV patients from February to May 2012 in Gondar University Hospital. Study subjects were divided in to two groups: 145 HIV positive treatment naïve and 145 on HAART. Data of socio demographic characteristics and clinical conditions of the study subjects was collected using structured pretested questionnaire at their follow up date. Hematological and immunological parameters were collected and processed by cell Dyne 1800 and BD FACS count respectively. The variables compared here were Hematological parameters (Total and differential WBC, RBC, Hgb, HCT, MCV, MCH, MCHC, RDW, PLT, and MPV) and CD4 count. In order to compare means independent sample T-test was conducted using SPSS version 20 statistical software. P- Value < 0.05 was considered as significant.ResultPrevalence of anemia, leucopenia, thrombocytopenia, neutropenia and lymphopenia were 11.7%, 35.9%, 4.1%, 28.3% and 2.1% in patients on HAART and 29.7%, 16.6%, 9%, 14.5% and 2.1% in HAART naïve patients respectively. There was a significant difference in total WBC, RBC, Hgb, MCV, MCH, MCHC, MPV and CD4 counts between patients on HAART and HAART naïve patients.ConclusionPrevalence of anemia was high in HAART naïve patients while leucopenia and neutropenia prevalence was higher in patients on HAART and their prevalence increased as the CD4 count decreased. HIV Patients should be investigated for hematological and immunological changes following with appropriate therapeutic interventions.

Comparative Evaluation between the New BD FACS Count System and Standard BD FACS Count System by Enumeration of Absolute TCD4 Lymphocytes in Adults: Preliminary Results

Background: The purpose of this study was to evaluate the performance of the new BD FACS Count System compared to the standard BD FACS Count system for count of lymphocytes TCD4. Methods: It was a comparative study conducted in Centre MURAZ research institute. The New BD FACS Count System dedicated to enumerate absolute and percentage of TCD4+ was compared to the standard BD FACS Count System dedicated to enumerate only absolute number of TCD4+, TCD8+, TCD3+ and the CD4/CD8 ratio. Results were analyzed by Meth Val software. Results: The New BD FACS Count System compared favorably with the BD FACS Count System for absolute TCD4+, resulting in an overall correlation coefficient of 0.99 for the patients evaluated. Conclusion: The New BD FACS Count System is simple to perform as the old system and was an excellent alternative method to manage adults HIV in resource limited settings.

Erratum to: Total Lymphocyte Count as surrogate marker for CD4 Cell Count in HIV-Infected Individuals in Gondar University Hospital, Northwest Ethiopia

The high cost of CD4 count estimation in resource-limited countries is a major challenge in initiating patients on highly active antiretroviral therapy (HAART). Therefore, assessment of inexpensive and simple laboratory diagnostic marker is mandatory to diagnose immuno-suppression. To evaluate utility of total lymphocyte count (TLC) as surrogate marker for CD4 count in HIV-infected patients. In this cross sectional study, 400 ART-naive HIV-positive patients enrolled in Gondar University Hospital, from March 2011 to May 2011, were tested for CD4 count & TLC. The cutoffs were determined as: 200 cells/μL for CD4 count and 1200 cells/μL for TLC by using BD FACS count and CELL DYN 1800 Flow Cytometrys respectively. Spearman correlation between TLC and CD4 cell count were assessed. Sensitivity, specificity, positive and negative predictive values for different age a group, TLC ≤1200 was computed for CD4 count ≤200 cells/cu.mm. Among 400 ART naive HIV infected patients, 278 (69.5%) were females. The mean age of the study participants was 33.7. TLC and CD4 count were positively correlated (r = 0.33, p = 0.001). A TLC of ≤1200 cells/m m3 was found to have a sensitivity (32.86%), specificity (95.33%), PPV (79.7%), and NPV (71.9%) for predicting a CD4 count of <200 cells/mm3. This study showed that low sensitivity and specificity of TLC as a surrogate measure for CD4 count. Moreover, CD4 cell counts of < 200 cells/mm3 were found in 96 cases (24%) with TLCs of ≤1200 cells/mm3. Thus, 1 in 4 individuals would have been deprived of needed treatment. Therefore, we recommend keep on expansion of access to CD4 counter.

Same-Day CD4 Testing to Improve Uptake of HIV Care and Treatment in South Africa: Point-of-Care Is Not Enough

Background. We evaluated whether a pilot program providing point-of-care (POC), but not rapid, CD4 testing (BD FACSCount) immediately after testing HIV-positive improved retention in care. Methods. We conducted a retrospective record review at the Themba Lethu Clinic in Johannesburg, South Africa. We compared all walk-in patients testing HIV-positive during February, July 2010 (pilot POC period) to patients testing positive during January 2008–February 2009 (baseline period). The outcome for those with a ≤250 cells/mm3 when testing HIV-positive was initiating ART <16 weeks after HIV testing. Results. 771 patients had CD4 results from the day of HIV testing (421 pilots, 350 baselines). ART initiation within 16 weeks was 49% in the pilot period and 46% in the baseline period. While all 421 patients during the pilot period should have been offered the POC test, patient records indicate that only 73% of them were actually offered it, and among these patients only 63% accepted the offer. Conclusions. Offering CD4 testing using a point-of-care, but not rapid, technology and without other health system changes had minor impacts on the uptake of HIV care and treatment. Point-of-care technologies alone may not be enough to improve linkage to care and treatment after HIV testing.

HIV elite controllers as a key to novel strategies in treatment of HIV infection

Purpose of the studyTo identify and primarily characterize the elite controllers (EC) in Moscow Regional HIV Living People Cohort (Russia).Methods2682 HIV‐1‐positive individuals with A1 (asymptomatic) stage of HIV infection were under regular physician observation continuously for at least 5 years. Verification antibody testing was performed with “New Love Blot” and “Autoblot 2000” (Biorad). Patients underwent scheduled HIV viral load and T‐lymphocyte subpopulation measurement (twice a year) and did not have indications to HAART (viral load less then 5 log10/ml, CD4+ counts more then 500 cells/mm3). HIV viral load was detected by PCR m2000rt Abbott Biosystems analyzer, “RealTime HIV‐1” sets with 20 copies per ml sensitivity) and major subpopulation of T‐lymphocytes were analyzed by flow cytometer BD FACSCount, sets of antibodies ÑD3/CD4/CD8/CD45) [1].imageSummary of resultsAverage log10 viral load was defined in each patient for 5‐year period, and the distribution appeared to have a bimodal character (Figure 1). 106 EC were primarily identified as having average viral load less then 1.7 log10 (50) HIV copies/ml.The incidence of EC appeared to be 3.95% (95% CI: 3.2%; 4.7%) of population with A1 (asymptomatic) disease with no indications to HAART, that corresponds to literary data [2]. Belonging to EC was then proved by laboratory dynamics. In EC 3 types of viral load dynamics were identified: 1) absence of detectable viremia, 2) single spikes, 3) episodic temporary elevation(s) (at mean 500‐900 copies) lasting half a year. All these emphasize the control of virus. In EC 3 types of ÑD4+T‐lymphocyte dynamics were defined: 1) CD4+ elevation (in case beginning from the acute stage of the disease), 2) stable ÑD4+ cells, 3) CD4+ cell depletion with very small velocity. 12 EC had “minimal change disease” defined additionally by the absence or trace appearance of pol 68/66, 52/51, 34/31 antibodies (Table 1) and non‐detectable PCR levels in all measurements. These represent 11.32% (95% CI: 5.17%; 17.47%) from EC and 0.45% (95% CI: 0.19%; 0.71%) from population with A1 (asymptomatic) HIV‐disease. Env Env Env Gag Gag Gag Gag Pol Pol Pol Patient number 160 120 41 55 40 24/25 18 68/66 52/51 34/31 1 + * + + + + − − − − 2 + + + + * + − * * + 3 + * + * ‐ * − * * * 4 + + + + + + − + * + 5 + + + + − + − + + − 6 + + + + + + * + + * 7 + + + + + + * * * + 8 + * + * ‐ * * * − * 9 + + + + ‐ + * − − − 10 + ‐ + * ‐ + − − − − 11 + + + + + + * − − − 12 + * + + + + − * − − Signs of antibodies: +, presence; *, traces; −, absence ConclusionsAmong EC patients with “minimal change disease” were identified. They may represent: (i) primarily persistent HIV infection (with reduced productive cycle), (ii) low dose (localized) HIV‐infection, (iii) rare successful immune‐mediated elimination of HIV that could be the model for novel elimination strategies.

Assessment of the impact of malaria on CD4+ T Cells and haemoglobin levels of HIV-malaria co-infected patients

The human immunodeficiency virus (HIV) and malaria destroy important cells required for proper immunological and haematological functioning of the body. This research therefore aimed to assess the effect of malaria on CD4+ and haemoglobin (Hb) levels of HIV-malaria co-infected patients. The study was performed by sampling 220 adult HIV patients on highly active anti retroviral therapy (HAART) who routinely visited the Tema General Hospital in Ghana. Blood samples were obtained for both blood film microscopy identification of malaria parasites and analysis using rapid diagnostic test kits. A BD Facscount Analyzer was used in the quantification of CD4+ levels. Of the 220 patients sampled, 34 (15.5%) were HIV-malaria co-infected, all of whom (34; 100%) had CD4+ counts below the normal range, while 23 (12.9%) of the HIV mono-infected patients had normal CD4+ counts. Almost all HIV-malaria co-infected patients (33; 97.1%) had low Hb levels, whereas 79 (42.5%) of the HIV mono-infected patients had normal Hb. Malaria infection strongly correlated positively and significantly with both low CD4+ count (χ2 = 0.828, P = 0.003) and Hb (χ2 = 0.817, P = 0.004) levels. Malaria co-infection with HIV decreases CD4+ T cells and Hb levels in patients. It is therefore recommended that HIV patients in malaria endemic areas should adhere to malaria preventive measures.

Total Lymphocyte Count as surrogate marker for CD4 Cell Count in HIV-Infected Individuals in Gondar University Hospital, Northwest Ethiopia

BackgroundThe high cost of CD4 count estimation in resource-limited countries is a major challenge in initiating patients on highly active antiretroviral therapy (HAART). Therefore, assessment of inexpensive and simple laboratory diagnostic marker is mandatory to diagnose immuno-suppression.ObjectiveTo evaluate utility of total lymphocyte count (TLC) as surrogate marker for CD4 count in HIV-infected patients.Materials and MethodsIn this cross sectional study, 400 ART-naive HIV-positive patients enrolled in Gondar University Hospital, from March 2011 to May 2011, were tested for CD4 count & TLC. The cutoffs were determined as: 200 cells/μL for CD4 count and 1200 cells/μL for TLC by using BD FACS count and CELL DYN 1800 Flow Cytometrys respectively. Spearman correlation between TLC and CD4 cell count were assessed. Sensitivity, specificity, positive and negative predictive values for different age a group, TLC ≤1200 was computed for CD4 count ≤200 cells/cu.mm.ResultsAmong 400 ART naive HIV infected patients, 278 (69.5%) were females. The mean age of the study participants was 33.7. TLC and CD4 count were positively correlated (r = 0.33, p = 0.001). A TLC of ≤1200 cells/m m3 was found to have a sensitivity (32.86%), specificity (95.33%), PPV (79.7%), and NPV (71.9%) for predicting a CD4 count of <200 cells/mm3.ConclusionThis study showed that low sensitivity and specificity of TLC as a surrogate measure for CD4 count. Moreover, CD4 cell counts of < 200 cells/mm3 were found in 96 cases (24%) with TLCs of ≤1200 cells/mm3. Thus, 1 in 4 individuals would have been deprived of needed treatment. Therefore, we recommend keep on expansion of access to CD4 counter.

CD4‐ and CD3‐T Lymphocyte Reference Values of Immunocompetent Urban and Rural Subjects in an African Nation

Studies on the reference values of CD4 and CD3 T cells in healthy individuals have continued to gain significance because of the importance of these immunological markers in the initiation of combination antiretroviral therapy (cART). The aim of the present study was to determine and compare the reference values of CD4 and CD3 T cells in urban and rural Nigerians who were human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) negative. After ethical clearance and informed consent, 1123 subjects who met the inclusion criteria [mean age = 24.4 (± 11.2) years] were recruited in this study. Blood samples were analysed using the BD FACScount cytometer according to the manufacturer's instructions. Of the overall 1123 subjects, reference means of CD4, CD3 and CD4/CD3 ratio were 1030 ± 367, 1757 ± 609 cells/μl and 0.59 ± 0.08, respectively. Five hundred and fifty-one (49.1%) were an urban population with the mean CD4, CD3 and CD4/CD3 T cell ratio of 1032 ± 369, 1761 ± 612 cells/μl and 0.59 (±0.08), respectively. The remaining 572 (50.9%) were of a rural population with the mean CD4, CD3 and CD4/CD3 T cell ratio of 1028 ± 459, 1753 ± 958 cells/μl and 0.59 ± 0.13, respectively. Subjects with higher CD4 and CD3 T cells were more likely to be female than male (P < 0.05). There was no significant difference between the T cell values of the two populations (P > 0.05). Our findings provide new insight in the CD4 and CD3 T cell reference values of Nigerians.

CD4+ T lymphocyte reference values of immunocompetent subjects in an African university

CD4(+) T cells play critical roles in the immune system and, being primary targets of HIV infection, they are used to measure disease progression and response to combination antiretroviral therapy (cART), alongside other parameters, in HIV/AIDS patients. The aim of this study was to determine the reference values of CD4(+) T cells in a student population that was HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) negative. After ethical clearance and informed consent, 500 subjects (mean age = 26 years) were recruited, of whom 56 (11.2%) had HIV, HBV or HCV and were excluded. Blood samples were collected from the remaining 444 subjects into vacutainer tubes and analysed using the BD FACScount cytometer according to the manufacturer's instructions. Of the 444 subjects, 266 (59.9%) were male and 178 (40.1%) were female. The mean (± standard deviation) CD4(+) T cell count was 987 cells/µL (± 336). The mean counts among males and females were 957 cells/µL (± 306) and 991 cells/µL (± 340), respectively. Values of CD4(+) T cells ranged from 651 cells/µL to 1705 cells/µL. Subjects with higher CD4(+) T Cells were more likely to be female than male. There was no direct correlation between CD4(+) T cell values and age of the participants. Our findings offer the first insight into the CD4(+) T cell reference values of a Nigerian student population and provide useful data that will guide future cART decisions and other immune-based therapies.

New BD FACSCount™ CD4 reagent system for simultaneous enumeration of percent and absolute CD4 T-lymphocytes in HIV-1-infected pediatric patients

Absolute CD4+ T-lymphocyte counts are used in the initiation and monitoring of antiretroviral therapy in HIV-infected patients. Becton Dickinson's (BD) FACSCount system was introduced 12 years ago as a dedicated instrument for enumeration of absolute CD4+ T-lymphocytes. However, this system does not provide percent CD4+ T-lymphocyte that is the required monitoring parameter in pediatric patients. We evaluated a new BD FACSCount CD4 software and reagents for simultaneous percent and absolute CD4+ T-lymphocytes in HIV-infected blood. Percent and absolute CD4+ T-lymphocytes in 149 HIV-infected blood samples were determined using a new FACSCount system. Results of percent and absolute CD4+ T-lymphocytes were compared between the dual-platform (DP) method, using BD FACScan flow cytometer plus hematology analyzer and the standard FACSCount system. Correlation and agreement were analyzed using linear regression and Bland-Altman analysis. Percent CD4+ T-lymphocyte values obtained from the new FACSCount system correlated well with DP FACScan method (r2 = 0.977, P < 0.0001). Mean bias was only -0.36% [limit of agreement (LOA): -2.52% to +1.80%] and percent similarity was 101.36%. For absolute CD4+ T-lymphocyte, the new system correlated highly with standard FACSCount system (r2 = 0.986, P < 0.0001), with a percent similarity of 98.2. Mean bias was +3.39 cells/microl with LOA of -52.53 cells/mul to +59.31 cells/microl. This new FACSCount system is a simple and reliable system for enumeration of absolute and percent CD4+ T-lymphocytes. Having one system giving both results should reduce the cost and thus increase access to CD4 testing for pediatric and adult patients.