Diagnostic Accuracy and Practicability of the VISITECT® Advanced HIV Disease CD4 Test Compared to the Partec® CyFlow Counter II for CD4 Enumeration in Nigeria’s HIV Program

doi:10.21203/rs.3.rs-7411110/v1

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Diagnostic Accuracy and Practicability of the VISITECT® Advanced HIV Disease CD4 Test Compared to the Partec® CyFlow Counter II for CD4 Enumeration in Nigeria’s HIV Program

https://doi.org/10.21203/rs.3.rs-7411110/v1

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We evaluated the diagnostic performance and practicability of the VISITECT® CD4 Lateral Flow Assay for identifying advanced HIV disease (AHD) across 334 facilities in Nigeria. In two phases, 468 participants were tested with VISITECT® CD4 and Partec® CyFlow. VISITECT® CD4 achieved a sensitivity of 100.0% and specificity of 89.8% for detecting CD4 ≤ 200 cells/mm³. The positive predictive value (PPV) was 91.3% (95% CI: 87.4–94.1), and the negative predictive value (NPV) was 100% (95% Cl: 98.1–100.0). Overall diagnostic accuracy was 95.1% (95% CI: 92.8–96.8). We recommend its use to enhance timely AHD detection and improve linkage to prophylactic interventions in resource-limited settings.

HIV

Advanced HIV Disease

CD4 testing

Point-of-care diagnostics

VISITECT

Flow cytometry

Despite significant progress in expanding access to antiretroviral therapy (ART), advanced HIV disease (AHD) remains a major contributor to HIV-associated morbidity and mortality, particularly in sub-Saharan Africa (1). Globally, an estimated 30–40% of people initiating or re-initiating ART present with advanced disease, and mortality remains unacceptably high within the first three months of treatment initiation (2–5). Nigeria, which bears the second-largest HIV epidemic globally, continues to see a significant proportion of its HIV-positive population presenting late to care. Programmatic data from Nigeria and other West African countries suggest that nearly half of newly diagnosed individuals still enter care with CD4 counts ≤ 200 cells/mm³, particularly among key populations, adolescents, and individuals with prior treatment interruption (6).

Timely identification of AHD is critical for initiating life-saving interventions such as cotrimoxazole prophylaxis, tuberculosis and cryptococcal screening, and enhanced ART support (6, 7). Historically, CD4 testing has relied on flow cytometry platforms like Partec® CyFlow, which are centralized, resource-intensive, and impractical for primary health care has steadily declined in many low- and middle-income countries (LMICs), particularly following the scale-up of viral load testing and the shift away from routine CD4 monitoring (8). To close this gap, WHO now recommends point-of-care CD4 assays (6). We therefore evaluated the VISITECT® CD4 test, a simple lateral flow assay, for diagnostic accuracy and practicability under routine conditions in Nigeria’s HIV program.

Study Design and Setting

We conducted a cross-sectional diagnostic accuracy study across APIN-supported health facilities in Northcentral and Southwest Nigeria, following STARD 2015 guidelines (9). The objective was to evaluate the diagnostic performance and practicability of the VISITECT® CD4 Advanced HIV Disease assay in identifying individuals with CD4 counts ≤ 200 cells/mm³, using the Partec® CyFlow Counter II as the reference standard.

Sampling and Participant Recruitment

A consecutive sampling strategy was employed during routine HIV clinic visits in two phases: Phase I (March–May 2023) and Phase II (January 2025). Eligible participants were aged ≥ 5 years, newly diagnosed with HIV, WHO clinical stage 3/4, re-engaging in care after ≥ 90 days interruption, or showing evidence of treatment failure requiring CD4 testing. Exclusion criteria were age < 5 years, refusal to consent, or individuals with known hematologic disorders affecting CD4 counts. HIV status was confirmed using Nigeria’s national HIV testing algorithm before CD4 was carried out. Written informed consent was obtained (plus assent for minors). Figure 1.

Sample Size

Sample size was calculated using Buderer’s formula, (10, 11) assuming 90% sensitivity/specificity, 5% precision, and 95% confidence at 40% prevalence of AHD. A minimum of 346 participants was required for sensitivity and 122 for specificity, yielding a total of 468 participants. Phase I enrolled 238 and Phase II 230 using identical procedures, enabling assessment of temporal consistency.

Sample Collection and Testing

For each participant, a 3 mL venous EDTA blood sample was collected and used for both index and reference tests. Using venous rather than finger-prick blood minimized patient discomfort and mirrored routine clinic practice. VISITECT® CD4 LFA was performed by trained staff following manufacturers’ instructions for use; two blinded readers interpreted each result. Partec® CyFlow Counter II served as the reference platform, following standard operating procedures with daily calibration. External quality assurance was incorporated through national proficiency testing panels (NEQUAL). Detailed procedural steps for both assays are published elsewhere (12, 13). Figure 2.

Blinding and Quality Control

Index and reference tests were performed by independent testers in separate rooms to ensure blinding. Repeat reads were performed to minimize interpretive bias. Kits were stored under manufacturer-recommended conditions throughout.

Data Management and Analysis

Demographic and clinical data were captured on standardized forms and entered into a password-protected database. Sensitivity, specificity, predictive values, and accuracy with 95% confidence intervals (Wilson method) were calculated. Agreement between VISITECT® and CyFlow was assessed using Cohen’s Kappa statistics, while McNemar’s test evaluated paired misclassification. Usability data from healthcare worker questionnaires were summarized as proportions.

Ethical Considerations

The study was approved by the National Health Research Ethics Committee of Nigeria (NHREC/01/01/2007) and the Institutional Review Board of APIN Public Health Initiatives (NHREC/APIN-HREC/2/12/24iii).

Baseline demographic and clinical characteristics of the study participants

A total of 468 participants was enrolled, including 254 (54.3%) females, 126 (26.9%) males, 48 (10.3%) external quality assurance (EQA) samples, and 40 (8.5%) with undocumented sex. The median age was 37 years [IQR: 28–45], and the median CD4 count was 224.5 cells/mm³ [IQR: 99.3–428.8]. Overall, 242 participants (51.7%) had CD4 ≤ 200 cells/mm³ (Table 1).

Diagnostic performance of the VISITECT® CD4 LFA compared to the Partec® CyFlow Counter II (reference standard)

Compared with the Partec® CyFlow Counter II, VISITECT® CD4 LFA achieved a sensitivity of 100.0% (95% CI: 97.9–100.0) and specificity of 89.8% (95% CI: 85.3–93.0). The PPV was 91.3% (95% CI: 87.4–94.1), NPV 100% (95% CI: 98.1–100.0), and overall accuracy 95.1% (95% CI: 92.8–96.8), with almost perfect agreement (κ = 0.90). Misclassifications were entirely attributable to false positives (p < 0.001, McNemar’s test).

Diagnostic performance of VISITECT® CD4 LFA at different CD4 cut-offs of the reference test

VISITECT® CD4 correctly identified all participants with CD4 < 100 cells/mm³ and 100–200 cells/mm³, as well as all with > 350 cells/mm³. Misclassification occurred in the 201–350 range, where 23/97 (23.7%) were classified as ≤ 200 cells/mm³, yielding specificity of 76.3% (95% CI: 66.9–83.9).

VISITECT® CD4 LFA Practicability of Use

Among 423 trained testers across all healthcare levels, 1,692 tests were performed with 99% success (1,675/1,692) and 17 invalid results. Turnaround time averaged 45 minutes. Ninety-three percent found the kit easy to use, gaining confidence after four tests. Interpretation challenges were noted among older testers and under poor lighting. 67 (15%) preferred the Partec® CyFlow for its faster, quantitative output. Overall, 90% considered VISITECT® a “game changer” for AHD, but two-thirds recommended it complement rather than replace conventional methods.

This multi-site evaluation demonstrated that the VISITECT® CD4 LFA achieved high diagnostic accuracy for ruling out people without advanced HIV diseases CD4 counts > 200 cells/mm³, meeting WHO’s performance benchmarks for rapid CD4 testing. Performance was consistent across two phases, with sensitivity highest at very low CD4 counts (< 100 cells/mm³) and specificity highest at very high counts (> 350 cells/mm³). This pattern underscores the test’s reliability in detecting those at greatest risk of advanced HIV disease (AHD) who require urgent Cryptococcal infection screening (CrAg Test) and tuberculosis screening (Urine TB LAM test) and in excluding those at least risk, thereby reducing unnecessary further investigations.

A small but important limitation was the 4.9% (23/468) misclassification rate, where patients with CD4 counts between 201–350 cells/mm³ were incorrectly classified as ≤ 200 cells/mm³. While this may trigger overuse of confirmatory AHD diagnostics, the balance of benefit favours early intervention given the high mortality risk in true AHD cases. Importantly, adherence to the manufacturer’s precise sequential incubation times (3, 17, and 20 minutes) was critical: shorter first incubation produced faint test lines (over-diagnosing AHD), while extended incubation could yield stronger lines (under-diagnosing AHD).

Operationally, the assay performed well across all tiers of care in Nigeria tertiary, secondary, and peripheral PHCs with a 99% valid result rate. Blinded dual reading, repeat reading, and inclusion of both ART-naïve and ART-experienced patients (including those returning to care or with treatment failure) enhance the robustness of these findings. Interpretation, however, was influenced by lighting and visual acuity, particularly among older testers, and faintness of the control and reference lines suggests a need for improved colour intensity to differentiate lines.

Our results align with, those from the multicountry study conducted across seven sub-Saharan African countries, which reported a pooled sensitivity of 95.0% (95% CI: 92.8–96.6) and specificity of 82.6% (95% CI: 80.2–84.8) for VISITECT® CD4 compared to flow cytometry (14). It also aligns with study done in Malawi, Zimbabwe and Democratic Republic of Congo with a sensitivity of 95.0% [95% CI: 91.3–97.5] and specificity of 81.9% [95% CI: 78.2–85.2%] (15). Our study like other studies demonstrate the test’s strength in reliably detecting CD4 ≤ 200 cells/mm³ and ruling out those without advanced HIV disease. Importantly, our study adds new evidence on temporal consistency, with comparable diagnostic performance across two phases (2023 and 2025), as well as operational insights from a practicability survey spanning Nigeria’s three tiers of health care.

These findings reinforce VISITECT® CD4 LFA as a promising tool to support timely AHD screening and linkage to care in resource-limited settings, especially at primary health care level where access to conventional flow cytometry is lacking. VISITECT® CD4 LFA offers a valuable decentralized option for early AHD detection, potentially reducing referrals and facilitating immediate linkage to care. It could also be deployed in mobile clinics, high-risk outreach, and community ART groups. Strengths of this study include its multi-tier implementation, inclusion of patients aged ≥ 5 years, and a substantial proportion with advanced immunosuppression. The main limitations was reduction in specificity in the CD4 201–350 cells/mm³ range, leading to some misclassification. This limitation may result in unnecessary downstream testing, which has both clinical and programmatic implications.

The VISITECT® CD4 LFA represents a valuable complementary tool for expanding access to AHD screening, especially in settings lacking flow cytometry. However, it should not replace conventional CD4 enumeration methods; instead, it can complement them at the primary and secondary health care levels.

Conflict of Interest

The authors declare that they have no competing interests

Funding

The authors received no funding for this study

Author Contribution

JON conceived the study. Protocol development was undertaken by JON, EAO, FEO, and EO. Data collection and laboratory testing were performed by DO, PCO, IOO, DCO, AMO, OSO, OO, AIC, JOO, and AA. Data analysis was conducted by DSA, II, JON, and AAA. The manuscript was drafted by JON, COU, EAO, FEO, OK, AAA, DSA, II, and DCO. All authors contributed to the review of the final version of the manuscript. JON, FEO, EAO, DCO, COU, II reviewed the final copy for submission. All authors agree to be accountable for all aspects of the work.

Acknowledgement

We sincerely thank the management and staff of the participating APIN-supported health facilities for their invaluable support during this study. We acknowledge the dedication of all laboratory scientists, laboratory technicians, laboratory assistants, and other healthcare workers who participated in the practicability assessment and ensured high-quality data collection. We are grateful to the patients and their caregivers for their willingness to participate in this study

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Table 1 and 2 are available in the Supplementary Files section.

No competing interests reported.

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You are reading this latest preprint version

Diagnostic Accuracy and Practicability of the VISITECT® Advanced HIV Disease CD4 Test Compared to the Partec® CyFlow Counter II for CD4 Enumeration in Nigeria’s HIV Program

Status:

Version 1

Abstract

Figures

Introduction

Methods

Study Design and Setting

Sampling and Participant Recruitment

Sample Size

Sample Collection and Testing

Blinding and Quality Control

Data Management and Analysis

Ethical Considerations

Results

Baseline demographic and clinical characteristics of the study participants

Diagnostic performance of VISITECT® CD4 LFA at different CD4 cut-offs of the reference test

VISITECT® CD4 LFA Practicability of Use

Discussion

Conclusion and Recommendations

Declarations

Conflict of Interest

Funding

Author Contribution

Acknowledgement

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1