LAMP-enabled diagnosis of Kaposi's sarcoma for sub-Saharan Africa

Abstract

Kaposi's sarcoma (KS) is an endothelial cancer caused by the Kaposi's sarcoma-associated herpesvirus (KSHV) and is one of the most common cancers in sub-Saharan Africa. In limited-resource settings, traditional pathology infrastructure is often insufficient for timely diagnosis, leading to frequent diagnoses at advanced-stage disease where survival is poor. In this study, we investigate molecular diagnosis of KS performed in a point-of-care device to circumvent the limited infrastructure for traditional diagnosis. Using 506 mucocutaneous biopsies collected from patients at three HIV clinics in Uganda, we achieved 97% sensitivity, 92% specificity, and 96% accuracy compared to gold standard U.S.-based pathology. The results presented in this manuscript show that LAMP-based quantification of KSHV DNA extracted from KS-suspected biopsies has the potential to serve as a successful diagnostic for the disease and that diagnosis may be accurately achieved using a point-of-care device, reducing the barriers to obtaining KS diagnosis while increasing diagnostic accuracy.

Fig. 1.. Flow diagram for traditional and proposed diagnosis techniques.

Comparison of traditional histopathologic diagnosis of KS and proposed molecular diagnostic method. After clinical examination and biopsy, traditional diagnosis is performed using a hematoxylin and eosin (H&E) stain and an anti-LANA stain if available. This is considered the gold standard for KS diagnosis yet is resource demanding. Molecular analysis using skin requires DNA extraction and purification before LAMP testing in TINY. This point-of-care approach may provide a diagnosis in only a few hours, as well as be accessible within limited-resource settings.

Fig. 2.. Study design flow for biopsy analysis.

Study design for analysis of LAMP as a diagnostic tool for KS. A section of biopsy is sent for analysis by at least two U.S. pathologists, where a third pathologist is used if a consensus diagnosis is not made. A different portion of the biopsy undergoes DNA extraction and purification before being tested in duplicate in the TINY. If those replicates disagree—one did not amplify and the other did, or they are among the top 5% of disparate threshold times—then additional replicates are performed. The output value used for KS diagnosis is the mean threshold time of all available (two or four total) replicates.

Fig. 3.. Multireplicate KSHV-LAMP reproducibility analysis.

A Bland-Altman plot (n = 356) of LAMP replicates 1 and 2 when both amplified. Dashed lines exclude replicates (n = 18) with top 5% largest difference (absolute threshold time difference of 2 min 40 s and greater) to be retested in duplicate. Darker data point color indicates a difference closer to zero.

Fig. 4.. Threshold time comparison of KS-present and KS-absent samples.

Mean KS-LAMP threshold time value for all available replicates organized by consensus pathology result; mean of two replicates used for n = 448 and mean of four replicates used for n = 43. Excluding samples indeterminate by pathology (n = 15). Darker color indicates later threshold time.

Fig. 5.. Diagnostic performance of KSHV-LAMP.

An ROC curve was generated from the sensitivity and specificity results for each cutoff time through the range of all possible values. The area under the curve (AUC) was 0.967 (95% CI: 0.948 to 0.985) with two cutoff times shown at maximum sensitivity or specificity while keeping the other above 90%. Local pathology had a reported 72% sensitivity and 84% specificity (19).