A Real-Time PCR Assay for the Diagnosis of Intestinal Schistosomiasis and Cure Assessment After the Treatment of Individuals With Low Parasite Burden

Abstract

The laboratorial diagnosis of the intestinal schistosomiasis is always performed using Kato-Katz technique. However, this technique presents low sensitivity for diagnosis of individuals with low parasite burden, which constitutes the majority in low endemicity Brazilian locations for the disease. The objective of this study was developed and to validate a real-time PCR assay (qPCR) targeting 121 bp sequence to detect Schistosoma spp. DNA for the diagnosis of intestinal schistosomiasis and a sequence of the human β-actin gene as internal control. Firstly, the qPCR was standardized and next it was evaluated for diagnosis and cure assessment of intestinal schistosomiasis in the resident individuals in Tabuas and Estreito de Miralta, two locations in Brazil endemic for intestinal schistosomiasis. The qPCR assay results were compared with those of the Kato-Katz (KK) test, examining 2 or 24 slides, Saline Gradient (SG) and “reference test” (24 KK slides + SG). The cure assessment was measured by these diagnostic techniques at 30, 90, and 180 days post-treatment. In Tabuas, the positivity rates obtained by the qPCR was 30.4% (45/148) and by “reference test” was of 31.0% (46/148), with no statistical difference (p = 0.91). The presumed cure rates at 30, 90, and 180 days post-treatment were 100, 94.4, and 78.4% by the analysis of 24 KK slides, 100, 94.4, and 78.4% by the SG, and 100, 83.3, and 62.1% by the qPCR assay. In Estreito de Miralta, the positivity obtained by qPCR was 18.3% (26/142) and with “reference test” was 24.6% (35/142), with no statistical difference (p = 0.20). The presumed cure rates were 93.3, 96.9, and 96.5% by the KK, 93.3, 96.9, and 100% by the SG, and 93.3, 93.9, and 96.5% by the qPCR at 30, 90, and 180 days post-treatment, respectively. This study showed that the diagnostic techniques presented different performance in the populations from the two districts (Tabuas and Estreito de Miralta) and reinforces the need of combining techniques to improve diagnosis accuracy, increasing the detection of individuals with low parasite burden. This combination of techniques consists an important strategy for controlling the disease transmission.

The Road to Elimination: Current State of Schistosomiasis Research and Progress Towards the End Game

“…Based on the results from the “reference test” (24 Kato-Katz slides + Saline Gradient method-SG) the qPCR presented 82.6%, 93.1%, and 89.8% of sensitivity, specificity, and accuracy rates, respectively. The positivity of qPCR was higher than that of the 24 Kato-Katz slides, SG techniques ( 103 ) or Kato-Katz and Spontaneous Sedimentation techniques combined ( 148 ) when tested in a low endemicity area. As the number of infection intensity reduces and the number of epg of feces becomes smaller ( 149 ), there is an increased likelihood of discrepant results between PCR-based assays and parasitological tests.…”

“…A cross-sectional study identified S. haematobium eggs in semen of fishermen, demonstrating the lodgment of eggs in the reproductive system with the implication that quantification of infection burden using urine alone may underestimate infections ( 91 ). Although the positivity rate of Kato-Katz is directly proportional to the number of slides and fecal samples examined ( 103 , 104 ), it is logistically challenging in large epidemiological studies to examine multiple replicate slides. Given that the WHO recommends that stool sampling be done on three consecutive days to have a reliable analysis brings to the fore how challenging this is in resource limited settings ( 105 ).…”

“…Polymerase chain reaction (PCR)-based assays use different targets to detect Schistosoma DNA, including the ribosomal subunits 18s rDNA, 28s rDNA and SSU-rRNA ( 133 , 134 ), mitochondrial genes (nicotinamide adenine dinucleotide hydrogen – NADH-I, NADH-3), internal transcriber-spacer-2 sequence – ITS2), and the cytochrome c oxidase-COX I ( 135 ). The most studied target is the 121 bp tandem repeat ( 103 , 136 – 138 ), across different species of schistosomes because of the high copy number (600, 000 per schistosome cell) making for high detection sensitivity even if only a few DNA fragments are present in the sample. Apart from whole eggs in stool, urine and tissue biopsies, schistosome DNA can also be amplified from cell-free parasite DNA (cfDNA) released from schistosome stages (schistosomula, adult worms and eggs), which could derive from dead or decaying parasites within the circulation and tissues, active shedding from the parasite or from disintegrating inactive eggs ( 139 – 141 ).…”

“…The sensitivity of PCR-based assays has been improved using q-PCR technique to a limit of detection of 0.38fg (equivalent of less than a single cell) of S. mansoni DNA ( 103 ), which corresponds to approximately 0.00065 times its genome ( 133 ). Compared to Kato-Katz technique with two slides (used in Schistosome control programs in Brazil) as a definitive diagnostic, qPCR had 95.7% sensitivity, 81.6% specificity and 83.8% accuracy ( 103 ). When the number of Kato-Katz slides were increased to 24, qPCR presented 96.7% sensitivity, 87.2% specificity and 89.2% accuracy.…”

Laboratory Evaluation of a Basic Recombinase Polymerase Amplification (RPA) Assay for Early Detection of Schistosoma japonicum

“…With the development of molecular diagnostic technology, nucleic acid detection methods including conventional polymerase chain reaction (PCR) [ 19 ], nested PCR [ 20 ], real-time PCR [ 21 , 22 , 23 ], digital PCR [ 24 ], and so on [ 25 ], presenting high detection specificity and sensitivity [ 26 , 27 ], provide a new idea for the diagnosis of schistosomiasis. However, these PCR-based techniques require expensive equipment and skilled personnel, which are rarely available in schistosomiasis endemic areas featured by poverty and scarce of resources [ 28 ].…”