Sensitivity and specificity of point-of-care circulating Cathodic antigen test before and after praziquantel treatment in diagnosing Schistosoma mansoni infection in adult population co-infected with human immunodeficiency virus-1, North-Western Tanzania

Background The effect of Human Immunodeficiency Virus-1 (HIV-1) on CD4+ Th2 cells is hypothesized to affect parasitological diagnosis of Schistosoma mansoni using Kato Katz technique. Thus, the use of more sensitive technique such as Point-of-Care Circulating Cathodic Antigen (POC-CCA) test is recommended. However, the sensitivity of this diagnostic test in diagnosing S.mansoni infection and the usefulness of it in monitoring efficacy of praziquantel drug in presence of HIV-1 co-infection remains inconclusive. The Primary objective of the present study was to assess accuracy of the POC-CCA test in diagnosing S.mansoni infection before and after praziquantel treatment in adult population co-infected with HIV -1. Methods A prospective longitudinal study was conducted among individuals aged 15–55 years at Igalagala village, north-western Tanzania. At baseline and 4 weeks after treatment, a single stool and urine samples were collected from each participants. Kato Katz (KK) technique and Point-of-Care Circulating Cathodic Antigen tests were used for diagnosis of Schistosoma mansoni. Results At baseline, based on KK and POC-CCA, the prevalence of S.mansoni was 57.8% (95%CI: 52.9–62.4) and 87.5% (95%CI: 83.9–90.4). Based on KK technique and POC-CCA test, 3.6% and 5.7% of the study participants were co-infected with S.mansoni and HIV-1. At baseline, in the general population, the sensitivities of POC-CCA test using KK technique and combine gold standard were 96.3%(95%CI: 93.1–98.3) and 97.6%(95%CI:95.5–98.9) respectively. In the HIV-1 seropositive group, at baseline, the sensitivities of POC-CCA test using KK technique and combined gold standards, were 93.3%(95%CI:68.1–99.8) and 96%(95CI%:79.6–99.9). Four weeks after treatment, in general population, the sensitivities of POC-CCA test using KK technique and combined gold standards were 47.8%(95%CI:26.8–69.4) and 84.4%(95%CI:74.4–91.7). In the HIV-1 seropositive group, using KK technique, the sensitivity was 100% (95%CI:2.5–100). Conclusion The sensitivity of POC-CCA in diagnosing S.mansoni infection was higher than KK technique in adult individuals likely to have low infection intensity and co-infected with HIV-1. However, its sensitivity decreases following praziquantel treatment but remained higher than Kato Katz technique. If the goal of the post-treatment is to identify uncured individuals, then POC-CCA test offers the best choice.


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Conclusion: The sensitivity of POC-CCA in diagnosing S.mansoni infection was higher than KK technique in adult individuals likely to have low infection intensity and co-infected with HIV-1. However, its sensitivity decreases following praziquantel treatment but remained higher than Kato Katz technique. If the goal of the post-treatment is to identify uncured individuals, then POC-CCA test offers the best choice.

Background
In the past three decades, epidemiological studies have demonstrated an overlap of Human Immunodeficiency (HIV-1) and Schistosoma mansoni infections in Sub-Saharan Africa [1,2], leading to co-infection in proportion of individuals living in highly endemic regions [3,4]. It is hypothesized that helminths infections such as Schistosoma mansoni may exacerbate HIV-1 transmission or progression to AIDS in Africa [2,3]. This is based on observations that infection with these parasites cause a chronic stimulation of the immune system with a strong bias towards a type 2 CD4 + helper T cell (Th 2 ) response [3,5]. This in turn affects the ability of the host's immune system to control HIV-1 replication [3]. Conversely, HIV-induced destruction of CD4 + T cells, especially those that secrete type 2 cytokines [6,7]; this may affect granuloma formation and alter egg excretion efficiency.
Studies in suppressed animal models has demonstrated that the excretion of S.mansoni eggs is immune dependent, and that T-cells, specifically anti-eggs Th 2 response [8] are necessary for the transposition and excretion of eggs from the host blood stream into the intestinal lumen [7]. HIV-1 infection has been demonstrated to affect S.mansoni eggs excretion efficiencies [9]. This observation have been described to impact on diagnosis of S.mansoni infection using Kato Katz technique, which depend on examination of the parasite eggs [9]. This may lead to underestimation of the prevalence and intensity of infection before and after praziquantel treatment in adult population co-infected or not with HIV-1 [9]. The potential for HIV-1 to affect S. mansoni eggs excretion makes the detection of circulating schistosome antigens released insitu particularly important in detecting S. mansoni infection [10]. In this context, a Point-of-Care Circulating Cathodic Antigen test has been developed as an alternative test to the Kato Katz technique. This test has been extensively assessed in sub-Saharan Africa [10,11]. However, the results on the sensitivity of circulating antigen detection tests in adult individuals co-infected or not with HIV-1 evaluated before and after praziquantel treatment have yielded contrasting results [12,13]. The discrepancies observed between these studies calls for further studies to further evaluate the sensitivity of POC-CCA test in the adult population and assess its effectiveness in evaluating mass drug administration treatment programs in adult population. In this context, the primary objective of this study was to assess the sensitivity and specificity of Point-of-Care Circulating Cathodic Antigens (POC-CCA) test in diagnosing S. mansoni infection in comparison to parasitological Kato Katz technique (regarded as "gold standard") and a combined gold standard (infection-positive by either Kato Katz technique or POC-CCA-positivity (assuming 100% specificity of the POC-CCA test) [14] among adult population living in a fishing village of north-western Tanzania, either co-infected or not with HIV-1. Secondarily, the study assessed the usefulness of Point-of-Care Circulating Cathodic Antigen test in monitoring the efficacy (CCA clearance at 4 weeks following treatment) of praziquantel drug in presence of HIV-1 infection among adult population.

Study area
The study area is described in details in [15,16], briefly, the present study was conducted at Ilemela district, Mwanza region, located at 32-34°E and 2-4 0 S, on the southern shores of the Lake Victoria, north-western Tanzania. Specifically, the study was conducted at Igalagala village located at shoreline of the Lake Victoria. The choice of this village was based on its close proximity to the lake, high prevalence and intensity of S. mansoni infection [16] and high HIV-1 prevalence [15]. The majority of the populations are of Sukuma tribe and other are migrating tribes such as Wakerewe, Wajita and Wakara. The main economic activities are fishing and farming. The Lake is the major source of water and mainly used for bathing, cooking, drinking and recreation. Because of high water contact, the residents have high risk of being infected with S.mansoni and carry highest infection intensity [15]. Annual mass drug administration (MDA) using praziquantel against S. mansoni infection mainly focus to schoolchildren and adult population is not included.

Study design, inclusion and exclusion criteria
This was a prospective longitudinal study, which was conducted in October-November 2016, was characterized with two cross-sectional surveys at baseline and 4 weeks after praziquantel treatment (Fig. 1). All adult individuals aged 15-55 years of age and lived in the study village for > 2 years were eligible for recruitment into the study. The length of stay in the risky areas for transmission of S.mansoni has been demonstrated to increase the chances for acquiring the infection and developing the related hepatosplenic morbidities [17]. The study excluded individuals who were on antiretroviral treatment (ART) due to the fact that excretion of S.mansoni eggs is a function of immune status as measured in term of CD4 + Th 2 cells [9] and taking antiretroviral therapy have effects on the level of CD4 + Th 2 cells [4]. Thus, including individuals who were on antiretroviral therapy would confound the results of Kato Katz technique among the HIV-1 infected group. The study also excluded individuals with CD4 + < 350cells/μL from the prospective survey. By the time this study was conducted, the country guideline for ART initiation required that all individuals diagnosed with HIV-1 infection and having CD4 + < 350cells/μL to be initiated treatment. Therefore, it was considered unethical to included HIV-1 infected individuals with CD4 + < 350cells/in a prospective study. Lastly, individuals with history of taking anti-schistosomiasis in the past 6-month were also excluded to remove the confounding effects of the treatment on the sensitivity of the diagnostic test especially parasitological technique.

Sample size and sampling procedures
For calculation of the sample size, we used the formula for comparing two independent proportional to estimate sample size for comparing sensitivity/specificity of the POC-CCA test and that of the Kato Katz technique (referred as a Gold standard) [18]. At 95% confidence interval and a power of 80% to detect a difference of 10% from sensitivity of 85% (i.e. P 1 = 85%, P 2 = 75% and P 0 = 77.5%) [18], the required sample size was 472 participants but only 419 participants were enrolled into the study. Although the sample size was below the estimated sample size, our power to detect the estimated difference remained > 90%.
Selection of the study participants has been describe in details in [15], briefly a random sampling procedure was used to select households and household members to participate in the study. Prior to study implementation, a list of households and household members was obtained to allow random sampling of eligible individuals from randomly selected households.

Data collections Questionnaires
A pre-tested questionnaire was used to collect demographic information of the study participants, past history of HIV testing, use of anti-schistosomiasis treatment and use of anti-retroviral therapy.

Examination of Schistosoma mansoni circulating cathodic (CCA) antigen and Schistosoma haematobium eggs
For screening of S.mansoni infection, a point-of-care Circulating-Cathodic-Antigen (CCA) (Rapid Medical Diagnostic-http://www.rapid-diagnostics.com/, Batch number: 33827) which uses urine sample was employed. A single urine sample was collected from each participating individuals [19] and the POC-CCA test was used to screen for the antigen. The POC-CCA test detects the presence of schistosome CCA released from worms excreted in the host's urine [19]. The test was performed as per manufacturer instructions (Rapid Medical Diagnostic-http://www.rapid-diagnostics.com/) and "trace" results were considered as positive. Qualified laboratory technician were blinded for the Kato Katz parasitological results and HIV-1 sero-status of the study participants used the test to screen for S.mansoni infection.
For screening of S.haematobium infection, the same urine sample collected for POC-CCA screening was used. Urine sample was grossly examined for the presence of gross heamaturia and using Hemastix® for presence of micro-haematuria [20]. Then, the urine sample was filtered (pore aperture 20 μm; Sefar AG, Heiden, Switzerland), filter placed on a slide and examined under a microscope for presence of S. haematobium eggs [20].

Examination of human immunodeficiency Virus-1
Human Immunodeficiency Virus-1 (HIV-1) testing was done according to Tanzanian National HIV algorithm [21]. This involved the use of Determine® and UNGold qualitative tests [21]. Before, HIV testing, participants received counseling from qualified HIV counselors. Then, finger prick blood sample was collected from each consenting study participants and screening for HIV-1 infection using both test. Results were recorded based on the manufacturer instructions. The quantification of CD4 + cells was done using a FACS Calibur machine (Becton and Dickinson, BD Biosciences, San Jose, CA, USA), following standard manufacturer procedures.

Parasitological examination of Schistosoma mansoni eggs in stool samples
For diagnosis of S. mansoni infection, a single stool sample was collected from each study participant at baseline and 4 weeks after praziquantel treatment. On the screening day, each participating individual was provided with a container for stool sample collection. Stool samples were collected fresh, labeled with a specific study number and processed using Kato Katz thick smear technique using a template of 41.7 mg per thick smear [22]. From each collected stool sample, four Kato Katz thick smears were prepared. Three-laboratory technician supervised by a senior laboratory technician examined all Kato Katz thick smears at the National Institute for Medical Research, Mwanza, Tanzania. For quality assurance purposes, 10% of all positive and negative slides were re-examined by an independent laboratory technician.

Data analysis
A Microsoft Excel system was used for double data entry and data analysis was performed using Stata Version 15 (StataCorp, 2017, Stata statistical software: release 13. College Station, TX: StataCorp LP, Taxes, USA). Numbers and percentages summarized categorical variables. Comparison of proportions/categorical variables was done using chi-square (χ 2 )/fisher exact where appropriate. For continuous variables descriptive statistics were reported as means with standard deviation for normally distributed variable and medians with interquartile ranges (IQR) for variables that were not normally distributed. The arithmetic mean of S. mansoni egg counts for each participant was calculated from the counts of four Kato Katz thick smears and multiplied by 24 to obtain individual eggs per gram of faeces. Schistosoma mansoni egg counts were logarithmically transformed to check for normality but only non-transformed means are presented. Mean egg counts for S. mansoni between sex and age were compared using Student-t-test (two groups) or ANOVA (more than two groups). Intensity of infection was categorized according to WHO criteria as: 1-99 epg, 100-399 epg, ≥400epg defined as low, moderate and heavy intensities of infection respectively [23].
The sensitivity, specificity, positive and negative predictive values of the POC-CCA test was calculated using two gold standards, (1) egg-positive by Kato Katz technique (as reference standard for diagnosis of S. mansoni infection) and (2) combined gold standard (infection-positive by either Kato Katz technique or POC-CCA-positivity (assuming 100% specificity of the POC-CCA test) [14]. Specificity, i.e. the percentage of negative individuals correctly identified as such, was calculated as described in [24]. Similarly, sensitivity was calculated as percentage of positive individuals correctly identified as such [24]. In addition, positive predictive value (PPV = proportion of positive test results that are truly positive) and negative predictive value (NPV = proportion of negative test results that are truly negative) were calculated after calculating sensitivity and specificity of the diagnostic tests [24]. Statistical significance was assigned at P < 0.05 for all analyses.

Ethical consideration and confidentiality
Ethical approval was sought from the joint Ethical and Review Committee of Bugando Medical Centre and Catholic University of Health and Allied Sciences. The study received further permission from the district administrative and division authorities. Kiswahili translated informed consent forms were used to obtain participants consent to participate in the study and assent for those who were aged 15-< 18 years. Written informed consent was obtained from guardians/parents of all participants aged 15-< 18 years. For illiterate participants, a thumbprint was used to sign the consent/assent forms after a clear description of the study objectives was given to them. To maintain confidentiality, all the demographic and other clinical data of the study participants were kept in a closed cabinet and whenever the data were accessed no participants name were disclosed, only identification number of the participants were used to identify participants. All study participants infected with S. mansoni were treated with praziquantel (40 mg/kg) according to WHO guidelines [25].

Demographic characteristics of study participants
A total of 419 study participants were enrolled in this study, of these 46.1% (n = 193/419) and 53.9% (n = 226/ 419) were female and male respectively. The median age of the study participants was 30 years (IQR: 22-42 years). Table 1 shows the age and sex distribution of the study participants from Igalagala village. All of the study participants were from Igalagala village located along the shoreline of the Lake Victoria. All of the study participants performed their economic activities within the village. The main economic activities of the study participants were farming, fishing and fish processing and small-scale business.

Pre-treatment baseline prevalence and intensity of
of them were available for re-examination, 4/6 (66.7%) had no infection and only 2/6(33.3%) remained with detectable eggs in their stool samples (Fig. 1).
In relation to HIV-1 serostatus, of the 15 HIV-1 seropositive who were co-infected with S.mansoni infection based on Kato Katz technique, eight (8) of them were available for re-examination, 25% (2/8) remained with S.mansoni eggs in their stool samples at 4 weeks after treatment (Fig. 1). Based on POC-CCA test, of the 24 HIV-1 seropositive who had POC-CCA positive test at baseline, 12 were available for re-examination, of these 33.3% (4/12) remained with CCA positive results at 4 weeks of follow-up (Fig. 1). There was no difference in age and sex differences between those who were present and those who were absent at 4 weeks of follow-up (χ 2 = 5.7496, P = 0.23). In addition, there was a mean difference in infection intensity between those who were present and those who were absent at 4 weeks of follow-up (t = 2.3329, P < 0.02), with those who were absent had a higher infection intensity.
Diagnostic characteristics of the point-of-care circulating cathodic antigen test before treatment  Table 2. Then, Using the combined gold standard (infectionpositivity by either egg-or-POC-CCA positivity, assuming 100% specificity of the POC-CCA test), the sensitivity of the POC-CCA test improved to 96.0% (95%CI: 79.6-99.9).

Diagnostic characteristics of the point-of-care circulating cathodic antigen test after treatment
The results obtained using Kato Katz technique as a gold standard and combined gold standard (infection-positivity by either egg-or-POC-CCA positivity, assuming 100% specificity of the POC-CCA test) 4 weeks after treatment are shown in Table 2

Discussion
The main findings from this study indicate that, pre-and-post treatment sensitivity and specificity of the point-of-care circulating Cathodic Antigen test using combine gold standard were higher than that of Kato Katz technique, both in the general population and the HIV-1 seropositive group. We observed declined in sensitivity of POC-CCA test 4 weeks after treatment, when   KK technique was used as a gold standard, but this changed to a higher sensitivity when a combined gold standard was used. We observed high praziquantel cure rates both in HIV-1 seropositive and seronegative groups infected with S.mansoni evaluated at 4 weeks after treatment.
At pre-treatment assessment, our data indicated that POC-CCA test achieved a sensitivity of 96.3% but had low specificity in detecting S.mansoni infection using Kato Katz technique as a gold standard in the general population. Similar findings were noted by previous similar studies conducted elsewhere in African setting using Kato Katz technique as a gold standard [26,27]. Conversely, at pre-treatment, the POC-CCA test achieved high sensitivity (93.3%) but low specificity in detecting S.mansoni infection in the HIV-1 seropositive group. A high pre-treatment intensity of S.mansoni infection appears to influence the high sensitivity of POC-CCA test [28]. Significant positive correlations between positivity of POC-CCA test and S. mansoni eggs counts have been noted in previous study in Brazil and Kenya [28,29]. This partly could explain a high pre-treatment sensitivity of the POC-CCA test in the two groups, which is reported to decrease in individuals with light infections (< 100 eggs/g of feaces) [26,28]. The low POC-CCA test specificities in part are explained by the low sensitivities of the Kato Katz technique [26]. Our pre-treatment findings on the sensitivity and specificity of POC-CCA test are comparable to previous findings published among primary school children in endemic areas of Uganda (94.8%) [26] but higher than 86.9% and 56.3% observed in Cote d'Ivoire [27]. The variation in prevalence and intensity of S.mansoni infection between geographical settings seems to influence the sensitivity of the POC-CCA test. In region with low prevalence such as in Cote d'Ivoire, the sensitivity of POC-CCA test was lower (56.3%) despite that three POC-CCAs were performed to increase sensitivity [27]. In our present study, 32.5% and 35% of the study participants identified by POC-CCA test having S.mansoni infection in the general population and in the HIV-1 seropositive group were missed by Kato Katz technique. This observation indicate that, if selective treatment is done based on the KK test, these individuals would be erroneously indicated not infected. Previous studies have demonstrated reduce efficiency of S.mansoni eggs excretion in HIV-1 individuals co-infected with S.mansoni infection [1,9,15,30]. Thus, the use of a more sensitive diagnostic test/technique than Kato Katz technique is justifiable in adult population likely to be co-infected with HIV-1 and S.mansoni and likely to have light to moderate intensity of infection [26].
At 4 weeks post-treatment using Kato Katz technique as a gold standard, the sensitivity of POC-CCA test decreased to below 50% but its specificity improved to almost to 75% in the general population. A similar findings have been reported in Uganda, in which at 1 and 4 weeks post-praziquantel treatment, the sensitivity of the POC-CCA test decreased from 91.7% to 66.7% and 73% [26]. A plausible explanation for this observation is the increase in POC-CCA false positives due to increase in specificity [26,29,31]. Alternatively, the decline in intensity of infection at post-praziquantel treatment affects the performance of POC-CCA tests [28]. A noted pre-treatment correlation between S.mansoni egg loads and the strength of positivity of POC-CCA test has been reported [26,29,32]. An interesting questions which needs follow-up is the relationship between parasites antigen levels at pre-and-post praziquantel treatment and reduced eggs intensity following praziquantel treatment. The fact that Circulating Cathodic Antigen are released by mature adult worms which are also responsible for producing eggs [19], it will be interesting to understanding this relationship.
In the HIV-1 seropositive group, the POC-CCA test attained a sensitivity of 100% using Kato Katz technique as a gold standard. Is worthwhile mentioning that at 4 weeks post-treatment evaluation, our analysis suffered from low number of HIV-1/S.mansoni co-infected individuals to give a meaningful statistical analysis on the sensitivity and specificity of the POC-CCA test. This is a limitation for this study. Evidence from our data indicate that for the HIV-1/S.mansoni co-infected individuals who returned after 4 weeks for re-examination, at least 60% were Kato-Katz negative/POC-CCA positive. Similar findings have been reported in rural Zimbabwe, where a low CCA clearance rates was observed in HIV-1/S.mansoni co-infected individuals [30]. In term of public health, this findings adds weight on the need to included highly sensitive diagnostic test especially when screening and evaluating MDA programs which involve adult individuals in areas which are co-endemic to HIV-1/S.mansoni infection.
Understanding the limitations of Kato Katz technique as gold standard diagnostic test [33], we developed an assumption that the POC-CCA test is 100% specific and constructed an artificial gold standard based on either Kato Katz technique egg positive or POC-CCA test seropositivity [14]. Using the combined gold standard resulted in improved in the sensitivity of POC-CCA test compared to what was observed when using Kato Katz technique as a gold standard, both at pre-treatment (96.3% versus 97.6%) and 4 weeks after treatment (47.8% versus 84.4%) in the general population. Conversely, for the HIV-1 seropositive group, the use of combined gold standard resulted in increased in sensitivity of POC-CCA test compared to what was recorded when using Kato Katz technique before treatment (93.3% versus 96%). However, using a combined gold standard, the sensitivity of Kato Katz technique before and 4 weeks after treatment, both in the general population and in the HIV-1 seropositive group remained lower than that of POC-CCA test. These findings indicates the needs to supplement Kato Katz technique in either epidemiological surveys or when assessing the efficacy of praziquantel drug especially in adult individuals likely to have light infection intensity or co-infected with HIV-1/ S.mansoni. The use of more than one diagnostic tests to screen parasitic diseases is highly recommended in order to improve the sensitivity of field based diagnostic techniques. This increases the chances of detecting individuals with different levels of infection intensity in endemic areas and allows precise measure of the burden of disease when planning for community based intervention measures [34,35].
Efficacy of praziquantel drugs as measured by parasitological cure rates between the HIV-1 seropositive groups co-infected with S.mansoni and HIV-1 seronegative group infected only with S.mansoni parasite did not vary significantly. Similar findings were reported in northwestern Tanzania, in which parasitological cure rates of 62.6% and 48.3% were reported among HIV-1 seronegative group infected with S.mansoni and HIV-1-S.mansoni co-infected group [36]. At 12 weeks assessment of praziquantel treatment, Kallestrup et al., [13] in Zimbabwe recorded parasitological cure rates of 86% and 85% in HIV-1 seropositive group co-infected with S.mansoni and those only infected with S.mansoni [13]. However, at 4 weeks of praziquantel treatment evaluation, Karanja et al., in [12] recorded lower cure rates of 53% and 59% among HIV-1 seronegative group infected with S.mansoni and those who were co-infected with HIV-1 and S.mansoni [22]. The variation in pre-treatment intensity of S.mansoni, intensity of transmission and low sensitivity to praziquantel in some of the strains of S.mansoni parasite can partly explains the observed differences in cure rates between the HIV-1 seronegative groups and groups co-infected with HIV-1 and S.mansoni [37].
The observation of Kato Katz-negative/POC-CCA positive results at 4 weeks post-praziquantel treatment may reflects existing of adult or juvenile stages not affected by treatment and newly acquired infection in high transmission setting, like that of the present study area. In general, the presence of Kato Katz negative/POC-CCA positive reflects continued infection in treated individuals (having worms which continue to release CCA). With majority of Kato Katz negative individuals remaining harboring light intensity of infections as shown by POC-CCA test in the present study and those of other authors [26,29], these findings clearly suggest the need for repeated rounds of treatment for persistent infection per year contrasting the current recommendation of once per year [23]. This means more costs to the MDA programs. To reduce costs, the currently used Kato Katz technique to evaluate MDA program need to be supplemented with a more sensitive assay such as POC-CCA test.
The present study was not conducted without limitation, the low number of HIV-1/S.mansoni co-infected individuals affected our analysis on the evaluation of sensitivity and specificity of POC-CCA test and Kato Katz technique at 4 weeks after treatment. Moreover, the use of a single stool sample to examine for S.mansoni infection owing the day to day variability of S.mansoni eggs output may have affected the performance of the Kato Katz technique.

Conclusion
Our findings indicate that the POC-CCA test had higher sensitivity at pre-and-post praziquantel treatment than Kato Katz technique using a combined gold standard. The sensitivity of POC-CCA test remained higher than Kato-Katz technique even in the presence of HIV-1 co-infection before and after praziquantel treatment. However, the POC-CCA test had low specificity at pre-treatment which increased at 4 weeks post-praziquantel treatment. Although POC-CCA test have shown promising results for its use in post-treatment evaluation of MDA program, further studies are needed to elucidate the full potential of the test in evaluating the impact of MDA programs in population living in areas with different transmission intensity.