A total of 516 subjects, visiting HIV/AIDS treatment centers located in different districts of Punjab, Pakistan, participated in this study. Out of these 152(29.3%) were females, 360 (69.7%) were males, and 4 (0.77%) were transgender. The average age of subjects was 32.10 years (range 4-67 years) with 296(57.36%) participants belonging to the age group 30- 42 years(Figure 1). 276 (53.4%) were on ART while 240 (46.5%) subjects were treatment-naïve.

Blood samples of all study participants were evaluated for HIV-1 antibodies using three ELISA kits (based on different principles and antigens) as a gold standard. Overall, 408 subjects tested positive and 108 tested negatives for HIV on these kits. On 03 kit HIV testing method, 276 patient which were on ART treatment showed reactivity against serum IgG antibodies for HIV  (100%) and none of them was HIV negative. In case of naïve patients and high risk groups suspected participants, 144 (60%) were determined to be HIV positive, and 96(40%) of participants were non-reactors. On Calypte HIV-1 Urine EIA test, 176 (63.76%) individuals that were on ART tested positive and 100 (36.23%) tested negative. However, among 240 antiretroviral-naïve/ high risk suspected participants HIV patients,132 (55%) and108 (45%) tested positive and negative, respectively.

However, when compared to the gold standard protocol, different results were observed. 312 (60.46%) of the patients tested positive on the Urine Elisa kit whereas 204 (39.53%) showed negative results. As 108 (52.9%) of the 204 individuals tested negative on both the urine Elisa kit and three serum ELISA test kits, they were termed true negatives. 96(47.5%) subjects were classified as false negatives since they only tested negative with the urine Elisa kit (Figure 2).

Calypte HIV-1 Urine EIA test was assessed for sensitivity and specificity. The sensitivity value turned out to be 76.47%, while 100% specificity was observed.  Likewise, the positive predictive value was also 100%. However, the negative predictive value was 52.94% (95% CI 48.57% – 57.27%) and overall accuracy result for this kit was observed to be 81.40% (Table 1).

The elevated need for HIV-specific antibodies has highlighted the importance of diagnostic assays that can be utilized in a variety of settings [12]. The ideal alternative in resource-limited countries is the development of cost-efficient assays with accurate results. The most preferable option is the use of tests that require easily collectible samples while yielding accurate results at the same time [13]. The presence of HIV-specific antibodies in serum or plasma governed the use of these samples for HIV testing for a long time. Alternative non-invasive samples, such as urine or saliva have also been considered. US Food and Drug Administration has approved the use of oral testing devices because the assays have demonstrated excellent accuracy in many studies [14-16]. HIV testing employs specific antigens that are usually obtained from lysates in purified form, synthetic peptides, or recombinant technology. Sensitivity and specificity result due to these antigens. Because of their relatively easy technique, inherent high sensitivity, and suitability for testing large quantities of samples, ELISA tests are the most routinely used assays to screen for HIV infection [12]. Antibody titer or antibody avidity-based methodologies have been developed to identify newly infected people from those who have been infected for a long time [5,17].

In 1998, HIV testing from urine was first proposed. It was proposed due to the considerable benefits over other testing methods since urine is easy to collect, poses less risk of HIV transmission, better compliance, and reduced costs as compared to serum [18]. In many recent studies, Urine immunoassays have proved many benefits. The success of the method lies in its ease of use and safe disposal of the materials involved. The sample collection of urine analysis does not require specialized personnel and adds to the existing benefits of using the method. A study has elucidated the ability of this technique for the detection of HIV-1 despite the non-appearance of HIV-1 p24 antigen. Sensitivity and specificity testing of urine immunoassays have also exhibited promising results. However, this is not always the case as some studies have also reported weak results. Thus, the need is to use the urine immunoassays in a specific population after close evaluation of its validity testing in that particular population [7]. In 1996, the US Food and Drug Administration approved the Calypte Urine HIV-1 enzyme immunoassay (EIA) (Calypte Biomedical Corporation, Berkeley, CA, USA) for the detection of HIV-1 antibodies in urine, as well as the one-band gp160 WB confirmation test in 1998. Even though these tests are routinely used for screening in the United States [19], none of the currently available urine-based assays have been employed for epidemiological reasons in resource-limited countries. IgG antibody concentration in urine is about 1mg/L that is why fairly sensitive techniques are required to detect antibodies against HIV-1 in urine. To date, urine sample testing has relied on ELISA and Western blot techniques, although attempts are being undertaken to create simple/quick procedures for the detection of HIV antibodies in urine specimens [20].

The goal of this study was to see if this commercial urine-based test might be used as a screening tool for HIV infection in Pakistani populations at risk. It was observed that the concentration of antibodies is substantially low as compared to serum. Other factors may alter antibody concentrations in urine samples, causing a sample to be nonreactive to the screening and confirmation techniques utilized in the study, even though the sample was obtained from an HIV-positive individual. Urine collection after a very short period of the last urination can lead to false-negative results since it takes time for antibody levels to return to normal set point. Also, the intake of anti-diuretic, liquids in large amounts, and immuno-compromised status of an individual consequence into varied quantity of antibodies; hence, false-negative test results [13,19,21].

Although collecting urine samples is simple, sample processing necessitates the use of qualified personnel because urine ELISA is a sophisticated technique. Furthermore, in developing countries, urine collection facilities may not be accessible easily. Urine analysis also cannot be used for testing of subjects from remote sites due to the shorter shelf life of the sample. In Pakistan, HIV testing is still a centralized system and individuals from remote sites have no access to HIV testing facilities. This necessitates the use of specialized personnel for sample collection from those areas. Thus, the use of urine for testing those subjects will not serve any purpose with a centralized testing facility.

There have been a large number of studies that have elucidated good sensitivity and specificity of Calypte-HIV-1 testing. However, all these studies were set in high prevalence populations. Pakistan has an HIV population prevalence of less than 0.1% and does not fall under the recommended criteria of using urine-based assays [22]. In addition to these, all these studies have recommended the use of urine-based assays in epidemiological settings instead of diagnostic ones [20].

The comparison of Calypte HIV-1 urine enzyme immune assay with the 3-kit method yielded uneven results in our study. On one hand, high specificity was observed whereas sensitivity was considerably low. Same was the case with positive predictive value and negative predictive value where the latter was much lower than the former.

In our results, the urine ELISA kit correctly identified all the subjects without the disease i.e., excellent specificity was observed. It was 100% with 95% CI. These results were on par with the results of a similar kind of past study [23]. Positive predictive value (PPV) obtained in this research i.e., 100% with a confidence interval of 95% was also per previous investigations. WHO guidelines for diagnostic assays to be used in the developing world suggest a high sensitivity and specificity. Even though our test results yielded a high specificity, the outcome of low sensitivity makes these assays rather doubtful in our population settings [24]. Additionally, other studies have also discouraged the use of HIV-1 EIAs to be used as single confirmatory assays where low specificities were observed [25]. Thus, the results in our study, WHO recommendations, and previous studies are all suggestive of not using only this kit in these particular settings for diagnostic purposes.

The dire need for quick, efficient, and reliable testing protocols for HIV diagnosis demands increased research on the subject. Developing countries face many issues of limited resources and compromised health infrastructure; therefore, the use of any testing means should be in accordance with the set standards. Even though Calypte-HIV-1 Urine EIA is an ideal alternative of invasive techniques in high prevalence regions, its use in developing countries should be carefully analyzed. The research aimed at examining the test results of Calypte HIV-1 Urine EIA by comparison with a routinely used 03-kit method. The low sensitivity of the technique in our results, centralized system of testing, and low prevalence of HIV in the country suggest against the use of technique in daily diagnostic procedures.