Urine-based diagnostic technologies

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Urine-based diagnostic technologies Howard B. Urnovitz, William H. Murphy, Toby D. Gottfried and Alvin E. Friedman-Kien The worldwide dissemination of infectious agents has created a demand for simple diagnostic

tests. Urine-based testing makes use of non-invasive collection

of

specimens, and there is no need for expensive facilities and equipment, or for highly trained personnel. As urine antibodies retain activity under normal conditions of transport and storage, such tests appear to have widespread application. Urinebased antibody tests have also indicated a compartmentalized

antibody response

to HIV-1 infection. Urine studies suggest that antibodies

to the products of

endogenous viral genes may be involved in the pathogenesis of chronic diseases of suspected viral etiology.

The rapid development of countries, along with accompanying population growth and industrialization, has resulted in an urgent demand for novel, simple and inexpensive methods to diagnose infectious diseases. In part, this has resulted from the worldwide dissemination of infectious agents by modern means of transportation’. Examples include the global distribution of HIV-l, Ebola virus, Lassa fever virus, dengue fever virus and hantaviruses. Equally important, but less recognized, is the worldwide distribution of simian viruses among human populations, which has resulted from the global use of vaccines made from monkey kidney cells contaminated with such viruses’+. For example, it has been considered that viral genes of simian origin may have been ‘archived’ in various human cells. Apparently, such genes can be maintained in lymphocytes for up to 40 years after an infection, and may be involved in the pathogenesis of some chronic diseases of suspected viral etiology, as in the case of ‘post-polio syndrome’s. There is a trend towards perceiving many chronic illnesses as ‘interactive infections’“, i.e. interactions among several infectious agents (their genes or gene products) in a single host, resulting in a unique clinical entity that cannot be duplicated by a single infectious agent acting alone. Examples would include AIDS, post-viral fatigue syndromes (Chronic Fatigue Syndrome) and the ‘Persian Gulf War Syndrome’. These syndromes appear to have a multifactorial basis H. B. Urnovitz ([email protected]) and T. D. Got!ftiin’cd are at Calyp BiotnnedicalCorporation, 1440 Fourth Street, Berkeley, Cz4 94710, US4. W. H. Muy,hy is at the Department (!f‘~~icrobio2oXI,/Intmttnolo*cy,JXversity qfMichigatz Medico2 School, 9nn ilrbar, LW 48 709, USA. A. E. Friedman-Kien is at thr Drpartmerzts qfDwmatolqg and Microbiology, New York Ut&errity, A’ew York, NY 10016, L&4.

involving one or more infectious agents or their derivatives. For the reasonsoutlined above, public health officialshave called for initiatives’ to detect infections that lead to chronic illnesses.Given these unprecedented demands on diagnostic capabilities and their global application, it is evident that the following criteria must be met for test purposes:testsmust (I) be accurate and sensitive; (2) be inexpensive; (3) involve noninvasive procurement of clinical specimens;(4) require minimal technical facilities, equipment and personnel; and (5) use analytesthat arestableunder ambient conditions. Ideally, the application of such technologies should provide new insights into the pathogenesisof infectious diseasesand be of epidemiological value. Serological testsmeet some of thesecriteria, but have not been generally applicable in Third-World countries. The discovery of antibodies to HIV-1 in the urine of infected subject9 provided an alternative to serological procedures. At the sametime, it provided a basisfor determining whether urine-based testsare generally applicable. In this article, we useurine assays for antibody to HIV-1 as a paradigm to discussthe general application of urine-based diagnostic testing. Basic elements of urine-based technologies Urine diagnostic procedures are based on noninvasive specimencollection, do not require complex laboratory facilities or highly trained personnel, and can provide accurate and sensitive results. The technology is basedon the useof unprocessedurine specimens that do not necessarilyrequire preservatives. It hasbeen demonstrated that HIV-l antibodies in urine are stablefor 55 daysat room temperature, and for one year at 2-8°C so they can be mailed or shippedwithout degradation (either with or without preservatives). -

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Freezing of samples is not recommended because this has led to false-positive reactions”. The key to urine test technologies, however, has been the development of recombinant and synthetic antigens to detect antibodies of diagnostic significance. Such tests are designed to be used on unprocessed urine samples of 200 ~1, which can be directly added to a microplate well. Microplate incubation takes place at 37°C in a standard laboratory incubator for one hour, and is followed by washing, incubation for an additional hour with an anti-human IgG/IgM-alkaline phosphatase conjugate, washing and development with substrate (p-nitrophenyl phosphate; pNPP) for 30 minutes. The results are read spectrophotometrically in a standard plate reader at a wavelength of 405 nm. Urine antibody assays to HIV-l In studies carried out since 1990 on more than 11000 paired urine and serum specimens, we have documented that urine-based tests have essentially the same sensitivity and specificity as conventional serological assays; such results have been verified”’ independently. A modified western blot was used to confirm repeatedly reactive enzyme-linked immunosorbent assay (ELISA)-positive serum specimens. The band found most consistently in the urine of patients exposed to HIV-l is that for the antibody to gpl6O (Ref. 11). Table 1 shows that the one-band criterion for antibody to gp160 in the urine corresponds with the two-band criteria” used for serum antibody tests.

Table 1. Correlation between urine HIV-1 western blot band criterion and serum HIV-1 two-band criteria Number Matched groupa of tests WB seropositive ELlSAseronon-reactive

825 390

One band urineb

Two band urinec

824 (99.9%) 0

789 (95.6%) Od

aELI.SA,enzyme-llnked immunosorbent assay; WB, western blot. bA reactive gp160 band present. Other bands may or may not be present. No band intensity criterion required. CTWOor more of the following bands present: ~24, gp41 and gp16O/gp120. dFour lndeterminates (1% of 390 urines).

Table 2. Human endogenous retrovirus (HERV)urine autoantibodies in serum HIV-1 western blot indeterminate subjects Antibodies to Subject category

Number of subjects

HERV4.1”

CKS-17”

ISI

;

:

:

aH, healthy, no evidence of HIV-1 infection; HSI, healthy sero-indeterminate. bNumber of subjects with at least one urine void positive to the synthetic antigen. CKS-17 used as a control is described in the text.

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Therefore, we counted the occurrence of an antibody band to gp160 in the urine as the interpretative criterion for a positive antibody response to HIV-l exposure. It is noteworthy (Table 1) that indeterminate results occurred with a frequency of about 1% for the urine-based tests in subjects from the ‘low-risk’ category, as compared with the conventionally higher percentage of 15’%1 indeterminates associated with serum HIV-l western blots’“. Compartmentalization of the immune response to HIV-l infection Two findings from the extensive studies of matched serum and urine samples required fLIrther analysis: (1) in some subjects, serum was negative for antibody, while urine was positive14,13: and (2) no ready explanation was evident for the serum-indeterminate results described above. Further tests confirmed that HIV-l urine antibodies were found in two patients with AIDS defining illnesses, low CD4 T-cell counts and serum indeterminate results’+ virus isolated from one subject was typed as HIV-l group ‘0’ (Ref. 13). These findingsL documented’” that HIV-l seronegative subjects had specific antibody to HIV-l in their urine. Moreover, the peripheral blood cells of these subjects had a Thl-type cytokine profile” to HIV-l envelope proteins. These results support the conclusion that there was a compartmentalized immune response to HIV-l infection, and support the belief that urinebased antibody assays provide vital information that cannot be obtained using serum assays. Urine-based analysis of HIV-l western blot indeterminate results Serutn antibodies to human endogenous retrovirus (HERV) gene products are common in subjects with various malignant, autoimmune and chronic diseases of suspected viral etioloby’x. It seemed reasonable, therefore, to test whether urine antibodies from subjects with indeterminate HIV-l antibody reactivity had ‘autoantibodies’ to HEW gene products. Because HERV 4.1 occurs commonly in the human genome, is widely distributedlx in human populations and has been fully sequenced, it has been possible to synthesize a HERV 4.1 envelope (env) peptide antigen to test whether antibodies of the western blot indeterminate category were made up, at least in part, of HERV 4.1 ‘autoantibodies’; a corresponding env peptide encoded by one of the mouse leukemia viruses was used as a control. Table 2 indicates that subjects that had indeterminate results also had antibodies to HERV 4.1 in their urine’“, but such antibodies were not detected in the HIV-l-negative controls. Although such results could account, at least in part, for results of the indeterminate results category, it is evident that such assays need to be confirmed and expanded to gain a better insight into their significance. In addition, studies must address the contribution of specific immune responses to HERV gene products on the immune responses to exogenous retrovirus infections such as HIV-l.

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Urine-based analysis of surrogate markers Because autoimmune responses are such a prominent feature of AIDS, it might be possible to use the defined autoantibodies that occur as surrogate markers for the determination of the clinical stage of the disease. To test this possibility, a cohort of symptomatic and asymptomatic HIV-l-infected subjects were surveyed using urine specimens collected over a 24-hour period. Table 3 indicates that at least one urine specimen from each symptomatic subject contained antibodies to HERV 4.1 (Ref. 19), whereas no antibodies were detected in asymptomatic individuals. This observation has been confirmed in a doubleblind study*“. Such results suggest that urine assays might be useful for determining the clinical stage of AIDS, or for monitoring the response to therapeutic modalities.

Urine-based tests for chronic infections Various microbial agents (e.g. Chlatnydia trachomatis, one of the classic sexually transmitted infections) have been implicated in the pathogenesis of chronic diseases*‘. The US Center for Disease Control and Prevention estimates that there are approximately four million new cases of C. trachomatis infections per year in the USA alone*?. We have used C. trachomatis infection as a model to determine whether urine-based testing is applicable to chronic diseases of an infectious etiology’3. Table 4 indicates the presence ofa relatively high frequency ofantibodies to C. trachotnatis in populations at high risk of sexually transmitted diseases. The common occurrence of antibodies in low-risk populations suggests that the occurrence of C. trachornatis infections in such populations may be underestimated significantly. Although these results are not definitive, they nevertheless indicate that urine-based diagnostic testing can provide important epidemiological data.

Conclusion The urine-based testing described in this article combines the advantages of simplicity, cost-effectiveness and application under circumstances in which serological testing is largely impractical. These advantages provide the opportunity for the diagnostic testing of large populations in impoverished areas, and the ability to transport and store urine specimens under ambient conditions facilitates application. The results summarized in this article provide reasonable evidence that diagnostic urine-based testing is practicable for HIV-l infection. There are three additional cogent findings: (1) testing for antibodies in urine is comparable in sensitivity and specificity to serum-based tests; (2) the antibody response to HIV-l, and its variants, appears compartmentalized; and (3) antibody responses to HIV-l of the indeterminate category may be ‘autoantibodies’ to HERV-encoded gene products. The results for chronic infections, in which C. tracl?omatis was used as a paradigm, are more difficult to interpret because they may, or may not, satisfactorily distinguish between recent infection or the exacerbation of persistent low grade disease. Nevertheless, it

Table 3. Human endogenousretrovirus (HERV)urine autoantibodies in HIV-l-infected subjects Antibodies to Subject categorya

Number of subjects

HIV-1

HERV4.1”

CKS-17”

Asymptomatic Symptomatic

18 8

18 8

Fi

Y

aAsymptomatic subjects had no AIDS-defining illnesses at the time of sample collection. Symptomatic subjects had at least one AIDSdefining illness at the time of sample collection. bNumber of subjects with at least one urine void positive to the recombinant or synthetic antigen. CKS-17 used as a control IS described in the text.

Table 4. Chlamydia trachomatis urine antibodies in low- and high-risk subjects

Risk categorya

Number of subjects

C. trachomatis-confirmed positive@

High Low

;A

10 3

asubjects at risk for sexually transmitted diseases. bC. trachomatis elementary-body preparations were used as target antigen. Confirmation of urine-positive screens was determined by western blot analysis of MOMP chlamydial antigen. would appear that ‘cut off’ points can be identified that distinguish between recent infection, as opposed to the recurrence of a chronic disease. Whatever the case, it is evident that urine-based tests for C. trachomatis infection have epidemiological value. The limits to which urine-based diagnostic testing may be applied remain to be defined. However, such tests are applicable to infectious agents such as poliomyelitis’-!, Helicobacter pylorPs and human hepatitis C virus26. The basic question appears to be whether the occurrence in urine of antibodies to infectious agents occurs with sufficient frequency that urinebased testing becomes generally applicable, and is capable of providing alternatives to serum-based technologies because of putative cost-effectiveness and simplicity of application. Other applications of this technology, such as for the diagnosis of autoimmune or allergic disease, or of other chronic diseases, should also be explored.

References Fldlrr, D. P., Jr (1996) Ewqin~ Ifif: Dir. 2, 77-W Gxrrtt. L. (1991) 7%~ Cornuz~ P/qx: ~Vw~y Emryirq Diwses in n Wol’ouldOur ofBa/mce, pp. 53-70, Farrar, Straus and G~roux Hull, l<. N., Mimer, J. R. and Mascoli, C. C. (1958) Am.j. Hy,. 68, 31-44 Shah. K. and Nathanson, N. (1976) Am,], Epdemiol. 103. 1-12 Leon-Monzon, M. E. and Dalakas, M. C. (1995) Ann. ?\ie~ York Ad Sci. 753, 208-218 Umovitz, H. U. (1992) Biot& Fwnm Europe 9. 1-5 Anon. (1992) LIFT 2000: Laboratory Inifiafrvec &r the Yeor 2000, Assomtion of State and Territorial Pubhc Health Laboratory TIBTECH OCTOBER 1996 WOL 14)

Evaluation of hepatitis C virus protein epitopes for vaccine development Rajen Koshy and Genevieve Inchamp Infection with hepatitis C virus (HCV) leads to viral persistence and chronic disease in a very high proportion of cases, despite a broad immunological

response to viral

proteins. These responses may be thwarted by the high rate of mutation, which leads to the generation of ‘escape’ variants of HCV that persist as a quasi-species in infected individuals. The specificity of the immune responses of infected patients suggests that responses directed at certain viral epitopes may be associated with less aggressive clearance.

disease and, possibly, good interferon

The identification

of such epitopes

response and virus

may hold the key for future

development both of prophylactic and therapeutic vaccines.

Hepatitis C virus (HCV) is the etiological agent of the majority of both blood-transfusion-associated and community-acquired non-A-non-B (NANB) hepatitis worldwide. Acute infections progress to chronicity in an astounding proportion (estimated at 180%) of cases, and frequently lead to cirrhosis and hepatocellular carcinoma (Fig. 3). At present, treatment with a-interferon (IFN-(x) is the only available therapy, but long-term benefit is only obtained in about 20% of treated cases’. With an estimated 500 million people infected worldwide, HCV constitutes a massive me& cal threat, underscoring the urgent need for anti-HCV vaccines and antiviral agents. The available clinical

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and molecular evidence indicate that the task ahead will be arduous because, although a certain degree of immunity appears to be induced following primary infection, it fails to control the infection. A number of possible reasons for the failure to mount a protective immune response are being studied and, in particular, there is growing evidence for the following observations: (1) typically, HCV infections are associated with low viral titres, i.e. lo’-10’ chimpanzee infectious doses (CID) per millilitre, which may account for low antigenic stimulus; (2) virus particles are closely associated with low-density lipoproteins or immunoglobulins, and this could result in the masking of antigenie determinants; and (3) rapid evolution of mutant viruses with altered B- and T-cell epitopes occurs (discussed below). Other suggested reasons, for which evidence is still lacking are: production of defective virus particles, downregulation by viral gene products Ltd. All rights resewed.

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