The World Health Organization estimates that at any point in 2008, 36.4 million adults 15 to 49 years of age worldwide had syphilis, with 10.6 million incident cases (http://www.who.int/reproductivehealth/publications/rtis/stisestimates/en/). Seventy-three percent of the prevalent and 60% of the incident syphilis cases came from Africa and Southeast Asia, regions of the world in which HIV is also highly prevalent and incident,1 and in which diagnostic resources may be limited. Neurosyphilis is a serious complication of syphilis that can be asymptomatic or symptomatic; when symptomatic, it can cause vision and hearing loss, meningitis, stroke, and cognitive impairment. Neurosyphilis may be more common in HIV-infected individuals than in those who are not infected with HIV.2
The laboratory diagnosis of neurosyphilis rests upon identifying cerebrospinal fluid (CSF) abnormalities, including CSF pleocytosis and reactivity of the CSF–Venereal Disease Research Laboratory (VDRL) test.3 A reactive CSF-VDRL confirms the diagnosis of neurosyphilis, but the test can be nonreactive in individuals with neurosyphilis.4 Treponemal tests such as the fluorescent treponemal antibody–absorption (FTA-ABS) have been used on CSF for neurosyphilis diagnosis; they are more sensitive than the CSF-VDRL, particularly in asymptomatic neurosyphilis, but they are less specific.5 Both the CSF-VDRL and the CSF–FTA-ABS are logistically complicated to perform. The CSF-VDRL requires specialized glass plates and a light microscope, and the CSF–FTA-ABS requires a fluorescent microscope.6 In addition, the reagents for the FTA-ABS must be refrigerated.6 As a consequence, these tests may not be available in the parts of the world where syphilis and neurosyphilis are most common.
Treponemal immunochromatographic strip tests, or ICSTs, have been developed as point-of-care tests for syphilis diagnosis. These can be used to test whole blood, plasma, or serum; do not require special equipment or refrigeration; are simple to perform; and have high diagnostic sensitivity and specificity.7 We investigated whether such tests could be used on CSF to diagnose neurosyphilis.
MATERIALS AND METHODS
Samples used in this study were collected from individuals with syphilis enrolled in a study of CSF abnormalities conducted in Seattle, WA.8 All participants underwent a structured history and neurological examination that included assessment of cranial nerves, motor strength, sensation, coordination, reflexes, and gait; lumbar puncture; and venipuncture. Individuals with CSF abnormalities who were treated for neurosyphilis underwent follow-up lumbar punctures at 3 months after therapy. Those with persistent CSF abnormalities at 3 months underwent repeat CSF examinations at 6 months after therapy; if CSF was abnormal at 6 months, the study was repeated at 12 months after therapy. The study protocol was reviewed and approved by the University of Washington Institutional Review Board, and all participants provided written informed consent.
Standard Laboratory Methods
Cerebrospinal fluid–VDRL tests and enumeration of CSF white blood cells (WBCs) were performed in a Clinical Laboratory Improvement Amendments–approved hospital clinical laboratory. Cerebrospinal fluid–FTA-ABS was performed in a single research laboratory according to standard methods,6 except that unheated cell-free CSF (centrifuged at 1000 rpm for 10 minutes at room temperature) was used rather than serum.
Optimization of Treponemal ICSTs for CSF
Cerebrospinal fluid samples were centrifuged at 1000 rpm for 10 minutes at room temperature, frozen, and thawed once before testing. Three commercial treponemal ICSTs were chosen for study: the Bioline SD Syphilis 3.0 test (Standard Diagnostics, Inc, Kyonggi-do, Korea; hereafter referred to as “Bioline”), the Visitect Syphilis test (Omega Diagnostics, Scotland, UK; hereafter referred to as “Visitect”), and the Syphicheck-WB test (Qualpro Diagnostics, Goa, India; hereafter referred to as “Syphicheck”). We used a panel of 19 CSF samples from patients with syphilis to optimize the 3 tests: 10 positive CSF controls (VDRL reactive with titers ranging from 1:1 to 1:64) and 9 negative CSF controls (CSF-VDRL and FTA-ABS nonreactive, WBCs <5/μL). We reasoned that the optimal conditions for each test on blood, plasma, or serum might not be optimal for CSF because the concentration of treponemal antibodies in CSF would be lower. We thus increased the amount of CSF and decreased the amount of diluent buffer, maintaining the total volume consistent with the manufacturer’s recommendation. We also varied the incubation times for each test compared with the manufacturer’s instructions for blood, plasma, or serum. The Bioline test recommends use of a standard laboratory pipette for measurement of sample and diluent volumes. The Visitect and Syphicheck tests use a proprietary disposable dropper for application of sample and diluent buffer on each test device. For these test kits, we determined the volume in microliters of 1 drop from each test dropper device by weighing the volume in 1 drop of water 10 times and by averaging the results. We identified the optimal volumes and incubation times that provided the fewest false-positive and false-negative results and were judged by the operator as “easiest to read” using our defined panel of 19 samples. A single operator performed all the tests and was blinded to the participants’ clinical and laboratory data.
Determination of the “Best” CSF Treponemal ICST for Neurosyphilis Diagnosis
Cerebrospinal fluid samples from 217 individuals with syphilis were tested using all 3 tests employing the optimal conditions as identified in the studies described above. Test results were compared with the result of the CSF-VDRL using the κ statistic. Serial 2-fold dilutions of CSF in normal saline from 152 individuals with syphilis were tested using the optimized Syphicheck method (the test that was ultimately deemed best); the highest reactive dilution was defined as the test titer. A receiver operator characteristic curve was derived using the CSF-VDRL as the gold standard, and the optimal test titer was determined. Sensitivity and specificity for a laboratory definition of neurosyphilis (CSF WBCs >20/μL regardless of clinical findings) and a clinical definition of neurosyphilis (vision or hearing loss regardless of CSF findings) were determined for the optimal test, the optimal test titer, the CSF-VDRL, and the CSF–FTA-ABS.
Normalization of the CSF ICST After Neurosyphilis Therapy
Cerebrospinal fluid was collected from 53 individuals after neurosyphilis therapy. Normalization for the CSF Syphicheck and the CSF-VDRL was defined as a 4-fold decline in titer or reversion to nonreactive. Time to normalization of the CSF-Syphicheck and of the CSF-VDRL was estimated by Kaplan-Meier analysis.
Modification and Performance of the CSF ICST for the Point of Care
We optimized the 3 tests in a research laboratory using centrifuged CSF delivered by laboratory pipettes, methods that may not be feasible at the point-of-care settings in which the tests might ultimately be used. As such, we investigated agreement between the CSF-Syphicheck conducted as above compared with modifying the method by measuring the CSF and diluent volumes using a tuberculin syringe (0.3 mL, with graduated markings indicating 10-μL increments; the needle was removed) on centrifuged and uncentrifuged CSF samples from 107 consecutive patients, 95 undergoing initial evaluation for possible neurosyphilis, and 12 undergoing a follow-up visit after neurosyphilis treatment. For 15 centrifuged and 14 uncentrifuged CSF samples, we also compared agreement between results obtained with CSF diluted 1:4 in saline using pipettes or syringes in the laboratory. Finally, in parallel with the laboratory studies described above, the study clinician tested 102 centrifuged and uncentrifuged undiluted CSF samples, and 11 centrifuged and 9 uncentrifuged samples diluted 1:4 in saline at the point of care (a US sexually transmitted diseases clinic). The clinician performed the centrifugation and dilution of their sample aliquots in the clinic. Results were compared with the results obtained using the syringe method in the laboratory.
SPSS version 19 (IBM Company, Armonk, NY) was used to calculate κ statistics and to perform Kaplan-Meier analysis. Sensitivity and specificity were calculated using standard formulas and expressed as percent (95% confidence intervals [CIs]); statistical differences between them were assessed using Stata version 11.2 (StataCorp, College Station, TX). Log-rank test was used to compare normalization of CSF-Syphicheck and CSF-VDRL titers using Prism 5 for Mac OS X (GraphPad Software Inc, San Diego, CA).
Optimization of Treponemal ICSTs for CSF
For all 3 tests, we varied the incubation times for the assays from 5 to 30 minutes in 5-minute increments. For the Bioline test, we varied the CSF volume from 10 to 130 μL in a total volume of 130 μL including diluent (at the highest CSF volume, no diluent was added for all tests). For the Visitect, we varied the CSF volume from 36 to 90 μL in a total volume of 90 μL, and for the Syphicheck, we varied the CSF volume from 38 to 142 μL in a total of 142 μL. Table 1 shows the manufacturers’ recommended conditions and the conditions that we determined to be optimal for CSF as defined above for each of the 3 tests. As expected, higher volumes of CSF than recommended for blood, plasma, or serum improved the performance of each test on CSF.
CSF Treponemal ICSTs for Neurosyphilis Diagnosis
We compared agreement between the results of the 3 optimized ICSTs on CSF and results of the CSF-VDRL test in a convenience sample of CSF from 217 individuals with syphilis. In keeping with the demography of syphilis in Seattle, these individuals were predominantly white (n = 145; 67%), HIV-infected (n = 152; 70%) men (n = 197; 91%). One hundred thirty-seven (63%) had early syphilis and 80 (37%) had late latent or unknown duration syphilis; CSF-VDRL was reactive in 54 (25%). Agreement with the results of the CSF-VDRL was moderate for all 3 ICSTs and was highest for the Syphicheck test (Bioline κ = 0.40, Visitect κ = 0.45, Syphicheck κ = 0.54). Compared with the CSF-VDRL, the diagnostic specificity of the CSF-Syphicheck was 79% (73%–85%) and the sensitivity was 83% (73%–93%).
To determine the reproducibility of the CSF-Syphicheck, we subsequently repeated the tests on 104 of the 217 samples using kits from a different lot. Agreement between the results was good (κ = 0.69): 5 (11%) of 44 samples that were negative on the first assessment were positive on the second assessment, and 11 (18%) of 60 that were positive on the first assessment were negative on the second.
One study showed that the specificity and sensitivity of a treponemal test on CSF (the Treponema pallidum hemagglutination assay or TPHA) for diagnosis of neurosyphilis were high if a titer threshold was used to define a positive result rather than simply considering the test to be reactive versus nonreactive.9 Accordingly, we pursued whether the performance of the CSF-Syphicheck could be improved by identifying a titer cutoff in a convenience sample of 152 patients that included 104 previously tested. As with the analysis of the best test samples, most participants were white (n = 116; 76%), HIV-infected (n = 123; 81%) men (n = 147; 97%). One hundred three (68%) had early syphilis and 49 (32%) had late latent or unknown duration syphilis; CSF-VDRL was reactive in 56 (37%). Using a receiver operator characteristic curve (not shown), we determined that a titer at or above 1:4 versus below 1:4 yielded 93% (95% CI, 88%–98%) specificity and 59% (99% CI, 46%–72%) sensitivity using the CSF-VDRL as the diagnostic gold standard.
We then compared the diagnostic performance of the CSF-Syphicheck with and without the titer cutoff to the CSF-VDRL using the laboratory and clinical definitions of neurosyphilis (see “Materials and Methods”) in the same 152 patients (Table 2). For purposes of comparison, we also determined the specificity and sensitivity of the CSF–FTA-ABS; results for this test were missing for 3 patients (Table 2). Sixty-three patients (41%) had CSF WBCs greater than 20/μL, and 36 (25%) of 145 individuals without preexisting abnormalities had vision or hearing loss. The diagnostic specificity of the CSF-Syphicheck was significantly poorer than the CSF-VDRL, but using the titer cutoff of below versus at or above 1:4, the specificity was equivalent to the CSF-VDRL for both neurosyphilis definitions. The diagnostic sensitivity for the CSF-Syphicheck was equivalent to the CSF-VDRL, but was significantly lower for the CSF-Syphicheck ≥1:4 for the clinical neurosyphilis definition, and bordered on being significantly lower for the laboratory definition of neurosyphilis. The diagnostic sensitivity and specificity of the CSF–FTA-ABS were similar to the CSF-Syphicheck for both neurosyphilis definitions (Table 2). The specificity of the CSF–FTA-ABS was significantly lower and the sensitivity significantly higher than the CSF-Syphicheck ≥1:4 for both neurosyphilis definitions (P < 0.005 for all comparisons). As expected, the diagnostic specificity of the CSF–FTA-ABS was significantly lower than the CSF-VDRL for both neurosyphilis definitions; the diagnostic sensitivity for the clinical definition of neurosyphilis was equivalent to the CSF-VDRL, and it bordered on being better than the CSF-VDRL for the laboratory definition of neurosyphilis.
Normalization of the CSF-Syphicheck After Neurosyphilis Therapy
Follow-up CSF samples were obtained for 53 patients treated for neurosyphilis. Median pretreatment CSF WBCs was 38/μL (interquartile range [IQR], 13–66) and median pretreatment CSF-Syphicheck and CSF-VDRL titers were 1:4 (IQR, 1:2–1:8) and 1:1 (IQR, nonreactive [n = 15]–1:4). The median estimated time to normalization of the CSF-Syphicheck after neurosyphilis therapy was 4.2 months (95% CI, 3.8–4.5 months). This compared favorably to the CSF-VDRL (median time to normalization 3.7 months; 95% CI, 3.5–3.8 months). Figure 1 shows Kaplan-Meier curves for normalization of the 2 tests; there was no statistically significant difference between them.
Modification and Performance of the CSF-Syphicheck for the Point of Care
We modified the optimized CSF-Syphicheck for use at the point of care by using a tuberculin syringe (with the needle removed) for sample delivery. In the research laboratory, we found that agreement between the results of the test using a laboratory pipette or using the syringe on 107 consecutive samples was very good for centrifuged (κ = 0.87) and uncentrifuged (κ = 0.80) CSF. We also compared the results of the CSF-Syphicheck with a sample diluted 1:4 in saline in the laboratory using a pipette or syringe on 15 consecutive cell-free and 14 consecutive uncentrifuged samples. Agreement was good (κ = 0.73) for cell-free CSF and was moderate for uncentrifuged CSF (κ = 0.58).
We made similar comparisons between results of tests conducted in the laboratory and by the study clinician at the point of care, both using a tuberculin syringe for CSF and diluent delivery, on 102 undiluted cell-free and uncentrifuged CSF samples, and 11 cell-free and 9 uncentrifuged CSF samples diluted 1:4 in saline. Agreement between results obtained in the laboratory and at the point of care for the undiluted CSF samples was very good for cell-free CSF (κ = 0.80) and was good for uncentrifuged CSF (κ = 0.63). Agreement between results of the diluted samples obtained in the laboratory and at the point of care for the cell-free CSF samples was moderate (κ = 0.46) but was only fair for uncentrifuged CSF (κ = 0.36).
The CSF-VDRL is considered the gold standard test for diagnosis of neurosyphilis. However, it is logistically complicated and is not available in many parts of the world. Several studies have examined whether the Toludine Red Unheated Serum Test or the rapid plasma reagin (RPR) test, nontreponemal tests that use the same antigen as the VDRL but do not require specialized glass plates or a light microscope, can be used in place of the CSF-VDRL.10–13 These studies have used different diagnostic criteria for neurosyphilis and overall have shown as good or better diagnostic specificity, but generally lower sensitivity, than the CSF-VDRL. Although these tests are less logistically complicated to read than the CSF-VDRL, like the CSF-VDRL, they require a mechanical rotator and a centrifuge, and the reagents for the RPR must be refrigerated.6 As yet, these tests have not been applied to point-of-care testing.
The goal of this study was to determine if a commercially available syphilis ICST, which requires no specialized equipment, could be adapted for use on CSF as an alternative to the CSF-VDRL to diagnose neurosyphilis. We optimized 3 ICSTs for CSF and chose one, the CSF-Syphicheck, with the best performance relative to the CSF-VDRL for further study. We found that the diagnostic sensitivity of a reactive CSF-Syphicheck and the diagnostic specificity of a CSF-Syphicheck titer at or above 1:4 were equivalent to the CSF-VDRL. We further show that the CSF-Syphicheck normalized after neurosyphilis therapy similarly to the CSF-VDRL. We modified the CSF-Syphicheck to be performed at the point of care and showed that it performed well, albeit with better performance on cell-free compared with uncentrifuged CSF. Better performance on cell-free CSF might be expected. The methods for the CSF-VDRL and CSF–FTA-ABS both specify that cell-free CSF should be used,6 presumably because red cells or WBCs or debris can inhibit the antigen-antibody reaction.
Several factors should be considered in interpreting our data. Most of our study participants were HIV-infected, consistent with the demographics of syphilis in the United States. Although there is no evidence that, compared with HIV-uninfected persons, HIV-infected individuals have impaired antibody responses to treponemal or nontreponemal antigens in serum,3 it is possible that these antibody responses are impaired in CSF. The number of HIV-uninfected individuals in our study was too small to allow for meaningful comparisons by HIV status. Our goal was to replace the CSF-VDRL, which is specific but lacks sensitivity for neurosyphilis diagnosis. Although we were not able to improve on this drawback of the CSF-VDRL, we reasoned that being able to replace the CSF-VDRL with an equivalently specific test would be a vast improvement over the situation in which the test is simply not available. Our definition of laboratory neurosyphilis was regardless of symptoms or signs, and our definition of clinical neurosyphilis was regardless of CSF abnormalities. These definitions recapitulate clinical care scenarios. Although we tested agreement between the results of the CSF-Syphicheck performed in the laboratory and at the point of care, our point of care, which was an air conditioned US sexually transmitted diseases clinic, may not be relevant to point-of-care conditions in other parts of the world. Moreover, we were only able to test the performance of the titer cutoff in the clinic in a small number of cases, limiting the accuracy of our κ estimates.
The CSF-Syphicheck, particularly using a titer cutoff, shows promise for neurosyphilis diagnosis. However, before the CSF-Syphicheck or any other CSF ICST can be used for point-of-care neurosyphilis diagnosis, further studies are required to determine test performance, including CSF dilution and use of uncentrifuged CSF samples, at the point of care in resource-limited point-of-care settings.
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