Making Sense of Serologies: HIV, Lyme, Hepatitis, and TB Testing in Primary Care

 

NP CHRONICLES

Clinical Education for NP Students & New Graduates

Making Sense of Serologies: HIV, Lyme, Hepatitis, and TB Testing in Primary Care

Infectious Disease  |  Board Prep  |  Test Interpretation

Serologic testing looks straightforward until you're staring at a reactive HIV screen in a low-risk patient, a Lyme ELISA drawn three days after a rash appeared, or an isolated hepatitis B core antibody before an infusion. This post distills a CE lecture from Daniel A. Solomon, MD, FIDSA, of the Division of Infectious Diseases at Brigham and Women's Hospital, covering four serologies that come up constantly in primary care: HIV, Lyme disease, hepatitis B/C, and latent TB. The focus is interpretation — not just what to order, but what the result actually means once it's back.

HIV: The Algorithm Matters as Much as the Result

Modern HIV screening uses a 4th-generation antigen/antibody (Ag/Ab) combination assay, which detects both HIV antibody and the p24 antigen — meaningfully shortening the “window period” compared to older antibody-only assays. But a reactive screen is never a diagnosis on its own. It triggers a defined confirmatory sequence.

Step	Test	Purpose
1	HIV Ag/Ab combination assay (screen)	Initial screen; detects antibody and p24 antigen
2	HIV-1/2 differentiation assay	Confirms and distinguishes HIV-1 vs. HIV-2 if the screen is reactive
3	HIV RNA PCR (viral load)	Resolves discordant results; confirms diagnosis in suspected acute infection

 

CASE FROM PRACTICE A 26-year-old pregnant woman, mutually monogamous and asymptomatic, has a reactive HIV Ag/Ab screen on routine prenatal testing. The HIV-1/2 differentiation assay comes back negative, and HIV viral load is undetectable. Interpretation: false-positive screen, possibly related to cross-reactive antibodies that can occur during pregnancy. In low-prevalence populations, a false-positive screening result is genuinely more common than acute HIV — but a false positive on both the screen AND the differentiation assay is rare, occurring in roughly 1 in 200,000 cases.

That case illustrates a core teaching point: as HIV prevalence in the tested population drops, the proportion of reactive screens that are false positives rises, even with a highly specific test (99.4% specificity in the example used). At 10% prevalence, most reactive screens are true positives; at 0.4% prevalence, false positives become common. This is Bayesian reasoning applied directly to a test result NPs order constantly.

When Acute HIV Is the Real Concern

CASE FROM PRACTICE A 22-year-old man who has sex with both men and women presents with fever, myalgias, pharyngitis, diarrhea, and cervical lymphadenopathy. His last HIV test, one month prior, was negative, and he has had one new partner since. HIV Ag/Ab is reactive, but the differentiation assay is negative — a pattern that would normally suggest a false positive. Because acute infection was clinically suspected, viral load was sent anyway: >10,000,000 copies/mL, with a CD4 count of 256 (13%). Diagnosis: acute HIV. He was started on ART at diagnosis and has since achieved an undetectable viral load with excellent adherence.

The clinical lesson: a reactive screen with a negative differentiation assay is not automatically a false positive if acute infection is on the differential. Antibody hasn't yet fully developed during the acute window, so the differentiation assay can be falsely negative even as the patient is acutely, highly infectious. Any time acute HIV is a real possibility — a compatible “mononucleosis-like” syndrome, recent high-risk exposure, or a discordant result in a symptomatic patient — send the viral load.

•     Acute HIV syndrome is highly variable: approximately 40% of cases are asymptomatic. Among those who are symptomatic, the most common features are fever (75%), fatigue (68%), myalgia (49%), rash (48%), headache (45%), pharyngitis (40%), and cervical adenopathy (39%).

•     Diagnosing acute HIV matters because the window period, while small, still exists; the syndrome resolves spontaneously (creating real risk of loss to follow-up if missed); acute infection is a period of maximum infectiousness; and early treatment reduces transmission risk, preserves CD4 counts, targets a less diverse viral population, and reduces the size of the HIV reservoir.

NUANCE TO FLAG Lifetime HIV risk is not evenly distributed, and the data are worth knowing for both clinical index of suspicion and for framing risk discussions without bias. Estimated lifetime risk in heterosexual men: 1 in 2,713 (White), 1 in 437 (Latinx), 1 in 97 (Black/African American). Among men who have sex with men: 1 in 11 (White), 1 in 5 (Latinx), 1 in 2–3 (Black/African American). These disparities reflect structural and systemic factors, not individual behavior differences, and should inform equitable testing practices — offering testing broadly rather than only to patients who disclose risk factors.

CLINICAL BOTTOM LINE — HIV False-positive HIV screens are common in low-prevalence populations. The standard confirmatory test is the HIV-1/2 differentiation assay, but any positive screen — and any case where acute infection is clinically suspected, regardless of differentiation assay result — should be confirmed or clarified with an HIV RNA viral load.

Lyme Disease: Match the Test to the Stage

Lyme testing performance depends heavily on how long the infection has been present, which is why clinical staging should drive both diagnosis and test selection rather than reflexively ordering serology on every rash or joint complaint.

Stage	Time from Bite	Key Features	Diagnostic Approach
Early localized	7–28 days	Erythema migrans; fatigue, headache, myalgias, fever	Clinical diagnosis preferred — serology often still negative
Early disseminated	Weeks to months	Meningitis, CN palsy, radiculopathy, peripheral neuropathy; Lyme carditis, AV block	Antibody test with reflex to confirmatory testing
Late disease	Months to years	Knee arthritis, acrodermatitis chronica atrophicans, encephalopathy	Antibody test with reflex to confirmatory testing
Post-treatment Lyme disease syndrome	3–12 months post-treatment	Headache, fatigue, arthralgias	Clinical diagnosis; no antibiotics indicated

 

Standard two-tier testing (an ELISA with reflex to Western blot, or a modified two-tier approach using two sequential EIAs) is designed to be used in early disseminated or late disease — not early localized disease, where sensitivity is genuinely poor.

Stage	Sensitivity	Specificity
Early localized	14–17%	>99%
Early disseminated	89.7%	>99%
Late disseminated	99.4%	>99%

 

CLINICAL BOTTOM LINE — LYME In early localized disease (classic erythema migrans, 7–28 days post-bite), diagnose clinically — serology sensitivity is only 14–17% at this stage and a negative result should not delay treatment. Reserve two-tier serologic testing for early disseminated or late disease, where sensitivity exceeds 89%. Don't use antibody testing as a “test of cure” — many patients remain seropositive long after successful treatment, and diagnosing reinfection serologically can be genuinely difficult.

Hepatitis B: The Triple Screen, Explained

CASE FROM PRACTICE A 37-year-old woman with no past medical history, born in Ho Chi Minh City and living in the US since 2007, presents for a routine visit. Should she be screened for hepatitis B? Yes — and per current CDC guidance, so should essentially every adult.

CDC now recommends universal hepatitis B screening: every adult aged 18 and older should be screened at least once in their lifetime, regardless of disclosed risk factors. Pregnant patients should be screened for HBsAg during every pregnancy, regardless of vaccination history or prior testing. Periodic risk-based testing should additionally cover incarcerated individuals, people with a history of STIs or multiple partners, people with hepatitis C, and anyone who simply requests testing.

The “triple screen” — HBsAg, HBsAb, and HBcAb — accomplishes three distinct goals at once, and understanding what each component tells you is the key to interpreting the panel correctly.

Goal	Test	Action if Positive/Result
Identify chronic HBV	HBsAg	Monitoring and/or antiviral treatment
Identify susceptibility	HBsAb (anti-HBs)	If negative — vaccinate
Identify reactivation risk	HBcAb (anti-HBc)	If positive and patient becomes immunosuppressed — monitor or give prophylaxis

 

The reactivation risk identified by an isolated positive HBcAb is not theoretical. HBV flares can occur even in patients who appear to have fully resolved infection, are often preceded by a rise in viral DNA, and classically occur at the start or withdrawal of immunosuppressive therapy. The 2025 AGA Clinical Practice Guidelines stratify management by reactivation risk category:

•     High risk: antiviral prophylaxis is recommended over monitoring alone (strong recommendation, moderate-certainty evidence).

•     Moderate risk: antiviral prophylaxis is suggested over monitoring alone (conditional recommendation, moderate-certainty evidence).

•     Low risk: monitoring alone is suggested over antiviral prophylaxis (conditional recommendation, moderate-certainty evidence).

CASE FROM PRACTICE A 42-year-old woman with newly diagnosed multiple sclerosis is about to start ocrelizumab, an anti-CD20 agent. Her hepatitis B panel: HBsAg negative, HBsAb negative, HBcAb positive. Interpretation: this is an isolated core antibody pattern — no current active infection, no protective surface antibody, but evidence of past exposure with a real risk of reactivation on B-cell-depleting therapy. Recommended action: preventive antiviral therapy for roughly one month before starting ocrelizumab, then continuing through the full treatment course.

Hepatitis C: One Antibody Test, Different Meanings Over Time

Hepatitis C follows a different logic than hepatitis B, and it trips people up for a specific reason: the antibody result doesn't change even after the virus is long gone.

•     HCV antibody remains positive for life once a person has been infected — whether they spontaneously cleared the virus, were successfully treated, or have ongoing chronic infection.

•     A positive HCV antibody does not confer immunity to reinfection. Patients with ongoing risk factors need periodic screening for reinfection using an HCV viral load, not repeat antibody testing.

•     If HCV antibody is positive, the next step is an HCV viral load (RNA). A negative viral load in an antibody-positive patient indicates either spontaneous clearance or prior successful treatment — not an active infection needing treatment now, but a patient who should be counseled on reinfection risk and re-screened as needed.

•     A positive viral load confirms current infection requiring further workup and treatment consideration.

Left untreated, chronic HCV follows a long natural history: roughly 20–25 years from infection to cirrhosis in a subset of patients, and 25–30 years to hepatocellular carcinoma, end-stage liver disease, or death in those who progress — underscoring why identifying chronic infection early, even in asymptomatic patients, changes the trajectory.

CLINICAL BOTTOM LINE — HEPATITIS B & C For hepatitis B, order the triple screen (HBsAg, HBsAb, HBcAb) to simultaneously assess for active infection, immunity, and reactivation risk — an isolated positive HBcAb before immunosuppressive therapy is a common, clinically important finding that changes management. For hepatitis C, remember the antibody is lifelong and does not indicate current infection or immunity; use HCV viral load to determine current infection status and to screen for reinfection in patients with ongoing risk factors.

Latent TB: TST vs. IGRA, and Why Pre-Test Probability Matters

TB has not disappeared from US practice — cases in foreign-born individuals have exceeded those in US-born individuals every year since 2001, and globally TB remains a major cause of morbidity and mortality. Age at immigration matters because it affects the likelihood of prior BCG vaccination and the duration and context of potential TB exposure, both of which shape how test results should be interpreted.

Tuberculin Skin Test (TST)

The TST involves an intradermal injection of purified protein derivative (PPD), read at 48–72 hours and measured in millimeters of induration — never simply recorded as positive or negative. The cutoff for a positive result depends on the patient's individual risk profile:

Induration Cutoff	Applies To
≥5 mm	HIV or other immunosuppression, close contacts of active TB cases, fibrotic changes on chest x-ray
≥10 mm	Other high-risk groups (e.g., recent immigrants from high-burden countries, congregate settings, certain comorbidities)
≥15 mm	Everyone else (no known risk factors)

 

TST has real limitations in both directions. False positives can result from non-TB mycobacteria exposure or prior BCG vaccination (more likely to cause persistent false positives with older age at vaccination) or simple misinterpretation. False negatives can occur with poor technique, the two-visit requirement (loss to follow-up), very recent infection, active TB disease itself, immunosuppression or anergy, very young age (under 6 months), live virus vaccination, or recent viral infection — measles being the classically described culprit.

Interferon-Gamma Release Assays (IGRAs)

IGRAs (QuantiFERON-TB Gold and T-SPOT.TB are the two commercially available options) measure interferon-gamma production after incubating whole blood with M. tuberculosis-specific antigens. Advantages over TST include requiring only one visit, more objective interpretation, and no confusion from prior BCG vaccination. Downsides include higher cost, the need for whole blood processing, variable availability, and cross-reactivity with M. marinum and M. kansasii.

Test	Sensitivity	Specificity	Negative Predictive Value
TST	90–100%	29–39%	99–100%
IGRA	80–90%	56–83%	99–100%

 

Note the specificity gap: TST has meaningfully lower specificity than IGRA, largely driven by BCG cross-reactivity, which is precisely why IGRA is often preferred in BCG-vaccinated populations. TST and IGRA can be discordant in either direction, and when they disagree, incorporating pre-test probability — not just picking whichever result you trust more — is the correct approach. The Online TST/IGRA Interpreter (tstin3d.com, or search “Online IGRA Interpreter”) is a free calculator designed for exactly this scenario, incorporating risk factors like HIV status, immunosuppression, chest x-ray findings, and age at TB exposure to estimate active TB risk from a discordant or borderline result.

Why Biologics Change the Calculus

TNF-alpha is critical to both in vitro and in vivo control of TB, which is why anti-TNF biologics carry a well-documented TB reactivation risk. Compiled data cited in the lecture found active TB rates of roughly 10.9 per 100,000 in the general population versus 1,503 per 100,000 in patients on biologics during the pre-screening era — a more than 100-fold difference. Among patients on TNF inhibitors who develop active TB, median time to onset is 3–6 months (closer to 12 months for etanercept specifically), disseminated disease occurs in roughly a third of cases (versus ~6% in the general population), and fatality is 12–18% (versus under 5% in patients not on biologics).

CASE FROM PRACTICE A patient with a positive TST, a borderline IGRA, and a history of BCG vaccination is being considered for etanercept. Using the online TST/IGRA interpreter with the patient's specific risk profile (including planned TNF-alpha inhibitor use) to estimate active TB risk, the recommended answer was: give preventive (latent TB) therapy for approximately one month before starting etanercept, then continue the full course of treatment concurrently. This reflects the standard approach of treating LTBI before or concurrent with initiating anti-TNF therapy rather than withholding the biologic indefinitely.

CLINICAL BOTTOM LINE — LATENT TB TST and IGRA measure different things and can legitimately disagree — TST's main weakness is lower specificity (BCG cross-reactivity), while IGRA trades some sensitivity for improved specificity and single-visit convenience. Interpret either test using the patient's pre-test probability, not in isolation. Before starting any anti-TNF biologic, screen for LTBI and treat it — reactivation risk on these agents is dramatically elevated and carries higher rates of disseminated disease and fatality than TB in the general population.

Putting It All Together

The thread connecting all four serologies is the same: a single test result rarely stands on its own. It has to be read against the pre-test probability the clinical picture creates — prevalence and exposure history for HIV, time since tick bite for Lyme, immunosuppression status for hepatitis B, and TB exposure risk for TST/IGRA interpretation. Ordering the right test, at the right time, for the right stage of disease is most of the battle; the rest is resisting the urge to treat a lab value as a verdict rather than one more piece of the clinical picture.

 

Reference

Solomon, D.A. (2026). Making sense of serologies: Ordering and interpreting HIV, hepatitis, Lyme, and TB tests in primary care. Pri-Med Institute CE activity.