What is the recommended blood sample collection and diagnosis protocol for a dengue patient?

Blood Sample Collection and Diagnostic Protocol for Dengue

Specimen Collection Based on Timing of Symptoms

For patients presenting within 7 days of symptom onset, collect serum for nucleic acid amplification testing (NAAT) and NS1 antigen detection, as these are the most sensitive diagnostic methods during the acute phase. 1, 2

Early Phase (≤7 Days After Symptom Onset)

• Collect serum as the primary specimen for NAAT/PCR and NS1 antigen testing, which have peak sensitivity (75-90%) during the first 3-5 days of illness 2, 3
• NAAT can also be performed on plasma, whole blood, cerebrospinal fluid, or urine as alternative specimens 1
• NS1 antigen remains detectable for up to 10 days after symptom onset, though sensitivity begins declining after day 5 2
• IgM antibodies typically appear 3-5 days after symptom onset, so early negative IgM does not rule out dengue 2

Late Phase (>7 Days After Symptom Onset)

• Collect serum for IgM and IgG antibody testing, as serologic methods become more sensitive than molecular methods after the first week 1, 2
• IgM antibody testing is the preferred diagnostic method for specimens collected 7 days to 12 weeks after symptom onset 1
• A negative IgM result during this timeframe effectively rules out recent dengue infection 1

Diagnostic Testing Algorithm

For Nonpregnant Patients

• Perform dengue NAAT on serum collected ≤7 days after symptom onset 1, 3
• If NAAT is positive, no antibody testing is indicated unless the diagnosis has epidemiologic or clinical significance (e.g., first local transmission, unusual clinical syndrome) 1
• If NAAT is negative, perform IgM and IgG antibody testing on the same specimen 1
• For specimens collected >7 days after onset, proceed directly to IgM and IgG antibody testing 1, 3
• Some laboratories may choose to perform NAAT and antibody testing simultaneously rather than sequentially 1

For Pregnant Women

• Test by NAAT for both dengue and Zika virus regardless of outbreak patterns, due to potential adverse outcomes including maternal death, hemorrhage, preeclampsia, and vertical transmission 1, 3
• Perform serology for evidence of infection with the virus causing any local outbreak 1
• Collect both serum and urine specimens for comprehensive testing 3

Interpretation of Results

NS1 Positive, IgM/IgG Negative

• Indicates acute primary dengue infection in the very early phase (typically days 1-5), before antibody development 2
• Confirms active viral replication and acute infection rather than past exposure 2
• No additional confirmatory testing (PRNT) is needed, as NS1 positivity already confirms acute dengue 2

NS1 Negative, IgM and IgG Positive

• Indicates either secondary dengue infection, late primary infection, or past dengue infection where IgM is still detectable 2
• Requires confirmatory plaque reduction neutralization testing (PRNT) to definitively distinguish dengue from other flavivirus infections 2
• Most likely represents specimen collection more than 7 days after symptom onset 2

NAAT Negative, IgM Negative (<7 Days)

• Suggests no recent flavivirus infection, though a negative acute IgM test might reflect specimen collection before antibody development 1
• Does not definitively rule out infection due to possible decline in viremia or inaccurate reporting of symptom onset dates 1

NAAT Negative, IgM Negative (7 Days to 12 Weeks)

• Rules out recent dengue infection, and testing for other etiologies should be pursued 1

Alternative Specimen Types

Capillary Blood (Fingerstick)

• Capillary blood samples on filter paper demonstrate 81.6% sensitivity and 90.7% specificity compared to venous blood during acute phase (days 1-4: 88.5% sensitivity, 93.8% specificity) 4
• NS1 detection in capillary blood shows 96% sensitivity and 100% specificity 5
• Particularly useful in community-level settings, remote areas, and pediatric populations where venous blood collection is challenging 5, 4

Dried Blood Spots (DBS)

• Display high sensitivity (IgM: 100%, IgG: 96%, NS1: 100%) and specificity (IgM: 75%, IgG: 93%) 6
• DENV RNA can be amplified from DBS with 95.6% sensitivity 6
• Convenient for storage and transport without cold chain requirements 4

Urine and Saliva

• Urine and saliva are less sensitive alternatives (urine RT-PCR: 41.6%, saliva RT-PCR: 39%) but may be justified when blood collection is not possible 7
• When tested concurrently, urine and saliva achieve 69.1% sensitivity for RNA detection 7
• IgM is never detected in urine but can be found in saliva (36% sensitivity) 7

Critical Pitfalls to Avoid

• Never assume acute infection based on positive antibodies alone, as IgM can persist for months after initial infection 2
• Do not rule out dengue based solely on negative NAAT, as viremia declines over time and symptom onset dates may be inaccurate 1
• Avoid testing only with IgM during the first 3-5 days of illness, as antibodies may not have developed yet 2
• Be aware of cross-reactivity with other flaviviruses (Zika, yellow fever, Japanese encephalitis) when interpreting serologic results 2, 3
• In areas with multiple circulating flaviviruses and low dengue prevalence, false-positive NS1 results may occur due to lower positive predictive value 2

Confirmatory Testing

• For epidemiologically significant cases or indeterminate results, perform PRNT against dengue and other endemic flaviviruses 1, 2
• PRNT titer ≥10 for dengue and <10 for other flaviviruses confirms recent dengue infection 2
• Repeat NAAT on newly extracted RNA from the same specimen to rule out false-positive results in critical cases 1, 2