Wastewater testing during the South African 2022-2023 measles outbreak demonstrates the potential of environmental surveillance to support measles elimination

This study demonstrates the potential of wastewater environmental surveillance to support measles elimination in South Africa by successfully detecting measles virus RNA in samples from districts where clinical surveillance failed to identify cases, despite challenges with RNA degradation in stored samples.

The Gist

Imagine the city's sewage system as a giant, underground river that carries a tiny, invisible "snapshot" of the health of everyone living above it. Every time someone uses the toilet or washes their hands, they leave behind microscopic traces of what's happening inside their body.

This paper is about scientists in South Africa who decided to use this "underground river" to catch a very sneaky virus: Measles.

Here is the story of their discovery, explained simply:

1. The Problem: The "Silent" Outbreak

Measles is a dangerous virus that the world is trying to eliminate. Usually, doctors find it by waiting for sick people to show up at clinics with a fever and a rash. But this method has a big hole in it:

• The "Silent" Gap: Not everyone who gets sick goes to the doctor. Some people think it's just a mild cold, or they can't afford to go.
• The Result: The official count of sick people often looks lower than the real number of infections. It's like trying to count fish in a lake by only looking at the ones that jump out of the water; you miss the ones swimming deep down.

2. The Solution: The "Sewage Spy"

The scientists asked a clever question: "If sick people pee and poop out the virus, can we find it in the sewage before we even know who is sick?"

They treated the wastewater system like a super-sensitive security camera. Instead of waiting for a person to report a crime (getting sick), they looked for the "footprints" (viral genetic material) left behind in the sewer.

3. The Experiment: Looking Back in Time

South Africa had a big measles outbreak starting in late 2022. The scientists didn't have a special "Measles Detector" ready at the start, but they had a goldmine of data: frozen sewage samples collected over three years for other viruses (like Polio and COVID).

• The Tool: They built a new, high-tech test called digital PCR. Think of this as a "molecular magnifying glass" that can find just a few drops of virus in a swimming pool of water.
• The Test: They took 2,149 frozen sewage samples from 47 different towns and cities and ran them through this new test.

4. The Big Discovery: Finding Ghosts

The results were fascinating:

• The Catch: They found measles virus in 43 samples (about 2% of the total).
• The Surprise: In half of the places where they found the virus in the sewage, no one had reported being sick to the doctors.

The Analogy: Imagine a town where the police (doctors) say, "We have zero burglars." But the security camera (sewage test) finds footprints in the alleyways. The police didn't see the burglars because the burglars were hiding, but the footprints proved they were there.

5. Why This Matters

This study proves that wastewater surveillance is a powerful new tool for public health.

• Early Warning: It can tell us an outbreak is happening before the hospitals get overwhelmed.
• Filling the Gaps: It catches the infections that the traditional system misses because people aren't going to the clinic.
• The Future: The scientists suggest that in the future, we should treat sewage testing like a routine check-up for the whole city, not just for Polio or COVID, but for Measles too.

The Catch (Limitations)

The scientists were honest about the challenges:

• The "Rot" Factor: Because the samples were frozen for a long time, some of the virus "rotted" (degraded), making it harder to find. If they had tested the sewage fresh (right after collection), they likely would have found even more cases.
• The "Vaccine" Confusion: It was hard to tell if the virus came from a wild infection or a recent vaccine shot, but since vaccine viruses don't spread easily, they are pretty sure the virus they found was the dangerous wild kind.

The Bottom Line

This paper is a victory for "detective work." By looking at the city's sewage, the scientists found hidden measles outbreaks that the traditional system missed. It shows that if we want to wipe measles off the face of the earth, we need to listen to what the sewers are telling us, not just what the clinics are reporting. It's a new way to keep our communities safe.

Technical Summary

1. Problem Statement

Measles remains a leading cause of death for children in low- and middle-income countries, despite global efforts toward elimination by 2030. Current surveillance relies heavily on clinical fever-rash reporting and laboratory confirmation (IgM serology and PCR). However, this clinical surveillance suffers from four critical limitations:

• Health System Factors: Inconsistent clinician awareness, testing propensity, and specimen submission.
• Patient Behavior: Caregivers often do not seek care for uncomplicated cases, particularly in older children or during recognized outbreaks.
• Data Gaps: Incomplete clinical data (e.g., rash onset dates, vaccination history) hinders case classification and contact tracing.
• Specimen Limitations: Urine and throat swabs are rarely submitted for PCR, making genomic sequencing and genotype determination difficult.

While Wastewater and Environmental Surveillance (WES) has been successful for polio and SARS-CoV-2, its systematic application to measles control has not been established. This study aimed to develop a robust assay for Measles Virus (MeV) in wastewater and evaluate its utility as a complementary surveillance tool during a national outbreak in South Africa.

2. Methodology

Study Setting and Sampling:

• Location: National sentinel surveillance network in South Africa, covering 47 sites (28 wastewater treatment plants and 19 community in-line sewer sites) across nine provinces.
• Sample Collection: Grab samples (1L) were collected monthly to biweekly. Raw sewage was concentrated via centrifugation and ultrafiltration (Centricon® Plus-70) to ~1 mL.
• Storage: Concentrates were stored at -20°C for up to 15 months (February 2021 – March 2024) before testing.

Assay Development (RT-dPCR):

• Platform: QIAcuity® One, 5plex System (Digital PCR).
• Targets:
  • Pan-MeV: Primers/probes targeting the N gene to detect all MeV.
  • Genotype Differentiation: Designed to distinguish Wild-Type (WT) genotypes (B3, D8, H1) from the Vaccine strain (Genotype A/Edmonston).
  • Normalization: Pepper Mild Mottle Virus (PMMoV) was used as a fecal load marker.
• Controls: Used WHO Global Measles Reference Laboratory Network (GMRLN) positive controls, commercial MMR vaccine (Genotype A), and clinical outbreak samples.
• Limit of Detection (LoD): Determined via serial dilutions. The LoD was defined as 1 positive partition in valid assays (70–100% valid partitions), calculated to be 0.346 – 0.494 genome copies/µL.

Data Analysis:

• Comparison: Wastewater results were compared against national clinical surveillance data (IgM-positive cases) by health district and epidemiological week.
• Concordance: Defined as "positive concordant" (both wastewater and clinical positive) or "discordant" (one positive, one negative).
• Normalization: MeV genome copies were normalized against PMMoV concentrations.

3. Key Contributions

1. Assay Development: Successfully optimized and validated a duplex RT-dPCR assay for MeV detection in complex wastewater matrices.
2. Retrospective Application: Applied the assay to over 2,100 archived wastewater samples from a national outbreak period, demonstrating the feasibility of using existing infrastructure for new pathogen surveillance.
3. Surveillance Gap Identification: Provided empirical evidence that wastewater surveillance can detect MeV transmission in districts where clinical surveillance failed to identify cases (false negatives in clinical data).
4. Genotype Differentiation Challenge: Highlighted the difficulty in distinguishing vaccine strains from wild-type strains in wastewater using N-gene primers due to high sequence homology, suggesting a need for M-gene targeting or sequencing for precise differentiation.

4. Key Results

• Detection Rates: Out of 2,149 wastewater samples tested, 43 (2.0%) were positive for MeV RNA.
• Concentrations: Positive samples ranged from 1.97 to 165.8 genome copies/mL. The median concentration was 2.12 gc/mL.
• Geographic Distribution: The majority of positive samples (31/43) were from Gauteng province, which also had the highest volume of samples tested.
• Concordance Analysis:
  • In 481 week-district pairs analyzed, MeV was detected by both methods in only 2.9% (14 pairs).
  • Discordance: In 25.8% of pairs, results were discordant. Notably, in 13 instances (48%) where wastewater was positive, no clinical cases were reported in that district for that week.
  • When expanding the window to include clinical cases from the week before and after the wastewater sample, the number of "wastewater-only" positive detections dropped to 10 (2.1%), but still indicated transmission missed by clinical surveillance.
• Vaccine vs. Wild-Type: The study found that the in-house wild-type primers lacked specificity (cross-reacting with vaccine strain) and sensitivity. However, the authors argue that since vaccine virus shedding is low and short-lived, positive wastewater detections likely represent wild-type transmission.
• RNA Degradation: The study noted that long-term storage (-20°C) likely caused RNA degradation, potentially underestimating viral loads. Despite this, detection was still possible.

5. Significance and Implications

• Complementary Surveillance: WES serves as a critical "safety net" for measles elimination. It can detect active transmission in communities where clinical surveillance is failing due to poor health-seeking behavior or system gaps.
• Early Warning System: In non-endemic settings or areas with high vaccination coverage, wastewater detection could serve as an early signal for the onset of an outbreak before clinical cases reach the threshold for intervention.
• Programmatic Value: While current WHO guidelines trigger Supplementary Immunization Activities (SIAs) based on clinical case thresholds, wastewater detection could identify vulnerable communities needing strengthened clinical surveillance or targeted interventions even before a clinical outbreak is declared.
• Future Directions:
  • Real-time Processing: Immediate processing (rather than long-term storage) is essential to prevent RNA decay and improve sensitivity.
  • Improved Genotyping: Development of better assays (e.g., targeting the M gene or using sequencing) to definitively distinguish vaccine strains from wild-type strains.
  • Integration: WES should be integrated into routine measles surveillance programs to accumulate data and refine response protocols.

Conclusion:
The study demonstrates that despite challenges like RNA degradation and assay specificity, wastewater surveillance is a viable and powerful tool for monitoring measles transmission. It offers a unique capability to uncover "silent" transmission that clinical systems miss, thereby supporting the global goal of measles elimination.