Table 2.

Chronological and comprehensive comparison of MR diagnostic methods with commercial kits, performance metrics, and cost-effectiveness analysis

Serial No.	Method	Year	Target	Commercial kits/manufacturers	Sensitivity (%)	Specificity (%)	PPV/NPV (%)a,b	LOD	Sample volume	Sample types	Cost per test (US$)c	Equipment cost (US$)	Time to result	Technical requirements	Pros	Cons
1	HI126	1940s	Antibodies	Laboratory protocols; no commercial kits	Moderate (60–75)	Moderate (75–85)	Variable by prevalence	N/A	50–100 μL	Serum	5–8	2,000–5,000	1–2 days	Low; basic serology training	Simple setup, historical importance	Outdated, poor sensitivity/specificity
2	PRNT127	1960s	Neutralizing antibodies	Laboratory protocols; no commercial kits	Very high (98–100)	Very high (99–100)	M: 98–100/99–100; R: 98–100/99–100	Variable	50–100 μL	Serum	50–80	15,000–40,000	3–5 days	Very high; specialized virology expertise	Gold standard for immunity; WHO reference	Labor intensive, requires BSL-2+, slow
3	ELISA (EIA)128,129,130	1971	IgM/IgG antibodies	Siemens Enzygnostd (discontinued 2020); Euroimmun; IBL129 International; SERION ELISA classic; NovaTec NovaLisa	M: 75–98; R: 78–99	M: 87–99; R: 52–100	M: 75–97/88–99; R: 0.2–1.4/94–99	N/A	10–100 μL	Serum, plasma	15–25	25,000–50,000	2–4 h	Moderate; trained laboratory technician	Widely available, WHO network standard	Lower PPV in elimination settings
4	CLIA131	1990s	IgM/IgG antibodies	DiaSorin LIAISON XL; Beckman Coulter Access; Abbott ARCHITECT; Siemens ADVIA Centaur	M: 94–97; R: 90–96129	M: 95–98; R: 93–98	M: 95–98/96–99; R: 94–97/95–98	N/A	25–50 μL	Serum, plasma	18–30	80,000–150,000	30–60 min	Moderate; automated platform training	High throughput, automated	Expensive equipment, platform specific
5	ICA	1980s	IgM/IgG antibodies	Research protocols; limited commercial availability	High (85–95)	High (90–98)	Variable by prevalence	N/A	50–100 μL	Serum	8–15	5,000–15,000	2–3 h	Moderate; immunology training	Visual detection, cost effective	Less standardized than ELISA
6	RT-PCR132,133	1990s	Viral RNA	Applied Biosystems TaqMan; Bio-Rad CFX; Roche LightCycler; QIAGEN Rotor-Gene; in-house protocols	M: 94–99; R: 98–100	M: 99–100; R: 100	M: 98–100/97–99; R: 99–100/98–100	10–1,000 copies/mLe	140–200 μL	Throat/NP swab, urine, serum	20–40	40,000–120,000	3–6 h	High; molecular biology expertise	Highly sensitive and specific, genotyping capable	Complex, expensive, requires skilled staff
7	FRNT134	1995	Neutralizing antibodies	Research protocols; immunocolorimetric methods	Very high (95–100)	Very high (98–100)	M: 98–100/98–100; R: 98–100/98–100	Variable	25–50 μL	Serum	30–60	20,000–50,000	2–3 days	Very high; cell culture expertise	More sensitive than PRNT, vaccine studies	Complex, requires cell culture facilities
8	AFRNT135	2005	Neutralizing antibodies	96-Well protocols; limited commercial systems	Very high (95–100)	Very high (98–100)	M: 98–100/98–100; R: 98–100/98–100	Variable	25–50 μL	Serum	25–50	30,000–80,000	2–3 days	High; automated systems	Reduced manual error, higher throughput	Expensive setup, specialized equipment
9	RT-LAMP136,137,138	2008	Viral RNA	Laboratory protocols; no commercial kits	M: 91–100; R: 95–100	M: 95–100; R: 95–100	M: 95–99/96–100; R: 88–98/95–100	M:30–50 copies/mL; R:380 copies/reaction	2–25 μL	Throat swab, urine, serum	8–15	5,000–15,000	30–60 min	Moderate; isothermal amplification training	Equipment-simple, rapid, cost effective	Contamination risk, primer design complexity
10	RT-RPA137	2012	Viral RNA	Laboratory protocols; no commercial kits	M: 94–100; R: 85–95	M: 95–100; R: 90–100	M: 96–99/97–100; R: 88–96/95–99	10–31 copies/reaction	1–5 μL	Multiple specimen types	12–25	2,000–8,000	15–30 min	Moderate; molecular training	Very rapid, high sensitivity	Higher reagent costs, proprietary
11	DMF-ELISA139	2016	IgM/IgG antibodies	MR Box (research prototype); custom platforms	M: 81–88; R: 81–88	M: 85–95; R: 85–95	M: 80–90/90–95; R: 80–90/90–95	N/A	<5 μL	Serum, oral fluid	10–20	1,000–5,000	30–60 min	Low; minimal training	Portable, minimal sample volume	Research stage, limited validation
12	Lateral flow RDT140,141	2020	IgM antibodies	Prototype developments (WHO/Gavi evaluation); no commercial products yet	M: 90–95f; R: Under development	M: 94–96f; R: N/A	M: 85–95/96–99f; R: N/A	N/A	5–10 μL	Capillary blood, serum, oral fluid	2–4	0	<30 min	Low; point-of-care use	Field deployable, immediate results	Limited validation, lower accuracy
13	MBA142	2021	Multiple antibodies	Luminex MAGPIX; Bio-Rad Bio-Plex; custom panels	M: 90–98; R: 92–99	M: 93–99; R: 95–100	M: 94–98/95–99; R: 96–99/97–100	N/A	1–5 μL	Serum, plasma	20–35	30,000–80,000	2–4 h	High; specialized platform	High throughput, multiplexing capability	Expensive platform, complex data analysis
14	Microfluidic two-stage amplification143	2021	Viral RNA	Research prototypes; custom chip fabrication	M: 100g; R: Under development	M: 100g; R: N/A	M: 100/100g; R: N/A	∼10 copies	2.1 μL (RPA) + 10.6 μL (LAMP)	NP swabs, saliva	20–35	10,000–30,000	<60 min	High; microfluidics expertise	Ultra-sensitive, integrated workflow	Complex fabrication, research stage
15	NGS amplicon sequencing144,145	2021	Viral genome	Illumina MiSeq/NextSeq; Oxford Nanopore; custom primer panels	M: 90–100; R: 85–100	M: 95–100; R: 90–100	M: 95–100/96–100; R: 90–100/95–100	Variable (Ct < 30 preferred)	50–200 μL	Multiple specimen types	80–150	50,000–250,000	1–3 days	Very high; bioinformatics required	Genotype specific, established protocols	Labor intensive, limited by primer design
16	NGS probe enrichment145	2022	Viral genome	Twist Bioscience; IDT xGen; custom probe panels	M: 95–100; R: 90–100	M: 98–100; R: 95–100	M: 98–100/98–100; R: 95–100/97–100	Variable (viral load dependent)	50–200 μL	Clinical specimens	75 (includes $7 enrichment)	50,000–250,000	2–3 days	Very high; NGS and bioinformatics	Genotype independent, cost-efficient	Probe maintenance, degraded sample failure
17	RT-ddPCR146,147	2023	Viral RNA	Research protocols; no commercial kits	M: 95–100; R: 90–100	M: 98–100; R: 95–100	M: 98–100 R: 95–100	M: 260; R: 460 copies/mL	10–50 μL	Throat/NP swab, urine, wastewater, serum	35–60	60,000–200,000	4–6 h	High; molecular biology and dPCR expertise	Absolute quantification without standards, strain differentiation, precision, multiplexing	Expensive equipment, no commercial kits, limited validation
18	CRISPR-Cas12a detection148	2024	Viral RNA	Research protocols; emerging commercial development	M: 96h; R: Under development	M: 100h; R: N/A	M: 98–100/97–99h; R: N/A	31 copies/reaction	1–5 μL	Throat swab, saliva	15–30	5,000–20,000	30–60 min	Moderate; CRISPR training	Ultra-specific, rapid, portable	Limited clinical validation, early development
19	Oxford nanopore sequencing149	2025	Viral genome	Oxford Nanopore MinION/GridION; custom library preparation	M: 95–100 (high viral load); R: Under development	M: 95–100; R: N/A	M: 95–100/96–100; R: N/A	Optimal >100 copies/μL	50–200 μL	Clinical specimens	60–120	10,000–50,000	6–24 h	High; real-time sequencing	Portable sequencing, real-time results	Error prone at low viral loads
20	Shotgun metagenomics145,149	2025	Total pathogen DNA/RNA	Illumina platforms; Oxford Nanopore; Ion Torrent	Moderate (70–85)	High (90–95)	Variable (pathogen dependent)	Variable	50–200 μL	Clinical specimens	120–200	50,000–250,000	2–5 days	Very high; advanced bioinformatics	Pathogen agnostic, discovers unknowns	Low efficiency, expensive, complex analysis

This table outlines key assays used for MR detection, including their diagnostic targets, commercial manufacturers, detailed performance characteristics (sensitivity, specificity, PPV/NPV), sample volume requirements, cost per test, turnaround time, technical requirements, advantages, and limitations, along with the year of introduction or significant application. The comparison reflects the progression from classical serology (1940s) to advanced molecular and point-of-care technologies (2025), incorporating recent systematic reviews, manufacturer specifications, and WHO guidelines to support evidence-based diagnostic platform selection.

AFRNT, automatable FRNT; ; BSL, biosafety level; HI, hemagglutination inhibition; LOD, limit of detection; M, measles; MR, measles and rubella; NP, nasopharyngeal; NPV, negative predictive value; PPV, positive predictive value; PRNT, plaque reduction neutralization test; CLIA, chemiluminescence immunoassay; R, rubella.

^aPPV/NPV values are highly dependent on disease prevalence and vary significantly between elimination and endemic settings.

^bPPV typically lower in elimination settings; NPV higher in endemic settings.

^cCosts vary by region, volume, and healthcare setting; ranges reflect global estimates.^81,252

^dWHO-endorsed until discontinuation; replacement evaluation ongoing.

^eDetection limit varies with specimen type and collection timing.

^fBased on limited field studies; broader validation ongoing.

^gLimited validation study (n = 40); requires larger clinical trials.

^hSingle study validation (n = 56); broader clinical validation needed.