The motivation for the review lies in the well-documented shortcomings of conventional diagnostic approaches. Microscopy, the historical mainstay, requires skilled microscopists, invasive sampling from lesions, bone marrow, lymph nodes or spleen, and it delivers sensitivity that falls sharply with parasite burden and operator experience. Serological tests, meanwhile, detect antibodies rather than the parasite itself, which means they cannot reliably distinguish active infection from past exposure, a critical limitation in endemic regions where a large share of the population may carry immunological memory without current disease. Parasite culture, although specific, is slow, technically demanding and insensitive. Together, these constraints have long argued for a shift toward methods that detect parasite nucleic acids directly, offering both higher analytical sensitivity and the ability to quantify parasite load for monitoring treatment response.

The quantitative core of the review centers on real-time quantitative PCR, or qPCR, targeting kinetoplast DNA. Kinetoplast DNA minicircles are small, circular mitochondrial DNA molecules present in thousands of copies per parasite, a feature that gives kDNA-targeted assays an enormous analytical advantage over single-copy nuclear targets. Pooling data across studies with a bivariate random-effects model, the review found that qPCR targeting kDNA achieved a pooled sensitivity of 95.8 percent, with a 95 percent confidence interval of 92.4 to 98.2 percent, and a pooled specificity of 98.8 percent, with a confidence interval of 96.7 to 99.7 percent. Summary receiver operating characteristic curves were generated using the hierarchical summary receiver operating characteristic model, following Cochrane guidance, and heterogeneity was quantified with the I² statistic. On the strength of these figures, the review positions kDNA-targeted qPCR as the reference standard among molecular approaches, albeit one whose performance comes with substantial infrastructure requirements.

That caveat matters. qPCR demands thermocycling instruments, continuous cold chains for reagents, reliable electricity, trained molecular biologists and quality assurance systems that are often absent precisely where leishmaniasis takes its greatest toll. Rural clinics in East Africa, South Asia and Latin America, where visceral and cutaneous disease overlap with poverty and weak laboratory networks, cannot routinely run quantitative real-time platforms. This is why the review devotes considerable attention to isothermal amplification technologies, which amplify nucleic acids at a constant temperature and therefore eliminate the need for expensive cycling equipment. The most mature of these is loop-mediated isothermal amplification, known as LAMP, which uses a set of four to six primers recognizing six to eight regions of the target sequence and a strand-displacing polymerase to generate amplification products that can be read visually by turbidity, fluorescence or color change.

The pooled performance of LAMP in the review was impressive: a sensitivity of 92.5 percent, with a 95 percent confidence interval of 87.1 to 96.4 percent, and a specificity of 98.9 percent, with a confidence interval of 96.2 to 99.9 percent. Yet the analysis also exposed a cautionary lesson about pooled diagnostic estimates. Heterogeneity among LAMP studies was extremely high, with an I² value of 98.3 percent, meaning that virtually all of the variability in reported accuracy reflected differences between studies rather than sampling variation within them. The review attributes much of this heterogeneity to primer mismatches for Leishmania aethiopica, a species that causes cutaneous disease in parts of East Africa and whose genetic divergence from better-studied species such as Leishmania donovani and Leishmania major can undermine primer binding. The finding underscores a recurring theme in molecular diagnostics: assay performance is not a property of the technology alone but of the fit between primers and the local parasite population.

Even faster than LAMP is recombinase polymerase amplification, or RPA, which operates at body temperature, typically 37 to 42 degrees Celsius, and can produce a result in as little as 15 to 20 minutes. RPA recruits recombinase enzymes to pair primers with their target sequence and a strand-displacing polymerase to extend them, making it uniquely tolerant of crude sample preparation and low-resource settings. In the review, RPA showed strong agreement with qPCR, with a Cohen’s kappa of 0.89, a level of concordance that supports its use as a point-of-care test. For field epidemiology, outbreak investigation and primary care in endemic villages, a test that returns an answer within a single patient visit, without a laboratory, could change the practical epidemiology of the disease, enabling same-day treatment decisions and reducing the losses to follow-up that plague current diagnostic pathways.

Another frontier highlighted by the review is the move away from invasive sampling. Traditional confirmation of visceral leishmaniasis may require splenic or bone marrow aspiration, procedures that carry real risk and require surgical skill. The review found that non-invasive samples, including urine, adhesive tape discs pressed against suspected skin lesions, and skin swabs, achieved a pooled molecular sensitivity of 93.9 percent, with a 95 percent confidence interval of 88.4 to 97.4 percent. Urine in particular has attracted interest because it can be collected repeatedly, safely and by minimally trained personnel, allowing parasite DNA detection and treatment monitoring without any needle. Tape discs and swabs open a similar door for cutaneous and mucocutaneous disease, where biopsy of facial lesions can be disfiguring. If non-invasive molecular sampling can be standardized, it would remove one of the last practical barriers to confirming diagnosis in primary care.

The review does not abandon serology entirely; instead it documents its modernization. Novel peptide-based enzyme-linked immunosorbent assays using the BT7 and EL1 peptides achieved an area under the curve of 0.971, approaching the discrimination of molecular tests while retaining the simplicity and low cost of antibody detection. Separately, a recombinant antigen derived from the Leishmania donovani Aurora kinase protein, designated LdAIRK, showed strong performance as a test of cure, addressing one of the most vexing problems in visceral leishmaniasis management: distinguishing patients who have been cured from those who will relapse or develop post-kala-azar dermal leishmaniasis. Conventional antibody tests remain positive for years after cure, making them useless for follow-up, so an antigen that tracks active infection status could transform post-treatment surveillance, particularly in South Asia where PKDL reservoirs sustain transmission.

The review is candid about what remains missing. Despite the proliferation of targeted amplification assays, next-generation sequencing has not yet been established for direct clinical diagnosis of leishmaniasis, leaving species identification, mixed infections and emerging drug resistance largely dependent on older tools such as restriction fragment length polymorphism analysis and internal transcribed spacer sequencing in reference laboratories. High-resolution melting analysis and miniature direct-on-blood PCR nucleic acid lateral flow immunoassays, abbreviated mini-dbPCR-NALFIA, appear among the innovations catalogued, but cost-effectiveness analyses, which health ministries need to justify procurement, are conspicuously absent from the literature. The review’s conclusion is therefore twofold: molecular methods have genuinely advanced, with qPCR setting the accuracy benchmark and isothermal platforms closing the accessibility gap, but heterogeneity across studies demands standardized protocols, shared primers and validated reference materials before these gains can be translated into routine practice across the 98 countries where the disease persists.

Subject of Research: Recent advances in molecular diagnosis of human leishmaniasis

Article Title: Recent advances in molecular diagnosis of human leishmaniasis

Article References: Asseri, J. I. (2026). Recent advances in molecular diagnosis of human leishmaniasis. BMC Infectious Diseases. https://doi.org/10.1186/s12879-026-14357-5

Image Credits: AI Generated

DOI: 10.1186/s12879-026-14357-5

Keywords: Recent, advances, molecular, diagnosis, human, leishmaniasis, scientific research