LCNTDR Research Afternoon: NTD Detection and Diagnostics, 6th June

With the WHO and global community targeting the control and elimination NTDs there has been an increased focus on strengthening the ability of control programmes to rapidly and accurately detect these pathogens. There is demand for high-quality, low-cost diagnostic and detection tools that can be used to monitor levels of infection and transmission especially in light of decreases in prevalence due to disease-control efforts. Appropriate tools also are needed to conduct surveillance for disease re-emergence after presumed elimination.

The Research Afternoon brought together researchers from across the LCNTDR to discuss some of the latest research on disease detection and diagnosis.

Research Presented

Mini-FLOTAC as an alternative, non-invasive diagnostic tool for Schistosoma mansoni and other trematode infections in wildlife reservoirs. 

Stefano Catalano (RVC) 

The zoonotic, multi-host nature of schistosomiasis and food-borne trematodiases is a significant challenge for disease control programmes in endemic areas. However, our understanding of the epidemiological role that animal reservoirs, particularly wild hosts, may play in the transmission of zoonotic trematodiases suffers a dearth of information, with few, if any, standardised, reliable diagnostic tests available.

Stefano and colleagues combined qualitative and quantitative data derived from post-mortem examinations, coprological analyses using the Mini-FLOTAC technique, and molecular tools to assess parasite community composition and the validity of non-invasive methods to detect trematode infections in 89 wild Hubert’s multimammate mice (Mastomys huberti) from northern Senegal. Parasites isolated at post-mortem examination were identified as Plagiorchis sp., Anchitrema sp., Echinostoma caproni, Schistosoma mansoni, and a hybrid between Schistosoma haematobium and Schistosoma bovis.

Comparison of prevalence estimates derived from parasitological analysis at post-mortem examination and Mini-FLOTAC analysis showed non-significant differences indicating comparable results between the two techniques. A Bayesian model, applied to estimate the sensitivities of the two tests for the diagnosis of Schistosoma infections, indicated similar median posterior probabilities of 83.1% for Mini-FLOTAC technique and 82.9% for post-mortem examination.

Their results showed that the Mini-FLOTAC could be applied as an alternative diagnostic technique for the detection of the zoonotic S. mansoni and other trematodes in rodent reservoirs. The implementation of non-invasive diagnostics in wildlife would offer numerous advantages over lethal sampling methodologies, with potential impact on control strategies of zoonotic helminthiases in endemic areas of sub-Saharan Africa and on fostering a framework of animal use reduction in scientific practice.

Download the presentation: Mini-FLOTAC as a diagnostic tool for Schistosoma mansoni

Understanding spatial patterns of NTD transmission using multiplex serological assays: examples from Malaysia and Northern Ghana. 

Dr Kimberly Fornace (LSHTM) 

Multiplex serological data is increasingly used to measure population-level and serological responses to neglected tropical diseases. These serological markers allow the collection of data on exposure to multiple pathogens simultaneously for relatively low cost and may be particularly useful in low transmission and elimination settings where the probability of detecting infections is low.

Combining these data with spatial information can provide new insights on spatial distribution of residual transmission and environmental drivers. However, analysis is complicated by the duration of antibody responses and the time and frequency of exposure. Kimberly presented examples of spatial analysis of multiplex serological data from Malaysian Borneo and Northern Ghana and discussed applications and further research needs.

Download the presentation: Understanding spatial patterns of NTD transmission using multiplex serological assays

QPCR-based diagnostics: how close are we to translating CQ values to intensity of infection?

Marina Papaiakovou (NHM)

Control and elimination programs tackling soil-transmitted helminth (STH) infections worldwide require sensitive, reliable and accurate diagnostic tools. For decades, microscopy-based methods have been used for the detection of intestinal parasites and monitoring due to their simplicity, low-cost and practicality. However, there is also a growing need for measures of infection intensity as programs approach STH elimination.

Even though the transmission breakpoint is yet to be defined epidemiologically, the adoption of molecular diagnostics has already been promoted. Real-time PCR and its analytical sensitivity lends itself to the detection of a DNA target present in stool even in low-prevalence settings. Combined with sensitivity, reproducibility and the ability to distinguish between species where microscopy fails, yield the ground for PCR to be an appealing diagnostic tool for detecting helminths in stool. Detecting low levels of STH infections is not only vital when the breakpoint of transmission is approached but, more importantly, once elimination is achieved, to monitor for recrudescence. The persistent barriers and misunderstandings (i.e nature of molecular target, DNA recovery during extractions, inhibition present) limit establishing the technique as a widely accepted and endorsed tool need to be highlighted. Quantitation of PCR analytically is trivial; its correlation to measurements biologically significant, however, is not.

Challenges remain in relying on qPCR as a diagnostic tool of choice for STH and of most concern is variation in molecular target copy number in the pathogens/parasites. New ideas and studies are required to accurately relate qPCR data (in relation to target copy number) to infection intensity. To employ PCR effectively for STH diagnostics we need to understand what PCR can do or measure before its deployment is endorsed as a cornerstone diagnostic tool.

Download the presentation: QPCR-based diagnostics: how close are we to translating CQ values to intensity of infection?
Presentation made on behalf of Marina by Dr Tim Littlewood

Complexities of snail xenomonitoring in relation to schistosomiasis transmission monitoring.

Dr Bonnie Webster (NHM)

Accurate diagnosis of urogenital schistosomiasis is vital for surveillance/control programmes. Amplification of schistosome DNA in urine by PCR is sensitive and specific but requires infrastructure, financial resources and skilled personnel, often not available in endemic areas.

Recombinase Polymerase Amplification (RPA) is an isothermal DNA amplification/detection technology. It is simple, rapid, portable and needs few resources making it a potential technology suited for the endemic setting. Here, a Schistosoma haematobium real time fluorescence RPA assay (RT-ShDra1-RPA) targeting the Dra1 genomic repeat region was developed and tested. Its utility and sensitivity were evaluated on 200 urine samples, with varying egg counts, from infected school children from Pemba Island, which had been stored as part of the Zanzibar Elimination of Schistosomiasis Transmission study (SCORE). Samples were prepared prior to RPA analysis using a quick crude field DNA extraction method, the Speed Extract Kit (Qiagen).

The assay successfully amplified S. haematobium DNA to a lower limit of 1fg with results obtained, within 10 minutes of running time, using a small portable battery powered tube scanner device that incubated reactions at 40°C whilst detecting changes in fluorescence over time. The assay gave positive results from urine samples with egg counts from 1 - 926 eggs/10ml, however results from certain samples with low and inconsistent egg counts were difficult to interpret. The RT-ShDra1-RPA assay proved robust for S. haematobium gDNA detection and gave promising results for the diagnosis of urogenital schistosomiasis using urine samples from infected patients.

RPA’s speed, simplicity and low resource requirements together with its ability to use crude sample preparations could advance molecular diagnostics for urogenital schistosomiasis at the point-of-need within endemic countries.

Further research is needed to evaluate the performance of the assay with lower intensity infections, which will inevitably be the main reason for implementing such an advanced diagnostic. The application is discussed in relation to other diagnostic needs in relation to urogenital schistosomiasis.

Download the presentation: Complexities of snail xenomonitoring in relation to schistosomiasis transmission monitoring.

POC-CCA vs. Kato-Katz in Brazil.

Dr Anna Phillips (Imperial)

Schistosomiasis control through mass treatment with Praziquantel is progressing rapidly, and elimination has recently been included on the agenda by the WHO in certain areas. For design of effective control programs, it is important to determine an accurate estimate of infection in a programme area.

Current diagnostic methods (detection of eggs in stool by Kato-Katz) are limited and may be particularly unreliable at low infection intensities, as would be expected after several rounds of treatment or where transmission is low. A particular problem is the high logistical cost and poor compliance to multiple days of Kato-Katz testing, which is required to accurately detect infection at low prevalence. In order to overcome some of the pitfalls of Kato-Katz, there has been interest in developing more sensitive tests for the diagnosis of intestinal schistosomiasis.

These tests often employ immunologic methods based on the detection of antibodies or antigens in blood or urine. Point-of-Care Circulating Cathodic Antigen (POC-CCA) detection in urine has been documented to be a sensitive and specific alternative to Kato-Katz through direct comparisons, however, data comparing multiple days of microscopy and antigen detection tests in low prevalence settings is currently lacking.

The data demonstrates the performance of POC-CCA with that of three days of duplicate Kato-Katz in low (Minas Gerais) and moderate (Bahia) prevalence settings. The results of this study will contribute to the literature as increasingly POC-CCA is being recognised as a mapping tool and therefore WHO guidelines need to be developed, particularly as many countries can realistically transition to achieving elimination. 

Imperial's Julia Dunn presented on behalf of Anna. 

Detection of Chlamydia trachomatis DNA from viable and non-viable organisms on different surfaces.

Dr Aalbertus Versteeg (LSHTM)

Chlamydia trachomatis (Ct) is the most common infectious cause of blindness (trachoma) worldwide. Fomites have been suggested to play a role in the ongoing transmission of Ct in trachoma endemic areas, but there is limited data available to show if Ct detected on different fomites is actual viable and can therefore actively contribute to ongoing transmission.

Aalbertus and colleagues are interesting in assessing the presence and viability of Ct DNA detected on different surfaces, including plastic, sleeping mat, cotton and pork skin.

Viable Ct DNA could be recovered from all surfaces except sleeping mat for up to 24 hours. These results show that viable Ct can be recovered from plastic, cloth and skin within a controlled environment, suggesting that these may indeed play a role in ongoing Ct transmission.