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Evaluating Detection Methods for Persistent Infection

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A pair of recent studies looked at the reliability of clinical observations and bacterial culture versus quantitative PCR (qPCR) in detecting clinically healthy carriers of the bacterium that causes strangles, who pose an infection risk to naïve individuals.Strangles, caused by the bacterium Streptococcus equi (S. equi), is the most frequently diagnosed infectious disease of horses worldwide and, aside from the pain and suffering caused, outbreaks can come with significant negative economic consequences. One of the key features identified as a significant contributor to the spread of disease is the development of what has been termed the “carrier state” or “silent carriers”. These animals appear clinically normal but harbor infectious S. equi that can then be passed on to infect naive animals. Identification and clearance of these animals is therefore of great importance for containing and preventing disease.

How are carrier animals identified?

Traditionally, carriers are associated with gross changes of the guttural pouch (part of the eustachian tube which is the typical site of carriage), from mild inflammation and thickening of the walls, the presence of purulent material (empyema), right up the presence of chondroids (dried inspissated pus). However, recent evidence suggests that even in animals where the guttural pouches appear normal and culture returns negative, there are still infectious S. equi lurking that may, at points, only be detected by qPCR. This therefore highlights the importance of appropriate testing before veterinarians declare an animal clear of infection or not. These studies took two outbreaks in which carrier animals developed to evaluate the efficacy of carrier detection methods. There has been some debate over the criteria that constitutes what is or is not a carrier, but for the purpose of these studies, “carriers” were defined as animals that still tested positive for S. equi by culture and/or qPCR on at least one sample at least six months after the index case.

Taking a look at persistence in two outbreaks

The first study, from Riihimäki et al. published in 2018, followed an outbreak for 13 months in which all 41 mature Icelandic horses in the group developed disease (38 of which were retained for the duration of the study) and subsequently 14 were identified as “carriers”, ten of whom were followed and repeat samplings performed.

Only one of the carriers had visually distinguishable changes that would identify it as a carrier. These animals were intermittently culture positive despite being constantly qPCR positive. However, strain sequence information was able to demonstrate that these animals were persistently infected with the same strain, it wasn’t a case of reinfection producing intermittent culture positives. These findings highlight that culture and visual inspection alone are not sufficient to pick up carriers consistently. Reliance on a single sampling site was also shown to reduce carrier detection.

The second study, from Pringle et al. published in 2019, included the same outbreak examined in the first study in addition to an outbreak of 112 weanlings in which morbidity was 53 % (98 of which were retained for the duration of the study) subsequent to which 15 were identified as “carriers”. This group was followed for 14 months post-index case.

As found in the earlier outbreak, qPCR detected carrier animals more reliably that were intermittently negative by culture, and more than half of the horses identified as carriers in both outbreaks did not differ from non-carriers based on endoscopic examination of the pouches. Whilst sampling the guttural pouch yielded the most qPCR positives, some animals at some samplings only tested positive on nasopharyngeal wash or nasal swab samples, indicating there is value in testing multiple sites if trying to capture all carriers in the group.

So, what is the take-home message?

In both studies, it became apparent that the appearance of visible evidence of infection in the guttural pouches and culture of live bacteria was not sufficiently sensitive to catch all carrier animals and the use of more sensitive methods, such as qPCR were key tools for combatting disease persistence and spread. Sampling of multiple sites (nasopharynx as well as the pouch itself) was also shown to be desirable for consistent detection of carriage.

Superior sensitivity of qPCR assays over culture and traditional PCR has been demonstrated in the detection of S. equi within clinical samples. These studies however take this one step further, illustrating their value in the detection of carriers in the clinical setting.

The value of genetic analysis was also evident in demonstrating that these animals were indeed persistently infected with the same strain, showing the intermittent culture positive results were not a product of reinfection with different strains from other animals in the group. The intermittence and recurrence of culture positives whilst qPCR results remain positive also point to fact that qPCR results are representing the presence of live bacteria, even at low levels, rather than DNA from dead or non-viable organisms as has been suggested in the past.

Professor John Pringle, first author of the 2019 publication commented “These two studies provide clinicians with key evidence-based advances in understanding of the strangles carrier state. Firstly, that horses culture negative yet qPCR positive for S. equi must be presumed to carry viable organisms until otherwise proven, and secondly that optimal carrier detection will require sampling from more than just the guttural pouch. The remaining unanswered question is whether these carriers that are only qPCR positive are infectious to new strangles-susceptible horses”.