Rapid point‐of‐care diagnostics for the detection of Chlamydia pecorum in koalas (Phascolarctos cinereus) using loop‐mediated isothermal amplification without nucleic acid purification

Abstract

Infectious disease, predominately chlamydiosis, contributes significantly to the decline in health of wild koala (Phascolarctos cinereus) populations in some regions of Australia. In this study, we describe the development and evaluation of a simple, sensitive, and specific loop‐mediated isothermal amplification (LAMP) assay for the detection of Chlamydia pecorum in koalas as a point‐of‐care diagnostic tool that can be used in any wildlife hospital and in the field on specialized instrumentation. A set of primers targeting a 188‐bp region of the C. pecorum genome was designed. 100% specificity of the LAMP assay was revealed by demonstrating no cross‐reactivity with 33 nontarget pathogens, and complete correlation with qPCR results for 43 clinical swabs collected opportunistically from wildlife hospitals. In sensitivity evaluations, the technique successfully detected serial dilutions of extracted C. pecorum DNA with a detection limit of 44 IFU/ml.

Real-Time Fluorometric Isothermal LAMP Assay for Detection of Chlamydia pecorum in Rapidly Processed Ovine Abortion Samples: A Veterinary Practitioner’s Perspective

“…This is also a limitation of currently available C. pecorum qPCRs [ 8 ], and is especially a concern for aborted material, which can easily be contaminated with non-pathogenic gastrointestinal C. pecorum strains. Nevertheless, the benefit of using C. pecorum detection by LAMP with the rapid swab processing protocol described by Jelocnik et al [ 12 ] is that residual swab suspension can be used for DNA extraction and molecular characterisation, unlike other protocols described for rapid swab processing for C. pecorum LAMP [ 13 ]. In addition to determining strain pathogenicity in positive samples, supportive histopathological data are recommended for a definitive diagnosis.…”

“…The initial study also described a rapid swab processing method, utilising vortexing to dislodge cells from clinical swabs, followed by heat lysis to release DNA to decrease sample processing time and use of commercial DNA extraction kits. Another isothermal C. pecorum assay for detection of koala C. pecorum infections was recently proposed by Hulse et al [ 13 ], who used KOH to lyse cells and a specific isothermal mastermix (Lyse N LAMP mix, Optigene, UK). However, the limitation of this assay is that, upon testing, the sample is no longer viable for DNA extraction and molecular characterisation of the infecting strains (where the sample tests positive for C. pecorum ).…”

“…However, the limitation of this assay is that, upon testing, the sample is no longer viable for DNA extraction and molecular characterisation of the infecting strains (where the sample tests positive for C. pecorum ). Nevertheless, the utility of these assays at clinical setting and point-of-care (POC) is evident in koala clinical practice, where several wildlife hospitals utilise the C. pecorum LAMP assays as the diagnostic toolkit [ 12 , 13 ] (personal communication Dr Amy Robbins, Endeavour Veterinary Ecology, and Dr Amber Gillet, Australia Zoo Wildlife Hospital). The utility of chlamydial rapid isothermal testing and rapid sample processing has also been demonstrated in equine clinical practice to detect C. psittaci in equine abortions, both at POC and in the diagnostic laboratories [ 14 ].…”

Pharmacokinetic Profile of Doxycycline in Koala Plasma after Weekly Subcutaneous Injections for the Treatment of Chlamydiosis

“…Chlamydial infection was confirmed via loop-mediated isothermal amplification (LAMP) positive swabs of the conjunctivae and penile urethra. Although the polymerase chain reaction (PCR) method is considered to be the 'golden standard' for its high sensitivity and specificity, LAMP is used to achieve rapid pathogen diagnosis in-house, which is critical to prevent delays in diagnosis and treatment, and is also reported to have high sensitivity and specificity in detecting C. pecorum in koalas [9].…”

Helping koalas battle disease – Recent advances inChlamydiaand koala retrovirus (KoRV) disease understanding and treatment in koalas

“… 2017 ), while the second assay, released in 2019, targets the mreC gene (coding for a cell shape determining protein) from C. pecorum (Hulse et al . 2019a ). Both assays report no cross-reactivity with a range of non-target organisms and rapid sample preparation protocols for minimal sample preparation, making them both candidates for POC diagnostics in the field or veterinary clinic (Jelocnik et al .…”