What is Cas-Find?

Cas-Find is a point-of-care diagnostic tool using the CRISPR-Cas9 system with the potential to radically change the speed of disease diagnosis, with a particular focus on the detection of sexually transmitted infections (STI’s).

Why STI's?

Sexually transmitted infections (STIs), including those caused by the human immunodeficiency virus (HIV) types 1 and 2, remain an important focus area for global public health. This is due to the high morbidity associated with STIs, such as the sequelae of reproductive tract infections, cervical cancer, congenital syphilis, ectopic pregnancy and infertility, as well as the morbidity of HIV-related illness and death from acquired immunodeficiency syndrome (AIDS).

More than 30 bacterial, viral, and parasitic pathogens are transmissible sexually and constitute a group of infections referred to as sexually transmitted infections (STIs). Although some of the pathogens can be acquired through routes other than sexual transmission, epidemiologically, sexual contact is more important for their transmission from one person to another (See Table 1 for information on the main sexually transmitted pathogens).

http://hai-sti.roche.com/images/hai-and-sti-epidemics.png

What is point-of-care testing?

Point-of-care testing (POCT): ‘tests designed to be used at or near the site where the patient is located, that do not require permanent dedicated space, and that are performed outside the physical facilities of the clinical laboratories’ i.e. testing at the time and place of patient care (Santrach 2007).

What are the potential benefits of point-of-care tests?

With the aim of POCT to bring the test conveniently and immediately to the patient it increases the likelihood of the physician and patient receiving results more quickly and allowing immediate clinical management decisions to be made. Rapid decision making, reduced number of outpatient clinic visits, reduced number of hospital beds required and occupied, overall ensuring the optimal use of professional time.

When used for diagnosis, the time required for test results to become available to guide management should be considered when choosing which test to use, since infected persons may transmit infections to others, may suffer complications of infection, or may be lost to follow-up in the interval between testing and notification of test results

How do POCT’s compare to other laboratory tests?

Laboratory-based tests, such as culture or nucleic acid amplification testing, may require special methods of specimen transport and specialized equipment and procedures for optimal performance, thus delaying the availability of results for immediate management decisions. Therefore laboratory-based diagnosis of STIs tends to be expensive in terms of equipment, reagents, infrastructure, and maintenance. Even more importantly, particularly in resource- constrained settings, the levels where most patients with STIs are processed have no laboratory facilities. (Peeling et.al. 2006)

Where can POCT’s have the greatest impact?

In countries with a high burden of sexually transmitted infections (STIs), where laboratory services for STIs are either not available or limited services are available and patients may not be able to physically access or to pay for these services.

Currently in many high risk 3^rd world countries the World Health Organisation (WHO) recommends the use of syndromic management where patients are treated for all the major causes of a particular syndrome. Syndromic management of STIs works well for urethral discharge, pelvic pain, and genital ulcer disease, but evaluations of the WHO flowcharts have shown that the algorithm for vaginal discharge lacks both sensitivity and specificity for the identification of women with Chlamydia trachomatis and Neisseria gonorrhoeae infection. Simple, affordable, rapid tests that can be performed at the point-of-care (POC) and enable treatment and case management decision to be made are urgently needed for these infections (Unemo 2013). Simple rapid POC tests are needed not only to increase the specificity of syndromic management, and reduce over-treatment of genital gonococcal and chlamydial infections but also to screen for asymptomatic STIs. A mathematical model estimated that a test for syphilis that requires no laboratory infrastructure could save more than 201,000 lives and avert 215,000 stillbirths per year worldwide. A similar test could save approximately 4 million disability- adjusted life years (DALYs), avert more than 16.5 million incident gonorrhoea and chlamydial infections and prevent more than 212,000 HIV infections per year (Unemo 2013).

What makes a good POCT?

Can I have an example of how Cas-Find would be useful?

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Lets take one of the most common sexually transmitted infections in the UK affecting on average 200,000 people each year, chlamydia (PHE 2015). Currently to test for this infection you take either a urine sample or swab to determine if the chlamydia bacteria (Chlamydia trachomatis) is present. Analysing the samples involves using nucleic acid amplification tests (NAAT) such as PCR or by growing a chlamydia culture. Cultures incur high costs, have varying sensitivity and limitations for widespread screening whilst both methods are laborious and often time consuming. Cas-Find aims to reduce the time to disease detection. Using the CRISPR-Cas9 system specific sequences on the suspect bacteria can be immediately detected, emitting a measureable fluorescence if present. This point-of-care nature of our project can significantly advantage those where lab access if limited, expensive and the speed of diagnosis is crucial in making the appropriate clinical decisions.

Table 1: Main sexually transmitted pathogens and the diseases they cause

Pathogen	Clinical manifestations and other associated diseases
Bacterial infections
Neisseria gonorrhoeae	GONORRHOEA Men: urethral discharge (urethritis), epididymitis, orchitis, infertility Women: cervicitis, endometritis, salpingitis, pelvic inflammatory disease, infertility, preterm rupture of membranes, perihepatitis; commonly asymptomatic
Chlamydia trachomatis	CHLAMYDIAL INFECTION Men: urethral discharge (urethritis), epididymitis, orchitis, infertility Women: cervicitis, endometritis, salpingitis, pelvic inflammatory disease, infertility, preterm rupture of membranes, perihepatitis; commonly asymptomatic Both sexes: proctitis, pharyngitis, Reiter’s syndrome Neonates: conjunctivitis, pneumonia
Chlamydia trachomatis (serovars L1–L3)	LYMPHOGRANULOMA VENEREUM Both sexes: ulcer, inguinal swelling (bubo), proctitis
Treponema pallidum	SYPHILIS Both sexes: primary ulcer (chancre) with local adenopathy, skin rashes, condylomata lata; bone, cardiovascular, and neurological damage Women: pregnancy wastage (abortion, stillbirth), premature delivery Neonates: stillbirth, congenital syphilis
Haemophilus ducreyi	CHANCROID Both sexes: painful genital ulcers; may be accompanied by bubo
Klebsiella (Calymmatobacterium) granulomatis	DONOVANOSIS (GRANULOMA INGUINALE) Both sexes: nodular swellings and ulcerative lesions of the inguinal and anogenital areas Men: urethral discharge (nongonococcal urethritis) Women: cervicitis, endometritis, probably pelvic inflammatory disease
Mycoplasma genitalium	Men: urethral discharge (nongonococcal urethritis) Women: cervicitis, endometritis, probably pelvic inflammatory disease
Viral infections
Human immunodeficiency virus (HIV)	ACQUIRED IMMUNODEFICIENCY SYNDROME (AIDS) Both sexes: HIV-related disease, AIDS
Herpes simplex virus type 2 Herpes simplex virus type 1 (less common)	GENITAL HERPES Both sexes: anogenital vesicular lesions and ulcerations Neonates: neonatal herpes (often fatal)
Human papillomavirus	GENITAL WARTS Men: penile and anal warts; carcinoma of the penis Women: vulval, anal, and cervical warts, cervical carcinoma, vulval carcinoma, anal carcinoma Neonates: laryngeal papilloma
Hepatitis B virus	VIRAL HEPATITIS Both sexes: acute hepatitis, liver cirrhosis, liver cancer
Cytomegalovirus	CYTOMEGALOVIRUS INFECTION Both sexes: subclinical or nonspecific fever, diffuse lymph node swelling, liver disease, etc.
Molluscum contagiosum virus	MOLLUSCUM CONTAGIOSUM Both sexes: genital or generalized umbilicated, firm skin nodules
Kaposi sarcoma associated herpesvirus (human herpesvirus type 8)	KAPOSI SARCOMA Both sexes: aggressive type of cancer in immunosuppressed persons
Protozoal infections
Trichomonas vaginalis	TRICHOMONIASIS Men: urethral discharge (nongonococcal urethritis); often asymptomatic Women: vaginosis with profuse, frothy vaginal discharge; preterm birth, low-birth-weight babies Neonates: low birth weight
Fungal infections
Candida albicans	CANDIDIASIS Men: superficial infection of the glans penis Women: vulvo-vaginitis with thick curd-like vaginal discharge, vulval itching, or burning
Parasitic infestations
Phthirus pubis Sarcoptes scabiei	PUBIC LICE INFESTATION SCABIES

References:

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Peeling, R.W., Holmes, K.K., Mabey, D. (2006) Rapid tests for sexually transmitted infections (STI’s): the way forward. Sex Transm Infect. 82:1-6.

Public Health England. (2015) Sexually transmitted infections and chlamydia screening in England. Infection Report. 10:22

Santrach, P.J. (2007) Current and Future Applications of Point of Care Testing. Mayo Clinic. Access online at: http://wwwn.cdc.gov/cliac/pdf/addenda/cliac0207/addendumf.pdf

Unemo M. (2013) Laboratory diagnosis of sexually transmitted infections, including human immunodeficiency virus. WHO.  

Design

'Cas-Find' is a novel bioluminescent system for point-of-care diagnostic testing.

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Laboratory-based tests, such as nucleic acid amplification (NAA) or culture, require special methods of specimen transport, alongside specalised equipment and procedures for optimal performance ^[2]. As such the utilisation of laboratory-based tests is generally expensive in terms of equipment, reagents, infrastructure and maintenance. This limits the availability of results for immediate use in management decisions, potentially impacting on patient prognosis. In addition the majority of STI testing is conducted in resource-constrained environments, where such laboratory facilities are unavailable ^[3].

Fig 1. Summary of Cas-Find project

Proof of concept in vitro system targeted to Escherichia coli 16S rRNA.

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Dr. Daniel Pass designed sgRNA constructs targeted to the E. coli 16S rRNA locus in order to facilitate proof-of-concept testing of our in vitro system. The design of these constructs was achieved using a Python script developed by Dr. Pass to test FASTA formatted genomic DNA for paired target sequences using guidelines from Takara Bio USA alongside additional sources. This script initially identifies a proto-spacer adjacent motif (PAM) sequence (5'-NGG-3') in this FASTA sequence. The sgRNA sequence is complementary to the 20 nucleotides upstream of the PAM sequence. This is passed to BLASTn to test for simple alignment against the reference dataset, which could include the remainder of the species genome, or multiple cross-reactive species. The output is a FASTA table of potential probes, and a table.txt file of the same information in a graphical representation.

Fig 2. Summary of sgRNA design

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Fig 3. Summary of Characterisation

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