Rapid and sensitive detection of Chlamydia trachomatis sexually transmitted infections in resource-constrained settings in Thailand at the point-of-care.

Abstract

Chlamydia trachomatis is the leading cause of sexually transmitted diseases (STDs) in females and males in both developed and developing countries, with more than 110 million cases annually. C. trachomatis resists antibiotic treatment and is a cofactor in HIV transmission and human cervical cancer [1]. Infection is often asymptomatic, causing the epidemiology to be underestimated. Therefore, we have developed a rapid, inexpensive, easy-to-interpret, sensitive and specific point-of-care (POC) C. trachomatis detection system, using loop-mediated isothermal amplification (LAMP) for target C. trachomatis DNA amplification, followed by gold nanoparticle probe (AuNP) for colorimetric C. trachomatis specific readout. The assay was evaluated using clinical samples and compared with polymerase chain reaction (PCR) of the same target gene, which is an outer membrane protein A (ompA) gene and a respected standard for C. trachomatis detection [1,2]. For nucleic acid amplification tests, recently LAMP has presented an attractive alternative to standard methods like PCR due to its low price, ease of use, rapid results, and lack of requirement for an expensive thermal cycler and specialized kits for DNA extraction and purification. A simple 5-minute boil for crude DNA lysis is sufficient for the LAMP reaction because the Bacillus stearothermophilus (Bst) DNA polymerase in LAMP has fewer inhibitor problems than Thermus aquaticus (Taq) DNA polymerase in PCR [3]. LAMP amplifies the target at a single temperature with high sensitivity (from 10 to 100 genome copies), and the product can be visualized as a white magnesium pyrophosphate precipitate. However, nebulous precipitate has the potential to cause misreading [3,4], so analysis by agarose gel-electrophoresis (GE), as with PCR, is standard. The positive LAMP reaction appears as multiple bands because several primers amplify amplicons of different sizes that are intercalated. Nevertheless, GE requires an electrophoresis apparatus, time, and often ethidium bromide exposure. In the current study, crude DNA lysis with combined LAMP-AuNP for POC C. trachomatis detection has been developed, and the specificity and limit of detection were validated by several positive and negative C. trachomatis genomes, as well as the possibility for POC diagnostic based on a statistical number of clinical endocervical swab sample tests (see Box 1). Box 1: Advantages and disadvantages of C. trachomatis LAMP-AuNP. Advantages Sensitive to as low as 11.25 copies of target DNA Total assay time is <1 hour Evaluation in independent replicates in 130 clinical samples showed 96% sensitivity and 99% to 100% specificity Disadvantages Cannot diagnose the severity of disease Reaction reagents are in liquid Color change observation may be difficult for low copy numbers of Chlamydia trachomatis Materials and methods Study design and clinical sample collections The criteria for symptomatic C. trachomatis was obtained from the description by the Centers for Disease Control and Prevention (CDC), such as abnormal cervical or vaginal discharge, elevated number of white blood cells in vaginal secretion, and co-infection with Neosseria gonorrhoeae [1]. Endocervical swab samples were randomly selected from a prospective study cohort of STD prevalence in symptomatic and healthy (which may include nonsymptomatic patients) Thai women aged 15 to 54 years in Bangkok and nearby areas. The samples were collected by clinicians during 2011 and 2012 from qualified volunteers attending the Buddhachinaraj Phitsanulok Hospital, and in 2013 and 2014 from Bangrak and Chonburi offices of Disease Prevention and Control [2]. The sample size of 130 (96 symptomatic and 34 healthy) was computed based on a standard statistical formula (http://www.calculator.net/sample-size-calculator.html), given the prevalence rates of C. trachomatis as 22% for symptomatic STDs and 3% for healthy people [2], with a margin of error of 0.05, and a 90% confidence interval. The samples were stored in 3 mL standard C. trachomatis collection medium M4RT (Thermo Fisher Scientific, Kansas, USA) at −80°C.