Development of a Dry-Reagent-Based qPCR to Facilitate the Diagnosis of Mycobacterium ulcerans Infection in Endemic Countries

Abstract

BackgroundBuruli ulcer is a neglected tropical disease caused by Mycobacterium ulcerans. This skin disease is the third most common mycobacterial disease and its rapid diagnosis and treatment are necessary. Polymerase chain reaction (PCR) is considered to be the most sensitive method for the laboratory confirmation of Buruli ulcer. However, PCR remains expensive and involves reagents unsuitable for use in tropical countries with poor storage conditions, hindering the development of reliable quantitative PCR (qPCR) diagnosis. We aimed to overcome this problem by developing a ready-to-use dry qPCR mix for the diagnosis of M. ulcerans infection.Methodology/Principal FindingsWe compared the efficiency of three different dry qPCR mixes, lyophilized with various concentrations of cryoprotectants, with that of a freshly prepared mixture, for the detection of a standard range of M. ulcerans DNA concentrations. We evaluated the heat resistance of the dry mixes, comparing them with the fresh mix after heating. We also evaluated one of the dry mixes in field conditions, by analyzing 93 specimens from patients with suspected Buruli ulcers. The dry mix was (i) highly resistant to heat; (ii) of similar sensitivity and efficiency to the fresh mix and (iii) easier to use than the fresh mix.ConclusionsDry qPCR mixes are suitable for use in the diagnosis of M. ulcerans infection in endemic countries. The user-friendly format of this mix makes it possible for untrained staff to perform diagnostic tests with a limited risk of contamination. The possibility of using this mix in either vial or strip form provides considerable flexibility for the management of small or large amounts of sample. Thus, dry-mix qPCR could be used as a reliable tool for the diagnosis of Buruli ulcer in the field.

Buruli Ulcer, a Prototype for Ecosystem-Related Infection, Caused by Mycobacterium ulcerans

“…PCR is considered the most sensitive method for the laboratory confirmation of Buruli ulcer. However, PCR remains expensive and involves reagents unsuitable for use in tropical countries with poor storage conditions, hindering the development of reliable qPCR diagnostic assays (274). It is highly sensitive and specific and is also reasonably rapid, but it requires trained personnel with specific equipment (274).…”

“…However, PCR remains expensive and involves reagents unsuitable for use in tropical countries with poor storage conditions, hindering the development of reliable qPCR diagnostic assays (274). It is highly sensitive and specific and is also reasonably rapid, but it requires trained personnel with specific equipment (274). Nevertheless, PCR is routinely performed in hospitals in countries such as Côte d'Ivoire, Ghana, Benin, Nigeria, Cameroon, and Togo, with the strengthening of laboratory capacity supported by national and international programs and nongovernmental organizations.…”

The Point-of-Care Laboratory in Clinical Microbiology

“…Reagents must be stored at the appropriate temperature, and storage in a refrigerator at 4°C is possible. PCR mixes can be packaged and delivered in a lyophilized form, guaranteeing their stability and intrinsic qualities, as recently shown for the molecular diagnosis of Buruli ulcer (78).…”

Parallel susceptibility testing of bacteria through culture-quantitative PCR in 96-well plates

“…All these features would help its use in clinical labs. Dry-reagent-based PCR has been reported in rapid detection of pathogenic bacteria because of its friendly format and limited risk of contamination (Al-Talib et al, 2014;Babonneau et al, 2015;Nagaraj et al, 2018;Khazani et al, 2017). However, its use in susceptibility test is hardly found.…”

“…This method is found to be not only faster (<8 h) than the traditional culture methods (24-48 h), but also applicable to the susceptibility testing of all the antibiotics. However, the multiple steps of DNA extraction needed by using either commercial extraction kits or the phenol-chloroform method not only prolong the overall test time, but also generate extraerrors in the amount of DNA extracted (Al-Talib et al, 2014;Babonneau et al, 2015;Nagaraj et al, 2018;Khazani et al, 2017). Furthermore, it is usually needed in clinical labs to test the susceptibility of an isolate against a panel of antibiotics and multiple samples simultaneously.…”