Antimicrobial susceptibility of Neisseria gonorrhoeae isolates and syndromic treatment of men with urethral discharge in Kingston, Jamaica, 2018-19.

OBJECTIVES: To quantitatively determine the antimicrobial susceptibility of clinical Neisseria gonorrhoeae isolates from men with urethral discharge in Jamaica and to describe the syndromic treatment therapies administered. METHODS: Urethral eSwabs (Copan) were collected from 175 men presenting with urethral discharge to the Comprehensive Health Centre STI Clinic, Kingston, Jamaica. Clinical information was collected and MICs of eight antimicrobials were determined for N. gonorrhoeae isolates (n = 96) using Etest and interpreted using CLSI criteria. RESULTS: The median age of the subjects was 28 years (range: 18-73 years) with a median of 2 sexual partners (range: 1-25) per male in the previous 3 months. All examined N. gonorrhoeae isolates were susceptible to ceftriaxone (96/96), azithromycin (91/91), cefixime (91/91) and spectinomycin (91/91). For ciprofloxacin and gentamicin, respectively, 98.9% (91/92) and 91.3% (84/92) of the isolates were susceptible and 1.1% (1/92) and 8.7% (8/92) showed intermediate susceptibility/resistance. For tetracycline and benzylpenicillin, respectively, 38.0% (35/92) and 22.0% (20/91) of the isolates were susceptible, 52.2% (48/92) and 74.7% (68/91) showed intermediate susceptibility/resistance and 9.8% (9/92) and 3.3% (3/91) were resistant. Syndromic treatment was administered as follows: 93.1% received 250 mg of ceftriaxone intramuscularly plus 100 mg of doxycycline orally q12h for 1-2 weeks and 6.9% received 500 mg of ciprofloxacin orally plus 100 mg of doxycycline orally q12h for 1 week. CONCLUSIONS: Ceftriaxone (250 mg) remains appropriate for gonorrhoea treatment in the examined population of men in Kingston, Jamaica. Surveillance of N. gonorrhoeae AMR should be expanded in Jamaica and other Caribbean countries to guide evidence-based treatment guidelines.

Use of loop-mediated isothermal amplification for detection of Ophiostoma clavatum, the primary blue stain fungus associated with Ips acuminatus.

Loop-mediated isothermal amplification (LAMP) is an alternative amplification technology which is highly sensitive and less time-consuming than conventional PCR-based methods. Three LAMP assays were developed, two for detection of species of symbiotic blue stain fungi associated with Ips acuminatus, a bark beetle infesting Scots pine (Pinus sylvestris), and an additional assay specific to I. acuminatus itself for use as a control. In common with most bark beetles, I. acuminatus is associated with phytopathogenic blue stain fungi involved in the process of exhausting tree defenses, which is a necessary step for the colonization of the plant by the insect. However, the identity of the main blue stain fungus vectored by I. acuminatus was still uncertain, as well as its frequency of association with I. acuminatus under outbreak and non-outbreak conditions. In this study, we employed LAMP technology to survey six populations of I. acuminatus sampled from the Southern Alps. Ophiostoma clavatum was detected at all sampling sites, while Ophiostoma brunneo-ciliatum, reported in part of the literature as the main blue stain fungus associated with I. acuminatus, was not detected on any of the samples. These results are consistent with the hypothesis that O. clavatum is the main blue stain fungus associated with I. acuminatus in the Southern Alps. The method developed in the course of this work provides a molecular tool by which it will be easy to screen populations and derive important data regarding the ecology of the species involved.

Detection of African swine fever virus by loop-mediated isothermal amplification.

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of African swine fever virus (ASFV). This assay targets the topoisomerase II gene of ASFV and its specificity was confirmed by restriction enzyme digestion of the reaction products. The analytical sensitivity of this ASFV LAMP assay was at least 330 genome copies, and the test was able to detect representative isolates of ASFV (n=38) without cross-reacting with classical swine fever virus. The performance of the LAMP assay was compared with other laboratory tests used for ASF diagnosis. Using blood and tissue samples collected from pigs experimentally infected with ASFV (Malawi isolate), there was good concordance between the LAMP assay and real-time PCR. In addition to detecting the reaction products using either agarose gels or real-time PCR machines, it was possible to visualise dual-labelled biotin and fluorescein ASFV LAMP amplicons using novel lateral flow devices. This assay and detection format represents the first step towards developing a practical, simple-to-use and inexpensive molecular assay format for ASF diagnosis in the field which is especially relevant to Africa where the disease is endemic in many countries.

Development of a One-Step Real-Time Polymerase Chain Reaction Assay for Diagnosis of Phytophthora ramorum.

ABSTRACT Phytophthora ramorum is a recently described pathogen causing bleeding cankers, dieback, and leaf blight on trees and shrubs in parts of Europe and North America, where the disease is commonly known as sudden oak death. This article describes the development of a single-round real-time polymerase chain reaction (PCR) assay based on TaqMan chemistry, designed within the internal transcribed spacer 1 region of the nuclear ribosomal (nr)RNA gene for detection of P. ramorum in plant material. Unlike previously described methods for the molecular detection of P. ramorum, this assay involves no post amplification steps or multiple rounds of PCR. The assay was found to have a limit of detection of 10 pg of P. ramorum DNA, and could detect P. ramorum in plant material containing 1% infected material by weight within 36 cycles of PCR. The assay also was used to test DNA from 28 other Phytophthora spp. to establish its specificity for P. ramorum. A quick and simple method was used to extract DNA directly from host plant material, and detection of P. ramorum was carried out in multiplex with an assay for a gene from the host plant in order to demonstrate whether amplifiable DNA had been extracted. Amplifiable DNA was extracted from 84.4% of samples, as demonstrated by amplification of host plant DNA. The real-time protocol was used to test 320 plant samples (from 19 different plant species) from which DNA extraction had been successful, and was shown to give results comparable with a traditional isolation technique for diagnosis of P. ramorum in plant material from common U.K. hosts.