Canada Communicable Disease Report

1 January 2008

Volume 34

Number 01

Absence of Prolyliminopeptidase-Negative Neisseria gonorrhoeae strains in Ontario, Canada

S Brown, BA, MLT (1), P Rawte, BSc, Ms, MLT, MIBMS (1), L Towns, MLT (1), F Jamieson, MD, FRCPC (1), RSW Tsang, M Med Sc, PhD (2)

1. Ontario Ministry of Health and Long Term Care, Central Public Health Laboratory, Etobicoke, Ontario, Canada.
2. Laboratory for Pathogenic Neisseria, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.

Background

Three commonly used approaches are used to identify oxidase positive, catalase positive Gram-negative cocci as Neisseria gonorrhoeae from cultures. The most traditional approach is based on carbohydrate utilization or acid production in cystine trytpicase agar (CTA) sugars⁽¹⁾. Newer and rapid methods include using monoclonal antibody to detect specific epitopes on the outer membrane proteins of N. gonorrhoeae, and biochemical assays to detect preformed enzyme such as the prolyliminopeptidase (also known as proline iminopeptidase) (PIP) which is thought to be universally present in all N. gonorrhoeae strains^(2,3). Another method for culture confirmation of N. gonorrhoeae is the DNA probe test⁽⁴⁾.

Between 2000 and 2003, PIP-negative N. gonorrhoeae strains began to emerge in several countries in Europe (England and Wales, Sweden, Denmark, Spain), New Zealand and Australia. Furthermore, phenotypic and genetic characterization of the PIP-negative gonococci from several countries suggested a global spread of a single strain^(5-10).

The Central Public Health Laboratory of the Ontario Ministry of Health and Long Term Care, and the Public Health Agency of Canada's National Microbiology Laboratory (NML) have been monitoring for the emergence of such PIP-negative N. gonorrhoeae strain in Ontario, Canada. The aim of this study was to determine if such strains exist in Canada, and to educate public health officials and laboratory scientists that some commercial test kits in the market may not be suitable for the identification of PIP-negative N. gonorrhoeae. This report also serves to notify laboratories performing primary isolation and identification of N. gonorrhoeae that PIP-negative N. gonorrhoeae control strains are available through the NML to ensure the methods used in the laboratory can detect this N. gonorrhoeae variant.

Methods

One hundred antibiotic sensitive (defined as susceptible to antibiotics commonly used for treatment of gonorrhoeae, including penicillin, ceftriaxone, cefixime, tetracycline, erythromycin, ciprofloxacin, and spectinomycin) and 100 antibiotic-resistant (defined as strains showing resistance to one or more of the above antibiotics) N. gonorrhoeae isolates recovered in 2006 from clinical cases in Ontario, Canada, were tested for PIP activity using the commercial test kit API-NH (Biomérieux Canada Inc., Brampton, Ontario). Seventy of the 100 antibiotic resistant isolates were also tested for PIP activity by Gonochek II (EY Laboratories Inc., San Mateo, California, USA).

Three PIP-negative control strains were provided by Professor Fernando Vazquez, Area de Microbiologia, Facultad de Medicina, Asturias, Spain. The identification of these isolates was verified by Gram stain, growth on modified Thayer-Martin medium, oxidase and catalase tests, CTA sugars, and Phadebact Monoclonal GC test (Boule Diagnostics AB, Sweden). PIP deficiency in these strains was confirmed using two commercial test kits: API-NH and Gonochek II.

Results and Discussion

Accurate identification of sexually transmitted infectious pathogens is important in the overall control of sexually transmitted diseases. Many laboratories now utilize test kits for the identification of N. gonorrhoeae, and some of these test kits (e.g. API NH, RapID NH, and Gonochek II) rely on the detection of preformed enzymes, such as gamma glutamyl transferase for identification of N. meningitidis, and PIP for identification of N. gonorrhoeae. When PIP-negative N. gonorrhoeae isolates are tested by such kits, a false-negative result may occur, with the potential for misdiagnosis.

Our current survey of 200 N. gonorrhoeae isolates from Ontario, Canada did not identify any PIP-negative strains, suggesting that if PIP-negative N. gonorrhoeae are present, they occur at a low prevalence rate of < 0.5%.

An increase in the detection of PIP-negative N. gonorrhoeae was first reported in 2001 in England(11). In 1991, only two (0.5%) of 398 N. gonorrhoeae cultures examined from England were PIP deficient; but in 2001, 17 PIP-negative N. gonorrhoeae isolates were encountered over a 5 week period and by 2004 an overall prevalence of PIP-negative N. gonorrhoeae isolates reported in England and Wales was 4.33%(5,11,12). Similar increases in PIP-negative gonococci were also detected in other countries with a prevalence ranging from 2% in New Zealand between 2002 to 2004, to 6.9% in northern Spain between 2003 to 2006, and an overall of 12.8% in Australia between the period of 2002 to 2005(7-9).

Laboratory characterization of the PIP-negative N. gonorrhoeae from different countries suggested many of the isolates were related according to a number of phenotypic and genetic markers, and hence the isolation of PIP-negative N. gonorrhoeae in different countries might be related to global spread of one or a few strains^(6,10). Epidemiological studies also suggested a possible association of infection by these strains in men who have sex with men (MSM)^(5,10). Also many of the PIP-negative N. gonorrhoeae isolates studied in these countries were susceptible to most of the commonly used antibiotics^(6,10).

The failure to detect any PIP-negative strains in this study may be related to the fact that PIP-negative N. gonorrhoeae isolates could have been missed at the primary isolation laboratory and therefore, only typical PIP-positive strains were submitted to the Ontario public health reference laboratory. Further studies at primary isolation laboratories as well as testing of isolates recovered from other provinces and MSM communities are required to rule out their presence in Canada. PIP-negative N. gonorrhoeae control strains are now available from the NML to any laboratories performing primary isolation and identification of gonococci to ensure their testing method(s) can identify PIP-negative strains.

Conclusion

A survey of 200 N. gonorrhoeae isolates, including 100 showing susceptibility towards antibiotics commonly used for treatment of gonorrhoea failed to detect any PIP-deficient strain. In order to detect any potential importation/emergence of PIP-negative gonococci into Canada, laboratories should exercise vigilance in their identification of N. gonorrhoeae. The current recommendation for definitive identification of N. gonorrhoeae requires a combination of at least two independent methods of testing, such as biochemical methods of either carbohydrate reaction test or enzymatic assays, and other assays such as immunological or nucleic acid-based methods⁽¹³⁾.

Acknowledgement

We thank Averil Griffith for her technical assistance.

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