Increased Incidence of Nitroimidazole-Refractory Giardiasis at the Hospital for Tropical Diseases, London: 2008–2013

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ORIGINAL ARTICLE PARASITOLOGY

Increased incidence of nitroimidazole-refractory giardiasis at the Hospital for Tropical Diseases, London: 2008–2013

L. E. B. Nabarro, R. A. Lever, M. Armstrong and P. L. Chiodini The Hospital for Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK

Abstract

Giardia intestinalis is the commonest gastrointestinal protozoal pathogen worldwide, and causes acute and chronic diarrhoea with malabsorption. First-line treatment is with a nitroimidazole, with a reported efficacy rate of 89%. Failure of treatment can occur in patients with hypogammaglobulinaemia or human immunodeficiency virus (HIV), or be due to nitroimidazole-resistant organisms. There is little evidence to guide the clinical management of nitroimidazole-refractory disease. We performed a retrospective audit of nitroimidazole-refractory giardiasis in returned travellers at the Hospital for Tropical Diseases, London between 2011 and 2013. Seventy- three patients with microscopy-proven or PCR-proven giardiasis in whom nitroimidazole treatment had failed were identified, and their management was investigated. In 2008, nitroimidazole treatment failed in 15.1% of patients. This increased to 20.6% in 2011 and to 40.2% in 2013. Patient demographics remained stable during this period, as did routes of referral. Of patients with giardiasis, 39.0% had travelled to India; this rose to 69.9% in patients with nitroimidazole-refractory disease. Of the patients with refractory disease, 44.6% had HIV serological investigations performed and 36.5% had immunoglobulin levels determined. Patients with refractory disease were treated with various agents, including albendazole, nitazoxanide, and mepacrine, alone or in combination. All 20 patients who received a mepacrine-containing regimen were cured. This data shows a worrying increase in refractory disease, predominantly in travellers from India, which is likely to represent increasing nitroimidazole resistance. Improved tools for the diagnosis of resistant G. intestinalis are urgently needed to establish the true prevalence of nitroimidazole-resistant giardiasis, together with clinical trials to establish the most effective second-line agent for empirical treatment regimens. Clinical Microbiology and Infection © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Keywords: Albendazole, Giardia intestinalis, giardiasis, India, mepacrine, nitroimidazole, refractory, resistance, travellers Original Submission: 14 January 2015; Revised Submission: 12 April 2015; Accepted: 27 April 2015 Editor: E. Bottieau Article published online: 12 May 2015

travellers is acute or chronic diarrhoea, which may be Corresponding author: L. E. B. Nabarro, The Hospital for Tropical accompanied by flatulence and weight loss. Although giardiasis Diseases, Mortimer Market, Capper Street, London, WC1E 6JB, UK E-mail: [email protected] is rarely life-threatening, chronic tiredness and gastrointestinal L. E. B. Nabarro and R. A. Lever are joint first authors. symptoms may continue following eradication [1]. First-line treatment for giardiasis is using a nitroimidazole. Metronidazole, 500 mg three times daily for one week, is fi Introduction commonly used. The reported ef cacy is 89% [2]. Single-dose tinidazole has similar efficacy but fewer side effects, and thus better compliance [2]. A study of 170 patients in Madrid be- Giardia intestinalis is the commonest gastrointestinal protozoal tween 1989 and 2004 found 5.8% failed a mean of 3 courses of pathogen worldwide. It causes a spectrum of clinical disease, metronidazole [3]. Between 2007 and 2009, a study of travel- ranging from asymptomatic carriage to acute or chronic diar- lers returning to Barcelona found that nitroimidazole treatment rhoea with malabsorption. The commonest presentation in failed in 22% [4].

Clin Microbiol Infect 2015; 21: 791–796 Clinical Microbiology and Infection © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rightsreserved http://dx.doi.org/10.1016/j.cmi.2015.04.019 792 Clinical Microbiology and Infection, Volume 21 Number 8, August 2015 CMI

Risk factors for treatment failure include IgA deficiency [5], 2013 at the HTD. Patients with parasitologically proven human immunodeficiency virus (HIV) [6], and coeliac disease. giardiasis were identified from parasitology laboratory re- Patients may become re-infected from a contaminated water cords of positive stool microscopy or PCR results. In 2008 supply, an animal reservoir, or household or sexual contact. and 2011, stool microscopy for cysts of G. intestinalis was G. intestinalis can become resistant to nitroimidazole treatment the only available method for diagnosis. In 2012, a multiplex and other antiprotozoal drugs [7]. PCR, performed on stools, was introduced for the diagnosis The second-line treatment for patients in whom nitro- of G. intestinalis, Cryptosporidium and Entamoeba histolytica imidazole treatment fails is unclear. Most drug efficacy studies infection. Thus, for 2012 and 2013, both stool microscopy have been conducted in treatment-naïve patients, and the re- and PCR were performed on stool specimens from patients sults have been extrapolated to patients in whom nitro- with a queried diagnosis of giardiasis. Patients were imidazole treatment has failed. A single randomized trial excluded if they had a previous diagnosis of giardiasis at the investigated 20 patients in whom metronidazole treatment had HTD, or if they did not receive treatment for giardiasis at failed; of ten patients treated with albendazole, only two were the HTD. cured, in comparison with nine patients cured when albenda- Cases were deemed to be nitroimidazole refractory if they zole was combined with metronidazole [8]. Mørch evaluated a fulfilled the following case definition: (a) patients referred to the treatment ladder for refractory giardiasis during a waterborne HTD with previously documented positive stool samples for outbreak in Norway [9]. Of 38 patients in whom metronidazole G. intestinalis, previous nitroimidazole treatment, and a positive treatment failed, 79% (30/38) were cured with 7 days of stool sample at the HTD at least 2 weeks following treatment; combination metronidazole–albendazole. A further three of six or (b) patients reviewed at the HTD with a positive stool patients were cured with 7 days of paromomycin. The sample for G. intestinalis at least 2 weeks after documented remaining three were cured with combination quinacrine– nitroimidazole treatment. metronidazole for 2–3 weeks. All patients experienced Once refractory cases had been identified, further data were quinacrine-related side effects [9]. collected from laboratory records, electronic letters, and case The Hospital for Tropical Diseases, London (HTD) is a notes. These included travel history and a complete history of tertiary referral centre for tropical disease, and is part of antiprotozoal treatment. If performed, immunoglobulin levels, University College London Hospitals. An emergency self- HIV status and coeliac status were recorded, together with re- referral clinic is run daily for travellers returning from the infection history. tropics with fever or diarrhoea. There are four general infec- Data were analysed with MS Excel 2010. Research approval tious disease clinics a week, in which referrals are received was not required, as this was an audit intended to document from general practitioners and hospitals around the UK. Diag- current practice and improve future outcomes. nostic parasitological investigation is undertaken in the HTD Department of Clinical Parasitology, which is also the Public Results Health England National Parasitology Reference Laboratory. Tinidazole is used as first-line treatment for giardiasis in our patients. Available second-line treatments include metronida- Between 2011 and 2013, 259 patients were diagnosed with zole, albendazole, paromomycin, nitazoxanide, and mepacrine giardiasis; 179 were seen in the emergency clinic, and 39 were (quinacrine). These are given at the discretion of individual referred from general practitioners. The source of referral was clinicians. unclear for 41 cases. In 2013, a number of clinicians at the HTD noticed an in- In 2008, nitroimidazole therapy failed in eight (15.1%) pa- crease in the number of patients with nitroimidazole-refractory tients diagnosed with giardiasis. This increased to 15 (20.6%) in giardiasis. This audit aimed to establish whether the frequency 2011, 23 (23.2%) in 2012, and 35 (40.2%) in 2013 (n = 259 of nitroimidazole-refractory giardiasis was increasing in our cases) (Fig. 1). cohort, and the ways in which we were managing these In 2011, all patients diagnosed with giardiasis, either sensitive patients. or refractory to nitroimidazoles, were diagnosed by microscopy. In 2012, 16 (28.6%) patients diagnosed with giardiasis by microscopy had nitroimidazole-refractory giardiasis, as Materials and methods compared with 14 (22.2%) diagnosed by PCR. In 2013, 21 (38.9%) patients diagnosed by microscopy had refractory giar- We performed a retrospective audit of patients diagnosed diasis, as compared with 26 (47.3%) diagnosed by PCR with giardiasis in 2008 and between the years 2011 and (Table 1).

Clinical Microbiology and Infection © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved, CMI, 21,791–796 CMI Nabarro et al. Increased incidence of nitroimidazole-refractory giardiasis 793

FIG. 1. Increasing rates of nitroimidazole-refractory giardiasis between 2008 and 2013. Only one sample per patient is included in this data.

Of the patients with giardiasis, 39 (15.1%) were referred in 2011, 31 (42.5%) patients acquired G. intestinalis in India, as from general practitioners. This remained stable between 2011 compared with 37 (37.4%) in 2012 and 33 (37.9%) in 2013. In and 2013 (Table 2). The proportion of refractory patients was contrast, of 73 nitroimidazole-refractory cases, 51 (69.9%) had consistently higher among external referrals (40.9%) than acquired G. intestinalis in India, five (6.8%) elsewhere in Asia, and among emergency clinic referrals (33.6%). nine (12.3%) in Africa (Table 3). Of the patients with nitroimidazole-refractory disease, 51% Treatment strategies varied greatly between clinicians. were female, with a mean age of 37 years. This remained Common regimens included repeat tinidazole (typically 2 g as a consistent between 2011 and 2013. Forty-four (60.2%) single dose), and tinidazole–albendazole combination treatment received tinidazole as first-line treatment and 29 (39.7%) and nitazoxanide (at varying doses). Mepacrine, alone or with received metronidazole. Twenty-seven (36.5%) patients had another agent, was used at 100 mg three times daily for a serum immunoglobulin (IgA, IgG, and IgM) levels determined, median of 7 days (range 5–21 days). Table 4 shows the number and five (6.8%) of these had abnormal levels. Thirty-three of clinical encounters in which a particular regimen was used, (44.6%) patients had an HIV test performed; two (2.7%) were and whether the patient required further treatment following positive, but both patients were already known to have HIV. this. One hundred per cent of those receiving mepacrine- Thirty (40.5%) patients had coeliac serological investigations containing regimens did not require further treatment. performed, but no new diagnoses were established. Only 15 Because of varying drug doses and lengths of treatment, we (20.3%) patients were asked about risk factors for re-infection. were unable to analyse these data further. In total, six different Between 2011 and 2013, 101 (39.0%) patients acquired G. intestinalis in India, 42 (16.4%) elsewhere in Asia, and 47 TABLE 2. Sources of referral for patients with refractory (18.1%) in Africa. Travel history remained stable across years; giardiasis in 2011–2013, with numbers and percentages of total and refractory cases, respectively TABLE 1. Refractory cases by diagnostic modality; the Sources 2011 2012 2013 increase in refractory disease is not an artefact of the introduction of Giardia intestinalis PCR in 2011 Refractory cases, no. (%) General practitioner 5 (33.3) 13 (56.5) 8 (22.9) Emergency clinic 10 (66.7) 10 (43.5) 25 (71.4) 2011 2012 2013 Other/unknown 0 (0) 0 (0) 2 (5.7) All cases, no. (%) Microscopy alone 15 9 9 General practitioner 9 (12.3) 19 (19.2) 11 (12.5) PCR alone 0 7 13 Emergency clinic 50 (68.5) 65 (65.7) 64 (73.6) Microscopy and PCR 0 7 13 Other/unknown 14 (19.2) 15 (15.2) 12 (13.8)

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TABLE 3. Travel history of patients presenting with giardiasis owing to the lack of an appropriate reference standard, it is between 2011 and 2013 in comparison with those presenting certainly more sensitive than stool microscopy. As PCR detects with nitroimidazole-refractory disease over the same period DNA of G. intestinalis rather than intact cysts, it may be less fi Total cases Refractory cases speci c. However, stool samples are reportedly PCR negative 7 days post-treatment. In our cohort, the increase in refractory No. % No. % disease was seen in patients diagnosed both by microscopy and India 101 39.0 51 69.9 by PCR, and so is not an artefact of the introduction of PCR. Other Asia 42 16.2 5 6.8 Africa 47 18.1 9 12.3 Similarly, between 2011 and 2013, the source of referral Europe 5 1.9 2 2.7 Central/South America 17 6.6 1 1.4 remained stable. The proportion of patients with refractory No travel 18 6.9 4 5.5 Unknown 27 10.4 1 1.4 giardiasis was higher for external referrals than for emergency Middle East 1 0.4 0 0.0 North America 1 0.4 0 0.0 clinic patients, probably because general practitioners initially Total 259 100 73 100 treat their patients with metronidazole, only referring if the patient remains symptomatic. Thus, we suspect that the increase in nitroimidazole- refractory disease is due to increasing drug resistance. In vitro, drugs were given alone or as combination therapy at a variety nitroimidazole resistance was demonstrated in 1988 by of doses for different periods of time, giving a total of 11 exposing G. intestinalis to intermittent, slowly increasing con- different regimens. centrations of metronidazole [10]. This led to suggestions that combination therapy should be used to avoid the development Discussion of multidrug-resistant strains [11]. Despite this, demonstration of nitroimidazole-resistant G. intestinalis in clinical specimens is unusual [12]. It is hypothesized that nitroimidazole resistance is This study shows a rapid increase in the frequency of associated with impaired mucosal attachment, and thus reduced nitroimidazole-refractory giardiasis in patients reviewed at the parasite fitness [13]. HTD between 2008 and 2013. The reason for this is unclear. There are several proposed mechanisms of nitroimidazole Patient demographics have remained stable since 2008, as have resistance in G. intestinalis. The most thoroughly described hy- the countries in which giardiasis has been acquired. During this pothesis is reduced pyruvate:ferredoxin oxidoreductase activity time, we have continued to use 2 g of tinidazole as a single dose [7]. Other possible resistance mechanisms may confer resis- fi as rst-line treatment, with no change in manufacturer or tance across several antiprotozoal classes; altered expression of administration. genes involved in the stress response may result in both In 2012, a multiplex protozoal PCR was introduced for the nitroimidazole and nitazoxanide resistance [14]. diagnosis of giardiasis. This assay was developed and validated in This increase in the frequency of nitroimidazole-refractory our laboratory. Although the sensitivity of this assay is unclear, giardiasis, which we think represents drug resistance, poses a significant clinical problem. Unlike bacterial diarrhoea, for TABLE 4. Agents used to treat refractory giardiasis between which the pathogen can be cultured and sensitivities obtained 2011 and 2013 within 48 h, G. intestinalis sensitivity testing cannot be performed in most laboratories. The organism is difficult and time- Patients who did Total Final not require further consuming to culture, with a success rate of between 20% and treatment treatment treatment at our Treatment episodes episodes centre (%) 44% [15]. There are no established antimicrobial breakpoints, and it is unclear whether in vivo response correlates with in vitro Tinidazole 39 23 58.9 Metronidazole 11 2 18.2 sensitivity [5]. Although G. intestinalis has a fully sequenced Albendazole 8 2 25 Nitazoxanide 18 9 50 genome [16], and there is some understanding of the mecha- Paromomycin 3 1 33.3 Mepacrine 11 11 100 nisms of nitroimidazole resistance, there are no known mo- Mepacrine and 7 7 100 albendazole lecular markers for predicting drug resistance in clinical Mepacrine and 2 2 100 specimens. tinidazole Tinidazole and 1 1 100 Seventy per cent of patients with refractory giardiasis had paromomycin Tinidazole and 20 12 60 travelled to India. This suggests that there is a significant albendazole Tinidazole and 2150 amount of nitroimidazole-resistant giardiasis in India, although nitazoxanide Unclear 2 2 100 there are no published data on resistance rates. There is evidence of metronidazole resistance in other pathogens that are

commonly treated with nitroimidazoles, including Trichomonas concerning, as giardiasis is associated with long-term conse- vaginalis [17] and Helicobacter pylori [18]. quences, including malnutrition and cognitive retardation [25]. There is significant overprescribing and overuse of antibi- Further work is needed to establish the prevalence of otics in healthcare in India, with a 40% increase in the amount of nitroimidazole-resistant G. intestinalis in India. Simplified antibiotics sold between 2005 and 2009 [19]. Factors driving methods for culture and sensitivity could allow routine testing this include the sale of antibiotics without prescription, and in clinical laboratories, and reduce the empirical treatment of limited access to healthcare and microbiological services [20]. resistant disease. Genetic markers of resistance should be Lack of appropriate knowledge, the desire to meet patient sought to predict drug resistance in clinical isolates. expectations, the fear of clinical failure and economic incentives Until routine sensitivities are available in clinical practice, from drug companies may influence doctors to prescribe an- empirical treatment of refractory disease will continue. Clinical tibiotics inappropriately [21]. trials are needed to establish the most effective, affordable and Fixed-dose combinations containing ciprofloxacin and tini- readily available second-line treatment. An observational, open- dazole are widely used in diarrhoea management. This is irra- label, multicentre study, GiardiaRef, has begun recruiting tional, as diarrhoea is usually caused by either bacteria or through TropNet centres. The aim of this study is to assess the protozoa, but not by both simultaneously [22]. Generic fixed- clinical and parasitological efficacy of combination chloro- dose combinations usually contain 500 mg of ciprofloxacin quine–albendazole vs. quinacrine (mepacrine) in patients in and 600 mg of tinidazole, taken twice daily. This gives an whom nitroimidazole therapy has failed, and to evaluate adequate dose of ciprofloxacin, but falls short of the thera- adverse effects and adherence as secondary outcomes [26]. peutic dose of tinidazole (2 g once daily), potentially driving Chloroquine is not widely used as a treatment for giardiasis. drug resistance. Most data on its efficacy come from small studies in children in There is little evidence to guide the clinical management of Cuba [27], and there is no evidence for its efficacy in combi- nitroimidazole-refractory giardiasis. Once nitroimidazole failure nation therapy or in patients with nitroimidazole-refractory has been parasitologically proven, we suggest investigating the disease. A clinical trial of quinacrine (mepacrine) vs. tinida- patient for risk factors for refractory disease, including immu- zole–albendazole combination therapy may be more relevant noglobulin levels, HIV, and coeliac serology. This was per- and evidence based [8]. formed in only 36–45% of our patients. We also advocate There are a number of limitations to our study. This was a taking a detailed history to establish the risk of re-infection retrospective audit of case notes and electronic records, so from a household or sexual contact, water source, or infec- data quality was dependent on documentation. This did not ted animal. affect our understanding of treatment or tests for the under- Although the failure rate of nitroimidazole therapy in our lying causes of refractory giardiasis. However, we suspect that patient cohort is 40%, we continue to use single-dose tini- more patients were asked about risk factors for re-infection dazole as first-line treatment, because it is well tolerated, than were documented. The absence of this data means that with few contraindications to use. As second-line treatment, we cannot exclude re-infection as a cause of treatment failure in we use 400 mg of albendazole twice daily for 7 days, with 2 our refractory patients. We were unable to draw conclusions g of tinidazole at the beginning and end of treatment. This is about the efficacy of individual drugs or combination therapy, based on Cacopardo’s small trial, which suggested synergism owing to the wide range of doses, courses and combinations between metronidazole and albendazole [8]. In addition, 60% used. Furthermore, we did not routinely test for cure following of patients given this combination in our cohort were cured. treatment, but relied on symptom resolution. Some patients If this fails, we offer 100 mg of mepacrine three times daily may have become asymptomatic carriers following treatment, for 7 days. The patient is counselled on the side effects of resulting in an underestimation of the prevalence of drug mepacrine, notably a 0.4% risk of psychosis [23]. This resistance. In contrast, as giardiasis may be self-limiting, some treatment is not offered to patients with a psychiatric his- patients may have been cured despite, rather than because of, tory. During the period of this audit, 20 patients with treatment. nitroimidazole-refractory giardiasis were treated with mepacrine; all were cured. Conclusion In our cohort of travellers, most of whom are well nourished with no underlying disease and good access to healthcare, a 40% first-line antiprotozoal failure rate is inconvenient but not life- This retrospective audit shows a worrying increase in the fre- threatening. In children in India, where the community preva- quency of nitroimidazole-refractory giardiasis, from 15% in lence can reach 30% [24], nitroimidazole resistance is more 2008 to 40.3% in 2014. We suspect that this is due to increasing

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