G Model JIPH-716; No. of Pages 3 ARTICLE IN PRESS Journal of Infection and Public Health xxx (2017) xxx–xxx Contents lists available at ScienceDirect Journal of Infection and Public Health journal homepage: http://www.elsevier.com/locate/jiph Molecular detection of leptospirosis and melioidosis co-infection: A case report a,b a Mohammad Ridhuan Mohd Ali , Amira Wahida Mohamad Safiee , a c d a Padmaloseni Thangarajah , Mohd Hashairi Fauzi , Alwi Muhd Besari , Nabilah Ismail , a,∗ Chan Yean Yean a Department of Medical Microbiology & Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia b Secretariat National Institutes of Health (NIH), Ministry of Health Malaysia, c/o Institut Pengurusan Kesihatan, Jalan Rumah Sakit Bangsar, 59000 Kuala Lumpur, Malaysia c Department of Emergency Medicine, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia d Department of Medicine, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia a r t i c l e i n f o a b s t r a c t Article history: Leptospirosis and melioidosis are important tropical infections caused by Leptospira and Burkholdheria Received 10 November 2016 pseudomallei, respectively. As both infections share similar clinical manifestations yet require differ- Received in revised form 5 February 2017 ent managements, complementary laboratory tests are crucial for the diagnosis. We describe a case of Accepted 22 February 2017 Leptospira and B. pseudomallei co-infection in a diabetic 40-year-old woman with history of visit to a freshwater camping site in northern Malaysia. To our knowledge, this is the first case of such double- Keywords: infection, simultaneously demonstrated by molecular approach. This case highlights the possibility of Leptospirosis leptospirosis and melioidosis co-infections and their underlying challenges in the rapid and accurate Melioidosis Co-infection detection of the etiologic microorganism. PCR © 2017 The Authors. Published by Elsevier Limited. This is an open access article under the CC Febrile BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Introduction Clinically, leptospirosis and melioidosis portray an almost sim- ilar wide spectrum of clinical features including fever, headache, Pathogenic Leptospira spp. and Burkholderia pseudomallei are myalgia, cough, diarrhoea, vomiting and jaundice [4,7]. As a con- emerging tropical diseases that cause leptospirosis and melioido- sequence, physicians rely significantly on the laboratory tests for sis, respectively. Leptospira infecting more than one million people a confirmatory diagnosis. A misdiagnosis may happen when only and cause 58,900 deaths annually [1]. Meanwhile, melioidosis inci- either of the tests is considered or available. dence is approximately one tenth of the leptospirosis’, but, as high as 89,000 patients succumbed to the disease [2]. Case report Rodents and ruminants are known leptospiral hosts and are presumed to excrete the organism to the environment. Similarly, A 40-year-old female school teacher, who has type 2 diabetes seroprevalence of B. pseudomallei been reported in several animals, mellitus for the past 3 years, poor compliance to medication, was including livestock, but contaminated soil remains as one of the referred to our centre with prolonged high grade fever for 2 weeks, main source of melioidosis [3]. Both organism are usually found in chills and rigor, headache, arthralgia and myalgia, poor appetite, moist environment with pH close to 7 [4,5]. Infections may occur associated with epigastric pain for 2 days duration. There was no by direct inhalation, ingestion, inoculation of the organism or indi- history of vomiting, diarrhoea, cough or sputum, rash, seizure, tea rectly from contaminated environment [4,6]. coloured urine and contact with tuberculosis. Even though patient resided in dengue endemic area, there was no fogging activity nearby her house at that time. She visited emergency department ∗ Corresponding author. of another tertiary hospital during her first week of illness, but was E-mail addresses: [email protected] (M.R. Mohd Ali), discharged with oral antibiotics. There were histories of visiting [email protected] (A.W. Mohamad Safiee), [email protected] nearby waterfall one month prior to admission and contact with (P. Thangarajah), [email protected] (M.H. Fauzi), [email protected] (A. Muhd Besari), [email protected] (N. Ismail), [email protected] (C. Yean Yean). rats and mice at home. http://dx.doi.org/10.1016/j.jiph.2017.02.009 1876-0341/© 2017 The Authors. Published by Elsevier Limited. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc- nd/4.0/). Please cite this article in press as: Mohd Ali MR, et al. Molecular detection of leptospirosis and melioidosis co-infection: A case report. J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2017.02.009 G Model JIPH-716; No. of Pages 3 ARTICLE IN PRESS 2 M.R. Mohd Ali et al. / Journal of Infection and Public Health xxx (2017) xxx–xxx Table 1 Table 2 Biochemistry and hematology investigation results. Serology and microbiology investigation results. Laboratory test Result Normal value Laboratory test Result Manufacturer Haemoglobin 9.1 (9.81–13.85) g/dl Dengue NS1 ELISA Non-reactive Panbio 9 WBC 8.6 (3.4–10.1) × 10 /l Dengue IgM Capture ELISA Non-reactive Panbio 9 Platelet 127 (158–410) × 10 /l Dengue IgG Capture ELISA Non-reactive Panbio Serum bilirubin 25 (5–17) mmol/l Hepatitis B surface antigen Non-reactive Roche Aspartate aminotransferase 173 (8–48) IU/l Hepatitis C antibody Non-reactive Roche Alanine aminotransferase 73 (7–55) IU/l HIV antibody Non-reactive Roche ALP 548 (45–115) IU/l Peripheral blood smear for malaria parasitesNo malaria parasite seenIn-house Urine culture No growth In-house Discussion Upon admission to our centre she was conscious, lethargy and Based on our extensive literature review, there are only looked dehydrated. Her blood pressure was 120/88 mmHg, pulse ◦ two reported cases of leptospirosis and melioidosis co-infections rate of 120 beats per minute and temperature of 39.5 C. The oxygen [9–11]. While this is the third report of such double infections, this saturation was 100% under room air. Capillary blood sugar level was case is unique because the presences of both etiologic agents (Lep- markedly elevated on glucometer. Physical examination revealed tospira spp. and B. pseudomallei) were demonstrated by Taqman tenderness at right hypochondriac region radiating to left iliac fossa real-time PCR, simultaneously. Results were made available to the and hepatosplenomegaly. Bedside ultrasonography revealed hep- emergency department on the same day; hence allowed immedi- atosplenomegaly and multiple splenic microabscesses with normal ate diagnosis and appropriate management. It is way faster than the kidney and no gall bladder stone. current B. pseudomallei culture and leptospiral MAT gold standard The initial investigations revealed low haemoglobin and platelet which may takes up to 4 and 14 days respectively [4,12]. This case count, normal total white blood cell count with predominant neu- highlights the potential lies within molecular platform for rapid trophil. Erythrocyte sedimentation rate and C-reactive protein was detection of leptospirosis and melioidosis aetilogic agents. In addi- elevated, serum total bilirubin was 25 mmol/L, liver profile showed tion, PCR has been shown to be superior with specificity of more elevated transaminases, however renal function was normal. Other 3 than 90% and limit of detection (LOD) of 5 × 10 genome equiva- blood investigations of this patient are shown in Table 1. The dif- lents per ml, hence should be applied in larger scale [13,14]. ferential diagnoses at this stage included leptospirosis, melioidosis This case report also showed that the bacterial genomic DNA was complicated with splenic microabscess and dengue fever. available in the patient’s blood on day 17 of illness. This observa- Molecular investigations for Leptospira and B. pseudomallei DNA tion is possible as a previous study had shown that leptospiral DNA were requested immediately. Following DNA extraction using were detected as late as day 22 of illness [15]. In addition, more than Nucleospin Blood Mini kit, 8 ␮l sample was added to reaction TM one third of MAT-positive Leptospirosis had detectable leptospiral mixture, consisted of 1X Biorad SsoAdvanced Universal Probes DNA, even though there is limited consistency between MAT and Supermix, 200 ␮M oligonucleotides, 100 ␮M probes and water, molecular test [16]. On the other hand, B. pseudomallei have been adjusted to a final volume of 20 ␮l. Reactions were subjected to Bio- ◦ known to remain in the blood stream up to 50 days, which even- rad CFX96 thermalcycler uder following conditions; 95 C for 5 min, ◦ ◦ tually may lead to dual positivity in both serological and molecular followed by 42 cycles of 95 C for 30 s, 61.3 C for 30 s. The tested investigation [7]. blood sample was positive for leptospiral DNA and B. pseudomallei DNA. The patient’s initial presentations failed to raise suspicion of the possibility of melioidosis, despite several risk factors such as Empirical antibiotic therapy and supportive management with uncontrolled diabetes and exposure to soil. This has led to inap- intravenous fluids were started. Based on initial assessment patient propriate oral antibiotic therapy, because B. pseudomallei is known was treated with intravenous ceftriaxone 1 g 12-hourly for lep- to be intrinsically resistant to commonly used oral antibiotics such tospirosis. Following positive PCR result and preliminary Gram as penicillin, amoxicillin, aminoglycosides and all of the first- and stain from blood culture vial, antibiotic was then changed to intra- second-generation cephalosporins [17]. venous ceftazidime 2 g 8-hourly. Gram negative bacilli from blood Upon admission, full blood count showed bicytopenias with culture vial were detected post 6-hours incubation (BD BACTEC white cell count in the normal range and predominant neutrophil.