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Contents lists available at ScienceDirect

Journal of Infection and Public Health

journal homepage: http://www.elsevier.com/locate/jiph

Molecular detection of leptospirosis and 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 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 , ,

Pathogenic Leptospira spp. and Burkholderia pseudomallei are , cough, diarrhoea, vomiting and [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 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, 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 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 . 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). 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 25 (5–17) mmol/l B surface antigen Non-reactive Roche

Aspartate aminotransferase 173 (8–48) IU/l Hepatitis C 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 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, 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 .

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 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 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 , , 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.

FX, Becton, Dickson and Company, USA). After 48 h, B. pseudomallei

Normal or slightly increased leukocyte numbers were observed in

was identified by Vitek 2 System GN panel (bioMerieux, Inc. USA).

the majority of leptospirosis cases and the severity of the disease

It was susceptible to ceftazidime, amoxicillin-clavulanate, doxy-

may be predicted by a neutrophilic response [18]. More over the

cycline, imipenem and sulfamethaxazole-trimepthoprim by MIC

neutrophilic response is overwhelmed by intrinsic resistance of B.

method based on the Clinical and Laboratory Standards Institute

pseudomallei to and polymorphonuclear (PMN) neu-

(CLSI) M45-A2 guideline [8]. The ImmuneMed Leptospira Rapid

trophil killing. The underlying diabetes mellitus in this patient also

immunochromatographic test for leptospira IgM was positive but

®

lead to impaired PMN cells migration and apoptosis [19]. This is a

Panbio Leptospira IgM ELISA was not reactive. Chest radiograph

common manifestation of an overwhelming , whereby there

revealed a clear lung fields with a slight cardiomegaly. Sinus tachy-

is a possibility of consumptions of leucocytes and platelets in the

cardia was noted on electrocardiogram (ECG) monitoring. Other

form of disseminated intravascular .

serology and microbiology investigations are listed in Table 2. Based

on clinical and laboratory findings, the final diagnosis was sep-

sis secondary to multiple splenic microabscess, due to melioidosis Conclusion

with concurrent leptospirosis.

During her second day of admission, patient developed respi- Patient succumbed to the infection because of multiple factors

ratory distress went into septic shock, intubated and ventilated in including underlying poorly controlled diabetes mellitus, delay in

ICU. Despite the aggressive intervention and escalation of antibi- the diagnosis of melioidosis & initiation of appropriate antibiotics

otic to intravenous meropenem 2 g 8-hourly, the patient’s condition during her first visits to the hospital and the bacteremic melioi-

continued to deteriorate then succumbed to death. dosis. This report emphasizes the possibilities of co-infection of

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

M.R. Mohd Ali et al. / Journal of Infection and Public Health xxx (2017) xxx–xxx 3

Leptospira and B. pseudomallei, especially in endemic tropical [5] Wang-Ngarm S, Chareonsudjai S, Chareonsudjai P. Physicochemical factors

affecting the growth of Burkholderia pseudomallei in soil microcosm. Am J Trop

regions and highlights the promising potential of molecular

Med Hyg 2014;90:480–5, http://dx.doi.org/10.4269/ajtmh.13-0446.

approach for a rapid, sensitive and specific detection of the causing

[6] Lim C, Peacock SJ, Limmathurotsakul D. Association between activities related

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in South Korea: a case series with a literature review. Osong Public Heal Res

Perspect 2015;6:363–8, http://dx.doi.org/10.1016/j.phrp.2015.10.014.

This work was funded by Research University Grant

[8] CLSI. Methods for Antimicrobial Dilution and Disk Susceptibility Testing of

(1001/PPSP/812144) and Long Term Research Grant Scheme Infrequently Isolated or Fastidious Bacteria. 3rd ed. CLSI guideline M45. Wayne,

PA: Clinical and Laboratory Standards Institute; 2015.

(203/PPSP/6770004). The first author is financially supported

[9] Lu P-L, Tseng S-H. Fatal septicemic melioidosis in a young mil-

by Public Service Department (JPA) Malaysia through the Yang

itary person possibly co-infected with and

di-Pertuan Agong Scholarship programme and the Ministry of . Kaohsiung J Med Sci 2005;21:173–8, http://dx.doi.org/10.1016/S1607-551X(09)70297-9.

Health Malaysia.

[10] Hin HS, Ramalingam R, Chunn KY, Ahmad N, Ab Rahman J, Mohamed MS. Fatal

co-infection–melioidosis ad leptospirosis. Am J Trop Med Hyg 2012;87:737–40,

Competing interests http://dx.doi.org/10.4269/ajtmh.2012.12-0165.

[11] Sapian M, Khairi MT, How SH, Rajalingam R, Sahhir K, Norazah A, et al. Outbreak

of melioidosis and leptospirosis co-infection following a rescue operation. Med

The authors declare that there is no conflict of interest.

J Malays 2012;67:293–7.

[12] Kelser EA. Melioidosis: a greater threat than previously suspected? Microbes

Acknowledgments Infect 2016;18:661–8, http://dx.doi.org/10.1016/j.micinf.2016.07.001.

[13] Novak RT, Glass MB, Gee JE, Gal D, Mayo MJ, Currie BJ, et al. Devel-

opment and evaluation of a real-time PCR assay targeting the type III

Special thanks to the staffs at the Department of Medical Micro-

secretion system of Burkholderia pseudomallei. J Clin Microbiol 2006;44:85–90,

biology & Parasitology and Department of Emergency, Universiti http://dx.doi.org/10.1128/JCM.44.1.85.

Sains Malaysia for the direct and indirectly contribution. The [14] Bourhy P, Bremont S, Zinini F, Giry C, Picardeau M. Comparison of real-time

authors would like to thank the Director of Health Malaysia for PCR assays for detection of pathogenic Leptospira spp. in blood and identi-

fication of variations in target sequences. J Clin Microbiol 2011;49:2154–60,

permission to publish this paper. The project was scientifically sup-

http://dx.doi.org/10.1128/JCM.02452-10.

ported by King Saud University, Deanship of Scientific Research, The

[15] Waggoner JJ, Balassiano I, Mohamed-Hadley A, Vital-Brazil JM, Sahoo MK,

Research Chairs and The Research Chair of Health Informatics and Pinsky BA. Reverse-transcriptase PCR detection of Leptospira: absence of

Promotion. agreement with single-specimen microscopic testing. PLoS One

2015;10:e0132988, http://dx.doi.org/10.1371/journal.pone.0132988.

[16] Ravara A, Cota M. Evaluation of MAT, IgM ELISA and PCR methods for

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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