Page 1 of 4 Case studies

Prolonged febrile illness due to CTX-M-15 extended-

spectrum β-lactamase-producing Klebsiella pneumoniae
infection in Nigeria
Authors: We report on an 8-year-old patient with septicaemia unresponsive to therapy for five weeks.
Oladipo A. Aboderin1,2
Undetected, extended-spectrum β-lactamase (ESBL) production by the infecting Klebsiella
Olufemi Adefehinti3
Babatunde W. Odetoyin1 strain was regarded as responsible for treatment failure. Intravenously administered imipenem
Amadin A. Olotu2 during the sixth week led to sustained resolution of fever. Resource-limited hospitals can
Iruka N. Okeke4
incur prohibitive costs from ESBL-producer infections because of diagnostic limitations and
Olugbenga O. Adeodu3,5
consequent treatment failure involving prolonged supportive therapy.
Affiliations:
1
Department of Medical
Microbiology and
Parasitology, Obafemi Introduction
Awolowo University, Ile-Ife,
Nigeria The first report of plasmid-encoded β-lactamase capable of hydrolysing the extended-spectrum
cephalosporins was published in 1983.1 Since then, extended-spectrum β-lactamases (ESBLs)
2
Department of Medical have become an increasingly important resistance mechanism among Enterobacteriaceae
Microbiology and
Parasitology, Obafemi worldwide. A 2006 report of the Infectious Diseases Society of America listed ESBL-producing
Awolowo University Teaching Klebsiella pneumoniae and Escherichia coli among drug-resistant microbes for which new therapies
Hospitals Complex, Ile-Ife, are urgently needed.2 Reports show that the ESBL problem is rapidly evolving and increasing in
Nigeria
severity and scope with the discovery of new ESBLs, particularly the CTX-M types, which have
3
Department of Paediatrics, become the most prevalent.3 The β-lactamases are the greatest threat to the usefulness of β-lactam
Obafemi Awolowo University
antibiotics such as the penicillins and cephalosporins. Of all the different types of β-lactamases,
Teaching Hospitals Complex,
Ile-Ife, Nigeria ESBLs currently have the greatest clinical impact in terms of diversity and distribution as well as
the ability to hydrolyse expanded-spectrum third generation cephalosporins. The earliest variants
Department of Biology,
4

Haverford College, Haverford, of ESBLs originated as a result of point mutations in the genes for broad-spectrum β-lactamases
United States whilst newer ones, including the most successful such as CTX-M, arose by acquisition from the
environmental metagenome through horizontal gene transfer.4 Cephalosporins as bactericidal,
5
Department of Paediatrics
and Child Health, Obafemi cell wall-active β-lactam agents were introduced in the 1980s and as a result of effectiveness
Awolowo University, Ile-Ife, against broad-spectrum β-lactamases became standard for treatment of severe conditions such
Nigeria as bloodstream infections, pneumonia and intra-abdominal infections, until ESBLs started
Correspondence to: compromising usefulness in response to overuse and selective pressure. Organisms that produce
Oladipo Aboderin ESBLs are an important reason for therapy failure with cephalosporins and have serious
consequences for infection control. Furthermore, CTX-M ESBL enzymes have been associated
Email:
aaboderi@oauife.edu.ng with coresistance to other agents including trimethoprim-sulphamethoxazole, tetracycline,
gentamicin, tobramycin and ciprofloxacin.5 It is essential that clinical microbiology laboratories
Postal address: rapidly and reliably detect and report ESBL-producing organisms.
Department of Medical
Microbiology and
Parasitology, College of
Health Sciences, Obafemi
Case report
Awolowo University, 220005, An eight-year-old girl presented with a two-week history of fever, abdominal pain, passage
Ile-Ife, Nigeria
of watery stool and recurrent vomiting. There was also a history of frequent micturition
Dates: with occasional dysuria but neither haematuria nor passage of dark-coloured urine. Prior to
Received: 25 June 2011 presentation in the teaching hospital, she had been admitted to a distant private hospital for five
Accepted: 19 Jan. 2012
days, where she was treated with amoxicillin, ciprofloxacin and artesunate (doses and duration
Published: 04 June 2012
of treatment are unknown). She was discharged from that hospital because her state of health
How to cite this article: was not improving significantly and also because of the need for better family support. There
Aboderin OA, Adefehinti was no other history of hospital admission or blood transfusion and no history suggestive of
O, Odetoyin BW, Olotu
AA, Okeke IN, Adeodu OO. haemoglobinopathy. Immunisation and nutritional history were essentially normal.
Prolonged febrile illness
due to CTX-M-15 extended- General physical examination revealed a conscious but ill-looking, somewhat pale, febrile
spectrum β-lactamase-
producing Klebsiella (temperature 38.5 °C) girl. She was moderately dehydrated and jaundiced. Her weight was 22 kg
pneumoniae infection in (86% of expected weight for age). There was no facial or pedal oedema. The respiratory rate was
Nigeria. Afr J Lab Med.
2012;1(1), Art. #16, 4 pages. Note: This report was presented as a poster (abstract C-119) at the 111th General Meeting, American Society for Microbiology, May
http://dx.doi.org/10.4102/ 21–24, New Orleans, Louisiana, United States.
ajlm.v1i1.16 Copyright: © 2012. The Authors. Licensee: AOSIS OpenJournals. This work is licensed under the Creative Commons Attribution License.

http://www.ajlmonline.org doi:10.4102/ajlm.v1i1.16
 Page 2 of 4 Case studies

32 cycles per min and breathing was regular; pulse regular The Klebsiella sp. isolate was identified as Klebsiella pneumoniae
at 120 beats per min and with good volume. Her blood subspecies pneumoniae using the API 20E identification strips
pressure was 90/50 mmHg. The significant systemic findings for Enterobacteriaceae (bioMérieux, Marcy-l’Étoile, France).
on examination at admission were severe suprapubic Presumptive ESBL phenotypic testing and confirmation
tenderness, moderate hepatosplenomegaly (firm, not tender) in the organism was performed by disc diffusion tests on
and negative renal angle tenderness. All other systems were Mueller Hinton agar by employing ceftazidime (30  µg)
normal. and cefpodoxime (10  µg) alone and in combination with
clavulanic acid as ceftazidime-clavulanic acid (30/10 µg) and
Conventional blood cultures were done at five different times cefpodoxime-clavulanic acid (10/1 µg) respectively. Results
after admission. There was a growth of Klebsiella sp. on three were interpreted using the Clinical and Laboratory Standards
occassions. Once, the isolate was sensitive to gentamicin and Institute (CLSI) criteria for disc diffusion.6 Antimicrobial
ceftriaxone but resistant to all available antibiotics tested susceptibility testing for the organism was carried out by
on the other two occasions. Urine and stool cultures did the disc diffusion technique according to the guidelines
not yield growth of any pathogens. Screening for human and recommendations of CLSI.6 The isolate was resistant
immunodeficiency virus (HIV), hepatitis C virus (HCV) and to streptomycin, gentamicin, chloramphenicol, tetracycline,
hepatitis B surface antigen (HBsAg) was negative. nalidixic acid, ciprofloxacin, ampicillin, trimethoprim,
sulphamethoxazole, ceftriaxone, cefepime and amoxicillin-
Full blood counts showed a haematocrit ranged between 14% clavulanic acid, but susceptible to imipenem.
and 30%, white cell counts of 8 x 109/L – 9.2 x 109/L and an
essentially normal platelet count (260 x 109/L). The erythrocyte Genomic DNA was extracted from the isolate using the
sedimentation rate was 80  mm/hr (Westergreen method) Wizard genomic extraction kit (Promega) according to the
and the haemoglobin phenotype (by electrophoresis) was manufacturer’s directions and used as template for PCR
AS. Serum biochemistry parameters were all normal except reactions targeting resistance elements and genes. Platinum
for conjugated hyperbilirubinaemia. Repeated abdominal PCR Supermix (Invitrogen) was used for all reactions,
ultrasonography showed findings that are consistent with and PCR cycle conditions were as recorded in the original
hepatosplenomegaly in a septicaemic patient. articles describing the primers (Table 2). We employed
oligonucleotides that prime the conserved ends of the
Whilst in hospital and when fever was uncontrolled cassette regions of class 1 and 2 integrons respectively to
and persistent, the patient was given fresh whole blood screen for these elements (Table 2).7,8 As shown in Figure 1,
transfusions thrice and exchange blood transfusions twice, we were able to determine that the strain harboured a class 1,
amongst other forms of treatment. but not a class 2 integron. Sequencing of the 1.6 kb amplified
class 1 cassette region revealed that it was identical to the
Fever remained persistent for five weeks following cassette region of plasmid pIP1206 (Genbank Accession
admission, despite different courses of antibiotics involving number NC_010558), containing two integrated cassettes:
ciprofloxacin, gentamicin, ceftazidime, ceftriaxone and a dfrA17 cassette encoding resistance to trimethoprim, and
amoxicillin-clavulanic acid (Table 1). It was only at this point an aadA4 aminoglycoside resistance cassette.9 Since the
that the possibility of infection with an ESBL-producing integron did not contain an ESBL cassette, we screened
organism was considered. ESBL-producers are not routinely the isolate for blaCTX-M type genes, employing primers that
sought in the diagnostic laboratory. During the sixth week, amplify an internal fragment from multiple blaCTX-M alleles
the Klebsiella sp. isolate from the patient was tested and (Table 2)10. The resulting 550 bp product shown in Figure 2
confirmed to be producing an ESBL. Immediately following was sequenced and found to be identical to the corresponding
this test result, treatment was commenced with imipenem (not region of blaCTX-M-15.
routinely available in the hospital) and there was dramatic
resolution of fever. The patient remained free of fever for The cost of repeated investigations (Table 3) was
one week after receiving imipenem and was subsequently N14 000.00 ($90.92), which is more than a tenfold increase
discharged. Two weeks later, when she reported for follow- on projected diagnostic expenses, had a diagnosis estimate
up, she was still fever-free and healthy. been made immediately on admission. Antibiotics and

TABLE 1: Treatment interventions.

S/No. Period Treatment Cost
NGN USD
1 First week I/V ciprofloxacin & I/M gentamicin 3780.00 24.55
2 Second week I/V ceftazidime & I/M gentamicin 10 920.00 70.91
3 Third/Fourth week I/V ceftriaxone & I/M gentamicin 8500.00 55.19
4 Fifth week I/V amoxicillin/clavulanate & I/M gentamicin 6800.00 44.16
5 Sixth week I/V imipenem/cilastatin 50 400.00 327.27
6 - Blood transfusions 9500.00 61.69
Total cost 89900.00 583.77
S/No., Serial number; NGN, Nigerian Naira; USD, United States Dollar; I/V, intravenous; I/M, intramuscular.

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 Page 3 of 4 Case studies

TABLE 2: Oligonucleotides for PCR reactions.

Target gene Primers Amplicon size Reference
Name Sequence
Class 1 integron Lev5’CS 5’-GGC ATC CAA GCA GCA AG-3’ Varies with cassette content (0.7 Kb for aadA in 7 (Lévesque et al.)
cassette region control strain 042)
Lev3’CS 5’ AAG CAG ACT TGA CCT GA-3’
Class 2 integron hep74 5′- CGG GAT CCC GGA CGG CAT GCA CGA Varies with cassette content (2.2 Kb for dfrA1-sat1- 8 (White et al.)
cassette region TTT GTA- 3′ aadA1 in control strain 17-2)
hep51 5′-GAT GCC ATC GCA AGT ACG AG-3′
CTX-M genes CTX-MA 5′-CGC TTT G CG ATG TGC AG-3′ 0.55 Kb 10 (Bonnet et al.)
  CTX-MB 5′-ACC GCG ATA TCG TTG GT-3′    
Note: Please see the full reference list of the article, Aboderin AO, Adefehinti O, Odetoyin BW, Olotu AA, Okeke IN, Adeodu OO. Prolonged febrile illness due to CTX-M-15 extended-spectrum
b-lactamase-producing Klebsiella pneumoniae infection in Nigeria. Afr J Lab Med. 2012;1(1), Art. #16, 4 pages. http://dx.doi.org/10.4102/ajlm.v1i1.16, for more information.

1 2 3 4 5 a b 1 2 3 4 5 c
kbp kbp
3 3

2 2

dfrA17 aadA4

1 1

0.5
0.5

FIGURE 1: PCR amplification of the variable regions of class 1 and class 2 integrons. (a) Class 1 integron-variable regions amplified using Lev5’CS and Lev3’CS primers.
Lane 1: No template; Lane 2: E. coli strain 042 bearing an aadA cassette within a class 1 integron; Lane 3: E. coli strain 17-2 bearing the dfrA1-sat-aadA cassette sequence
within a class 2 integron; Lane 4: K. pneumoniae subsp. pneumoniae isolate K01 from this study; Lane 5: 1 kb Ladder plus (Invitrogen). Marker size fragments are indicated
to the right of the gel in kilobase pairs. (b) Class 2 integron-variable regions amplified using hep51 and hep74 primers with samples shown in (a) loaded on to the gel. (c)
Cassette content and orientation of the K01 integron amplified in (A), as predicted from the DNA sequence.

fresh whole blood transfusions (thrice) as well as exchange highlighting the presence of CTX-M genes in Africa even
blood transfusions (twice) cost N89 900.00 ($583.77). These though there is a scarcity of reports in the literature. Here,
and other treatment interventions effectively doubled we describe a case of prolonged, uncontrolled fever found to
the cost of treatment as compared to that for what be due to ESBL-producing K. pneumoniae. To the best of our
would normally have been appropriate therapy after knowledge, this is the first documented clinical course and
admission. Finally, prolonged hospital accommodation, outcome of ESBL-producing bacterial infection in Nigeria.
feeding and nursing care over 48 days amounted to
Failure to recognise and initially diagnose the presence of an
N20 400.00 ($132.46) in contrast to N2975.00 ($19.32) for
ESBL-producing organism resulted in considerable expense
admission for one week, an almost tenfold increase.
in the management of the infection. This includes the cost
of different courses of ineffective antibiotics for five weeks,
Discussion exchange blood transfusions and whole blood transfusions,
The occurrence and spread of infections resulting from as well as hospital charges resulting from prolonged stay in
ESBL-producing organisms have been well documented in hospital. The estimated avoidable cost of supportive therapy
countries of Europe, Asia and North America.11 In contrast, and investigation related to possible alternative diagnoses
was almost $600 in a country where the average annual per
data on the epidemiology of ESBL enzymes is very limited
capita income is $2300 and health care resources are severely
in Nigeria. Molecular analysis of eight Nigerian ESBL-
limited.
producing Enterobacter species in 2001 detected only TEM
and SHV-like ESBLs and no CTX-M types.12 In a study of
The clinical diagnostic microbiology laboratory plays a
Klebsiella pneumoniae isolates associated with community-
crucial part in the detection and reporting of ESBL-producing
acquired urinary tract infections between 2002 and 2003 in bacteria, and it is important that laboratories be fully aware
Ibadan, Nigeria, CTX-M group 1, -like enzymes were found in of the significance of ESBL-producing organisms and the
17 (57%), but CTX-M-15 was identified in only two isolates.13 best methods for detecting them, as in our case. Resource-
Olowe et al. investigated the occurrence of CTX-M ESBL- limited hospitals can incur prohibitive costs associated with
producing E. coli and found nine of 79 ampicillin-resistant ESBL-producer infections because of prolonged supportive
hospital isolates to be ESBL producers.14 More recently, a case therapy and treatment failure following the use of readily
of necrotising fasciitis was reported in a Nigerian patient in available antibiotics. Diagnostic improvements to allow
the UK.15 Morganella morganii and Citrobacter freundii carrying routine detection and reporting of ESBL production in
the CTX-M-15 ESBL gene were isolated from the patient, Enterobacteriaceae will help greatly in avoiding these costs.

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 Page 4 of 4 Case studies

1 2 3 4 Authors’ contributions
kbp
O.A.A. (Obafemi Awolowo University) coordinated the
2 research. O.A. (Obafemi Awolowo University Teaching
Hospitals Complex) A.A.O. (Obafemi Awolowo University
Teaching Hospitals Complex), O.A.A. and O.O.A. (Obafemi
Awolowo University Teaching Hospitals Complex) managed
the patient (case) clinically. A.A.O., O.A.A. and B.W.O.
1 (Obafemi Awolowo University) performed microbiological
testing whilst I.N.O. carried out molecular experiments.
A.O.A. and I.N.O. (Haverford College) drafted the paper, to
which O.A., B.W.O., O.O.A. and A.A.O. contributed. O.A.A.
and I.N.O. undertook revision of the manuscript. All authors
approved the final version.
0.5

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