# FatalPlasmodium falciparum, Clostridium perfringens, and Candida spp.  Coinfections in a Traveler to Haiti

Authors: Gillian L. Genrich; Julu Bhatnagar; Christopher D. Paddock; Sherif R. Zaki
Journal: Journal of Tropical Medicine
 (2009)

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## Abstract

Malaria is one of the most common causes of febrile illness in travelers. Coinfections with bacterial, viral, and fungal pathogens may not be suspected unless a patient fails to respond to malaria treatment. Using novel immunohistochemical and molecular techniques,Plasmodium falciparum, Clostridium perfringens, and Candida spp. coinfections were confirmed in a German traveler to Haiti. Plasmodium falciparum-induced ischemia may have increased this patient's susceptibility to C. perfringens and disseminated candidiasis leading to his death. When a patient presents with P. falciparum and shock and is unresponsive to malaria treatment, secondary infections should be suspected to initiate appropriate
treatment.


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## Body

## 1. Introduction

Plasmodium falciparum, an
intraerythrocytic  parasite that causes the most severe form of   human malaria,
is endemic to Haiti where it caused 32 739 infections and contributed to an
estimated 741 malaria deaths in 2006 [1–3]. Malaria infection poses a risk to
the 140 000 travelers from nonendemic countries that visit Haiti
each year [4]. Progression to
severe illness from initial symptoms of fever, headache, chills, and myalgia
occurs rapidly through the phenomenon of sequestration, in which P. falciparum-infected erythrocytes
attach to blood vessels and impede normal blood flow, particularly in the brain
[5]. Poor perfusion leads to tissue ischemia, which may subsequently increase a
patient’s susceptibility to secondary infections [5, 6] such as C. perfringens, and Candida spp. C. perfrignens is an anaerobic spore-forming bacillus that produces a virulent
hemolytic alpha toxin [7] and causes gas gangrene, a potentially
deadly infection characterized by fever, pain, edema, myonecrosis, and gas
production [8].  And in a setting of
tissue ischemia and necrosis, endogenous gut microflora such as Candida may
translocate across the epithelial border and gain access to systemic
circulation resulting in disseminated candidiasis [9].Malaria
coinfections withLeptospira spp., Coxiella burnetti, Brucella melitensis, and Streptococcus
pneumoniae as well as with enteric bacteria (Eschericha coli and Salmonella,
and Acinetobacter baumannii) have
previously been reported [10–14].  To our
knowledge, this report describes the first
fatal coinfection of P. falciparum,
C. perfringens, and
Candida spp.


## 2. Materials and Methods

### 2.1. Case Presentation

On October 29th 2005, a
56-year-old German tourist living in Haiti, with a history of heavy
alcohol use and 2 malaria infections treated with chloroquine, developed
symptoms of fever, headache, nausea, and diarrhea.  On November 3rd he visited a local
hospital in Côtes des Arcadins and was diagnosed
withP. falciparum infection
by peripheral blood smear. The patient was treated with chloroquine and
doxycycline the same day but developed altered mental status and was
hospitalized.  Despite falling parasite
counts, the
patient’s neurological condition continued to worsen and he was mechanically
ventilated.  Seizures, bloody stools,
hematemesis, and decreased urinary output were also documented.  Due to his worsening condition, he was
transferred to a hospital in Miami
the following day, November 4th.
At the time of hospital admission in Miami, the patient had thrombocytopenia,
diffuse intravascular coagulation, electrolyte imbalance, and acute renal
failure. Blood smears showed few malaria parasites, but treatment with
antimalarial agents was continued.
Bronchoalveolar lavage was positive for Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomas aeruginosa, and yeast forms.Blood culture on
November 4th was positive forC.
perfringens. Vancomycin, amphotericin,
piperacillin, phenytoin, lorazepam, and hydrocortisone were added to the
treatment regimen.  On November 5th blood culture revealed Candida tropicalis.  An encephalitis panel was also negative. The
patient continued to decline clinically and he expired on November 7th,
10 days after symptom onset.


### 2.2. Tissue Samples and Immunohistochemical Analyses

Formalin-fixed,
paraffin-embedded tissue (FFPET) sections of trachea, lung, heart, liver,
spleen, kidney, small intestine, and central nervous system (CNS) were
submitted to the Infectious Diseases Pathology Branch (IDPB) at the Centers for
Disease Control and Prevention for diagnostic consultation.Tissue sections
were evaluated by routine hematoxylin and eosin (H&E), GMS, gram and
Steiner stains, and were subsequently tested by immunohistochemistry (IHC) for
suspected pathogens using immunoalkaline phosphatase technique as described
previously [15, 16].  In brief, FFPET
sections were deparaffinized in xylene and rehydrated in a graded alcohol
series. IHC pretreatment conditions varied by primary antibody, and as deemed
appropriate, consisted of one of the following 2 methods. (1) Antigen retrieval was performed
by incubating sections submerged in Antigen Retrieval Citra Solution (BioGenex,
San Ramon, Calif, USA) in a steamer for 10 minutes or (2) sections were digested in 0.1 mg/mL proteinase K (Boehringer-Mannheim, Indianapolis, Ind, USA) solution for 15
minutes. Sections were then blocked with 20% normal sheep serum in
Tris-saline-Triton (NSS/TST) and incubated with the primary antibody (see
below) for 60 minutes on the autostainer (DAKO, Carpinteria, Calif, USA).
Detection of the bound antibody was performed using a secondary biotinylated
antimouse antibody (DAKO), alkaline phosphatase-conjugated streptavidin, and
naphthol phosphatase-fast red chromogen reagent. Slides were rinsed,
counterstained in Mayer’s hematoxylin, and mounted with aqueous mounting medium.The primary
antibodies used in immunohistochemical assays includedP. falciparum histidine rich protein-2 (HRP-2, Biodesign, Saco,
Maine, dilution 1:1000), a protein only produced by P. falciparum  [16]; a polyclonal antibody reactive with various Clostridium species including C. perfringens, C. botulinum, C. sordelii, C. novyii, and C. subterminale (Biodesign, dilution 1:1000) [15];  a polyclonal
antibody reactive with Candida species
(Meridian Life Sciences; dilution 1:200). Appropriate positive and negative
controls were run in parallel.


### 2.3. Molecular Analyses

DNA was extracted from FFPET sections of
small intestine using the QIAamp DNA minikit (Qiagen, Valencia,
Calif, USA), following the tissue extraction protocol. AC. perfringens-specific PCR assay was performed using the High
Fidelity PCR kit (Roche Diagnostics, Indianapolis, Ind, USA) according to the manufacturer’s
instructions to amplify 283-bp fragment of the phospholipase C (alpha toxin)
gene. For further evaluation, two P.
falciparum- specific PCR assays targeting the 18S rRNA gene were performed.
Primers used in PCR assays were published previously [17, 18] and are described
in Table 1. PCR assays were modified for the FFPET. For each primer set,
annealing temperature was adjusted accordingly. PCR was carried out on a
GeneAmp PCR System 9700 thermocycler (Perkin-Elmer).Table 1
Oligonucleotide primers used in the PCR assays.
Primer sequence (5′-3′)Gene targetProduct size (bp)Anneling temperature (C°)ReferenceClostridium perfringens specific PCRPL3 AAG TTA CCT TTG CTG CAT AAT CCCPhospholipase C28355Fach and Popoff  1997  [17]PL7 ATA GAT ACT CCA TAT CAT CCT GCTPhospholipase CPlasmodium falciparum  specific PCRrFAL1 TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT18S rRNA20558Perandin et al.   2004  [18]rFAL2 ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC18S rRNAFAL-F CTT TTG AGA GGT TTT GTT ACT TTG AGT AA18S rRNA9858Perandin et al.   2004 [18]FAL-R TAT TCC ATG CTG TAG TAT TCA AAC ACA A18S rRNAAmplified
PCR products were separated on 1.8% agarose gel, extracted from the gel by
using QIAquick gel extraction kit (Qiagen), and cycle sequenced by CEQ 2000 dye
terminator cycle sequencing with quick start kit (Beckman Coulter, Fullerton,
Calif, USA) and the respective primers. Postreaction cleanup was done by centri-sep
spin columns (Princeton Separations, Adelphia,
NJ). The samples were sequenced
on a CEQ 2000 XL sequencer (Beckman Coulter, Fullerton, Calif, USA).
Search for homologies to known sequences was done using the nucleotide database
of the Basic Local Alignment Search Tool (BLAST) athttp://www.ncbi.nlm.nih.gov/BLAST.


## 2.1. Case Presentation

On October 29th 2005, a
56-year-old German tourist living in Haiti, with a history of heavy
alcohol use and 2 malaria infections treated with chloroquine, developed
symptoms of fever, headache, nausea, and diarrhea.  On November 3rd he visited a local
hospital in Côtes des Arcadins and was diagnosed
withP. falciparum infection
by peripheral blood smear. The patient was treated with chloroquine and
doxycycline the same day but developed altered mental status and was
hospitalized.  Despite falling parasite
counts, the
patient’s neurological condition continued to worsen and he was mechanically
ventilated.  Seizures, bloody stools,
hematemesis, and decreased urinary output were also documented.  Due to his worsening condition, he was
transferred to a hospital in Miami
the following day, November 4th.
At the time of hospital admission in Miami, the patient had thrombocytopenia,
diffuse intravascular coagulation, electrolyte imbalance, and acute renal
failure. Blood smears showed few malaria parasites, but treatment with
antimalarial agents was continued.
Bronchoalveolar lavage was positive for Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomas aeruginosa, and yeast forms.Blood culture on
November 4th was positive forC.
perfringens. Vancomycin, amphotericin,
piperacillin, phenytoin, lorazepam, and hydrocortisone were added to the
treatment regimen.  On November 5th blood culture revealed Candida tropicalis.  An encephalitis panel was also negative. The
patient continued to decline clinically and he expired on November 7th,
10 days after symptom onset.


## 2.2. Tissue Samples and Immunohistochemical Analyses

Formalin-fixed,
paraffin-embedded tissue (FFPET) sections of trachea, lung, heart, liver,
spleen, kidney, small intestine, and central nervous system (CNS) were
submitted to the Infectious Diseases Pathology Branch (IDPB) at the Centers for
Disease Control and Prevention for diagnostic consultation.Tissue sections
were evaluated by routine hematoxylin and eosin (H&E), GMS, gram and
Steiner stains, and were subsequently tested by immunohistochemistry (IHC) for
suspected pathogens using immunoalkaline phosphatase technique as described
previously [15, 16].  In brief, FFPET
sections were deparaffinized in xylene and rehydrated in a graded alcohol
series. IHC pretreatment conditions varied by primary antibody, and as deemed
appropriate, consisted of one of the following 2 methods. (1) Antigen retrieval was performed
by incubating sections submerged in Antigen Retrieval Citra Solution (BioGenex,
San Ramon, Calif, USA) in a steamer for 10 minutes or (2) sections were digested in 0.1 mg/mL proteinase K (Boehringer-Mannheim, Indianapolis, Ind, USA) solution for 15
minutes. Sections were then blocked with 20% normal sheep serum in
Tris-saline-Triton (NSS/TST) and incubated with the primary antibody (see
below) for 60 minutes on the autostainer (DAKO, Carpinteria, Calif, USA).
Detection of the bound antibody was performed using a secondary biotinylated
antimouse antibody (DAKO), alkaline phosphatase-conjugated streptavidin, and
naphthol phosphatase-fast red chromogen reagent. Slides were rinsed,
counterstained in Mayer’s hematoxylin, and mounted with aqueous mounting medium.The primary
antibodies used in immunohistochemical assays includedP. falciparum histidine rich protein-2 (HRP-2, Biodesign, Saco,
Maine, dilution 1:1000), a protein only produced by P. falciparum  [16]; a polyclonal antibody reactive with various Clostridium species including C. perfringens, C. botulinum, C. sordelii, C. novyii, and C. subterminale (Biodesign, dilution 1:1000) [15];  a polyclonal
antibody reactive with Candida species
(Meridian Life Sciences; dilution 1:200). Appropriate positive and negative
controls were run in parallel.


## 2.3. Molecular Analyses

DNA was extracted from FFPET sections of
small intestine using the QIAamp DNA minikit (Qiagen, Valencia,
Calif, USA), following the tissue extraction protocol. AC. perfringens-specific PCR assay was performed using the High
Fidelity PCR kit (Roche Diagnostics, Indianapolis, Ind, USA) according to the manufacturer’s
instructions to amplify 283-bp fragment of the phospholipase C (alpha toxin)
gene. For further evaluation, two P.
falciparum- specific PCR assays targeting the 18S rRNA gene were performed.
Primers used in PCR assays were published previously [17, 18] and are described
in Table 1. PCR assays were modified for the FFPET. For each primer set,
annealing temperature was adjusted accordingly. PCR was carried out on a
GeneAmp PCR System 9700 thermocycler (Perkin-Elmer).Table 1
Oligonucleotide primers used in the PCR assays.
Primer sequence (5′-3′)Gene targetProduct size (bp)Anneling temperature (C°)ReferenceClostridium perfringens specific PCRPL3 AAG TTA CCT TTG CTG CAT AAT CCCPhospholipase C28355Fach and Popoff  1997  [17]PL7 ATA GAT ACT CCA TAT CAT CCT GCTPhospholipase CPlasmodium falciparum  specific PCRrFAL1 TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT18S rRNA20558Perandin et al.   2004  [18]rFAL2 ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC18S rRNAFAL-F CTT TTG AGA GGT TTT GTT ACT TTG AGT AA18S rRNA9858Perandin et al.   2004 [18]FAL-R TAT TCC ATG CTG TAG TAT TCA AAC ACA A18S rRNAAmplified
PCR products were separated on 1.8% agarose gel, extracted from the gel by
using QIAquick gel extraction kit (Qiagen), and cycle sequenced by CEQ 2000 dye
terminator cycle sequencing with quick start kit (Beckman Coulter, Fullerton,
Calif, USA) and the respective primers. Postreaction cleanup was done by centri-sep
spin columns (Princeton Separations, Adelphia,
NJ). The samples were sequenced
on a CEQ 2000 XL sequencer (Beckman Coulter, Fullerton, Calif, USA).
Search for homologies to known sequences was done using the nucleotide database
of the Basic Local Alignment Search Tool (BLAST) athttp://www.ncbi.nlm.nih.gov/BLAST.


## 3. Results

No hemozoin
pigment was observed by routine hematoxylin-eosin (H&E) stain and no
malaria parasites were seen in red blood cells on careful examination of
multiple tissues. Histopathology of the colon and small intestine mucosa showed
extensive necrosis with diffuse, extensive, submucosal edema and multifocal
inflammatory cell infiltrates comprised predominately of neutrophils
(Figure1(a)). The serosa was moderately thickened and contained mixed inflammatory cell
infiltrates. The liver showed autolysis with no significant inflammatory cell
infiltrates.  Spleen sections were congested
and necrotic. The mucosal surface of the larynx was also extensively necrotic
with abundant neutrophilic infiltrates.
There were diffuse autolysis in the kidneys and intra-alveolar edema in
the lungs.  The heart showed interstitial
edema. There were focal hemorrhages in the white matter of the cerebral cortex,
but no conspicuous inflammatory cell infiltrates observed in the hippocampus,
pons, cerebellum, and spinal cord.Histopathologic and immunohistochemical findings in representstive tissues of study patient. (a) Extensive necrosis of intestinal mucosa with diffuse, extensive, submucosal edema and multifocal inflammatory cell infiltrates (H&E, original magnification X13). (b) Abundant, diffuse immunostaining ofClostridium spp. antigens in intestine (original magnification X13; inset X100). (c) Immunohistochemical detection of P. falciparum HRP-2 antigens in pRBCs in spleen (original magnification X100). (d) HRP-2 antigen immunostaining in endothelium of CNS blood vessels (original magnification X50).
(a)(b)(c)(d)The HRP-2 IHC assay
revealed discrete immunostaining of intra-erythrocytic parasites in CNS,
kidney, liver, heart, and spleen
(Figure1(c)). HRP-2 antigens were also detected
in endothelium of systemic and CNS blood vessels
(Figure 1(d)), and in renal
tubular epithelium and renal casts. The Clostridia
spp. IHC assay revealed abundant immunostaining in necrotic areas of the
small intestine (Figure 1(b)); other tissues were negative. Small budding yeasts
in the alveolar space of the lung were identified by an IHC stain for Candida spp.Amplification
products of expected sizes were  generated by both theC. perfringens and P.falciparum PCR assays using DNA
extracted from FFPET sections. Sequence analysis of positive amplicons also
confirmed the infections of P. falciparum and
C. perfringens.


## 4. Discussion

Immunohistochemical
and molecular analysis of FFPET sections from the study patient confirmed
coinfections  withP. falciparum, C. perfringens, and Candidia spp. Malaria is one of the  most common causes of fever in
travelers [19] and nosocomial coinfections may occur with a frequency of 25%,
according to a recent study of 96 fatal P.
falciparum cases [20].  However,
malaria coinfections are difficult to diagnose clinically and may only be
suspected when a patient fails to respond to malaria treatment [10]. Several
observations suggest that the patient described here, who presented to hospital
with classic malaria symptoms, was successfully treated for malaria infection
with chloroquine and doxycycline: (1) declining parasite counts on peripheral
blood smear were documented during his hospital stay; (2) absence of hemozoin,
a birefringent pigment produced by plasmodium in correlation with parasite
density [21, 22], on H&E evaluation;   (3) rare HRP-2 antigens detected by
IHC in CNS, heart, lung, and liver sections as described in detail above. Rare
staining was anticipated in this patient, considering that HRP-2 antigens are
slow to clear from the blood and may persist in treated patients for up to two
weeks [23].  The presence of
amplification products of P. falciparum using PCR assays is not inconsistent with the H&E and immunohistochemical findings
of resolving malaria infection. The primers used are highly sensitive, shown to
detect as few as 0.7 parasites/mL [18]. In this context, and as our patient
illustrates, PCR results must be correlated with the patient’s clinical
history.The observation of
necrosis in the small intestine supports the finding ofC. perfringens in blood cultures found on November 4th,
(day 7 of illness) after the patient was hospitalized in Miami. C. perfringens infection was
subsequently confirmed by IHC testing and by PCR and sequencing analysis. C. perfringens is associated with
several human diseases, including necrotizing enterocolitis [24], gas gangrene
[25], antibiotic-associated diarrhea, and food poisoning outbreaks worldwide
[26]. The alpha toxin is the most virulent of the 12 toxins produced by C. perfringens [9] because it
destroys cell membranes, including those of red blood cells, platelets, and
muscles. The bacterium also has sphingomyelinase activity that causes damage to
the nerve-sheath in the central nervous system [8]. Deaths due to C. perfringens infections are rare in
humans, and the portals of entry are usually surgical wounds [27]. However
concomitant ischemia, or low oxygen tension in necrotic tissue, is a trigger
for bacterial spore germination [28], and subsequent
toxin production leads to anaerobic cellulitis or myonecrosis (gas
gangrene) that rapidly progresses to severe sepsis [8]. The source of this
patient’s C. perfringens infection is
unknown as C. perfringens is
widespread in the environment and can be a component of normal human flora, but
broad spectrum antibiotic use is suspected.
It is likely that P. falciparum infection increased susceptibility to C. perfringens and Candidaspp.
in this patient.C. perfringens infection causing intestinal myonecrosis may have begun with tissue ischemia
due to P. falciparum sequestration,
which is characterized by the attachment of parasitized erythrocytes (pRBCs) to
endothelial cells lining blood vessels via a variety of constitutive
receptors.  Sequestration causes sluggish
blood flow and disruption of microcirculation [6, 29] leading to ischemia. The
infection subsequently leads to hyperlactemia, hypoglycemia, and metabolic
acidosis, creating a dependence on anaerobic glycolysis for energy production [30].  Studies suggest that obstruction of the
splanchnic blood vessels by pRBCs facilitates the entry of endotoxins and
bacteria like C. perfringens from the
digestive tract into the bloodstream [31]. While limited clinical data on this
patient is available, this mechanism seems plausible and is supported by the
tissue-based and molecular testing performed.This patient’s
chronic alcohol consumption may also have contributed to the severity of
multiple infections.  Ethanol has been
shown to decrease the respiratory burst activity of neutrophils [32], and heavy
alcohol consumption (> or =  5 drinks per day) is significantly associated
with ICU-acquired bacterial infection, even when controlling for duration of
mechanical ventilation and other risk factors [33].  Further, the oral flora of heavy alcohol
drinkers  has been shown to differ significantly from the flora of
nonalcoholics. One study showed that anaerobes, including
Clostridrium spp.,
are present in 84.5% of heavy drinkers, compared with 30.5% of nonalcoholics
and similarly, Candida spp. were found in 34.5% of heavy drinkers whereas
only 5.5% of nonalcoholics carried the microbiota [34]. The pathogens detected
by bronchoalveolar lavage, Klebsiella
pneumoniae, and Acinetobacter
baumannii are particularly common causes of pneumonia in chronic
alcoholics; while Pseudomas aeruginosa is associated with mechanical ventilation
[35].Disseminated
candidiasis contributed to our patient’s demise;C. tropicalis was detected by bronchoalveolar lavage, and our IHC
analysis revealed Candidaspp. in FFPET of small intestine. Animal
studies have shown that mucosal damage and broad-spectrum antibiotics are
important factors in opportunistic candidiasis [30].  In this case, we suggest that the use of
broad spectrum antibiotic coverage in the setting of tissue ischemia and
mucosal erosion secondary to P. falciparum and C. perfringens infections facilitated disseminated infection.To our knowledge,
this is the first report ofP. falciparum, C. perfringens, and Candida spp. coinfections. In the case
presented here, P. falciparum may
have increased susceptibility to C.
perfringens infection by inducing a state of hypoxia-ischemia.  The patient’s chronic alcohol consumption may
have increased his susceptibility to intestinal necrosis and ischemia, which
created a suitable environment for translocation and dissemination of
Candida
spp., particularly in the setting of broad-spectrum antibiotic
administration. P.falciparum remains a significant threat
to travelers to Haiti
and other areas where the parasite is endemic. This case highlights the
importance of suspecting bacterial and fungal
coinfections in patients refractory to malaria treatment.



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*Source: 969070-2009-05-05.xml*