Opportunistic infections by bacteria, fungi, and parasites often produce
diseases with similar symptoms and therefore require careful differential
diagnosis since treatment is usually different for each type of organism.
Clinically significant gastrointestinal disease, for example, is present
in up to 90% of HIV infected patients, and may be caused by Salmonella,
Shigella, Campylobacter, the mycobacteria, Clostridium difficile,
Candida, Histoplasma, Cytomegalovirus, Rotavirus, HIV enteropathy,
Norwalk virus, as well as a number of parasites. Non-infectious causes
can be lymphomas, Kaposi's sarcoma, and drug toxicity. A brief review
of parasitic infections that are common in the HIV positive host is
given in the following pages. The reader should refer to other resources
for detailed instructions of identification procedures and diagnostic
tests.
A. Strongyloides stercoralis
Strongyloides stercoralis is an intestinal roundworm
of humans, found worldwide, that causes diarrhea and a wide range
of other symptoms. As the organism goes through its migratory cycle
in the normal host, symptoms may resemble those of bronchitis, pneumonia,
and peptic ulcer disease as well as more typical intestinal infection.
Asymptomatic infection can last 30 to 40 years. Strongyloidiasis can
be a serious and life threatening disseminated disease in immunocompromised
patients due to hyperinfection and massive tissue invasion by the
parasite. The condition is further complicated by gram negative rod
septicemia and meningitis. Strongyloides infection begins when filariform
larvae penetrate the skin. The complicated life cycle includes filariform
larvae in the lung phase, adults in the mucosa of the small bowel,
and non-infectious rhabditiform larvae in the stool. In some cases
(autoinfection) rhabditiform larvae in the intestines molt to infectious
filariform larvae that penetrates the skin or mucosa and repeat the
infectious cycle. Autoinfection progresses rapidly in the immunocompromised
host, leading to
disseminated infection. The pulmonary barrier is penetrated by the
larvae, which then enter the arterial circulation and disseminate
to many organs (hyperinfection syndrome).
Hyperinfection in the immunocompromised host can lead
to septicemia and meningitis with enteric bacteria. The enteric bacteria
are carried into the blood stream as the larvae migrate through the
intestinal wall. Migrating larvae may also cause tissue and brain
damage, and respiratory failure. A mortality rate approaching 50%
is reported in immunocompromised patients with hyperinfection syndrome
(Koneman et al, 1992).
Thiabendazole is the drug most commonly used. Ivermectin
and albendazole have also been used experimentally and may be approved
for use.
LABORATORY DIAGNOSIS
Laboratory diagnosis is based upon identification of
the larval forms of the worm in the stool, duodenal drainage, and
sputum. The form seen most often is the non-infectious rhabditiform
larvae. In hyperinfection, infectious filariform larvae may be present
in the stool and sputum. If stool specimens are negative, duodenal
drainage should reveal the larvae, and possibly eggs and adults. The
Entero-Test, consisting of weighted nylon string in a gelatin capsule,
is used to sample the duodenal contents. One
end of the string is taped to the patient's face and the capsule is
swallowed. The string is retrieved after 4 hours in the duodenum,
and mucous is scraped off and examined. The mucous should be examined
immediately by wet mount, or preserved in l0% formalin and PVA in
case giardia is also present. Sputum specimens should not be concentrated,
but examined in a manner similar to that used for the examination
of duodenal contents.
Specimens should be examined immediately or preserved
in formalin, since the larvae could molt or free living nematode contaminants
(Rhabditis) could multiply, making identification more difficult.
Hookworm larvae are very rare in stools, but when they are present
they must be distinguished from Strongyloides larvae. Charcot-Leyden
crystals, formed from disintegrated eosinophils, may also be present.
An eosinophil count should also be done, as eosinophil
counts usually range from l0 to 50%. Declining or low eosinophil counts
indicate a poor prognosis in strongyloidiasis (Howard et al, l994)
Safety measures are extremely important in handling
all specimens. Filariform larvae, if present, could penetrate the
skin of the laboratory worker and cause infection.
B. Entamoeba histolytica
Entamoeba histolytica is a protozoan parasite of humans
found throughout the world, causing about 500,000 infections annually.
Asymptomatic carriers are largely responsible for spread of infection
through food or water contaminated by the cyst stage.
Ninety percent of infections are asymptomatic or very
mild. In acute amebiasis, Entamoeba histolytica lyses the tissues
of the colon and forms flask shaped ulcers. The patient may have few
serious symptoms or develop severe bloody dysentery, leading to weight
loss, fever, dehydration, and electrolyte imbalance. Chronic disease
symptoms can be similar to those of ulcerative colitis, hepatic carcinoma,
or diverticulitis. Extra-intestinal disease occurs when the organisms
migrate through the intestinal mucosa into the blood or lymphatic
system. The amoeba disseminate primarily to the liver, forming abscesses.
Liver abscess leads to increased leukocyte count, abdominal pain,
and fever. Liver function tests may remain normal, and jaundice is
rare. The abscess may rupture into the pleural cavity, resulting lung
infection characterized by chest pain, cough and fever. Migration
of amoeba to the brain can cause focal amebic encephalitis. Dissemination
of the amoeba in the immunocompromised host may
be enhanced if amebiasis is present but not diagnosed, and the patient
may be treated with corticosteroids for inflammatory bowel disease.
Amebiasis is treated by agents that affect the trophozoite (amoeboid)
stage, and treatment depends upon the severity of the infection and
its location. Large abscesses may have to be drained surgically for
antibiotics to be effective. Metronidazole and iodoquin are two drugs
that are commonly used in treatment, however metronidazole is not
recommended for pregnant women.
LABORATORY DIAGNOSIS
Laboratory diagnosis is made by microscopic observation
of the amoeba trophozoites and cysts in stool or tissue. An enzyme
immunoassay to detect antigen in stool specimens is also available
(Xia et al, l994) Indirect hemagglutination tests are positive in
almost 87% to l00% of all liver abscesses and 85% to 95% of acute
amoebic dysentery (Howard, l994). Serologic tests for antibody are
unreliable in asymptomatic or non-invasive intestinal disease, or
in immunocompromised patients who can no
longer mount an antibody response (Neva and Brown, l994). Where amebiasis
is endemic, positive serology may indicate past infection rather than
current infection.
Up to six stool specimens, collected over a l5 day period,
should be examined. If bloody mucous is present, it should be selected
for examination. A direct saline mount done within 30 minutes of passage
may show motile amoebas. Concentration and a permanent stained smear
such as trichrome should always be done. Aspirates from sigmoidoscopy
should be examined immediately for motiletrophozoites.
The amoeba form, or trophozoite, is seen more often
in liquid stools. The trophozoite usually ranges from l5 to 30 microns
in size, and may have ingested red blood cells within the cytoplasm.
The cysts, l0 to 20 microns in diameter, are more often found in formed
stool specimens and can have up to four nuclei. Charcot-Leyden crystals
may be present in the specimen.
C. Giardia lamblia
Giardia lamblia is a flagellated protozoan parasite
of humans and other mammals, and is generally more prevalent in children.
It is common worldwide, and one of the most common parasitic diseases
in the United States. Prevalence is increasing in the homosexual population
due to anal and oral sexual practices, but the main route of transmission
is through water or food that has been contaminated by fecal material.
Outbreaks have been attributed to municipal water supplies as well
as to water from
mountain streams. The disease does not seem to be any more severe
in immunocompromised than in immunocompetent hosts. Giardia lamblia
infects the duodenum. Infection ranges from asymptomatic to malabsorption
syndrome, mucosal irritation, and increased mucous secretion. Patients
may have pain, flatulence, bloating, and frothy, foul smelling stools.
They may have non-bloody diarrhea with increased fat and mucous. Infection
may involve the gall bladder, with resulting jaundice.
Quinacrime (Atabrine) or metronidazole is used for treatment.
LABORATORY DIAGNOSIS
Laboratory diagnosis is made by microscopic observation
of Giardia trophozoites and cysts in stool or duodenal drainage. Enzyme
immunoassays and fluorescent antibody assays that detect antigen in
stool specimens are available and positive results indicate current
infection (Garcia and Bruckner, l994). Presence of serum antibodies
is not a good diagnostic aid because a positive result may indicate
either past or present infection. Up to six stool specimens over a
l5 day period may be necessary to
detect the parasite since Giardia adheres tightly to the duodenal
mucosa, and tends to appear in "showers". Either the Entero-Test string
capsule described for Strongyloides diagnosis, or biopsy may be necessary
for detection of Giardia.
The trophozoite stage is described as having a "falling
leaf" motility but this may be difficult to detect because of intestinal
mucous. Using the trichrome stain the cysts are usually easy to see,
however the trophozoites may be more difficult as they do not stain
as readily. The pear shaped trophozoite ranges from l0 to 20 microns
long and 5 to l5 micronswide. It contains two nuclei, and may be described
as resembling a wide-eyed old man. On side view the trophozoite is
spoon shaped. The cyst is egg shaped, 11 to l4 microns long by 7 to
l0 microns wide. It contains four nuclei and a jumble of rod shaped
bodies. Some cysts may be distorted; also the cytoplasm may pull away
from the cyst wall leaving an empty space. The trophozoite is seen
more often in liquid stools, the cyst in formed stools.
Part III Specific Parasites
D. Cryptosporidium parvum
Cryptosporidia are protozoa that infect the cells of
the small bowel. They were first seen in veterinary medicine as a
cause of diarrhea in calves. Today Cryptosporidiuym parvum is recognized
as a worldwide cause of diarrhea in man, causing major outbreaks when
water systems are contaminated, especially by animal wastes. High
risk groups include workers and children in day care centers, animal
handlers, and travelers, as well as the immunocompromised and malnourished.
The small bowel of humans and animals is the most common
site of infection, but all areas of the gastrointestinal tract can
become involved, including the esophagus, stomach, colon, and rectum,
especially in the immunocompromised. Patients can have 5 to l0 watery
(non-bloody), frothy, mucous-flecked stools a day, and in severe cases
there may be a loss of up to l5 liters of fluid a day. There may also
be nausea, fever, cramps, and loss of appetite. Severity of diarrhea
is related to the number
of parasites infecting the host and the immune status of the host,
particularly the number of CD4+ cells. The organism is capable of
autoinfection, so the parasite burden can increase rapidly in the
immunocompromised host..
Dissemination to bronchial epithelial cells can lead
to respiratory infection characterized by an unremitting cough. Malnourished
children as well as AIDS patients can develop respiratory disease
along with diarrhea due to Cryptosporidium.
Gall bladder, biliary tract disease, and liver involvement
is also possible. In gall bladder disease, cholecystectomy may be
necessary to prevent rupture and peritonitis.
Currently there is no effective treatment for cryptosporidiosis.
Clinical remission seems to be related to the degree of
immunocompetence. Debilitated patients with disseminated infection
usually die from overwhelming gastrointestinal disease.
LABORATORY DIAGNOSIS
The diagnostic and infective form of the parasite is
the oocyst. Oocysts are round, 4 to 5 microns in diameter, and most
often found in watery stools. Stool or sputum specimens should be
placed in l0% formalin immediately upon collection. The stool or sputum
specimen should be processed using a concentration or sedimentation
procedure.
The most common test for identification is a modified
acid fast stain of the formalin-ethyl acetate concentrated stool or
sputum for the oocyst. Several specimens and at least five or six
acid fast stains should be examined. Enzyme immunoassays and direct
fluorescent assays increase sensitivity and specificity but are more
expensive than the acid fast stain. They may be more useful when the
parasite is present in small numbers, as when tracing the source of
an outbreak.
Safety should be a primary concern both during collection,
processing, and testing as the oocysts are infectious in unpreserved
material.
E. Isospora belli
This protozoan parasite is more likely to be seen in
patients from the Caribbean (Libman and Witzburg, l993).
Transmission is through contaminated food or water containing
the oocytes, and there is some postulation that Isospora may also
be sexually transmitted (Forthal and Guest, l984). Oocytes may survive
in the environment for months if they are kept cool and moist.
Isospora belli infects the cells of the duodenum and
jejunum of humans. Infection in the immunocompetent may be asymptomatic,
mild, and transient. In AIDS patients it causes a syndrome similar
to cryptosporidiosis, with up to l0 watery or soft, foamy and foul
smelling stools per day. Malabsorption syndrome can also occur due
to mucosal lesions and infiltration of mucosal tissue by eosinophils
and other white blood cells. There may be pain and loss of appetite.
In the immunocompromised patient, diarrhea can be profuse, leading
to weakness, loss of appetite, and weight loss. The diarrhea may be
prolonged and recur after treatment. This may be due to autoinfection
of the organism.
Rest and bland diet may cure mild infections. In more
severe cases, trimethoprim and sulfamethoxazole is usually effective.
In AIDS patients relapse rates can be as high as 50% when antibiotic
therapy is discontinued; therefore antibiotics must be administered
for prolonged periods. Other drugs are effective also, including combinations
of pyrimethamine and sulfadiazine.
LABORATORY DIAGNOSIS
The diagnostic and infective form of the parasite in
the stool is the oocyst. The immature oocyst is most commonly seen,
ellipsoidal, 20 to 30 microns long, containing a large round mass.
The mature oocyst, containing two spherical sporocysts may also be
seen, as well as Charcot-Leyden crystals.
Stool specimens should be preserved in l0% formalin,
and concentrated. The organism may be observed on iodine stained wet
mount. It is more easily seen on modified acid fast stained concentrated
specimens, where the central mass of protoplasm and the sporocysts
appear bright red.
F. Microsporidia
The microsporidia are obligate intracellular protozoa
that are widely distributed in insects and other animals. The group
is made up of several genera, Encephalitozoon, Nosema, Pleistophora,
Enterocytozoon, Septata intestinalis, and Microsporidium. In AIDS
patients the three reported most commonly are Enterocytozoon bieneusi,
Encephalitozoon hellem, and Septata intestinalis. Spores, 2 to 7 microns
in size, can be transmitted to other hosts through ingestion, inhalation,
or direct inoculation.
Reports of infections are increasing, but the number of unrecognized
cases is probably higher since the parasite is difficult to identify.
Microsporidia infects the enterocyte of the small bowel,
causing diarrhea. Spores released from the host cell pass in the stool,
or may directly reinfect the enterocyte. In the normal host the intestinal
disease is mild, short, and self limiting. The most common disease
in the immunocompromised host is severe enteritis with chronic watery
diarrhea (four to eight non bloody stools per day), fever, nausea,
anorexia and weight loss. Microsporidia in some patients may spread
from the intestines
throughout the body. They have been found in the kidney, gall bladder,
heart, muscle, diaphragm, liver, lungs, sinus, nasal epithelium, and
eyes (Garcia and Bruckner, l993; Neva and Brown, l994). Other symptoms
can include myositis and muscle degeneration, hepatitis, convulsion,
vomiting, headache, fever, and unconsciousness.
Treatment results have been variable with a number of
antibiotics and agents, such as metronidazole, sulfasalazine and lomotil.
Disease can reactivate since organisms persist in the tissues.
LABORATORY DIAGNOSIS
Electron microscopy of biopsy specimens is the standard
for identification of microsporidia, but may not be sensitive enough
to detect small numbers of organisms. The organism has been stained
with modified trichrome for stool and sputum, Giemsa for touch preparations
of tissue, or acid fast stains for plastic-embedded tissue (Garcia
and Bruckner, l994). The spore should be l to l.5 microns long, with
a pink to red wall. It contains a polar tube which may appear as an
interior stripe or the interior may be
clear. Bacteria, yeast, and debris can be confused with microsporidia,
and positive controls must be stained and examined with the specimen
stains, and these may be difficult to obtain.
AIDS patients with microsporidium infections generally
have a CD4 count below 200 cells/mm3 and often below l00 cells/mm3.
Specific, reliable serological tests to indicate infection have not
been developed. The organisms are common in nature and a positive
serological test may not indicate disease even in an immunocompetent
host.
G. Toxoplasma gondii
Toxoplasma are tissue protozoa that can infect tissues
of a wide variety of vertebrate animals, including humans. The organism
exists as cysts (bradyzoites) in muscle and brain and as intracellular
trophozoites (tachyzoites).
Transmission occurs through ingestion of raw meat or
cat feces containing infective stages of the parasite. Infection in
immunocompetent individuals is generally asymptomatic or mild, but
congenital infection may have devastating consequences to the fetus
and newborn. In the immunocompromised, the nervous system is most
often involved. Toxoplasmosis is the moscommon cause of opportunistic
infection in the brain in HIV positive patients. Toxoplasma is also
the second most common cause of retinitis in the HIV infected host,
second only to cytomegalovirus.
Intracellular tachyzoites multiply and lyse the cells,
creating continually expanding lesions. In the immunocompetent hose,
the parasite then forms cysts, halting the spread. Also in the immunocompromised
host, the organism can spread to other organs and tissues through
the bloodstream or lymphatics. Existing cysts can become reactivated.
The mild form of the disease resembles mononucleosis,
with lymphadenopathy, muscle pain, and fever. Skin rash, high fever
and chills, myocarditis, hepatitis, pneumonia, retinitis, and central
nervous system (CNS) involvement can result from disseminated infection.
Central nervous system infections result in encephalopathy, meningoencephalitis,
cerebral mass lesions, altered mental state, motor impairment, seizures,
abnormal reflexes, and other signs of neurologic damage.
Low doses trimethoprim with sulfamethoxazole as prophylaxis
or treatment can protect against toxoplasmosis as well as Pneumocystis
carinii pneumonia. Once infected, another choice of treatment is pyrimethamine
and sulfadiazine. Lifelong suppressive therapy is also required, as
the drugs inhibit folate metabolism and do not kill the parasite.
LABORATORY DIAGNOSIS
Serology is commonly used to indicate infection with
Toxoplasma. Indirect fluorescent antibody (IFA) tests and various
types of ELISA tests are used for detection of IgG and IgM antibody.
Rising IgG titers and specific IgM titers of l:64 (IFA) or l:256 (ELISA)
indicate probable infection in the immunocompetent host. Antibody
levels peak early in infection, and a single high
serum titer of l:l000 or more may indicate a possible diagnosis. Serology
may be difficult to interpret in HIV positive patients since antibody
levels may be suppressed.
The presence of crescent shaped tachyzoites, 4 to 8
microns long, in tissue or spinal fluid is a significant finding microscopically.
Demonstration of the parasite on Wright or Giemsa stain of lung biopsy
material is required for diagnosis of pulmonary disease.
The organism may be observed in histologic preparations of tissue
or cultured in tissue culture or mice. They have been
detected by immunofluorescence within 2 days of inoculation into MRC-5
cells.
In the immunocompetent host, positive histology or culture
may not be diagnostic for the disease because the cysts can be present
without active infection. In the immunocompromised host with clinical
symptoms, observation or isolation of the parasite along with interpretation
of serologic results can be an important aid in diagnosis.
H. Pneumocystis carinii
Pneumocystis infects the alveolar spaces of the lungs
in a wide variety of mammals, including humans. It has been classified
as a protozoan, but recent studies indicate that it may be more closely
related to the fungi.
In the immunocompetent host there is usually an asymptomatic
or mild infection. Symptoms include shortness of breath, non-productive
cough, and low grade fever. In immunocompromised patients onset is
generally rapid, but AIDS patients may have an incubation period lasting
up to a year, with weight loss, diarrhea, non-productive cough, and
low grade fever. Pneumocystis is the major cause of pneumonia (PCP)
in HIV infected patients (over 80%). As the disease progresses, cyanosis
can develop and progress to asphyxiation and death. Extra-pulmonary
infections may result following damage to the endothelial tissues,
especially by methods used to aerosolize pentamidine. Choroiditis
may be an earlyindication of disseminated disease.
In AIDS patients, combined trimethoprim and sulfamethoxazole
is given when the CD4+ cell count drops below 200/mm2, and is the
treatment of choice following infection. A more toxic drug, pentamidine
isethionate can be given intramuscularly, intravenously, or as an
aerosol. Oxygen and assisted ventilation may also be necessary.
LABORATORY DIAGNOSIS
Diagnosis is based on observation of the organism in
lung tissue or secretions. Specimens include lung biopsy, broncho-alveolar
lavage, and brush biopsy using bronchoscopy. Sputum is generally not
recommended, but may be positive in AIDS patients since they usually
have abundant organisms present.
A variety of stains may be used to visualize the organism.
Silver stains and toluidine O can be used to stain the cysts. With
silver stains, the organism appears as a brown, often crumpled disk
about 5 microns in diameter. Toluidine O stains the cyst wall and
trophozoites blue. Giemsa will stain the trophozoites only, as pink
dots within a clear space, but is more difficult to read and interpret
than the silver stain. Monoclonal antibody with immunofluorescence
has been reported to improve detection of the parasite (Garcia and
Bruckner, l993) . Serologic tests are not reliable for diagnosis.
Part IV. Conclusion
Parasitic infections that cause relatively mild diseases in the normal
host can cause severe, recurrent, and life threatening infections
in the immunocompromised host. Many of the parasitic diseases prevalent
in HIV infected patients can have unusual manifestations and cause
a wide range of non-specific symptoms; therefore, physicians may have
difficulty making a clinical diagnosis. Treatment is challenging for
a variety of reasons, primarily because of the lack of host immune
function. With impaired antibody production, serologic tests for identification
become unreliable. Other laboratory tests, therefore, are
important aids to diagnosis, not only in the detection and identification
of the parasite, but also the determination of the immune status of
the host.
This issue has discussed eight parasites that are frequently
seen in HIV-infected and other immunocompromised patients. Table I
gives an overview of the diseases and laboratory tests for each. The
reader should note that opportunistic parasitic disease is not limited
to these organisms. Others such as Acanthamoeba, Blastocystis, Anisakis,
and Taenia are examples of parasites that the laboratorian may find
associated with disease in the immunocompromised. As medical treatments
and diagnostic methods become more sophisticated, more patients will
be effectively treated, but there may be increasing numbers of opportunistic
infections that will require the expertise of laboratory personnel.
All levels of laboratory personnel should be aware of these diseases
and should be trained to expect the unexpected.
Medical College of Georgia - 1993
