Page 2568 - Hematology_ Basic Principles and Practice ( PDFDrive )

Chapter 158 Hematologic Aspects of Parasitic Diseases 2291

should be swiftly inserted to a depth of approximately 2 or 3 cm at
an angle of roughly 45 degrees to the skin, applying negative pressure.
The needle should be withdrawn immediately while maintaining
negative pressure and taking only a few seconds for the entire proce-
dure. The splenic tissue may be expelled from the needle using a small
amount of air and by fixing the stain using standard procedures.

Culture

Amastigotes may be grown as motile promastigotes from aspirates on
the classic Novy-MacNeal-Nicolle (NNN) medium or other suitable
media at 22°C to 24°C, and populations double only every 2 to 3
days. 163,164 Inoculation of material into the susceptible golden hamster
is no longer used to grow parasites.
Fig. 158.8 LEISHMANIA INFANTUM IN MACROPHAGE FROM
BONE MARROW. Although typically found in macrophages as shown here, Molecular Methods
isolated extracellular amastigotes from disrupted host cells are commonly seen
in such preparations.
The PCR may detect low levels of infection and has been used for
diagnosis and monitoring of treatment as well as to determine the
genotype of the parasites. 165,166 Blood from filter paper can be used
and/or increased splenic clearance. After treatment, hematologic to prepare DNA, but as for direct morphology, PCR from bone
recovery is slow, and full recovery may take many months. 155 marrow samples is a more sensitive method to demonstrate para-
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sites. It may be particularly useful for monitoring treatment and
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relapse in HIV- and Leishmania-infected patients. Isothermal DNA
Laboratory Features amplification methods are being applied to develop tests to be used
where sophisticated equipment is not available. 168
The epidemiologic context and clinical features can suggest a diag-
nosis of leishmaniasis. Hematologic and biochemical tests are non-
specific; biochemical tests may show mild elevation of bilirubin and Serologic Tests
transaminase levels and more commonly a raised level of alkaline
phosphatase. Previously the nonspecific formol-gel test was used to Serologic tests have to be interpreted in light of the clinical findings.
indicate hypergammaglobulinemia. Diagnosis is confirmed by direct The tests are useful to screen suspected cases or populations for VL.
examination of parasites by microscopy, culture, or polymerase chain However, they are unreliable in immunocompromised patients, who
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reaction (PCR) (for detailed review, see Boelaert et al and Srividya frequently are seronegative in spite of patent infection. Conversely,
162
et al ). those harboring a subclinical infection may be seropositive, but
samples may fail to yield evidence of parasites. Finally, serologic
findings are positive many years after treatment or self-cure.
Morphology An indirect IFAT or ELISA using freeze-dried Leishmania antigen
has sensitivity and specificity greater than 95%. A DAT is also avail-
The pathognomic amastigotes of Leishmania spp. can be found in a able with purified, freeze-dried antigen and is a highly sensitive and
169
variety of sources. The diagnosis is often made by splenic puncture specific test. The freeze-dried antigen is very stable even when
or by bone marrow aspiration where large numbers of amastigotes stored in extreme conditions.
may be seen within macrophages. Amastigotes are stained using The ELISA and immunochromatographic tests using Leishmania
Giemsa or other Romanowsky stains and demonstrate a purple recombinant K39 antigen are 100% sensitive and more than 98%
nucleus and the large anterior kinetoplast (see Fig. 158.8). specific in Asia. 170,171 The sensitivity and specificity of the DAT and
In nonimmunocompromised patients with VL, splenic aspirates K39 immunochromatographic test are broadly similar, although the
are positive in more than 95%, bone marrow aspirates positive in K39 test was less sensitive in the Sudan than in Asia. 161,172 An alterna-
more than 85%, buffy coat macrophages positive in more than 70%, tive target antigen for serologic tests such as recombinant K28 and a
and lymph nodes positive in more than 60%. In practice, samples rapid point-of-care test are now being developed. 21
are taken from the least invasive sites first, although lymphadenopathy Treatment in patients without HIV coinfection may be monitored
is not invariable. using urine antigen tests for detection of the K39 and K26 Leishmania
Where coinfection of HIV and leishmania occurs, amastigotes are antigens. These test results become negative 3 weeks after commenc-
more reliably found in bone marrow than in peripheral blood and ing treatment.
may also be demonstrated in biopsy specimens from a wide variety
of sites. Biopsy material can be stained with polyclonal anti-Leishmania
serum and indirect immunofluorescence. However, direct demonstra- Management
tion of parasites is less sensitive than culture or PCR.
General supportive care is important and includes correction of
nutritional and hematinic deficiencies, blood transfusion in severe
Splenic Aspiration anemia, and antibiotics for secondary infections (for review of diag-
nosis, therapy, and management, see report from the World Health
173
Splenic aspiration is rarely performed in North America and Europe Organization ).
but is a common diagnostic procedure in endemic areas. Aspiration Specific chemotherapy for VL uses a number of potentially toxic
is contraindicated in the presence of a bleeding tendency, portal drugs. Intercurrent infections must be identified and treated, and
hypertension, or a splenic hydatid cyst. Splenic aspirates are obtained general measures include improvement of nutritional status (for
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from the middle of the long axis of the spleen. After the skin is review of treatment, see Sundar et al and Copeland et al ). Use
cleaned, a 21-gauge needle attached to a 5-mL syringe is inserted of pentavalent antimonials, including sodium stibogluconate (Pento-
subcutaneously in line with the long axis of the spleen. The needle stam) and meglumine antimonate (Glucantime) are now limited,