(Looareesuwan 1999 ). These symptoms were
compared between patients with and without
malaria in the study population. Malaria was
diagnosed microscopically by staining thick and
thin blood films on a glass slide to visually iden-
tify the malaria parasites. Following skin disin-
fection (74.1 % ethanol and propan-2-ol 10 %)
with aerosol Softasept®N (B. Braun Melsungen
PLC, Melsungen, Germany), a minimum of
2.7 mL blood in EDTA Monovette®
(SARSTEDT, Nümbrecht, Germany) (red top)
was taken through venipuncture with a blood-
collection needle (Safety-Multifly®,
SARSTEDT). A thick blood film was prepared
from each patient. The blood spot was smeared
using a circular motion with the corner of a
spreader slide, being careful not to make the
preparation too thick, and permitting the smear
to dehydrate without fixing. After drying, the
spot was sullied with diluted Giemsa (1:20,
vol/vol) for 20 min and washed by placing the
film in buffered water for 3 min. The glass slide
was allowed to air dry in a vertical position and
was examined using a light microscope. As the
specimens were unfixed, the red cells lysed when
a water-based stain was applied. A thin blood
film was prepared by immediately placing the
smooth edge of a spreader slide in a drop of
blood, adjusting the angle between the slide and
spreader to 45, and then smearing the blood with
a swift and steady sweep along the surface. The
film was then permitted to air dry and was fixed
with complete methanol. After ventilation, the
sample was stained with thinned Giemsa (1:20,
vol/vol) for 20 min and then washed by briefly
plunging the glass slide in and out of a jar of
buffered water, as overdone washing would
decolorize the film. The glass slide was then
allowed to air dry in a vertical position and was
inspected under a light microscope (Chotivanich
et al. 2007 ).
The diagnosis of malaria was made according
to the gold standard blood smear. The polymer-
ase chain reaction (PCR) technique was used to
confirm malaria infection, to monitor follow-up
therapeutic response, and to detect drug resis-
tance. The classification of malaria was specified
in each case according to the latest edition of the
International Classification of Disease (ICD
B50-B54). The use of chemoprophylaxis was
compared in both groups.2.5 Laboratory EvaluationAfter the sample collection, the amount of
C-reactive protein (CRP) in the human serum
and plasma was measured in lithium heparin
SARSTEDT Monovette®4.7 mL (orange top)
using a standard immuno-turbidimetric assay on
the COBAS®INTEGRA system (normal value
<6 mg/L). Simultaneously, total bilirubin (nor-
mal range 0.1–1.0 mg/dL), creatinine (normal
range 0.7–1.2 mg/dL), and glucose (normal
range 55–110 mg/dL) were measured in the
plasma. Blood leukocyte count (normal range
4000–10,000/μL) was carried out by flow
cytometry after collection in EDTA Monovette®
2.7 mL. Along with the leukocyte count, hemo-
globin (normal range 14–18 g/dL), hematocrit
(normal range 41–53 %), and platelet (normal
range 140,000–400,000/μL) were determined
for all patients simultaneously. Thrombocytope-
nia was defined as a platelet count under
140,000/μL.2.6 Virological EvaluationVirological-serological tests were done on the
serum of all of the patients to exclude hepatitis
infection. For this purpose, two SARSTEDT
serum Monovette®4.7 mL (brown top) and two
EDTA Monovette®2.7 mL were used after a
venipuncture. The initial round of examination
consisted of hepatitis B serology using enzyme-
linked immunosorbent assay (ELISA), hepatitis
B surface antigen (HBsAg, anti-HBsAg, and
anti-HBcAg), antibodies against hepatitis C
virus (including a serological confirmatory test
if screening was positive), antibodies against
hepatitis A virus immunoglobulin (IgG and
IgM), antibodies against cytomegalovirus
(CMV) (IgG and IgM); and antibodies against
Epstein-Barr-Virus (EBV) (VCA IgG and IgM).
The subsequent investigation involved38 J. Yayan and K. Rasche