Mohamad, Amira Wahida
(2018)
Isolation of leptospira spp. and serological diagnoses in patients with acute febrile illness in Hospital Universiti Sains Malaysia.
Masters thesis, Universiti Sains Malaysia.
Abstract
Leptospirosis is an acute febrile illness and re-emerging disease that occurs
worldwide and most incidence in tropical countries such as Malaysia. Leptospirosis is
caused by the pathogenic Leptospira species. The disease is maintained in the nature by
chronic renal infection of reservoir host particularly rodents and human transmission
occurs through indirect or direct contact with the urine of infected animals. Leptospirosis
is difficult to diagnose because of the unspecific symptoms and serological tests results
that need to be interpreted carefully. There is much overlap in the clinical presentation of
undifferentiated febrile illnesses, which includes leptospirosis, malaria, rickettsioses, and
arboviral diseases, it is not possible to reliably predict the pathogen based on clinical signs
and symptoms. Therefore, the aim of this study is to isolate the Leptospira spp. and to
perform serological diagonoses from patients with acute febrile illness in Hospital
Universiti Sains Malaysia (HUSM). This is a cross sectional descriptive study. All patients
(n= 109) were recruited from the emergency department of HUSM with the symptoms of
acute febrile illness. The blood samples were taken and inoculated in the modified
Ellinghausen McCullough Johnson Harris (EMJH) media with addition of different
concentration of 5-Fluorouracil. The cultures were incubated in incubator shaker at 30°C
for 6 months and examined weekly under dark-field microscopy for presence of
Leptospira. Serology tests which were immunochromatography test (ICT) and
microscopic agglutination test (MAT) were carried out to determine the presence ofspecific antibodies against Leptospira in the recruited patients. The positive cultures were
amplified and identified by PCR on 16S rRNA gene by sequencing. The presence of the
pathogenic genes also was determined by using nine pathogenic genes which are lfb1,
flaB, OmpL1, ligA, ligB, ligC, lipL21, lipL32 and lipL4. A total of 109 samples from
patients whose seek treatments at emergency department of HUSM were collected. Based
on microscopic observation under dark field microscope, 1.85% (n= 2/109) of the samples
were positive with Leptospira isolation which were labelled as B004 and B208. Only
2.75% (n=3/109) were positive when tested with ICT. All samples with positive and
intermediate ICT tested with MAT were all negative. In addition, sample with positive
culture (B004) was tested negative for ICT meanwhile, B208 was tested intermediate with
ICT and negative with MAT. Isolates B004 and B208 were identified by 16S rRNA as
Leptospira interrogans and Leptospira weilli respectively. Both of the isolates were
classified under pathogenic Leptospira and were determined by the presence of nine and
five pathogenic genes respectively. The constructed phylogenetic tree confirms the
genetic relationships between the two species which arised from different species under
pathogenic group. The optimization of different culture supplementation and type of
samples were conducted by using different type of serovars for isolation improvements.
The results showed whole blood and EMJH without addition of others supplement were
the best among others which were human serum and rabbit serum. In conclusion, two
pathogenic Leptospira isolates were successfully cultivated from patients with acute
febrile illness in HUSM and both were characterized by nine pathogenic genes. Further
study with comprehensive approaches need to be conducted to improve the isolation rate
and molecular study could be more explored.
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