Miscellaneous articles
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Human Rabies in a Romanian boy |
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M. Luminos1, G. Barboi2,
A. Draganescu1, A. Streinu Cercel1,
F. Staniceanu1, G. Jugulete1, A.
Visan1, C. Negulescu1,
M.A. Turcitu2
1
Matei Bals National Institute for Infectious Disease
2
National Reference Laboratory for Rabies, Institute for
Diagnosis and Animal Health
Rabies is endemic in the Balkan region, including Romania,
with the main reservoir being the red fox (Vulpes Vulpes).
However, a substantial number of wildlife cases other than
foxes have also been reported in Romania (Avram et al,
2006). Genetic characterization of rabies viruses
characterized showed a high degree of genetic diversity,
indicating several introduction from neighbouring countries
(Turcitu et al, 2010). Besides wildlife species, most rabies
cases in domestic animals are diagnosed in pets, i.e. dogs
and cats (WHO Rabies Bulletin Europe), therefore antirabic
vaccination is compulsory but only for dog owners. The close
contact to these animals and perhaps insufficient public
awareness about rabies increases the possibility of human
exposure to rabies virus (Barboi et al, 2007)
Rabies is inevitably fatal once clinical symptoms of the
disease are observed. Recently, a form of induced-coma is
suggested to have contributed to the survival of a teenager
infected with rabies and was used as “Milwaukee protocol” in
several human rabies patients with limited success (Hunter
et al., 2010). Here, we describe a human rabies case in a 11
years old female child from the southern part of Romania, In
the doctors’ fight to safe the life of the girl a similar
protocol was used.
Anamnesis
showed that the patient was bitten 6 weeks before developing
clinical signs by an unvaccinated domestic cat on the left
hand thumb region; in the same day, another 14 years old
male child was attacked by the same cat. Following these
events, but only for the 14 years old boy, proper antirabic
specific therapy was conducted after 48 hours.
Clinical signs
were represented by altered general condition, fever
(38-39ºC body temperature) and dysfagia starting 48 hours
prior to Slatina county hospital submission on 8th
of august. During the next day the patient status got worse,
with the installation of marked
psychomotor agitation, hydrofobia, laringeal spasm and
hiperosmia. Imagistic computer thomograph investigation did
not revealed any anathomopathologic lesions on the central
nervous system (data not shown).
Due to the suspicion of rabies and the rapid deterioration
of clinical status, the patient was transferred to Matei
Bals National Institute for Infectious Disease, Bucharest,
on 10th of august. Here, the following clinical
signs were registered: fever (38ºC),
marked
psychomotor agitation that alternated with somnolent
periods, aerophobia, hydrophobia, hiperosmia,
hypersalivation, dryness of mucous membranes and skin,
tachycardia (AV 170 beats/minute, arterial blood pressure
100/70 mmHg), without signs of inflamatory meningitis.
After 24 hours, the patient condition agravates, showing
spontaneous laryngospasm and respiratory disorders,
hipersalivation, tachycardia (170 beats/minute) and
mydriasis.
Treatment:
Initial treatment consisted of specific antirabic serum
administration (Favirab, 40U/kg body weight) and vaccination
(Verorab, 6 doses), along with protective antibiotherapy -
ceftriaxone 100mg/kgbw/24h, ciprofloxacin 25mg/kgbw/24h and
later on meropenem 120mg/kgbw/day with the evidentiation of
pulmonary implication (radiographic examination), hydration
treatment (glucose and electrolytes – intravenous route),
sedation with diazepam and/or phenobarbital.
As the situation got worse, it was decided to swich to
orotracheal intubation and assisted ventilation together
with drug induced coma
(midazolam, thiopental, ketamine), antiviral treatment (amantadine
100mg/dose administered twice per day, ribavirin 16mg/kgbw/dose
administered four times per day), symptomatic treatment
(anti-inflammatory/antipyretic
– ibuprofen, H2 antagonist - ranitidin)
and supplements (Q10 coenzyme, magnesium, vitamins B6 and
C), according to Milwaukee international protocol guidance.
Laboratory findings:
Biochemical examination:
Hyperuremia identified in the second biochemical screening
(102,7mg/dl) seems to be of pre-renal origin; this findings
are supported by normal creatinine levels identified in both
tests performed but also by normal specific gravity of the
urine and absence of proteinuria that can be attributed to
kidney failure (acute and/or chronic). Moreover, elevated
activity of
Creatine Kinase (CK) and Lactate Dehydrogenase (LDH)
shows clear implication of the muscle in terms of acute
myositis, situation that also supports the elevated levels
of BUN (blood urea nitrogen) and clearly indicates an
intense catabolic state that occurs in marked
psychomotor
situations, including rabies.
Another interesting finding is the elevated activity of
heart fraction of
Creatine Kinase (CKMB, most likely attributed to the
inflammation of the myocardium, supported also by clinical
findings in terms of
tachycardia and in concordance with the evolution of rabies.
Regarding serum transaminases (GOT, GPT and GGT), high
degree of activity can be observed, situation compatible
with severe hepatic injuries, most likely due to the
prolonged administration of sedatives and/or anesthetics
(diazepam, phenobarbital, ketamine).
Besides the above mentioned biochemical abnormalities,
several others were identified, however with low degree of
implications in the disease evolution: hypoalbuminemia
(2,5g/dl) identified in the second biochemical screening
might be a consequence of poor exogenous intake of protein
sources, directly chorelated with the imposibility of
feeding, hyponatremia observed in the first screening can be
atributed to vomiting episodes that might occur at this
stage, whereas hypernatremia from the second screening is
most likely atributed to the parenteral route administration
of electrolytes.
Biochemistry results
|
Test |
Screening date |
Reference values |
|
15.08.2010 |
03.09.2010 |
|
Glucose |
118 |
113 |
65-105 mg/dl |
|
Urea (BUN) |
12,1 |
102,7 |
15-36 mg/dl |
|
Creatinine |
0,3 |
0,8 |
0,2-0,7 mg/dl |
|
Natrium |
126 |
154 |
137-145 mmol/L |
|
Potassium |
2,7 |
3,8 |
3,6-5 mmol/L |
|
Amylase |
73 |
ND |
30-100 U/L |
|
Lipase |
364 |
ND |
23-300 U/L |
|
AST/GOT |
678 |
687 |
10-40 U/L |
|
ALT/GPT |
186 |
968 |
10-30 U/L |
|
GGT |
ND |
254 |
17-28 U/L |
|
Ionised Calcium |
3.8 |
ND |
3,36-4,8 mg/dl |
|
LDH |
1995 |
3711 |
380-700 U/L |
|
CK |
22876 |
544 |
80-230 U/L |
|
CKMB |
122 |
34 |
1-16 U/L |
|
Albumin |
|
2,5 |
3,7-5,6 g/dl |
|
Total Protein |
6,3 |
ND |
6,3-8,6 g/dl |
|
Total Calcium |
8,2 |
ND |
8,9-10,1 mg/dl |
|
Fe (iron) |
58 |
ND |
37-170 µg/dl |
ND = not determined
Haematological investigation:
In general, haematological findings seem to be
unspecific for the disease evolution, possibly with one
single exception of monocytosis in the first screening
that can suggest viral implication. Apart from this, and
only for the first screening, increase in white blood
cells and neutrophiles numbers can orientate to a
secondary bacterial infection (neutrophilic
leukocytosis), since for the second screening those
values were normal again due to the administration of
general antibiotherapy.
Interestingly, along with disease evolution, mild to
moderate anemia has been detected (second screening,
with low number of red blood cells, hemoglobin and
hematocrit levels), situation that might be attributed
to prolonged infection. Moreover, the chorelation
between low number of trombocytes (PLT), fibrinogen
decrease and increase in D dimers can be attributed to
disseminated intravascular coagulation.
Haematological results
|
Test |
Date |
Reference values |
|
10.08.2010 |
03.09.2010 |
|
WBC |
20,1 |
8,2 |
3,9-9,6 x 103/µL |
|
NE % |
74,4 |
55,4 |
37-73% |
|
LY % |
13,9 |
35,5 |
20-55% |
|
MO % |
10,7 |
7 |
2,5-10% |
|
EO % |
1 |
2 |
0,6-11% |
|
BA % |
0 |
0,3 |
0-2% |
|
NE # |
15 |
4,5 |
1,4-6,5 x 103/µL |
|
LY # |
2,7 |
2,9 |
1,2-3,4 x 103/µL |
|
MO # |
2.2 |
0,6 |
0-0,7 x 103/µL |
|
EO # |
0,2 |
0,2 |
0-0,7 x 103/µL |
|
BA # |
0 |
0 |
0-0,2 x 103/µL |
|
RBC |
4,67 |
3,6 |
3,9-5,7103 x 103/µL |
|
HGB |
13 |
11 |
12,1-17,2 g/dl |
|
HCT |
39,6 |
33,2 |
36,1-50,3% |
|
MCV |
84,8 |
92,3 |
82,2-97,4 fL |
|
MCH |
27,8 |
30,6 |
27,6-33,3pg |
|
MCHC |
32,8 |
33,2 |
33-34,8 g/dl |
|
RDW |
13 |
16 |
11,6-13,7% |
|
PLT |
309 |
138 |
200-400 x 103/µL |
|
MPV |
4,7 |
9 |
7,8-11 fL |
|
PCT |
0,15 |
ND |
0-0,99% |
|
PDW |
15,9 |
ND |
0-99,9% |
ND = not determined
Anatomo-pathological examination:
Macroscopic findings
showed meningeal and cerebral hyperaemia, rabies
encephalopathy, enlargement (congestion) of liver
(correlated with the increased activity of serum
transaminases), spleen (possibly due to prolonged
anaesthesia and infection) and kidney, pulmonary oedema,
enlargement of right atrium and ventricle (correlated with
the increased activity of CKMB).
Histopathological findings:
vast areas of brain tissue showing disruption of brain
substance with marked edema and fragmentation of nerve
fibers, sometimes with neuronal necrosis; rare
multinucleated astrocytes (gemistocytes), along with the
existence of eosionophilic inclusions in basal nuclea and
cerebellum (Babes-Negri bodies); moderate inflammatory
response through limfoplasmocitary infiltrations, reactive
microgliosis, marked microvascular proliferation.
RT-PCR:
Biological material
subjected to analysis was represented by cerebrospinal fluid
as well as saliva.
RNA isolation
was performed from cerebrospinal fluid using commercial kits
available – PureLink RNA Mini Kit (Invitrogen),
protocol recommended by the manufacturer, using 200µl of
sample and RNA elution in 50µl of water. For saliva samples,
an initial lysis with Trizol reagent (Invitrogen) was
performed using 250µl of sample, acording to manufacturer
instructions, followed by centrifugation and phases
separation. Aproximately 600µl of the upper RNA aqueos phase
was further purified and concentrated using PureLink RNA
Mcro Kit (Invitrogen), with elution in 20µl of water
Revers transcription and amplification
The near complete nucleoprotein open reading frame was
amplified using primers described by Bourhy (not published),
commercial OneStep RT-PCR Kit (Qiagen), protocol
recommended by the manufacturer, with final reaction volume
of 50µl and primer concentration of 0,6µM. Thermal profile
consist of one 50minutes at 50°C for revers -
transcription, 15 minutes at 95°C for inactivation of revers
transcriptases, initial denaturation and Taq activation, 40
PCR cycles of 95°C for 35 seconds, 55°C for 40 seconds and
72°C for 2 minutes, 8 minutes at 72°C final extension and
4°C upon gel loading (Figure 1).
For cerebrospinal fluid, no specific amplicons were obtained
at this stage, due to the low amount of viral RNA;
therefore, a second heminested PCR was conducted, using the
forward primer JW6 described by Heaton (Figure 2). The
partial amplification of the rabies nucleoprotein was
performed with the FastStart Taq DNA polymerase kit (Roche
Applied Science), following the manufacturer
recommendations, in a final concentration of 0.6µM primers,
2mM MgCl2, 200µM of each dNTP and 2 units of DNA
polymerase enzyme. A volume of 10µl of first stage PCR was
used in a final volume of 50µl per reaction.
PCR products purification and sequencing was conducted using
QIAquick Gel Extraction Kit (Qiagen), protocol
recommended by the manufacturer and subjected to direct
sequencing using BigDye Terminator V1.1 Cycle Sequencing
Kit on 3130 Genetic Analyzer (both Applied Bioscience).

Figure
1. RT-PCR results for saliva samples, Lane 1,9 – molecular
ladders, 2-4 – analyzed samples
5,7 – negative controls (NC), 6, 8 - positive controls (PC)

Figure 2. RT-PCR results for cerebrospinal fluid samples,
Lane 1– analyzed sample, 2, 3 – NC, 4, 5 – PC
Phylogenetic investigation
Sequence aligning and reconstruction was performed using
Bio Edit and Clustal W softwares,
resulting in partial but significant 1308bp (base pairs)
fragment length of the nucleoprotein gene. Phylogenetic
tree was obtained using MEGA 4.0 software,
Neighbour Joining algorithmic method, bootstrap value of
3000 replicates and Kimura 2 parameter (Figure 3).
Results showed that the human sequence falls into
previously described lineage RO#2, along with sequences
isolated mainly from the south-western part of the
country.
Figure 3. Phylogenetic tree of the Romanian rabies
isolates obtained using NJ algorithm, bootstrap support
of 3000 replicates. Laboratory strains SAD, Pasteur and
CVS were used as an outgroup. Human sequence is marked.
Discussion
Pathological and laboratory diagnostics confirmed the
clinical suspicion on rabies as a cause for the disease
and death in this girl. Especially molecular tools such
as RT-PCR proved their sensitivity in the early ante
mortem diagnosis as described before (Hunter et al.
2010). The genetic characterization supported the
exposure by a rabies virus from the southeast of
Romania.
The case reiterates the need for public health awareness
in respect to the risk of contracting rabies, especially
for the rural and suburban areas, where probability for
domestic animals to come in contact with infected wild
animals is higher. Moreover, for this particular areas,
aditional effort from the authorities to further
emphasize the risk seems to be needed, since not all
such events are treated with the same respect – proper
PEP was initiated only for the young boy. Finally, by
strengthening the response to such cases through early
diagnostic methods and
specific measures from the authorities in order to have the
same approach in case of rabies suspicion hopefully will
conduct in decreasing the period of time from the event to
proper treatment. Timely and adequate PEP as recommended by
WHO remains the mainstay for the prevention of human rabies.
Using the Milwaukee protocol offers clinicians a treatment
option, however, this will not prevent the majority of human
deaths. Nevertheless, each attempt allows for a discussion
on the pros and cons on using this protocol, perhaps
providing further insights into rabies pathogenesis and host
response to specific and/or symptomatic treatment.
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