Miscellaneous articles

Human Rabies in a Romanian boy

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 8^th 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 10^th 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

ND = not determined

Haematological results

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.

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.

References: 

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3. Paul R. Heaton, Pamela JohnstonE, Lorraine M. MCElhinney, Roy Cowley, Elizabeth O’Sullivan, Janice E. Whitby. Heminested PCR Assay for Detection of Six Genotypes of Rabies and Rabies-Related Viruses. Journal Of Clinical Microbiology, 0095-1137/97/$04.0010 Nov. 1997, p. 2762–2766.

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Contents of this Bulletin

• The Journal
• Editorial
• Miscellaneous Articles
• Surveillance of Rabies
• Rabies Dynamics
• Trend Tables
• Maps
• List of Contributors