J Clin Microbiol. 2004 March; 42(3): 1337–1340.
doi: 10.1128/JCM.42.3.1337-1340.2004.
PMCID: PMC356840
Copyright © 2004, American Society for Microbiology
Rapid Detection of Klebsiella pneumoniae from Blood Culture Bottles by Real-Time PCR
Prathiba Kurupati,1 Carol Chow,2 Gamini Kumarasinghe,2 and Chit Laa Poh1*
Department of Microbiology, National University of Singapore, Singapore 117597,1 Department of Laboratory Medicine, National University Hospital, Singapore 119074, Republic of Singapore2
*Corresponding author. Mailing address: Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Republic of Singapore. Phone: (65) 687-43674. Fax: (65) 677-66872. E-mail: micpohcl@nus.edu.sg.
Received July 14, 2003; Revised August 27, 2003; Accepted November 17, 2003.
This article has been cited by other articles in PMC.
ABSTRACT
A LightCycler real-time PCR hybridization probe-based assay which detects a partial Klebsiella pneumoniae 16S rRNA gene was developed for the rapid identification of K. pneumoniae directly from growth-positive blood culture bottles (BACTEC 9240 system) within 2 h. No cross-reactivity was observed with 65 negative-control blood cultures that grew bacteria other than K. pneumoniae and 48 negative blood cultures from double-blind experiments, thus demonstrating 100% specificity when compared to results of conventional biochemical characterization. The assay also showed 100% sensitivity, as it correctly identified all 142 positive-control blood cultures and 4 from double-blind trials.
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Klebsiella pneumoniae is an important hospital-acquired pathogen that causes severe morbidity and mortality among the newborn, the elderly, and immunocompromised patients (3, 4, 15). K. pneumoniae infections acquired in hospitals can be difficult to treat, as many strains are highly resistant to treatment with broad-spectrum cephalosporins and aminoglycosides (1, 7, 8, 10, 13). The isolation of bacteria from blood culture bottles is usually indicative of a serious invasive infection (bacteremia) which requires urgent antimicrobial therapy. Microorganisms isolated from patients usually have different antimicrobial susceptibilities, and successful treatment is dependent on the prompt administration of the appropriate antibiotic (6, 9, 11, 14). K. pneumoniae is identified by various automated and manual products such as Vitek (BioMérieux, Inc.) and the Analytab Products (API) system (BioMérieux, Inc.) or by traditional biochemical tests, and the whole process requires at least 24 to 48 h.
In recent years, real-time PCR has emerged as a valuable tool for the rapid testing of various biological specimens and body fluids for the presence of microorganisms (2, 5, 12, 17). The development of highly sensitive and specific PCR assays has alleviated the problems typically associated with microorganisms that are found in low numbers in tissues or body fluids, that are difficult to culture, or that are serologically similar. The LightCycler system (Roche Molecular Biochemicals, Mannheim, Germany) offers two different fluorescence detection formats. The first employs SYBR Green I, which is a dye that binds nonspecifically to double-stranded DNA (16); the second uses hybridization probes which allow sequence-specific detection by using fluorescence energy transfer (FRET) between two fluorophores. This result is achieved by attaching the two fluorophores to two oligonucleotide probes designed to hybridize to a complementary region of the target gene, leaving a 1-nucleotide-wide gap. The LightCycler instrument has been developed for fast cycling and real-time monitoring of the amplification process and thus eliminates the need to carry out gel electrophoresis of the samples after PCR.
In this study, we describe a real-time PCR assay with specific primers and hybridization probes targeting the 16S rRNA gene for the direct detection of K. pneumoniae from positive blood culture bottles. The assay also provides specific and sensitive quantification of K. pneumoniae DNA.
We studied 142 blood cultures that were known to be positive controls for K. pneumoniae, 65 that were negative controls for K. pneumoniae, and 52 double-blind samples (Table 1) that showed positive growth in BACTEC Plus aerobic medium in the BACTEC 9240 system. DNA extractions were performed in all experiments by using the QIAamp blood and tissue kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions. DNA extracted from type strains which acted as positive and negative controls was stored at −20°C until use. Oligonucleotide primers were designed to amplify a 126-bp target sequence of the 16S rRNA gene spanning nucleotide positions 44 to 170 of the K. pneumoniae strain with EMBL databank accession no. X93214. The forward primer K16SF (15-mer; position 44 to 58) and reverse primer K16SR (16-mer; position 155 to 170) were used. Within the 16S rRNA gene, fluorescence-labeled probes were designed to hybridize to the species-specific region of K. pneumoniae. The sensor probe used was the K. pneumoniae sensor (19-mer; melting temperature [Tm] = 62.7°C) oligonucleotide labeled with LightCycler Red 640 at the 5′ end and phosphorylated at the 3′ end to block extension. The corresponding fluorescein isothiocyanate-labeled anchor probe at the 3′ end was the K. pneumoniae anchor (30-mer, Tm = 69.9°C) oligonucleotide that binds to the template strand at a distance of one base to the bound sensor probe.
TABLE 1.
Real-time PCR hybridization probe-based testing of microorganisms
REAL-TIME PCR SAMPLE PREPARATION.
The master mixture contained 2 μl of 10× LightCycler-FastStart DNA master hybridization probes (Roche Diagnostics), 3 mM MgCl2 (final concentration), 0.5 μM final concentration of K. pneumonia-specific primers, and 0.2 μM final concentration of hybridization probes. To complete the PCR mixtures, 18 μl of master mixture and 2 μl of a DNA preparation were added into the LightCycler glass capillaries. After a short centrifugation (700 × g for 10 s), the sealed capillaries were placed into the LightCycler.
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REAL-TIME PCR AMPLIFICATION AND MELTING-CURVE ANALYSIS.
Thermocycling conditions were optimized to one cycle of denaturation at 95°C for 10 min, followed by 30 amplification cycles (temperature transition rate of 20°C/s), each comprising denaturation (95°C for 10 s), annealing (55°C for 15 s), and extension (74°C for 10 s). PCR amplification was followed by melting-curve analysis from 40 to 95°C (temperature transition rate of 0.1°C/s) with continuous fluorescence readings. Fluorescence was measured continuously during the slow temperature rise to monitor the dissociation of the LightCycler Red 640-labeled sensor probe at the F2 channel. Fluorescence signals from F2 were plotted automatically in real time versus temperature (T) to produce melting curves for amplicons. Melting curves were then converted into melting peaks by plotting the negative derivative of fluorescence versus T (−dF2/dT versus T and −dF3/dT versus T). Water was used as the negative control and was included in each set of melting peak determinations. The entire process took approximately 40 min. The presence of the 126-bp amplicon was verified by agarose gel electrophoresis.
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SENSITIVITY OF REAL-TIME PCR ASSAY FOR K. PNEUMONIAE.
The sensitivity of the real-time PCR assay was optimized to 10 fg of purified K. pneumoniae DNA. Agarose gel electrophoresis of the amplicons showed that the band intensity correlated well with the calculated concentrations of amplicons (Fig. (Fig.11 A), and the sequence was analyzed (data not shown). Real-time PCR sensitivity optimization performed by using the K. pneumoniae type strain (ATCC 13883) resulted in a linear-regression curve across 107 to 1 genomic equivalents (10 ng to 1 fg of DNA) with an error rate of <1% and a correlation coefficient at −1 (Fig. (Fig.1B).1B). The intraassay variation of the threshold/cycle (CT) values was evaluated by using K. pneumoniae DNA dilutions containing 107, 106, 105, 104, 103, 102, and 10 copies. CT values were established between 10 and 27. The CT values, which are inversely related to the quantity of organisms, ranged between 10 and 27 corresponded to 107 to 10 organisms (Fig. (Fig.1B1B).
FIG. 1.
Standard curve for K. pneumoniae DNA quantification. (A) Agarose gel electrophoresis of a 126-bp PCR product. Lane W represents the negative control (water). Lane M represents the 100-bp ladder DNA marker; lanes 1 to 8 represent the 10-fold-dilution series (more ...)
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SPECIFICITY OF THE REAL-TIME PCR ASSAY.
The specificity of the real-time PCR assay for K. pneumoniae with primers specific for the 16S rRNA gene was investigated by testing 1 ng of purified DNA from each of the 65 negative-control organisms (Table 1). Amplification of Pseudomonas aeruginosa, Streptococcus spp., Staphylococcus aureus, Enterococcus faecalis, Haemophilus influenzae, and Neisseria meningitidis remained negative when tested at all concentrations (10 ng to 1 fg), whereas the CT values for Klebsiella oxytoca, Escherichia coli, and Enterococcus faecium were greater than 29 at a concentration of 1 ng/μl. By comparison, a CT value of ≤13 was achieved for K. pneumoniae at the same concentration of 1 ng/μl (Fig. (Fig.2).2). Upon agarose gel electrophoresis of PCR mixtures derived from negative-control organisms, no amplicon was ever observed. The target sequence was shown to be highly specific for K. pneumoniae, as it failed to detect any other bacteria that are known to cause bacteremia (Fig. (Fig.22).
FIG. 2.
Specificity of the real-time PCR assay for K. pneumoniae. Different bacterial species were subjected to DNA extraction and quantitation by the real-time PCR assay. Data for fluorescence versus cycle number plots for clinical samples are shown.
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MELTING-CURVE ANALYSIS OF REAL-TIME PCR ASSAY FOR K. PNEUMONIAE.
When the temperature reached the Tm of the probes, a rapid loss of fluorescence was observed as the two adjacently bound probes dissociated from their complementary targets and FRET stopped. This was plotted as the negative derivative of fluorescence versus temperature to define the template-specific melting curves (Fig. (Fig.3).3). Without changing the MgCl2 concentration, we could still maintain a sensitivity of 10 organisms per sample, as shown by a 10-fold dilution series from 107 to 1 genome equivalent(s) of our reference strain. With the hybridization probes, the PCR products were found to reproducibly yield a melting peak of 66°C (Fig. (Fig.33).
FIG. 3.
Melting curve for the LightCycler KPS and KPA FRET hybridization probes. The specific melting peak can be observed with the quantified number of copies of K. pneumoniae DNA.
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DOUBLE-BLIND TESTING OF BLOOD CULTURE BOTTLES.
Four of 52 double-blind samples tested positive for K. pneumoniae, with a CT value of <20, and 48 samples displayed no detectable fluorescence above the background level. Double-blind PCR-based testing results were concordant with those of biochemical identification.
PCR-based testing of all 259 blood samples by real-time PCR demonstrated a sensitivity of 100% (146 of 146) and a specificity of 100% (113 of 113). The molecular assay was completed within 2 h upon receiving BACTEC 9240 positive blood culture bottles. No contamination was observed at any time during the study. No false-positive signal was detected when the negative control (which comprised sterile water, primers, and probes) was amplified in the reaction capillaries. In contrast to conventional PCR methods, they are less prone to contamination because postamplification handling is eliminated (12). Our real-time PCR-based approach for identification of K. pneumoniae resulted in significant time saving in comparison to traditional biochemical identification of K. pneumoniae from BACTEC 9240 positive blood cultures.
In conclusion, real-time PCR offers a fast tool with high sensitivity and specificity for the identification of K. pneumoniae from positive blood cultures. If in the future it becomes possible to detect more pathogens by multiplex real-time PCR, this method could become a suitable technology for implementation in routine diagnostic laboratories. It is also suitable for laboratories which encounter high isolation rates for K. pneumoniae or in outbreak situations.
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Kurupati, P.
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Kumarasinghe, G.
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Rapid Detection of Klebsiella pneumoniae from Blood Culture Bottles by Real-Time PCR
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PubMed
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Molecular epidemiology of plasmid spread among extended broad-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a pediatric hospital.
[J Clin Microbiol. 1993]
Multiresistant Klebsiella pneumoniae in a neonatal nursery: the importance of maintenance of infection control policies and procedures in the prevention of outbreaks.
[J Hosp Infect. 1992]
Outbreak of ceftazidime resistance caused by extended-spectrum beta-lactamases at a Massachusetts chronic-care facility.
[Antimicrob Agents Chemother. 1990]
Outbreak of nosocomial infections due to Klebsiella pneumoniae producing SHV-4 beta-lactamase.
[Eur J Clin Microbiol Infect Dis. 1990]
See more articles cited in this paragraph
Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes.
[J Clin Microbiol. 2001]
Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR.
[J Clin Microbiol. 2001]
Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR.
[J Clin Microbiol. 2001]
Molecular beacons: probes that fluoresce upon hybridization.
[Nat Biotechnol. 1996]
Analysis of double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection using the monomeric dye SYBR green I.
[Anal Biochem. 1995]
Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR.
[J Clin Microbiol. 2001]
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Cefotaxime Dosage
H. Sumano1, Lilia Gutierrez2, L. Ocampo3
1Department of Physiology and Pharmacology, College of Veterinary Medicine, National Autonomous University of Mexico 04510 Mexico City, Mexico
2Department of Physiology and Pharmacology, School of Veterinary Medicine, National Autonomous University of Mexico Mexico City 04510, Mexico
3Department of Physiology and Pharmacology, College of Veterinary Medicine, National Autonomous University of Mexico 04510 Mexico City, Mexico
Abstract
Considering the already known pharmacological features of cefotaxime, a study with two approaches of pharmacokinetics and clinical efficacy in septicaemic dogs was carried out. Pharmacokinetic variables were defined for doses of 10 mg/kg, and 20 mg/kg, utilising a quantitative bacteriological analysis. Values for half-life (T½ß) at 10 mg/kg were 0.8, 1.48 and 1.52 h for the i.v., s.c. and i.m. routes, respectively. Corresponding values for the 20 mg/kg dose for the same routes were 0.8, 1.49 and 1.53 h, respectively. Relatively fast clearance (ranging from 0.58 to 0.64 L/kg/h) allowed a maximum dose interval of 12 h. The above-stated doses of cefotaxime were administered i.v. to 40 cases of septicaemia, clinically divided into 20 moderately severe cases treated with 10 mg/kg i.v., of cefotaxime bid, and 20 severe ones, treated with 20 mg/kgi.v. of cefotaxime bid. Injections continued until a previously defined criterion of 'clinically recovered' was obtained. Thereafter, a follow-up treatment was established using the same dose and dose-interval but through the s.c. route. Due to the apparent volumes of distribution obtained (ranging from 0.48 to 0.51 L/kg), considering the overall clinical efficacy obtained (90% for the 10 mg/kg dose and 75% for the 20 mg/kg dose), and due to the rapid improvement observed after a few doses of the drug (1.8 to 2.5 doses to 'clinical improvement'), it is safe to postulate such doses of cefotaxime as excellent choices for the treatment of septicaemia in dogs.
Keywords
pharmacokinetics, pharmacokinetics, pharmacokinetics, dogs, Cefotaxime, septicaemia, efficacy
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[CrossRef] [PubMed]
Trudel, J. L., Wittnich, C. and Brown, R. A. (1994): Antibiotics bioavailability in acute experimental pancreatitis. J. Am. College. Surg. 178, 475-479.
Tsai, T. H., Chen, Y. F., Chen, K. C., Shum, A. Y. and Chen, C. F. (2000): Concurrent quantification and pharmacokinetic analysis of cefotaxime in rat blood and brain by microdialysis and microbore liquid chromatography. J. Chromatogr. 738, 75-81.
[CrossRef]
Viladrich, P. F., Cabellos, C., Pallares, R., Tubau, F., Martinez-Lacasa, J., Linares, J. and Gudiel, F. (1996): High doses of cefotaxime in treatment of adult meningitis due to Streptococcus pneumoniae with decreased susceptibilities to broad-spectrum cephalosporins. Antimicrob. Agents Chemother. 40, 218-220.
[PubMed]
Wang, R., Fang, Y. and Du, L. H. (1998): Influencing factors of postantibiotic effect of 4 cephalosporins. Chin. J. Clin. Pharmacy 7, 77-80.
Withrow, S. J. (1979): Dental extraction as a probable cause of septicemia in a dog. J. Am. Anim. Hosp. Assoc. 115, 345-346.
Ziv, G., Cohen, R. O., Winkler, M. and Saran, A. (1996): Antimicrobial susceptibility of Escherichia coli and Streptococcus sp. recovered from the uterus of dairy cows with post parturient metritis. Israel J. Vet. Med. 51, 63-66.
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1Department of Physiology and Pharmacology, College of Veterinary Medicine, National Autonomous University of Mexico 04510 Mexico City, Mexico
2Department of Physiology and Pharmacology, School of Veterinary Medicine, National Autonomous University of Mexico Mexico City 04510, Mexico
3Department of Physiology and Pharmacology, College of Veterinary Medicine, National Autonomous University of Mexico 04510 Mexico City, Mexico
Abstract
Considering the already known pharmacological features of cefotaxime, a study with two approaches of pharmacokinetics and clinical efficacy in septicaemic dogs was carried out. Pharmacokinetic variables were defined for doses of 10 mg/kg, and 20 mg/kg, utilising a quantitative bacteriological analysis. Values for half-life (T½ß) at 10 mg/kg were 0.8, 1.48 and 1.52 h for the i.v., s.c. and i.m. routes, respectively. Corresponding values for the 20 mg/kg dose for the same routes were 0.8, 1.49 and 1.53 h, respectively. Relatively fast clearance (ranging from 0.58 to 0.64 L/kg/h) allowed a maximum dose interval of 12 h. The above-stated doses of cefotaxime were administered i.v. to 40 cases of septicaemia, clinically divided into 20 moderately severe cases treated with 10 mg/kg i.v., of cefotaxime bid, and 20 severe ones, treated with 20 mg/kgi.v. of cefotaxime bid. Injections continued until a previously defined criterion of 'clinically recovered' was obtained. Thereafter, a follow-up treatment was established using the same dose and dose-interval but through the s.c. route. Due to the apparent volumes of distribution obtained (ranging from 0.48 to 0.51 L/kg), considering the overall clinical efficacy obtained (90% for the 10 mg/kg dose and 75% for the 20 mg/kg dose), and due to the rapid improvement observed after a few doses of the drug (1.8 to 2.5 doses to 'clinical improvement'), it is safe to postulate such doses of cefotaxime as excellent choices for the treatment of septicaemia in dogs.
Keywords
pharmacokinetics, pharmacokinetics, pharmacokinetics, dogs, Cefotaxime, septicaemia, efficacy
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Sumano, L. H. and Ocampo, C. L. (1992): Farmacología Veterinaria. 2nd edition, 1992. McGraw-Hill, México, DF. pp. 125-138.
Sumano, L. H., Ocampo, C. L. and Pulido, E. (2000): Manual de farmacología clínica para pequeñas especies. 2nd edition, 2000. Laboratorios Virbac, México, DF. pp. 35-41.
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Turnidge, J. D. (1995): Pharmacodynamic (kinetic) considerations in the treatment of moderately severe infections with cefotaxime. Diagn. Microbiol. Infect. Dis. 22, 57-69.
[CrossRef] [PubMed]
Trudel, J. L., Wittnich, C. and Brown, R. A. (1994): Antibiotics bioavailability in acute experimental pancreatitis. J. Am. College. Surg. 178, 475-479.
Tsai, T. H., Chen, Y. F., Chen, K. C., Shum, A. Y. and Chen, C. F. (2000): Concurrent quantification and pharmacokinetic analysis of cefotaxime in rat blood and brain by microdialysis and microbore liquid chromatography. J. Chromatogr. 738, 75-81.
[CrossRef]
Viladrich, P. F., Cabellos, C., Pallares, R., Tubau, F., Martinez-Lacasa, J., Linares, J. and Gudiel, F. (1996): High doses of cefotaxime in treatment of adult meningitis due to Streptococcus pneumoniae with decreased susceptibilities to broad-spectrum cephalosporins. Antimicrob. Agents Chemother. 40, 218-220.
[PubMed]
Wang, R., Fang, Y. and Du, L. H. (1998): Influencing factors of postantibiotic effect of 4 cephalosporins. Chin. J. Clin. Pharmacy 7, 77-80.
Withrow, S. J. (1979): Dental extraction as a probable cause of septicemia in a dog. J. Am. Anim. Hosp. Assoc. 115, 345-346.
Ziv, G., Cohen, R. O., Winkler, M. and Saran, A. (1996): Antimicrobial susceptibility of Escherichia coli and Streptococcus sp. recovered from the uterus of dairy cows with post parturient metritis. Israel J. Vet. Med. 51, 63-66.
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canine parvo viral treatment
Canine Parvovirus Enteritis
by Dr. Randy Wetzel
Canine parvovirus (CPV) enteritis is a common cause of acute, severe gastroenteritis in young dogs. Dogs at greatest risk are less than 6 months of age and either unvaccinated or incompletely vaccinated for canine parvovirus. Dogs vaccinated with over-the-counter vaccines also appear to have a higher incidence of parvovirus likely due to ineffective vaccine or improper use. Early clinical signs of parvovirus enteritis include anorexia, lethargy, diarrhea and vomiting.
Parvovirus enteritis has a survival rate of 85-90% with aggressive treatment and recovery generally occurs in 3-7 days. For patients who do not receive aggressive and appropriate treatment, the mortality rates increase substantially. Death is generally due to sepsis and/or dehydration. Correcting dehydration and electrolyte imbalances and reducing further fluid loss is critical to successfully managing these patients. For most patients this requires hospitalization on IV fluids, IV antibiotics, antiemetics, and GI protectants as the mainstay of treatment. Once a patient is hydrated, we find synthetic colloids such as Hetastarch (20ml/kg/day CRI) beneficial in replacing intravascular volume, maintaining hydration and managing shock. Synthetic colloids are excellent in their ability to increase COP which is often a concern in patients with hypoalbuminemia and increased capillary permeability as is often the case with so many CPV enteritis patients. Use of synthetic colloids generally permits you to decrease crystalloid fluid rates by 40-60%. Natural colloids such as fresh frozen plasma (FFP) or fresh whole blood (FWB) may be beneficial for anemic patients or those needing further immune support. In addition to providing oncotic components, both whole blood and plasma contain antibodies and serum protease inhibitors which may be beneficial in neutralizing circulating virus and controlling the systemic inflammatory response associated with the disease.
Parvovirus patients are often suffering from bone marrow suppression and their immune systems are overwhelmed by the viral infection making them at high risk of sepsis. We use broad-spectrum antibiotics such as Ampicillin, 22mg/kg IV TID, and amikacin 10mg/kg, IV TID, once the patient is hydrated. For patients with leukopenia, enrofloxacin at 5mg/kg IV BID diluted 1:1, is considered safe to use short term (3-5 days). Butorphanol 0.3mg/kg IV is recommended for abdominal pain, and a lower dose (0.15mg/kg) has been suggested in the medical management of patients suspected of having intussusception.
Tamilflu (oseltamivir phosphate) (1mg/kg PO BID for 5 days) used to treat influenza in people has anecdotally shown to be beneficial in treating CPV enteritis and thought to reduce viral replication.
Monitoring a complete blood count (CBC) is beneficial in evaluating a patient's condition. Patients with severe leukopenia raise concern about bone marrow suppression and risk of sepsis. I often find a poor correlation between the physical exam and attitude of the patient compared to the leukogram count. However, I do find positive correlation in patients with a low leukogram count and increased morbidity. These patients may benefit from more aggressive treatment immediately.
Treating patients with CPV enteritis is labor intensive and costly. A proper isolation ward and isolation protocol is important for reducing contamination to other patients. The profuse amount of vomiting and diarrhea makes proper isolation protocol challenging.
Parvovirus is shed in the feces by infected animals and this virus can remain stable in the environment for 7 months or longer. These viral particles can be transferred on the shoes, hands and clothes of people who then may expose an unprotected dog. It is very important to understand the ease of transmission and the fact that direct contact between the unprotected dog and the infected dog is not necessary for a pet to become exposed. Reducing risk of exposure is necessary, but it is vital to have pets inoculated with an effective vaccine at the appropriate time and properly administered. As an emergency clinician, I commonly see cases of parvovirus enteritis in patients who were vaccinated with vaccines attained through feed stores. The frequency at which this occurs raises a concern that the vaccines being used are ineffective or perhaps inappropriately administered. I commonly see mistakes in the timing of the vaccination when they are administered at home. It is important to understand the cycle of a puppy’s immune system with regard to maternal antibodies and duration of response to vaccination. Therefore initiating vaccination at 6 weeks of age by a veterinarian is strongly recommended.
References:
Rebecca Kirby, DVM, DACVIM, DACVECC
David Miller, BVSc, MMedVet
Douglass K. Macintire, DVM, MS, DACVIM, DACVECC
Alice M. Wolf, DVM, DACVIM, DABVP
by Dr. Randy Wetzel
Canine parvovirus (CPV) enteritis is a common cause of acute, severe gastroenteritis in young dogs. Dogs at greatest risk are less than 6 months of age and either unvaccinated or incompletely vaccinated for canine parvovirus. Dogs vaccinated with over-the-counter vaccines also appear to have a higher incidence of parvovirus likely due to ineffective vaccine or improper use. Early clinical signs of parvovirus enteritis include anorexia, lethargy, diarrhea and vomiting.
Parvovirus enteritis has a survival rate of 85-90% with aggressive treatment and recovery generally occurs in 3-7 days. For patients who do not receive aggressive and appropriate treatment, the mortality rates increase substantially. Death is generally due to sepsis and/or dehydration. Correcting dehydration and electrolyte imbalances and reducing further fluid loss is critical to successfully managing these patients. For most patients this requires hospitalization on IV fluids, IV antibiotics, antiemetics, and GI protectants as the mainstay of treatment. Once a patient is hydrated, we find synthetic colloids such as Hetastarch (20ml/kg/day CRI) beneficial in replacing intravascular volume, maintaining hydration and managing shock. Synthetic colloids are excellent in their ability to increase COP which is often a concern in patients with hypoalbuminemia and increased capillary permeability as is often the case with so many CPV enteritis patients. Use of synthetic colloids generally permits you to decrease crystalloid fluid rates by 40-60%. Natural colloids such as fresh frozen plasma (FFP) or fresh whole blood (FWB) may be beneficial for anemic patients or those needing further immune support. In addition to providing oncotic components, both whole blood and plasma contain antibodies and serum protease inhibitors which may be beneficial in neutralizing circulating virus and controlling the systemic inflammatory response associated with the disease.
Parvovirus patients are often suffering from bone marrow suppression and their immune systems are overwhelmed by the viral infection making them at high risk of sepsis. We use broad-spectrum antibiotics such as Ampicillin, 22mg/kg IV TID, and amikacin 10mg/kg, IV TID, once the patient is hydrated. For patients with leukopenia, enrofloxacin at 5mg/kg IV BID diluted 1:1, is considered safe to use short term (3-5 days). Butorphanol 0.3mg/kg IV is recommended for abdominal pain, and a lower dose (0.15mg/kg) has been suggested in the medical management of patients suspected of having intussusception.
Tamilflu (oseltamivir phosphate) (1mg/kg PO BID for 5 days) used to treat influenza in people has anecdotally shown to be beneficial in treating CPV enteritis and thought to reduce viral replication.
Monitoring a complete blood count (CBC) is beneficial in evaluating a patient's condition. Patients with severe leukopenia raise concern about bone marrow suppression and risk of sepsis. I often find a poor correlation between the physical exam and attitude of the patient compared to the leukogram count. However, I do find positive correlation in patients with a low leukogram count and increased morbidity. These patients may benefit from more aggressive treatment immediately.
Treating patients with CPV enteritis is labor intensive and costly. A proper isolation ward and isolation protocol is important for reducing contamination to other patients. The profuse amount of vomiting and diarrhea makes proper isolation protocol challenging.
Parvovirus is shed in the feces by infected animals and this virus can remain stable in the environment for 7 months or longer. These viral particles can be transferred on the shoes, hands and clothes of people who then may expose an unprotected dog. It is very important to understand the ease of transmission and the fact that direct contact between the unprotected dog and the infected dog is not necessary for a pet to become exposed. Reducing risk of exposure is necessary, but it is vital to have pets inoculated with an effective vaccine at the appropriate time and properly administered. As an emergency clinician, I commonly see cases of parvovirus enteritis in patients who were vaccinated with vaccines attained through feed stores. The frequency at which this occurs raises a concern that the vaccines being used are ineffective or perhaps inappropriately administered. I commonly see mistakes in the timing of the vaccination when they are administered at home. It is important to understand the cycle of a puppy’s immune system with regard to maternal antibodies and duration of response to vaccination. Therefore initiating vaccination at 6 weeks of age by a veterinarian is strongly recommended.
References:
Rebecca Kirby, DVM, DACVIM, DACVECC
David Miller, BVSc, MMedVet
Douglass K. Macintire, DVM, MS, DACVIM, DACVECC
Alice M. Wolf, DVM, DACVIM, DABVP
Amikacine Dose rate Canines
Description
Amikacin sulfate is a semi-synthetic aminoglycoside antibiotic derived from kanamycin. It is C22H43N5O13•2H2SO4,D-streptamine, 0-3-amino-3-deoxy- α- D-glucopyranosyl-(1→6)-0-[6-amino-6-deoxy- α - D-glucopyranosyl-(1→4)]-N1-(4-amino-2- hydroxy-1- oxobutyl)-2-deoxy-,(S)-, sulfate (1:2)(salt).
The dosage form supplied is a sterile, colorless to straw colored solution containing, in addition to amikacin sulfate, 2.5% sodium citrate, pH adjusted with sulfuric acid, 0.66% sodium bisulfite added, and 0.1 mg benzethonium chloride per mL as a preservative.
Each Ml Of Solution Contains
Amikacin (as the sulfate)
50 mg
Sodium citrate, USP (as buffer)
25.1 mg
Sodium bisulfite
6.6 mg
Benzethonium chloride, USP (as preservative)
0.1 mg
Water for injection, USP
q.s.
pH adjusted with sulfuric acid.
Action
Amikacin, like other aminoglycoside antibiotics, is a bactericidal agent that exerts its action at the level of the bacterial ribosome. Amikacin has been shown to be effective against many aminoglycoside-resistant strains due to its ability to resist degradation by aminoglycoside inactivating enzymes known to affect gentamicin, tobramycin and kanamycin(1).
Microbiology
Amikacin has been shown to be effective in the treatment of skin and soft tissue infections caused by Pseudomonas sp and Escherichia coli and in urinary tract infections caused by Escherichia coli and Proteus sp. The susceptibility of veterinary isolates to amikacin is summarized in the following table:
Organism (no. Of Isolates) Minimum Inhibitory Concentration (mcg/ml)
Range Mic90*
Escherichia coli (50)
1-32
4
Proteus mirabilis (50)
1-128
6
Enterobacter sp. (50)
0.5-128
4
Staphylococcus aureus (50)
1-128
2
Klebsiella pneumoniae (50)
0.5-16
2
Pseudomonas aeruginosa (50)
1-64
8
*Concentration at which 90% of the isolates are susceptible.
In addition, the following microorganisms have been shown to be susceptible to amikacin in vitro(2), although the clinical significance of this action has not been demonstrated in animals:
Serratia marcescens, Salmonella sp, Shigella sp, Providencia sp, Citrobacter freundii, Listeria monocytogenes
The aminoglycoside antibiotics in general have limited activity against gram-positive pathogens, although Staphylococcus aureus and Listeria monocytogenes are susceptible to amikacin as noted above.
Pharmacokinetics
Amikacin is well absorbed following intravenous, subcutaneous, or intramuscular injection but is not appreciably absorbed orally. The serum half-life (T1/2) averages from 1 to 2 hours in dogs depending on the route of administration(3). Amikacin is excreted unchanged in urine, concentrations in excess of 1,000 mcg/mL typically being achieved within three hours in dogs. Serum concentration-time profiles in dogs following subcutaneous administration are illustrated graphically in Figure 1.
Figure 1
Amikacin Concentration-Time Curves in Dogs (n=6) Following Subcutaneous Injection
Toxicology
The intravenous and intramuscular LD50 in dogs is greater than 250 mg/kg. Like other aminoglycosides, amikacin has nephrotoxic, neurotoxic, and ototoxic potential. In dogs, minimal to mild renal changes were detectable histopathologically after amikacin dosage of 45 mg/kg/day for two weeks, and dogs receiving a dosage of 30 mg/kg/day for 90 days had minimal renal alterations which were believed to be entirely reversible. Urinary casts were not observed in dogs receiving a 30 mg/kg dose for 90 days. In efficacy studies involving 80 infected dogs treated with amikacin at the recommended dosage rate of 10 mg/kg b.i.d. for 8-21 consecutive days, no evidence of nephrotoxicity or any other toxicity was encountered. Regarding ototoxicity, studies in cats reveal that amikacin has less potential for ototoxicity than gentamicin(4).
Amikacin K-9 Injection Indications
Amikacin K-9 Injection (Amikacin Sulfate Injection) is indicated for the treatment of the following conditions in dogs: Genitourinary tract infections (cystitis) caused by susceptible strains of Escherichia coli and Proteus sp. Skin and soft tissue infections caused by susceptible strains of Pseudomonas sp and Escherichia coli.
While nearly all strains of Escherichia coli, Pseudomonas sp, and Proteus sp, including those that are resistant to gentamicin, kanamycin or other aminoglycosides, are susceptible to amikacin at levels achieved following treatment, it is recommended that the invading organism be cultured and its susceptibility demonstrated as a guide to therapy. Amikacin susceptibility discs, 30 mcg, should be used for determining in vitro susceptibility.
Contraindications
Systemic aminoglycoside therapy is contraindicated in dogs with seriously impaired renal function.
Precautions
The following conditions have been found to contribute to the toxicity of aminoglycosides in dogs:
-Prior renal damage (most commonly found in dogs of advanced age) and dogs infected with heartworm microfilaria.(5)
-Hypovolemic dehydration (dehydrated patients should be rehydrated prior to initiating therapy).
In dogs where decreased renal function is suspected prior to treatment, BUN or serum creatinine levels may not indicate the degree of kidney impairment. A creatinine clearance determination may be more useful.
Monitoring of renal function during treatment is recommended. Although there is not a completely reliable monitoring program for aminoglycoside toxicity, urinalysis may indicate early nephrotoxicity. Unfavorable changes in the urinalysis which may indicate toxicity include:
-Decreased specific gravity in the absence of fluid therapy.
-Appearance in the urine of casts, albumin, glucose or blood.
Continued use of aminoglycosides where any functional renal impairment has occurred may lead to enhanced renal damage as well as increased likelihood of ototoxicity and/or neuromuscular blockade.(6)
Concurrent or sequential use of topically or systemically administered nephrotoxic, ototoxic, or neuromuscular blocking drugs, particularly other aminoglycosides such as streptomycin, gentamicin, kanamycin and neomycin should be avoided because of the potential for additive effects.
Concurrent administration of furosemide with aminoglycosides may enhance nephrotoxicity.(7)
Not for use in breeding dogs as reproductive studies have not been conducted.
Neurotoxic and nephrotoxic antibiotics may be absorbed in significant quantities from body surfaces after local irrigation or application. The potential toxic effect of antibiotics administered in this fashion should be considered.(8)
If hypersensitivity develops, treatment with Amikacin K-9 Injection (Amikacin Sulfate Injection) should be discontinued and appropriate therapy instituted.
Warning
Amikacin should be used with extreme caution in dogs, in which hearing acuity is required for functioning, such as seeing eye, hearing ear or military patrol, as the auditory and vestibular impairment tends not to be reversible.(6)
Aminoglycosides, including amikacin, are not indicated in uncomplicated episodes of cystitis unless causative agents are susceptible to them and are not susceptible to antibiotics having less potential for toxicity.
Early signs of ototoxicity can include ataxia, nausea and vomiting. Auditory and vestibular impairment may be reversible in the very early stages, but if treatment is continued, the conditions will become irreversible.(6)
Adverse Reactions
In clinical studies in dogs, transient pain on injection has been reported as well as rare cases of vomiting or diarrhea following amikacin therapy. In 90 day intramuscular toxicology studies, evidence of muscle damage was detected histologically as well as by elevated creatinine phosphokinase.
Amikacin K-9 Injection Dosage And Administration
Amikacin K-9 Injection (Amikacin Sulfate Injection) should be administered subcutaneously or intramuscularly at a dosage of 10 mg/kg (5 mg/lb) twice daily. Dogs with skin and soft tissue infections should be treated for a minimum of 7 days and those with genitourinary infections should be treated for 7 to 21 days or until culture negative and asymptomatic. If no response is observed after three days of treatment, therapy should be discontinued and the case re-evaluated. Maximum duration of therapy should not exceed 30 days.
Amikacin K-9 Injection Caution
Federal law restricts this drug to use by or on the order of a licensed veterinarian.
Supply
50 mL vial, 50 mg/mL, Amikacin K-9 Injection (Amikacin Sulfate Injection).
Store vials between 15° and 30°C, (59° and 86°F). At times the solution may become pale yellow in color. This does not indicate a decrease in potency.
References
1.Davies, J., Courvalin, P.: Mechanisms of Resistance to Aminoglycosides. Am. J. Med. 62: 868-872, 1977.
2.Price, K.E., et al: Microbiological Evaluation of BB-K8, a New Semisynthetic Aminoglycoside. J. Antibiot. 25: 709-731, 1972.
3.Baggot, J.D., Ling, G.V., and Chaffield, R.C.: The Pharmacokinetics of Amikacin in Dogs. Am. J. Vet. Res., 46:1793-1796, 1985.
4.Christensen, E.F., Reiffenstein, J.C., and Madissoo, H.: Comparative Ototoxicity of Amikacin and Gentamicin in Cats. Antimicrob. Agents Chemother. 12: 178-184, 1977.
5.Casey, H.W., D.K. Obeck and G.A. Splitter, "Immunopathology Studies on Canine Heartworm", Canine Heartworm Disease, the Current Knowledge, University of Florida Press, 31-32, 1973.
6.Kirk, R.W., ed., Current Veterinary Therapy VIII Small Animal Practice, W.B. Saunders Company, Philadelphia, PA, 119, 731, 1040, 1983.
7.Raisbeck, M.F., W.R. Hewitt and W.B. McIntyre, "Fatal Nephrotoxicosis Associated with Furosemide and Gentamicin Therapy in a Dog", Journal of American Veterinary Medical Association, 183: 892-893, 1983.
8.Riviere, J.E., and L.E. Davis, 1964, Renal Handling of Drugs in Renal Failure, Harwal Publishers, 666-667, 1984.
600072
Iss. 11-01
Amikacin sulfate is a semi-synthetic aminoglycoside antibiotic derived from kanamycin. It is C22H43N5O13•2H2SO4,D-streptamine, 0-3-amino-3-deoxy- α- D-glucopyranosyl-(1→6)-0-[6-amino-6-deoxy- α - D-glucopyranosyl-(1→4)]-N1-(4-amino-2- hydroxy-1- oxobutyl)-2-deoxy-,(S)-, sulfate (1:2)(salt).
The dosage form supplied is a sterile, colorless to straw colored solution containing, in addition to amikacin sulfate, 2.5% sodium citrate, pH adjusted with sulfuric acid, 0.66% sodium bisulfite added, and 0.1 mg benzethonium chloride per mL as a preservative.
Each Ml Of Solution Contains
Amikacin (as the sulfate)
50 mg
Sodium citrate, USP (as buffer)
25.1 mg
Sodium bisulfite
6.6 mg
Benzethonium chloride, USP (as preservative)
0.1 mg
Water for injection, USP
q.s.
pH adjusted with sulfuric acid.
Action
Amikacin, like other aminoglycoside antibiotics, is a bactericidal agent that exerts its action at the level of the bacterial ribosome. Amikacin has been shown to be effective against many aminoglycoside-resistant strains due to its ability to resist degradation by aminoglycoside inactivating enzymes known to affect gentamicin, tobramycin and kanamycin(1).
Microbiology
Amikacin has been shown to be effective in the treatment of skin and soft tissue infections caused by Pseudomonas sp and Escherichia coli and in urinary tract infections caused by Escherichia coli and Proteus sp. The susceptibility of veterinary isolates to amikacin is summarized in the following table:
Organism (no. Of Isolates) Minimum Inhibitory Concentration (mcg/ml)
Range Mic90*
Escherichia coli (50)
1-32
4
Proteus mirabilis (50)
1-128
6
Enterobacter sp. (50)
0.5-128
4
Staphylococcus aureus (50)
1-128
2
Klebsiella pneumoniae (50)
0.5-16
2
Pseudomonas aeruginosa (50)
1-64
8
*Concentration at which 90% of the isolates are susceptible.
In addition, the following microorganisms have been shown to be susceptible to amikacin in vitro(2), although the clinical significance of this action has not been demonstrated in animals:
Serratia marcescens, Salmonella sp, Shigella sp, Providencia sp, Citrobacter freundii, Listeria monocytogenes
The aminoglycoside antibiotics in general have limited activity against gram-positive pathogens, although Staphylococcus aureus and Listeria monocytogenes are susceptible to amikacin as noted above.
Pharmacokinetics
Amikacin is well absorbed following intravenous, subcutaneous, or intramuscular injection but is not appreciably absorbed orally. The serum half-life (T1/2) averages from 1 to 2 hours in dogs depending on the route of administration(3). Amikacin is excreted unchanged in urine, concentrations in excess of 1,000 mcg/mL typically being achieved within three hours in dogs. Serum concentration-time profiles in dogs following subcutaneous administration are illustrated graphically in Figure 1.
Figure 1
Amikacin Concentration-Time Curves in Dogs (n=6) Following Subcutaneous Injection
Toxicology
The intravenous and intramuscular LD50 in dogs is greater than 250 mg/kg. Like other aminoglycosides, amikacin has nephrotoxic, neurotoxic, and ototoxic potential. In dogs, minimal to mild renal changes were detectable histopathologically after amikacin dosage of 45 mg/kg/day for two weeks, and dogs receiving a dosage of 30 mg/kg/day for 90 days had minimal renal alterations which were believed to be entirely reversible. Urinary casts were not observed in dogs receiving a 30 mg/kg dose for 90 days. In efficacy studies involving 80 infected dogs treated with amikacin at the recommended dosage rate of 10 mg/kg b.i.d. for 8-21 consecutive days, no evidence of nephrotoxicity or any other toxicity was encountered. Regarding ototoxicity, studies in cats reveal that amikacin has less potential for ototoxicity than gentamicin(4).
Amikacin K-9 Injection Indications
Amikacin K-9 Injection (Amikacin Sulfate Injection) is indicated for the treatment of the following conditions in dogs: Genitourinary tract infections (cystitis) caused by susceptible strains of Escherichia coli and Proteus sp. Skin and soft tissue infections caused by susceptible strains of Pseudomonas sp and Escherichia coli.
While nearly all strains of Escherichia coli, Pseudomonas sp, and Proteus sp, including those that are resistant to gentamicin, kanamycin or other aminoglycosides, are susceptible to amikacin at levels achieved following treatment, it is recommended that the invading organism be cultured and its susceptibility demonstrated as a guide to therapy. Amikacin susceptibility discs, 30 mcg, should be used for determining in vitro susceptibility.
Contraindications
Systemic aminoglycoside therapy is contraindicated in dogs with seriously impaired renal function.
Precautions
The following conditions have been found to contribute to the toxicity of aminoglycosides in dogs:
-Prior renal damage (most commonly found in dogs of advanced age) and dogs infected with heartworm microfilaria.(5)
-Hypovolemic dehydration (dehydrated patients should be rehydrated prior to initiating therapy).
In dogs where decreased renal function is suspected prior to treatment, BUN or serum creatinine levels may not indicate the degree of kidney impairment. A creatinine clearance determination may be more useful.
Monitoring of renal function during treatment is recommended. Although there is not a completely reliable monitoring program for aminoglycoside toxicity, urinalysis may indicate early nephrotoxicity. Unfavorable changes in the urinalysis which may indicate toxicity include:
-Decreased specific gravity in the absence of fluid therapy.
-Appearance in the urine of casts, albumin, glucose or blood.
Continued use of aminoglycosides where any functional renal impairment has occurred may lead to enhanced renal damage as well as increased likelihood of ototoxicity and/or neuromuscular blockade.(6)
Concurrent or sequential use of topically or systemically administered nephrotoxic, ototoxic, or neuromuscular blocking drugs, particularly other aminoglycosides such as streptomycin, gentamicin, kanamycin and neomycin should be avoided because of the potential for additive effects.
Concurrent administration of furosemide with aminoglycosides may enhance nephrotoxicity.(7)
Not for use in breeding dogs as reproductive studies have not been conducted.
Neurotoxic and nephrotoxic antibiotics may be absorbed in significant quantities from body surfaces after local irrigation or application. The potential toxic effect of antibiotics administered in this fashion should be considered.(8)
If hypersensitivity develops, treatment with Amikacin K-9 Injection (Amikacin Sulfate Injection) should be discontinued and appropriate therapy instituted.
Warning
Amikacin should be used with extreme caution in dogs, in which hearing acuity is required for functioning, such as seeing eye, hearing ear or military patrol, as the auditory and vestibular impairment tends not to be reversible.(6)
Aminoglycosides, including amikacin, are not indicated in uncomplicated episodes of cystitis unless causative agents are susceptible to them and are not susceptible to antibiotics having less potential for toxicity.
Early signs of ototoxicity can include ataxia, nausea and vomiting. Auditory and vestibular impairment may be reversible in the very early stages, but if treatment is continued, the conditions will become irreversible.(6)
Adverse Reactions
In clinical studies in dogs, transient pain on injection has been reported as well as rare cases of vomiting or diarrhea following amikacin therapy. In 90 day intramuscular toxicology studies, evidence of muscle damage was detected histologically as well as by elevated creatinine phosphokinase.
Amikacin K-9 Injection Dosage And Administration
Amikacin K-9 Injection (Amikacin Sulfate Injection) should be administered subcutaneously or intramuscularly at a dosage of 10 mg/kg (5 mg/lb) twice daily. Dogs with skin and soft tissue infections should be treated for a minimum of 7 days and those with genitourinary infections should be treated for 7 to 21 days or until culture negative and asymptomatic. If no response is observed after three days of treatment, therapy should be discontinued and the case re-evaluated. Maximum duration of therapy should not exceed 30 days.
Amikacin K-9 Injection Caution
Federal law restricts this drug to use by or on the order of a licensed veterinarian.
Supply
50 mL vial, 50 mg/mL, Amikacin K-9 Injection (Amikacin Sulfate Injection).
Store vials between 15° and 30°C, (59° and 86°F). At times the solution may become pale yellow in color. This does not indicate a decrease in potency.
References
1.Davies, J., Courvalin, P.: Mechanisms of Resistance to Aminoglycosides. Am. J. Med. 62: 868-872, 1977.
2.Price, K.E., et al: Microbiological Evaluation of BB-K8, a New Semisynthetic Aminoglycoside. J. Antibiot. 25: 709-731, 1972.
3.Baggot, J.D., Ling, G.V., and Chaffield, R.C.: The Pharmacokinetics of Amikacin in Dogs. Am. J. Vet. Res., 46:1793-1796, 1985.
4.Christensen, E.F., Reiffenstein, J.C., and Madissoo, H.: Comparative Ototoxicity of Amikacin and Gentamicin in Cats. Antimicrob. Agents Chemother. 12: 178-184, 1977.
5.Casey, H.W., D.K. Obeck and G.A. Splitter, "Immunopathology Studies on Canine Heartworm", Canine Heartworm Disease, the Current Knowledge, University of Florida Press, 31-32, 1973.
6.Kirk, R.W., ed., Current Veterinary Therapy VIII Small Animal Practice, W.B. Saunders Company, Philadelphia, PA, 119, 731, 1040, 1983.
7.Raisbeck, M.F., W.R. Hewitt and W.B. McIntyre, "Fatal Nephrotoxicosis Associated with Furosemide and Gentamicin Therapy in a Dog", Journal of American Veterinary Medical Association, 183: 892-893, 1983.
8.Riviere, J.E., and L.E. Davis, 1964, Renal Handling of Drugs in Renal Failure, Harwal Publishers, 666-667, 1984.
600072
Iss. 11-01
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