Dr Shivanee Shah
An 8-year old male child presented to Rainbow Children’s Hospital, Vijaywada, with on and off fever for the past three months. His parents also mentioned that he had had 2 episodes of seizures and complained of shortness of breath over the last 2 weeks. The patient then underwent a two-dimensional echocardiography (2D ECHO) to monitor the heart and great vessels and to determine if there are any anomalies in the appearance of the valves or the functioning of the heart muscles. 2D ECHO showed a heavy vegetation in both mitral and tricuspid valves, suggesting infective endocarditis Infective endocarditis can be caused by a myriad of agents that could be bacterial, fungal, or even viral. A biopsy was done and sent for routine microbial culture to determine the causative agent. As another alternative, to help reach a quicker diagnosis, the doctors decided to confer with the diagnostic team at a diagnostics lab in Mumbai, for molecular diagnostic testing. The lab suggested that the biopsy sample be sent to them for their infective endocarditis panel testing. Thus, a portion of the biopsy was also sent to the lab. The infective endocarditis panel consists of various strains that are routinely implicated in endocarditis, including the usual suspects Streptococcus, Enterococcus, and Staphylococcus. Thus,if the sample has any of these strains present, the panel test will pick out the causative pathogen. Interestingly, while the microbial culture tests came back negative, results from the genetic lab came back positive for Staphylococcus aureus.
Staphylococcus aureus is known to cause many diseases including sepsis and endocarditis. Antibiotics are the most effective treatment for such infections. However, due to inappropriate antibiotic usage over the years, antibiotic resistant strains are now rampant, and doctors need to ensure prescribing the correct antibiotic treatment for effectively treating Staphylococcus aureus infections. Correct antibiotic treatment is typically identified using antibiotic sensitivity assays, in which, small wafers containing antibiotics are placed onto a culture plate on which the bacteria are already growing.
Antibiotics that prevent the growth of the bacteria on the plate are then chosen as the treatment of choice. In the case of this patient, since the microbial cultures were negative, no culture plates were available and carrying out an antibiotic sensitivity assay was not possible. The molecular diagnostic lab came to the rescue again and use done of their innovative in-house developed PCR-based assays to detect antibiotic resistance. PCR or polymerase chain reaction is a molecular biology technique used to amplify a particular DNA sequence from a single copy to millions of copies.
Antibiotic resistance is conferred due to the presence of certain genes. There are many different genes that can confer antibiotic resistance, including those for beta-lactamases which break down beta-lactam present in many antibiotics such as penicillin and cephalosporin. An antibiotic resistance assay uses this information and has created a panel that looks for the presence of specific antibiotic resistance genes in the DNA from the sample. It screens for all classes of beta-lactamases, in addition to genes that confer methicillin and vancomycin resistance. Results from this assay indicated that the Staphylococcus aureus infection was caused by a methicillin-resistant strain and the causative agent was diagnosed as methicillin-resistant Staphylococcus aureus (MRSA).This strain is also resistant to other known antibiotics such as amoxicillin, penicillin, and oxacillin. The patient was therefore put on an alternative antibiotic therapy– vancomycin, in combination with ceftriaxone–which proved to be effective for the patient who recovered well.
In this case, not only was molecular testing extremely important to identify
the causative agent, it also helped in charting out the effective treatment route. According to Dr. Aparna Kotekar, HOD Molecular Department, iGenetic Diagnostics, Mumbai, while about 20-30% results come positive via microbial culture testing, about 50-60% can be identified via PCR-based assays due to their high sensitivity. The results for molecular testing also come back much quicker than microbial cultures and should especially be used when time is of major concern. Of course, the major drawback of using molecular testing is the cost, which may currently not be affordable for many. However, as better and newer technology becomes available,diagnostic labs are constantly trying to make this testing more economical. Currently, they also offer smaller panels instead of their wide screening panels when the doctors
are relatively sure of the probable
causative agent. This can considerably drop the prices and make diagnostic testing