Evaluation of the Efficacy of DNA Macro Chips in Early Detection of Septicemia in Children Receiving Cancer Chemotherapy

INTRODUCTION: Infection is a major cause of morbidity and mortality in pediatric oncology patients 1. Febrile episodes occur in approximately one‐third of neutropenic episodes in children with chemotherapy‐induced neutropenia 2. The possible infectious origin of fever is a central point in the management of neutropenic patients. A rapid microbiological diagnosis could therefore confirm an infectious cause of fever (thereby excluding non‐infectious causes) and help in the choice of a specific therapy.The current gold standard for the detection of bacterial pathogens in blood is blood culture. However, all blood culture systems suffer from several limitations, such as lack of rapidity and low sensitivity, especially when the patient has already received antibiotics and when fastidious micro‐organisms are involved 3. From this perspective, the diagnosis of bloodstream infections could prove really challenging in hemato‐ oncological patients, who routinely receive prophylactic antibiotics and whose blood cultures therefore often remain negative 4,5,6,7. Even after the detection of growth in cultured blood (usually not before 6–12 h of incubation), conventional blood cultures require at least a further 24–48 h for the definitive identification of the pathogen and the evaluation of its sensitivity to antibiotics 3,8. Other parallel approaches are therefore needed, and among them well‐designed molecular assays could prove really useful. Several molecular techniques have already been successfully used in routine microbiology laboratories for direct detection of viral, bacterial, mycotic and protozoan pathogens However, their use on whole blood samples for detection of sepsis has been hampered by several factors, including insufficient sensitivity, presence of PCR inhibitors in blood, and the difficulty of setting up an assay capable of detecting a wide range of potential pathogens 3. The DNA Macro Chip is a new concept, that allows for the simultaneous identification of multiple organisms like bacteria, viruses, fungi and parasites in a single test from a single sample. It involves the concept of syndrome based diagnosis, which allows for simultaneous detection of all probable causative agents which can cause sepsis, obviating multiple sequential tests and loss of time. AIMS AND OBJECTIVES: 1.To evaluate the efficacy of DNA Macrochips in early detection of septicaemia in children receiving cancer chemotherapy. OBJECTIVE OF THE STUDY: 1. To assess the sensitivity, specificity and predictive value of DNA Macrochips in the diagnosis of septicaemia 2. To compare the results of the DNA Macrochip with blood cultures in children with various malignancies during febrile episodes 3. To assess whether the DNA Macrochip can be used as an added / alternate modality in detecting bacteremia during febrile episodes in children undergoing cancer chemotherapy. DISCUSSION: In this study we aimed to evaluate the efficacy of DNA macrochip, a new PCR based technology, in early detection of septicaemia in children undergoing cancer chemotherapy in our centre. Blood culture and DNA macochip tests were done 121 children on 157 occasions. The incidence of septicaemia in our study was 9.6%. This is less than our previous observations44 and those published in the literature by Erten et al (20%)55 and Dubey et al (36%)56. The spectrum of organisms isolated in blood culture were Coagulase negative Staphylococcus(4), NFGNB(3), Pseudomonas aeruginosa(2), alpha hemolytic streptococci (1), E coli(1), Enterococci (1), Hemophilus influenzae (1), gram negative bacilli (1), aerobic spore forming organism (1), Klebsiella pneumonia (1) and Candida (1) with equal proportion of gram negative and gram positive infections. This observation was similar to that of Erten et al55, where the proportion of Gram positive and Gram negative bacteremia was similar. However, in the study conducted bt Dubey et al56, they found a higher proportion of Gram negative bacteremia in 83% of cases. An obvious focus of infection was present in 79 of 157 cases; these were lower respiratory tract infection in 31, oral mucositis 21, acute gastroenteritis 17 and local infections such as cellulitis, ear infection and throat infection in 11. This incidence is similar to that reported by Jimeno A et al57 in a study conducted on adult patients with febrile neutropenia, where the incidence of pneumonia was 23%, acute gastroenteritis 12.8% and ENT infections 7.7%. They found a higher incidence of oral mucositis (23%), in their group. DNA Macrochip test identified various microbes in 85 /157 samples (54.% ) The spectrum of organisms isolated were Acinetobacter baumanii (25), Candida (25) Pseudomanas aeruginosa (14), Streptococcus species (other than Streptococcus pneumonia)(12), Klebsiella pneumoniae (11), Staphylococcus aureus (9), Streptococcus pneumonia (8), Aspergillus species (8), Enterococcus(5), Bacteroides (2), Neisseria meningitides (1). This higher rate of isolation of organisms may be because of previous subclinical infections, cross reaction of the primer used in the technique for PCR amplification with the genome of other organisms, or due to contamination of the samples with organisms. However, when we compared the corresponding reports of the blood culture and DNA Macrochip on the same sample, in only four cases both tests was positive for the same organism. SUMMARY AND CONCLUSIONS: • We studied 157 febrile episodes in 121 febrile children undergoing chemotherapy for various malignancies and compared the results of blood culture and the DNA Macrochip in this group. 1. Blood culture was positive in 15 cases whereas DNA macrochip identified 120 organisms from 85 blood samples with multiple isolates in 28 cases. Both tests identified same organism only in 4 cases. 2. Sensitivity and specificity of the DNA Macrochip was calculated using blood culture as gold standard. The sensitivity was 26.7% and specificity was 47.37%, positive predictive value was 5.4% and negative predictive value was 85.13%. These were too low for a diagnostic test, therefore DNA Macrochip concept in it’s present stage is not an useful methodology for the detection of septicemia. 3. In the standardization study where 14 blood samples spiked with known organisms were sent to XCyton lab for DNA Macrochip analysis, it was found that the DNA Macrochip identified organisms in all 14 samples including 4 negative controls. In eight samples correct organism was detected, but in four samples additional organisms were isolated. The possibilities of the DNA primer used for PCR amplification cross reacting with the genome of other organisms, thereby giving false positive results as well as the contamination of the blood samples may explain this observation. 4. When we did a comparative analysis of the blood culture and DNA Macrochip against foci of infection and severity of illness, it was found that in 60% of cases with foci of infection was positive by DNA test and the organisms identified are known to cause these infections. Severity of illness was similar in both groups, suggesting equivalence between the two groups. However, sample sizes were too small to draw any conclusions from this.