Utility of Panfungal PCR in the diagnosis of invasive fungal infections in febrile neutropenia.

BACKGROUND: The prevalence of invasive fungal infections (IFIs) is increasing due to the increasing population of immunocompromised patients. Fungal culture is the gold standard for diagnosis but not sensitive and the turnaround time is long. Samples for histopathology are difficult to obtain because of profound cytopenias. We conducted this study with the aim to evaluate panfungal PCR for the diagnosis of IFIs in patients of febrile neutropenia. METHODS: This was a single-centre, cross-sectional observational study. Patients of febrile neutropenia suspected of having IFI were included in the study. Panfungal PCR was performed on the blood of included patients along with other investigations for diagnosis of IFI. The sensitivity, specificity, positive predictive value, and negative predictive value of panfungal PCR were calculated using EORTC/MSG 2008 criteria as the gold standard. RESULTS: Fifty patients of febrile neutropenia were included in the study, of which 52% were diagnosed positive by panfungal PCR assay. The sensitivity, specificity, positive predictive value, and negative predictive value of panfungal PCR assay was found to be 82.76%, 90.48%, 92.31% and 79.17% respectively. CONCLUSION: Panfungal PCR is a promising and highly sensitive diagnostic test for screening at-risk patients suspected to have IFIs. The use of panfungal PCR assay in combination with other diagnostic modalities and clinical judgment can be very helpful in the early diagnosis of IFI.

Epidemiology and Antifungal Susceptibility of Infections Caused by Trichosporon Species: An Emerging Non-Candida and Non-Cryptococcus Yeast Worldwide.

Introduction: Over the past four decades, there has been an increase in the number of fatal opportunistic invasive trichosporonosis cases especially in immunocompromised hosts. Objective: The objective of the study is to evaluate the epidemiological, clinical details and antifungal susceptibility pattern of the patients with Trichosporon infections. Materials and Methods: Twenty-four clinical isolates of Trichosporon species isolated from blood, samples, pleural fluid and nail were included in this study, over a period of 12 years (2005-2016) in a tertiary hospital in North India. The isolates were characterised phenotypically and few representative isolates were sequenced also. The minimum inhibitory concentration (MIC) was determined as per Clinical and Laboratory Standards Institute, 2012. Results: Trichosporon spp. from blood culture (57.78%), nail (37.5%) and pleural fluid (4.17%). On phenotypic tests, 79.16% of the isolates were Trichosporon asahii, followed by Trichosporon dermatis (8.33%), Trichosporon japonicum (4.17%), Trichosporon ovoides (4.17%) and Trichosporon mucoides (4.17%). The MIC range of Trichosporon species from invasive infections were fluconazole (0.06-256 µg/ml), amphotericin B (0.125-16 µg/ml), voriconazole (0.0616-8 µg/ml), posaconazole (0.0616-32 µg/ml) and caspofungin (8-32 µg/ml). The isolates from superficial infection were resistant to fluconazole (0.06-256 µg/ml) and itraconazole (0.125-32 µg/ml), all were susceptible to ketoconazole and while only two were resistant to voriconazole (0.25-4 µg/ml). Conclusion: T. asahii was the most common isolate. Disseminated trichosporonosis is being increasingly reported worldwide including India and represents a challenge for both diagnosis and species identification. Prognosis is limited, and antifungal regimens containing triazoles appear to be the best therapeutic approach. In addition, accurate identification, removal of central venous lines and voriconazole-based treatment along with control of underlying conditions were associated with favourable outcomes.

High mTOR expression independently prognosticates poor clinical outcome to induction chemotherapy in acute lymphoblastic leukemia.

In acute lymphoblastic leukemia (ALL), limited data are available on mTOR gene expression in clinical samples and its role in predicting response to induction chemotherapy. mRNA expression of mTOR gene was determined quantitatively by real-time PCR in 50 ALL patients (30 B-ALL and 20 T-ALL) and correlated with clinical outcome after induction chemotherapy. Expression level of mTOR was upregulated in more than 50% of cases of ALL. In T-ALL, high expression of mTOR was commonly seen, more in adults than children (82 vs. 55% cases), while in B-ALL it was same (~ 63% cases) in both adults and children. Mean fold change of mTOR expression was significantly higher in non-responders compared to responders of both adult B-ALL (7.4 vs. 2.7, p = 0.05) and T-ALL (13.9 vs. 2.4, p = 0.001). Similar results were seen in pediatric non-responders when compared to responders of both B-ALL (14.5 vs. 2.5, p = 0.006) and T-ALL (24.2 vs. 1.7, p = 0.002). Interestingly, we have observed that mTOR expression was two times higher in non-responders of children compared to adults in both B-ALL (14.5 vs. 7.4, p = 0.05) and T-ALL (24.2 vs. 13.9, p = 0.01). Multivariate analysis with other known prognostic factors revealed that mTOR expression independently predicts clinical response to induction chemotherapy in ALL. This study demonstrates that high mTOR expression is associated with poor clinical outcome in ALL and can serve as a potential target for novel therapeutic strategies.

Mutation of NPM1 and FLT3 genes in acute myeloid leukemia and their association with clinical and immunophenotypic features.

BACKGROUND: Mutations in NPM1 and FLT3 genes represent the most frequent genetic alterations and important diagnostic and prognostic indicators in patients with acute myeloid leukemia (AML). OBJECTIVE: We investigated the prevalence and clinical characteristics of NPM1 and FLT3 mutations in 161 patients of de novo AML including adults and children. RESULTS: NPM1 mutation was found in 21% and FLT3 mutation in 25% of the AML patients. Thirteen (8%) samples were positive for both NPM1 and FLT3/ITD mutations. Adult patients had significantly higher frequency of NPM1 mutation than children (25.8% versus 8.8%; P = 0.02). Further, NPM1 mutation was found to be more frequent in patients above 45 years of age (P = 0.02). NPM1 mutation was significantly associated with higher platelet count (P = 0.05) and absence of hepatosplenomegaly (P = 0.01), while FLT3/ITD mutation was associated with higher white blood count (P = 0.01). Immunophenotypically, NPM1 mutation was associated with the lack of CD34 (P < 0.001) and HLD-DR expression (P < 0.001), while FLT3/ITD mutation was positively associated with the expression of CD7 (P = 0.04). No correlation was found between NPM1 mutation and fusion gene. Interestingly, FLT3/ITD mutation was found to be inversely associated with AML/ETO fusion gene (P = 0.04). CONCLUSIONS: The results suggest that distinct clinical and immunophenotypic characteristics of NPM1 and FLT3/ITD mutations present further insight into the molecular mechanism of leukemogenesis.

Expression of genes related to multiple drug resistance and apoptosis in acute leukemia: response to induction chemotherapy.

Resistance to chemotherapy is a major impediment to the successful treatment of acute leukemia (AL). Expression of genes involved in drug resistance and apoptosis may be responsible for this. This study aimed to investigate the expression of drug resistance (MDR1, MRP1, LRP, BCRP, GSTP1, DHFR) and apoptotic genes (p53, BCL-2, Survivin) in adult acute leukemias and compare them with clinical and hematological findings and response to induction chemotherapy. Eighty-five patients with AL [45 with acute myeloid leukemia (AML) and 40 with acute lymphoblastic leukemia (ALL)] were used as a study group. Real-time PCR results showed that expression level of MDR1 was significantly higher in AML whereas expression of DHFR, BCRP and Survivin was significantly higher in ALL patients. In AML, significant correlation was observed between LRP and MRP1 (r(s)=0.44, p=0.016), LRP and DHFR (r(s)=0.41, p=0.02), MDR1 and BCL-2 (r(s)=0.38, p=0.03). Expression of GSTP1 and LRP correlated with high white blood count (p=0.03 and p=0.03) and BCL-2 with high peripheral blast count (p=0.009). MDR1 expression was significantly associated with the expression of immature stem cell marker CD34 (p=0.002). In ALL, significant association was found between LRP gene and female sex (p<0.0001), LRP and B-ALL patients (p=0.04) and LRP and BCR/ABL positive patients (p=0.004). High expression of MDR1 and BCL-2 in AML and MRP1 gene in ALL was associated with response to induction chemotherapy (p=0.001, p=0.02 and p=0.007 respectively). These results showed the potential clinical relevance of MDR1, MRP1 and BCL-2 in adult patients with acute leukemia in the context of induction chemotherapy.

Association of glutathione S-transferase, EPHX, and p53 codon 72 gene polymorphisms with adult acute myeloid leukemia.

Polymorphisms in genes encoding detoxification enzymes have been suggested as susceptibility factors for many solid tumors. However, their association with hematological malignancies is controversial. A case-control study was done to determine the association between glutathione S-transferase M1 (GSTM1), GSTT1, GSTP1, EPHX1, and p53 codon 72 polymorphisms as risk factors in 120 adult acute myeloid leukemia (AML) cases and 202 healthy controls by polymerase chain reaction-restriction fragment length polymorphism techniques. Data were analyzed using χ(2) and conditional logistic regression model. None of the polymorphisms studied alone was associated with increased risk for AML. However, the frequency of GSTT1 null genotype was higher among controls (28.7%) than AML cases (21.6%), which showed a protective effect of the null genotype (odds ratio = 0.58, 95% confidence interval: 0.33-1.05, p = 0.07). In a combined analysis, both EPHX1 (His113His) and GSTP1 (Ile/Val) genes imparted a fourfold risk for adult AML but did not reach statistical significance (odds ratio = 4.22, 95% confidence interval: 0.992-17.99, p = 0.05). These findings suggest that the etiology of adult AML cannot be explained by polymorphism at a single locus, perhaps because of complexity involved in the metabolism of diverse xenobiotic compounds, and therefore, multiple gene-gene interactions should be investigated to predict the risk of AML.