| Abstract: |
Infections caused by Candida species in general are referred to as candidiasis. Candida albicans is the commonest and most pathogenic species. Candida is an opportunistic pathogen so it needs many predisposing factors in order to cause disease.Almost any organ or system in the body can be affected by candidiasis. It may be superficial involving skin , nails and mucosal surfaces, deep seated infections in internal organs or disseminated disease.The term candidemia indicates the presence of Candida species in the blood, this severe infection occurs when there are certain risk factors such as immunosuppression , steroids , cytotoxic drugs , broad spectrum antibiotics, central venous catheters (CVC), neutropenia and major surgery.Candidemia is caused mainly by C. albicans but recently other species have been identified particularly C. tropicalis , C. glabrata and C. krusei. The incidence of candidemia has increased over the last two decades and has been associated with high morbidity and mortality.Although blood culture has been considered as the gold standard for diagnosis of candidemia, it is useful to find a rapid and more reliable method for early diagnosis and hence early initiation of treatment.These new diagnostic methods include serological tests for detection of antibodies and antigens by different techniques and molecular diagnosis such as PCR and FISH techniques.We had done our study for evaluation of real-time PCR for detection of C. albicans DNA and ELISA for detection of C.albicans mannan antigen and specific IgM antibodies as diagnostic tools for candidemia in immunocompromised patients. These methods have been compared with blood culture as a gold standard.The study included 80 persons divided into 3 groups : group I included 20 apparently healthy persons with age ranging from 7-66 years as a control group , group II included 30 patients under chemotherapy for hematological malignancy with age ranging from 6-69 years and group III included 30 patients admitted to the ICU for different causes with age ranging from 3-71 years. All patients have been carefully selected to be highly suspected clinically to suffer from candidemia.Blood culture for Candida was done using Sabouraud broth and Sabouraud dextrose agar (for subculture), and blood culture for bacteria was done using ready commercial blood culture bottles (EDM) and different selective media (for subculture).For real-time PCR , extraction of DNA of C. albicans was done using MagNA Pure Compact nucleic acid isolation kit I with the automated MagNA Pure Compact instrument. Amplification was done using LightCycler Fast Start DNA Master PLUS SYBR Green I.We applied ELISA technique for detection of C.albicans mannan antigen [Platelia Candida Ag] and C.albicans IgM antibody [DRG Candida albicans IgM ELISA kit].There was highly significant difference between control group (I) and patients group (II+III) as regard positive blood culture for Candida , real-time PCR for C. albicans DNA , presence of C. albicans mannan antigen and IgM antibody by ELISA and urine culture for Candida. But there was no significant difference between the 2 groups as regard blood and urine culture for bacteria. All subjects of control group were negative for these tests except 2 cases who had positive urine culture for bacteria.On comparing hematological malignancy patients and ICU patients as regard blood culture for Candida or bacteria , real-time PCR , ELISA for antigen and antibody and urine culture for Candida or bacteria , there was no significant difference.Regarding blood culture for Candida , 25 out of the 60 patients were positive , 19 of these isolates were identified as C. albicans , 4 were identified as C. tropicalis and 2 cases as C. glabrata. So , the frequency of isolation of C. albicans among other species was 76 %.The majority of positive cases of candidemia (68 %) occurred at age ≥ 45 years.Our study showed a strong association between patients with candidemia and presence of C.V.C. (78.6 % in ICU patients and 72.7 % in hematological malignancy patients with total incidence of 76 % in the 2 groups together).Other risk factors include ventilatory support which was present in 57.1 % of candidemic cases in ICU group , and exposure to major surgery which was present in 42.8 % of positive cases in ICU group and 45.4 % of positive cases in hematological malignancy group with total incidence of 44 %.As regard hematological malignancy patients , 11 patients were positive for candidemia by blood culture and 8 out of them had acute leukemia, and the total number of acute leukemia patients in this study was 18, so the percentage of positive cases among this category is 44.4 % which is higher than other categories of hematological malignancy (NHL and MM).We compared recent techniques for diagnosis of candidemia with the gold standard (blood culture) and we applied this comparison to cases positive for C. albicans by culture and excluded other species.We found that 19 cases were positive for C. albicans by culture , 11 cases among ICU cases and 8 among hematological malignancy cases. All these cases were positive for C. albicans DNA by real-time PCR , but there are 2 cases negative by culture proved to be positive by PCR.Real-time PCR had a sensitivity of 100 % , specificity of 95.1 % , PPV of 90.5 % , NPV of 100 % and accuracy of 96.6 %.As regard detection of circulating C. albicans mannan antigen by ELISA, we found that 16 out of the 19 positive cases by culture are also positive for C. albicans antigen while the remaining 3 cases are negative. On the other hand all cases proved to be negative by culture were also negative by ELISA for antigen.Detection of mannan antigen by ELISA was found to have sensitivity of 84.2 % , specificity of 100 % , PPV of 100 % , NPV of 93.1 % and accuracy of 95 %.We also studied the detection of specific C. albicans antibody (IgM) by ELISA , 14 cases among the 19 positive cases by culture were also positive for C. albicans IgM , while the other 5 cases were negative. On the other hand there were 3 cases negative by culture but positive for IgM.Detection of IgM antibody by ELISA was found to have sensitivity of 73.7 % , specificity of 92.7 % , PPV of 82.3 % , NPV of 88.4 % and accuracy of 86.7 %.We also studied the presence of bacterial septicemia and we found that the number of positive bacterial blood culture was higher in cases that were positive for candidemia than cases which were negative.ELISA technique for C. albicans mannan antigen was done by the quantitative mode , and we found a significant positive correlation between mannan antigen load (ng/ml) and number of copies of C. albicans DNA detected by real-time PCR (copies/ml).Urine culture was done to detect urinary candidiasis, 23 positive cases were detected among the 60 patients examined, 18 cases were identified as C. albicans , 3 were identified as C. glabrata and 2 cases as C. tropicalis. There were 12 cases positive for both candidemia and urinary candidiasis while 11 cases had urinary candidiasis without candidemia.
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