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Comparative evaluation of staining techniques and polymerase chain reaction for diagnosis of intestinal microsporidiosis in immunocompromised patients.

Ghoshal U, Khanduja S, Agarwal V, Dhole TN, Ghoshal UC - Trop Parasitol (2015 Jul-Dec)

Bottom Line: Absence of Microsporidia by all three techniques was taken as true negative.Though Calcoflour white stain is a highly sensitive, but it is nonspecific technique.Furthermore, PCR is useful for species identification, which has therapeutic implications.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India.

ABSTRACT

Context: Microsporidia, which causes chronic diarrhoea in immunocompromised hosts, are often missed. The commonest diagnostic techniques include modified trichrome (MT) stain; however, it requires expertise and does not identify the species, which is important therapeutically. Other diagnostic techniques include Calcoflour white staining and polymerase chain reaction (PCR). Data on comparative utility of different diagnostic techniques are scanty.

Aim: Comparison of Calcoflour white, MT staining and PCR for the diagnosis of intestinal microsporidiosis.

Subjects and methods: Fecal samples of consecutive immunocompromised patients were evaluated for Microsporidia using Calcoflour white, MT stain and PCR. Species were identified by restriction fragment length polymorphism using HindIII and HinfI. Presence of Microsporidia by two or more techniques was considered true positive. Absence of Microsporidia by all three techniques was taken as true negative.

Results: Of 730 patients, Microsporidia was detected in 28 (3.8%), 250 (34.2%) and 30 (4.1%) patients by MT, Calcoflour white stains and PCR, respectively. Enterocytozoon bieneusi was identified in all 30 (4.1%) patients. 30 (4.1%) and 479 (65.6%) patients were true positive and true negative, respectively. Sensitivity and specificity of Calcoflour white, MT stains and PCR were 100%, 93.8%, 96.8% and 68.5%, 100% and 99.8%, respectively. Diagnostic accuracy of MT stain and PCR was superior to Calcoflour white (99.6% vs. 69.8%; P < 0.05).

Conclusions: Though Calcoflour white stain is a highly sensitive, but it is nonspecific technique. MT stain and PCR with high sensitivity, specificity and diagnostic accuracy are useful diagnostic techniques. Furthermore, PCR is useful for species identification, which has therapeutic implications.

No MeSH data available.


Related in: MedlinePlus

Digestion pattern of polymerase chain reaction products using restriction enzyme HinfI and Hind III. Lane M, 100 bp ladder; Lane PC, positive control for Enterocytozoon bieneusi using HinfI; Lane 2 and 4, samples positive for E. bieneusi using HindIII; Lane 3, samples positive for E. bieneusi using HinfI
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Figure 3: Digestion pattern of polymerase chain reaction products using restriction enzyme HinfI and Hind III. Lane M, 100 bp ladder; Lane PC, positive control for Enterocytozoon bieneusi using HinfI; Lane 2 and 4, samples positive for E. bieneusi using HindIII; Lane 3, samples positive for E. bieneusi using HinfI

Mentions: Polymerase chain reaction was positive for Microsporidia in 30/730 (4.1%) patients and restriction fragment length polymorphism (RFLP) using restriction enzymes Hinf I and Hind III identified E. bieneusi as the causative species of intestinal microsporidiosis in all of them. E. intestinalis, E. cuniculi and E. hellem were not identified in any patient. All the positive samples were confirmed by sequencing. Figures 2 and 3 show the representative gel pictures of patients positive for Microsporidia and enzymatic digestion of amplified products using restriction enzyme Hinf I and Hind III.


Comparative evaluation of staining techniques and polymerase chain reaction for diagnosis of intestinal microsporidiosis in immunocompromised patients.

Ghoshal U, Khanduja S, Agarwal V, Dhole TN, Ghoshal UC - Trop Parasitol (2015 Jul-Dec)

Digestion pattern of polymerase chain reaction products using restriction enzyme HinfI and Hind III. Lane M, 100 bp ladder; Lane PC, positive control for Enterocytozoon bieneusi using HinfI; Lane 2 and 4, samples positive for E. bieneusi using HindIII; Lane 3, samples positive for E. bieneusi using HinfI
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4557148&req=5

Figure 3: Digestion pattern of polymerase chain reaction products using restriction enzyme HinfI and Hind III. Lane M, 100 bp ladder; Lane PC, positive control for Enterocytozoon bieneusi using HinfI; Lane 2 and 4, samples positive for E. bieneusi using HindIII; Lane 3, samples positive for E. bieneusi using HinfI
Mentions: Polymerase chain reaction was positive for Microsporidia in 30/730 (4.1%) patients and restriction fragment length polymorphism (RFLP) using restriction enzymes Hinf I and Hind III identified E. bieneusi as the causative species of intestinal microsporidiosis in all of them. E. intestinalis, E. cuniculi and E. hellem were not identified in any patient. All the positive samples were confirmed by sequencing. Figures 2 and 3 show the representative gel pictures of patients positive for Microsporidia and enzymatic digestion of amplified products using restriction enzyme Hinf I and Hind III.

Bottom Line: Absence of Microsporidia by all three techniques was taken as true negative.Though Calcoflour white stain is a highly sensitive, but it is nonspecific technique.Furthermore, PCR is useful for species identification, which has therapeutic implications.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India.

ABSTRACT

Context: Microsporidia, which causes chronic diarrhoea in immunocompromised hosts, are often missed. The commonest diagnostic techniques include modified trichrome (MT) stain; however, it requires expertise and does not identify the species, which is important therapeutically. Other diagnostic techniques include Calcoflour white staining and polymerase chain reaction (PCR). Data on comparative utility of different diagnostic techniques are scanty.

Aim: Comparison of Calcoflour white, MT staining and PCR for the diagnosis of intestinal microsporidiosis.

Subjects and methods: Fecal samples of consecutive immunocompromised patients were evaluated for Microsporidia using Calcoflour white, MT stain and PCR. Species were identified by restriction fragment length polymorphism using HindIII and HinfI. Presence of Microsporidia by two or more techniques was considered true positive. Absence of Microsporidia by all three techniques was taken as true negative.

Results: Of 730 patients, Microsporidia was detected in 28 (3.8%), 250 (34.2%) and 30 (4.1%) patients by MT, Calcoflour white stains and PCR, respectively. Enterocytozoon bieneusi was identified in all 30 (4.1%) patients. 30 (4.1%) and 479 (65.6%) patients were true positive and true negative, respectively. Sensitivity and specificity of Calcoflour white, MT stains and PCR were 100%, 93.8%, 96.8% and 68.5%, 100% and 99.8%, respectively. Diagnostic accuracy of MT stain and PCR was superior to Calcoflour white (99.6% vs. 69.8%; P < 0.05).

Conclusions: Though Calcoflour white stain is a highly sensitive, but it is nonspecific technique. MT stain and PCR with high sensitivity, specificity and diagnostic accuracy are useful diagnostic techniques. Furthermore, PCR is useful for species identification, which has therapeutic implications.

No MeSH data available.


Related in: MedlinePlus