ms defence - dr visnu pritom chowdhury

A statistical correlation between virulence genes and clinical features among patients with shigellosis in

Mirzapur, Bangladesh

Dr. Visnu Pritom ChowdhuryID#13176003

Session: Spring – 2013

MS in Biotechnology ProgramDepartment of Mathematics and Natural Science

BRAC University

Background

Shigella episodes:

Kotloff et al., 1999

● 164.7 million – worldwide

● 163.2 million – developing countries (with 1.1 million deaths)

Kotloff et al., 2013

● 0.8 million fatalities

– In, sub-Saharan Africa & south Asia

– Along with, Rotavirus, Cryptosporidium and ETEC

Classification

Genus: Shigella

Species:

1. Shigella dysenteriae (16 serotype)

2. Shigella flexneri (19)

3. Shigella boydii (20)

4. Shigella sonnei (1)

(Jakhetia et al., 2014)

S. sonnei

S. flexneri

S. flexneri with intermittent S. dysenteriae Type 1 epidemics

Epidemiology

Aim of this Study

Correlation between the presence of virulence factors and the clinical features observed in patients with shigellosis

Methodology

140 MD

105 MD

2.7 MD

35.6 MD

2.1 MD

1.8 MD

1.4 MD

62 MD

23 MD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Plasmid Isolation

140 MD plasmid:

79% (+)ve (n = 48)

Conserved Region in 140 MD Virulence Plasmid

T3SS NC of Shigella spp.

PCR Assay

Genes:

1. Virulence – ipaH, ial

2. Toxin – set1A, set1B, sen

3. T3SS – 18 genes

– Regulator – virB, mxiE

– Effector – ipgB1, icsB, ipgD

– Translocator – ipaBCD

– Chaperone – ipgC, ipgA, ipgE, spa15,

– Machinery – ipgF, mxiH, mxiI, mxiK, , mxiC, spa47, spa32, spa24

Primer Designing

1. spa15 – chaperone

2. mxiE – T3SS regulator

Steps for Primer Designing

1. Sequence retrieval ← NCBI

Steps for Primer Designing

1. Sequence retrieval ← NCBI

2. Sequence input → Primer3Plus

Steps for Primer Designing

1. Sequence retrieval ← NCBI

2. Sequence input → Primer3Plus

3. Parameter optimization Primer length – 18 – 20 nt Melting temperature (Tm) – 57 – 63 °C GC content – 45 – 60% GC clamp – sticky end and prevent mispriming

Gene Primer Gene Primer

spa15 1 ACAGCCATTCAGCGATTACC mxiE 1 GCATAGCCATGCTGAAAAATG

spa15 2 AACGATGAAGCTCTACCAGCTC mxiE 2 TCCGACACACCATAATGCTC

Predicted Sequence

Good temperature:Solid bands and no non-specific bands

Temperature too low:Reduced band intensity

Temperature too high:Fainting bands

Gene Cycle Td (°C)

Ta (°C) Gradient

Te (°C) Final Extn (°C)

Lowest Ta

Highest Ta

mxiE 32 94°C, 60 sec 48°C, 60 sec 64°C, 60 sec 72°C, 90 sec 72°C, 600 sec

spa15 32 94°C, 60 sec 48°C, 60 sec 64°C, 60 sec 72°C, 90 sec 72°C, 600 sec

Confirmation of predicted primer sequence

ipaBCD500bp

icsB1568bp

ipgD560bp

spa24547bp

set1B147bp

set1A309bp

set2799bp

Agarose gel electrophoresis image of few genes

ipaBCD ial ipgC ipgE virB ipgA sen mxiH mxiI spa15 spa47 ipgD ipgF spa32 ipgB1 mxiK spa24 mxiE set icsB mxiC0

10

20

30

40

50

60

70

80

90

100

PCR Assay

pedal edemaseverity

abdominal painblood in stool

bloody mucoidfever

rectal strain vomiting

coughsunken eye

dry mouthdehydration

convulsion mental status

0

10

20

30

40

50

60

70

80

90

100

Clinical Feature Analysis

Statistical Analysis

1. Presence of virulence genesVs.

2. Presence of clinical features

Gene Clinical FeaturesChi-Squared Test

Value df P ( <0.01 )

set Blood in stool 9.244 1 .002

sen Blood in stool 9.244 1 .002

set Rectal strain 8.306 1 .004

sen Rectal strain 8.306 1 .004

ipgD Pedal edema 7.358 1 .007

Highly Statistically Significant Associations (P < 0.01)

Gene Clinical FeatureChi-Squared Test

Value df P ( <0.05 )

set Stool consistency (Bloody mucoid) 9.514 3 .023

sen Stool consistency (Bloody mucoid) 9.514 3 .023

spa24 Rectal strain 4.378 1 .036

ial Abdominal pain 7.778 2 .020

ipaBCD Abdominal pain 7.378 2 .025

set Mouth dryness 4.118 1 .042

sen Mouth dryness 4.118 1 .042

set Cough 4.253 1 .039

sen Cough 4.253 1 .039

ipgD Cough 4.476 1 .034

set Fever 5.435 1 .020

sen Fever 5.435 1 .020

icsB Fever 3.840 1 .050

set Diarrheal disease severity 4.118 1 .042

sen Diarrheal disease severity 4.118 1 .042

Statistically Significant Associations (P < 0.05)

In conclusion

● Shigella enterotoxins (set, sen) were mainly found associated with major clinical features (blood in stool and bloody mucoid stool consistency, rectal strain, dehydration, cough, fever, severity) observed in shigellosis

● The effector gene, ipgD was found to be correlated with pedal edema and cough

● And lastly, spa24 and icsB were associated with rectal strain and fever respectively; and ial, ipaBCD both were found associated with abdominal pain

Limitations

1. Plasmids were isolated with only alkaline lysis method

2. Clinical data were insufficient to exclude associated co-morbidities or co-infections

3. Small sample size

Recommendation for future work

1. Plasmid isolation confirmation through Sereny test

2. Exploring possibilities of chromosomal integration of plasmid borne virulence genes

3. Determination of any common conserved regions among Shigella and other immune system mediated cross-reacting pathogens

4. A more comprehensive study with larger sample size and extended clinical information

Reference

Kotloff, K.L., Winickoff, J.P., Ivanoff, B., Clemens, J.D., Swerdlow, D.L., Sansonetti, P.J., Adak, G.K., and Levine, M.M. (1999). Global burden of Shigella infections: implications for vaccine development and implementation of control strategies. Bull. World Health Organ. 77, 651–666.

Kotloff, K.L., Nataro, J.P., Blackwelder, W.C., Nasrin, D., Farag, T.H., Panchalingam, S., Wu, Y., Sow, S.O., Sur, D., Breiman, R.F., et al. (2013). Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 382, 209–222.

Jakhetia, R., Marri, A., Ståhle, J., Widmalm, G., and Verma, N.K. (2014). Serotype-conversion in Shigella flexneri: identification of a novel bacteriophage, Sf101, from a serotype 7a strain. BMC Genomics 15, 742

Khan, W.A., Dhar, U., Salam, M.A., Griffiths, J.K., Rand, W., and Bennish, M.L (1999). Central nervous system manifestations of childhood shigellosis: prevalence, risk factors, and outcome. Pediatrics 103, E18.

Thank you