Molecular Approach For Molecular Approach For Diagnosis Of Rickettsia:Diagnosis Of Rickettsia:

A Collaborative Initiative By A Collaborative Initiative By Dept. Of Medicine & Dept. Of Medicine &

MicrobiologyMicrobiology

Dr. Faria FerdouseDr. Faria Ferdouse Thesis part student of Thesis part student of

M.Phil (Microbiology) M.Phil (Microbiology) Mymensingh Medical Mymensingh Medical

CollegeCollege

Rickettsial diseases are a group of infections Rickettsial diseases are a group of infections caused by the obligate intracellular bacteria caused by the obligate intracellular bacteria Rickettsia. Rickettsia. ItIt is a Gram-negative, non flagellated, non-is a Gram-negative, non flagellated, non-motile, non-spore forming, highly motile, non-spore forming, highly pleomorphic bacteria. pleomorphic bacteria. They are small, cocco-bacilli that can They are small, cocco-bacilli that can present as cocci or bacilli. They comprise a present as cocci or bacilli. They comprise a group of microorganisms that group of microorganisms that phylogenetically occupy a position between phylogenetically occupy a position between bacteria and virusesbacteria and viruses

The name Rickettsiaceae honors Haword Taylor Ricketts for his brilliant experiments. Ricketts, as well as another famous rickettsiologist, Von Prowazek, died of rickettsia during their study period

•From 1873 to 1920, 66% of 431 reported cases resulted in death in USA by Rocky mountain spotted fever.During 1983-1998, Five to thirty-nine deaths were reported annually to public health authorities.

In 1918 In 1918 R. felis R. felis was first detected in European cat fleas was first detected in European cat fleas ((Ctenocephalides felis) Ctenocephalides felis)

In 1990 rediscovered in the United States.In 1990 rediscovered in the United States.

In 2003 In Asia, the first case of In 2003 In Asia, the first case of R. felis R. felis infection infection reported.reported.

Recently has been demonstrated in Brazil, France, Recently has been demonstrated in Brazil, France, Germany, Texas, Mexico, Africa, SpainGermany, Texas, Mexico, Africa, Spain

In 1993, WHO reported that, these are In 1993, WHO reported that, these are major causes of febrile illnesses major causes of febrile illnesses throughout the Asia-Pacific region, also throughout the Asia-Pacific region, also present in several parts of the Indian present in several parts of the Indian subcontinent.subcontinent.

From India in 2010 reported that 45.6% From India in 2010 reported that 45.6% had spotted fever group and 30.7% had spotted fever group and 30.7% scrub typhus & untreated cases can scrub typhus & untreated cases can have fatality rates as high as 30-35%.have fatality rates as high as 30-35%.

For India, the reported numbers are an For India, the reported numbers are an underestimate due to lack of community underestimate due to lack of community based data and non-availability of based data and non-availability of confirmatory laboratory tests.confirmatory laboratory tests.

Rickettsial disease in India has been Rickettsial disease in India has been documented from Jammu, Kashmir, documented from Jammu, Kashmir, Himachal Pradesh, Uttaranchal, Himachal Pradesh, Uttaranchal, Rajasthan, Assam, West Bengal, Rajasthan, Assam, West Bengal, Maharashtra, Kerala and Tamil NaduMaharashtra, Kerala and Tamil Nadu

Since Bangladesh is very adjacent to Since Bangladesh is very adjacent to the West Bengal of India, it is very the West Bengal of India, it is very likely that the same likely that the same RickettsiaRickettsia would would be responsible for certain illnesses be responsible for certain illnesses such as PUO, however there is no such as PUO, however there is no scientific studies in Bangladesh so far.scientific studies in Bangladesh so far.

With this concept in the background, With this concept in the background, we conducted this study aiming at the we conducted this study aiming at the following objectives:following objectives:

General objective:General objective: Diagnosis of Rickettsial infection by PCR and other Diagnosis of Rickettsial infection by PCR and other

conventional diagnostic tool.conventional diagnostic tool.

Specific objectives:Specific objectives: To screen the Rickettsial fever by Weil-Felix test.To screen the Rickettsial fever by Weil-Felix test. To detect IgM antibody against Rickettsia specific To detect IgM antibody against Rickettsia specific

antigen by ELISA. antigen by ELISA. To diagnose Rickettsial fever by PCR using genus To diagnose Rickettsial fever by PCR using genus

specific gene gltA (citrate synthetase) and 16s specific gene gltA (citrate synthetase) and 16s rDNA. rDNA.

A total of 155 clinicaliy suspected cases A total of 155 clinicaliy suspected cases of febrile illness was selected from of febrile illness was selected from inpatient and outpatient department of inpatient and outpatient department of medicine and pediatrics of MMCH medicine and pediatrics of MMCH during the period from july 2012 to during the period from july 2012 to september 2013september 2013. .

Blood was collected and from each of Blood was collected and from each of the patient with standard procedure in the patient with standard procedure in the department of microbiology MMCH the department of microbiology MMCH for Weil felix test ,ELISA and PCR.for Weil felix test ,ELISA and PCR.

• All the samples are tested by Weil felix test. In Weil felix test OXK,OX2 and OX19 strains of proteus are used to detect rikettsial antibody

• ELISA was done only Weil felix test positive cases.

• Rikettsial outer membrane lipopolysaccharide antigen were used for detection of antibody.

•PCR was done for all the specimens (155)

AgeAgegroupgroup

MaleMale

(n = 62)(n = 62)Female Female (n = 74)(n = 74)

TotalTotal(n = 136)(n = 136)

Up to 15 Up to 15 yearsyears

0909 1717 2626

15 to 30 years15 to 30 years 3636 3535 717130 to 45 years30 to 45 years 2323 2222 4545

>45 years>45 years 0606 0707 1313TotalTotal 74 (48%)74 (48%) 81 (52%)81 (52%) 155 (100%)155 (100%)

TABLE IAge & sex distribution

of the cases

ClinicalClinicalfeaturesfeatures

No. of presenting No. of presenting casescases

Percentage (%)Percentage (%)

FeverFever 155155 100100

HeadacheHeadache 9898 6363

Body ache Body ache 6565 4141

CoughCough 1212 7.77.7

RashRash 0303 0202

Conjunctival HgeConjunctival Hge 0101 0.60.6

Table IIClinical features of the cases

Table IIIResult of Weil-Felix test

Weil-FelixWeil-FelixAntigenAntigen

OX-2OX-2 OX-KOX-K OX-19OX-19OX-2OX-2

++OX-OX-KK

OX-2OX-2++

OX-OX-1919

OX-OX-1919++

OX-KOX-K

AllAllthreethree

Weil-FelixWeil-FelixTestTest

PositivePositive

4747

All160,All160, 1-3201-320

0909(All (All 160,160, 1 – 1 – 320)320)

0606 (All (All 160)160)

2525 1717 1010 2222

Total testedTotal tested 155 (100)155 (100)

Weil-Felix positiveWeil-Felix positive 136 (88%)136 (88%)

TABLE IVResult of ELISA for IgM

Total testedTotal tested 129 (100%)129 (100%)

ELISA for IgM PositiveELISA for IgM Positive 31 (24%)31 (24%)

Total testedTotal tested 155 (100%)155 (100%)

PCR positivePCR positive 61 (39%)61 (39%)

Table VResult of PCR

TestTest SensitivitySensitivity SpecificitySpecificity

Weil-FelixWeil-Felix 96.7296.72 43.3843.38

PCRPCR 100100 100100

Table VICorrelation of Weil-Felix and PCR

PCRPCRWeil-Felix testWeil-Felix test

PositivePositive NegativeNegativePositivePositive 5959 0202

NegativeNegative 7777 1616

Considering PCR as gold standard

TestTest SensitivitySensitivity SpecificitySpecificity

ELISAELISA 32.7832.78 60.6160.61

PCRPCR 100100 100100

Table VIICorrelation of ELISA and PCR

PCRPCRELISAELISA

PositivePositive NegativeNegativePositivePositive 2020 4141

NegativeNegative 1313 8181

Considering PCR as gold standard

lanelane 0101 0202 0303 0404 0505 0606 0707 0808 0909 1010 1111 1212 1313 1414 1515 1616 1717

Sl Sl no.no.

0101 0202 0303 0404 0505 0606 0707 0808 0909 LaddLadderer 1010 1111 1212 1313 1414 1515 1616

PosPos PosPos NegNeg NegNeg PosPos NegNeg PosPos PosPos NegNeg PosPos PosPos PosPos PosPos NegNeg NegNeg

Rickettssia PCR

Date : 11/09/13

Primer : 17 K Da Product size: 232 bp

232 bp

For confirmation of PCR product, For confirmation of PCR product, Sequencing was done at Sapporo Sequencing was done at Sapporo Medical University using Big Dye Medical University using Big Dye terminator.terminator.

On Wednesday, October 23, 2013 3:48 PM, Nobumichi Kobayashi On Wednesday, October 23, 2013 3:48 PM, Nobumichi Kobayashi <nkobayas@sapmed.ac.jp> wrote:<nkobayas@sapmed.ac.jp> wrote:

Dear Dr.Shyamal K. Paul,Dear Dr.Shyamal K. Paul,

How are you?How are you?Salma arrived at Sapporo safely on Monday and started experiments on her Salma arrived at Sapporo safely on Monday and started experiments on her study.study.I received a souvenir from you, through Salma.I received a souvenir from you, through Salma.Thank you very much for it. We will try to taste it in our lunch or tea time.Thank you very much for it. We will try to taste it in our lunch or tea time.

Today Souvik tried to Today Souvik tried to determine sequences of five PCR products determine sequences of five PCR products from suspected rickettsiosis brought by Salma. These five from suspected rickettsiosis brought by Salma. These five sequences were 99% identical to that of Rickettsia felis.This sequences were 99% identical to that of Rickettsia felis.This Rickettsia distributes to cats, and infect to human via cat Rickettsia distributes to cats, and infect to human via cat flea.R. felis infection has been reported in some countries, but flea.R. felis infection has been reported in some countries, but not in many countries, thus seems to be still rare.not in many countries, thus seems to be still rare.I guess probably all the specimens are clonal. This is very interesting finding.I guess probably all the specimens are clonal. This is very interesting finding.Next week, Souvik will complete sequencing of all the pcr products and send Next week, Souvik will complete sequencing of all the pcr products and send you results.you results.Anyhow. please suggest Faria to search for literatures on R.felis as well as Anyhow. please suggest Faria to search for literatures on R.felis as well as other common Rickettsia, to facilitate preparation of thesis.other common Rickettsia, to facilitate preparation of thesis.

Sincerely,Sincerely,N.KobayashiN.Kobayashi

1st email from Prof. Nobumichi Kobayashi

Dear Prof.Akram Hossain, Dr.Shyamal Paul,Dear Prof.Akram Hossain, Dr.Shyamal Paul, Thank you very much for your message. Thank you very much for your message.In my department, Souvik performed In my department, Souvik performed sequencing of 13 samples (2nd PCR products), sequencing of 13 samples (2nd PCR products), out of 16 samples. Three samples could not be sequenced and these products may out of 16 samples. Three samples could not be sequenced and these products may be nonspecific amplicons. Attached please find the original sequence data and be nonspecific amplicons. Attached please find the original sequence data and alignment of 13 samples with Rickettsia felis strain, GenBank KF241854, which alignment of 13 samples with Rickettsia felis strain, GenBank KF241854, which shows 99% identity, with only one mismatch. (shown in color in the file)shows 99% identity, with only one mismatch. (shown in color in the file)Me and Souvik are now very busy, so further phylogenetic analysis will be done later.Me and Souvik are now very busy, so further phylogenetic analysis will be done later.Please send these data to the Mphil student, Faria.Please send these data to the Mphil student, Faria.I think that present study will be good enough for M Phil thesis as well as a good research I think that present study will be good enough for M Phil thesis as well as a good research paper in international journal.paper in international journal.I have an I have an idea for further study. Although detection of R. felis gene by nested PCR idea for further study. Although detection of R. felis gene by nested PCR is highly interesting, 16S rRNA gene was not amplified, probably due to very less is highly interesting, 16S rRNA gene was not amplified, probably due to very less amount of Rickettsial cell in the blood samples. Therefore, I would like to analyze amount of Rickettsial cell in the blood samples. Therefore, I would like to analyze more genes, to confirm and characterize R. felis in Bangladesh. For this purpose, more genes, to confirm and characterize R. felis in Bangladesh. For this purpose, I would like to collect fleas from cats, i.e., wondring cats or those fed in houses of I would like to collect fleas from cats, i.e., wondring cats or those fed in houses of patients.patients. Of course because Rickettsia is very dangerous bacteria, we should pay high Of course because Rickettsia is very dangerous bacteria, we should pay high level of attention for biohazards. Of course we never use them for culture. We should level of attention for biohazards. Of course we never use them for culture. We should extract DNA from suspension of fleas and do PCR only. This is just introduction of my extract DNA from suspension of fleas and do PCR only. This is just introduction of my idea.idea.Anyhow I hope to have good discussion and research activity when I visit MMC again.Anyhow I hope to have good discussion and research activity when I visit MMC again.If you have any plan of PCR for next time (in my next visit in January), please tell me in If you have any plan of PCR for next time (in my next visit in January), please tell me in advance, because I will prepare primers for it.advance, because I will prepare primers for it.Sincerely,Sincerely,

N.KobayashiN.Kobayashi

2nd email from Prof. Nobumichi Kobayashi

Multiple alignment of 17 kDa antigen protein gene, partial cds, of Rickettsia felis strain, GenBank accession no. Multiple alignment of 17 kDa antigen protein gene, partial cds, of Rickettsia felis strain, GenBank accession no. KF241854, with those of KF241854, with those of Rickettsia strains detected in humans Rickettsia strains detected in humans inBangladesh.inBangladesh.  R_felis R_felis --- ---TACTTGGTTCTCAATTCGGCAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 577 --TACTTGGTTCTCAATTCGGCAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 577 --TTACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5813 --TTACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5813 --TTACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5814 -TTACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5814 -ATTACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5925 -ATTACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5925 -ATTACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5927 -ATTACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5927 -ATTACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5932 ----ATTACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5932 ----ACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5636 ---ACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5636 ---TACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5740 ---TACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5740 ---TACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5749 ---TACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5749 ---TACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5767 --TACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5767 --TTACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5871 TTACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5871 CATTACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 6072 ---CATTACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 6072 ---TACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5784 TACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 5784 CATTACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 60 CATTACTTGGTTCTCAATTCGGTAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 60

****************** ************************************* ****************** ************************************* R_felis R_felis CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 1177 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 1177 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11813 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11813 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11814 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11814 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11925 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11925 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11927 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11927 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11932 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11932 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11636 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11636 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11740 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11740 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11749 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11749 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11767 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11767 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11871 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11871 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 12072 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 12072 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11784 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 11784 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 120 CATTACTTGGAGCAGTTCTTGGTGGACAAATAGGTGCAGGTATGGATGAGCAGGATAGAA 120 ************************************************************    ************************************************************    R_felisR_felis

Sequence Alignment

Multiple alignment of 17 kDa antigen protein gene, partial cds, of Rickettsia felis strain, GenBank accession no. KF241854, Multiple alignment of 17 kDa antigen protein gene, partial cds, of Rickettsia felis strain, GenBank accession no. KF241854, with those of Rickettsia strains detected in humans inBangladesh. R_felis ---with those of Rickettsia strains detected in humans inBangladesh. R_felis ---TACTTGGTTCTCAATTCGGCAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 577 -- TACTTGGTTCTCAATTCGGCAAGGGCAAAGGACAGCTTGTCGGAGTAGGTGTAGGTG 577 -- ************************************************************    R_felis ************************************************************    R_felis GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 1777 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 1777 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17813 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17813 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17814 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17814 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17925 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17925 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17927 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17927 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17932 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17932 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17636 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17636 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17740 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17740 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17749 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17749 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17767 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17767 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17871 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17871 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 18072 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 18072 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17784 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 17784 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 180 GACTTGCTGAACTCACTTCACAAAGAGCTTTAGAAGCAACACCTAGCGGCACTAGCGTAG 180 ************************************************************ R_felis ************************************************************ R_felis

AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAAA-- 2277 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAAA-- 2277 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAA--- 22713 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAA--- 22713 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAAAAC 23014 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAAAAC 23014 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAAAA- 23025 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAAAA- 23025 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAA--- 22827 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAA--- 22827 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATA---- 22732 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATA---- 22732 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAA--- 22536 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAA--- 22536 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAAA-- 22740 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAAA-- 22740

AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAAC------------ 21749 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAAC------------ 21749 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAA--- 22667 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAA--- 22667 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAA--- 22771 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAA--- 22771 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAA--- 22972 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAA--- 22972 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAA--- 22684 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAA--- 22684 AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAA--- 229 **************************************** AATGGCGTAATCCGGATAACGGTAATCATGGTTACGTAACACCTAATAA--- 229 ****************************************    

Sequence Alignment

SummarySummaryAndAnd

ConclusionConclusion

A total of 155 clinically suspected febrile patients were enrolled in the study. Out of them, 136 (88%), 31 (23%) and 61 (43%) were positive by Weil-Felix

test, ELISA and PCR respectively.

Out of the 61 PCR positive products, 16 were sequenced in Sapporo Medical University, Japan

where 13 were found to be 99.9% consistent with Rickettsia felis.

Considering PCR as gold standard, Weil-Felix test is found highly sensitive (96.72%) but it

lacks good specificity (43.38%); whereas ELISA has a moderate specificity (60.61%) but

its sensitivity is poor (32.78%).

PCR is considered as gold standard since currently there is no provision for microscopy, culture &/or

IFA tests at MMC.

On the basis of these finding, we are now keen to explore the relationship between the cat-flea and

the R. felis.

This study is now being conducted byDr. Rajib Ahmed,

Student of MPhil (Thesis part), Dept. of Microbiology, MMC.

Further studyFurther study

RECOMMENDATIONIn our study, Rickettsia is found as an important causative agent of febrile illness especially the PUO.

So we recommend PCR as the confirmatory diagnostic option of choice for those cases of febrile illnesses which is not confirmed to be caused by other agents and when a preceding Weil-Felix test is positive.

Prof. Nobumichi KobayashiProf. Nobumichi Kobayashi Assoc. Prof. Shyamal Kumar PaulAssoc. Prof. Shyamal Kumar Paul Dr. Md. Chand Mahmud & Syeda Anjuman Dr. Md. Chand Mahmud & Syeda Anjuman

NasrinNasrin Dr. Syed Ahmed Abdullah & Rajib AhmedDr. Syed Ahmed Abdullah & Rajib Ahmed All faculty members, fellow students, All faculty members, fellow students,

Lab. technician & departmental staffLab. technician & departmental staff Physicians of Medicine Dept. who Physicians of Medicine Dept. who

referred the patientsreferred the patients..

ACKNOWLEDGEMENT

Overview of

Rickettsial fever

Classification

A genus of small, rod-shaped, round to A genus of small, rod-shaped, round to pleomorphic microorganisms in the order pleomorphic microorganisms in the order Rickettsiales. Rickettsiales.

They are true bacteria, gram-negative, They are true bacteria, gram-negative, and cultivable only in living tissues. and cultivable only in living tissues.

Transmitted by lice fleas and ticks, they Transmitted by lice fleas and ticks, they cause disease in humans and domestic cause disease in humans and domestic animals animals

Also found in the cytoplasm of tissue cells Also found in the cytoplasm of tissue cells of lice, fleas, ticks and mites, which may of lice, fleas, ticks and mites, which may act as reservoirs and vectorsact as reservoirs and vectors

Rickettsiae

Variable shape, Variable shape, coccobacillicoccobacilli

Gram negativeGram negative Microcapsule and slim Microcapsule and slim

layerlayer Culture : in yolk sacs of Culture : in yolk sacs of

embryonated eggs/ embryonated eggs/ Tissue cultureTissue culture

Biological featuresBiological features

Maintain in animal and arthropod Maintain in animal and arthropod reservoirs (ticks, mites, lice, fleas by reservoirs (ticks, mites, lice, fleas by transovarian transmission).transovarian transmission).

Transmitted to humans by Transmitted to humans by arthropod arthropod vectorsvectors

Humans are accidental hosts: acquired by Humans are accidental hosts: acquired by arthropod bite or contact of arthropod arthropod bite or contact of arthropod excreta with abraded skinexcreta with abraded skin

Rickettsia Transmission

No toxins, no immunopathologyNo toxins, no immunopathology RickettsiaRickettsia replicate in endothelial replicate in endothelial

cellscells, cause cell damage and blood , cause cell damage and blood leakage, vasculitis, microthrombi, leakage, vasculitis, microthrombi, focal ischemia, hemorrhage, focal ischemia, hemorrhage, skin skin rashrash..

Hypovolemia, hypoproteinemia, Hypovolemia, hypoproteinemia, reduced perfusion, organ failure.reduced perfusion, organ failure.

Pathogenesis

PathophysiologyPathophysiology

Laboratory DiagnosisLaboratory DiagnosisSpecimen:Specimen:

Blood & skin biopsy.Blood & skin biopsy. Culture:Culture:

Tissue culture or chick embryo.Tissue culture or chick embryo. Direct detection in clinical specimen:Direct detection in clinical specimen:

PCRPCR Immunofluorescence Immunofluorescence

Serological diagnosis:Serological diagnosis: Detection of Detection of rising titerrising titer of anti-rickettsial of anti-rickettsial

antibodies by antibodies by ELISAELISA.. Weil-Felix reaction: no longer used because it is Weil-Felix reaction: no longer used because it is

non specific.non specific.

IFA reaction of a positive human serum on Rickettsia rickettsii grown in chicken yolk sacs, 400X