A Study on Rickettsial Infections and its Isolation by Serological and Molecular Methods

 

S. Mohammed Shahid¹, Dr. Siddesh B. Sirwar²*, Dr. R. Vijayaraghavan³

¹Assistant Professor, Dept. of Microbiology, Khaja Bandanawaz Institute of Medical Sciences,

Gulbarga- 585104 and Ph.D Scholar, Saveetha University, Chennai - 602105.

²Professor and HOD, Dept. of Microbiology, Khaja Bandanawaz Institute of Medical Sciences,

Gulbarga-585104, Karnataka.

³Director of Research, Saveetha University, Thandalam, Chennai, 602105. India

 

ABSTRACT:

Background and objectives: Prevalence of re-emerging of rickettsial infections are increased the major clinical manifestations present times in India. Aim: To study the incidence, prevalence and isolation by serological and molecular methods.Methodology: All the suspected cases clinically were subjected for Weil-Felix test and further confirmation by isolation by molecular method.Results: The study depicts pattern in clinically signs and symptoms with fever, chills and rash in majority cases, along with incidence predominant in above 30 years in male and female group. Out of 262 cases 116 positive by Weil- Felix test and among them 69 showed positive for gltA gene by PCR.Conclusion: Detection of gltA gene is ideal target for the diagnosing of rickettsial infections in early phase of the infection because of its high sensitivity and specificity.

 

 

 

INTRODUCTION:

Rickettsial infections are acute febrile illness and under diagnosed group of diseases. The majority of places in India there is re-rise in rickettsial infections¹. Rickettsial diseases caused by Rickettsiae are gram negative bacilli, coccobacilli, obligate intracellular and transmitted by such as mites, fleas, lice and ticks like arthropod vectors. They originate in the digestive tract of these vectors². Majorly they infect the vascular endothelium and reticuloendothelial cells. Common symptoms are fever with chills, severe headache, history of insect bite, rashes, eschar, myalgia, sore throat, lymphadenopathy and hepatic syndromes etc³. Specific diagnosis and community based studies are needed to get rid from the burden of rickettsial infections⁴. Misdiagnosed and untreated cases become fatal and may increase the mortality rate as high as 30-35% and more⁵.

 

Prevalence of rickettsiosis seen in major states of         India^6-8.

 

Because of its improper symptoms diagnosis of rickettsial infections have became tuff in diagnosing early phase of illness. Preliminary confirmation by Weil-Felix (WF) and ELISA tests are helpful for the empirical treatment in the rickettsial infections. Molecular diagnosis by PCR test gives accurate diagnosis as a point of care strategy included to molecular tools⁹. All the group of rickettsial infections are confirmed by citrate synthase (gltA) gene^10-11.

 

The present study depicts the incidence, prevalence and its isolation by serology and molecular methods.

 

MATERIALS AND METHODS:

The study was conducted in Khaja Bandanawaz Institute of Medical Sciences, Gulbarga during the period from May-2016 to June-2017. Blood samples were collected from the patients who are having thesymptoms and signs of history of insect bite, headache, fever, rash, eschar, and myalgia are as an inclusion criteria. A total number of 262 samples were tested by Weil- Felix test and results were noted down within one minute as agglutination obtained, later to the positive samples performed the PCR test for further confirmation.

 

Ethical consideration:

The present study was approved by the Institutional Ethics Committee (IEC) of Khaja Banda Nawaz Institute of Medical Sciences, Gulbarga-Karnataka, India (IEC No KES/KBNIMS/IEC/2016-17/24; meeting held on 21-08-2016). All samples were processed as per standard operating procedures.

 

DNA extraction and Sequencing:

Whole blood samples were used to extract the DNA and used citrate synthase gene (gltA) gene for identification of rickettsial infections. The sequence of the nucleotide was done by using Basic Local Alignment Search Tool (BLAST) program¹². Procedure of PCR method performed with the instructions of the manufactures manual. The gltA gene was selected for the rickettsial infections because of its higher variability. The DNA was amplified with designed primers of both forward and backward primers Rtp-F(5′-TTCGGATTGCTGGCTCATCA-3′) and Rtp-R (5′-AAATGGATCATTCTTATCTTTAGCTTTAGC-3′). Master mix was prepared by adding 12.5 μL of QiagenQuantiTect Multiplex PCR Master Mix with 0.4 μM of each of the forward and reverse primers, probe 0.2μM, DNA extract 2.5 μL in 25μL volume. By using thermal cycler (GeNeiTM, Merk specialties Pvt. Ltd) cycling of 1 cycle was donefor 15 minutes at 95°C and next 45 cycles for 60 seconds at 95°C .By using UV illuminator (agarose gel) the final products were observed and recorded.

 

RESULTS:

A total 262 whole blood samples collected from patients presenting history of fever with chills, insect bite, rash, eschar and multiple clinical manifestations. Rash and fever with chills were major presentation in males and females. Table no: 1 described the pattern of Rickettsia isolated from variousinfections among male and female. In table no: 2the above 30 years of age group 71 % (49) of incidence was seen in both genders. Less incidence rate was observed in both genders below 30 years of age group. In table no: 3 out of 262 samples analysed and tested by the preliminary test Weil-Felix test in which positive samples were 116 (44.27 %) in that males were 61.2 % (71/116) and females were 38.8 %(45/116). Further these samples were run on PCR method. In PCR method 69 samples found positive in that males were 58 %(40/69) and females were 42%(29) observed.

 

Table 1: Pattern of Rickettsia isolated from variousinfections among male and female.

Clinical features	Male (n=40)		Female (n=29)		Total n=(69)
	No	Percentage	No	Percentage	No	Percentage
History of insect bite	01	2.5 %	02	7 %	03	4.34 %
Fever with chills	10	25 %	10	34.48 %	20	29.00 %
Eschar	06	15 %	02	7 %	08	11.59 %
Rash	16	40 %	11	38 %	27	39.13%
Multiple clinical manifestations	07	17.5 %	04	14 %	11	15.94 %
Total	40	100	29	100	69	100

 

Table 2: Age and sex wise distribution of rickettsial infections

Age group	Male		Female		Total
	No	Percentage	No	Percentage	No	Percentage
<30 years	15	(37.5%)	05	(17.24 %)	20	(29%)
> 30 years	25	(62.5%)	24	(82.75%)	49	(71%)
Total	40	58%	29	42%	69	(100 %)

 

 

Table 3: Rickettsial diagnosis by Weil-Felix test and PCR

Sex	Weil-Felix test (n=262)				PCR (n=116)
	Positive		Negative		Positive		Negative
	No	Percentage	No	Percentage	No	Percentage	No	Percentage
Male	71	(61.2%)	91	(62.32%)	40	(58%)	28	(60%)
Female	45	(38.8%)	55	(37.67%)	29	(42%)	19	(40.42%)
Total	116	100 %	146	100 %	69	100 %	47	100 %

 

DISCUSSION:

In present study highest prevalence was seen in rash (40%) followed by the symptom fever with chills (25%). From multiple clinical manifestations Rickettsia bacteria was isolated about 17.5%. Paddock et al.,¹³ was isolated Rickettsia from eschar clinical feature.

 

 

In a study by Aure lie Renvoiseet al., 2012 observed high prevalence rate in isolated bacteria from cutaneous biopsy samples (68.9%) than cutaneous swabs (17.8%) from rash and eschar¹⁴. About 4.4 % samples in whole blood and 8.9% were from serum samples. S. Santibanezet al, reported the gltA was isolated from whole blood samples (33.33 %).¹⁵ from serum samples 71 cases isolated for the detection of rickettsial infections by PCR. Theodore Tzianabos study et al study used blood clots from 9 cases for the diagnosing Rickettsia¹⁶. Other studies like Furuya et al, Hanaoka N et al used whole blood, serum, blood clots for diagnosis of rickettsial infections^17-18.

 

Another study by Walker et al 2003 observed that all rashes (maculopapular, erythematous and petechial lesions) are the main important multiplication sites for Rickettsia¹⁹. Cutaneous swabs and biopsy samples from ecshar and rash category helps in confirming the rickettsial infections without risk²⁰. In above 30 years of age group shown high prevalence rate when compared to less than 30 years of age group in present study. A study by Prakashet al., were found the majorgroup (58.6%) constituted in below 6 years of age²¹.

 

Present study showed the incidence of males were 40(58%) and females were 29(40.42%) by the PCR test. Prakashet al., were observed incidence of 58.6% in their study²¹. Wood et al and Wiegand. G et al study also used gltA gene for the detection of rickettsial infections^22-23. The citrate synthase gene (gltA) is a highly conserved gene among the genus Rickettsia and present widely among Rickettsia bacteria. The highly conserved nature of the gltA gene makes it an ideal target for real-time PCR^10,24.

 

The mode of transmission of Rickettsia are characterized generally by transovarial were in high vector population. By using gltA gene in PCR assay high rate of sensitivity was obtained and very good diagnostic tool for the confirmation of rickettsial infections^25-26.

 

CONCLUSION:

Rickettsial infections are diagnosed by Weil-Felix testsince a long time along with clinical features but in specificityand sensitivity the test is very poor. RT- PCR establishes accurate and specific diagnostic test and also diagnose early where improper symptoms are present and also provide the specific clinical distribution of rickettsial infections characteristics. PCR assays (gltA gene) are very good diagnostic tools for the detection of rickettsial infections in molecular methods when serology results were variable. Early diagnosis helps to reduce the morbidity and mortality of rickettsial infections and these infections are co-endemic along with bacterial and viral diseases. With the widespread availability of the new tools with which one can confirm the diagnosis of rickettsial diseases.

 

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Accepted on 10.04.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12 (8):3687-3689.