Clinical Performance Analysis and Evaluation of Quantitative Real Time PCR for Diagnosis of Scrub Typhus in North East India

Scrub typhus being a life threatening infectious disease and always creating diagnostic dilemma in terms of rapid turnaround time and accuracy, qRT PCR can become a very good option to achieve the desired result with molecular level of accuracy and boost up the rapid patient management. This study was performed to evaluate the performance of qRT PCR in comparison to commonly used IgM ELISA and Weil-Felix tests to diagnose scrub typhus, as well as to look for the demographic and clinical profile of the disease in North-East India. It was a hospital-based prospective study conducted in a tertiary care hospital of north-east India, over a period of 1 year, in which all the samples from suspected scrub typhus cases were screened by Weil-Felix test as per institute’s diagnostic protocol after which IgM ELISA for Scrub Typhus was performed. All the IgM positive samples and 20 highly suspected but ELISA negative samples were subjected to qRT PCR, targeting 56 kDa type specific gene of O. tsutsugamushi. Statistical analysis was done by MS-Excel for Windows v2013® and MedCalc® v17.9 for Windows (MedCalc Software, Acacialaan 22, B-8400 Ostend, Belgium). In this study we have successfully evaluated the performance of qRT PCR kit for diagnosis of scrub typhus. Out of 54 samples tested, 24 IgM ELISA positive samples and 3 IgM ELISA negative samples have shown the presence of bacterial DNA with quantification of DNA copies. It has also been observed that 21 out of 27 PCR positive samples (77.8%) were detected within the 1 7 days of illness. All the demographic as well as clinical data were also analysed. The performance of the commercial qRT PCR kit used in our study is satisfactory, which provides extra advantage of quantification of DNA copies and increases diagnostic accuracy within the 1 week of fever.


INTRODUCTION
study was conducted, this disease is known to be endemic, responsible for significant amount of mortality and morbidity every year [13][14][15].
Isolation of the organism from blood is laborious and time-consuming process as it takes more than a month using cell culture. Nonspecific Weil-Felix test is extensively used everywhere but it lacks in both sensitivity and specificity. The presence of scrub typhus based on this nonspecific Weil-Felix test, had been documented in Jammu and Kashmir, Himachal Pradesh, Uttaranchal, Rajasthan, Assam, West Bengal, Maharashtra, Puducherry, Kerala and Tamil Nadu [16,17]. Immunochromatographic card test is also a very popularly used tool, which also lacks in specificity. The Immunofluorescence antibody assay (IFA) has subsequently replaced the Weil-Felix assay as a "Gold Standard" test for the detection of antibodies induced by Orientia [18]. However, the lack of standardization of IFA assays among laboratories around the world has brought into question, its applicability in the diagnosis of scrub typhus [19]. A more standardized methodology is the ELISA, which has been used for years in surveillance studies, where large number of sera can be assayed at one time.
The utilization of molecular methods in the past 20 years have increased the sensitivity and specificity of diagnosis of scrub typhus and other rickettsial diseases along with their detection in vertebrate hosts and invertebrate vectors [20]. Recent nucleic acid-based techniques like conventional PCR, nested PCR and Real time PCR are useful for early and specific diagnosis.
Because of the low index of suspicion, nonspecific signs and symptoms, emergence in nonendemic areas as well as re-emergence in previously endemic areas and absence of widely available sensitive and specific diagnostic tests, Scrub typhus is really imposing a diagnostic challenge.
Hence, this study was planned to evaluate the diagnostic performance of quantitative real time PCR to detect the presence of Orientia tsutsugamushi in patients with febrile illness, presenting to a tertiary care hospital of North-East India, where Scrub-Typhus is highly endemic.

MATERIALS AND METHODS
Study design -This was a hospital based prospective study conducted in the Department of Microbiology, of a tertiary care center of North-East India over a period of 1 year (June 2016 -May 2017).

Inclusion Criteria -
Case -Patients are included in the study if they have -1) Febrile illness (fever > 37.8º C) and a clinical suspicion of scrub typhus, presenting to the department of Medicine and Pediatrics. 2) Given written informed consent to participate in the study, either by themselves or by parents/guardians (for pediatric age group). Controls -Twenty volunteers with no history of fever and no history of scrub typhus before, otherwise healthy were also included in the study as controls.
Exclusion Criteria -Patients were excluded if a definitive diagnosis of malaria, enteric fever, tuberculosis, any other infectious or non-infectious etiology was made during patient work-up.
Sample size -One hundred patients with acute febrile illness, without any definitive diagnosis and clinical suspicion of scrub typhus were included in the study.
Ethical approval -Ethical approval was taken from the institution Ethics Committee, letter no. -NEIGR/IEC/2015/0040. History taking and Sample collection -A detailed history taking of all the patients along with physical examination was performed. All the patients were further followed up for their clinical progression and outcome.
Blood samples were collected in plain vacutainers (without anticoagulant) from 100 patients of acute febrile illness (cases) and from 20 healthy volunteers (controls), taking both inclusion and exclusion criteria under consideration.
Samples were further processed and analyzed for the detection of Orientia tsutsugamushi.
Collection of serum samples and storage -Serum had been collected from plain vials clotted blood after centrifuging at 2000 RPM for 10 minutes and then separating serum without touching the leukocyte layer.
Aliquots were made from serum samples into 3 parts for different purposes.
Part 1 -For screening by Weil-Felix test.
Part 2 -For doing ELISA to detect IgM against Orientia tsutsugamushi.
Part 3 -For DNA extraction from the serum samples and PCR. Part 2 and 3 of aliquots were stored at 2 to 8º C for 48 hours, then at -20º C for 1 month and for further storage of >1-month aliquots were stored at -80º C.
Screening by Weil-Felix test -All the serum samples were screened first for scrub typhus by using Weil-Felix test. "PROGEN" Proteus antigen suspensions by Tulip Diagnostics (P) LTD. India, were used for Weil-Felix test by semi-quantitative slide method following the exact manufacturer's instructions and titers of OXK antigen were obtained.
IgM ELISA -"Scrub Typhus Detect TM IgM ELISA System" by INBIOS International, Inc. was used for performing ELISA to detect IgM antibodies against Orientia tsutsugamushi derived recombinant antigen in stored serum samples.
ELISA procedure -All the serum samples from 20 healthy volunteers and all the 100 stored serum samples from suspected scrub typhus patients were subjected to IgM ELISA for scrub typhus following the manufacturer's instructions exactly. All the ELISA readings were obtained by using "Merilyzer EIAQuant Microplate Reader"by Meril Life Sciences.
Cut-off calculation -Cut off was calculated as per manufacturer's instructions by adding the mean of OD plus three times of the Standard Deviation (SD) of normal human serum. Serum samples from 20 healthy volunteers were used to calculate the cut-off. The mean and SD of all healthy volunteers corresponded to OD values of 0.1445 and 0.0437, respectively. Using the manufacturer's cutoff formula of the final ELISA cutoff corresponded to an OD of 0.2756.
Interpretation of the results were done by following the below mentioned criteria -1. Samples with spectrophotometric readings > Cut off are "Reactive" and samples below this criterion are considered to be "Non-Reactive".
2. Values near the Cut off are doubtful and the assays were repeated DNA extraction -From all the IgM ELISA reactive samples as well as 20 highly suspected but ELISA negative samples DNA was extracted using "QIAamp DNA Mini Kit" (Qiagen, Germany) as per the manufacturers' instructions. Extracted DNA samples were kept frozen at −80°C till the time of testing.
Real time Polymerase Chain Reaction -Extracted DNA samples were subjected to quantitative real-time PCR by using "Geno-Sen's Scrub Typhus (Rotor Gene) real time PCR kit", Genome Diagnostics Pvt. Ltd, Solan, Himachal Pradesh, India.

Principle of Real-Time PCR kit used -
The kit targets 56 kDa Type Specific gene of O. tsutsugamushi. The robust assay exploits the so-called TaqMan principle. During PCR, forward and reverse primers hybridize to a specific sequence product. A TaqMan probe, which is contained in the same reaction mixture and which consists of an oligonucleotide labeled with a 5'-reporter dye and a downstream, 3'-quencher dye, hybridizes to a target sequence within the PCR product. A Taq polymerase which possesses 5' -3' exonuclease activity cleaves the probe. The reporter dye and quencher dye are separated upon cleavage, resulting in an increase in fluorescence for the reporter. Thus, the increase in fluorescence is directly proportional to the target amplification during PCR.
The primer details are as follows: • OtsuF: 5'-AATTGCTAGTGCAATGTCTG-3', 15 μl of the Master Mix/Premix was pipetted into each labelled PCR tube. Then 10 μl of the earlier extracted DNA was added to each sample tube and mixed well by pipetting up and down. Correspondingly, 10 μl of the Standards (SCRUB TYPHUS S1-5) was added as a positive control and 10 μl of water (Water, PCR grade) as a negative control. PCR tubes were closed and transferred the tubes into the rotor of the RotorGene™ Q (QIAGEN Real-time PCR Cycler). Locking Ring was placed on top of the rotor to prevent accidental opening of the tubes during the run.
Then PCR run was started following the optimum cycling conditions mentioned below as per the manufacturer's instructions.

Cycle
Cycle Point Hold (Initiation): 95º C for 10 minutes Cycling (45 repeats) Step 1 (Denaturation): 95º C for 15 seconds Step 2 (Annealing): 55º C for 20 seconds Step 3 (Extension): 72º C for 15 seconds Other instructions for ideal PCR run condition were followed exactly as mentioned in the user manual provided with the PCR kit.

Quantitation -
The quantitation standards provided in the kit (SCRUB TYPHUS S 1-5) has been treated in the same way as extracted samples and the same volume is used i.e. (10μl) instead of the sample.
To generate a standard curve in the RotorGene™ Q system, all 5 Standards were used as defined in the menu window Edit Samples of the RotorGene™ software. The standard curves generated were used for quantitation in subsequent runs, provided that at least one standard was used in the current run.
The standards are defined as copies/μl. The following formula can be applied to convert the values determined using the standard curve into copies/ml of sample material:

Result (Copies/μl) x Elution Volume (μl) Result (Copies/ml) = ______________________________________ Sample Volume (ml)
In our study direct conversion formula was used as the starting volume of the sample while using the Qiagen QIAamp DNA Mini extraction kit was 200μl & the final Eluted Volume was 50μl.
Following values for the standards was fed directly into the operating software which was again based on the above formula and yielded direct conversion of per ml of patient sample data. S1: 10 5 Copies/μl = 25000000 Copies/ml S2: 10 4 Copies /μl = 2500000 Copies/ml S3: 10 3 Copies /μl = 250000 Copies/ml S4: 10 2 Copies /μl = 25000 Copies/ml S5: 10 1 Copies /μl = 2500 Copies/ml Statistical analysis -The data was collected and recorded using MS-Excel for Windows v2013®. The basic descriptive statistics, frequency charts graphs, trend analysis and related graphs were computed using the same. Summary statistics and ROC curve analysis were done using MedCalc® v17.9 for Windows (MedCalc Software, Acacialaan 22, B-8400 Ostend, Belgium). The comparison of single and two proportions were done using Chi-square test. The threshold for significance was considered at p<0.05.
The data analysis and generation of graphs for the PCR was performed using Rotor-Gene Q Series software v2.3.1 (Build 49).

RESULTS
During the study period of one year 100 patients with febrile illness were evaluated for scrub typhus (ST). The study was conducted not only to evaluate the performance of Quantitative Real-Time (qRT) PCR as an accurate and quick diagnostic procedure for ST but also to find out different etiologies of febrile illness cases other than ST. All the collected serum samples were subjected to Weil-Felix for the screening and IgM ELISA for confirmation of ST. All the IgM ELISA positive samples and 20 highly clinically suspected samples which were IgM ELISA negative were subjected to qRT PCR. Based on IgM ELISA and PCR findings 37 patients were diagnosed to be having ST, according to STIC criteria [21]. Other patients were followed up for the alternate final diagnosis, based on other laboratory investigation findings done by the respective departments. Different aetiologies of fever other than ST were sepsis (n=17), malaria (n=4), enteric fever (n=4), tubercular meningitis (n=4), Japanese encephalitis (n=3), pneumonia (n=1), meningitis (n=1) and dengue (n=1). In few patients (n=29) any diagnosis could not be established as some of them lost follow up due to death before any established diagnosis or discharge against advice or non-admission as an outpatient. Diagnostic profile of all the 37 diagnosed ST patients are described here on.
For the screening of scrub typhus cases an easy to do and widely used serological test Weil-Felix was performed. For confirming the scrub typhus cases IgM ELISA was used, which is well established and widely performed. Molecular test -Quantitative Real Time PCR is a relatively new, fast and accurate diagnostic modality which is yet to be evaluated. In our study we tried to see the usefulness and utility of Real Time PCR in comparison to a standard methodology for diagnosing scrub typhus -IgM ELISA. Results of all the 3 tests are as follows:
Different OXK titers of all the 100 samples are as follows:  Table   2)  (Table).
Very low count -DNA copies in multiplies of 10 3 Low count -DNA copies in multiplies of 10 4 Medium count -DNA copies in multiplies of 10 5 High count -DNA copies in multiplies of 10 6 Relation between different bacterial DNA loads and day of fever when sample was collected were analysed which was statistically not significant (see Table 4).   have been mentioned in various literatures [22,23]. Being in an endemic zone, scrub typhus is very much prevalent in India, causing multiple outbreaks every year [24,25], especially in the North-Eastern part of the country [13,14,26,27]. Scrub typhus is a well-known disease in the state of Meghalaya, which has a local name also "Niangsohot". Every year lots of cases and deaths due to scrub typhus are being reported from Meghalaya and it has become an important cause of acute febrile illness and related mortality and morbidity. Most of the time the disease remains undiagnosed due to non-specific clinical presentation and low index of suspicion among physicians. Regarding diagnosis, the most widely used screening test Weil-Felix suffers lack of sensitivity and specificity, whereas gold standard test IFA lacks in standardization and availability. Presently many studies and case reports on scrub typhus are coming out from different parts of the country, revealing new pockets and true scenario of this dreaded disease.
PCR as a molecular test scores more in the aspect of sensitivity and specificity but warrants more stringent conditions. Till now most of the studies have used Nested PCR, which has shown good sensitivity and specificity but currently Real-Time PCR is on the trends with a boon of quantification.
This study was a venture to know different aetiologies of acute febrile illness and to evaluate Real-Time Quantitative PCR as an accurate, less labour intensive and with an extra benefit of quantification; for diagnosing an important cause of acute febrile illness -Scrub Typhus.  [29].

Aetiologies of AFI
From the diagnostic point of view scrub typhus will always be a challenging task, because of its divergent presentations and scope of clinical misjudgement. In a resource poor set up like India, especially in the corner most part of the countrythe north-eastern region, easy to do and cheaper ways of diagnostics will always be more preferred. Therefore, in an endemic zone of scrub typhus Weil-Felix will be a preferred tool for screening and its results can be confirmed by serological tools like IgM ELISA or Molecular tests like Real-time PCR. Sensitivity, which is the parameter of choice to know the performance of a test, suffers a lot, if high cut off titers of antibodies against OXK is used. Therefore, seroprevalence of scrub typhus and thereby determining the cut off titer for OXK is especially important. Moreover, Weil-Felix has a good PPV and NPV despite having low sensitivity as shown in our study. So, it remains as a good tool for the screening of scrub typhus, especially in our set up.
IgM ELISA test -As Immunoflourescence assay (IFA)the gold standard test for scrub typhus, was not available and moreover its lack of standardization worldwide [19], IgM ELISA was considered as the standard methodology test in our study. All the 34 positive samples by IgM ELISA were true positive samples and were used to compare the results of the other 2 tests.
In a recent study to evaluate diagnostic accuracy of the InBios Scrub Typhus Detect ELISA kit for the detection of IgM Antibodies by Blacksell et al., which was also used in our study, a sensitivity of 93% and specificity of 91% was reported when cut off OD value for IgM ELISA was calculated according to manufacturer's instructions [30].
Jang et al., evaluated IgM ELISA for the diagnosis of scrub typhus and reported sensitivity of 100% and specificity of 99% for IgM IFA-positive samples [31]. The performance and diagnostic accuracy of this IgM ELISA kit was validated by different authors in India [29,[32][33][34][35][36] and the results were quite satisfactory. Even though IFA is the gold standard test for diagnosing scrub typhus, it is difficult to perform, cumbersome, requires a skilled observer, mandates inclusion of several antigenic types, subjective, and too expensive to be imported [36].
The sensitivity and specificity of ST IgM ELISA are almost equivalent to those of IFA, and it can be performed by most laboratories [18,37]. Therefore for confirmation of the screening positive samples IgM ELISA is a very good alternative to IFA.
However, few false positive results were obtained by Prakash et al., in patients with falciparum malaria, pulmonary tuberculosis, Streptococcus viridans septicaemia, and typhoid fever [33].
But we in our study did not get any such instances of cross reactivity. Out of 77 ELISA positive samples 42 (54.54%) were PCR positive. In that study the sample of choice was buffy coat and IgM ELISA was used as an alternative to IFA for serological confirmation, which was like our study [38].

Real Time PCR assay
Difference between positivity rates amongst both the studies can be attributed to the different prevalence rates of the study location.
In another study by Singhsilarak et [40].
In a study by Usha et al.,34.69% patients were nested PCR positive targeting 56kDa gene [41].
A study by Mahajan et al. where eschar samples were collected from 3 regions of the country in which 79.5% of positive samples for 56kDa gene using conventional PCR were reported [42].
Molecular diagnosis always takes the upper hand from serological tests, as using PCR tests  [43]. Compared to other types of PCR, real-time PCR using the above-mentioned kit is fasterresults can be obtained within 2 hours. Weil-Felix has shown low sensitivity in our study, considering its good PPV and NPV we can use this test as screening tool of scrub typhus in a resource poor setup. But cut off titer for antibody against OXK should be re-evaluated in this geographical area based on the prevalence of scrub typhus cases. The newly available Real-time PCR kit can be used as a confirmation tool for scrub typhus, but its performance can be re-evaluated using other patient samples like buffy coat, blood clot or eschar tissues.

CONCLUSION
Scrub typhus being a diagnostic challenge to the clinicians as well as to the clinical microbiologists, will always continue to raise questions regarding its correct, accurate and faster diagnostic approach. Even though its treatment is plain sailing, provided diagnosed on time. But situations may turn out to be detrimental for the patients if determination of the correct one is delayed. In this purpose accurate and cost-effective screening along with flawless confirmation can lead us to the desired outcome for the patients.
In a place like North-East of India, Weil-Felix can still serve the purpose of screening out the cases of scrub typhus but for confirmation IgM ELISA or Real-Time PCR can be used. If we can combine the power of both the tests then there will be less chances of missing a case.
The performance of the commercial quantitative real-time PCR kit used in our study is satisfactory. Molecular diagnosis should be carried out preferably during the 1 st week of febrile illness for better results. Due to the reasonable levels of sensitivity and specificity of Real-time PCR, its continued usage as a standard methodology in developing countries like India may be a good choice, as utility and availability of IFA kits are still questionable and culturing Orientia is a tough nut to crack.