Role of TaqMan array card in determining causative organisms of acute febrile illness in hospitalized patients

Abstract Background Acute febrile illness (AFI) is a prevalent disease in developing countries that is difficult to diagnose due to the diversity of infectious organisms and the poor quality of clinical diagnosis. TaqMan array card (TAC) can detect up to 35 AFI‐associated organisms in 1.5 h, addressing diagnostic demands. In this study, we aimed to evaluate the role of TAC in determining the causative organisms in hospitalized AFI patients. Methods The study had a cross‐sectional design and enrolled 120 admitted patients with persistent fever for three or more days from the medicine ward of Chittagong Medical College Hospital (CMCH) and Bangladesh Institute of Tropical and Infectious Diseases Hospital (BITID). Blood samples were collected and then subjected to automated BacT/Alert blood culture, microbial culture, TAC assay, and typhoid/paratyphoid test. Results The total number of study participants was 120, among them 48 (40%) samples showed a positive result in TAC card, 29 (24.16%) were TP positive and nine (7.51%) were culture positive. The number of organisms detected by the TAC card was 13 bacteria, three viruses, one protozoan, and one fungus. The sensitivity and specificity of the TAC assay for different bacterial pathogen compared to blood culture was 44.44%, and 90.99%, respectively. In contrast, the TP test had a sensitivity and specificity of 100% and 80%, respectively, compared to the blood culture test. Conclusion TAC can be a handful tool for detecting multiple organisms in AFI with high specificity which can facilitate early diagnosis of different pathogens contributing to AFI.

endemic and epidemic diseases globally. 3,4 In Bangladesh, major causes of AFI include dengue, malaria, typhoid fever, leptospirosis, and rickettsial infection. [5][6][7][8][9] The case fatality rate varies between 5% and 24% among hospitalized adults with AFI. 10,11 Identifying the specific infectious agent causing AFI is challenging due to the nonspecific clinical presentation, diverse causes, various specimen types, and the need for multiple laboratory techniques. Prompt diagnosis is crucial in preventing a negative outcome of the illness, as delayed appropriate antibiotic treatment can decrease survival chances by 7.6% per hour. 12 The blood culture test is the current standard for detecting bacterial infection in patients with AFI. However, it has limitations in detecting slow-growing, intracellular, and fastidious microorganisms and in patients who have received antibiotics. 13,14 Due to its poor sensitivity of about 60%, a blood culture test may cause patients to skip timely antibiotic doses. [15][16][17] Nucleic acid amplification (NAA) based approaches might reduce the turnaround time, and multiplex polymerase chain reaction (PCR) could simultaneously detect multiple pathogens. 18 Although PCR assays can detect multiple organisms in a single reaction through multiplexing, screening a large number of samples for several pathogens simultaneously can be extremely labor-intensive and impractical. Therefore, the development of a rapid and efficient nucleic acid-based molecular diagnostic method capable of identifying multiple microorganisms of AFI could greatly improve patient care. The TaqMan array card (TAC) is a multiplex real-time PCR with 384 wells, based on microfluidics, and is especially suitable for samples of only 1/2 μL volume. It can analyze 24-384 pathogenic targets simultaneously and its closed system structure reduces the possibility of contamination. 19 In Bangladesh, limited data are available regarding the etiology of AFI. As of today, few studies have been conducted using TAC technology to identify the causative agents of AFI in hospitalized patients. Therefore, our objective was to evaluate the effectiveness of TAC in detecting infectious bacteria, viruses, protozoa, and fungi in the blood samples of hospitalized AFI patients compared with blood cultures.

| Study design
The cross-sectional study included 120 patients with an unknown etiology of fever who were admitted to the Department of Medicine

| Inclusion criteria
All patients attending the hospitals with a diagnosis of AFI were at first assessed for eligibility. Patients of either sex with documented fever (≥38°C/100.4°F) or on admission oral temperature (≥38°C/100.4°F) for 3-14 days and age above 12 years with unknown etiology of fever were enrolled in this study.

| Excluding criteria
Patients who were under the age of 12 and had incomplete medical records during the study period were excluded.

| Blood collection
Blood samples were collected from enrolled patients by a trained nurse by maintaining proper precautions and aseptic conditions. A total of 10 mL of venous blood were collected from each patient after physical examination for different assay purposes, and distributed accordingly: 5 mL in aerobic FAN bottle for automated BacT/ Alert blood culture system, 1 mL in sodium heparin tube for typhoid/ paratyphoid (TP) test, 2 mL in EDTA tube for TAC assay and 2 mL in EDTA tube for complete blood count (CBC). Following the blood collection, EDTA, or sodium heparin tubes were flipped 2-3 times for homogenous mixing. Blood in the EDTA tube for TAC assay was stored at −80°C.

| Blood culture
Blood culture was performed in an automated BacT/Alert blood culture system. Briefly, 5 mL of blood samples were collected from each patient and immediately inoculated into aerobic FAN blood culture bottles. These bottles were then inserted into BacT/Alert incubator unit for 5/6 days. A positive or negative FAN bottle for bacterial growth was subjected to subculture on MacConkey (MC) agar, chocolate agar, and blood agar (which contained 5% sheep blood) for 2 days. The MC agar plates were then aerobically incubated at 35°C, while the chocolate and blood agar plates were incubated in microaerophilic growth conditions (5% CO 2 and 35°C). Microbial isolates were identified using the Clinical and Laboratory Standards Institute (CLSI) guidelines. 20

| TPTest for detecting Salmonella Typhi/ Paratyphi
Besides the automated BacT/Alert blood culture system, TPTest was also performed for identifying Salmonella-specific IgA responses in the lymphocyte culture supernatant as previously described. 21 In summary, unstimulated peripheral blood mononuclear cells (PBMCs) were cultured at a concentration of 10 7 cells/mL in RPMI 1640 medium, with heat-inactivated fetal bovine serum (10%), streptomycin (100 μg/mL), penicillin (100 U/mL), pyruvate (100 mM), and L-glutamine (200 mM). The culture was incubated at 37°C with 5% CO 2 for several time periods. Following this, the culture supernatants were collected and analyzed for IgA antibodies that were specific to Salmonella Typhi membrane preparation (MP) using the enzyme-linked immunosorbent assay (ELISA) method.

| Total nucleic acid extraction from blood samples for TAC assay
Total nucleic acid is extracted from blood specimens using the High

| Identifying bloodstream pathogens by TAC assay
Nucleic acid extract was mixed with TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems) in a 100 μL reaction mixture and then pipette the mixture into the inlet port of each channel. The card was centrifuged twice for 1 min at 97 g to fill the well, sealed the card to close the well, and the inlet ports were removed. Within 10 min card was ready to run on the QuantStudio™ 7 Flex instrument (Applied Biosystems, Thermo Fisher Scientific). Primers and probes targeting AFI-associated microorganisms developed by Liu et al., 22 were used in this study (Table S1). The TAC was run using PCR cycling conditions comprising 10 min at 50°C and 20 s at 95°C followed by 45 two-step cycles of 3 s at 95°C and 30 s at 60°C. A universal bacterial 16S assay was also included on the card for inferring the presence of bacterial pathogens that were not interrogated on the card. After 1.5 h the results were ready for analysis. The amplification data were analyzed by the conventional cycle-threshold (C t ) method. A C t threshold value of 40 cycles was employed to distinguish between positive and negative samples for both clinical specimens and the negative control on the TAC.

| Data analysis
Data were analyzed using statistical software (Statistical Package for Social Science, SPSS Version 23.0, IBM Corporation). Continuous variables were given as means ± SD, whereas categorical variables were presented as frequency and percentages. Sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy for each test were computed, as were 95% confidence intervals. The gold standard for analytical comparison was blood culture. Statistical significance was defined as p < 0.05 and a confidence interval was set at 95%.

| Characteristics of study participants
A total of 120 hospitalized AFI patients [70 (58.3%) from CMCH and 50 (41.7%) from BITID] were enrolled in the study for evaluating the role of the TAC test in diagnosing hospitalized AFI patients.
The calculated mean age of the patients was 31.88 (±14.9) years with male predominance (57.5%) and a majority of them were from rural areas. The most prevalent symptom other than fever was a headache in 67 patients followed by nausea, vomiting, headache, back pain, abdominal pain, rash, cough, red eye, retro-orbital pain, etc. Anemia was the commonest sign followed by dehydration, red eye, jaundice, conjunctival hemorrhage, skin rash, cervical lymphadenopathy, hepatosplenomegaly, etc.
Characteristics of the fever of the AFI patients showed that the mean temperature on admission was 102.04 ± 1.21°F. In most cases, temperature was high. The average duration of fever was 9 days, which indicates patients are suffering from fever for a long duration without specific treatment. The majority of the patients had a history of receiving both oral and injectable antibiotics before blood sample collection (Table 1).

| The positivity rate of different tests among patients with AFI
To determine the etiology of AFI TAC, blood culture and TP test were done. Among these three tests, TAC shows the highest sensitivity followed by the TP test and blood culture. All groups of organisms of AFI such as bacteria, viruses, fungi, and protozoa were identified by TAC assay and the major cause was bacteria. Out of 120 AFI cases, at least 1 pathogen was identified in 40% (48) specimens by TAC test.
In blood culture 7.5% (9) specimens were positive. The commonest culture-positive organism is Salmonella, followed by Acinetobacter and Escherichia coli. TP test was found positive in 24.16% (29) specimens (Table 2).

| Detection of co-infection using TAC
Of 120 cases, in 48 cases either single or multiple organisms were detected. Besides, the detection of one organism, in eight cases codetection of more than one organism was observed. Two pathogens were detected in 5% (6). Three and four pathogens were detected in 0.8% (01) specimens. The most common co-pathogen was dengue followed by Salmonella and Rickettsia (Table 3).

| The sensitivity and specificity of TAC compared to blood culture
Among 120 samples TAC test was positive for bacteria in 14 samples and blood culture was positive in only nine samples (Table 4).
Of these nine positive culture-positive cases, four cases were TACpositive revealing a sensitivity of the TAC test of 44.4% considering blood culture as the gold standard. The specificity of the TAC test and overall accuracy of the TAC test in comparison to blood culture was, respectively, 90.99% and 87.50%. PPV was 28.57% which means the probability that the culture is present when the TAC test is positive. NPV was 95.28% indicating the probability that culture is negative when the TAC test is negative.

| Diagnosis of enteric fever using TP test
TP Test was positive in 29 samples out of 120 and blood culture was positive in only six samples ( Table 5). All of the culture-positive cases were also TP-positive revealing a sensitivity of TP test 100% considered blood culture as the gold standard. The specificity and overall accuracy of the TP test in comparison to blood culture were, respectively, 79.82% and 80.83%.

| DISCUSS ION
This study provides us with a valuable and informative diagnostic assessment of AFI cases in tertiary hospitals. Of 120 enrolled patients, at least one organism was identified in 48 (40%) cases by TAC assay.
Bacteria, viruses, protozoa, and fungi all types of organisms were detected by TAC assay from AFI patients. The most prevalent bacteria was Salmonella Typhi (15%) and the virus was Dengue (10%). We also found Rickettsia, E. coli, Pseudomonas, TA B L E 1 Description of the fever of the patients (n = 120). 90.99%, respectively. We anticipated that the sensitivity of PCR for bacterial pathogens on a single blood specimen, particularly with TAC and its small sample volume may be mediocre versus that of culture. A meta-analysis of commercial PCR tests for bacterial detection in samples of sepsis patients showed a sensitivity of only 61%-80% versus blood culture. 24 This finding is specifically well known for Salmonella Typhi. In another study, the sensitivity of PCR in blood was 50% versus blood culture. 25 In a study for AFI outbreak investigation and surveillance TAC was 71% sensitive versus that of blood culture. 18 Our study is in line with these observations. However, organism-specific serological tests, ELISA, or individual real-time PCR were not performed for any AFI patient in our study.
We have co-detected up to three organisms. Co-detection of one organism was found in six cases but two and three organisms in one case each. These results of co-detection are consistent with another AFI study 23 where no organism was found in 50% of cases Although the patients presented with clinical symptoms of infection but 111 samples showed negative results in the blood culture test. Among these 111, bacteria were identified in 28 specimens by the TAC assay. In previous studies, 18 to verify this type of discrepancy, TAC results were verified by PCR/sequencing and found that the results were the same as those of TAC but we could not confirm these organisms by individual PCR/sequencing. Most of our cases received several doses of antibiotics before blood collection. As a result, bacterial load was low in the blood. So, the findings were not the same as we thought.
According to our study, bacterial pathogens, specifically,

| CON CLUS ION
TAC assay is useful for the detection of multiple organisms of AFI.
It provides us a diversity of previously undetected pathogens of AFI which was missed by blood culture. We documented that this test was highly specific so, there was no false-positive result. We may use this newer molecular technique as a diagnostic tool for better characterization and early diagnosis of multiple contributing agents of AFI. Greater awareness will be developed among clinicians about etiology of AFI which will help in early diagnosis and management of AFI.

S U PP O RTI N G I N FO R M ATI O N
Additional supporting information can be found online in the Supporting Information section at the end of this article.