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Letter to Editor
16 (
2
); 238-240
doi:
10.25259/JLP_9_2023

Laboratory diagnosis of non-scrub rickettsiosis – An Indian scenario

Department of Microbiology, Institute of Medical Sciences and SUM Hospital, Bhubaneswar, Odisha, India.

*Corresponding author: Sarita Otta, Department of Microbiology, Institute of Medical Sciences and SUM Hospital, Bhubaneswar, Odisha, India. saritaotta@gmail.com

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This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

How to cite this article: Rout D, Otta S. Laboratory diagnosis of non-scrub rickettsiosis – An Indian scenario. J Lab Physicians. 2024;16:238-40. doi: 10.25259/JLP_9_2023

Dear Editor,

In a setup of reducing the prevalence of malaria, a recent study from Uganda[1] ascertains spotted fever and typhus fever as a cause of acute undifferentiated febrile illness in 33.8% of cases. Yet, in India, rickettsiosis apart from scrub typhus is rarely investigated. Rickettsiosis is caused by organisms of genus Rickettsia and Orientia that are obligate intracellular Gram-negative coccobacilli maintained in nature in arthropods. Very few studies in India have used immunofluorescent assay (IFA) and they have focused on R. conorii causing Indian tick typhus and R. typhi causing flea-borne endemic typhus, while other species remain uninvestigated.[2] Genus Rickettsia is classified into four groups: An ancestral group with Rickettsia bellii and Rickettsia canadensis; spotted fever group (SFG) containing Rickettsia rickettsii, Rickettsia conorii (R. conorii) etc.; typhus group (TG) having Rickettsia prowazekii and Rickettsia typhi (R.typhi); and transitional group with Rickettsia akari, Rickettsia australis, and Rickettsia felis. Recent studies depicting seroepidemiology of the SFG and TG group rickettsial illness in the Indian subcontinent are depicted in Table 1.[3-10]

Table 1: Recent studies from India showing seroepidemiology of spotted fever and typhus fever groups in India.
Area Year Study settings Test used Number of patients Type of patients Common age group Sex predilection Season SFG TG Reference
North India
Lucknow 2013–2015 Prospective IgM/IgG ELISA
IFA
432 AUFI 0–15 M (54%)>F (46%) July–October 26.6% - Tripathi
et al., 2017[3]
Chandigarh April and May 2019 Prospective glt nested PCR 200 AUFI - F (57%)>M (43%) - 4% 3% Biswal et al., 2020[4]
Gorakhpur 2016 Retrospective IgM/IgG ELISA
using R. conorii
IgM/IgG R. typhi
294 >6 years AUFI during an AES outbreak 15–24 – IgM SFG seropositivity>45 years -IgG SFG seropositivity F (55%)>M (45%) for SFG
No sex predilection in TG
- IgM- 13.6%
IgG- 36.7
IgM- 7.1%
IgG- 15.3%
Mane et al., 2019[5]
South India
Puducherry 2012–2015 Prospective ELISA by paired sera using R. conorii IgM/IgG 320 paired sera
195 adults (>18 years)
125 children (<18 years)
AUFI - - - Adult- 46.6%
Children- 40.8%
- Stephen et al., 2019[6]
Tamil Nadu 2017–2020 Prospective IgG ELISA
using R. conorii
IgG ELISA R. typhi
2565 Healthy adults 46–60 years for SFG - - 6% 2.1% D’Cruz
et al., 2022[7]
Tamil Nadu 2020 Crosssectional IgG ELISA
using R. conorii
IgG ELISA R. typhi
1353 Healthy adults in scrub affected area No age predominance SFG-No gender predominance
TG-F>M (RR=1.8)
- 10.4% 5.4% Devamani et al., 2020[8]
North Eastern states
North Eastern states 2016–2018 Cross sectional R. typhi IgG ELISA 762 AES and 1437 with FUO AES and AUFI >15 years M>F in Manipur and Tripura
M=F in Nagaland
May–July in Manipur and Tripura
March-June in Nagaland
- 2.7% in AUFI
3.93% in AES
Khan et al., 2019[9]
Assam, Arunachal Pradesh and Nagaland 2016 Cross sectional IgM/IgG ELISA
using R. conorii
IgM/IgG R. typhi
1265 Healthy people in scrub affected area - - - 13.8% 4.2% Khan et al., 2016[10]

SFG: Spotted fever group, TG: Typhus fever group, AUFI: Acute undifferentiated fever illness, AES: Acute Encephalitis syndrome, FUO: Fever of unknown origin, M: Male, F: Female, RR: Risk ratio, ELISA: Enzyme-linked immunosorbent, PCR: Polymerase chain reaction, IgM: Immunoglobulin M, IgG: Immunoglobulin G, R. conorii: Rickettsia conorii, R. typhi: Rickettsia typhi, IFA: immunofluorescent assay.

Serological tests form the mainstay of laboratory diagnosis. However, they have many limitations as follows: (1) Diagnosis is possible to serogroup level only (SFG and TG), (2) confirmed diagnosis rests on seroconversion or four-fold rise in immunoglobulin G (IgG) antibody titers of paired sera taken 7–14 days apart because of missing baseline titers as well as persistence of immunoglobulin M (IgM) antibodies after clinical cure. A single rise of IgG aids clinical diagnosis without confirming infection, (3) appropriate antigen as per prevailing species should be used for these assays, and (4) IgM antibodies may be demonstrated in patients for whom no other clinical signs of a recent rickettsiosis.

Weil-Felix test is the oldest and cheapest serological assay but has low sensitivity with fair specificity of detection and a titer of >1:80 taken for positivity in Indian studies. Enzyme-linked immunosorbent assay (ELISA) (IgM and IgG) is the most widely used qualitative assay because of good sensitivity, specificity, and ease of use. Elongation factor thermal unstable (EF-Tu), 60 KDa chaperonin (groEL), and putative outer surface protein (adr2) are non-specific antigens, while recombinant truncated outer membrane proteins A and B (omp A and omp B) antigens are 100% specific and 90% sensitive. For murine typhus, R. typhi IgG ELISA (Fuller Laboratory, Fullerton, CA) covers specific protein Omp B, and for spotted fever, R. conorii ELISA IgG/IgM (Vircell, Granada, Spain) has been extensively evaluated in India. Immunofluorescent and immunoperoxidase assays are gold-standard quantitative assays with high sensitivity and specificity and can aid in species detection among serogroups but are technically demanding. Micro-immunofluorescence assay (IFA) is a modification of IFA where multiple species can be detected at once using indirect immunofluorescence. An IFA cut-off titer of >1:128 for SFG with R. conorii IgG IFA test (Fuller Laboratories, Fullerton, California, USA) is adopted in Indian studies. A new Dip-S-Ticks (INDX Integrated Diagnostics Inc., USA) test is available as a screening tool for rickettsia infections before performing the IFA as a confirmatory test.

Molecular tests in conventional polymerase chain reaction (PCR)/nested PCR/quantitative PCR formats in samples such as whole blood, buffy coat, serum, tissue biopsies (such as skin), eschar scrapings, swabs from a deroofed eschar, and cerebrospinal fluid are different samples to detect infection in the early stage of disease. Sensitivity of detection in the blood is reduced in view of low bacteremia in the early stages of disease. PCR with multiple targets, first to detect rickettsial genes (citrate synthase [gltA], 16Svedberg ribosomal ribonucleic acid [16S rRNA], and 17 Kilodalton [17kDa] lipoprotein outer membrane antigen genes) followed by SFG gene (omp A and omp B), improves sensitivity of detection. The omp A gene being specific for the SFG rickettsia is used to exclude a TG organism. The sensitivity and specificity of detection by molecular techniques vary with sample, timing of collection, and targeted primer.

Loop-mediated isothermal amplification (LAMP) assay having omp B target, a rapid molecular-based test was evaluated in China and had 100% specificity and 73% sensitivity when using whole blood samples. Recombinant polymerase amplification test based on the detection of 23S rRNA for SFG and 16S rRNA for all Rickettsia species, a rapid test format has been evaluated. The test products can further be biotin and fluorescein amidite (FAM) labeled and detected in a lateral flow assay has also been used for the detection of R. rickettsii. In the Indian setup, we are yet to validate and develop a reliable Point-of-care testing (POCT) tool for rickettsial infections apart from scrub typhus.

Antigen detection by immunohistochemistry and immunofluorescence techniques in paraffin blocks of biopsy of eschar is 100% specific and 70% sensitive for diagnosis. Culture of this organism with clinical samples processed through shell vial technique requires a BSL-3 setup (particularly for R. rickettsii) in cell line that has lower sensitivity and longer time to positivity and is reserved for reference laboratories.

With climatic change, there is emerging evidence of newer rickettsiosis in the Asia-Pacific region. Thus, large-scale studies should be undertaken in India to determine the actual burden. Further, antigenic, phenotypic, or genotypic variation and geographic differences in rickettsiosis apart from Scrub typhus should be ascertained.

Ethical approval

The Institutional Review Board approval is not required.

Declaration of patient consent

Patient’s consent was not required as there were no patients in this study.

Conflicts of interest

There are no conflicts of interest.

Use of artificial intelligence (AI)-assisted technology for manuscript preparation

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

Financial support and sponsorship

Nil.

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