Spotted fever: early diagnosis and its relevance

Scientific Electronic Library Online (SCIELO), Latin American and Caribbean Literature on Health Sciences (LILACS), Virtual Health Library (BVS) and PubMed. The articles were selected through the delimitation of keywords by the Descriptors in Health Sciences (DeCS-BIREME) described in the keywords. Relevant articles were selected, published between 2011 and 2022, written in Spanish, English or Portuguese. Results and Discussion: The laboratory diagnosis of BSF can be performed by Indirect Immunofluorescence Assay (IFA), Enzyme Immunoassay (ELISA), Immunohistochemistry (IHC), Polymerase Chain Reaction (PCR) or Culture with bacterial isolation. Complementary exams include blood count, coagulogram, evaluation of enzymes such as lactic dehydrogenase (LDH), liver transaminases (ALT and AST), creatine kinase (CK) and pancreatic enzymes. The procalcitonin test (PCT) can be useful to select suspected cases, since procalcitonin is an important laboratory marker in systemic bacterial infections and its elevation can guide the clinical diagnosis. Conclusion: Factors such as delay in laboratory diagnosis or inaccuracy in the clinical diagnosis of this neglected disease result in high lethality, which remains a challenge for public health institutions. The present work demonstrated that IFA is considered the gold standard test for laboratory diagnosis, but there are problems such as cross-reactions with other exanthematous diseases, which causes inaccuracy in the results. The data obtained underscore the need to implement new technologies for clinical and laboratory diagnosis in order to speed up the diagnosis and appropriate treatment.


RESUMO
of ticks, which contributes to a wide transmission area 2,3 .In Brazil, the main vector of Rickettsia rickettsii are ticks of the Amblyomma cajennense species, known as carrapatoestrela (star-tick), one of the most common and significant species when it comes to diseases that can be transmitted to humans, usually as accidental hosts.Ticks parasitize horses, rodents, capybaras, possums, rabbits, dogs and other domestic and wild animals 1 .Scarce knowledge about the disease tends to delay diagnosis, resulting in a number of deaths that can reach up to 80% of untreated cases.In Brazil, this disease tends to be more frequent in the States of Santa Catarina, São Paulo, Minas Gerais, Rio de Janeiro and Espírito Santo 1,4 .
The clinical diagnosis can be masked by nonspecific symptoms similar to other exanthematous diseases.The main symptom is rash, but it is not always present in patients affected by the disease, which leads to a complexity and delay in clinical diagnosis [1][2][3][4] .Characterized as an acute febrile condition, it has nonspecific symptoms, such as intense myalgia, high fever, general malaise, nausea, vomiting, abdominal pain and diarrhea, which can cause confusion with other exanthematic diseases that have similar symptoms, such as: dengue, leptospirosis, malaria and meningoencephalitis 4,5 .In more severe cases, hemorrhagic manifestations such as cutaneous, digestive and pulmonary bleeding can be noticed 4 .
The laboratory diagnosis of BSF occurs through specific exams (direct investigation of rickettsia, investigation of genetic material by molecular biology and investigation of anti-rickettsial antibodies) associated with complementary nonspecific laboratory evaluation (hematological exams and quantification of serum enzymes).Currently, the serological test of Indirect Immunofluorescence Assay (IFA) is the gold standard for diagnosing rickettsiosis.However, IgM antibodies are easily triggered in cross-reaction with other diseases and IgG antibodies are only detected from 7 to 10 days after the appearance of signs and/or symptoms, requiring the collection of a serum sample in the first days of the disease and another serum sample within a period of 14 to 21 days after the first collection 4 .
This work aims to analyze the challenges of clinical and laboratorial diagnosis of BSF, addressing epidemiological aspects and describing the signs and symptoms presented by patients affected by the disease in clinical cases, with the aim of highlighting the importance of an early clinical diagnosis, as well as exploring alternatives for laboratory diagnosis, with the aim of reducing the period for correct identification of this zoonosis.

Methods
The present study is a narrative review about BSF and its clinical and epidemiological aspects, as well as its laboratory diagnosis methods.The following databases were used for consultation: Scientific Electronic Library Online (SciELO), Latin American and Caribbean Literature in Health Sciences (LILACS), Biblioteca Virtual em Saúde (BVS) and PubMed.Articles were selected by the delimitation of keywords by Descritores em Ciências da Saúde (DeCS-BIREME).Key words were: Brazilian Spotted Fever (D012373); diagnosis (D012698); Rickettsia rickettsii (D012284); Rickettsia infections (D012282) and Spotted Fever Group Rickettsiosis (D000073605).The articles were selected according to the year, language and relevance of the subject.Articles published between 2011 and 2022 were selected.There were few works in Portuguese, so it was decided to also include articles in English and Spanish.Epidemiological bulletins were used in order to contribute with aspects about the expansion of information about the disease.Other inclusion criteria involved research on case reports that illustrated the high lethality of BSF, compulsory notification, areas of coverage, research on the etiological agent and vector, research on clinical and laboratorial diagnosis, in addition to possible technologies for early diagnosis.Exclusion criteria were: articles published outside the American continent, duplicate articles, articles without free access and articles outside the chosen time range.

PathoPhysIology of Bsf
Infection occurs through the introduction of the bacterium Rickettsia rickettsii, under the posterior dermis, adhesion to the endothelial surface and dissemination of the infection by the endothelial cells.The bacterium is gramnegative, bacillary in shape, has an approximate size of 0.3 to 0.5μm by 0.8 to 2.0µm and is obligatorily intracellular 4,6,7 .As it is an infectious agent with tropism for endothelial cells, BSF can be considered a kind of systemic vasculitis, as it is associated with a disseminated inflammatory vascular state, triggering the phagocytic defense cells, which are directed to the focus of the infection, resulting in the activation of procoagulatory vasoactive mediators and proinflammatory cytokines that increase vascular permeability and other effects that can culminate in hemorrhage, microthrombi and, in many cases, rapid evolution to generalized edema and multiple organ failure.By infecting endothelial cells, the bacterium induces the activation of proteins of the complexes responsible for the innate immune response, which is directly linked to the process of autophagy, and, paradoxically, the beginning of this process favors the replication of the bacteria 6 .transMIssIon Rickettsia rickettsii is found in the salivary glands and ovaries of BSF -transmitting arthropods.The main transmitting ticks are those of the genus Amblyomma, of the species A. sculptum, formerly known as Amblyomma cajennense.This tick species has a wide distribution in America.The vector has a preferred host, the horse, but in the larval and nymph stages, the parasite specificity is low, which can lead to the occurrence of other hosts/reservoirs, such as: oxen, sheep, goats, dogs, pigs, deer, capybaras, wild dogs, rabbits, agoutis, raccoons, armadillos and anteaters.The vectors are popularly known as "star-tick", "horsetick" or "rodoleiro", which are always hematophagous and become infected the moment they suck wild animals.They are considered reservoirs, since they can transmit the bacteria in eggs and larvae and/or transmission between vector generations, in order to continue the disease cycle.Transmission to the host occurs through the bite of the infected tick, and it is not necessary to take into account the length of time the tick remained adhered to the dermis.After the bite, the time for the installation of the bacteria in the endothelial cells can vary from six to ten hours [7][8][9][10][11] .

ePIdeMIology and clInIcal asPects of Bsf
The Brazilian Ministry of Health determined BSF would be an infectious disease of compulsory notification in 2001 and, subsequently, of immediate compulsory notification in 2014 10 .A "suspected case" of BSF is defined as any patient who presents nonspecific initial signs and symptoms, such as: acute high fever of sudden onset, headache, severe myalgia, general malaise, nausea, vomiting, appearance of maculopapular rash between the second and sixth day after the onset of infection, hemorrhagic manifestations and/or patient who has reported a tick bite or who has visited areas considered at risk in the last 15 days.These are important points to take into account during the patient's anamnesis.The summary of clinical manifestations is described in Chart 1 below 4,[8][9][10] .
SINAN -Sistema de Informação de Agravos de Notificação (Notifiable Diseases Information System) is the official system to report cases by the Brazilian Ministry of Health.It collects information from records of confirmed cases, clinical suspicions and notifications expressed in a number of days.For the definition of a "confirmed case", laboratory criteria are necessary, in addition to the inclusion of individuals who present the signs, symptoms, past history that fulfill the definitions of a "suspected case", in addition to confirmation of Rickettsia infection in one of the diagnostic tests: Immunofluorescence Assay (IFA), Immunohistochemistry (IHC), Isolation in culture of the etiologic agent and/or Molecular biology techniques (PCR).In cases where death occurred without clinical/laboratory confirmation, investigative clinical-epidemiological criteria are used where individuals are scored; who have presented signs and symptoms compatible with the disease; frequented BSF transmission areas; presented tick bites; who have reported contact with domestic and wild animals; contact with people who have had laboratorial confirmation of the infection.Considering the confirmed cases in Brazil, the predominance occurs in the South and Southeast regions 4,[10][11][12] .

case rePort
In order to discuss better the laboratorial diagnosis of BSF, a preliminary analysis of case reports was carried out in different places of the American continent, being notorious the severity and lethality of the disease, which is often confused with other conditions due to the nonspecific symptoms presented by the patients affected by BSF, which can be seen in Table 1 [13][14][15][16][17][18][19] .
In an endemic region, where individuals have tick bites, rash, fever and headaches, occurring during the expected season (dry season in Brazilian Southeast Region), the clinical diagnosis is easier.On the other hand, for the reported cases in patients who present the disease in the initial phase, without previous history of contact with ticks, not showing a rash, all these factors can cause confusion with other viral diseases.Because it is a disease that has nonspecific symptoms, it is difficult to have a definitive clinical diagnosis, and also the patient may be symptomatic with clinical manifestations compatible with other diseases, or with inconsistent characteristics and even patients who recover without treatment, with subclinical infections or even there may be asymptomatic patients.In all these examples, incorrect diagnosis may occur 1,4,7,8,11 .
Chart 1. Clinical manifestations and symptoms of patients with BSF.
Source: Elaborated by authors.Adding to the difficulty in clinical and laboratorial diagnosis, another aggravating factor -which can be seen on Table 2 -has to do with incorrect treatment.The delay of clinical suspicion directly impacts the laboratory diagnosis.In the case reports, serological and molecular tests, for the most part, only yielded results when the patients had already died or were in an advanced stage of the disease, which led to a delay in correct treatment.Such delay in starting appropriate antibiotic therapy is related to the difficulty/ delays in concluding the results of laboratory tests, which, in most cases, can lead to death due to the delay in treatment or failure administrating doxicicline during the first days of the infection 9,[14][15][16][17][18][19] .
results and dIscussIon

a dIsease wIth challenges In clInIcal and laBoratory dIagnosIs
Currently, the laboratorial diagnosis of BSF is performed by Indirect Immunofluorescence Assay (IFA); also the following methods may be used: Immunoenzymatic Assay (ELISA), Immunohistochemistry (IHC), Polymerase Chain Reaction (PCR) or Culture with isolation of the bacterium.Complementary laboratory tests, other than serological, can help to define or elucidate certain hematological or biochemical parameters presented by the affected patients such as blood count, clotting tests, evaluation of enzymes such as lactate dehydrogenase (LDH), liver transaminases (AST, ALT), creatine kinase (CK), and pancreatic enzymes 20 .
Diagnosis using IFA reaction has some limiting characteristics, namely: immunoglobulins M (IgM) are easily triggered in cross-reaction with other diseases, which can lead to false positive results, with low sensitivity and specificity, due to reactions with non-specific bacterial lipopolysaccharides.IgG-type antibodies are detected from the 7th day of infection onwards, requiring paired serum collections: an initial one in the first days of the disease and another sample between the 14th and 21st day after the first collection 3 .IFA can show low specificity for the detection of anti-Rickettsia rickettsii specific antibodies due to the sharing of similar surface proteins with other species; therefore, antibodies that react with different antigens can be detected, and, in this way, false-positive results may occur 20 .
Traceability studies to detect anti-Rickettsia rickettsii antibodies (IgG) in confirmed cases, demonstrated that despite being seropositive by the IFA method with a title equal to or greater than 1:64, the laboratory diagnosis of BSF might represent a challenge to different population profiles.For example, confounders were found among cases notified in the period from 2010 to 2015, in blood donors in 2016, and in people showing acute cases of infection by Dengue and Chikungunya among individuals with seropositivity for anti-Rickettsia rickettsii antibodies, being difficult to confirm the disease by IFA [20][21][22] (Table 3).
Regarding the other laboratory methods for BSF's laboratory diagnosis, ELISA is also a serological method that presents similar challenges presented by the IFA assays, related to the low specificity for the detection of anti-Rickettsia rickettsii antibodies, in addition to the possibility of cross-reactivity with other pathogens, since ELISA detects antibodies formed after pathogen exposure 4,[20][21][22][23] .
IHC, in turn, search for Rickettsia rickettsii antigens, and because of this, presents an even more important complicating factor than the serological tests: it requires tissue samples obtained from biopsy of skin lesion, necropsy or fragments of organs and tissues.In most of the cases, the execution of an invasive procedure to obtain not completely well located tissues (that needs to be in some way compromised by the pathogen) is impracticable and even unnecessary 4,[20][21][22][23] .Molecular techniques, such as PCR, use whole blood samples as the starting material, or clots formed after centrifugation of the collected blood, or biopsy, or necropsy tissues.PCR can be very useful for the characterization of the Rickettsia groups, mainly in the context of research laboratories.However, for the routine laboratory diagnosis, these molecular techniques present important difficulties, such as the standardization of the primers and the establishment of adequate positive control samples 4,[20][21][22][23] .
Bacteriological culture of samples (clotted blood, or tissue, or organ fragments) followed by the isolation of the pathogen -a method that would presumably be considered as ideal for laboratory diagnosis -has the limiting effect of empiric treatment with antibiotics inhibiting the bacterial growth.Besides this, in many cases, the biosafety requirements for this cultivation would be an obstacle in many Brazilian territorial regions that lack the adequate laboratory structure and the properly trained staff for interpreting the results 4,[20][21][22][23] .
Either way, confirmatory assays in tropical settings will probably only be available in zoonotic diseases Reference Centers.That being said, confirmatory tests, as serology or nucleic acid amplification, may take weeks to return and, in most cases, will not help to direct the acute treatment.Therefore, providers must use clinical and epidemiological clues to make presumptive diagnosis and start empiric antibiotic treatment while awaiting confirmatory test results 24 .
Another aggravating factor in obtaining an accurate laboratory diagnosis is empiric treatment, which can mask the disease, reducing the production of antibodies detectable by IFA, or even preventing the appearance of skin eruptions and/or typical signs of BSF.On the other hand, empiric treatment can reduce the severity and duration of the disease, and help to save lives 19,20,24 .

IMPortant laBoratory tests In the dIagnosIs of Bsf
The implementation of new methods or technologies to be used concomitantly with the serological methods of diagnosing BSF may improve the speed and accuracy of the laboratory diagnosis of the disease, since it is an infectious disease of compulsory notification, with high lethality and late diagnosis.In a molecular study carried out using the direct immunofluorescence technique, with the aim of unveiling the mechanistic actions involved in a specific signalization pathway, the presence of the Rickettsia rickettsia were in the lysate of endothelial cells infected by it, proving that, with a specific spot for samples collection, it is possible to retrieve the pathogen antigens 6 .The direct method, nevertheless, is still restricted to molecular research, and, if transposed from bench to bed hospital, the main difficulty would be obtaining representative biological material, since the pathogen is necessarily intracellular.Precise ways to obtain the most representative area with the possible presence of the pathogen need further investigations.
Another promising laboratory test, although still not very explored, comprises the procalcitonin (PCT) test, since this marker can be useful to better discriminate suspected cases: PCT is an important laboratory marker that has been studied as a serological marker of systemic infection and sepsis.PCT is a 116 amino acid peptide that can be elevated by many orders of magnitude in systemic inflammation accompanying sepsis.In systemic bacterial infections, PCT elevation would rule out viral infections.PCT levels above 1ng/mL indicate systemic bacterial infections or sepsis 15,19,23 .

conclusIon
Factors such as the delay in laboratorial diagnosis and inaccuracy in the clinical diagnosis of BSF, a neglected disease, result in high mortality, which remains a challenge for public health institutions.The present work demonstrated that IFA, even though it is the gold standard for laboratory diagnosis, is dependent on the generation of antibodies by the host, which hinders the speed of the diagnostic process, in addition to the possibility of cross-reactions with other exanthematic diseases, causing inaccuracy in the results.The clinical manifestations are nonspecific and can be easily confused with some arboviruses, making the diagnosis even more difficult.Other situations include the fact that it is an endemic disease, however, contact with the vector is not necessarily reported, which results in delayed treatment.The data obtained underscore the need for studies of new diagnosis technologies, preferably for direct detection of the pathogen, in addition to PCT evaluation, with the aim of helping in the earlier diagnosis of the disease and, therefore, in adequate treatment.Late adequate antibiotic therapy is associated with worse prognosis for patients with severe bacterial infection.

Table 1 .
BSF case reports detailed, with major clinical and laboratory findings.

Table 2 .
Antibiotic treatment for patients described in some case reports.
Legend: Gender of the patients: M = Masculine; F = Feminine.