**7. Laboratory diagnostic tools**

As occurs for all infectious diseases, the most definitive diagnostic method is the rickettsial isolation in culture. The main problem is that *Rickettsia* spp. are strictly intracellular bacteria, conventional growth media cannot be used, and a laboratory with P3 safety level (not generally available in clinical microbiology labs) is necessary. Furthermore, culture is not very sensitive and the yield decreases when clinical samples are taken after antibiotic treatment or when samples are not processed within 24 hours. It is a slow technique that is used for research purposes but not for the routine clinical practice. Centrifugation shell-vial technique is a commercially available adaptation of cell cultures that is easier to handle, faster and less hazardous. Isolation attempts on cell cultures may be performed using buffy coat or tissue samples (eschar biopsies when possible). If not processed within 24 h, samples must be frozen at -70ºC or in liquid nitrogen.

Detection of rickettsiae by Giménez or Giemsa staining from blood and tissue samples would allow the confirmation of the diagnosis, but these techniques are non-specific and their sensitivity is very low.

In some laboratories molecular biology tools, such as polymerase chain reaction (PCR) and sequencing, are also available. PCR-based assays from anticoagulated blood, biopsies and arthropod tissue samples targeting *Rickettsia* spp. genes are quite sensitive and useful for a quick diagnosis of these infections. The evaluation of several primer sets for the molecular diagnosis of rickettsioses demonstrated that the performance of three sequential PCRs (nested or semi-nested ones) allowed the detection and identification of *Rickettsia* species in a high percentage of the samples with previous clinical diagnosis or microbiological confirmation (serological analysis) of rickettsiosis (Santibáñez et al., 2011). Blood and tissue samples should be stored at -20ºC or lower if PCR-based diagnosis is delayed for more than 24 hours. The European guidelines for the diagnosis of tick-borne bacterial diseases contain useful information for clinicians and microbiologists (Brouqui et al., 2004).

Indirect diagnostic tests and specifically, immunofluorescence assays (IFA) are considered the standard tests. Besides, since most traveller patients are investigated after returning, IFA are the most available tools for diagnosis. Acute and convalescent sera (collected 4-6 weeks after the onset of the illness) should be taken. In many cases we cannot observe seroconvertion but a high titre of antibodies. Cross-reactions among *Rickettsia* spp. make very difficult to definitively identify the causative agent by means of IFA. This can only be achieved in reference centres in which different antigens and other serological assays, such as western-blot, are available. Serum samples can be preserved at -20ºC or lower for several months without significant degradation of antibodies.

Rickettsialpox is a worldwide (North America, Eastern Europe, Korea and southern Africa) rickettsiosis caused by *Rickettsia akari* and transmitted by the bite of the mouse mite *Lyponyssoides sanguineus.* We can consider it a remerging infection since several cases have been detected in New York City after September 11 attacks (Paddock et al., 2003). Patients have fever, a prominent eschar -which is the best sign of the disease- and rash that, as occurs in ATBF and Queensland tick typhus, may be vesicular. Palms and soles are not involved. The presence of regional lymphadenopathy is common. Patients recover without treatment

As occurs for all infectious diseases, the most definitive diagnostic method is the rickettsial isolation in culture. The main problem is that *Rickettsia* spp. are strictly intracellular bacteria, conventional growth media cannot be used, and a laboratory with P3 safety level (not generally available in clinical microbiology labs) is necessary. Furthermore, culture is not very sensitive and the yield decreases when clinical samples are taken after antibiotic treatment or when samples are not processed within 24 hours. It is a slow technique that is used for research purposes but not for the routine clinical practice. Centrifugation shell-vial technique is a commercially available adaptation of cell cultures that is easier to handle, faster and less hazardous. Isolation attempts on cell cultures may be performed using buffy coat or tissue samples (eschar biopsies when possible). If not processed within 24 h, samples

Detection of rickettsiae by Giménez or Giemsa staining from blood and tissue samples would allow the confirmation of the diagnosis, but these techniques are non-specific and

In some laboratories molecular biology tools, such as polymerase chain reaction (PCR) and sequencing, are also available. PCR-based assays from anticoagulated blood, biopsies and arthropod tissue samples targeting *Rickettsia* spp. genes are quite sensitive and useful for a quick diagnosis of these infections. The evaluation of several primer sets for the molecular diagnosis of rickettsioses demonstrated that the performance of three sequential PCRs (nested or semi-nested ones) allowed the detection and identification of *Rickettsia* species in a high percentage of the samples with previous clinical diagnosis or microbiological confirmation (serological analysis) of rickettsiosis (Santibáñez et al., 2011). Blood and tissue samples should be stored at -20ºC or lower if PCR-based diagnosis is delayed for more than 24 hours. The European guidelines for the diagnosis of tick-borne bacterial diseases contain

Indirect diagnostic tests and specifically, immunofluorescence assays (IFA) are considered the standard tests. Besides, since most traveller patients are investigated after returning, IFA are the most available tools for diagnosis. Acute and convalescent sera (collected 4-6 weeks after the onset of the illness) should be taken. In many cases we cannot observe seroconvertion but a high titre of antibodies. Cross-reactions among *Rickettsia* spp. make very difficult to definitively identify the causative agent by means of IFA. This can only be achieved in reference centres in which different antigens and other serological assays, such as western-blot, are available. Serum samples can be preserved at -20ºC or lower for several

useful information for clinicians and microbiologists (Brouqui et al., 2004).

months without significant degradation of antibodies.

**6. Rickettsialpox** 

in most cases (Raoult, 2010b).

**7. Laboratory diagnostic tools** 

must be frozen at -70ºC or in liquid nitrogen.

their sensitivity is very low.

Ticks removed from patients can be used as tools for the diagnosis of tick-borne rickettsioses. The strategy includes the identification of the tick to the species level, and the detection or isolation of rickettsias (Table 3).


Table 3. Strategy for detecting and/or isolating rickettsias from ticks

Diagnostic scores with epidemiological, clinical and laboratory tests for some tick-borne rickettsioses (ATBF and MSF) have been proposed (Tables 4 and 5).


Positive serology against spotted fever group rickettsiae *or* 

c. Clinical and epidemiological features consistent with a spotted fever group rickettsiosis such as fever and or any cutaneous rash and or single inoculation eschar after travel to sub-Saharan Africa or French West Indies *and* 

Serology specific for a recent *R. africae* infection (seroconversion or presence of IgM ‡ 1:32), with antibodies to *R. africae* greater than those to *R. conorii* by at least two dilutions, and or a Western blot or cross-absorption showing antibodies specific for *R. africae*

Table 4. Diagnostic criteria for African-tick bite fever (ATBF). A patient is considered to have ATBF when criteria A, B or C are met

Rickettsiosis as Threat for the Traveller 19

There are two questions that physicians have linked up with tick-bites: How must I remove

The first question is easy to answer. The most useful method to remove an attached tick is using forceps. Smooth forceps (without teeth) must be introduced between the tick's head and the skin in a 90º angle and then pull (Oteo et al., 1996). Other traditional methods as

The other question is the use of prophylactic drugs after arthropod bites. There are no studies to answer this question. The transmission of rickettsias may be very quick, so we cannot extrapolate the recommendations for Lyme disease. Anyway, when people have been bitten by several ticks in an endemic area for a determinate disease (i.e.: Kruger National Park in South Africa and ATBF) and if the patient is anxious, we can offer doxycycline. It has been demonstrated that 3 doses of 100 mg. every 12 hours is safety and sufficient as treatment for the majority of rickettsioses. We must be cautious with the sun to avoid photo-sensibility. Children can take doxycycline for a short period of time. It is only contraindicated for

Vaccine approaches for prevention of rickettsial diseases have been developed since the past century, but currently no vaccine is available. Major surface protein antigens (OmpA and OmpB) of *R. rickettsii* and *R. conorii* are candidate vaccine antigens. Molecular biology techniques such as selection, cloning and expression of genes encoding *R. prowazekii* virulence-associated proteins, offer the opportunity to develop new rickettsial vaccines against typhus group rickettsiae. Further research is needed to develop effective vaccines

The treatment of rickettsiosis should be initiated as soon as possible. Antibiotics are very effective and may avoid severe complications and death. In all cases if rickettsiosis is suspected, samples should be sent for laboratory confirmation. In DEBONEL/TIBOLA, in which the clinical signs and symptoms are less severe, recovery without antimicrobials occurs but the use of antibiotics shortens the clinical course and improves the clinical picture

Doxycycline is the most useful drug in children and adults. Doxycycline can be administered in short course (100 mg. every 12 hours for one day) for the treatment of typhus and scrub typhus. In the case of MSF, 2 doses of 200 mg./12 hous are also very effective (in children, 5 mg./kg./12hours); although most physicians use 100 mg. every 12 hours for 3-7 days after fever disappears. The same can be recommended for ATBF. This antibiotic regimen could probably be followed in other tick-borne rickettsioses but there are not good evidences (clinical assays) to support a recommendation. In RMSF the administration of doxycycline for 7 days is recommended. Other drugs that can be prescribed when not using doxycycline (allergy or pregnancy) are chloramphenicol (50-75 mg./kg./day given in 4 doses for 7-10 days) and azythromycin (500 mg./day for 5 days). Doxycycline for 7 days is the treatment of choice for rickettsialpox. Although there is *in vitro*

5. The contact with parasitized pets and wild animals must be avoided.

pregnant women and in this case we can use macrolides (i.e. azythromycin).

without undesirable toxic reactions (Azad & Radulovic, 2003; Walker, 2009).

removal of the tick has to be done.

the tick? Must I take prophylactic drugs after a tick-bite?

using oil, burning or freezing must be forgotten.

**9. Treatment** 

(Ibarra et al., 2005).

effective for the prevention of Lyme disease. The tick needs at least 24-48 hours for the transmission of *Borrelia burgdorferi.* This measure can be less efficient for *Rickettsia* spp. because these microorganisms can be transmitted since the first hours. But, anyway, the


SGOT, serum glutamate–oxaloacetate transaminase; SGPT, serum glutamate–pyruvate transaminase. aA positive diagnosis is made when the overall score is ≥ 25.

Table 5. Diagnostic criteria for Mediterranean spotted fever caused by *Rickettsia conorii* 
