Abstract
Plague still poses a significant threat to human health and as a reemerging infection is unfamiliar to the majority of the modern medical doctors. In this chapter, the plague is described according to Dr. Nikiforov’s experiences in the diagnosis and treatment of patients, and also a review of the relevant literature on this subject is provided. The main modern methods and criteria for laboratory diagnosis of plague are briefly described. The clinical presentations include the bubonic and pneumonic form, septicemia, rarely pharyngitis, and meningitis. Early diagnosis and the prompt initiation of treatment reduce the mortality rate associated with bubonic plague and septicemic plague to 5–50 %; although a delay of more than 24 h in the administration of antibiotics and antishock treatment can be fatal for plague patients. Most human cases can successfully be treated with antibiotics.
Keywords
11.1 Clinical Forms of Plague and Their Manifestations
It is difficult to describe precisely the clinical manifestations of plague. This is because a definite near-mythical concept of this disease, based on the works of previous clinicians in the pre-antibiotic age, had acquired some canonical features in the academic literature. The descriptions from the end of the nineteenth century and the beginning of the twentieth century may be more reliable than earlier literature on this subject. In earlier publications, plague patients and those suffering from other severe infections were in many cases indistinguishable, indicating that in ancient times the plague was a term encompassing a collective of different infectious diseases.
It is written in Galen’s Commentaries on Hippocratic Epidemics that if a disease affects many people in a region, it can be referred to as an epidemic disease, and if this disease causes many human deaths, it is then a “plague.” The majority of modern authors have limited clinical experience of the plague because it is currently a relatively rare infectious disease. In addition, the clinical manifestations of the disease in endemic areas vary considerably owing to “natural immunization” of the local population [1] and the various host specificities of Yersinia pestis strains from different natural plague foci [2]. It is clear that the data on plague manifestations typical for the residents of Vietnam or India, for example, cannot unconditionally be extrapolated to Europeans. We can only speculate as to how different Y. pestis clones would interact with nonimmune European populations. In this chapter, we will describe the plague according to our experiences in the diagnosis and treatment of patients, and we will also provide a comprehensive review of the relevant literature on this subject.
In 1940, G. P. Rudnev [3] classified plague according to its epidemiological features, and this classification is still used to date in Russia:
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(A)
Mainly local forms (typically peripheral with relatively rare external dissemination):
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(i)
Cutaneous
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(ii)
Bubonic
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(iii)
Cutaneous-bubonic
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(i)
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(B)
Internally disseminated or generalized forms:
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(i)
Primary septicemic
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(ii)
Secondary septicemic
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(i)
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(C)
Externally disseminated forms (often with abundant external dissemination):
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(i)
Primary pneumonic
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(ii)
Secondary pneumonic
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(iii)
Intestinal
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(i)
This classification, in general, meets the practical public health requirements; however, the “pure” cutaneous and intestinal forms of the plague have never been observed in practice.
From a clinician perspective, primary and secondary forms of pneumonic plague may be considered as primary and secondary septicemic (generalized) forms, mainly affecting the lungs. Accordingly, the intestinal form could be considered a septicemic form with a primary lesion of the intestine.
The incubation period for the plague is usually 2–3 days, occasionally 1–5 days, and, rarely, up to 6 days. In the case of insufficient prophylactic antibiotic therapy, the delitescence may increase up to 10 days [4]. However, if the infection is caused by a laboratory accident (a high-dose aerosol infection), the incubation period is only several hours. It is widely believed that a longer incubation period is associated with milder clinical symptoms and better outcomes. Hence, as early as 1926, the international sanitary convention proposed that persons who had come into contact with plague patients should be isolated for medical observation for 6 days [5].
In most cases, the onset of plague is sudden. In rare cases, patients display some preliminary nonspecific symptoms, such as anorexia, pain in the sacral area, weakness, and exhaustion. In some cases, patients appear healthy the day before disease onset but experience feelings of nervousness, anxiety, or depression, falling seriously ill the following day.
The real onset of plague is marked with fever, chills, and headaches. Headaches so closely accompany plague that it became commonly referred to as “head disease” [3]. Patients become anxious and their gait is unsteady, with characteristic hand waving. The skin becomes dry and hot. The face appears red, swollen, and masklike, losing the expression of emotions. There is conjunctival injection, making sparkling eyes appear lustrous but still, and the pupils become slightly dilated. Patients often refuse to be physically evaluated. Speech becomes slurred. Hearing is impaired. In general, the patient gives the impression of being intoxicated. Thus, one patient in the Central Asian region of the former Soviet Union was refused admission by a physician-therapist because of strange behavior thought to be linked to intoxication with alcohol or drugs. That patient was found dead on a bench near the hospital later the same day, and the diagnosis at postmortem was the plague.
Other symptoms include a dry, swollen tongue, covered with chalklike, white limescale, and dry lips. Herpes labialis is not characteristic of plague patients. Patients sometimes experience burning pains in the upper and lower abdomen that cannot be relieved by drinking large quantities of cold water. The dissonance between the patient’s thirst and shaking chills is also noteworthy. There may be repeated vomiting and/or tachycardia and a decrease in blood pressure. At this stage, plague patients look similar in appearance to typhoid fever patients with status typhosus.
The initial stages of the disease do not exhibit any unique symptoms. Clinicians can therefore often only diagnose a severe general intoxication syndrome. Plague may be suspected due to epidemiological anamnesis, but no definitive diagnosis can be made. However, as the disease progresses, the skin lesion caused by Y. pestis may develop into a carbuncle (plague ulcer), and this primary carbuncle (at the site of the bite), secondary carbuncles (due to hematogenous dissemination) may also develop. At first, an intensive crimson fleck that looks like an insect bite emerges on the skin. The skin in this area becomes very painful. Over the following 24 h, the skin hardens (first only detectable by touch), and then the affected area increases in size and becomes visible, rising above the surface (forming a papule). The pain intensifies, and a so-called plague flictena, a small pea-sized bubble filled with fluid that is yellowish or darker in appearance if blood is present, develops at the center of the papule. In most patients, the envelope of the bubble becomes torn, and a skin ulcer is formed, the edges of which begin to swell and increasingly protrude above the skin surface, forming a pinkish-red inflammatory torus that soon becomes cyanotic. The ulcer becomes deeply embedded in the skin and at the surface becomes encased by a dark scab. The skin lesion is now referred to as a plague carbuncle (Fig. 11.1a). New vesicles may be formed along the edge of this ulcer, making plague carbuncles appear similar to the ulcers associated with anthrax. The inflammation expands to the periphery over a period of less than 2 days, causing eccentric growth of the ulcer, with carbuncles considerably varying in size from 0.5 to >5.0 cm in diameter. There may be only one or several primary and/or secondary carbuncles. Two or more secondary skin lesions may form due to hematogenous dissemination of the pathogen, whereas the presence of several primary carbuncles may result from multiple simultaneous infections caused by repeated bites by one or several infected fleas.
The healing of carbuncles occurs extremely slowly. It begins with the appearance of a demarcation zone characterized by the release of moderate amounts of pus at the edges of the scab. The subsequent casting off of the scab and the outcrop of a granulating surface is followed by the formation of rough scars.
Although the existence of “pure” skin plague has been proposed, whereby the pathogen does not spread to the nearby lymph nodes, we have no evidence for this in clinical practice, and there is no consistent data on this proposed phenomenon in the literature.
As a rule, cutaneous lesions in plague patients develop at the same time as, or a little earlier than, a typical bubo, which is located close to the ulcer. For example, crus ulcers are accompanied by inguinal buboes, while hand ulcers are accompanied by axillary lymphadenitis. So, the illness initially presents in cutaneous-bubonic form (Fig. 11.1b), which clinically is identical in terms of its main manifestations to the solely bubonic form that will be discussed below.
Bubonic plague is the most frequent and well-described form of the disease. The early signs of an emerging bubo are painful sensitivity of the inguinal, axillary, cervical or submandibular areas that can be detected by careful medical examination within the first few hours of the disease. Usually there are no obvious insect bite marks at the site of infection or signs of lymphangitis. In the regional lymph nodes, however, one or two swollen and extremely painful lymph nodes may be noted. As the disease progresses, a “tumor” (typically, singular) grows and lymph nodes cluster into a dense conglomerate, which appears linked to the skin, making it impossible to palpate individual nodes. The bubo varies in size from a hazelnut to a hen’s egg or even larger (according to historical descriptions, it may reach the size of a newborn’s head). It has sharp borders and is fixed and very painful. The severe pain often compels the patient to remain in a forced position. During the first days of the disease, the skin over the bubo remains unchanged (Fig. 11.1c). The most frequent locations of the buboes are the inguinal region (>50 % of all observations), the axillary area (c. 20 %) (Fig. 11.1d), and the neck (c. 5 %), with buboes on the elbow and popliteal region occurring rarely and even less frequently elsewhere on the body (Fig. 11.1e). Usually a patient displays only one bubo; however, there are exceptions as in the case of plague carbuncles (Fig. 11.1f). The simultaneous development of buboes at the groin, axilla, and neck is very rare. Most often, the affected area in the groin involves the lymph nodes lying 2–3 fingers below the femoral ring in the triangular space between the sartorius and adductor longus muscles. The higher frequency of femoral-inguinal buboes can be explained by the large quantity of lymph collected here due to the considerable skin surface area encompassed compared with other draining nodes. The general condition of patients progressively worsens as a bubo approaches the head. During the development of periadenitis, the skin over the bubo becomes denser making it impossible to fold, and inflammatory swelling of the skin gradually develops. The true size and boundaries of the bubo become uncertain because of compulsory periadenitis (Fig. 11.1g, h). A doughlike edema may spread a considerable distance from the bubo (e.g., above the inguinal ligament if the bubo is femoral). If one presses the swollen region, an indented impression is clearly observed (Fig. 11.2a–c). For axillary lymphadenitis that easily develops into secondary pneumonic plague, the edema can expand to the breast area, sometimes producing trembling from palpation of the “gelatinous tumor” [4]. Buboes located in the neck area generally correlate with severe symptoms and poor patient outcomes. The lymph glands enlarge rapidly and the hypodermis swells, forming a doughlike tumor, which becomes infiltrated and indurated during the course of periadenitis. Swelling can spread at the front down to the mammary glands and from behind down to the scapular area. The disease peaks 4–6 days after onset. Fever remains at 39–40 °C, with symptoms of intoxication reaching their maximum. The face becomes contorted, sometimes with an expression of anxiety and horror. Pronounced tachycardia, dull heart sounds, and low blood pressure are all characteristics of this stage of the disease. The liver and spleen become enlarged. If the patient survives this period, the buboes may follow a different clinical course. The buboes rarely disappear completely, but rather become sclerotized or suppurate. In the latter case, which often occurs around days 6–8, the skin over the bubo reddens and then becomes cyanotic (Fig. 11.2d). Bubo palpation, which in this period becomes less painful, begins to detect some movement within it. On days 8–10, the bubo bursts with the release of green-yellow homogeneous pus (Fig. 11.2e), sometimes mixed with blood, but with no strong odor. The epidemiological risk from suppurative masses is minimal, as bacteria were difficult to isolate after the first day. The mature purulent cavity becomes gradually filled with granulations and the process of cavity healing can take up to 3–4 weeks or even more. The purulent fistula continues to discharge during this period (Fig. 11.2f). The bubo eventually turns into a rough deep scar. In general, suppuration of a bubo is followed by a significant improvement in the general health of the patient. However, suppuration does not contribute to the favorable course of the disease, as the time necessary for a bubo to reach the stage of purulent melting is also sufficient for the patient to overcome the most difficult period of the illness. Furthermore, the purulent fistula is a potential site of entry for a secondary infection that can cause the development of phlegmonous adenitis.
All of the above applies to the so-called primary or early buboes, i.e., developing at the lymph nodes nearest to the site of infection. The occurrence of secondary or late buboes is possible during the course of any form of plague and at almost any time point owing to the hematogenous dissemination of Y. pestis to a distant lymph gland. Secondary buboes are smaller in size than primary buboes, are less painful, and generally do not suppurate, being dissolved in the case of recovery.
Cutaneous-bubonic and bubonic forms of plague, unless treated quickly with effective antibiotics, can cause systemic infection. Secondary pneumonia, secondary buboes, and meningitis, along with other manifestations, can develop when Y. pestis enters and multiplies in any secondary organs or target tissues (e.g., the lungs, lymph nodes irrelevant to the primary bubo, meninges). In such cases, the so-called secondary septicemic (secondary generalized) form of the plague develops. However, infectious-toxic shock syndrome (ITSS), which is manifested by a sharp increase in the signs of general intoxication and the emergence of new and specific septic shock symptoms, in particular skin and mucous bleeding, often develops in patients instead of the formation of bacterial metastasis.
In the case of systemic infection without the formation of a detectable primary lesion (e.g., bubo), the patient is usually diagnosed with primary septicemic plague. However, if the development of secondary septicemic plague is suspected from the pathogenetic profile, the reason for the development of primary generalized forms of plague is unclear. The term “primary septicemic plague” implies the absence of a preliminary stage of Y. pestis accumulation in a regional bubo prior to its breakthrough into the blood. Whereas, real primary systemic plague may result from (i) a large infecting dose, (ii) unusually high virulence of the pathogen (both may be due to a laboratory-acquired infection or a bioterrorist attack), as well as (iii) the immunocompromised status of the patient. Other cases of primary generalized forms that do not adhere to these criteria remain to be clarified.
The issue of appearance of Y. pestis in the blood at different stages of the disease warrants further examination. In Vietnam during the 1980s, Y. pestis was often isolated from the blood of patients with the bubonic form of plague (or observed by microscopy of blood smears), whose general condition could be considered neither critical nor even severe. The presence of Y. pestis in the blood, regardless of the severity of the disease, was also noted earlier by other clinicians [6]. Thus, the detection of Y. pestis in the blood of a patient is not reason alone for diagnosing the septic form of the disease. In this regard, it remains a mystery where the distinction lies between a “simple” bacteremia and a systemic infectious bacteremia as the pathogenic basis for the generalized forms of plague (septicemia). This issue requires further clarification.
In the past, different terminology has been used to describe the cause of death in plague patients, especially during the first week of the disease with either the bubonic or pneumonic form; such terms have included “progressive heart failure,” “sudden loss of strength,” and “acute collapse.” Such terms are still cited in some modern manuals [7]. However, plague and all that it involves clearly need demystification. So from the point of view of a modern clinician, all clinical manifestations of the terminal phase of plague are consistent with an ITSS caused by Gram-negative bacteria with some plague-specific features (see below).
Meningitis can appear at any stage of any form of plague infection. However, meningitis usually occurs either during severe forms of the disease, aggravating the main pathogenic process, or during the period of reversal of the symptoms. In the pre-antibiotic era, meningitis was frequently the cause of death, whereas today, the prognosis is somewhat more optimistic [8, 9]. The development of meningitis is associated with worsening of the patient’s condition, increasing headaches, loss of consciousness, appearance of meningeal symptoms, and the characteristic positioning of patients (the classic “meningeal” posture), which may persist even after death. However, we have observed cases in which meningitis developed during the convalescent period of bubonic plague and was considered to be a relapse, without being deadly or particularly severe. Cerebrospinal fluid analysis indicates the purulent nature of inflammation, and Y. pestis can often be isolated from cerebrospinal fluid.
Secondary pneumonic plague is more common, developing in 8–10 % of bubonic plague patients. In addition to a worsening prognosis, the development of pneumonia increases the risk of pathogen transmission via coughing and breathing. As in the case of plague meningitis, the development of secondary plague pneumonia is possible at any stage of the infectious process and leads to a significant worsening of the patient’s condition. Even when the fever subsides, the body temperature may return to 39 °C or higher. The involvement of the lungs in the infectious process is accompanied by the appearance of a cough and chest pain while breathing due to the development of pleurisy. It should be emphasized that pain (including headaches) is a major component of the plague infectious process and the absence of pain makes a diagnosis of plague unlikely, or even completely rejected.
Dry coughing is rarely seen in pneumonic plague. Scant and glassy sputum begins to excrete almost immediately. Then after about a day, the sputum quantity increases and it becomes contaminated with blood. On examination, lobar pneumonia of any etiology may be detected with localization of the lesions mostly in the middle lobe of the right lung or the upper lobes of both lungs. Such “atypical” localization of inflammatory foci allows the clinician to suspect that the pneumonia is caused by hematogenous dissemination of the pathogen, as more trivial secondary infections of the lungs caused by adhering microorganisms have the tendency to be localized mainly in the lower lobes.
More or less similar descriptions of the clinical profile of primary pneumonic plague can be found in many textbooks, manuals, and monographs. In Russian literature, the original detailed description of primary pneumonic plague was provided by G. P. Rudnev in 1940 [3]. Because there are few observations of this type of plague in the literature, this text is important, and we insert below practically word-for-word translation of its most recent adapted version [4].
G. P. Rudnev divided the disease into three stages: an initial stage, a peak stage, and a terminal stage (with progressive dyspnea, cyanosis, and sometimes coma). The second stage was considered the most dangerous in terms of transmission because patients develop a cough, expelling the pathogens as a respiratory spray.
The clinical picture, especially during the first hours of the disease, may be quite different. Usually, the disease has a rapid onset involving an abrupt and repeated fever, rapidly rising body temperature, an extremely severe headache, and frequent repeated vomiting. This may be followed by “knife-cutting” chest pain, palpitation, rapid pulse, severe dyspnea, and delirium. Later, the patient may collapse and fall into a coma, and this may subsequently lead to death. Coughing is associated with all stages of the disease, with variations in the quantity of sputum, from several spits to tens of liters. For a few patients, sputum production does not occur. Between the two types of pneumonic plague, wet (with sputum production in large quantities) and dry (without sputum production), there is a lot of variability in terms of sputum amount and appearance (sputum with odor, vitreous, liquid, or blood stained). In the final stage, the sputum is usually pure blood. In general, sputum viscosity is used as one of the diagnostic markers. In some nontypical cases, sputum may be rusty in appearance, for example, in croupous pneumonia or tuberculosis-like pneumonia. In extremely severe cases, palpation and auscultation are not particularly informative.
At the peak of primary pneumonic plague, patients usually manifest with depression followed by excitement, delirium, high fever, associated mild pneumonia, frequent coughing with bloody sputum, muffled heart sounds, excessive tachycardia, arrhythmia, and often vomiting of blood. Finally, sopor develops, shortness of breath increases, and the patient’s faces become cyanotic as if suffocating, exhausted by a back-breaking struggle. At this stage, the physical strength of the patient is fading and the pulse becomes faster and weaker. Some patients fall into a coma; some die during repeated attempts to stand up and run away. These attempts to stand and run are very characteristic of plague delirium. The disease lasts only 3–5 days and without treatment is lethal. Just before death, the body temperature of some patients drops sharply to normal.
Analysis of the clinical profile described above from a contemporary point of view indicates that the final stages of the development of primary pneumonic plague are consistent with ITSS, along with all of the ensuing negative consequences for the patient. Recognition of this fact is extremely important because it emphasizes that any reduction in plague mortality is unlikely without significant progress in the treatment of ITSS [10].
As for primary septicemic plague, it differs clinically from primary pneumonic plague only by the absence of changes in the lungs and by a shorter clinical course (usually, <3 days from onset to death). We fully support the assertion of N. N. Zhukov-Verezhnikov [11] that this diagnosis is based only on laboratory data, because this form of plague has no specific clinical manifestations. We can speculate that both of these disease forms have identical pathogenic mechanisms, the only difference being that death in the case of primary septicemic plague occurs prior to the development of inflammatory lesions in the lungs.
Functional gastrointestinal disorders such as vomiting and diarrhea are frequently associated with severe and generalized forms of plague. It is often possible to isolate Y. pestis cultures from the feces of such patients. In some cases, the symptoms (e.g., nausea, vomiting, abdominal pain, diarrhea with mucus and blood) may, at some stage of the disease, become the dominant characteristic, tempting the classification of an “intestinal” form of plague. However, the generalized signs and symptoms associated with the infectious process in the end prevail over intestinal symptoms. So, we should speak not about a separate form of the disease, but about a systemic plague, mainly affecting the intestine.
It is well established that ITSS plays a crucial role in the mortality associated with the plague, and it displays features common to all types of septic shock caused by Gram-negative bacteria as well as some specific features. As is the case in other similar types of shock, tachycardia and tachypnea increase, body temperature and blood pressure decrease, and urination ceases. Other possible symptoms are nasal and gastrointestinal bleeding, nephrorrhagia, and metrorrhagia, but these are not common in uncomplicated cases of the plague. The peculiarities of the clinical manifestations of the plague are the unusually early and abundant multiple bluish-black hemorrhages (bruises) with a leaden hue that appear on the skin and mucosa. Their size may vary from a dot (Fig. 11.2g) or line to a much wider area, and they are commonly referred to as “spots of death” or maculae mortis (Fig. 11.2h). The similar signs can be seen on mucosal membranes and in the internal organs [12] (Fig. 11.2i). This particular characteristic feature together with the high mortality rate was the reason why the disease became known as the “Black Death.” In addition, the facial expression of a person dying from the plague is distinctive. G. P. Rudnev (1940) described the facies pestica as suffering and cyanotic, with an expression of horror and with sharp features and abundant sweat drops on the forehead (“dew of death”).
Another characteristic feature of patients in the final stages of the plague is their behavior. As mentioned earlier, if patients are able to move, they always try to escape their house and die curled up at the gate of their own home [3], just as animals always die at the entrance of their burrows [13]. It is therefore important for medical staff in emergency care units to restrict patients from leaving.
In the absence of effective treatment, the human plague generally always leads to severe symptoms. However, some plague patients display relatively mild clinical symptoms, and such a variant of bubonic plague is named “pestis minor” or “pestis ambulans.” Also, the possibility of asymptomatic (subclinical) plague should be considered and even the existence of a carrier state [3]. It may be that the plague actually occurs more frequently than it is diagnosed.
Figure 11.3 is a sketch map for the main clinical and pathogenic stages of the plague.
Recovery from the plague is a long process, often interspersed with periods of improvement and deterioration in the patient’s general state of health. Complications of a nonspecific nature, such as nonspecific pneumonia, phlebitis, stomatitis, gingivitis, otitis, and erysipelas, as well as exacerbation of preexisting chronic diseases are possible.
It is not difficult to diagnose clinically cutaneous-bubonic or bubonic plague, but diagnosis of primary pneumonic plague is more challenging and diagnosis of primary septicemic plague is impossible. We do not see the benefit in discussing the differential diagnosis of the plague because the list of possible nosological forms of the disease would be considerable (from tularemia, anthrax, and glanders to meningococcemia). If a clinician is familiar with infectious pathology in general and in particular the clinical manifestations of plague, differentiating the specific form of the disease is not necessary. By contrast, if a clinician is not familiar with such rare diseases, there is also no benefit of a differential diagnosis.
For plague patients, blood and urine examinations are rarely performed. In routine blood examinations, the only change usually detected is an increase in white blood cells (25,000/mm3), which is not normally seen in other infectious diseases. In routine urine examinations, elevated protein levels may be observed (“fever urine”). Microscopic examination of a whole blood smear could confirm the presence of Y. pestis unequivocally.
11.2 Rapid Detection of Y. pestis on Site
Alexandre Yersin was the first to discover the plague bacillus and paved the way for modern laboratory diagnosis of the disease. In 1894, he performed isolation of Y. pestis pure culture and identified it as the causative agent of plague using (i) examination of smears prepared from autopsy material (buboes) and pure cultures, (ii) bacterial culture on solid media, and (iii) infection of laboratory animals with autopsy material and pure cultures [14].
Laboratory diagnosis is problematic in many plague-affected countries, especially in the remote areas of developing countries, where the logistics, infrastructure, and resources are limited. Rapid and reliable detection of Y. pestis on site is essential for timely initiation of medical treatment and postexposure prophylactic measures when plague cases are suspected. However, the reference standard for confirmation of plague remains the isolation of Y. pestis, which is time-consuming and not possible in the field. Simple tools that require limited resources and allow for the rapid detection of Y. pestis on site are needed for the diagnosis of plague cases.
11.2.1 Immunological Diagnosis: Development of an Immunochromatographic Assay
Immunochromatographic assays (ICA) have become popular bedside or point-of-care (POC) diagnostic tools because they are sensitive, simple to perform, inexpensive to manufacture, and well suited for rapid on-site detection and can be performed in a variety of settings by nontechnical personnel. The Institute Pasteur of Madagascar has developed an ICA that uses monoclonal antibodies to the F1 antigen of Y. pestis for the rapid diagnosis of pneumonic and bubonic plague [15]. Samples from patients suspected of plague were tested using the assay both in the laboratory and by health workers at 26 pilot sites in Madagascar. The assay detected concentrations of F1 antigen as low as 0.5 ng/mL within 15 min. With the combination of bacteriological methods and F1 ELISA as a reference standard, the positive and negative predictive values of the test were 90.6 % and 86.7 %, respectively. The agreement rate between assays performed at remote centers and those carried out in the laboratory was 89.8 %. Like other ICA assays using colloidal gold particles as the reporter, it is a semiquantitative test involving manual reading and a subjective threshold. The test must therefore be performed by professional staff.
Up-converting phosphor (UCP) technology-based ICA (UPT-ICA) has attracted considerable attention because of its advantages of quantification, reliability, and robustness [16]. UCPs are a type of rare-earth-containing crystal particles with the unique property of up-converting infrared excitation light (980 nm) to emit visible light. These unique optical properties render UCPs ideal labels and are more sensitive than other conventional reporters. UPT-ICAs for the rapid diagnosis of Y. pestis have been developed with high specificity and a low limit of detection of 104 CFU/mL (100 CFU/test) [17, 18]. This type of assay is applicable in the field with excellent tolerance to various complex samples, such as blood, viscera, (fresh and decomposed) and powders. Samples with a wide pH range (2–12), high ion strength, high viscosity, or the presence of bio-macromolecules could also be directly detected through simple dissolution or homogenization. The detection and quantification of Y. pestis with UPT-ICA can be performed using a biosensor reader in 15 min. Furthermore, it can be operated by nonprofessional staff.
11.2.2 Molecular Diagnosis by Real-Time PCR
Real-time PCR tests that target the structural gene for F1 antigen (caf1), the plasminogen activator gene (pla), and the gene encoding the specific chromosomal fragment (3a) have been developed and may prove useful as specific and rapid tests for plague diagnosis [19, 20]. The time required for the assay and the detection limit are less than those of conventional PCR amplification, and the risk of cross-contamination between amplification products is also reduced because post-amplification procedures are not required. Moreover, the assay could be adapted for use in the field by employing portable real-time PCR instruments and reagents able to be stored at room temperature. More recently, it was shown that a pla- and 3a-based PCR method could not reliably identify Y. pestis [2, 21–23] and that multilocus detection methods should be developed for this pathogen.
A novel portable real-time PCR thermocycler, PikoReal™ (Thermo Fisher Scientific Inc., USA), has been developed for detecting Y. pestis in the field [24]. The PikoReal system weighs less than the common PCR instruments (about 10 kg) and is equipped with five LED-illuminated optical channels, which increase durability when compared with standard light technologies, facilitating multiplexing. Analyses of Y. pestis with the PikoReal system proved to be simple and time-efficient compared with assays using the Applied Biosystems® 7300 equipment (Life Technologies Ltd., USA). In field trials, reliable results were achieved in approximately 90 min, from the beginning of sample preparation (45 min) to the completion of the diagnosis. All assay-specific primers and probes, in both the PikoReal and the ABI 7300 systems, correctly identified all tested isolates of Y. pestis, while no cross-reactivity was observed.
Real-time PCR reagents able to be stored at room temperature have also been developed using primers and probes targeting the 3a sequence and caf1 in Y. pestis [20]. Carbohydrate mixtures were added to the PCR reagents, which were later vacuum-dried and evaluated for stability. The vacuum-dried reagents were stable at 37 °C for at least 49 days at a lower concentration of template DNA (10 copies/μl) and up to 79 days at a higher concentration (≥102 copies/μl). Soil samples spiked with Y. pestis (5 × 104 CFU/g) could be detected with the dried reagents, indicating they may be used at room temperature for field application.
In the absence of a national strategy for plague diagnosis [25–27], it is necessary to use the WHO recommendations [28].
11.3 Identification and Source Tracing of Y. pestis in the Laboratory
When the plague is suspected, clinical specimens should be collected immediately and sent to a professional laboratory for confirmation. Diagnosis can be made from a variety of specimens, including blood, aspirates from suspected buboes, pharyngeal swabs, cerebrospinal fluid, and sputum samples. All specimens sent from suspected cases should be labeled as high risk and handled in a biosafety cabinet.
11.3.1 Bacteriologic Tests (The Reference Standard Method)
The reference standard method for laboratory diagnosis of Y. pestis infection is based on the isolation and identification of the organism from clinical specimens [29]. The organism can be cultured and grows on most routine solid and liquid culture media (e.g., brain-heart infusion broth, sheep blood agar, or MacConkey agar). The optimal growth temperature of Y. pestis is 28 °C. However, standard 35–37 °C incubation is necessary for development of the F1 antigen of Y. pestis and for recovery of other pathogens that may be present. Y. pestis grow as gray-white, translucent colonies on solid media that are visible after incubation at 25–37 °C for 48 h. Colonies are about 1–2 mm in diameter and may have a raised center with a flat periphery (referred to as a “fried egg” or “hammered copper” appearance). Y. pestis is a pleomorphic Gram-negative rod. On staining of a smear with Giemsa or Wayson stain, this organism appears as bipolar coccobacilli. However, bipolar staining is not unique for Y. pestis and therefore is considered only suggestive of a diagnosis. Cultures can be conclusively identified as Y. pestis by specific phage lysis. Unlike F1 antigen expression, Y. pestis is susceptible to phage lysis at both 25 °C and 37 °C. Conventional biochemical identification systems can be used to assist in identification, but misidentification with other organisms, such as Y. pseudotuberculosis or other Enterobacteriaceae, is possible [30].
11.3.2 F1 Antigen Detection and Serological Diagnostic Techniques
In the absence of a definite Y. pestis isolate, a diagnosis can also be made using serological diagnostic techniques. A fourfold or greater change in the titer of antibodies to Y. pestis F1 antigen in a passive hemagglutination test of paired serum specimens is confirmatory for Y. pestis. The specificity of a positive passive hemagglutination test requires confirmation with the F1 antigen hemagglutination-inhibition test. Hemagglutinating antibodies directed against the F1 antigen of Y. pestis can appear as early as 5 days after the onset of symptoms but more commonly appear between 1 and 2 weeks after onset [30].
Detection of the F1 antigen in tissues or fluids by direct fluorescent antibody testing is considered an indicator of the presence of Y. pestis. Similarly, detection of an elevated F1 antibody titer (more than 1:10) in a single serum sample from a patient displaying plague-like symptoms who has not been vaccinated previously and has no history of Y. pestis infection is a positive diagnostic indicator. PCR analysis using primers specific for the gene encoding F1 antigen has also been reported as a method of Y. pestis identification. Furthermore, enzyme-linked immunosorbent assays (ELISAs) can be employed to detect Y. pestis and measure F1 antigen levels or levels of serum antibodies to F1 antigen [31]. An ELISA is available to measure antibodies against F1 antigen that involves both immunoglobulin M (IgM), indicative of a recent or current infection, and immunoglobulin G (IgG), indicative of a past infection.
11.4 Diagnostic Criteria for Plague
The standard case definition of plague (according to the WHO, weekly Epidemiological Record, no 28, July 2006) is as follows:
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1.
Suspected case: compatible clinical presentation and consistent epidemiological features, including exposure to infected animals or humans, and/or evidence of flea bites, and/or residence in or travel to a known endemic focus area within the previous 10 days
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2.
Presumptive case: meeting the definition of a suspected case plus:
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(A)
Putative new or reemerging focus: at least two of the following tests proved positive:
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Microscopy: material from bubo, blood, or sputum contains Gram-negative coccobacilli, bipolar after Wayson, or Giemsa staining
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F1 antigen detected in bubo aspirate, blood, or sputum
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A single anti-F1 serology without evidence of previous Y. pestis infection or immunization
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PCR detection of Y. pestis in bubo aspirate, blood, or sputum
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-
(B)
Known endemic focus: at least one of the following tests proved positive:
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Microscopic evidence from bubo, blood, or sputum sample of Gram-negative coccobacilli or bipolar coccobacilli observed after Wayson or Giemsa
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A single anti-F1 serology without evidence of previous plague infection or immunization
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F1 antigen detected in bubo aspirate, blood, or sputum
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PCR detection of Y. pestis in bubo aspirate, blood, or sputum
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-
3.
Confirmed case: meeting the definition of suspected case plus:
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An isolate from a clinical sample identified as Y. pestis by colony morphology and a positive result for at least two of the following four tests: phage lysis of cultures at 20–25 °C and 37 °C, F1 antigen detection, PCR, Y. pestis biochemical profile
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A fourfold rise in the anti-F1 antibody titer in paired serum samples
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In endemic areas when no other confirmatory test can be performed, a positive rapid diagnostic test with immunochromatography to detect F1 antigen
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11.5 Treatment of Plague
In the absence of a national strategy for plague treatment [27], it is necessary to use the WHO recommendations [32].
Plague treatment involves two components: specific antibiotic treatment and nonspecific supporting therapy, such as antishock therapy. As mentioned previously, before the age of antibiotics, the mortality rate from bubonic plague was more than 50 %, with almost 100 % lethality reported for untreated cases of primary plague meningitis, pneumonia, or septicemia. Early diagnosis and the prompt initiation of treatment reduce the mortality rate associated with bubonic plague and septicemic plague to 5–50 % [33, 34], although a delay of more than 24 h in the administration of antibiotics and antishock treatment can be fatal for plague patients.
Antimicrobial therapy is the simplest component of the complex therapy required for plague patients. Streptomycin at a dose of 1.0 g twice a day (30 mg/kg/day) for 10 days continues to be considered the most effective anti-plague remedy. There is also reason to believe that gentamicin will be highly effective at standard dosage for the management of patients with severe sepsis. In randomized comparative studies in Tanzania, a 7-day course of intramuscular therapy with gentamicin was equally effective against bubonic, septicemic, and pneumonic plague, as the oral use of doxycycline in both adults and children [35, 36]. Chloramphenicol is recommended for the treatment of plague meningitis. In all cases, antibiotic therapy should be continued for 10 days.
In the absence of clinical trials of various antimicrobials for the treatment of primary pneumonic plague in humans, the existing recommendations are based solely on the results of in vitro and animal experiments. The remedies of choice both in adults and children are the aminoglycosides, streptomycin, or gentamicin, at age-appropriate doses [35, 37] (Table 11.1).
Doxycycline, chloramphenicol, and ciprofloxacin can also be used as alternatives. When treating a limited number of plague patients, all of these drugs are administered parenterally, whereas during a plague outbreak involving a large number of patients and people who may have come into contact with the plague, oral administration of antibiotics is employed. In this situation, preference is given to doxycycline, which is administered at an oral dose of 100 mg twice a day or ciprofloxacin at an oral dose of 500 mg twice a day (Table 11.2). Chloramphenicol is administered orally at a dose of 25 mg/kg four times a day; however, this drug is not permitted for use in pregnant women owing to the high risk of toxic effects on the fetus [35, 37].
An adequate intensive pathogenetic treatment of plague [38, 39], and especially antishock therapy, requires much more knowledge and skills. ITSS is initially triggered by bacterial toxic substances but is then a self-sustaining, progressive process. With simplification, the relationship between Y. pestis and ITSS can be explained using the analogy of a match and a fire. The match can easily light the fire, but quenching the match alone will not extinguish the fire. Removal of the causative agent is necessary for effective treatment of the plague but does not quell ITSS. The aim of intensive therapy is the maintenance of a patient’s core indicators at the following levels:
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Central venous pressure: 8–12 mm Hg
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Mean arterial pressure: ≥65 mm Hg
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Urination: ≥0.5 ml/kg/h
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Degree of blood oxygen saturation: ≥70 %
Comprehensive antishock therapy for plague patients should include antibiotic treatment, infusion therapy, administration of vasopressors and corticosteroids, inotropy, and respiratory support (if necessary). Infusion therapy is carried out using a combination of crystalloids (e.g., physiological saline solution or plasma-Lyte 148) and colloids (modified fluid gelatin: gelofusine 4 %, polygeline, hydroxyethyl starch 200–500 ml of a 6 % or 10 % solution) at a ratio of 1:1.
Vasopressors, norepinephrine and dopamine, are the first choice drugs. The initial dosage for dopamine is 5 ml of a 4 % solution added to 200 ml of physiological saline, which is infused until the mean arterial pressure reaches 65 mm Hg. The dosage for norepinephrine is 2–5 μg/min, and this can be used in combination with dopamine.
Inotropic dobutaminum could be used for patients with reduced cardiac output at a dosage of 250 mg in 500 ml of physiological saline, potentially in combination with vasopressors at a calculated dosage is 2.5–40.0 μg/kg/min. Steroids (hydrocortisone, 240–300 mg/day for 5–7 days) can hasten hemodynamics to a normal level. We speculate that the use of extracorporeal purification methods, such as plasmapheresis or hemo-/plasma filtration, as used in cases of extreme hemorrhagic fever, or transfusion of erythrocytes en masse, would be an appropriate therapeutic strategy in the case of plague.
During intensive care, patients should be provided 25–30 kilocalories/kg/day. The level of blood glucose should be maintained at 4.5–6.1 mM/L. Phlebothrombosis and stress-induced gastrointestinal ulcers should be prevented during the entire treatment process. For all types of plague, only fully recovered patients with negative bacteriological results can be discharged. Patients who have suffered from bubonic plague can be discharged after repeated lymph node examinations at a 2-day interval, and pneumonic plague patients should undergo triplicate sputum examinations.
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Nikiforov, V.V., Gao, H., Zhou, L., Anisimov, A. (2016). Plague: Clinics, Diagnosis and Treatment. In: Yang, R., Anisimov, A. (eds) Yersinia pestis: Retrospective and Perspective. Advances in Experimental Medicine and Biology, vol 918. Springer, Dordrecht. https://doi.org/10.1007/978-94-024-0890-4_11
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