Neutropenia induced by high-dose intravenous benzylpenicillin in treating neurosyphilis: Does it really matter?

Background Prompt therapy with high-dose intravenous benzylpenicillin for a prolonged period is critical for neurosyphilis patients to avoid irreversible sequelae. However, life-threatening neutropenia has been reported as a complication of prolonged therapy with high doses of benzylpenicillin when treating other diseases. This study aimed to investigate the incidence, presentation, management and prognosis of benzylpenicillin-induced neutropenia in treating neurosyphilis based on a large sample of syphilis patients in Shanghai. Methodology/Principal findings Between 1st January 2013 and 31st December 2015, 1367 patients with neurosyphilis were treated with benzylpenicillin, 578 of whom were eligible for recruitment to this study. Among patients without medical co-morbidities, the total incidence of benzylpenicillin-induced neutropenia and severe neutropenia was 2.42% (95% CI: 1.38–4.13%) and 0.35% (95% CI: 0.06–1.39%), respectively. The treatment duration before onset of neutropenia ranged from 10 to 14 days, with a total cumulative dose of between 240 and 324 megaunits of benzylpenicillin. Neutropenia was accompanied by symptoms of chills and fever (5 patients), fatigue (2 patients), cough (1 patient), sore throat (1 patient), diarrhea (1 patient) and erythematous rash (1 patient). The severity of neutropenia was not associated with age, gender or type of neurosyphilis (p>0.05). Neutropenia, even when severe, was often tolerated and normalized within one week. A more serious neutropenia did not occur when reinstituting benzylpenicillin in patients with mild or moderate neutropenia nor when ceftriaxone was used three months after patients had previously experienced severe neutropenia. Conclusions/Significance Benzylpenicillin-induced neutropenia was uncommon in our cohort of patients. Continuation of therapy was possible with intensive surveillance for those with mild or moderate neutropenia. For severe neutropenia, it is not essential to aggressively use hematopoietic growth factors or broad-spectrum antibiotics for patients in good physical condition after withdrawing anti-neurosyphilis regimen. We did not see an exacerbation of neutropenia in patients with the readministration of benzylpenicillin.

Syphilis has returned to china with a vengeance in the 21st century [8,9]. The epidemiology of neurosyphilis (NS) has largely mirrored that of early infective syphilis [10]. Prompt therapy of NS is critical for avoiding irreversible sequelae such as general paresis and tabes dorsalis [11]. The current recommended regimen is high-dose intravenous benzylpenicillin (18 to 24 megaunits daily) for a prolonged period (10 to 14 days) [12,13]. It is worth considering how to balance the benefit of treating NS with benzylpenicillin and harm if drug-induced neutropenia arises. We analyzed the clinical data of NS patients during three continuous years in order to investigate the incidence, presentation, management and prognosis of benzylpenicillin-induced neutropenia in order to provide helpful experience for other regions with a high burden of syphilis.

Study population and criteria
This retrospective study was approved by the medical ethics committee of the Shanghai Skin Disease Hospital, and conducted according to the principles expressed in the Declaration of Helsinki at the Sexually Transmitted Disease Institute of the Shanghai Skin Disease Hospital from January 1, 2013 to December 31, 2015. We recruited NS patients who (1) underwent their first therapy of high-dose intravenous benzylpenicillin, (2) did not have a recent history of other infections (e.g.: viral, bacterial, protozoal), (3) denied a past and family medical history of autoimmune diseases, underlying hematological diseases, nutritional deficiencies, splenic sequestration or congenital leukopenia, (4) did not receive chemotherapy, radiotherapy, immunotherapy, oral /intravenous /intramuscular usage of antibiotics, or other new medications in the past three months [14,15], (5) had no history of alcohol abuse, and (6) had negative HIV status. Patients were excluded if they were under 18 years of age or had a pre-treatment complete blood count (CBC) outside the normal reference range. Written informed consent was obtained before the laboratory test and NS treatment for clinical care and research.
A CBC, urinalysis, routine stool studies for infection and occult blood, biochemical profile, electrolytes, chest radiography and electrocardiograph were performed in all patients before benzylpenicillin therapy. CBC monitoring was performed every other day for mild or moderate neutropenia and every day for severe neutropenia until the value normalized. Other essential tests, including blood culture, sputum culture, biochemical profile, or virus antibody, were also performed when neutropenia occurred. The NS treatment regimen was 4 megaunits of benzylpenicillin as a freshly prepared bolus and slow infusion intravenously every 4 hours for 14 days [12,13].

Data extraction and analysis
Clinical data were recorded in terms of age, gender, diagnosis, cumulative dose of benzylpenicillin, days to onset of neutropenia, accompanying symptoms when ANC nadir occurred, clinical management, recovery time and readministration of benzylpenicillin. All data were independently double-coded with Epidata software(version 3.1; Denmark), then transferred into SPSS software (version 18.0; Chicago, IL, USA) for analyses. Descriptive statistics were used to calculate median, percentage, and incidence with 95% confidence interval (CI). A chisquare test (p<0.05 indicating statistical significance) was applied to analyze the potential factors associated with neutropenia. The continuous variable "age" was categorized into two subgroups, including age<55 years and age!55 years. Multivariate logistic regression was used to further identify factors independently associated with neutropenia when significant factors were found by chi-square test.

Results
A total of 1,367 NS patients were treated with a standard regimen of benzylpenicillin during the study period, 613 of whom received treatment for the first time. Of these, 578 patients underwent repeat CBC during the treatment and were included according to the study criteria. Fourteen patients, all of whom had prior normal CBCs, had a repeat ANC below 1.5×10 9 /L. The median age of these patients was 55 years (range: 27 to 79). Nine were male, and 12 had neurologic complications with a diverse spectrum of diagnoses, including syphilitic meningitis and parenchymatous neurosyphilis. (Fig 1, Tables 1
For the majority (13/14) of patients, the duration of treatment before onset of neutropenia ranged from 10 to 14 days, and the cumulative dose of benzylpenicillin varied from 240 to 324 megaunits. A single patient received 120 megaunits over five days of treatment.The range of nadir total white blood cell (WBC) counts was 0.60 to 3.69×10 9 /L, with nadir ANC from 0.04 to 1.49×10 9 /L. Three patients had concurrent thrombocytopenia. The accompanying symptoms were chills and fever (38.5-39.7˚C, 5 patients), fatigue (2 patients), cough (1 patient), sore throat (1 patient), diarrhea (1 patient) and erythematous rash (1 patient). Blood, sputum ANC, absolute neutrophil count. a One patient did not receive a repeat CBC due to some personal reasons. b See detailed criteria in the text. c 1×10 9 /L ANC<1.5×10 9 /L d 0.5×10 9 /L ANC<1×10 9 /L e ANC<0.5×10 9 /L https://doi.org/10.1371/journal.pntd. 0005456.g001 and throat swab cultures did not reveal an infectious etiology (e.g.: bacterium and fungus) among the febrile patients. (Table 2) One patient with syphilitic meningitis, ocular and otic syphilis (case 12) had an itchy rash on the trunk on day 4 and fever (maximum 38.5˚C) on day 5. Repeat laboratory testing revealed that his ANC declined to 1.13×10 9 /L. Thus, antihistamine and methylprednisolone (40mg daily) were commenced instead of benzylpenicillin. His symptoms and CBC count normalized on day 8. Subsequently, an alternative regimen of intravenous ceftriaxone (1.0 g every 12 hours for 15 days) (13) was reinstituted uneventfully three months later. (Table 2) Another two patients (case 1 with general paresis and case 13 with syphilitic meningitis) did well until they had fever, and repeat CBC revealed thrombocytopenia and severe neutropenia (ANC of case 1: 0.04×10 9 /L; ANC of case 13: 0.21×10 9 /L) near the end of therapy. Benzylpenicillin was discontinued. Despite mild symptoms, their fever in the context of a severe neutropenia caused a high level of concern for underlying life-threatening infection, and both patients were transferred to the emergency department. The results of bone marrow examination, Coombs' test, and cytomegalovirus and rubella virus IgM antibodies were not significant. They were given symptomatic relief and supportive treatment rather than human granulocyte colony-stimulating factor, glucocorticosteroid or other prophylactic broad-spectrum antibiotics. Both patients' CBCs returned to normal within four and five days, respectively, after withdrawing benzylpenicillin. Initiation of intravenous ceftriaxone did not induce neutropenia three months later in either patient. (Table 2) The other 11 patients with mild or moderate neutropenia finished the 14-day therapy with close monitoring of CBC and observation for sequelae of neutropenia. None experienced any severe complication of therapy and all had recovery of a normal ANC within seven days. Some of these patients received a second round of therapy with benzylpenicillin three months later and either had no neutropenia or experienced similar neutropenia without symptoms. ( Table 2)

Discussion
The neutrophil is the most abundant WBC in the peripheral blood and plays a critical role in preventing infections as part of the innate immune system [16]. It has been documented that the offending medications associated with severe neutropenia are methimazole, ticlopidine, Table 1

No. patients with neutropenia (%) (n = 14) No. patients without neutropenia (%) (n = 564)
Age (  Blood culture was performed in all patients with a recorded oral temperature greater than 38.5˚C.Case 9, who also had sore throat and cough, received a throat swab and sputum culture at the same time.
c Days to recovery is the number of days between stopping benzylpenicillin and normalization of ANC. clozapine, sulfasalazine, trimethoprim-sulfamethoxazole and dipyrone in descending order of likelihood [17][18][19].The hematologic complication of hypersensitivity to penicillin is rare, with an overall acute neutropenia of 2.4 to 15.4 cases per million populations over the last 20 years [14]. Based on this large clinical dataset, we concluded a total incidence of 2.42% among healthy NS patients receiving benzylpenicillin. It is noteworthy that the likelihood of acute neutropenia caused by intravenous benzylpenicillin for NS is much higher than that by penicillin for other diseases [14]. Penicillin agents are thought to be able to cause granulopoiesis inhibition [20,21], and benzylpenicillin-induced neutropenia is dose related more than a pure immunological reaction [15,22]. As indicated earlier, the duration of beta-lactam therapy prior to the start of neutropenia always exceeded 15 days [21]. We saw cases of neutropenia caused by benzylpenicillin within 14 days probably due to the higher daily dose used for NS than for other diseases. In the 1980s, Al-Hadramy and his colleagues [6] summarized 28 reported cases of benzylpenicillin-induced neutropenia for diseases such as infective endocarditis, bowel obstruction, cellulitis, gangrenous appendix, pneumonia, hemangioma, septic arthritis, and pleural empyema. Therein, 71% patients developed neutropenia after taking 200 megaunits or more, and neutropenia developed in 82% of patients on treatment for two or more weeks, which is consistent with our findings of neutropenia being associated with high-dose and prolonged treatment [6]. Some studies have proposed the hypothesis that genetic and epigenetic modifications predispose an individual to idiosyncratic drug sensitivity [23,24]. The genetic susceptibility might be associated with an increased risk of neutropenia induced by high-dose benzylpenicillin which needs to be further investigated.
According to previous reports, acute neutropenia was often well tolerated and normalized rapidly [2]. In our study, fever accompanied by general malaise was the first and often the only manifestation in patients. No patients experienced life-threatening complications. Withdrawing benzylpenicillin rapidly led to a recovery in the patient who had an ANC of 0.04×10 9 /L at nadir but no other high-risk symptoms. Even though potential antibody cross-reactivity existed, we found that it was relatively safe when benzylpenicillin was reinstituted in patients with mild or moderate neutropenia, and ceftriaxone in patients with severe neutropenia, three months later.
Previous research has identified that older age (>65 years), septicemia or shock, metabolic disorders such as renal failure, and an ANC under 0.1×10 9 /L were poor prognostic factors associated with drug-induced neutropenia [14]. Thus, in patients with these factors, the empirical use of hematopoietic growth factors, glucocorticosteroid and/or broad-spectrum antibiotics may positively impact the prognosis [18]. Among the 14 NS patients with acute neutropenia in this study, none had metabolic disorders, severe infections, septicemia or septic shock. No patients were given hematopoietic growth factors or broad-spectrum antibiotics, even though three patients were older than 65 years, and one patient had an ANC of 0.04×10 9 / L. We also found the severity of neutropenia had no significant association with age, gender or the type of NS.
Syphilis is far from eradicated, especially in the resource-limited areas worldwide, and it can affect any part of the neuraxis at any stage of infection [25,26]. There is a growing consensus that NS patients can benefit from regular benzylpenicillin therapy, and high-dose benzylpenicillin is of proven efficacy at the early stage of NS [27]. Here, we outlined benzylpenicillininduced neutropenia as a complication of NS treatment. Some limitations should be acknowledged. First, due to limited published data on when to obtain surveillance CBCs during treatment, we arranged the first repeat CBC on day 10 unless any clinical symptom occurred beforehand. Thus, asymptomatic neutropenia may have been present in the two patients (case 8 and 9) earlier than day 10. Second, prompt NS therapy was limited to patients whose other medical conditions (e.g. uncontrolled hypertension or diabetes) were stable in order to minimize risk of therapy. Meanwhile, HIV co-infected patients were not included in the analysis because of possible confounding of leukopenia caused by HIV. These factors might limit the generalizability of our findings.
In conclusion, benzylpenicillin-induced neutropenia was well tolerated in our cohort of patients with mild or moderate type. It also normalized rapidly without aggressive management for those with severe neutropenia after withdrawing anti-neurosyphilis regimen. We did not see an exacerbation of neutropenia in patients with the readministration of benzylpenicillin.