An unusual autopsy case in which the consumption of organophosphate insecticide was not the direct cause of death

In acute poisoning cases involving the ingestion of organophosphate insecticides such as fenitrothion and malathion, serum cholinesterase (ChE) activity is remarkably decreased, thus representing a useful indicator of the direct cause of death. In the present case, a man in his early 70s tried to committed suicide via the oral ingestion of both fenitrothion and malathion. Fenitrothion and malathion concentrations in cardiac blood were 2.63–2.98 and 0.31–0.58 µg/mL, respectively. However, the serum ChE level was 200 IU/L, which was not considerably lower than the normal range in males (242–495 IU/L). Conversely, we conrmed a positive reaction for Streptococcus pneumoniae using a urinary antigen detection kit. Moreover, histopathological analysis of both the left and right lungs revealed extensive inammatory cell inltration into the alveolar space. The autopsy and histopathological ndings indicated that the direct cause of death was severe bacterial pneumonia caused by the infection of S. pneumoniae. This is an unusual autopsy case in which the oral ingestion of both fenitrothion and malathion was not the direct cause of the death, and might have rapidly exacerbated respiratory decline.


Introduction
Organophosphates (OPs) have been used as agricultural insecticides globally [1][2][3]. In humans, the inhibition of acetylcholinesterase (AChE) by OP insecticides, such as fenitrothion and malathion, causes the accumulation of acetylcholine (ACh), and leads to the overstimulation of the muscarinic receptors that induces miosis [2,4]. Although patients' pupils were miotic (1-2 mm) in clinical emergency cases of OP poisonings [5,6], in forensic autopsy cases, miosis was not always been observed owing to the elapsed time after death [7][8][9]. Conversely, the serum cholinesterase (ChE) activity markedly decreased in cases of fatal OP poisonings [7][8][9]; thus, the measurement of the deceased's serum ChE levels is used to determine the direct cause of death in acute poisoning cases caused by the ingestion of OPs [10].
In Japan, pneumonia is the fth leading direct cause of death, and death attributable to pneumonia most commonly occurs in the elderly [11. 12]. In elderly Japanese patients, Streptococcus pneumoniae (S. pneumoniae) is the most frequently detected pathogenic species [13]. Extensive in ammatory cell in ltration into the alveolar space is a typical histopathological nding in the lungs of patients with bacterial pneumonia caused by S. pneumoniae [14,15].
Here, we report an unusual autopsy case of a man in his 70s who died of bacterial pneumonia caused by S. pneumoniae, and the oral ingestion of both fenitrothion and malathion was believed to have hastened his death.

Case Report
One day, a man in his early 70s was diagnosed with dementia in a hospital. Following the diagnosis, he called his wife and expressed suicidal ideation, and went missing on the same day. Five days after his disappearance, he was found dead on a forest road approximately 17 km away from his house.

Autopsy Findings
On external examination, the man was 167 cm tall, and he weighed 57.2 kg. The diameters of his left and right pupils could not be measured because of severe corneal opacity. On internal examination, a volatile odor typical of organic solvents was detected from the tongue and bronchi. On the trachea and both the left and right bronchial mucosa, a milky white liquid was attached. A 275-mL of coagulated cardiac blood was noted to be dusky red in color. In the stomach, 250 mL of a greenish white muddy uid with a volatile odor were found. The left and right lungs weighed 730 and 390 g, respectively, and the inferior lobes of both lungs were muddy white in color. The remaining organs exhibited no pathological ndings highlighting the direct cause of death.
Hematoxylin-eosin (HE) staining of both the left and right lungs revealed the extensive in ltration of in ammatory cells into the alveolar space of the inferior lobes (Fig. 1A). Additionally, detachment of the bronchiolar mucosal epithelial cells was observed in the lungs (Fig. 1B).
The serum ChE level was 200 IU/L (normal range in males: 242-495 IU/L). For the detection of S. pneumoniae antigens in urine to diagnose S. pneumoniae infection, an IMMUNOCATCH™ S. pneumoniae rapid diagnostic test kit (Eiken Chemical Co., Ltd, Tokyo, Japan) was used, and a positive result was obtained. The alcohol concentration in the cardiac blood and urine was <0.1 mg/mL, as determined by gas chromatography. Drug screening using cardiac blood was performed with the LC/MS/MS Rapid Toxicology Screening System Ver. 3 (Shimadzu, Kyoto, Japan) and the results revealed the presence of caffeine, fenitrothion, and malathion.

Chemicals and Reagents
Fenitrothion, fenthion, and malathion were obtained from Sigma-Aldrich (Tokyo, Japan). Acetonitrile, ammonium formate, formic acid (abt. 99%), methanol, and ultrapure water were all analytical grade, and they were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). A methanolic solution of fenthion was used as the internal standard (IS) for both fenitrothion and malathion. Stock solutions of fenitrothion, fenthion, and malathion (mg/mL) were prepared in methanol.

Sample Preparation
For drug analyses at the time of autopsy, whole blood was obtained from both the left and the right cardiac chambers. Blank whole human blood, which was purchased from KAC Co., Ltd. (Kyoto, Japan), was screened for fenitrothion, fenthion, and malathion, none of which was detected in the samples.

LC-MS/MS conditions
Qualitative and quantitative analyses were performed using a Nexera X2 HPLC system coupled with an LCMS-8045 triple quadrupole mass spectrometer (Shimadzu). Chromatographic separation was achieved using a Kinetex® XB-C18 column (100 × 2.1 mm i.d.; particle size, 2.6 µm; Phenomenex, Torrance, CA, USA) with a Security Guard ULTRA cartridge system (UHPLC C18 for 2.1 mm ID column; Phenomenex) maintained at 40°C. The mobile phases consisted of 10 mM ammonium formate with 0.1% formic acid in water (A) and in methanol (B). The ow rate and the elution gradient were conducted as described previously [16]. The mass spectrometer was operated on the positive mode with an electrospray ionization interface. The ionization source conditions were conducted as described previously [16]. The multiple reaction monitoring (MRM) mode was used to detect analytes. Two product ions (m/z), namely a quanti er and a quali er, were monitored for each compound in the MRM transitions (Table 1). Both the product ions and collision energy were optimized via the post-column infusion of each compound's methanolic solution (Table 1). Labsolutions Insight Ver. 3.10 SP1 software (Shimadzu) was used for the quantitative analysis of all data.

Method Validation
The limit of detection (LOD) and LLOQ, which corresponded to signal/noise ratios of ≥ 3 and ≥ 10, respectively, were calculated as the concentrations of analytes in QC samples. The recoveries of fenitrothion or malathion were determined by comparing the peak area of fenitrothion or malathion extracted from the spiked blood samples with the mean peak area of the recovery standards. The matrix effects of fenitrothion or malathion were determined by comparing the peak area of the recovery standard with the mean peak area of the standard solution at the same concentration. To determine the accuracy and precision, QC samples at each concentration of the analytes were analyzed over 3 days. The percentage deviations of the means from the true values were determined as the relative error and relative standard deviation and served as measures of the method's accuracy and precision. Table 2, all the results obtained with this method using whole blood were linear and sensitive for each analyte. Additionally, validated data (Tables S1 and S2) met the criteria indicated in the US Food and Drug Administration guidelines [17]. Table 3 summarizes the quantitative results for fenitrothion, and malathion, which were detected in left and right cardiac blood from the autopsy.

Discussion
Generally, both fenitrothion and malathion are rapidly metabolized, and their elimination half-lives in blood are 0.8-4.5 h and 3-6 h, respectively [18,19]. In addition, 10 µg/mL concentration of fenitrothion or malathion decreased by > 25% or 100%, respectively, after 24 h at room temperature [20]. Therefore, in forensic autopsy cases of fenitrothion or malathion poisoning, the detected blood concentrations of these compounds may be lower than the ingested antemortem blood concentrations because a certain period has passed since death. Several previously reported fenitrothion or malathion fatal poisoning cases based on cadaveric blood or serum collected from the femoral vein, or the cardiac chambers [21][22][23][24][25] were not presented (Table 3). Therefore, it was di cult to determine the fatal cardiac blood concentrations of fenitrothion or malathion. However, the cardiac blood fenitrothion concentrations in our case was ranged of 2.63-2.98 µg/mL. In view of the time elapsed since his death, we thought that these values were within the range of fenitrothion intoxication concentrations. Conversely, the cardiac blood malathion concentrations were relatively low compared with the ndings in other fatal malathion poisoning cases (Table 3). In our case, the outside temperature of the area in which he was found was 24.5°C; thus, malathion might have decomposed after his death.
In humans, butyrylcholinesterase (BuChE), also known as serum ChE, is produced is secreted into serum [10,26]. Acute poisoning of fenitrothion or malathion in humans is often fatal and life-threatening, and the serum ChE activities were remarkably reduced to a few % of the normal values [18,21]. Cadaveric ChE levels based on blood samples collected within 24 h after death were slightly decreased [27]. However, in fatal OP poisoning cases, serum ChE levels were decreased considerably below the normal range [7][8][9]. In our case, the serum ChE level was 200 IU/L, which was not reduced compared with that in previous reported cases of OPs acute poisoning. Serum ChE levels in males gradually decrease with age, ranging 200-450 IU/L in 70-year-olds [28]. Considering the age of the studied subject, this nding appeared to re ect age-related declines in serum ChE activity rather than OP-related decreases.
In Japan, the causative microorganisms of pneumonia in the elderly are more diverse than those in the young [14,29]. As a parameter indicating the severity of pneumonia, lung weight on autopsy has been considered useful [30]. In the present case, the left lung weight on autopsy was 730 g, which was more obviously weighted than that of non-pneumonia. To identify the causative pathogens in fatal autopsy cases of pneumonia, histopathological examination using lung tissue can identify speci c pathogens and corroborate the microbiological diagnosis [31]. In autopsy cases of patients who died of pneumonia, the inferior lobes of the left and right lungs were the most common sites of pneumonia [32]. As microscopic ndings in the patients with pneumococcal pneumonia, intra-alveolar brinous exudates with neutrophils and mononuclear cells and marked capillary congestion have been observed [15]. We observed white turbid inferior lobes in both lungs during the autopsy. Also, the remarkable in ammatory cell in ltration, distended capillaries, and congestion with prominent neutrophilic in ltration and erythrocytes were found within the alveolar space of both lung lobes. Because the presence of desquamated bronchiole mucosal epithelial cells in the lungs on histopathology was observed (Fig. 1B), it was believed to re ect the oral ingestion of fenitrothion and malathion, and these compounds were aspirated intratracheally. In OP poisoning, the impairment of the diaphragm and thoracic skeletal muscles cause respiratory paralysis, and high ACh concentrations in the central nervous system cause