Prevalence of Signs of Severity Identified in the Thai Population with Malaria: A Systematic Review and Meta-Analysis.

Understanding the prevalence of signs of severity identified in the Thai population with malaria could aid clinical management and disease control efforts, decrease mortality, and promote malaria elimination in Thailand. This systematic review aimed to collate the evidence regarding signs of severity identified in the Thai population with malaria. MEDLINE, Web of Science, and Scopus were searched for potentially relevant studies. The quality of the included studies was assessed using the Joanna Briggs Institute critical appraisal tools. The pooled prevalence of signs of severity among patients with severe malaria and the pooled proportion of each sign of severity among all signs of severity were estimated using random-effects models. Heterogeneity among included studies was assessed using Cochran's Q test. A subgroup analysis was performed to evaluate whether differences in pooled estimates between different study sites. Publication bias was assessed by visualizing funnel plot asymmetry and using Egger's test. Among 741 studies identified by literature searching, 12 studies of a total of 2900 patients with severe malaria, in 7 Thai hospitals, met the eligibility criteria. Results of meta-analyses showed that the signs of the severity of malaria with the highest prevalence in Thailand were jaundice (54%), hyperparasitemia (47%), impaired consciousness/coma (21%), acidosis (18%), renal impairment (13%), shock (10%), convulsions (9%), severe anemia (8%), pulmonary edema/acute respiratory distress syndrome (ARDS) (8%), hypoglycemia (4%), and bleeding/disseminated intravascular coagulation (DIC) (2%). The signs of the severity of malaria that made up the highest proportion of all signs of severity identified in the Thai population with malaria were hyperparasitemia (33%), jaundice (33%), impaired consciousness/coma (12%), acidosis (9%), renal impairment (7%), severe anemia (6%), convulsions (5%), shock (5%), pulmonary edema/ARDS (3%), bleeding/DIC (1%), and hypoglycemia (1%). The present study revealed the prevalence of signs of severity identified in the Thai population with malaria. Jaundice, hyperparasitemia, and impaired consciousness/coma were the most common signs of severity identified. These results may inform the management of patients with severe malaria and promote malaria-elimination efforts in Thailand.

Ultrastructure of male terminalia of Boettcherisca peregrina and Boettcherisca nathani (Diptera: Sarcophagidae), flesh fly species of forensic importance.

Flesh flies are received greater attention due to their use as evidence in forensic investigations of decomposing human remains. Investigators will often use age of immature insects associated with such remains as the minimal postmortem interval (PMImin) given certain assumptions. Boettcherisca peregrina is a significant flesh fly species of forensic importance in several countries, whereas B. nathani is potentially a species of forensic importance. Distinguishing these two species is difficult and relies on close examination of the male terminalia. For genus Boettcherisca belonging to subfamily Sarcophaginae, identification is primarily based on the morphological structures associated with male terminalia. Using light microscopy and scanning electron microscopy, similarities and differences were determined for these two species. In this study, B. peregrina was collected from an urban area of Mueang District, while B. nathani was collected from the highland forest area of Doi Saket District, Chiang Mai province, northern Thailand. The morphological observations indicated similarities in the morphology of vesica (large, rounded anterior margin, bi-lobed, half upper-outer margins serrated), harpes (well-developed, elongated, broad basally with bifurcated apically) and lateral styli (two parallel tube-like with open ends with microserration apically) in both species. However, differences were found with the cercus and juxta. With regards to the cercus, B. peregrina has bristles or setae only along the posterior surface and the cercal tip is pointed, while B. nathani has bristles or setae on both the anterior and posterior surfaces, and the cercal tip is hook-shaped. Morphometric measurements of three criteria of the cerci (length from top to middle, internal distance at narrow part and distance between apex of cercal prong) revealed that the average length of each criteria of B. nathani cerci (n = 30) were significantly greater than B. peregrina cerci (n = 30). As for the juxta, the juxtal lateral plate was different: primarily bifurcated in B. peregrina and non-bifurcated in B. nathani. The medial part of juxta was different: V-shaped 95.8% (46/48) and, to a lesser extent, W-shaped 4.2% (2/48) in B. peregrina, only W-shaped 100% (52/52) in B. nathani. Such information is useful for entomologists to distinguish between adult males of these two species, which could be valuable when determining colonization patterns in association with decomposing human remains.

Daily and seasonal variation of muscid flies (Diptera: Muscidae) in Chiang Mai province, northern Thailand.

Flies of the family Muscidae, or muscids, are of medical and veterinary importance worldwide due to their recognition as nuisance pests and myiasis-producing agents. Effective control of muscids requires biological information on population dynamics daily and across seasons. In this study, such patterns were investigated in three different microhabitats (e.g., forest area, palm plantation and longan orchard) in a suburban area of Chiang Mai Province, northern Thailand. Adult fly samplings were conducted for 24-h intervals using semiautomatic traps and 1-day old beef offal as bait. Samplings were carried out twice per month from July 2013 to June 2014. A total of 3,419 muscids were trapped, comprising nine species, with Musca domestica Linnaeus accounting for the majority (n = 1,329; 38.9%) followed by Hydrotaea spinigera Stein (n = 770; 22.5%) and Musca ventrosa Wiedemann (n = 740; 21.7%). The greatest overall abundance was in the longan orchard location (n = 1,508; 44.1%). Community structure peaked during the rainy season (mid-May to mid-Oct). Peak activity during the day was late morning (9.00 to 12.00 h) for M. domestica, early morning (6.00 to 9.00 h) for H. spinigera, and early afternoon (12.00 to 15.00 h) for M. ventrosa. Temperature had no significant effect on the abundance of M. domestica (rs= -0.030, p = 0.576) or H. spinigera (rs = 0.068, p = 0.200), but had a weak negative correlation with M. ventrosa (rs = -0.238, p = 0.0001). Relative humidity had a weak negative correlation with M. domestica (rs = -0.263, p = 0.0001), H. spinigera (rs = -0.107, p = 0.043) and M. ventrosa (rs = -0.344, p = 0.0001). More females (n = 2,078) were trapped than males (n = 761). These results provide baseline information of daily and seasonal dynamic activity of muscid flies under natural conditions, which is the prerequisite information for effective control measures.

Mitochondrial DNA-Based Identification of Forensically Important Flesh Flies (Diptera: Sarcophagidae) in Thailand.

Flesh flies (Sarcophagidae) are necrophagous insects initially colonizing on a corpse. The species-specific developmental data of the flies collected from a death scene can be used to estimate the minimum postmortem interval (PMImin). Thus, the first crucial step is to correctly identify the fly species. Because of the high similarity among species of flesh flies, DNA-based identification is considered more favorable than morphology-based identification. In this study, we demonstrated the effectiveness of combined sequences (2216 to 2218 bp) of cytochrome c oxidase subunit I and II genes (COI and COII) for identification of the following 14 forensically important flesh fly species in Thailand: Boettcherisca nathani Lopes, Fengia ostindicae (Senior-White), Harpagophalla kempi (Senior-White), Liopygia ruficornis (Fabricius), Lioproctia pattoni (Senior-White), Lioproctia saprianovae (Pape & Bänziger), Parasarcophaga albiceps (Meigen), Parasarcophaga brevicornis (Ho), Parasarcophaga dux (Thomson), Parasarcophaga misera (Walker), Sarcorohdendorfia antilope (Böttcher), Sarcorohdendorfia inextricata (Walker), Sarcorohdendorfia seniorwhitei (Ho) and Seniorwhitea princeps (Wiedemann). Nucleotide variations of Thai flesh flies were evenly distributed throughout the COI-COII genes. Mean intra- and interspecific variations ranged from 0.00 to 0.96% and 5.22% to 12.31%, respectively. Using Best Match (BM) and Best Close Match (BCM) criteria, identification success for the combined genes was 100%, while the All Species Barcodes (ASB) criterion showed 76.74% success. Maximum Likelihood (ML) and Bayesian Inference (BI) phylogenetic analyses yielded similar tree topologies of monophyletic clades between species with very strong support values. The achieved sequences covering 14 forensically important flesh fly species including newly submitted sequences for B. nathani, F. ostindicae and S. seniorwhitei, can serve as a reliable reference database for further forensic entomological research in Thailand and in other areas where those species occur.

Wing morphometric analysis of forensically important flesh flies (Diptera: Sarcophagidae) in Thailand.

Flesh flies are insects of forensic importance as the larvae associated with human remains can be used to estimate a minimum post-mortem interval (PMImin) in most cases. And, because life-history traits can vary across species, correct identification is a mandatory step before being used as evidence. Adult flesh flies are extremely similar in general appearance, which causes difficulty in species identification as it is largely based on the morphology of the male genitalia; this also makes it difficult to identify females. Currently, landmark-based geometric morphometric analysis of insect wings has proven to be a valuable tool for species identification. Herein, we applied wing morphometric analysis of 524 flesh fly specimens comprising 12 species from Thailand. The right wing of each specimen was removed, mounted on a microscope slide, photographed, and digitized using 18 landmarks. Wing shape variation among genera and species were analyzed using canonical variate analysis, while wing shape variation between sexes of each species was analyzed using discriminant function analysis. A cross-validation test was used to evaluate the reliability of classification. Results of this study demonstrate wing shape can be used to separate genera and species, and distinguish between sexes of the same species, with high reliability. Therefore, the landmark-based geometric morphometric analysis of wings is a useful additional method for species and sex discrimination of flesh flies.

Chrysomya chani Kurahashi (Diptera: Calliphoridae), a blow fly species of forensic importance: morphological characters of the third larval instar and a case report from Thailand.

Blow flies are worldwide the most important insects from a forensic point of view. In Thailand, aside from the two most common species, Chrysomya megacephala (F.) and Chrysomya rufifacies (Macquart), Chrysomya chani Kurahashi was also found to be of forensic importance. We present a case of a human female cadaver in its bloated stage of decomposition, discovered at Pachangnoi Subdistrict, northern Thailand. Entomological sampling during the autopsy displayed an assemblage of numerous dipteran larvae. Macroscopic observations showed the coexistence of third instar larvae of the three blow flies C. megacephala, Chrysomya villeneuvi Patton, an unknown blow fly species and one muscid, Hydrotaea sp. The minimum post-mortem interval was estimated to be six days, based on the developmental rate of C. megacephala. The ID of the unknown larva, which is the focus of this report, was revealed later as C. chani by DNA sequencing, using a 1205 bp of cytochrome c oxidase subunit I (COI). The occurrence of C. chani on a human body revealed the need to analyse and describe the morphology of its immature stage, to enable forensic entomologists to identify this fly species in future cases. The morphological examination of the third instar was performed, revealing peculiar characteristics: protuberant tubercles encircling abdominal segments; 9-11 lobes on the anterior spiracle; six prominent pairs of tubercles along the peripheral rim of the eighth abdominal segment; a heavily sclerotized complete peritreme of the posterior spiracles. A key to differentiate the third instar of blow flies of forensic importance in Thailand is provided.

Morphology of immature blow fly Hypopygiopsis infumata (Bigot) (Diptera: Calliphoridae), a potential species of forensic importance.

Blow flies of the genus Hypopygiopsis are forensically-important, as their larvae are commonly associated with human corpses. Within a forensic entomology context, species identification of specimens collected from human corpses is the initial mandatory step in the investigation. Without identification, complete interpretation of entomological evidence is challenged. In this study, the ultrastructures of eggs, all instars, and puparia of Hypopygiopsis infumata (Bigot) are presented based on assessment with scanning electron microscopy (SEM) and light microscopy (LM). Distinctive features used for species identification of all stages are highlighted. Eggs have a slightly widening median area extending almost the entire length. Larvae are vermiform-shaped, creamy white, and have a smooth integument. The pseudocephalon of larvae bears sensory structures (i.e., antennal dome, maxillary palpus and ventral organ). In the first instar, two tufts of cirri are observed along the dorsal margin of the mouth opening. In the second and third instars, six minute tubercles are present along the peripheral rims of the last abdominal segment. The anterior spiracle of the second, third instar, and puparia is fan-shape of single row, comprising 9-11 papillae. The cuticular spines between the 1st and 2nd thoracic segments of the third instar possess many rows of posteriorly-projecting acuminate spines in clusters. In puparia, at the latero-dorsal edge of the 1st abdominal segment, a cluster of ∼92 bubble membranes is present in young puparia (20-24 h). The peristigmatic tufts adjacent to the posterior spiracle of the second instar, third instar, and puparia are heavily branches of long, fine hairs. Our results demonstrate the morphology of eggs, larvae, and puparia of H. infumata are similar to other species in Hypopygiopsis. This study highlighted the main features of cephaloskeleton of H. infumata larvae as observed under LM. Particular attention is given to oral sclerite and rough surface of dorsal cornua which can distinguish between H. infumata and H. tumrasvini.

Bionomics of the oriental latrine fly Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae): temporal fluctuation and reproductive potential.

BACKGROUND: Chrysomya megacephala is a blow fly species of medical and forensic importance worldwide. Understanding its bionomics is essential for both designing effective fly control programs and its use in forensic investigations. METHODS: The daily flight activity, seasonal abundance related to abiotic factors (temperature, relative humidity and rainfall) and reproductive potential of this species was investigated. Adult flies were sampled twice a month for one year from July 2013 to June 2014 in three different ecotypes (forest area, longan orchard and palm plantation) of Chiang Mai Province, northern Thailand, using semi-automatic funnel traps. One-day tainted beef offal was used as bait. RESULTS: A total of 88,273 flies were sampled, of which 82,800 flies (93.8%) were caught during the day (from 06:00 to 18:00 h); while 5473 flies (6.2%) were caught at night (from 18:00 to 06:00 h). Concurrently, the abundance of C. megacephala was higher in the forest area (n = 31,873; 36.1%) and palm plantation (n = 31,347; 35.5%), compared to the longan orchard (n = 25,053; 28.4%). The number of females was significantly higher than that of males, exhibiting a female to male sex ratio of 2.36:1. Seasonal fluctuation revealed the highest abundance of C. megacephala in summer, but low numbers in the rainy season and winter. Fly density was significantly positively correlated with temperature, but negatively correlated with relative humidity. No correlation between numbers of C. megacephala with rainfall was found. Activity occurred throughout the daytime with high numbers from 06:00 to 18:00 h in summer and 12:00 to 18:00 h in the rainy season and winter. As for the nocturnal flight activity, a small number of flies were collected in summer and the rainy season, while none were collected in the winter. Dissection of the females indicated that fecundity was highest during the rainy season, followed by winter and summer. CONCLUSIONS: Since the assessment of daily, seasonal activity and the reproductive potential of C. megacephala remains a crucial point to be elucidated, this extensive study offers insights into bionomics, which may be considered for integrated fly control strategies and forensic entomology issues.

Field evaluation of a semi-automatic funnel trap targeted the medically important non-biting flies.

Bait-trapping is a useful approach for monitoring fly population dynamics, and it is an effective tool for physical control of pest species. The aim of this study was to test a newly developed semi-automatic funnel fly trap with some modifications of the former prototype fly trap to study medically important fly population density. The efficacy of the semi-automatic funnel trap was assessed by field sampling during July 2013-June 2014 using 1-day tainted beef offal as bait. The modified semi-automatic funnel traps were able to capture a total of 151,141 adult flies, belonging to the families: Calliphoridae (n=147,248; 97.4%), Muscidae (n=3,124; 2.1%) and Sarcophagidae (n=769; 0.5%), which are the medically important fly species. Among the total of 35 species collected, Chrysomya megacephala (Diptera: Calliphoridae) (n=88,273; 59.95%), Musca domestica (Diptera: Muscidae) (n=1,324; 42.38%) and Boettcherisca peregrina (Diptera: Sarcophagidae) (n=68; 33.01%) were the predominant species of each family. High number of flies was captured in forest area, representing 42.47% (n=64,197) of total specimens. Female flies were trapped more than male with total sex ratio of 0.37 male/female. Flies were trapped throughout the year with peak population in summer. Peak activity was recorded in the afternoon (12.00-18.00h). In summary, the modified semi-automatic funnel fly trap can be used for field collection of the adult fly. By setting the timer, population dynamics, diversity, and periodic activity of adult flies were determined.

Forensically Important Blow Flies Chrysomya pinguis, C. villeneuvi, and Lucilia porphyrina (Diptera: Calliphoridae) in a Case of Human Remains in Thailand.

This is the first study to report Chrysomya pinguis (Walker) and Lucilia porphyrina (Walker) (Diptera: Calliphoridae) as forensically important blow fly species from human cadavers in Thailand, in addition to Chrysomya villeneuvi (Patton) already known in Thailand. In 2016, a fully decomposed body of an unknown adult male was discovered in a high mountainous forest during winter in Chiang Mai province. The remains were infested heavily with thousands of blow fly larvae feeding simultaneously on them. Morphological identification of adults reared from the larvae, and molecular analysis based on sequencing of 1,247 bp partial mitochondrial cytochrome c oxidase subunit 1 gene (CO1) of the larvae and puparia, confirmed the above mentioned 3 species. The approving forensic fly evidence by molecular approach was described for the first time in Thailand. Moreover, neighbor-joining phylogenetic analysis of the CO1 was performed to compare the relatedness of the species, thereby affirming the accuracy of identification. As species of entomofauna varies among cases in different geographic and climatic circumstances, C. pinguis and L. porphyrina were added to the list of Thai forensic entomology caseworks, including colonizers of human remains in open, high mountainous areas during winter. Further research should focus on these 3 species, for which no developmental data are currently available.

DERMAL MYIASIS CAUSED BY THE FLESH FLY, PARASARCOPHAGA (LIOSARCOPHAGA) DUX (THOMSON, 1869) (DIPTERA: SARCOPHAGIDAE) AT THE SITE OF A MALIGNANT MELANOMA: A CASE REPORT.

Dermal myiasis due to the flesh fly has not been commonly reported in Thailand. A 64-year-old man came to the hospital with a 6-month history of a black tumor on the dorsum of the right great toe. The tumor was biopsied and determined to be a malignant melanoma. Second instar larvae were also seen in the wound and one larva was reared until the adult stage wherein it was identified as the flesh fly, Parasarcophaga (Liosarcophaga) dux (Thomson, 1869) (Diptera: Sarcophagidae). This case shows myiasis due to synanthropic sarcophagid flies occurs in Thailand and can occur in a malignant melanoma.

Morphology of immature stages of flesh flies, Boettcherisca nathani and Lioproctia pattoni (Diptera: Sarcophagidae).

The flesh flies are medically-important because the larvae found in the human corpses can provide evidence in forensic investigations through larva identification and their developmental rate. Firstly, we thoroughly described the larval morphology of Boettcherisca nathani and Lioproctia pattoni, using scanning electron microscopy (SEM). The third instar of the two species differed markedly in two characters: (1) spines between the prothorax and mesothorax-B. nathani has more or less slender triangular spines, with those at the posterior region more slender than the anterior region; whereas L. pattoni has stout triangular spines with one or two tips anteriorly, with smaller and tapered triangular shape, grouped two to four laterally in the posterior end, and (2) morphology of the peristigmatic tufts at the posterior spiracle-B. nathani has extensively branched long, fine hairs, whereas tufts in L. pattoni have moderately branched long, fine hairs. The anterior spiracle displayed similarity; B. nathani has two irregular rows of 21-27 papillae, while L. pattoni has a single irregular row of 20-28 papillae. Secondly, we use light microscopy to compare morphology of the third instar of the two species and additional three species, i.e., Bercaea africa, Parasarcophaga dux and Liopygia ruficornis. Particular attention was paid to the features of anterior spiracle, spines between prothorax and mesothorax and posterior spiracle. These results are useful in species identification and estimation of age of larvae found associated with corpses.

Surface ultrastrucure of larva and puparia of blow fly Hypopygiopsis tumrasvini Kurahashi (Diptera: Calliphoridae).

Flies of the genus Hypopygiopsis are forensically important, as their larvae are found to associate with human corpses. In this study, the ultrastructure of larvae and puparia of Hypopygiopsis tumrasvini Kurahashi is presented using scanning electron microscopy (SEM). The larvae are vermiform-shaped, creamy white, and have a smooth integument. The pseudocephalon of larvae bears sensory structures, i.e., antenna, maxillary palpus, and ventral organ. Two tufts of fresh outgrowths (or cerri) were observed along the dorsal margin of the mouth opening of the first instar; whereas a strong mouth hook was apparent in the second and third instars. Keilin's organ, the sensory structure, was noticeable on the ventral surface of the thoracic segments in all instars. In the second and third instars, six conspicuous tubercles were present along the peripheral rims of the last abdominal segment. The puparia were relatively large, measuring 7.77-9.51 mm in length and 3.10-3.97 mm in width. At the latero-dorsal edge of the first abdominal segment, a cluster of ~125 bubble membranes was seen in young puparia; whereas a minute pupal respiratory horn was observed in old one. An SEM image revealed antler-like projections lined within a chamber of a broken pupal respiratory horn. Comparison on number of the bubble membranes of the other blow fly species was shown and the role of pupal respiratory horn compared with other dipterans was discussed.