Diverse effects of clarithromycin and proposal of its clinical application for treating COVID-19 as a repurposing drug.

Outbreak of SARS-CoV-2 has been declared a pandemic, which is a serious threat to human health. The disease was named coronavirus disease 2019 (COVID-19). Until now, several vaccines and a few drugs have been approved for the prevention and treatment for COVID-19. Recently, the effect of some macrolides including clarithromycin (CAM) on COVID-19 has attracted attention. CAM is known to have diverse effects including immunomodulatory and immunosuppressive effects, autophagy inhibition, steroid sparing effect, reversibility of drug resistance, antineoplastic effect, antiviral effect as well as bacteriostatic/bactericidal effect. Many patients with COVID-19 died due to an overwhelming response of their own immune system characterized by the uncontrolled release of circulating inflammatory cytokines (cytokine release syndrome [CRS]). This CRS plays a major role in progressing pneumonia to acute respiratory distress syndrome (ARDS) in COVID-19 patients. It is noteworthy that CAM can suppress inflammatory cytokines responsible for CRS and also has anti-SARS-CoV-2 effect. Considering the rapidly progressive global disease burden of COVID 19, the application of CAM for treating COVID-19 needs to be urgently evaluated. Recently, an open-labeled non-randomized trial using CAM for treating COVID-19 (ACHIEVE) was initiated in Greece in May, 2020. Its results, though preprint, indicated that CAM treatment of patients with moderate COVID-19 was associated with early clinical improvement and containment of viral load. Thus, treatment with CAM as a single agent or combined with other anti-SARS CoV-2 drugs should be tried for treating COVID-19. In this article, we discussed the significance and usefulness of CAM in treating COVID-19.

Discovery of cockroach specimens of the genus Squamoptera (Ectobiidae: Pseudophyllodromiinae) from Okinawa, Japan, showing wing polymorphism.

Cockroach specimens of the genus, Squamoptera were collected from the Iriomote island of Okinawa prefecture, Japan. The morphological features of the specimens were characterized as having a white band on the dorsal surface of its thorax, its tegmen reduced into a tiny scale-like structure and the hindwing was absent. Ocelli was also absent and the small compound eyes not extending to apex of the head nor to the frontal face but extend further lower than the base of the antennae. When the specimens were reared in the laboratory, besides the short wing form, the long wing form began to appear in the rearing colony. In our reproductive biological study, we observed that hatching of the ootheca from the short wing female takes about 30 days, with an average of 6.6 nymphs being hatched from one ootheca. The male to female ratio of the offspring was 36:30. However, the frequency appearance of the offspring from the ootheca of the short wing female was 98.5% short wing and 1.5% long wing form. Our specimens occasionally show body polymorphism in the form of individuals having long wings instead of the usual short one. The long wing form does not show the white band on the dorsal surface of its thorax.

A new species of cockroach, Periplaneta gajajimana sp. nov., collected in Gajajima, Kagoshima Prefecture, Japan.

We described a new species of cockroach, Periplaneta gajajimana sp. nov., which was collected in Gajajima, Kagoshima-gun Toshimamura, Kagoshima Prefecture, Japan, on November 2012. The new species is characterized by its reddish brown to blackish brown body, smooth surface pronotum, well developed compound eyes, dark brown head apex, dark reddish brown front face and small white ocelli connected to the antennal sockets. In male, the tegmen tip reach the abdomen end or are slightly shorter, while in the female, it does not reach the abdominal end and exposes the abdomen beyond the 7th abdominal plate. We confirmed the validity of this new species by breeding the specimens in our laboratory to demonstrate that the features of the progeny were maintained for several generations. For comparison and easy identification of this new species, the key to species identification of the genus Periplaneta that had been reported in Japan to date are also presented.

Serum liver enzyme profile in Timor deer (Cervus timorensis) with fascioliasis in Indonesia.

Timor deer (Cervus timorensis) at Surabaya zoo, Indonesia, that were found to be naturally infected with Fasciola, showed elevated level of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). Of a total of 75 deer examined, 12 (25%) of the 47 adult deer and 8 (29%) of the 28 juvenile deer were found to be infected with fascioliasis, as evidenced by the shedding of the parasite eggs. The level of ALT, AST and ALP were significantly elevated (p<0.05) in all the infected deer. Only Fasciolainfected deer showed elevated serum liver enzyme. Deer with elevated enzyme level show a trend that positively correspond with higher Egg per gram of feces (EPG). The average size of the parasite eggs at 169.0±11.1 × 96.0±3.5µm, correspond well with that of Fasciola gigantica. No other trematode eggs were observed besides that of F. gigantica. There was no significant difference in the enzyme profile between the two sexes in both the infected and the uninfected group. This is the first report of the elevation of serum liver enzyme in Timor deer that is associated with not only fascioliasis and also correspond positively with the EPG.

Diverse effects of clarithromycin and proposal of its clinical application for treating COVID-19 as a repurposing drug

@#Outbreak of SARS-CoV-2 has been declared a pandemic, which is a serious threat to human health. The disease was named coronavirus disease 2019 (COVID-19). Until now, several vaccines and a few drugs have been approved for the prevention and treatment for COVID-19. Recently, the effect of some macrolides including clarithromycin (CAM) on COVID-19 has attracted attention. CAM is known to have diverse effects including immunomodulatory and immunosuppressive effects, autophagy inhibition, steroid sparing effect, reversibility of drug resistance, antineoplastic effect, antiviral effect as well as bacteriostatic/bactericidal effect. Many patients with COVID-19 died due to an overwhelming response of their own immune system characterized by the uncontrolled release of circulating inflammatory cytokines (cytokine release syndrome [CRS]). This CRS plays a major role in progressing pneumonia to acute respiratory distress syndrome (ARDS) in COVID-19 patients. It is noteworthy that CAM can suppress inflammatory cytokines responsible for CRS and also has anti-SARS-CoV-2 effect. Considering the rapidly progressive global disease burden of COVID- 19, the application of CAM for treating COVID-19 needs to be urgently evaluated. Recently, an open-labeled non-randomized trial using CAM for treating COVID-19 (ACHIEVE) was initiated in Greece in May, 2020. Its results, though preprint, indicated that CAM treatment of patients with moderate COVID-19 was associated with early clinical improvement and containment of viral load. Thus, treatment with CAM as a single agent or combined with other anti-SARSCoV- 2 drugs should be tried for treating COVID-19. In this article, we discussed the significance and usefulness of CAM in treating COVID-19.

Discovery of cockroach specimens of the genus Squamoptera (Ectobiidae: Pseudophyllodromiinae) from Okinawa, Japan, showing wing polymorphism

@# Cockroach specimens of the genus, Squamoptera were collected from the Iriomote island of Okinawa prefecture, Japan. The morphological features of the specimens were characterized as having a white band on the dorsal surface of its thorax, its tegmen reduced into a tiny scale-like structure and the hindwing was absent. Ocelli was also absent and the small compound eyes not extending to apex of the head nor to the frontal face but extend further lower than the base of the antennae. When the specimens were reared in the laboratory, besides the short wing form, the long wing form began to appear in the rearing colony. In our reproductive biological study, we observed that hatching of the ootheca from the short wing female takes about 30 days, with an average of 6.6 nymphs being hatched from one ootheca. The male to female ratio of the offspring was 36:30. However, the frequency appearance of the offspring from the ootheca of the short wing female was 98.5% short wing and 1.5% long wing form. Our specimens occasionally show body polymorphism in the form of individuals having long wings instead of the usual short one. The long wing form does not show the white band on the dorsal surface of its thorax.

A new species of cockroach, Periplaneta gajajimana sp. nov., collected in Gajajima, Kagoshima Prefecture, Japan

@#We described a new species of cockroach, Periplaneta gajajimana sp. nov., which was collected in Gajajima, Kagoshima-gun Toshimamura, Kagoshima Prefecture, Japan, on November 2012. The new species is characterized by its reddish brown to blackish brown body, smooth surface pronotum, well developed compound eyes, dark brown head apex, dark reddish brown front face and small white ocelli connected to the antennal sockets. In male, the tegmen tip reach the abdomen end or are slightly shorter, while in the female, it does not reach the abdominal end and exposes the abdomen beyond the 7th abdominal plate. We confirmed the validity of this new species by breeding the specimens in our laboratory to demonstrate that the features of the progeny were maintained for several generations. For comparison and easy identification of this new species, the key to species identification of the genus Periplaneta that had been reported in Japan to date are also presented.

Serum liver enzyme profile in Timor deer (Cervus timorensis) with fascioliasis in Indonesia

@#Timor deer (Cervus timorensis) at Surabaya zoo, Indonesia, that were found to be naturally infected with Fasciola, showed elevated level of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). Of a total of 75 deer examined, 12 (25%) of the 47 adult deer and 8 (29%) of the 28 juvenile deer were found to be infected with fascioliasis, as evidenced by the shedding of the parasite eggs. The level of ALT, AST and ALP were significantly elevated (p<0.05) in all the infected deer. Only Fasciolainfected deer showed elevated serum liver enzyme. Deer with elevated enzyme level show a trend that positively correspond with higher Egg per gram of feces (EPG). The average size of the parasite eggs at 169.0±11.1 × 96.0±3.5μm, correspond well with that of Fasciola gigantica. No other trematode eggs were observed besides that of F. gigantica. There was no significant difference in the enzyme profile between the two sexes in both the infected and the uninfected group. This is the first report of the elevation of serum liver enzyme in Timor deer that is associated with not only fascioliasis and also correspond positively with the EPG.

Experimental Infection with Baylisascaris Potosis in Chickens.

The larvae of the genus Baylisascaris can cause larva migrans in mammals and birds. This study investigated the larval migration of Baylisascaris potosis, the roundworm of kinkajou (Potos flavus), in chickens and the associated clinical manifestations of the host. Thirty-six 3-week-old chickens divided into 6 groups were orally inoculated with 3,000 B. potosis eggs/chick. Each group of chicken was necropsied at days 1, 2, 3, 7, 30 and 90 PI (post inoculation), and the number of larvae in various organs were counted until day 90 PI. No clinical signs were observed in chickens during the study. Larvae were detected from the liver, lungs or breast-muscles of 13/36 (36.1%) chickens. The mean total number of larvae in the liver, lungs and breast-muscles at days 1, 2, 3, 7, 30 and 90 PI were 0.34, 0.17, 1.66, 1.01, 0.17 and 0, respectively. No larvae were found in the brain, eyes, hid-limb muscles, heart, kidneys and spleen. Although infectivity of larvae in egg-inoculated chickens was low, the present study demonstrated that B. potosis larvae can migrate in chickens tissues up to day 30 PI. The result suggests that chickens can serve as a paratenic host for B. potosis and may underline a public health importance of B. potosis infection as a potential foodborne disease in humans.

Baylisascaris Potosis Larvae in Mice of Different Strains and Infectivity of Tissue Larvae.

Migration of Baylisascaris potosis larvae in different mouse strains were compared, and infectivity of the persisting larvae in mice tissues were investigated. Five strains of mice, BALB/c, C57BL/6, AKR, B10.BR, and ICR were inoculated with 1,000 B. potosis eggs/mouse, and necropsied at week 13 post inoculation (PI). The other uninfected ICR mice (secondary host) were inoculated with 43 larvae/ mouse recovered from mice at week 13 PI with eggs, and necropsied at day 21 PI. Larvae in organs or tissues were counted at necropsy. One AKR mouse showed torticollis and circling at day 56 PI. At necropsy at week 13 PI, larvae were recovered from all mice. A mean total larvae recovered were 124.1 (n=40). Majority of larvae were found in the carcass (mean 113.9) and some in the viscera (mean 9.9). Zero to 1 larva were found in the brain or eyes of some mice. There were no differences among the mouse strains in the number of larvae, except in the viscera; more larvae were seen in BALB/c or ICR than in B10.BR mice. No larvae were found in the secondary host mice. Present study demonstrated that B. potosis larvae migrate well in the carcass of any strains of mice, however, the tissue larvae did not infect the secondary host. Results of our present study suggest that B. potosis larvae is less aggressive for the nervous tissue migration than that of B. procyonis larvae which is commonly known to migrate in central nervous system of mammals and birds.

Species clarification of Ogasawara cockroaches which inhabit Japan.

The so-called "Ogasawara cockroaches" were examined by morphological observations and by breeding experiments to elucidate their actual taxonomical status. Fourteen groups (isolate) of "Ogasawara cockroaches" collected from Iwoto-A, Iwoto-B, Hahajima, Chichijima, Nishijima, Nakodojima, Tokunoshima-A, Tokunoshima-B, Okinawato- A, Okinawa-B, Amamiooshima, Miyakojima, Ishigakijima and Hawaii, were bred and passaged in our laboratory. Cockroaches collected from the field were first reared individually and the sexes of their offspring examined. Cockroaches collected from Iwoto, Tokushima and Okinawa, were found to consist of two groups; those whose offspring were all female and the other whose offspring consist of both male and female. Cross-breeding experiments showed that individuals from the group that did not produce any male but only female offspring were parthenogenetic. On the contrary, the group that have bisexual individuals produced both male and female offspring in a ratio of 1:1. Our results show that the so-called "Ogasawara cockroaches" consist of 2 species, namely, Pycnoscelus surinamensis and Pycnoscelus indicus. There are areas in which both species co-habitated together and there are also areas in which either only one of the two species can be found. The group that reproduces only female offspring and only through parthenogenesis was identified as P. surinamensis. The group that reproduces heterosexually and produce male and female offspring was identified as P. indicus. Thus, the so-called "Ogasawara cockroaches" found in Japan actually consist of 2 species, namely, P. surinamensis and P. indicus, which can be differentiated using the solitary breeding method to demonstrate parthenogenesis in the former and the need for sexual reproduction in the latter.

Development of Taenia asiatica cysticerci to infective stage and adult stage in Mongolian gerbils.

The development of metacestodes and adult worms of Taenia asiatica in Mongolian gerbils (Meriones unguiculatus) were observed. Cysticerci were recovered from gerbils subcutaneously injected with hatched oncospheres. The recovery rate ranged from 0.1 to 3.2%. No cysticerci were recovered from the orally inoculated gerbils. The infectivity of the cysticerci recovered at 48 weeks post-infection was evaluated. Tapeworms were recovered on day 14 post-infection from the small intestine of 5 of 11 gerbils, with a recovery rate of 27% (6 worms recovered/22 worms inoculated). Three and four adult worms were recovered from two human volunteers who ingested five cysticerci after 4 months post-infection. In worms recovered from gerbils, segmentation and genital primordia in the posterior proglottids and hooklets in the residual rostellum were observed. The results indicate that gerbils can serve as an alternative intermediate host and that partial development of the adult worm stage occurs in gerbils.

Development of Taenia saginata asiatica metacestodes in SCID mice and its infectivity in human and alternative definitive hosts.

Development of Taenia saginata asiatica metacestodes in SCID mice, and its infectivity in humans, golden hamsters, and Mongolian gerbils as alternative definitive hosts, were investigated. Cysticerci were recovered from SCID mice that were subcutaneously injected with hatched oncospheres of T. s. asiatica. The morphological changes of metacestodes were observed. The recovered cysticerci were fed to gerbils, hamsters and humans, to check for their infectivity. Tapeworms were recovered from gerbils and hamsters fed with 20 to 45 week-old cysticerci, and proglottids excretions were observed in human volunteers fed with 45 week-old cysticerci. However, no tapeworms were recovered from gerbils fed with 10 week-old cysticerci. Our results suggest that T. s. asiatica oncospheres needed more than 20 weeks to develop to maturity in SCID mice to be infective to both their natural and alternative definitive hosts.

Retarded gastric acid secretion in rats infected with larval Taenia taeniaeformis.

The influence of hepatic larval Taenia taeniaeformis infection on gastric acid secretory activity and gastric mucosal integrity was investigated. After 12 weeks of infection with 2,000 T. taeniaeformis eggs, the gastric pH values of control and infected rats were 4.1+/-0.6 (mean +/- SD) and 8.4+/-0.2, respectively. There was no difference in the basal acid secretion between control (1.7+/-0.7 micro Eq.H(+)/15 min) and infected (1.9+/-0.3) rats. However, infected rats failed to respond to histamine stimulation, the maximum acid output level being 2.8+/-0.4 in the infected rats, compared to 12.9+/-3.3 in control rats. Larval T. taeniaeformis infection resulted in the suppression of gastric acid secretion leading to hypergastrinemia.

Susceptibility of mosquitoes in central Taiwan to natural infections of Dirofilaria immitis.

From October 1997 to September 1998, 3085 Culex quinquefasciatus (Say) (Diptera: Culicidae), 584 Cx. tritaeniorhynchus (Giles) (Diptera: Culicidae), 392 Cx. annulus (Theobald) (Diptera: Culicidae), 374 Aedes albopictus (Skuse) (Diptera: Culicidae) and 102 Armigeres subalbatus (Coquillet) (Diptera: Culicidae) were collected and examined for Dirofilaria immitis (Leidy) (Spirurida: Filariidae) infection. However, only Cx. quinquefasciatus and Ae. albopictus were infected, with a prevalence of 4.28% and 3.74%, respectively. The intensity of D. immitis found in Ae. albopictus (3.43 larvae/mosquito) was higher than that found in Cx. quinquefasciatus (2.89 larvae/mosquito). After being fed with canine blood containing 7500 microfilariae (mf) per mL, Cx. quinquefasciatus ingested approximately two times as many mf as Ae. albopictus (mean of 31.73 in comparison to 16.47). However, almost three times as many third-stage infective larvae developed in Ae. albopictus as in Cx. quinquefasciatus (mean of 3.25 as compared with 1.10), with a vector efficiency index (VEI) of 19.73 and 3.47, respectively. The results showed that Cx. quinquefasciatus and Ae. albopictus served as natural vectors of D. immitis in central Taiwan. Although Ae. albopictus was more efficient for heartworm transmission, Cx. quinquefasciatus may play a more prominent role on the transmission of dirofilariasis in central Taiwan.

Abnormal distribution of anionic sites in the glomerular basement membrane in glomerulonephritis of dogs infected with Dirofilaria immitis.

Ultrastructural alteration of anionic sites (ASs) in the glomerular basement membrane (GBM) was studied in glomerulonephritis characterized by linear capillary IgG deposition in four dogs infected with Dirofilaria immitis and two normal control dogs using polyethyleneimine. ASs were identified as small dense particles distributed regularly in the lamina rara externa (LRE), but there were no ASs in the lamina densa (LD) of the GBM of the control dogs. In the glomeruli of the infected dogs, ASs were distributed regularly or irregularly in the thickened LD. ASs were in addition localized over the characteristic continuous bands of subendothelial dense deposits. The number of ASs of the LRE increased in all four infected cases as compared to the controls (p<0.01).

Survey of Spirometra erinaceieuropaei in frogs in Taiwan and its experimental infection in cats.

Eighteen of 56 (32.1%) wild Rana limnocharis from central and south Taiwan were found to contain plerocercoids of Spirometra erinaceieuropaei. This is the first report of S. erinaceieuropaei infections in frogs in Taiwan, with the plerocercoids being recovered from the thigh and back muscles or under the skin. Other species of frogs examined, including nine wild R. latouchii, one wild Buergeria robustus and 110 cultured R. rugulosa were free of infection. The plerocercoids were orally inoculated into four cats; three of which were each given a single plerocercoid and one a dose of three plerocercoids. Daily faecal examination showed that two cats started shedding eggs of S. erinaceieuropaei on day 8 postinfection (PI) and the other two on day 10 PI. The highest eggs per gram and eggs per day for a single worm was found to be 428,000 and 14,416,000 respectively. Only the cat inoculated with three plerocercoids shed proglottids in its faeces during the 2 month observation period.

Serological survey and first finding of Neospora caninum in Taiwan, and the detection of its antibodies in various body fluids of cattle.

A serological survey for antibodies against Neospora caninum in cattle, goats and farm dogs in Taiwan was carried out. Sera of 613 cattle from 25 dairy farms, 24 goats from six goat farms and 13 dogs from six dairy cattle farms were tested for antibodies against N. caninum using indirect fluorescence antibody test (IFAT). The same sera were also tested for antibodies against Toxoplasma gondii using latex agglutination test. Of the 613 cattle sera, 44.9% (275/613) were found to have antibodies against N. caninum. Among these 275 positive cattle, 77 also possessed antibodies against T. gondii. Nevertheless, 92 cattle which were negative for N. caninum showed antibodies against T. gondii. Of the 24 goat sera tested, none was found to be positive for N. caninum but 50% (12/24) were positive for T. gondii. Of the 13 farm dogs tested, three were found to possess antibodies against N. caninum, two of which tested negative for T. gondii antibodies. Besides sera, antibodies to N. caninum in cattle could be observed in the milk, vaginal secretion and saliva. However, the order of higher frequency of antibodies detection is in sera, milk, vaginal secretion and saliva. This is the first demonstration of the presence of antibodies to N. caninum in vaginal secretion and saliva of cattle. A 50microm cyst was observed in the brain of one of the 13 prednisolone-treated SPF ICR mice which had been peritoneally inoculated 4 months earlier with the brain homogenate of a serologically N. caninum positive but T. gondii negative cattle. Thus, we have confirmed for the first time the presence of N. caninum in Taiwan and also observed that it is widespread among dairy cattle and farm dogs.

Competence of Aedes albopictus and Culex quinquefasciatus as vector of Dirofilaria immitis after blood meal with different microfilarial density.

Aedes albopictus and Culex quinquefasciatus were fed canine blood with different microfilarial density of Dirofilaria immitis ranging from 2500 to 25,000 mff/ml. Larval development in these two mosquito species did not differ significantly. Although C. quinquefasciatus ingested more microfilariae, the number of larvae which developed in A. albopictus was invariably greater than in C. quinquefasciatus. Mortality of the engorged A. albopictus was significantly greater than that of C. quinquefasciatus, and higher microfilarial density raised the mortality in both species. The vector efficiency index of A. albopictus was greater than C. quinquefasciatus at all microfilarial densities, but its survival time was much reduced. Thus, dogs with low microfilarial density are implicated as the main source for the transmission of D. immitis from dogs to mosquitoes.

Natural mass infection by heterophyid metacercariae in aquacultured Japanese eel in Taiwan.

A natural mass infection of heterophyid metacercariae in aquacultured Japanese eel Anguilla japonica in Taiwan was observed. Of the 28,000 adult eels in 2 ponds, about 25,000 (90%) showed swollen, cloudy and white eyes. Although morbidity was about 90%, there was no mortality among the affected eels. Histopathological sections showed edema and hemorrhage of the eye. Numerous metacercariae were observed in the muscle tissues around the eyeball, the subcutaneous tissue and even in the cartilage. Of the 6 eels digested with artificial gastric juice, all were found to contain metacercariae in their muscle tissues. The average number of metacercariae recovered from the 6 eels was 1219, with a range of 50 to 3762. These metacercariae, when fed orally to immunodeficient (scid) mice, developed into adult worms which were identified as Procerovum cheni Hsu 1950. The naturally infected eels were transferred to a new pond without snails and their eye lesions were not apparent anymore after 2 wk. In a follow-up investigation, 19 of 20 apparently healthy eels in a nearby aquaculture farm were found to harbour metacercariae in their muscles. However, the number of the metacercariae ranged from 1 to 14, with an average of 4.21. This is the first report of heterophyid metacercariae causing mass morbidity in aquacultured eels.