Evidence of human leptospirosis cases in a cohort of febrile patients in Bangui, Central African Republic: a retrospective study, 2012-2015.

BACKGROUND: In spite of a local favorable environment, leptospirosis has never been described in Central African Republic so far mainly because of the weakness of diagnostic tests and differential diagnostic strategy for febrile jaundice cases negative for yellow fever virus. Here we bring a complementary insight to conclusions of Gadia CLB et al. regarding the presence of leptospirosis in Central African Republic in YFV-negative febrile icteric patients. METHODS: Our study included 497 individuals presenting with fever and jaundice but negative for yellow fever infection, retrospectively selected from the national surveillance biobank for yellow fever in Institut Pasteur de Bangui, Central African Republic. A combination of serological (ELISA, agglutination) and molecular biology techniques (quantitative real-time polymerase chain reaction) was used to identify Leptospira or the patient's immune response to the bacteria. Statistical analyses were done using the non parametric Mann-Withney U test with a 5% statistical threshold. RESULTS: ELISA test results showed 46 positive serum samples while 445 were negative and 6 remains equivocal. In addition, the reference microscopic agglutination test for leptospirosis diagnostic confirmed that 7 out of 32 samples tested were positive. Unfortunately, all 497 serum samples tested for leptospirosis were negative using the molecular techniques. CONCLUSIONS: Unlike Gadia et al., we confirmed that leptospirosis is circulating in Central African Republic and therefore may be responsible for some of the unexplained cases of febrile jaundice in the country. Thus, leptospirosis needs to be investigated to improve identification of aetiological pathogens. Our study also suggests a need to improve sample transportation and storage conditions.

Leptospirosis as a cause of fever associated with jaundice in the Democratic Republic of the Congo.

BACKGROUND: Fever with jaundice is a common symptom of some infectious diseases. In public health surveillance within the Democratic Republic of the Congo (DRC), yellow fever is the only recognized cause of fever with jaundice. However, only 5% of the surveillance cases are positive for yellow fever and thus indicate the involvement of other pathogens. Leptospira spp. are the causative agents of leptospirosis, a widespread bacterial zoonosis, a known cause of fever with jaundice. This study aimed to determine the seropositivity of anti-Leptospira antibodies among suspected yellow fever cases and map the geographical distribution of possible leptospirosis in the DRC. METHODS: We conducted a retrospective study using 1,300 samples from yellow fever surveillance in the DRC from January 2017 to December 2018. Serum samples were screened for the presence of IgM against Leptospira spp. by a whole cell-based IgM ELISA (Patoc-IgM ELISA) at the Institut National de Recherche Biomedicale in Kinshasa (INRB) according to World Health Organization (WHO) guidance. Exploratory univariable and multivariable logistic regression analyses were undertaken to assess associations between socio-demographic factors and the presence of Leptospira IgM. RESULTS: Of the 1,300 serum samples screened, 88 (7%) showed evidence of IgM against Leptospira spp. Most positive cases (34%) were young adult males in the 20-29-year group. There were statistically significant associations between having Leptospira IgM antibodies, age, sex, and living area. Observed positive cases were mostly located in urban settings, and the majority lived in the province of Kinshasa. There was a statistically significant association between seasonality and IgM Leptospira spp. positivity amongst those living in Kinshasa, where most of the positive cases occurred during the rainy season. CONCLUSIONS: This study showed that leptospirosis is likely an overlooked cause of unexplained cases of fever with jaundice in the DRC and highlights the need to consider leptospirosis in the differential diagnosis of fever with jaundice, particularly in young adult males. Further studies are needed to identify animal reservoirs, associated risk factors, and the burden of human leptospirosis in the DRC.

[Detection of yellow fever virus by reverse transcriptase polymerase chain reaction in wild monkeys: a sensitive tool for epidemiologic surveillance]. / Detección por reacción en cadena de la polimerasa de transcriptasa inversa del virus de la fiebre amarilla en monos silvestres: una herramienta sensible para la vigilancia epidemiológica.

INTRODUCTION: Yellow fever is a zoonotic infection maintained in nature by non-human primates. Appropriate surveillance with sensitive laboratory techniques is necessary to evidence viral activity in the tropical forest habitats of these primates. OBJECTIVE: Yellow fever virus was detected in hepatic tissue samples from non-human primates by reverse transcriptase polymerase chain reaction (RT-PCR) technique using specific primers for diagnosis. MATERIALS AND METHODS: Hepatic tissue samples were processed from five monkeys belonging genus Alouatta spp found dead in sylvatic areas of Cesar and Magdalena Provinces, Colombia, between December 2003 and June 2004. Samples were treated with lysis buffer prior to the isolation of viral RNA, which was then subjected to reverse transcriptase polymerase chain reaction (RT-PCR) using yellow fever-specific primers. Simultaneously, viral proteins were identified by immunohistochemistry on parafin-embedded hepatic tissue. RESULTS: The PCR method amplified fragments of the expected size (424 bp) in four of the tested samples. In addition, these samples showed a positive reaction by immunohistochemistry, supporting the evidence that the virus was present. CONCLUSION: The detection of yellow fever virus in wild monkeys was clear evidence of enzootic activity in northern Colombia. Increased probability of yellow fever transmission among human populations is indicated due to urbanization processes as a consequence of forced migration and displacement of the human populations. Molecular tests for rapid and specific detection of yellow fever in tissue samples of non-human primates is an important tool for epidemiologic surveillance. Rapid virus identification will permit the timely activation of control systems for prevention of further cases and epidemic situations.

Geographical distribution of the red howler monkey (Alouatta seniculus) and yellow fever in Colombia.

INTRODUCTION: Colombia is a country with an important diversity of non-human primates, of which the red howler monkey (Alouatta seniculus) stands out because of its distribution and the role it plays in the occurrence of yellow fever.  OBJECTIVE: To describe the geographic co-occurrence of Alouatta seniculus and the reported presence of yellow fever.  MATERIALS AND METHODS: We conducted a descriptive study. The reported presence of yellow fever in Colombia was obtained from the reports and bulletins issued by the Instituto Nacional de Salud, and the study by Segura, et al. (2013). The occurrence of A. seniculus was determined based on the data from the Global Biodiversity Information Facility and the Colombian Biodiversity Information System. A map of the occurrence was developed using the DIVA-GIS program, and the ecological niche model under current conditions was created with the Maxent program.  RESULTS: The departments with the highest occurrence of A. seniculus were Antioquia, Meta and Casanare; 69.5% of the departments with reported history of yellow fever had co-occurrence with A. seniculus. The ecological niche model showed that Antioquia, Bolívar, La Guajira, Magdalena, Meta, Santander, Norte de Santander and Vichada had geographical portions with a probability rate nearing to 0.9 (90%).  CONCLUSIONS: In 69.5% of the departments with a history of yellow fever there was co-occurrence with A. seniculus, which is relevant because non-human primates play a well-known role as natural reservoirs of the virus, and they might contribute to the occurrence of the yellow fever, which makes them very useful as sentinels.

Combining the sterile insect technique with the incompatible insect technique: I-impact of wolbachia infection on the fitness of triple- and double-infected strains of Aedes albopictus.

The mosquito species Aedes albopictus is a major vector of the human diseases dengue and chikungunya. Due to the lack of efficient and sustainable methods to control this mosquito species, there is an increasing interest in developing and applying the sterile insect technique (SIT) and the incompatible insect technique (IIT), separately or in combination, as population suppression approaches. Ae. albopictus is naturally double-infected with two Wolbachia strains, wAlbA and wAlbB. A new triple Wolbachia-infected strain (i.e., a strain infected with wAlbA, wAlbB, and wPip), known as HC and expressing strong cytoplasmic incompatibility (CI) in appropriate matings, was recently developed. In the present study, we compared several fitness traits of three Ae. albopictus strains (triple-infected, double-infected and uninfected), all of which were of the same genetic background ("Guangzhou City, China") and were reared under the same conditions. Investigation of egg-hatching rate, survival of pupae and adults, sex ratio, duration of larval stages (development time from L1 to pupation), time to emergence (development time from L1 to adult emergence), wing length, female fecundity and adult longevity indicated that the presence of Wolbachia had only a minimal effect on host fitness. Based on this evidence, the HC strain is currently under consideration for mass rearing and application in a combined SIT-IIT strategy to control natural populations of Ae. albopictus in mainland China.

Passage of yellow fever virus: its effect on infection and transmission rates in Aedes aegypti.

The effect of successive lytic passage of yellow fever virus on mosquito infection and transmission rates in the vector, Aedes aegypti, was determined. Three strains of yellow fever virus from Trinidad and Peru were passaged five times in suckling mouse brains and seven times in BHK-21 cells. Mosquitoes were fed meals containing passaged and unpassaged viruses and infection and transmission rates were compared. Rates were similar for all but one of the three virus strains grown in both substrates with the exception of virus strain 1899/81 (human isolate from Peru) passaged seven times in BHK-21 cells. Infection rates declined from 62% (109/177) to 35% (61/176), and transmission rates declined from 64% (60/94) to 45% (22/49). The oligonucleotide fingerprint of strain 1899/81 passaged seven times in BHK-21 cells shared 98% (45/46) of its large, T1-resistant oligonucleotides with the parent strain, indicating limited biochemical differences. The data suggest that uncloned yellow fever virus populations, passaged a limited number of times, and exhibiting some phenotypic changes, are representative of the original virus strain and can be used with a reasonable degree of confidence in vector competence studies.

An epidemic of yellow fever in central Brazil. 1972-1973. I. Epidemiological studies.

An epidemic of jungle yellow fever occurred in Goiás State, Brazil, between December 1972 and March 1973. Laboratory confirmed cases were observed in 36 counties located in the central and southern parts of the State. Seventy-one cases were proved, of which 44 were fatal. The diagnosis was made on the basis of pathology, serology, and virus isolation. Besides yellow fever, malaria and viral hepatitis were present, and in two fatal cases there was malarial pigment in the liver in addition to the specific lesions associated with yellow fever virus infection. The fact that male patients strikingly outnumbered females (9:1) and that young adults were predominantly affected indicates that transmission occurred mainly inside or adjacent to the forests. The lack of cases in urban areas can be attributed to the absence of Aedes aegypti in these areas. Yellow fever complement-fixing antibody in high titers was found in 18 of 1,201 (1.4%) persons living in eight counties of the affected area. This finding suggests that at least 21,000 persons out of the 1.5 million rural inhabitants of the three districts where the epidemic occurred had been infected by the virus. The epidemic subsided following an intensive vaccination campaign, and the last four cases were observed in March 1973.

Arboviruses and lemurs in Madagascar: experimental infection of Lemur fulvus with yellow fever and West Nile viruses.

In previous serological surveys of lemurs in Madagascar, antibodies against flaviviruses were frequently detected. To examine the epidemiological role of Lemur fulvus, experimental infections with yellow fever (YF) virus and West Nile (WN) virus were performed. YF and WN infections were clinically unapparent. A 3 to 4-day-long viremia, with moderate levels was observed with YF virus. WN virus, especially the strain isolated in Madagascar, provoked a 4 to 6-day-long viremia sufficient to infect Aedes aegypti. In all experiments, the antibody response was studied during the following weeks by 3 methods. The results led to the conclusion that Malagasy lemurs could act as amplifying hosts for WN virus present in Madagascar, and as hosts for YF virus if it were introduced on the island. The epidemiological role of these primates is discussed according to their ecology and their contact with potential mosquito vectors in forest areas of Madagascar.

Comparative immunoelectron microscopy with monoclonal antibodies on yellow fever virus-infected cells: pre-embedding labelling versus immunocryoultramicrotomy.

To study the intra- and extracellular distribution of yellow fever virus 17D (YFV)-specific antigens, pre-embedding immunoelectron microscopy (IEM) and IEM on ultrathin frozen sections were carried out comparatively using monoclonal antibodies (MAB) and YFV-infected cells. In addition, three electron-dense marker systems (IgG-ferritin and IgG-gold and protein A-gold) were compared for their efficiency in detecting bound MAB. Pre-embedding immuno-labelling was performed in microtest plates followed by in situ embedding and immunocryoultramicrotomy was performed using pellets of sucrose-infused cells. In both procedures, cells were prefixed with different concentrations of glutaraldehyde (GA). In pre-embedding IEM virus-specific antigens could be detected on the envelopes of extracellular virions with YFV-neutralizing MAB. Using immunocryoultramicrotomy, neutralizing MAB bound to intracellular mature virions as well as to viral antigens incorporated into cytoplasmic membranes. A concentration of 1% GA destroyed antigenicity entirely, while with 0.25% and 0.1% GA immunoreactivity was retained for more than 3 mth. Some highly reactive MAB labelled antigen significantly in pre-embedding IEM, when used at concentrations of 1 ng/ml. Immunocryoultramicrotomy was 10-100 times less sensitive. On cryosections colloidal gold was the marker of choice, due to the fact that it showed less nonspecific sticking to intracellular components and that it was easily detectable on highly contrasted cryosections. Owing to their higher sensitivity, IgG-ferritin conjugates were preferred in pre-embedding IEM.

Sylvatic yellow fever activity in Trinidad, 1988-1989.

Of a total of 18,068 mosquitoes (361 pools) collected in south-eastern Trinidad forests from December 1988 to May 1989, 47 species belonging to 14 genera were identified. Five yellow fever virus isolates were made from Haemagogus janthinomys and one from Sabethes chloropterus. All the other pools of mosquitoes examined were negative for the virus. The mosquito isolates were made in December and January. In addition, in late February and early March, 2 infected howler monkeys (Alouatta sp.) were detected. Since March, despite continued surveillance, no yellow fever virus has been detected in mosquitoes or monkeys. There has been no reported human infection.

Virions and virus-associated structures within dendrites in an experimental flavovirus encephalomyelitis.

In experimental yellow fever virus encephalomyelitis of adult albino mice, virions, and virus-associated structures were observed not only inside neuronal perikarya but also within dendrites of varied size. The finding permits the following explanations: (1) either the viral agent is synthesized in the nerve cell bodies and transported intradendritically in a proximodistal direction; or (2) virus morphogenesis takes place in neuronal perikarya and dendrites as well; or (3) both possiblities are equally valid. Some incidental findings were suggestive of virus release at postsynaptic dendrite membranes. They are discussed with reference to a hypothetical long-distance pathway of viral dissemination involving endocytosis of the agent by presynaptic axon terminals, intraaxonal virus decoating, and retrograde axoplasmic transport of the infectious nucleic acid to the cell soma.

Human fatal yellow fever. Immunohistochemical detection of viral antigens in the liver, kidney and heart.

An immunohistochemical method to detect yellow fever antigen was developed using immune sera from rabbits and hamsters and hyperimmune ascitic fluid from mice. A search for the antigen was carried out in liver, kidney and heart in three fatal cases of yellow fever. In the liver it was present in the cytoplasm of hepatocytes, Councilman bodies and Kupffer cells. Yellow fever antigen was also detected in renal tubular epithelium and in groups of myocardial fibers. These findings suggest that viral replication occurs at sites other than the liver. Since yellow fever shares many features with other haemorrhagic fevers the use of immunohistochemistry can impart a significant improvement in the accuracy of its histopathological diagnosis.

Biological characterization of plaque-size variants of yellow fever virus in mosquitoes and mice.

We isolated plaque-size variants of a South American strain of yellow fever virus, and compared their ability to infect orally and be transmitted by vector Aedes aegypti mosquitoes with that of the uncloned, parental virus. We analyzed the same clonal isolates in mouse virulence experiments. No significant differences could be demonstrated in the capacities of the variants to infect and be transmitted by mosquitoes or in mouse virulence tests. The 17D vaccine virus (derived from the African Asibi strain) was, however, markedly attenuated in mosquitoes; also, a variant virus (Asibi strain) derived by continuous passage in HeLa cells (and attenuated for monkeys and mice) was markedly attenuated in mosquitoes when compared with its parent virus. The results suggest that vector competence and mouse virulence are similar in plaque-size variants of the South American strain of yellow fever virus. However, if vigorous and appropriate selection pressures are applied (as established by the derivation of the 17D vaccine and HeLa passaged viruses), attenuated variants can be discovered; their role and prevalence within a virus population in nature is unknown.

[Yellow fever virus, dengue 2 and other arboviruses isolated from mosquitos, in Burkina Faso, from 1983 to 1986. Entomological and epidemiological considerations]. / Virus amaril, dengue 2 et autres arbovirus isolés de moustiques, au Burkina Faso, de 1983 a 1986. Considérations entomologiques et épidémiologiques.

An arbovirus surveillance was carried out in Burkina Faso from 1983 to 1986. It was based on crepuscular catches of mosquitoes on human bait in some wooded areas and in one town. The total collection was 228 catches with an average of 8 men per catch. The total number of mosquitoes caught was 44,956 among which 32,010 potential vector of yellow fever; all these mosquitoes were analysed for arbovirology. In the south-western part of the country (region of Bobo-Dioulasso), surveillance was conducted each year from August to November, whilst the circulation of Aedes-borne arboviruses is well known to be favoured. In 1983, 1984 and 1986, seven strains of yellow fever virus were isolated in circumstances remarkably similar. They came from selvatic areas and never from the town. They concerned only Aedes (Stegomyia) luteocephalus which is the very predominant potential vector of yellow fever in the region. They were obtained in low figure, between 1 and 4 per year. They occurred from 27th of October to 21th of November. These observations confirm that the southern portion of the Sudan savanna zone of West Africa is the setting of a customary circulation of yellow fever virus and therefore belongs to the endemic emergence zone. In 1986, two strains of dengue 2 virus were isolated. One concerned Ae. luteocephalus from the selvatic area, the other Ae. (St.) aegypti from the heart of town. These data suggest two distinct cycles for dengue 2 virus, one urban and one selvatic, which could coexist simultaneously in the same region. In the south-eastern part of the country (region of Fada-N'Gourma) a yellow fever epidemic occurred between September and December 1983; its study has enable to precise their entomological aspects. The entomological inoculation rate of yellow fever virus has been evaluated to 22 infected bites per man during the month of october, for a man living close to forest gallery. 25 strains of yellow fever virus strains was isolated from Ae. (Diceromyia) furcifer which is the potential vector the most abundant in this region: the main role of this species in an epidemic was confirmed. An investigation in September 1984 had not permitted isolation of the virus therefore it is suspected that the large epizootic circulation of virus in 1983 has not been renewed the year after. In total 59 viral strains belonging to 10 different viruses were isolated from 9 species of mosquitoes.(ABSTRACT TRUNCATED AT 400 WORDS)

[Estimation of the survival rate, the relative density and the infection rate of a population of Haemagogus janthinomys Dyar (Diptera, Culicidae) from which strains of yellow fever were isolated in Brazilian Amazon]. / Estimation du taux de survie, de la densité relative et du taux d'infection d'une population d'Haemagogus janthinomys Dyar (Diptera, Culicidae) ayant fourni des souches de fièvre jaune en Amazonie Brésilienne.

The conditions of maintenance of YF virus in brazilian Amazonia are not yet elucidated. Generally, the presence of the virus is attested by human cases of sylvatic origin. During a survey done at the exact place where a man have probably been contaminated, it was possible for the first time in South America, to estimate the mean parity rate of a population of the potential vector Haemagogus janthinomys, from which the YF virus was actually isolated. The survival rate (Ts = 0.96), the biting rate (0.60 mosquitoes/man x hour), and the infection rate (1.71%) were also determinated for the same mosquitoes and have values compatible with the probable conditions of the human contamination. However, more data are needed, in particular in relation with other possible human contaminations and/or circulation of the YF virus in the monkey population (extension and duration of the epizootic episode), in order to know what maintenance cycle is prevalent in this region: a low level transmission, with the mosquito being a "vector-reservoir", or a "constantly moving epizootic wave".

[Epidemic of yellow fever in the southeastern region of Upper Volta (October-December, 1983). Epidemiological study. Preliminary results]. / L'épidémie de fièvre jaune du sud-est de la Haute-Volta (octobre-décembre 1983). Etude épidémiologique. Résultats préliminaires.

An epidemic of yellow fever raged during the last three months of 1983 in South East of Upper Volta. It spread on about ten thousand square kilometers, in a bushy savanna area, affecting only populations living in contact with forest galleries, belonging especially to the peul ethnical group. The transmission of the virus was effected by sylvatic vectors, essentially Aedes furcifer. Serological tests showed that about 50 % of the population living in contact with forest galleries was affected, that is to say 15.000 to 17.500 people. The average death rate on the whole area was 4 % (800 to 1.700 deaths); the lethality rate was estimated between 6 and 10 % of affected people. On the whole, 54 strains of yellow fever virus were isolated from human blood samples, and 26 strains from batches of mosquitoes. We called this epidemic "intermediate sylvatic epidemic". The epidemic quickly decreased in the sylvatic area, owing to climatic conditions. A mass campaign of vaccinations prevented it from spreading to near urban centres. On this particular case, the thermostability on field of the vaccine 17D provided by the Institute Pasteur of Dakar was proved to be effective.