Position statement for adult and paediatric spirometry in South Africa: 2022 update.

Spirometry is required as part of the comprehensive evaluation of both adult and paediatric individuals with suspected or confirmed respiratory diseases and occupational assessments. It is used in the categorisation of impairment, grading of severity, assessment of potential progression and response to interventions. Guidelines for spirometry in South Africa are required to improve the quality, standardisation and usefulness in local respiratory practice. The broad principles of spirometry have remained largely unchanged from previous versions of the South African Spirometry Guidelines; however, minor adjustments have been incorporated from more comprehensive international guidelines, including adoption of the Global Lung Function Initiative 2012 (GLI 2012) spirometry reference equations for the South African population. All equipment should have proof of validation regarding resolution and consistency of the system. Daily calibration must be performed, and equipment quality control processes adhered to. It is important to have standard operating procedures to ensure consistency and quality and, additionally, strict infection control as highlighted during the COVID-19 pandemic. Adequate spirometry relies on a competent, trained operator, accurate equipment, standardised operating procedures, quality control and patient co-operation. All manoeuvres must be performed strictly according to guidelines, and strict quality assurance methods should be in place, including acceptability criteria (for any given effort) and repeatability (between efforts). Results must be categorised and graded according to current guidelines, taking into consideration the indication for the test.

Development and analytical validation of a group-specific RT-qPCR assay for the detection of the Simbu serogroup orthobunyaviruses.

The Simbu serogroup within the genus Orthobunyavirus belongs to the family Peribunyaviridae and comprises 32 recognised three-segmented negative-sense single-stranded RNA viruses, with a cosmopolitan distribution. This group of arthropod-borne viruses includes important pathogens of humans and domestic animals e.g. Oropouche orthobunyavirus and Schmallenberg virus. Sensitive and specific diagnostic tools are required for recognition and control of outbreaks. A novel TaqMan® RT-qPCR assay was developed, optimised and analytically validated for the broad detection of the Simbu serogroup orthobunyaviruses. A region in the S segment, which encodes the nucleocapsid protein, was used to design a group primer set and a pair of differently labelled TaqMan® minor groove binder probes to distinguish phylogenetic clade A and B of the serogroup. Efficiencies determined for seven members of the group were 99% for Akabane orthobunyavirus (AKAV), 96% for Simbu orthobunyavirus (SIMV), 96% for Shuni orthobunyavirus (SHUV), 97% for Sathuperi orthobunyavirus (SATV), 84% for Shamonda orthobunyavirus (SHAV), 93% for Ingwavuma virus (INGV, now classified as Manzanilla orthobunyavirus) and 110% for Sabo virus (SABOV, now classified as AKAV). The 95% limit of detection (TCID50/reaction) was 10-3.61 for AKAV, 10-2.38 for SIMV, 10-3.42 for SHUV, 10-3.32 for SATV, 10-1.67 for SHAV, 100.39 for INGV and 10-2.70 for SABOV.

Phylogenetic analysis of Bunyamwera and Ngari viruses (family Bunyaviridae, genus Orthobunyavirus) isolated in Kenya.

Orthobunyaviruses, tri-segmented, negative-sense RNA viruses, have long been associated with mild to severe human disease in Africa, but not haemorrhagic fever. However, during a Rift Valley fever outbreak in East Africa in 1997-1998, Ngari virus was isolated from two patients and antibody detected in several others with haemorrhagic fever. The isolates were used to identify Ngari virus as a natural Orthobunyavirus reassortant. Despite their potential to reassort and cause severe human disease, characterization of orthobunyaviruses is hampered by paucity of genetic sequences. Our objective was to obtain complete gene sequences of two Bunyamwera virus and three Ngari virus isolates from recent surveys in Kenya and to determine their phylogenetic positioning within the Bunyamwera serogroup. Newly sequenced Kenyan Bunyamwera virus isolates clustered closest to a Bunyamwera virus isolate from the same locality and a Central African Republic isolate indicating that similar strains may be circulating regionally. Recent Kenyan Ngari isolates were closest to the Ngari isolates associated with the 1997-1998 haemorrhagic fever outbreak. We observed a temporal/geographical relationship among Ngari isolates in all three gene segments suggesting a geographical/temporal association with genetic diversity. These sequences in addition to earlier sequences can be used for future analyses of this neglected but potentially deadly group of viruses.

Rift valley Fever in Kruger national park: do buffalo play a role in the inter-epidemic circulation of virus?

Rift Valley fever (RVF) is a zoonotic mosquito-borne virus disease of livestock and wild ruminants that has been identified as a risk for international spread. Typically, the disease occurs in geographically limited outbreaks associated with high rainfall events and can cause massive losses of livestock. It is unclear how RVF virus persists during inter-epidemic periods but cryptic cycling of the virus in wildlife populations may play a role. We investigated the role that free-living African buffalo (Syncerus caffer caffer) might play in inter-epidemic circulation of the virus and looked for geographic, age and sex patterns of Rift Valley fever virus (RVFV) infection in African buffalo. Buffalo serum samples were collected (n = 1615) in Kruger National Park (KNP), South Africa, during a period of 1996-2007 and tested for antibodies to RVF. We found that older animals were more likely to be seropositive for anti-RVFV antibody than younger animals, but sex was not correlated with the likelihood of being anti-RVFV antibody positive. We also found geographic variation within KNP; herds in the south were more likely to have acquired anti-RVFV antibody than herds farther north - which could be driven by host or vector ecology. In all years of the study between 1996 and 2007, we found young buffalo (under 2 years of age) that were seropositive for anti-RVFV antibody, with prevalence ranging between 0 and 27% each year, indicating probable circulation. In addition, we also conducted a 4-year longitudinal study on 227 initially RVFV seronegative buffalo to look for evidence of seroconversion outside known RVF outbreaks within our study period (2008-2012). In the longitudinal study, we found five individuals that seroconverted from anti-RVFV antibody negative to anti-RVFV antibody positive, outside of any detected outbreak. Overall, our results provide evidence of long-term undetected circulation of RVFV in the buffalo population.

Next-generation sequencing of southern African Crimean-Congo haemorrhagic fever virus isolates reveals a high frequency of M segment reassortment.

Crimean Congo haemorrhagic fever virus (CCHFV) is a bunyavirus with a single-stranded RNA genome consisting of three segments (S, M, L), coding for the nucleocapsid protein, envelope glycoproteins and RNA polymerase, respectively. To date only five complete genome sequences are available from southern African isolates. Complete genome sequences were generated for 10 southern African CCHFV isolates using next-generation sequencing techniques. The maximum-likelihood method was used to generate tree topologies for 15 southern African plus 26 geographically distinct complete sequences from GenBank. M segment reassortment was identified in 10/15 southern African isolates by incongruencies in grouping compared to the S and L segments. These reassortant M segments cluster with isolates from Asia/Middle East, while the S and L segments cluster with strains from South/West Africa. The CCHFV M segment shows a high level of genetic diversity, while the S and L segments appear to co-evolve. The reason for the high frequency of M segment reassortment is not known. It has previously been suggested that M segment reassortment results in a virus with high fitness but a clear role in increased pathogenicity has yet to be shown.

Encephalomyocarditis virus mortality in semi-wild bonobos (Pan panicus).

BACKGROUND: Fatal myocarditis from encephalomyocarditis virus (EMCV) infection has previously been identified in sporadic and epidemic forms in many species of captive non-human primates probably including one bonobo (Pan paniscus). METHODS: We investigated the deaths of two bonobos that were suspicious of EMCV using a combination of histopathology, immunohistochemistry and, for one of the two bonobos, reverse transcription PCR. RESULTS: Histopathological examination of heart tissue from the two bonobos showed changes characteristic of EMCV. Immunohistochemical studies confirmed the presence of EMCV antigen in heart tissue of both and in kidney and intestine of one of the bonobos. EMCV RNA was also isolated from the serum of the bonobo tested. CONCLUSION: Together, these findings confirm that EMCV was responsible for deaths of the two bonobos. Strict separation of bonobos in particular and captive primates in general from potential sources of EMCV contamination should be maintained to prevent mortality caused by EMCV.

An alphavirus replicon-derived candidate vaccine against Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a mosquito-transmitted bunyavirus (genus Phlebovirus) associated with severe disease in livestock and fatal encephalitis or haemorrhagic fever in a proportion of infected humans. Although live attenuated and inactivated vaccines have been used in livestock, and on a limited scale in humans, there is a need for improved anti-RVFV vaccines. Towards this goal, Sindbis virus replicon vectors expressing the RVFV Gn and Gc glycoproteins, as well as the non-structural nsM protein, were constructed and evaluated for their ability to induce protective immune responses against RVFV. These replicon vectors were shown to produce the RVFV glycoproteins to high levels in vitro and to induce systemic anti-RVFV antibody responses in immunized mice, as determined by RVFV-specific ELISA, fluorescent antibody tests, and demonstration of a neutralizing antibody response. Replicon vaccination also provided 100% protection against lethal RVFV challenge by either the intraperitoneal or intranasal route. Furthermore, preliminary results indicate that the replicon vectors elicit RVFV-specific neutralizing antibody responses in vaccinated sheep. These results suggest that alphavirus-based replicon vectors can induce protective immunity against RVFV, and that this approach merits further investigation into its potential utility as a RVFV vaccine.

Genetic relationship in southern African Crimean-Congo haemorrhagic fever virus isolates: evidence for occurrence of reassortment.

Crimean-Congo haemorrhagic fever (CCHF) is a tick-borne viral zoonosis widely distributed in Africa, Asia and eastern Europe. Reassortment of CCHF genome segments has been shown to occur in nature. We therefore investigated the genetic relationship of southern African isolates using partial sequence data for each RNA segment, S, M and L, and comparing the tree topologies constructed using a neighbour joining method. A total of 21 southern African isolates were studied. The incongruencies which were identified in S, M and L sequence datasets involved group switching implying reassortment for 15 isolates. A higher fatality rate occurred in patients infected with isolates which had apparently acquired M segments from a group in which predominantly Asian strains are usually found. This suggests that reassortment may affect the pathogenicity of the virus.

Development and evaluation of a real-time reverse transcription-loop-mediated isothermal amplification assay for rapid detection of Rift Valley fever virus in clinical specimens.

This paper reports on the development and validation of a real-time reverse transcription-loop-mediated isothermal amplification assay (RT-LAMP) targeting the genomic large RNA segment of Rift Valley fever virus (RVFV). The set of six designed RT-LAMP primers identified strains of RVFV isolated in geographically distinct areas over a period of 50 years; there was no cross-reactivity with other genetically related and unrelated arboviruses. When testing serial sera and plasma from sheep experimentally infected with wild-type RVFV, there was 100% agreement between results of the RT-LAMP, a TaqMan-based real-time PCR, and virus isolation. Similarly, the assay had very high levels of diagnostic sensitivity and specificity when testing various clinical specimens from humans and animals naturally infected with the virus during recent outbreaks of the disease in Africa. The detection of specific viral genome targets in positive clinical specimens was achieved in less than 30 min. As a highly accurate, rapid, and very simple nucleic acid detection format, the RT-LAMP has the potential to be used in less-well-equipped laboratories in Africa and as a portable device during RVF outbreaks in remote areas, and it can be a valuable tool for the differential diagnosis of viral hemorrhagic fevers.

Epidemiology and pathogenicity of African bat lyssaviruses.

Lyssaviruses belonging to all four known African Lyssavirus genotypes (gts) have been reported and isolated from SouthAfrica over the past few decades. These are: (1) Duvenhage virus (gt4), isolated again in 2006 from a human fatality; (2) Mokola virus (gt3), isolated irregularly, mostly from cats; (3) Lagos bat virus (gt2) continually isolated over the past four years from Epomophorus fruit bats and from incidental terrestrial animals and (4) Rabies virus (gt1) - with two virus biotypes endemic in mongoose and in canid species (mostly domestic dogs, jackals and bat-eared foxes), respectively. Only two of these are associated with bats in Southern Africa, viz. Duvenhage virus and Lagos bat virus (gts 4 and 2). For both these genotypes the authors have embarked on a programme of comparative study of molecular epidemiology. Duvenhage virus nucleoprotein nucleotide sequence analysis indicated a very low nucleotide diversity even though isolates were isolated decades apart. In contrast, individual isolates of Lagos bat virus were found to differ significantly with respectto nucleoprotein gene nucleotide sequence diversity as well as in pathogenicity profiles.

Molecular epidemiology of African and Asian Crimean-Congo haemorrhagic fever isolates.

Phylogenetic relationships were examined for 70 Crimean-Congo haemorrhagic fever (CCHF) isolates from southern, central and West Africa, the Middle East and Greece using sequence data determined for a region of the S segment of the genome. Analysis revealed up to 18% genetic differences. Tree topology supports previous evidence for the existence of three groups of genetically related isolates, A, B and C. Within group A there are two clades: an African clade and a predominantly Asian clade comprising isolates from Pakistan, China, Iran, Russia and Madagascar. Group B includes isolates from southern and West Africa and Iran, and group C includes a single isolate from Greece. Despite the potential which exists for dispersal of the virus between Africa and Eurasia, it appears that circulation of the virus is largely compartmentalized within the two land masses, and the inference is that the geographic distribution of phylogenetic groups is related to the distribution and dispersal of tick vectors of the virus.

West Nile virus infection of Thoroughbred horses in South Africa (2000-2001).

REASONS FOR PERFORMING STUDY: West Nile virus (WNV) infection is endemic in southern Africa. With the recent emergence of WNV infection of horses in Europe and the USA the present study was performed to estimate the risk of seroconversion to WNV in a cohort of 488 young Thoroughbred (TB) horses. OBJECTIVES: To estimate the risk of seroconversion to WNV among a cohort of South African TB yearlings sold at the 2001 National Yearling Sales (NYS) and to determine whether the risk varied geographically. Two horses were also infected with a recent South African isolate of WNV to evaluate its virulence in horses. METHODS: Serum samples were collected from the cohort of 488 TB yearlings at the 2001 NYS. Serum samples that were collected from the same horses at the time that they were identified were sourced from our serum bank. Sera from 243 of the dams that were collected at the time that the foals were identified were also sourced from our serum bank. These sera were subjected to serum neutralisation (SN) tests for antibody to WNV. RESULTS: Approximately 11% of yearlings seroconverted to WNV on paired serum samples collected from each animal approximately 12 months apart. Studfarms with WNV-seropositive yearlings were widely distributed throughout South Africa and SN tests on sera from their dams indicated that exposure to WNV was even more prevalent (75%) in this population. Neurological disease was not described in any of the horses included in this study and 2 horses inoculated with a recent lineage 2 South African isolate of WNV showed no clinical signs of disease after infection and virus was not detected in their blood. CONCLUSIONS: Infection of horses with WNV is common in South Africa, but infection is not associated with neurological disease. POTENTIAL RELEVANCE: In contrast to recent reports from Europe, North Africa, Asia and North America, the results of our field and experimental studies indicated that exposure of horses to the endemic southern African strains of WNV was not associated with neurological disease.

Indirect enzyme-linked immunosorbent assay for the detection of antibody against Rift Valley fever virus in domestic and wild ruminant sera.

An indirect enzyme-linked immunosorbent assay (I-ELISA) for the detection of specific IgG immunoglobulins against Rift Valley fever virus (RVFV) was validated in-house. A total of 3055 sera from sheep (n = 1159), goats (n = 636), cattle (n = 203), African buffalo (n = 928), and other wild ruminants (n = 129), including eland, kudu, and black wildebeest, was used. Sera from domestic ruminants were collected in West (n = 10), South (n = 1654) and East Africa (n = 334), and sera from wild ruminants (n = 1064) were collected in South Africa. In addition, 136 sera from eight experimentally RVFV-infected sheep, taken during a period of 28 days post infection (dpi), were used to study the kinetics of RVFV antibody production. Field sera were tested by the serum neutralization (VN) test and experimental sera by VN and haemagglutination-inhibition (HI) test. Based on VN test results, negative sera were regarded as reference controls from RVFV-free, and positive sera were regarded as reference controls from RVFV-infected subpopulations of animals. ELISA data were expressed as the percentage positivity (PP) of an internal high positive control. The two-graph receiver operating characteristics approach was used for the selection and optimization of I-ELISA cut-offs including the misclassification costs term and Youden index (J). In addition, cut-off values were determined as the mean plus two-fold standard deviation of the result observed with the RVFV-free subpopulations. Established optimal cut-offs were different for each of the data sets analyzed, and ranged from 1.65 PP (buffalo) to 9.1 PP (goats). At the cut-off giving the highest estimate of combined measure of diagnostic accuracy (highest J value), the I-ELISA test parameters were determined as follows: (1) Diagnostic sensitivity (%): cattle--84.31, buffalo--94.44, sheep--98.91, goats--99.18. (2) Diagnostic specificity (%): cattle--99.34, buffalo--98.28, sheep--99.16, goats--99.23 and other game ruminants--99.26. In the group of RVFV-experimentally infected sheep, seroconversion In all individuals was detected by VN on 4-6 dpi, by HI on 5-7 dpi, and by I-ELISA on 6-7 dpi. All tests showed the same kinetic pattern of immunological response. Antibody levels were low for a very short period before increasing to high titres, after which it was easily detectable by all tests. Compared to traditional tests, the lower sensitivity of I-ELISA in the detection of the earliest stage of immunological response may be practically insignificant, particularily when this assay is used in population-based, disease-surveillance programmes. The high sensitivity and specificity of I-ELISA established in this study, especially for the statistically more representative subpopulations of animals tested, seem to support this prediction. Test parameters determined in this study should, however, be regarded as in-house diagnostic decision limits, for which further updating is recommended, particularly for specimens from other countries, and preferably by applying a standardized method for sampling of new subpopulations of animals to be targeted by the assay.

The 2000 epidemic of Rift Valley fever in Saudi Arabia: mosquito vector studies.

In mid-September 2000, Rift Valley fever (RVF) virus was diagnosed as the cause of infection in humans and livestock in Jizan Region, Saudi Arabia. This is the first time that this arbovirus has been found outside Africa and Madagascar. Collections of mosquitoes (Diptera: Culicidae) were therefore undertaken (from 25 September to 10 October) at eight sites during the epidemic to obtain mosquitoes for attempted RVF virus isolation. Among 23 699 mosquito females tested, six isolations of RVF virus were made from 15 428 Culex (Culex) tritaeniorhynchus Giles and seven from 8091 Aedes (Aedimorphus) vexans arabiensis Patton [corrected]. Minimum mosquito infection rates per 1000 at sites with infected mosquitoes were 0.3-13.8 Cx. tritaeniorhynchus and 1.94-9.03 Ae. v. arabiensis. Viral activity moved northwards as collecting was in progress and collectors 'caught up' with the virus at the two most northerly sites on the last two trapping evenings. Other species occurred in small numbers and were identified but not tested. Both Cx. tritaeniorhynchus and Ae. v. arabiensis were susceptible to RVF virus and transmitted between hamsters, and an additional quantitative test with Cx. tritaeniorhynchus showed that 71-73% of mosquitoes became infected after ingesting 6.9-7.9 log10 FFU/mL of virus; transmission rates were 10% (post-infection day 14) and 26% (post-infection day 20). It was concluded that both species were vectors on grounds of abundance, distribution, preference for humans and sheep, the virus isolations and vector competence tests.

Unexpected Ebola virus in a tertiary setting: clinical and epidemiologic aspects.

OBJECTIVES: To describe the clinical manifestations of viral hemorrhagic fever, and to increase clinicians' awareness and knowledge of these illnesses. DESIGN: Retrospective study of the clinical and laboratory data and management of two cases of Ebola virus infection with key epidemiologic data provided. SETTING: Two tertiary care hospitals. PATIENTS: Two adult patients, the index case and the source patient, both identified as having Ebola, one of whom originated in Gabon. INTERVENTIONS: One patient was admitted to the intensive care unit. The other was managed in a general ward. MEASUREMENT AND MAIN RESULTS: Clinical and laboratory data are reported. One patient, a healthcare worker who contracted this illness in the course of her work, died of refractory thrombocytopenia and an intracerebral bleed. The source patient survived. Despite a long period during which the diagnosis was obscure, none of the other 300 contacts contracted the illness. CONCLUSIONS: Identification of high-risk patients and use of universal blood and body fluid precautions will considerably decrease the risk of nosocomial spread of viral hemorrhagic fevers.

Experimental detection of Rift Valley fever virus by reverse transcription-polymerase chain reaction assay in large samples of mosquitoes.

A reverse transcription-polymerase chain reaction (RT-PCR) was assessed in laboratory tests to detect the presence of single Aedes aegypti (L.) or Eretmapodites quinquevittatus Theobald mosquitoes infected with Rift Valley fever virus in pools of mosquitoes, 50-600 in size, from laboratory colonies or mixed field collections. The viral RNA was detected in all pools containing infected mosquitoes and was shown to be as sensitive as infant mice but more sensitive than Vero cell cultures for virus detection. Pools diluted down to the equivalent of 1:16 000 mosquitoes were also positive by RT-PCR. RNAs from 4 other phleboviruses were negative, there were no false positives and the procedure followed, with the 2 particular primers chosen, gave consistently clear bands of the PCR products on agarose gels without nested PCR being necessary.

A novel immunohistochemical assay for the detection of Ebola virus in skin: implications for diagnosis, spread, and surveillance of Ebola hemorrhagic fever. Commission de Lutte contre les Epidémies à Kikwit.

Laboratory diagnosis of Ebola hemorrhagic fever (EHF) is currently performed by virus isolation and serology and can be done only in a few high-containment laboratories worldwide. In 1995, during the EHF outbreak in the Democratic Republic of Congo, the possibility of using immunohistochemistry (IHC) testing of formalin-fixed postmortem skin specimens was investigated as an alternative diagnostic method for EHF. Fourteen of 19 cases of suspected EHF met the surveillance definition for EHF and were positive by IHC. IHC, serologic, and virus isolation results were concordant for all EHF and non-EHF cases. IHC and electron microscopic examination showed that endothelial cells, mononuclear phagocytes, and hepatocytes are main targets of infection, and IHC showed an association of cellular damage with viral infection. The finding of abundant viral antigens and particles in the skin of EHF patients suggests an epidemiologic role for contact transmission. IHC testing of formalin-fixed skin specimens is a safe, sensitive, and specific method for laboratory diagnosis of EHF and should be useful for EHF surveillance and prevention.

The reemergence of Ebola hemorrhagic fever, Democratic Republic of the Congo, 1995. Commission de Lutte contre les Epidémies à Kikwit.

In May 1995, an international team characterized and contained an outbreak of Ebola hemorrhagic fever (EHF) in Kikwit, Democratic Republic of the Congo. Active surveillance was instituted using several methods, including house-to-house search, review of hospital and dispensary logs, interview of health care personnel, retrospective contact tracing, and direct follow-up of suspect cases. In the field, a clinical case was defined as fever and hemorrhagic signs, fever plus contact with a case-patient, or fever plus at least 3 of 10 symptoms. A total of 315 cases of EHF, with an 81% case fatality, were identified, excluding 10 clinical cases with negative laboratory results. The earliest documented case-patient had onset on 6 January, and the last case-patient died on 16 July. Eighty cases (25%) occurred among health care workers. Two individuals may have been the source of infection for >50 cases. The outbreak was terminated by the initiation of barrier-nursing techniques, health education efforts, and rapid identification of cases.

Serologic survey among hospital and health center workers during the Ebola hemorrhagic fever outbreak in Kikwit, Democratic Republic of the Congo, 1995.

From May to July 1995, a serologic and interview survey was conducted to describe Ebola hemorrhagic fever (EHF) among personnel working in 5 hospitals and 26 health care centers in and around Kikwit, Democratic Republic of the Congo. Job-specific attack rates estimated for Kikwit General Hospital, the epicenter of the EHF epidemic, were 31% for physicians, 11% for technicians/room attendants, 10% for nurses, and 4% for other workers. Among 402 workers who did not meet the EHF case definition, 12 had borderline positive antibody test results; subsequent specimens from 4 of these tested negative. Although an old infection with persistent Ebola antibody production or a recent atypical or asymptomatic infection cannot be ruled out, if they occur at all, they appear to be rare. This survey demonstrated that opportunities for transmission of Ebola virus to personnel in health facilities existed in Kikwit because blood and body fluid precautions were not being universally followed.

Clinical virology of Ebola hemorrhagic fever (EHF): virus, virus antigen, and IgG and IgM antibody findings among EHF patients in Kikwit, Democratic Republic of the Congo, 1995.

Ebola hemorrhagic fever (EHF) patients treated at Kikwit General Hospital during the 1995 outbreak were tested for viral antigen, IgG and IgM antibody, and infectious virus. Viral antigen could be detected in virtually all patients during the acute phase of illness, while antibody was not always detectable before death. Virus was also isolated from patients during the course of their febrile illness, but attempts to quantify virus in Vero E6 cells by standard plaque assay were often unsuccessful. IgG and IgM antibody appeared at approximately the same time after disease onset (8-10 days), but IgM persisted for a much shorter period among the surviving convalescent patients. IgG antibody was detectable in surviving patients through about 2 years after onset, the latest time that samples were obtained. Detection of Ebola virus antigens or virus isolation appears to be the most reliable means of diagnosis for patients with suspected acute EHF, since patients with this often-fatal disease (80% mortality) may not develop detectable antibodies before death.