Clinical epidemiology and high genetic diversity amongst Cryptococcus spp. isolates infecting people living with HIV in Kinshasa, Democratic Republic of Congo.

Neuromeningeal cryptococcosis (NMC) is a life-threatening opportunistic infection in advanced HIV disease patients (AHDP). It is caused by Cryptococcus spp. complexes and mainly occurs in sub-Saharan Africa. In this study, we performed molecular characterization and antifungal susceptibility profiling of Cryptococcus isolates from AHDP in Kinshasa (DRC). Additionally, we investigated a possible association between NMC severity factors and the Cryptococcus neoformans (Cn) multilocus sequence typing (MLST) profiles. We characterized the isolates using PCR serotyping, MALDI-TOF MS, internal transcribed spacer (ITS) sequencing, and MLST. Susceptibility testing for the major antifungal drugs was performed according to the EUCAST guidelines. Parameters associated with NMC severity, such as hypoglycorrhachia (< 50 mg/dL), increased cerebral spinal fluid opening pressure (> 30 cm H2O), and poor therapeutic outcome were compared with the Cn MLST sequences type (ST). Twenty-three out of 29 Cryptococcus isolates were identified as serotype A using PCR serotyping (79.3%; 95% IC: 65.5-93.1), while six (20.7%; 95% IC: 6.9-34.5) were not serotypable. The 29 isolates were identified by ITS sequencing as follows: Cryptococcus neoformans (23/29, 79.3%), Cutaneotrichosporon curvatus (previously called Cryptococcus curvatus) (5/29, 17.2%), and Papiliotrema laurentii (Cryptococcus laurentii) (1/29, 3.5%). Using the ISHAM MLST scheme, all Cn isolates were identified as molecular type VNI. These comprised seven different STs: ST93 (n = 15), ST5 (n = 2), ST53 (n = 1), ST31 (n = 1), ST4 (n = 1), ST69 (n = 1), and one novel ST that has not yet been reported from other parts of the world and was subsequently assigned as ST659 (n = 2). Of the included strains, only Papiliotrema laurentii was resistant to amphoterin B (1/29, 3.5%), 6.8% (2/29) were resistant to 5-flucytosine (the single Papiliotrema laurentii strain and one Cryptococcus neoformans isolate), and 13.8% (4/29) to fluconazole, including two of five (40%) Cutaneotrichosporon curvatus and two of 23 (8.7%) C. neoformans strains. We found a significative association between poor therapeutic outcome and a non-ST93 sequence type of causative strains (these concerned the less common sequence types: ST53, ST31, ST5, ST4, ST659, and ST69) (87.5% versus 40%, p = 0.02). Molecular analysis of Cryptococcus spp. isolates showed a wide species diversity and genetic heterogenicity of Cn within the VNI molecular type. Furthermore, it is worrying that among included strains we found resistances to several of the commonly used antifungals.

Biennial surveillance of Plasmodium falciparum anti-malarial drug resistance markers in Democratic Republic of Congo, 2017 and 2019.

BACKGROUND: Because of the loss of chloroquine (CQ) effectiveness, the Democratic Republic of Congo (DRC)'s malaria treatment policy replaced CQ by sulfadoxine-pyrimethamine (SP) as first-line treatment of uncomplicated malaria in 2003, which in turn was replaced by artemisinin-based combination therapies (ACT) in 2005. The World Health Organization (WHO) recommends monitoring of anti-malarial drug resistance every 2 years. The study aimed to provide baseline data for biennial molecular surveillance of anti-malarial drug resistance by comparing data from a study conducted in 2019 to previously published data from a similar study conducted in 2017 in the DRC. METHODS: From July to November 2019, a cross-sectional study was conducted in ten sites which were previously selected for a similar study conducted in 2017 across the DRC. P. falciparum malaria was diagnosed by a rapid diagnostic test (RDT) or by microscopy and dried blood samples (DBS) were taken from patients who had a positive test. Segments of interest in pfcrt and pfk13 genes were amplified by conventional PCR before sequencing. RESULTS: Out of 1087 enrolled patients, 906 (83.3%) were PCR-confirmed for P. falciparum. Like in the 2017-study, none of the mutations known to be associated with Artemisinine (ART) resistance in Southeast Asia was detected. However, non-synonymous (NS) mutations with unknown functions were observed among which, A578S was detected in both 2017 and 2019-studies. The overall prevalence of pfcrt-K76T mutation that confers CQ-resistance was 22.7% in 2019-study compared to 28.5% in 2017-study (p-value = 0.069), but there was high variability between sites in the two studies. Like in 2017-study, the pfcrt 72-76 SVMNT haplotype associated with resistance to amodiaquine was not detected. CONCLUSION: The study reported, within 2 years, the non-presence of molecular markers currently known to be associated with resistance to ART and to AQ in P. falciparum isolated in the DRC. However, the presence of polymorphisms with as-yet unknown functions was observed, requiring further characterization. Moreover, an overall decrease in the prevalence of CQ-resistance marker was observed in the DRC, but this prevalence remained highly variable from region to region.

Assessment of Plasmodium falciparum anti-malarial drug resistance markers in pfk13-propeller, pfcrt and pfmdr1 genes in isolates from treatment failure patients in Democratic Republic of Congo, 2018-2019.

BACKGROUND: The national policy for malaria treatment of the Democratic Republic of Congo recommends two first-line artemisinin-based combinations for the treatment of uncomplicated malaria: artesunate-amodiaquine and artemether-lumefantrine. This study investigated the presence of markers associated with resistance to the current first-line artemisinin-based combination therapy (ACT) in isolates of Plasmodium falciparum from treatment failure patients in the Democratic Republic of Congo. METHODS: From November 2018 to November 2019, dried blood spots were taken from patients returning to health centres for fever within 28 days after an initial malaria treatment in six sentinel sites of the National Malaria Control Programme across Democratic Republic of Congo. The new episode of malaria was first detected by a rapid diagnostic test and then confirmed by a real-time PCR assay to define treatment failure. Fragments of interest in pfk13 and pfcrt genes were amplified by conventional PCR before sequencing and the Pfmdr1 gene copy number was determined by a TaqMan real-time PCR assay. RESULTS: Out of 474 enrolled patients, 364 (76.8%) were confirmed positive by PCR for a new episode of P. falciparum malaria, thus considered as treatment failure. Of the 325 P. falciparum isolates obtained from 364 P. falciparum-positive patients and successfully sequenced in the pfk13-propeller gene, 7 (2.2%) isolates carried non-synonymous mutations, among which 3 have been previously reported (N498I, N554K and A557S) and 4 had not yet been reported (F506L, E507V, D516E and G538S). Of the 335 isolates successfully sequenced in the pfcrt gene, 139 (41.5%) harboured the K76T mutation known to be associated with chloroquine resistance. The SVMNT haplotype associated with resistance to amodiaquine was not found. None of the isolates carried an increased copy number of the pfmdr1 gene among the 322 P. falciparum isolates successfully analysed. CONCLUSION: No molecular markers currently known to be associated with resistance to the first-line ACT in use were detected in isolates of P. falciparum from treatment failure patients. Regular monitoring through in vivo drug efficacy and molecular studies must continue to ensure the effectiveness of malaria treatment in Democratic Republic of Congo.

The lack of K13-propeller mutations associated with artemisinin resistance in Plasmodium falciparum in Democratic Republic of Congo (DRC).

Artemisinin-based combination therapies (ACTs) have been recommended by the World Health Organization (WHO) as first-line treatment of uncomplicated Plasmodium falciparum (P. falciparum) malaria since 2005 in Democratic Republic of Congo (DRC) and a regular surveillance of the ACT efficacy is required to ensure the treatment effectiveness. Mutations in the propeller domain of the pfk13 gene were identified as molecular markers of artemisinin resistance (ART-R). This study investigated the pfk13-propeller gene polymorphism in clinical isolates of P. falciparum collected in the DRC. In 2017, ten geographical sites across DRC were selected for a cross-sectional study that was conducted first in Kinshasa from January to March, then in the nine other sites from September to December. Dried blood samples were collected from patients attending health centers for fever where diagnosis of Malaria was first made by rapid diagnostic test (RDT) available on site (SD Bioline malaria Ag Pf or CareStart Malaria Pf) or by thick blood smear and then confirmed by a P. falciparum real-time PCR assay. A pfk13-propeller segment containing a fragment that codes for amino acids at positions 427-595 was amplified by conventional PCR before sequencing. In total, 1070 patients were enrolled in the study. Real-time PCR performed confirmed the initial diagnosis of P. falciparum infection in 806 samples (75.3%; 95% CI: 72.6%- 77.9%). Of the 717 successfully sequenced P. falciparum isolates, 710 (99.0%; 95% CI: 97.9% - 99.6) were wild-type genotypes and 7 (1.0%; 95% CI: 0.4% - 2.1%) carried non-synonymous (NS) mutations in pfk13-propeller including 2 mutations (A578S and V534A) previously detected and 2 other (M472I and A569T) not yet detected in the DRC. Mutations associated with ART-R in Southeast Asia were not observed in DRC. However, the presence of other mutations in pfk13-propeller gene calls for further investigations to assess their implication in drug resistance.

A Recurrent Mutation at Position 26340 of SARS-CoV-2 Is Associated with Failure of the E Gene Quantitative Reverse Transcription-PCR Utilized in a Commercial Dual-Target Diagnostic Assay.

Control of the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires accurate laboratory testing to identify infected individuals while also clearing essential staff to continue to work. At the current time, a number of quantitative real-time PCR (qRT-PCR) assays have been developed to identify SARS-CoV-2, targeting multiple positions in the viral genome. While the mutation rate of SARS-CoV-2 is moderate, given the large number of transmission chains, it is prudent to monitor circulating viruses for variants that might compromise these assays. Here, we report the identification of a C-to-U transition at position 26340 of the SARS-CoV-2 genome that is associated with failure of the cobas SARS-CoV-2 E gene qRT-PCR in eight patients. As the cobas SARS-CoV-2 assay targets two positions in the genome, the individuals carrying this variant were still called SARS-CoV-2 positive. Whole-genome sequencing of SARS-CoV-2 showed all to carry closely related viruses. Examination of viral genomes deposited on GISAID showed this mutation has arisen independently at least four times. This work highlights the necessity of monitoring SARS-CoV-2 for the emergence of single-nucleotide polymorphisms that might adversely affect RT-PCRs used in diagnostics. Additionally, it argues that two regions in SARS-CoV-2 should be targeted to avoid false negatives.

Molecular surveillance of anti-malarial drug resistance in Democratic Republic of Congo: high variability of chloroquinoresistance and lack of amodiaquinoresistance.

BACKGROUND: The loss of chloroquine (CQ) effectiveness has led to its withdrawal from national policies as a first-line treatment for uncomplicated malaria in several endemic countries, such as the Democratic Republic of Congo (DRC). The K76T mutation on the pfcrt gene has been identified as a marker of CQ resistance and the SVMNT haplotype in codons 72-76 on the same gene has been associated with resistance to amodiaquine (AQ). In the DRC, the prevalence of K76T has decreased from 100% in 2000 to 63.9% in 2014. The purpose of this study was to determine the prevalence of K76T mutations in circulating strains of Plasmodium falciparum, 16 years after CQ withdrawal in the DRC and to investigate the presence of the SVMNT haplotype. METHODS: In 2017, ten geographical sites across the DRC were selected. Dried blood samples were collected from patients attending health centres. Malaria was first detected by a rapid diagnostic test (RDT) available on site (SD Bioline Malaria Ag Pf or CareStart Malaria Pf) or thick blood smear and then confirmed by a P. falciparum species-specific real-time PCR assay. A pfcrt gene segment containing a fragment that encodes amino acids at positions 72-76 was amplified by conventional PCR before sequencing. RESULTS: A total of 1070 patients were enrolled. Of the 806 PCR-confirmed P. falciparum positive samples, 764 were successfully sequenced. The K76T mutation was detected in 218 samples (28.5%; 95% CI 25.4%-31.9%), mainly (96%) with the CVIET haplotype. Prevalence of CQ resistance marker was unequally distributed across the country, ranging from 1.5% in Fungurume to 89.5% in Katana. The SVMNT haplotype, related to AQ resistance, was not detected. CONCLUSION: Overall, the frequency of the P. falciparum CQ resistance marker has decreased significantly and no resistance marker to AQ was detected in the DRC in 2017. However, the between regions variability of CQ resistance remains high in the country. Further studies are needed for continuous monitoring of the CQ resistance level for its prospective re-use in malaria management. The absence of the AQ resistance marker is in line with the use of this drug in the current DRC malaria treatment policy.

Evaluation of the Abbott RealTime quantitative CMV and EBV assays using the maxCycle protocol in a laboratory automation context.

Real-time PCR are often used for the diagnosis and monitoring of Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections in susceptible populations. In this context, we evaluated the analytical performances of the Abbott RealTime CMV/EBV maxCycle protocol automated on the m2000 platform (Abbott). It was compared to our routinely-used procedure consisting of a NucleoMag® DNA extraction automated on a STARlet platform followed by manually processed CMV and EBV quantitative real-time PCR (Diagenode). In this study, we showed that both EBV assays exhibited a similar sensitivity but with a better precision for the EBV Abbott RealTime assay. For the CMV performances, the Abbott assay was more sensitive and more precise than our routine method. The use of WHO International Standards also indicated a slight underestimation of the viral loads (-0.25 log10 IU/mL and -0.21 log10 IU/mL for CMV and EBV assays respectively) while these were rather overestimated with the Starlet/Diagenode method (0.48 log10 IU/mL and 0.19 log10 IU/mL for CMV and EBV assays respectively). These trends were confirmed using relevant whole-blood clinical samples and external quality controls. The workflows were also compared and we highlighted a significant technician hands-on time reduction (-63%) using the Abbott CMV/EBV maxCycle automated protocol.

Clinical evaluation of the DermaGenius® Nail real-time PCR assay for the detection of dermatophytes and Candida albicans in nails.

Onychomycosis represents one of the most frequent mycoses in the world. Causative agents are mainly dermatophytes, but yeasts and nondermatophyte moulds can also be involved. Conventional diagnostic methods include direct microscopy (or histology) and culturing. However, molecular methods are becoming increasingly popular in this field. The DermaGenius® (DG) Nail multiplex assay (PathoNostics, The Netherlands) is a new commercial real-time polymerase chain reaction (PCR) kit, which can detect Trichophyton rubrum, Trichophyton interdigitale and Candida albicans directly in nails. The present study is a retrospective evaluation of the kit applied to 138 finger and toenail clippings in comparison to histology and culture methods. The sensitivity and specificity of the PCR assay are 80% (76/95) and 74.4% (32/43), respectively, when histology and culture are used as reference to define onychomycosis. DG performance is not different from histology combined with culture (P = .11) but the best diagnostic efficacy (88.4%, 122/138) is obtained by the combination of histology and DG. In conclusion, this study emphasizes the clinical usefulness of the DG in diagnostics. The high specificity of this test guarantees a better identification compared to culture that can lead to dermatophyte misidentifications. It is a reliable PCR assay that shortens the time to diagnosis and can unmask the presence of nongrowing fungal pathogens in nails.

Effectiveness of a Malaria Surveillance Strategy Based on Active Case Detection during High Transmission Season in the Peruvian Amazon.

Background: Faced with the resurgence of malaria, malaria surveillance in the Peruvian Amazon incorporated consecutive active case detection (ACD) interventions using light microscopy (LM) as reactive measure in communities with an unusual high number of cases during high transmission season (HTS). We assessed the effectiveness in malaria detection of this local ACD-based strategy. Methods: A cohort study was conducted in June⁻July 2015 in Mazan, Loreto. Four consecutive ACD interventions at intervals of 10 days were conducted in four riverine communities (Gamitanacocha, Primero de Enero, Libertad and Urco Miraño). In each intervention, all inhabitants were visited at home, and finger-prick blood samples collected for immediate diagnosis by LM and on filter paper for later analysis by quantitative real-time polymerase chain reaction (qPCR). Effectiveness was calculated by dividing the number of malaria infections detected using LM by the number of malaria infections detected by delayed qPCR. Results: Most community inhabitants (88.1%, 822/933) were present in at least one of the four ACD interventions. A total of 451 infections were detected by qPCR in 446 participants (54.3% of total participants); five individuals had two infections. Plasmodium vivax was the predominant species (79.8%), followed by P. falciparum (15.3%) and P. vivax-P. falciparum co-infections (4.9%). Most qPCR-positive infections were asymptomatic (255/448, 56.9%). The ACD-strategy using LM had an effectiveness of 22.8% (detection of 103 of the total qPCR-positive infections). Children aged 5⁻14 years, and farming as main economic activity were associated with P. vivax infections. Conclusions: Although the ACD-strategy using LM increased the opportunity of detecting and treating malaria infections during HTS, the number of detected infections was considerably lower than the real burden of infections (those detected by qPCR).

Prevalence of Campylobacter among goats and retail goat meat in Congo.

BACKGROUND: The prevalence of Campylobacter jejuni and Campylobacter coli was determined in goat and goat meat sold at retail outlets in Lubumbashi, Democratic Republic of Congo (DR Congo). METHODOLOGY: A total of 644 samples, including 177 goat meat, 86 goat stomachs, 139 ready to eat (RTE) goat skewers, and 242 goat faecal samples were examined for the presence of Campylobacter jejuni and Campylobacter coli using polymerase chain reaction. RESULTS: Overall, Campylobacter spp. were found in 34.6% of the examined samples. C. jejuni was isolated in 10.1% and C. coli in 26.7% of samples. Only 2.2% of all samples were positive for both species. There was a significant association between the prevalence of C. coli and the type of sample (p < 0.05). The overall prevalence of Campylobacter in different sample groups was 41.2%, 37.2%, 23.7%, and 35.1% for goat meat, goat stomachs, RTE goat skewers, and goat faecal samples, respectively. There was no significant difference (p > 0.05) between the prevalence observed in the rainy season (16.7%) and the dry season (20.0%). Moreover, the overall prevalence of Campylobacter in slaughter sites, open-air markets, warehouses, and semi-open-air markets was 28.2%, 34.2%, 35.4%, and 42.9%, respectively. Statistically, there was no influence of the sample collection site on the frequency of isolation of Campylobacter (p > 0.05). CONCLUSION: This study shows that, considering the relatively high prevalence of this pathogen, live goat and goat meat are major sources of human and environmental contamination by Campylobacter spp. in Lubumbashi.

Assessment of pfcrt 72-76 haplotypes eight years after chloroquine withdrawal in Kinshasa, Democratic Republic of Congo.

BACKGROUND: In 2001, the World Health Organization (WHO) has recommended the use of artemisinin-based combination therapy (ACT) as the first-line treatment of uncomplicated malaria cases, as monotherapies had become ineffective in many parts of the world. As a result, the Democratic Republic of Congo (DRC) withdrew chloroquine (CQ) from its malaria treatment policy in 2002 and an artesunate (AS)-amodiaquine (AQ) combination became the ACT of choice in DRC in 2005. AQ-resistance (AQR) has been reported in several parts of the world and mutations in codons 72-76 of the Plasmodium falciparum chloroquine-resistance transporter (pfcrt) gene have been strongly correlated with resistance, especially mutations encoding the SVMNT haplotype. This haplotype was first identified in Southeast Asia and South America but was recently reported in two African countries neighbouring DRC. These facts raised two questions: the first about the evolution of CQ resistance (CQR) in DRC and the second about the presence of the SVMNT haplotype, which would compromise the use of AQ as a partner drug for ACT. METHODS: A total of 213 thick blood films were randomly collected in 2010 from a paediatric clinic in Kinshasa, DRC. Microscopy controls and real-time polymerase chain reaction (RT-PCR) were performed for Plasmodium species identification. Haplotypes of the pfcrt gene were determined by sequencing. RESULTS: The K76T mutation was detected in 145 out of 198 P. falciparum-positive samples (73.2%). In these 145 resistant strains, only the CVIET haplotype was detected. CONCLUSIONS: This study is the first to assess the molecular markers of resistance to CQ and AQ after the introduction of ACT in DRC. The results suggest first that CQR is decreasing, as wild-type pfcrt haplotypes were found in only 26.8% of the samples and secondly that the SVMNT haplotype is not yet present in Kinshasa, suggesting that AQ remains valid as a partner drug for ACT in this region.

Molecular epidemiology of norovirus infections in symptomatic and asymptomatic children from Bobo Dioulasso, Burkina Faso.

BACKGROUND: Noroviruses (NoV) are a leading cause of gastroenteritis worldwide. Few epidemiological data regarding the NoV strains circulating in African countries are available. OBJECTIVES: To determine the prevalence of NoV in Bobo Dioulasso (Burkina Faso) in both symptomatic and asymptomatic gastroenteritis patients. STUDY DESIGN: Patients both with and without gastro-intestinal disorders were selected. Clinical and epidemiological data, as well as stool samples, were collected through March to April 2011. NoV molecular detection (genogrouping and genotyping) and viral load quantification were also performed for all samples. RESULTS: NoV were detected in 22.2% of the 418 collected stool samples (21.2% and 24.8% from the 293 symptomatic patients (SP) and the 125 asymptomatic patients (ASP) respectively). Genogroup (G) distribution was 7.5%, 10.2% and 3.4% for GI, GII and both GI/GII respectively among SP and 12.0%, 11.2% and 1.6% for GI, GII and both GI/GII, respectively, among ASP. Average viral load values were higher in SP than in ASP for GI (p = 0.03) but not for GII. Phylogenic analysis showed a high degree of genotype diversity in SP and ASP. One recombinant GII.7/GII.6 sequence was, to the best of our knowledge, detected for the first time. CONCLUSIONS: This study enabled identification of the specific molecular epidemiology of NoV strains circulating in a representative country in Eastern Africa, and additionally showed that ASP could play an important "reservoir" role. A high strain diversity was detected with a surprisingly high proportion of NoV GI compared to the common genotypes usually reported in comparable epidemiological studies.

Proportion of extended-spectrum ß-lactamase-producing Enterobacteriaceae in community setting in Ngaoundere, Cameroon.

BACKGROUND: There is no information regarding the resistance mechanisms of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae in community setting in Cameroon. The current study aimed to determine the proportion of ESBLs in Enterobacteriaceae isolated in the community and to analyse some risk factors associated with ESBL carriage. METHODS: Faecal samples were collected from 208 different outpatients and 150 healthy student volunteers between 3 January and 3 April 2009. Enterobacterial isolates resistant to third-generation cephalosporins were screened for ESBL production by the double-disk synergy test. Presumptive ESBL-producing isolates with positive synergy test were identified by Mass Spectrometry using the BioTyper MALDI-TOF. For such ESBL positive isolates, antibiotic susceptibility was determined by the Vitek 2 system. PCR and sequencing were performed for the detection of different types of ESBL genes in presumptive ESBL-producing isolates. Statistical methods were used for the univariate calculation of risk factors. RESULTS: During the study period, a total of 358 faecal samples were analysed; 58 of such samples (16%) showed an ESBL phenotype and were confirmed by PCR. The proportion of ESBL producers in faecal carriage was statistically different between outpatients and student volunteers (23.1% vs. 6.7%: p < 0.000). According to a univariate analysis, previous use of antibiotics (ciprofloxacin) appeared to be a risk factor for ESBL carriage (p < 0.05).Escherichia coli was the species most frequently isolated among the ESBL producers in outpatients (66.7%) and student volunteers (90%). Isolates showed additional resistance to gentamicin, ciprofloxacin and trimethoprim/sulfamethoxazole but none of them was resistant to temocillin, amikacin or meropenem. Most of the strains (97%) produced a CTX-M group 1 enzymes [CTX-M-15 (98%) or CTX-M-1 (2%)] and the remaining strains produced SHV-12 enzyme (3%). CONCLUSIONS: The use of drugs such as amoxicillin, ciprofloxacin and trimethoprim/sulfamethoxazole does not seem appropriate for empirical treatment because of emerging resistance. The implementation in Cameroon or in other African countries of methods of screening ESBL-producing organisms in routine laboratories is of great importance in order for us to offer patients appropriate treatment and for infection control efforts to succeed.

Use of a serum-free medium to produce in vitro Neospora caninum and Toxoplasma gondii tachyzoites on Vero cells.

Neospora caninum and Toxoplasma gondii are cyst-forming coccidian parasites of human and veterinary clinical relevance. In vitro cultivation of the protozoans using Vero cells is usually performed in order to produce antigenic materials. Quantitative and qualitative comparisons of Vero cells grown in RPMI medium supplemented either with foetal calf serum (FCS), horse serum (HS) or a specific serum-free additive (DefCell) were performed. A serum-free cell culture system used to propagate N. caninum (NC-1 isolate) and T. gondii tachyzoites (Rh stain) were compared with the other two cell culture systems. FCS supplemented media was found to be more effective than the others in promoting Vero cells and N. caninum tachyzoites. However, it was found unable to support adequate T. gondii tachyzoite proliferation. Vero cells, T. gondii and N. caninum tachyzoite production gave similar growth patterns with either HS or DefCell supplemented media. Defcell was considered as a good alternative to supplement culture medium.