Objective:To determine the genetic diversity of Plasmodium(P.)knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia,targeting the S-type SSU rRNA gene and including aspects of natural selection and hap...Objective:To determine the genetic diversity of Plasmodium(P.)knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia,targeting the S-type SSU rRNA gene and including aspects of natural selection and haplotype.Methods:Thirty-nine blood samples infected with P.knowlesi were collected in Sabah,Malaysian Borneo and Peninsular Malaysia.The S-type SSU rRNA gene was amplified using polymerase chain reaction,cloned into a vector,and sequenced.The natural selection and haplotype of the S-type SSU rRNA gene sequences were determined using DnaSP v6 and illustrated using NETWORK v10.This study's 39 S-type SSU rRNA sequences and eight sequences from the Genbank database were subjected to phylogenetic analysis using MEGA 11.Results:Overall,the phylogenetic analysis showed no evidence of a geographical cluster of P.knowlesi isolates from different areas in Malaysia based on the S-type SSU rRNA gene sequences.The S-type SSU rRNA gene sequences were relatively conserved and with a purifying effect.Haplotype sharing of the S-type SSU rRNA gene was observed between the P.knowlesi isolates in Sabah,Malaysian Borneo,but not between Sabah,Malaysian Borneo and Peninsular Malaysia.Conclusions:This study suggests that the S-type SSU rRNA gene of P.knowlesi isolates in Sabah,Malaysian Borneo,and Peninsular Malaysia has fewer polymorphic sites,representing the conservation of the gene.These features make the S-type SSU rRNA gene suitable for comparative studies,such as determining the evolutionary relationships and common ancestry among P.knowlesi species.展开更多
Supported by The Ministry of S&T, “Construction of the Platform of Natural Resourses”(No.2004DKA30480) LM Malaria remains one of the most devasting infectious diseases in the world. The annual case number of the...Supported by The Ministry of S&T, “Construction of the Platform of Natural Resourses”(No.2004DKA30480) LM Malaria remains one of the most devasting infectious diseases in the world. The annual case number of the disease is estimated to be 350～500 million, and it causes about 1～3 million deaths each year, most of them are children. Due to the emergence of the drug resistance of the parasite and the insecticide resistance of mosquitoes, it becomes more difficult to control malaria. Furthermore, Simian malaria parasites infecting human were reported many times,which might cause severe public health problem. Since prevention of malaria epidemics hasn’t achieved tremendous success, emergence of primate malaria infection in human makes the situation even more complicated. Recently when we examine retrospectively the smears of “P. vivax” collected from Yunnan Province(the patient had spent sometimes in China-Myanmar border,logging), we found the similarity in morphology between the smears of the Yunnan patient and the naturally acquired P. knowlesi infection of human reported in Thailand, which belongs to primate malaria. P. knowlesi usually infects monkeys in nature and is shown to be infective to human occasionally from the report of oversea. It hadn’t drawn much attention of scientists, because its clinic symptoms were neither specific nor severe. The morphology of P. knowlesi in human infection could not easily differentiate with other Plasmodium species, especially for atypical form of P. vivax and P.malariae. We observed all stages of the erythrocytic cycle of the parasite in Giemsa-stained blood films. Early trophozoites appeared as ring forms as early trophozoites of P. falciparum in multi-infection,except the rings were a little larger. Frequently, more than one ring forms were noted in erythrocytes and double or tri-chromatin dots were also seen. Late trophozoites were usually amoeboid, as that of P. vivax. Sometimes, the cytoplasm was compact, which occupied no more than two-third of the erythrocytes. Some late trophozoites had a trend to form “band” that are typical for P. malariae. Mature schizonts had a central cluster of 8 to 16 merozoites and did not fill the whole erythrocyte. Late trophozoites and schizonts were densely pigmented with dark brown/black malaria pigment. Gametocytes filled most part of the erythrocytes and malaria pigment was scattered. The structure of gametocytes was compatible with that of P. vivax,but smaller. From the observation above, we can’t conclude which species of Plasmodium the patient was infected with. A DNA-based diagnosis with the polymerase chain reaction (PCR) method targeting the small subunit ribosomal RNA (SSU rRNA) genes of two human malaria species and P. knowlesi was introduced. The blood films were scraped using a clean blade. All the films were incubated overnight at 37℃ and extracted with pheonl-chlorolform and DNA was precipitated with isopropanol. Using primers targeting SSU rRNA of different Plasmodium species, the extacted DNA was amplified by PCR with different primes for P.falciparum, P.vivax and P. knowlesi,respectively. The primer pair specific for P. knowlesi produced a single fragment of 150 bp. The amplified fragments were digested by endonuclease, cloned into T-vector, and then transformed into E. coli XL-10. Analysis of sequences showed that the amplified fragment was identical to the sequence of P. knowlesi. This result confirmed that the patient was infected with P. knowlesi, not P. vivax. A pair of primer was designed according to the reported msp-1 partial sequence of P. knowlesi. The partial msp-1 encoding gene was amplified by PCR from the extracted DNA. The amplified fragments were digested by endonuclease, cloned into pET-32a vector, and then transformed into E. coli XL-10. Analysis of sequences showed that the partial msp-1 gene was identical to the sequence of P. knowlesi, which was deposited in the GenBank previously. The recombinant plasmid pET-32a/msp-1 was transformed into E. coli BL21(DE3), and then the positive recombinant clone was expressed by induction with IPTG. The expressed E. coli BL21(DE3) was analyzed by SDS-PAGE gel electrophoresis. A 31 kDa fusion protein band could be discerned. E. coli BL21(DE3) containing recombinant msp-1 was disintegrated with supersonic, then centrifuged. SDS-PAGE gel electrophoresis showed that the msp-1 is expressed in E. coli as precipitation. The protein was purified by Ni-NTA affinity chromatography column. Western blot result showed that the purified protein could react with the blood serum from the patient infected with P.falciparum and its molecular weight accorded with the theoretical expectation. This is the first report of naturally human infection of P. knowlesi in China,and also established the basis for molecular diagnosis and differentiation of Plasmodium species. Further working idea for identifying the P. vivax multinucleatum, and the importance of human infection of simian malaria are discussed.展开更多
Objective:To develop a new technique for diagnosis of Plasmodium knowlesi and at the same time to be able to discriminate among the diverse species of Plasmodium causing human malaria.Methods:In this study the nested ...Objective:To develop a new technique for diagnosis of Plasmodium knowlesi and at the same time to be able to discriminate among the diverse species of Plasmodium causing human malaria.Methods:In this study the nested multiplex malaria PCR was redesigned,targeting the 18S rR NA gene,to identify the fifth human Plasmodium species,Plasmodium knowlesi,together with the other human Plasmodium(Plasmodium falciparum,Plasmodium vivax,Plasmodium ovale and Plasmodium malariae)by amplified fragment size using only two amplification processes and including an internal reaction control to avoid false negatives.Results:The technique was validated with 91 clinical samples obtained from patients with malaria compatible symptoms.The technique showed high sensitivity(100%)and specificity(96%)when it was compared to the reference method employed for malaria diagnosis in the Instituto de Salud Carlos栿and a published real-time PCR malaria assay.Conclusions:The technique designed is an economical,sensitive and specific alternative to current diagnosis methods.Furthermore,the method might be tested in knowlesi-malaria endemic areas with a higher number of samples to confirm the quality of the method.展开更多
Objective:To determine the genetic diversity,natural selection and mutations in Plasmodium(P.)knowlesi drug resistant molecular markers Kelch 13 and dhps gene in clinical samples of Malaysia.Methods:P.knowlesi full-le...Objective:To determine the genetic diversity,natural selection and mutations in Plasmodium(P.)knowlesi drug resistant molecular markers Kelch 13 and dhps gene in clinical samples of Malaysia.Methods:P.knowlesi full-length gene sequences Kelch 13 gene(PkK13)from 40 samples and dhps gene from 30 samples originating from Malaysian Borneo were retrieved from public databases.Genetic diversity,natural selection,and phylogenetic analysis of gene sequences were analysed using DNAsp v5.10 and MEGA v5.2.Results:Seventy-two single nucleotide polymorphic sites(SNPs)across the full-length PkK13 gene(63 synonymous substitutions and 9 non-synonymous substitutions)with nucleotide diversity ofπ~0.005 was observed.Analysis of the full-length Pkdhps gene revealed 73 SNPs andπ~0.006(44 synonymous substitutions and 29 non-synonymous substitutions).A high number of haplotypes(PkK13;H=37 and Pkdhps;H=29)with haplotype diversity of Hd~0.99 were found in both genes,indicating population expansion.Nine mutant alleles were identified in PkK13 amino acid alignment of which,7(Asp3Glu,Lys50Gln,Lys53Glu,Ser123Thr,Ser127Pro,Ser149Thr and Ala169Thr)were within the Plasmodium specific domain,2(Val372Ile and Lys424Asn)were in the BTB/POZ domain and no mutation was observed within the kelch propeller domain.The 29 non-synonymous mutations in the Pkdhps gene were novel and only presented in exon 1 and 2.Conclusions:Monitoring the mutations from clinical samples collected from all states of Malaysia along with clinical efficacy studies will be necessary to determine the drug resistance in P.knowlesi.展开更多
To determine the distribution of Plasmodium (P) species including Plasmodium knowlesi and to compare the specificity and sensitivity of microscopy with nested PCR in malaria diagnosis.MethodsThe study was conducted in...To determine the distribution of Plasmodium (P) species including Plasmodium knowlesi and to compare the specificity and sensitivity of microscopy with nested PCR in malaria diagnosis.MethodsThe study was conducted in Kawthaung, southern Myanmar. Ninety clinically suspected malaria patients were screened for malaria by Giemsa stained microscopy and confirmed by nested PCR.ResultsAmong the participants, 57 (63.3%) were positive and 33 (36.7%) were negative by microscopy. Of positive samples, 39 (68.4%) were Plasmodium falciparum, 17 (29.8%) Plasmodium vivax and 1 (1.8%) Plasmodium malariae, whereas 59-amplified by PCR were 40 (67.8%), 18 (30.5%) and 1 (1.7%) respectively. PCR amplified 2 microscopy negative samples. Two samples of P. falciparum detected by microscopy were amplified as P. vivax and vice versa. All samples were negative for Plasmodium ovale, P. knowlesi and mixed infections. Microscopy had a very good measure of agreement (κ = 0.95) compared to nested PCR. Sensitivity and specificity of microscopy for diagnosis of P. falciparum were 92.5% (95% CI: 79.6-98.4) and 96.0% (95% CI: 86.3-99.5) respectively, whereas for P. vivax were 83.3% (95% CI: 58.6-96.4) and 97.2% (95% CI: 90.3-99.7).ConclusionsP. knowlesi was not detected by both microscopy and PCR. Giemsa stained microscopy can still be applied as primary method for malaria diagnosis and is considered as gold standard. As to the lower sensitivity of microscopy for vivax malaria, those with previous history of malaria and relapse cases should be diagnosed by RDT or PCR combined with microscopy. Inaccuracy of species diagnosis highlighted the requirement of training and refresher courses for microscopists.展开更多
Objective To study the histopathological changes of relevant intemal organs of Macaca mulatta infected with Plasmodium knowlesi ( P. knowlesi).Methods Histopathological examination of 3 monkeys who died of P. knowles...Objective To study the histopathological changes of relevant intemal organs of Macaca mulatta infected with Plasmodium knowlesi ( P. knowlesi).Methods Histopathological examination of 3 monkeys who died of P. knowlesi infection, 2 P. knowlesi infected monkeys who died of treatment failure with artesunate suppository and 1 P. knowlesi infected monkey that was cured by piperaquine phosphate (PQP) but died of trauma and necrosis of the fore limb.Results The heart, liver, spleen, lung, kidney, brain, pancreas, parathyriod, pituitary and lymph nodes showed severe pathological changes in 3 monkeys (No. 1, 7 and 12) who died of P. knowlesi infection and 1 infected monkey (No. 72) who died of treatment failure with artesunate suppository. Red blood cells containing rmalarial parasites and pigments were concentrated in the capillaries of these organs. Malarial pigments were deposited in many organs or phagocytized by macrophages in 1 monkey (No. 131 ), it was cured by piperaquine phosphate but died of trauma and necrosis of the fore limb; cellular atrophy and disappearance of pancreatic islets, parathyroid and pituitary cells were also observed. One monkey (No.33) treated with artesunate suppository, showed that blood parasites became negative but recrudesced and pituiary later died from a gavage accident. Its organs showed a significant difference to those of the infected monkeys receiving no treatment. Only the liver Kupffer cells and cerebral matrix contained malarial parasites and pigments; many relevant intemal organs showed repair.Conclusion The pathological changes of relevant internal organs of Macaca mulatta infected with P. knowlesi were examined in detail, especially cellular atrophy and the disappearance of pancreatic islets, parathyoid and pituitary cells and myolysis of cardiac muscles. These changes have not previously been reported elsewhere.展开更多
基金This study was supported by the Ministry of Higher Education,Malaysia(FRGS0322-SG-1/2013)Universiti Malaysia Sabah(GUG0521-2/2020).
文摘Objective:To determine the genetic diversity of Plasmodium(P.)knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia,targeting the S-type SSU rRNA gene and including aspects of natural selection and haplotype.Methods:Thirty-nine blood samples infected with P.knowlesi were collected in Sabah,Malaysian Borneo and Peninsular Malaysia.The S-type SSU rRNA gene was amplified using polymerase chain reaction,cloned into a vector,and sequenced.The natural selection and haplotype of the S-type SSU rRNA gene sequences were determined using DnaSP v6 and illustrated using NETWORK v10.This study's 39 S-type SSU rRNA sequences and eight sequences from the Genbank database were subjected to phylogenetic analysis using MEGA 11.Results:Overall,the phylogenetic analysis showed no evidence of a geographical cluster of P.knowlesi isolates from different areas in Malaysia based on the S-type SSU rRNA gene sequences.The S-type SSU rRNA gene sequences were relatively conserved and with a purifying effect.Haplotype sharing of the S-type SSU rRNA gene was observed between the P.knowlesi isolates in Sabah,Malaysian Borneo,but not between Sabah,Malaysian Borneo and Peninsular Malaysia.Conclusions:This study suggests that the S-type SSU rRNA gene of P.knowlesi isolates in Sabah,Malaysian Borneo,and Peninsular Malaysia has fewer polymorphic sites,representing the conservation of the gene.These features make the S-type SSU rRNA gene suitable for comparative studies,such as determining the evolutionary relationships and common ancestry among P.knowlesi species.
文摘Supported by The Ministry of S&T, “Construction of the Platform of Natural Resourses”(No.2004DKA30480) LM Malaria remains one of the most devasting infectious diseases in the world. The annual case number of the disease is estimated to be 350～500 million, and it causes about 1～3 million deaths each year, most of them are children. Due to the emergence of the drug resistance of the parasite and the insecticide resistance of mosquitoes, it becomes more difficult to control malaria. Furthermore, Simian malaria parasites infecting human were reported many times,which might cause severe public health problem. Since prevention of malaria epidemics hasn’t achieved tremendous success, emergence of primate malaria infection in human makes the situation even more complicated. Recently when we examine retrospectively the smears of “P. vivax” collected from Yunnan Province(the patient had spent sometimes in China-Myanmar border,logging), we found the similarity in morphology between the smears of the Yunnan patient and the naturally acquired P. knowlesi infection of human reported in Thailand, which belongs to primate malaria. P. knowlesi usually infects monkeys in nature and is shown to be infective to human occasionally from the report of oversea. It hadn’t drawn much attention of scientists, because its clinic symptoms were neither specific nor severe. The morphology of P. knowlesi in human infection could not easily differentiate with other Plasmodium species, especially for atypical form of P. vivax and P.malariae. We observed all stages of the erythrocytic cycle of the parasite in Giemsa-stained blood films. Early trophozoites appeared as ring forms as early trophozoites of P. falciparum in multi-infection,except the rings were a little larger. Frequently, more than one ring forms were noted in erythrocytes and double or tri-chromatin dots were also seen. Late trophozoites were usually amoeboid, as that of P. vivax. Sometimes, the cytoplasm was compact, which occupied no more than two-third of the erythrocytes. Some late trophozoites had a trend to form “band” that are typical for P. malariae. Mature schizonts had a central cluster of 8 to 16 merozoites and did not fill the whole erythrocyte. Late trophozoites and schizonts were densely pigmented with dark brown/black malaria pigment. Gametocytes filled most part of the erythrocytes and malaria pigment was scattered. The structure of gametocytes was compatible with that of P. vivax,but smaller. From the observation above, we can’t conclude which species of Plasmodium the patient was infected with. A DNA-based diagnosis with the polymerase chain reaction (PCR) method targeting the small subunit ribosomal RNA (SSU rRNA) genes of two human malaria species and P. knowlesi was introduced. The blood films were scraped using a clean blade. All the films were incubated overnight at 37℃ and extracted with pheonl-chlorolform and DNA was precipitated with isopropanol. Using primers targeting SSU rRNA of different Plasmodium species, the extacted DNA was amplified by PCR with different primes for P.falciparum, P.vivax and P. knowlesi,respectively. The primer pair specific for P. knowlesi produced a single fragment of 150 bp. The amplified fragments were digested by endonuclease, cloned into T-vector, and then transformed into E. coli XL-10. Analysis of sequences showed that the amplified fragment was identical to the sequence of P. knowlesi. This result confirmed that the patient was infected with P. knowlesi, not P. vivax. A pair of primer was designed according to the reported msp-1 partial sequence of P. knowlesi. The partial msp-1 encoding gene was amplified by PCR from the extracted DNA. The amplified fragments were digested by endonuclease, cloned into pET-32a vector, and then transformed into E. coli XL-10. Analysis of sequences showed that the partial msp-1 gene was identical to the sequence of P. knowlesi, which was deposited in the GenBank previously. The recombinant plasmid pET-32a/msp-1 was transformed into E. coli BL21(DE3), and then the positive recombinant clone was expressed by induction with IPTG. The expressed E. coli BL21(DE3) was analyzed by SDS-PAGE gel electrophoresis. A 31 kDa fusion protein band could be discerned. E. coli BL21(DE3) containing recombinant msp-1 was disintegrated with supersonic, then centrifuged. SDS-PAGE gel electrophoresis showed that the msp-1 is expressed in E. coli as precipitation. The protein was purified by Ni-NTA affinity chromatography column. Western blot result showed that the purified protein could react with the blood serum from the patient infected with P.falciparum and its molecular weight accorded with the theoretical expectation. This is the first report of naturally human infection of P. knowlesi in China,and also established the basis for molecular diagnosis and differentiation of Plasmodium species. Further working idea for identifying the P. vivax multinucleatum, and the importance of human infection of simian malaria are discussed.
基金supported by AESI-ISC Ⅲ grant number PI14C Ⅲ/00014
文摘Objective:To develop a new technique for diagnosis of Plasmodium knowlesi and at the same time to be able to discriminate among the diverse species of Plasmodium causing human malaria.Methods:In this study the nested multiplex malaria PCR was redesigned,targeting the 18S rR NA gene,to identify the fifth human Plasmodium species,Plasmodium knowlesi,together with the other human Plasmodium(Plasmodium falciparum,Plasmodium vivax,Plasmodium ovale and Plasmodium malariae)by amplified fragment size using only two amplification processes and including an internal reaction control to avoid false negatives.Results:The technique was validated with 91 clinical samples obtained from patients with malaria compatible symptoms.The technique showed high sensitivity(100%)and specificity(96%)when it was compared to the reference method employed for malaria diagnosis in the Instituto de Salud Carlos栿and a published real-time PCR malaria assay.Conclusions:The technique designed is an economical,sensitive and specific alternative to current diagnosis methods.Furthermore,the method might be tested in knowlesi-malaria endemic areas with a higher number of samples to confirm the quality of the method.
基金supported by the institutional funding committee of Najran University,Najran,Saudi Arabia(Project code:NU/IFC/ENT/01/007).
文摘Objective:To determine the genetic diversity,natural selection and mutations in Plasmodium(P.)knowlesi drug resistant molecular markers Kelch 13 and dhps gene in clinical samples of Malaysia.Methods:P.knowlesi full-length gene sequences Kelch 13 gene(PkK13)from 40 samples and dhps gene from 30 samples originating from Malaysian Borneo were retrieved from public databases.Genetic diversity,natural selection,and phylogenetic analysis of gene sequences were analysed using DNAsp v5.10 and MEGA v5.2.Results:Seventy-two single nucleotide polymorphic sites(SNPs)across the full-length PkK13 gene(63 synonymous substitutions and 9 non-synonymous substitutions)with nucleotide diversity ofπ~0.005 was observed.Analysis of the full-length Pkdhps gene revealed 73 SNPs andπ~0.006(44 synonymous substitutions and 29 non-synonymous substitutions).A high number of haplotypes(PkK13;H=37 and Pkdhps;H=29)with haplotype diversity of Hd~0.99 were found in both genes,indicating population expansion.Nine mutant alleles were identified in PkK13 amino acid alignment of which,7(Asp3Glu,Lys50Gln,Lys53Glu,Ser123Thr,Ser127Pro,Ser149Thr and Ala169Thr)were within the Plasmodium specific domain,2(Val372Ile and Lys424Asn)were in the BTB/POZ domain and no mutation was observed within the kelch propeller domain.The 29 non-synonymous mutations in the Pkdhps gene were novel and only presented in exon 1 and 2.Conclusions:Monitoring the mutations from clinical samples collected from all states of Malaysia along with clinical efficacy studies will be necessary to determine the drug resistance in P.knowlesi.
基金supported by the CERVIE (Centre of Research and Excellence for Research,Value Innovation and Entrepreneurship),UCSI University (Research Grant Scheme (RGS)[No.:Proj-In-FMS-020]support of CERVIE,UCSI University
文摘To determine the distribution of Plasmodium (P) species including Plasmodium knowlesi and to compare the specificity and sensitivity of microscopy with nested PCR in malaria diagnosis.MethodsThe study was conducted in Kawthaung, southern Myanmar. Ninety clinically suspected malaria patients were screened for malaria by Giemsa stained microscopy and confirmed by nested PCR.ResultsAmong the participants, 57 (63.3%) were positive and 33 (36.7%) were negative by microscopy. Of positive samples, 39 (68.4%) were Plasmodium falciparum, 17 (29.8%) Plasmodium vivax and 1 (1.8%) Plasmodium malariae, whereas 59-amplified by PCR were 40 (67.8%), 18 (30.5%) and 1 (1.7%) respectively. PCR amplified 2 microscopy negative samples. Two samples of P. falciparum detected by microscopy were amplified as P. vivax and vice versa. All samples were negative for Plasmodium ovale, P. knowlesi and mixed infections. Microscopy had a very good measure of agreement (κ = 0.95) compared to nested PCR. Sensitivity and specificity of microscopy for diagnosis of P. falciparum were 92.5% (95% CI: 79.6-98.4) and 96.0% (95% CI: 86.3-99.5) respectively, whereas for P. vivax were 83.3% (95% CI: 58.6-96.4) and 97.2% (95% CI: 90.3-99.7).ConclusionsP. knowlesi was not detected by both microscopy and PCR. Giemsa stained microscopy can still be applied as primary method for malaria diagnosis and is considered as gold standard. As to the lower sensitivity of microscopy for vivax malaria, those with previous history of malaria and relapse cases should be diagnosed by RDT or PCR combined with microscopy. Inaccuracy of species diagnosis highlighted the requirement of training and refresher courses for microscopists.
文摘Objective To study the histopathological changes of relevant intemal organs of Macaca mulatta infected with Plasmodium knowlesi ( P. knowlesi).Methods Histopathological examination of 3 monkeys who died of P. knowlesi infection, 2 P. knowlesi infected monkeys who died of treatment failure with artesunate suppository and 1 P. knowlesi infected monkey that was cured by piperaquine phosphate (PQP) but died of trauma and necrosis of the fore limb.Results The heart, liver, spleen, lung, kidney, brain, pancreas, parathyriod, pituitary and lymph nodes showed severe pathological changes in 3 monkeys (No. 1, 7 and 12) who died of P. knowlesi infection and 1 infected monkey (No. 72) who died of treatment failure with artesunate suppository. Red blood cells containing rmalarial parasites and pigments were concentrated in the capillaries of these organs. Malarial pigments were deposited in many organs or phagocytized by macrophages in 1 monkey (No. 131 ), it was cured by piperaquine phosphate but died of trauma and necrosis of the fore limb; cellular atrophy and disappearance of pancreatic islets, parathyroid and pituitary cells were also observed. One monkey (No.33) treated with artesunate suppository, showed that blood parasites became negative but recrudesced and pituiary later died from a gavage accident. Its organs showed a significant difference to those of the infected monkeys receiving no treatment. Only the liver Kupffer cells and cerebral matrix contained malarial parasites and pigments; many relevant intemal organs showed repair.Conclusion The pathological changes of relevant internal organs of Macaca mulatta infected with P. knowlesi were examined in detail, especially cellular atrophy and the disappearance of pancreatic islets, parathyoid and pituitary cells and myolysis of cardiac muscles. These changes have not previously been reported elsewhere.
